A KOPONYATRAUMA ÁLTAL KIVÁLTOTT DIFFÚZ AXONÁLIS ÉS NEURONÁLIS KÁROSODÁS PATHOMECHANIZMUSÁNAK VIZSGÁLATA ÉS TERÁPIÁS BEFOLYÁSOLÁSÁNAK LEHETŐSÉGEI

Átírás

1 A KOPONYATRAUMA ÁLTAL KIVÁLTOTT DIFFÚZ AXONÁLIS ÉS NEURONÁLIS KÁROSODÁS PATHOMECHANIZMUSÁNAK VIZSGÁLATA ÉS TERÁPIÁS BEFOLYÁSOLÁSÁNAK LEHETŐSÉGEI Doktori (PhD) értekezés Dr. Farkas Orsolya Témavezető: Dr. Gallyas Ferenc Dr. Büki András Elméleti Orvostudományok, Doktori Iskola vezetője: Dr. Szolcsányi János Kísérletes Neurológia, Programvezető: Dr. Gallyas Ferenc ( ig) Klinikai Orvostudományok, Doktori Iskola vezetője: Dr. Nagy Judit Klinikai Idegtudományok, Programvezető: Dr. Komoly Sámuel Pécsi Tudományegyetem, Általános Orvostudományi Kar Pécs, 2007

2 TARTALOMJEGYZÉK RÖVIDÍTÉSEK... 4 AZ ÉRTEKEZÉST KÉPEZŐ SAJÁT KÖZLEMÉNYEK LISTÁJA BEVEZETÉS A baleseti agysérülések epidemiológiai jelentősége A baleseti agysérülések osztályozása IRODALMI HÁTTÉR A diffúz axonális károsodás (diffuse axonal injury, DAI) Az axonduzzadás/axonballon-képződés (AD/B) Az axonduzzadás/axonballon-képződés felfedezése Az axonduzzadás/axonballon-képződés mechanizmusa Az ultrastrukturális (neurofilament) kompakció (UC, NFC) A DAI terápiájának, illetve klinikai-kémiai diagnosztizálhatóságának biokémiai alapjai Az axolemmális permeabilitási zavar okozta Ca 2+ beáramlás A calpain aktiváció és a calpain-mediált spektrin bontás A mitokondriális károsodás és a caspase-3 aktiváció A DAI terápiás befolyásolását célzó vizsgálatok A diffúz neuronális károsodás A sötét -idegsejt képződés A neuronális mechanoporáció CÉLKITŰZÉSEK ANYAGOK ÉS MÓDSZEREK A diffúz agysérülés modellezése Az impact-accelerációs koponyatrauma modell Szelektív ultrastrukturális kompakciót kiváltó koponyatrauma modell Szövettani feldolgozás Az argyrophil-iii típusú ezüstözési eljárás Immunhisztokémiai technikák Elektronmikroszkópos előkészítés Western blot Permeabilitás-vizsgálat

3 5. EREDMÉNYEK ÉS KÖVETKEZTETÉSEK A traumás axonkárosodás vizsgálata Az ultrastrukturális axon-kompakció vizsgálata Az ultrastrukturális axon-kompakció és az argyrophil axonkárosodás kapcsolata Ultrastrukturális kompakció szelektív előidézésére alkalmas koponyatrauma modell kidolgozása A kompaktálódott axonok sorsának vizsgálata szelektív ultrastrukturális kompakciót kiváltó koponyatrauma modellben Az ultrastrukturális axon- illetve idegsejt-kompakció mechanizmusának tisztázása Terápiás beavatkozások tesztelése Egy sejt-permeábilis calpain-inhibitor, az MDL-28170, hatásának vizsgálata a diffúz axonkárosodásra A trauma előtt illetve után iv- illetve icv-adott PACAP hatásának vizsgálata; valamint a hatásos dózis megállapítása A terápiás ablak meghatározása icv PACAP-kezelés esetén A neuronális mechanoporáció, a resealing és a CMSP vizsgálata A calpain- és caspase-aktiválódás szerepe a humán traumás agykárosodásban A TÉMÁBAN ELÉRT, NEMZETKÖZI-JELENTŐSÉGŰ EREDMÉNYEK IRODALOMJEGYZÉK KÖSZÖNETNYILVÁNÍTÁS AZ ÉRTEKEZÉST KÉPEZŐ SAJÁT KÖZLEMÉNYEK

4 RÖVIDÍTÉSEK AD/B APP CMSP CsA CSF CSpT DAB DAI EM GCS GOS IAT ICP icv iv KIR MLF MPT NF NFC PACAP SBDP TAI UC Axonduzzadás/axonballon-képződés Amyloid Precursor Protein Calpain-mediált spektrin-bontás (Calpain-mediated Spectrin Proteolysis) Cyclosporin A Cerebrospinális folyadék (liquor) Kortikospinális pálya (Corticospinal Tract) Diamino-benzidin Diffúz axonális károsodás (Diffuse Axonal Injury) Elektronmikroszkóp Glasgow kóma skála (Glasgow Coma Scale) Glasgow kimenetel skála (Glasgow Outcome Scale) Axonális transzport-zavar (Impaired Axonal Transport) Intrakraniális nyomás (Intracranial Pressure) intracerebroventrikuláris intravénás Központi Idegrendszer Mediális hosszanti köteg (Medial Longitudinal Fascicle) Mitokondriális permeabilitási átmenet (Mithochondrial Permeability Transition) Neurofilament Neurofilament kompakció (Neurofilament Compaction) Hipofízis adenilát-cikláz aktiváló polipeptid (Pytuitary Adenilate Cyclase Activating Polypeptide) Spektrin degradációs termék (Spectrin Breakdown Product) Traumás axonkárosodás (Traumatic Axonal Injury; a DAI állatkísérletes megfelelője) Ultrastrukturális kompakció (Ultrastructural Compaction) 4

5 AZ ÉRTEKEZÉST KÉPEZŐ SAJÁT KÖZLEMÉNYEK LISTÁJA I. Gallyas F, Farkas O, Mazlo M. Traumatic compaction of the axonal cytoskeleton induces argyrophilia: histological and theoretical importance. Acta Neuropathol (Berl) Jan;103(1): IF: II. Pal J, Toth Z, Farkas O, Kellenyi L, Doczi T, Gallyas F. Selective induction of ultrastructural (neurofilament) compaction in axons by means of a new head-injury apparatus. J Neurosci Methods Jun 15;153(2): Epub 2005 Dec 27. IF: III. Gallyas F, Pal J, Farkas O, Doczi T. The fate of axons subjected to traumatic ultrastructural (neurofilament) compaction: an electron-microscopic study. Acta Neuropathol (Berl) Mar;111(3): IF: IV. Gallyas F, Farkas O, Mazlo M. Gel-to-gel phase transition may occur in mammalian cells: Mechanism of formation of "dark" (compacted) neurons. Biol Cell May;96(4): IF:2.233 V. Farkas O, Lifshitz J, Povlishock JT. Mechanoporation induced by diffuse traumatic brain injury: an irreversible or reversible response to injury? J Neurosci Mar 22;26(12): IF: VI. Buki A, Farkas O, Doczi T, Povlishock JT. Preinjury administration of the calpain inhibitor MDL attenuates traumatically induced axonal injury. J Neurotrauma Mar;20(3): IF: VII. Farkas O, Tamas A, Zsombok A, Reglodi D, Pal J, Buki A, Lengvari I, 5

6 Povlishock JT, Doczi T. Effects of pituitary adenylate cyclase activating polypeptide in a rat model of traumatic brain injury. Regul Pept Dec 15;123(1-3): IF: VIII. Tamas A, Zsombok A, Farkas O, Reglodi D, Pal J, Buki A, Lengvari I, Povlishock JT, Doczi T. Postinjury administration of pituitary adenylate cyclase activating polypeptide (PACAP) attenuates traumatically induced axonal injury in rats. J Neurotrauma May;23(5): IF: IX. Farkas O, Polgar B, Szekeres-Bartho J, Doczi T, Povlishock JT, Buki A. Spectrin breakdown products in the cerebrospinal fluid in severe head injury--preliminary observations. Acta Neurochir (Wien) Aug;147(8): IF:

7 1. BEVEZETÉS 1.1. A baleseti agysérülések epidemiológiai jelentősége A balesetek során bekövetkező agysérülés gyakorisága a motorizációval párhuzamosan emelkedik; a fejlett ipari országokban a 40 év alatti lakosság körében a vezető halálokot képezi. A WHO felmérései szerint 10 millió baleseti agysérülésből következő haláleset, vagy kórházi ápolás történik világszerte minden évben, és a világon kb. 57 millió ember szenvedett élete során legalább egyszer traumás agysérülést [64]. A National Institute of Health felmérései alapján az USA-ban 1.4 millió baleseti agysérülés történik évente, ebből halálos kimenetelű, kórházi kezelést igényel, és 5.3 millió ember él baleseti agysérülések következtében kialakult tartós korlátozottsággal [64]. Követéses vizsgálatok igazolták, hogy a baleset után 1-3 évvel a normál populációhoz képest a baleseti agysérülést szenvedettek körében 1.8-szoros a rizikó alkohol-abúzus kialakulására [50], míg 11-szeres epilepszia (P. L. Ferguson, written communication, February 2006), 7.5-szörös halál, 1.5-szörös depresszió [49], illetve szörös Alzheimer kór előfordulására [103]. A silent epidemic -ként emlegetett népbetegség, a baleseti agysérülés, Magyarországon is hasonló nagyságrendű epidemiológiai probléma. Egy 2002-ben készült, a 2001-es év adataira vonatkozóan retrospektív, valamint egy három-hónapos időszakra vonatkozóan prospektív esetelemző tanulmány [24] szerint hazánkban a kórházba kerülő koponya-agysérültek évi száma közel , ebből 71.3% enyhe, 19.4% közepes, és 9.4% a súlyos kategóriába tartozik. A sérültek 67%-a férfi, 33%-a nő, 78%-a 60 év alatti, 36%-a 40 éves kor alatti. A prospektív adatok szerint a kórházakba súlyosként (GCS 8) került koponya-agysérültek 55%-a, míg a felvételkor közepesen súlyosnak (GCS=9-12) illetve enyhének (GCS=13-15) ítélt, de az első 24 órában tovább súlyosbodó állapotú sérültek 35%-a hal meg az akut ellátás során; ami jóval magasabb, mint a világ fejlett részén közölt halálozási adatok. Ennek hátterében a prehospitális és a hospitális ellátás közötti kommunikációs zavar, a definitív ellátásig, illetve az első CT-vizsgálatig eltelő extrém hosszú idő, továbbá az állhat, hogy a magyar és a nemzetközi ellátási irányelvek nem kerülnek széles körben alkalmazásra. A túlélők közül az elbocsátáskor 40% tartós vegetatív állapotú, vagy súlyos maradványtüneteket mutat, ami szintén jóval magasabb, mint a nemzetközi irodalomból ismert hasonló adatok. Az akut ellátást 7

8 túlélőknek csak 45%-a gyógyul enyhe maradványtünetekkel vagy maradványtünetek nélkül. A túlélők hosszú távú életminőségéről nem állnak rendelkezésre pontos adatok, de becslések szerint hazánkban a súlyos koponyasérülteknek csak mintegy negyede illeszkedhet vissza a társadalomba [24]. A fenti ijesztő epidemiológiai adatok hangsúlyozzák a baleseti agysérülések klinikai és társadalmi jelentőségét, és ezáltal a koponyatrauma különféle vonatkozásai irányába történő kutatások fontosságát A baleseti agysérülések osztályozása A koponyatrauma által kiváltott agysérülések igen heterogén betegségcsoportot képviselnek. Osztályozásuk számos szempont szerint történhet. A klinikai kép súlyossága alapján megkülönböztetünk enyhe, középsúlyos és súlyos sérüléseket. A koponyatrauma mechanizmusa alapján beszélhetünk nyílt illetve zárt sérülésekről. A koponyatrauma által okozott intrakraniális elváltozások lehetnek elsődlegesek, vagyis a mechanikai behatás közvetlen következményei, illetve másodlagosak; ezek a koponyatrauma következményeként kialakult különböző agyi szövődményekből eredeztethetők [45, 85, 119]. Az osztályozás szempontjait a jelen dolgozat tárgyát képező kutatási területeket vastag betűvel kiemelve az alábbiakban foglalhatjuk össze: I. A baleseti agysérülések osztályozása a klinikai kép súlyossága szerint (Glasgow Coma Scale alapján): Enyhe: GCS = Közepesen súlyos: GCS = 9-12 Súlyos: GCS 8 II. A baleseti agysérülések osztályozása a koponyatrauma mechanizmusa alapján: 1. Nyílt vagy penetráló: a dura mater sérül 2. Zárt vagy tompa: a dura mater intakt marad A.) Statikus: lassú erőbehatás B.) Dinamikus: hirtelen erőbehatás a.) Impakt típusú: hirtelen tompa ütés a fejre, amely ennek hatására alig mozdul el; általában kontakt erők okozzák az agysérülést b.) Impulzív típusú: a fej hirtelen mozgásba kerül vagy 8

9 mozgásból hirtelen megáll; általában tehetetlenségi erők okozzák az agysérülést (accelerációdeceleráció) III. A baleseti agysérülések osztályozása a koponyatrauma hatására kialakuló morfológiai elváltozások alapján: 1. A koponyatrauma hatására kialakuló elsődleges elváltozások A.) Fokális (gócos) Laceráció Kontúzió Intrakraniális vérzések (állományi, epidurális, subdurális vagy subarachnoidális vérzés B.) Diffúz Axonális károsodás Axonduzzadás/axonballon-képződés Ultrastrukturális (Neurofilament) kompakció Neuronális károsodás 2. A koponyatrauma hatására kialakuló másodlagos patológiás állapotok és következményeik (fokális vagy diffúz) Ödéma Ischemia Emelkedett intrakraniális nyomás A disszertáció első része e rendkívül összetett kórkép-csoportnak csak egyetlen alcsoportjával, az elsődleges diffúz morfológiai károsodásokkal foglalkozik, és vizsgálataink elsősorban állatkísérletes aspektusokra szorítkoznak. Kutatásaink másik része pedig állatkísérletes adatokra támaszkodva a humán baleseti agysérülések közvetlen vizsgálatát tűzte ki céljául. A jelen dolgozat a témához kapcsolódó szakirodalom összefoglalása (2. fejezet) és a célkitűzések ismertetése (3. fejezet) után röviden tárgyalja a kísérletek során alkalmazott módszereket (4. fejezet), a kísérletek eredményeit és legfontosabb következtetéseit (5. fejezet), külön kiemelve a témában elért legfontosabb új eredményeket (6. fejezet). Az egyes kísérletekben alkalmazott módszerek, eredmények és következtetések részletesen a kísérletekből született és a jelen értekezés alapját képező különlenyomatokban kerülnek ismertetésre. 9

10 2. IRODALMI HÁTTÉR 2.1. A diffúz axonális károsodás (diffuse axonal injury, DAI) A diffúz axonális károsodás (diffuse axonal injury, DAI) a koponyatrauma hatására kialakult primer axonális elváltozások összessége, melyek az agy egész állományára kiterjedve, ép axonok között elszórtan figyelhetők meg, egyébként többnyire ép parenchimális környezetben. A DAI klinikai jelentőségét alátámasztó adatok szerint e kórkép felelős 50 %-ban a tartós tudatzavarért, illetve 35 %-ban a mortalitásért, a nemtérfoglaló jellegű koponyasérülésekben [39]. A diffúz agysérülések kialakulásában acceleráció-deceleráció hatására ébredő nyíróerők játszanak szerepet, amelyek tipikusan motorbicikli, személygépkocsi, vagy gázolásos balesetek során fordulnak elő [1]. A kórkép klinikai megjelenésére a tudatzavart magyarázó térfoglaló elváltozás vagy metabolikus zavar nélkül fennálló komatózus tudatállapot a jellemző [39, 41, 44]. A CT-n csak kb. 20 %-ban detektálható elváltozás kicsi, petechiális vérzések formájában, elsősorban a szürke- és a fehérállomány határán, illetve a corpus callosumban és a hosszú pályák agytörzsi szakaszán [3]. A korai stádiumban végzett MRI a CT-nél hatékonyabb az elváltozások kimutatására. A nem-haemorrhagiás léziók a T2-súlyozott és a protondenzitású képeken kis, ovális vagy kerek hiperintenzív jelek formájában láthatók, míg a vérzéses léziók centrális területén hipodenzitás figyelhető meg. Ezek az elváltozások a krónikus fázisban nagyon alacsony intenzitású léziókként jelennek meg, mert hemosziderint tartalmaznak [47]. Annak ellenére, hogy a fejlettebb MRI eljárások (diffúzió-súlyozott képalkotás, diffúziós tensor képalkotás 3 dimenziós tractográfiával) megkönnyítik a kimutatást [3], a diffúz axonális károsodás teljes bizonyossággal még ma is elsősorban szövettani vizsgálattal (általában post mortem) diagnosztizálható. A diffúz axonális károsodásnak két, morfológiailag jól elkülöníthető megjelenési formája ismert: az axonduzzadás/axonballon-képződés és az ultrastrukturális (neurofilament) kompakció. A DAI-kutatások kezdetben az axonduzzadás vizsgálatára irányultak; az ultrastrukturális kompakció jelensége később került csak leírásra [98]. A klasszikus elképzelés szerint e két morfológiai jelenség kiváltásában ugyanazok a tényezők játszanak szerepet, ezáltal ugyanazokat az axonokat érintik; a legújabb nézetek szerint azonban a legtöbb esetben a kétféle elváltozás két jól-elkülöníthető axonpopulációban figyelhető meg [73, 135]. 10

11 Meg kell jegyezni, hogy a traumás axonkárosodás kutatások túlnyomó többsége különböző állatkísérletes modelleket alkalmaz a kórkép feltérképezésére és befolyásolásának vizsgálatára. Ezek a modellek természetesen nem képesek a humán viszonyokat és ezáltal a humán DAI teljes spektrumát, kiterjedését és időbeni lefolyását maradéktalanul reprodukálni, ezért helyesebb az állatkísérletekben modellezett axonkárosodást traumás axonkárosodásként (traumatic axonal injury, TAI) említeni, a DAI kifejezést pedig a humán esetekre alkalmazni [80]. Korlátai ellenére azonban az állatkísérletes modellek egyedülálló mértékben járultak hozzá a humán DAI kialakulása alapvető elemeinek feltérképezéséhez, segítségükkel ugyanis a humán DAI számos aspektusa fokális elváltozásokkal kísérve vagy anélkül megbízhatóan vizsgálható Az axonduzzadás/axonballon-képződés (AD/B) Az axonduzzadás/axonballon-képződés felfedezése A diffúz axonkárosodás fogalma régóta ismert az irodalomban. Több, mint 50 éve Strich és munkatársai fedezték fel és írták le, mint a súlyos traumás agysérülések következetes velejáróját. Súlyos koponya-agysérülésben meghalt betegek post mortem vizsgálata során számottevő fokális léziók hiányában az etiológiát kutatva Strich ballonszerű axontágulatokat figyelt meg a fehérállományban normális axonok között elszórtan, amely tágulatoktól disztális axonszakasz a túlélési idő növekedésével Wallerféle degenerációt és myelin-degradációt szenvedett [137, 138]. Ezt az axonballonképződést és az azt követő fehérállományi degenerációt Strich a trauma pillanatában kialakult azonnali axonszakadás következményének tartotta. Hipotézise szerint a trauma által okozott mechanikus erők az axon szakadásához, az érintett proximális axonvég következményes retrakciójához és az axoplasma kiboltosulásához vezetnek, létrehozva ezzel a retrakciós axonballont a kórkép korai szakaszában; ehhez társul a későbbiekben a disztális axonszakasz Waller-féle degenerációja [137]. E korai megfigyelések óta számos kutató megerősítette, hogy a traumás fehérállományi károsodás a koponyasérülések konzekvens velejárója azokban az esetekben, amelyekben térfoglaló agyi elváltozások nem mutathatóak ki, a beteg mégis klinikailag súlyos állapotban van. Strich a megfigyelt elváltozásokat még mint a fehérállomány diffúz degenerációját vagy traumás idegrost szakadást említi. A kórkép ma is használatos elnevezése, a diffúz axonális károsodás, Adamstől származik [1], és arra utal, hogy a károsodott axonok elszórtan, ép axonok 11

12 között az agy egész állományára kiterjedve fordulnak elő. Adams és kollégái hangsúlyozták Strich óta először, hogy a kórkép kialakulásában az axonkárosodás a koponyasérülés elsődleges következménye, szembeszállva ezzel sok támadással, amelyek az axonális károsodást másodlagos ödéma, hypotensio és/vagy hypoxia következtében kialakult elváltozásoknak tartották [44]. Adams és munkatársai ugyancsak úttörő munkát végeztek a humán diffúz axonkárosodás anatómiai lokalizációjának, illetve súlyossági fokozatainak leírásában [1, 2]. E szerint a DAI típusosan a féltekei fehérállományban, a corpus callosumban, az agytörzsi hosszúpályákban, illetve kisebb gyakorisággal a kisagyban fordul elő fokális károsodás nélkül (1-es fokozat) vagy fokális léziókkal a corpus callosumban (2-es fokozat) és a rostralis agytörzsben (3-as fokozat). A kórkép Strich és Adams szerinti pathomechanizmusa azaz, hogy az axonszakadás a trauma pillanatában azonnal bekövetkezik és emiatt terápiásan nem befolyásolható évtizedekig elfogadott volt, ami a kórkép kutatását és kezelését hosszú ideig hátráltatta [6] Az axonduzzadás/axonballon-képződés mechanizmusa Az axonduzzadás/axonballon-képződés kutatásának következő mérföldkövét Povlishock és munkatársai 1983-ban közölt munkája jelentette, amely szemben a korábban elfogadott elképzeléssel kimutatta, hogy a DAI nem a trauma pillanatában azonnal és véglegesen kialakuló axonszakadásként jelentkezik, hanem a károsodott axonok döntő többségében egy, időben fokozatosan progrediáló folyamatként [106]. Azóta ez az elmélet számos állatfajban és traumamodellben, valamint humán szövetmintákon is igazolódott [6, 7, 17, 19, 30, 31, 39, 42, 43, 84, 98, , 128, 129]. A TAI kialakulásának Povlishock-féle elmélete szerint ezen axonális károsodást a középsúlyos illetve a súlyos koponyatrauma által kiváltott nyíróerők indítják el, amelyek számos fény- és elektronmikroszkóp segítségével vizsgálható intraaxonális elváltozást eredményeznek, mégpedig: A károsodott axonszakaszok a sérülést követően azonnal (<5 perc) fokális axolemmális permeabilitási zavart mutatnak ( mechanoporáció ), amely folyamatot az szemlélteti és igazolja, hogy a károsodott axonok nagy molekulasúlyú anyagokat (pl. tormagyökér-peroxidázt, vagy dextránokat) vesznek fel, amelyek az ép axolemmán keresztül az ép axonokba illetve ép neuronokba nem tudnak bejutni [98, 99, , 136]. Az axolemma fokális sérülését egyéb morfológiai jellemzők kísérik: mitokondrium duzzadás [77, 98, 99], neurotubulus veszteség [78, 79, 99], neurofilamentmódosulás [51, 52, 93, 99, 112, 113, 134], axonális transzportzavar [99, 112, 114, 136], 12

13 amely utóbbi az előre irányuló intraaxonális transzport egy ponton való fokális leállásában nyilvánul meg, és az ilyen módon szállítódó sejtalkotók illetve egyéb anyagok felhalmozódásához (organellum-akkumuláció) és következményes axonduzzadáshoz vezet [75, 79, 106, 107]. A traumától eltelt idő előrehaladtával az axonduzzadás egyre nagyobb mértékű lesz, és végül az érintett axonszakasz ballon formájában lefűződik, létrehozva ezzel a kórkép korai leírásaiból ismert proximális axonballonokat, míg a disztális axonszakasz Waller-féle degenerációt szenved [80, 106, 109, 110, 114, 115]. Az axonális elváltozások kezdetétől az axonballon kialakulásáig illetve az axonszakadásig eltelt idő a trauma súlyossága és a species függvényében változik; emberben tipikusan órákban, napokban mérhető [128, 129, 148]. Ez a tény elvi lehetőséget biztosít az axonszakadás terápiás megelőzésére (terápiás ablak). Hangsúlyozni kívánjuk, hogy a TAI-ra jellemző fenti morfológiai elváltozások a humán DAI későbbi tanulmányozása során szintén leírásra kerültek [5, 6, 19, 42, 43, 92, 128, 129]. A szóban-forgó AD/B kimutatása az előre irányuló axontranszport által szállítódó és annak károsodásakor fokálisan felhalmozódó anyagok elleni antitestek alkalmazásával végzett immunhisztokémiai módszerekkel lehetséges. A legelterjedtebb ezek közül a béta amyloid precursor protein (APP) felhalmozódásának kimutatása, amely viszonylag egyszerű, ezért a DAI diagnózisának legszélesebb körben alkalmazott módjává vált [42, 67, 128, 129], annak ellenére, hogy a legújabb vizsgálatok szerint valószínűleg alulbecsüli a DAI mértékét [73, 115, 135] Az ultrastrukturális (neurofilament) kompakció (UC, NFC) A nagy-múltú axonballon képződéssel ellentétben az intraaxonális citoszkeleton kompakciója (a neurofilamentumok közötti távolságok lényeges csökkenése, neurofilament kompakció, NFC), a mikrotubulusok számának csökkenése, a neurofilamentumok oldalkarjainak módosulása csak a 90-es években kerültek leírásra [19, 43, 51, 52, 98, 99, 113, 150]. Kezdetben úgy gondolták, hogy a citoszkeletális elváltozások ugyanazokat az axonokat érintik, mint az AD/B. A hipotézis szerint az NFC következményeként áll le az axonduzzadáshoz vezető előreirányuló intraaxonális transzport [79, 80, 98, 99, 112]. Ezt támasztották alá azok a kísérletek is, melyek szerint az intraaxonális transzportzavart mutató APP-pozitív axonok és a neurofilament kompakciót mutató RMO-14-pozitív axonok ugyanazon anatómiai régiókban fordulnak elő és befolyásolásuk ugyanazon intervenciókkal volt lehetséges [8, 9, 58, 94, 95]. Mára azonban 13

14 már világossá vált, hogy TAI két jól elkülöníthető morfológiai jellemzője, tehát az AD/B, illetve az NFC két különböző axonpopoulációt érint az esetek túlnyomó többségében, és csak kis részben figyelhető meg egyazon károsodott axonon belül, leginkább a lemniscus medialis nagy kaliberű axonjaiban, és itt is csak immunhisztokémiai módszerekkel [73, 135]. Az NFC kimutatására a közepes méretű NF alegység (NF-M) elleni RMO-14 antitestet alkalmazzák, amely az axonális citoszkeleton strukturális átrendeződése folyamán (a calpain által mediált strukturális fehérjebontás következményeként, részletesen lásd később) szabaddá vált NF-M rod domén -hez képes kötődni [65, 113]. Az immunhisztokémiai technikát alkalmazó vizsgálatok minimum 15 perccel a koponyatrauma kiváltása után tudtak axon-kompakciót kimutatni. Ugyanakkor egy speciális ezüstözési eljárással a trauma után azonnal leölt, illetve post mortem koponyatraumát szenvedett állatokban is kimutatható egy hosszú axonszakaszok megvékonyodásával járó argyrophil károsodás, amely fénymikroszkópos megjelenése lehet az NFC-nek [33-35]. E jelenség más megvilágításba helyezi az NFC kialakulásának mechanizmusában feltételezett enzimatikus reakciók kizárólagos szerepét. Az ultrastrukturális kompakció jelensége tehát ismert, azonban kialakulása pontos mechanizmusáról, a humán neuropathológiában betöltött morfológiai és neurológiai szerepéről, illetve a kompakciót mutató axonok sorsáról viszonylag keveset tudunk. Itt kívánjuk megjegyezni, hogy a nemzetközi szakirodalom az ultrastrukturális kompakció megjelölésére axonok esetén a neurofilament kompakció kifejezést használja. Ez a terminus technikus idegsejtek és dendritek esetén azonban nem használható. Ezért a továbbiakban axonális kompakció esetére fenntartjuk az NFC megjelölést, hangsúlyozva, hogy ez megegyezik az általunk egyébként használt UC-vel A DAI terápiájának, illetve klinikai-kémiai diagnosztizálhatóságának biokémiai alapjai A fentebb részletezett morfológiai vizsgálatok kiderítették, hogy a koponyatrauma által kiváltott axonszakadás nem azonnal megy végbe, hanem a trauma pillanatától számított órák, napok múlva, ami lehetőséget biztosít a terápiás megelőzésre. Ehhez a morfológiai elváltozások hátterében lejátszódó biokémiai folyamatok megismerése elengedhetetlenül szükséges. 14

15 Az axolemmális permeabilitási zavar okozta Ca 2+ beáramlás A mára elfogadottá vált nézet szerint, az axolemma sérülése (mechanoporáció) és a következményes permeabilitási zavar a trauma által kiváltott erők hatására azonnal bekövetkezik [115, 133]. Ez az érintett axonokban az ion-homeosztázis felborulásához vezet, és lehetővé teszi, hogy az extracelluláris térből Ca 2+ -ionok áramoljanak az intraaxonális térbe. A megnövekedett intraaxonális Ca 2+ -koncentráció különböző fehérjebontó enzimrendszerek aktiválódásához vezet, illetve a mitokondriumokban akkumulálódva azok duzzadását, funkcionális károsodását okozhatja. Az intracelluláris térben megnövekedett Ca 2+ -koncentráció jelenlétére eleinte csak közvetlen bizonyíték létezett a Ca 2+ -függő enzimrendszerek aktiválódásának bizonyításával, mára azonban már közvetlenül is igazolt a folyamat lejátszódása [149] A calpain aktiváció és a calpain-mediált spektrin-bontás (calpain mediated spectrin proteolysis, CMSP) A Ca 2+ -dependens folyamatok közül egyik legfontosabb a calpain aktiválódása, amelynek számos idegrendszeri kórkép kialakulásában köztük az utóbbi évek kutatásai alapján a traumás agysérülések fokális [46, 54, 87, 88, , 105, 122] illetve diffúz [8, 10, 61, 62, 125] típusában is fontos szerepe van. A calpain a cisztein proteázok családjába tartozó enzim. Több fajtája ismert, ezek közül a calpain-1 vagy µ-calpain és a calpain-2 vagy m-calpain a központi idegrendszerben mindenhol előfordul [147]. Számos szubsztrátja ismert (pl. citoszkeletális fehérjék) [15, 18, 147], és élettani szerepe van az idegsejt fejlődés és a szinaptikus struktúra kialakításában [18]; túlműködése azonban a sejt számára végzetes lehet [18, 147]. Szerepe elsősorban a necrotikus sejthalál kialakulásában nagy jelentőségű, de a legújabb kutatások szerint egyes apoptotikus folyamatok során is aktiválódik [86]. Az agyi elváltozások vizsgálata során legnagyobb figyelmet kapott calpainspecifikus folyamat a citoszkeletális fehérjék, elsősorban a spektrin specifikus bontása (calpain-mediated spectrin proteolysis, CMSP). E folyamat eredményeként a citoszkeletont alkotó nem-erythroid alfa-ii spektrin calpain-specifikus 145 kd és 150 kd nagyságú lebontási termékei (spectrin breakdown products, SBDP) a bontás helyén felszaporodnak, és specifikus antitestek segítségével kimutathatóak [120, 130]. Traumás axonkárosodás során a sérült axolemmán beáramló Ca 2+ a CMSP aktiválódásához vezet. A spektrin bontása kezdetekben csak a subaxolemmális térre korlátozódik (15-30 perc); a subaxolemmális citoszkeletális hálózat emésztődése később 15

16 további permeabilitási zavart okoz, és órák alatt egy generalizált intraaxonális proteolitikus kaszkád aktiválásához vezet, ami végső soron az axon visszafordíthatatlan károsodását eredményezi [10, 11]. A calpain-specifikus spektrin-degradációs termékek kimutathatóak a sérülés helyén a fokális koponyatrauma állatkísérletes modelljeiben [46, 53, 87, 100, 122], diffúz traumás agysérülések esetén a károsodott axonokban [10, 62, 63, 125], fokális traumát szenvedett állatok cerebrospinális folyadékában [102], illetve traumás agysérülés következtében elhunyt egyének agyában a corpus callosum területén [83]. Az a tény, hogy a spektrin-degradációs termékek nemcsak a trauma helyén, hanem a cerebrospinális folyadékban is kimutathatóak, megteremti a lehetőséget olyan potenciális biomarkerek azonosítására koponyatraumát szenvedett betegekben, melyek specifikusan kapcsolódnak a baleseti agysérülések kialakulása során lejátszódó patobiokémiai folyamatokkal, és így potenciálisan összefüggésben lehetnek az agysérülés klinikai képével, például a súlyosságával vagy kimenetelével A mitokondriális károsodás és a caspase-3 aktiváció A megnövekedett intraaxonális Ca 2+ -koncentráció a fehérjebontó folyamatok aktiválása mellett további káros folyamatokat indukálhat. Ismert, hogy az intraaxonális mitokondriumok fiziológiás körülmények között nem akkumulálják a Ca 2+ -ionokat. Ha azonban az intraaxonális Ca 2+ -koncentráció meghaladja az 5 µm-t a Ca 2+ -többlet a mitokondriumokban szekvesztrálódik [80]. Ez a folyamat a mitokondriális transzmembrán-potenciál összeomlásához és az ún. mitokondriális tranziciós permeabilitási (mitochondrial permeability transition, MPT) pórus kinyílásához vezet, lehetővé téve víz beáramlását, a mitokondrium duzzadását és szétrepedését, valamint a lokális energiaháztartás zavarát [94, 80, 151]. A mitokondriumokban akkumulálódó Ca 2+ nemcsak a lokális energiaháztartást veszélyezteti, hanem e folyamat során lehetővé válik, hogy a károsodott mitokondriális membránon keresztül citokróm-c áramoljon a citoplazmába, amely a caspase enzimcsalád aktiválódásához vezet [11, 12, 60]. A caspaseok a calpainhoz hasonlóan a cisztein proteázok családjába tartoznak és az apoptotikus sejthalál szabályozásában és végrehajtásában játszanak szerepet [147]. A caspase-3, a közös apoptózis végrehajtó enzim, képes a calpain hatásának felerősítésére a calpain sejten belüli inhibitorának, a calpastatinnak gátlásával [145], illetve irreverzibilisen és a calpainhoz hasonlóan specifikusan hasítja a spektrint [146]. A mitokondriumok károsodása következtében kialakult citokróm-c felszabadulás, illetve a calpain és a 16

17 caspase-3 egyidejű aktiválódása TAI során is megfigyelhető [11]. A caspase-specifikus spektrin-degradációs termékek (egy 150 kd és a 120 kd molekulasúlyú SBDP) specifikus antitestek segítségével szintén kimutathatóak a koponyatrauma in vitro modelljében [101], továbbá állatkísérletesen az agyszövetben mind fokális, mind diffúz traumás agysérülések esetén [11, 63, 100], illetve állatkísérletekben a cerebrospinális folyadékban fokális traumás agysérülés esetén [102] A DAI terápiás befolyásolását célzó vizsgálatok Miután egyértelművé vált, hogy a DAI nem azonnal létrejövő axonszakadás, hanem egy órák alatt progrediáló folyamat, a komplex pathomechanizmusnak megfelelően, több támadásponton is kutatások kezdődtek a károsodás mérséklésére, illetve kivédésére. Az első sikeres beavatkozás a hypothermia volt [70]. A trauma után rövid időn belül (< 25 perc) alkalmazott moderált hypothermia (32 Celsius) csökkentette az AD/B-t mutató axonok számát kontúziós modellben. A korai kontrollált hypothermia szignifikáns axonális védelmet nyújtó hatása később a diffúz agykárosodást kiváltó modellekben is igazolást nyert mind az AD/B (APP-pozitivitás) [58, 81], mind a CMSP és az NFC tekintetében [8, 81]. Ugyanakkor az is bizonyítottá vált, hogy a felmelegedési periódusnak lassú, gradált formában kell történnie, a túl gyors visszamelegítés ugyanis az axonkárosodás újbóli felerősödését okozza [82, 139]. A terápiás jellegű kutatások egy másik válfaja a mitokondriumok integritásának megőrzését, ezáltal a lokális energiaháztartás fenntartását, illetve a citokróm-c felszabadulásának és a következményes caspase aktivációnak a megakadályozását célozta. E tekintetben a figyelem egy, a klinikumban már immunszupresszánsként bevált szernek, a cyclosporine-a-nak (CsA) irányába fordult; e vegyület ugyanis képes kötődni a MPTpórushoz, megakadályozza annak kinyílását, ezáltal csökkentve a mitokondriális károsodást [94]. A további kísérletek azt is igazolták, hogy a mitokondriumok funkciójának fennmaradása esetén az excesszív mértékben beáramló Ca 2+ eltávolítását végző folyamatok energiaigénye fedezhető, és így a Ca 2+ -indukálta strukturális fehérjebontás fékezhető, következményesen az axonális citoszkeletális elváltozások megelőzhetőek [9, 95]. Az NFC befolyásolásában megfigyelt jótékony hatás mellett, a CsA az intraaxonális transzportzavart mutató axonok számát is szignifikánsan csökkentette [9, 94, 96]. Az MPT-pórusokra kifejtett hatása mellett a CsA és számos 17

18 egyéb, hozzá hasonlóan az immunophillinek családjába tartozó vegyület képes a calcineurin gátlására is. Ismert, hogy a citoszkeleton integritásának fenntartásában elengedhetetlenül fontos a neurofilamentek foszforilált állapotának fenntartása. A calcineurin egy foszfatáz, amely olymódon segíti az axonális citoszkeleton károsodását, hogy a neurofilamenteket defoszforilásuk útján hozzáférhetőbbé teszi a calpain számára [97], így gátlása jótékonyan befolyásolhatja az axonkárosodás folyamatát [26, 27, 126]. E vegyületek közül az FK506 -ról sikerült kimutatni, hogy az axonduzzadást mutató axonok számának csökkentése révén kedvező hatása van diffúz traumás axonkárosodásra [74, 131]. A diffúz axonkárosodás mechanizmusának ismeretében további terápiás lehetőségnek tarthatjuk a calpain és a calpain-mediált spektrin proteolízis gátlását. Korábban kontúziós traumamodellben a calpain gátlásával az NFC és spektrin-degradációs termékek csökkent jelenléte volt megfigyelhető [105]. A calpain gátlása ugyancsak javította a trauma utáni neurológiai (magatartási) kimenetelt fokális agykárosodás esetén [123, 124], ugyanakkor e jótékony hatás mögött nem sikerült Western blottal és hisztológiai módszerekkel csökkent CMSP-t kimutatni [124]. Hasonlóan ellentmondásosak az eredmények a diffúz koponyatrauma egér-modelljében, amennyiben ez esetben sem kísérte a javult neurológiai kimenetelt csökkent neuronális CMSP az agykéregben illetve a hippocampusban [61]. A calpain-gátlás közvetlen axonális hatását azonban sem az idézett tanulmányok sem mások idáig még nem vizsgálták A diffúz neuronális károsodás Az utóbbi évtizedekben számos kutatócsoport foglalkozott a koponyasérülések során kialakuló diffúz axonkárosodás vizsgálatával. Ugyanakkor kevés figyelmet szenteltek annak, hogy ugyanazok az erők, melyek axonkárosodást váltanak ki, okoznak-e diffúz eloszlásban, egyébként ép parenchimális környezetben neuronális sérüléseket. Fokális traumás agykárosodás esetén kialakuló regionális nekrotikus és apoptotikus neuron pusztulás jól ismert az irodalomban [20, 22, 28, 89, 116]. Fokális agysérülésekhez társuló, de a kontúziós régiótól távol eső idegsejt-károsodásokat is leírtak már [21, 23, 48], ugyanakkor a diffúz agysérülések során kialakuló neuronális károsodás szerepét kevesen vizsgálták. A meglévő kutatási eredmények is inkább leíró jellegűek és nem terjednek ki a sejtkárosodás kialakulásának mechanizmusára [16, 69, 121]. A fent említett kísérletek azt 18

19 feltételezték, hogy a megfigyelt sejthalál a trauma által indukált neuroexcitáció, oxigénszabadgyök képződés vagy egyéb másodlagos elváltozások következménye, és nem vették figyelembe azt a lehetőséget, hogy a koponya trauma által kiváltott erők közvetlenül okoznának idegsejt-károsodást A sötét -idegsejt képződés Kontúziós és nem-kontúziós koponyatrauma által kiváltott agyi morfológiai elváltozások vizsgálata során Gallyas és munkatársai hívták fel a figyelmet először arra, hogy a trauma képes axonális károsodás mellett diffúz idegsejt-és dentritikuskárosodás közvetlen létrehozására is; ugyanis bizonyos argyrophil morfológiai elváltozások a trauma kiváltása után 1 percen belül leölt kíséreti állatokban már jelen voltak a behatás helyétől távoli agyterületeken is, míg a kontrol állatokból hiányoztak [33, 34]. E megfigyelés ellene szól annak az elképzelésnek, amely szerint a neuronális károsodás másodlagosan alakul ki a trauma által generált egyéb elváltozások (pl. ischemia) következtében. A trauma hatására kialakult korai diffúz eloszlású idegsejtelváltozások fénymikroszkóppal vizsgálva a sejttest-dendrit egység zsugorodásában és bennük argyrophilia, illetve hyperbasophilia megjelenésében nyilvánulnak meg. Az ilyen morfológiai jeleket mutató idegsejteket tradicionálisan sötét -idegsejteknek nevezik [13]. Ultrastrukturálisan a sötét -idegsejtekre emelkedett elektrondenzitás, UC, a Golgi ciszternák tágulata és a nukleáris kromatin nem-apoptotikus aggregációja jellemző. Hasonló sejt-elváltozások nemcsak trauma, hanem egyéb tényezők hypoglicaemia, ischemia, status epilepticus hatására is kialakulnak [32], sőt képződésük post mortem is kiváltható [35], ami felveti annak lehetőségét, hogy a sötét -idegsejt képződés nem enzim-mediált pathobiokémia folyamatok eredménye. A kísérletek során azt is megfigyelték, hogy az idegsejt-dendrit egységben, valamint az axonokban létrejött elváltozások egymástól függetlenek, ami azt jelezi, hogy a sejt különböző doménjei szelektíven érzékenyek a kiváltó traumából eredő hatásokra. A sötét -idegsejtek további sorsát vizsgálva az is leírásra került, hogy a kiváltó tényezők hatására azonnal kialakult sötét idegsejteknek csak egy hányada pusztul el, többségük azonban regenerálódik, és a kialakulás után 4 órán belül visszanyeri normális morfológiai és festődési tulajdonságait [25]. 19

20 A neuronális mechanoporáció In vitro módszerek alkalmazásával megfigyelték, hogy sejtkultúrák idegsejtjei nyújtás hatására nagy molekulasúlyú anyagokat vesznek fel a sejthártya sérülésének következtében. Ez az idegsejthártya-sérülés és permeabilitás-változás a kiváltó trauma után azonnal észlelhető volt, ami újra megkérdőjelezte az idegsejtkárosodás kialakulásában feltételezett másodlagos tényezők kizárólagos kóroki szerepét [38]. E kísérlet során a plazmamembrán-permeabilitás növekedése (mechanoporáció) az erőbehatás nagyságával és az alkalmazott anyagok molekulasúlyával arányosnak és időben átmenetinek bizonyult: a legkisebb anyagok felvétele a sérülést követően 5 perc múlva is megfigyelhető volt, míg a legnagyobb mólsúlyú anyagok csak a sérülés kiváltását követően azonnal jutottak át a sejthártyán [38]. A mechanoporáció jelensége in vivo modellben is megfigyelhető volt. A diffúz koponyatrauma egy patkány-modelljében nagy molekulasúlyú anyagok intracerebroventrikuláris alkalmazásával igazolódott, hogy az axonok mellett az agykéregben, a hippocampusban és a thalamusban is találhatók plazmamembrán permeabilitási zavart szenvedett neuronok; e sejtek közvetlenül a kiváltó trauma után (túlélési idő < 5 perc) felvesznek az ép sejthártyán keresztül át nem jutó nagy molekulasúlyú anyagokat [133]. Az idegsejt-elváltozások további vizsgálata során az is bebizonyosodott, hogy a kiváltó trauma nem egyformán érinti az idegsejteket, hanem a neuronkárosodás különböző, egymástól függetlenül kialakuló elváltozások egymás melletti (azonos agyi régiókban megtalálható) megjelenéséből tevődik össze. Meglepő megfigyelés volt, hogy a perisomatikus axonkárosodást szenvedett APP-pozitív axonok neuronjai nem mutattak sejthártya-permeabilitási zavart vagy súlyos sejtkárosodásokra utaló ultrastrukturális elváltozásokat [132, 133]. Ez annyit jelent, hogy az axonkárosodás és axonszakadás nem vezet az érintett idegsejt gyors pusztulásához, szemben a korábbi feltételezésekkel [4, 76]. A megfigyelt neuronális elváltozások spektruma a mechanoporációt mutató neuronok körében a gyors nekrózistól az ultrastrukturálisan ki nem mutatható elváltozásokig terjedt [133]. Ez utóbbi jelenség hátterében azaz abban, hogy a sejtmembrán-sérülés nem feltétlenül vezet az érintett idegsejt pusztulásához a szerzők az idegsejt-membrán gyors helyreállítódását (resealing) feltételezték. Ez irányú kísérletek azonban vizsgálatainkig nem történtek. 20

21 3. CÉLKITŰZÉSEK A dolgozat célkitűzéseit a kísérleti eredményeket megjelentető tudományos közlemények felsorolásával együtt az alábbiakban foglaljuk össze: 1. A traumás axonkárosodás vizsgálata 1.1. Az ultrastrukturális axon-kompakció vizsgálata Az ultrastrukturális kompakció és az argyrophil axonkárosodás kapcsolatának tisztázása (Gallyas F, Farkas O, Mazlo M. Traumatic compaction of the axonal cytoskeleton induces argyrophilia: histological and theoretical importance. Acta Neuropathol (Berl) Jan;103(1):36-42.) Olyan koponyatrauma modell kidolgozása, melynek segítségével az ultrastrukturális kompakció szelektíven idézhető elő (Pal J, Toth Z, Farkas O, Kellenyi L, Doczi T, Gallyas F. Selective induction of ultrastructural (neurofilament) compaction in axons by means of a new headinjury apparatus. J Neurosci Methods Jun 15;153(2):283-9.) A kompaktálódott axonok sorsának vizsgálata az előző pontban ismertetett modell segítségével (Gallyas F, Pal J, Farkas O, Doczi T. The fate of axons subjected to traumatic ultrastructural (neurofilament) compaction: an electron-microscopic study. Acta Neuropathol (Berl) Mar;111(3): ) Az ultrastrukturális axon- és idegsejt-kompakció mechanizmusának tisztázása (Gallyas F, Farkas O, Mazlo M. Gel-to-gel phase transition may occur in mammalian cells: Mechanism of formation of "dark" (compacted) neurons. Biol Cell May;96(4): ) 21

22 1.2. Terápiás beavatkozások tesztelése Egy sejt-permeábilis calpain inhibitor (MDL-28170) hatásának vizsgálata a diffúz axonkárosodásra (Buki A, Farkas O, Doczi T, Povlishock JT. Preinjury administration of the calpain inhibitor MDL attenuates traumatically induced axonal injury. J Neurotrauma Mar;20(3):261-8.) A más központi idegrendszeri betegségekben már hatásosnak bizonyult PACAP (Hypophysis Adenilát Cikláz Aktiváló Polypeptid) hatásának vizsgálata a diffúz axonkárosodásra, a trauma előtt illetve után iv illetve icv adva, továbbá hatásos dózisának megállapítása (Farkas O, Tamas A, Zsombok A, Reglodi D, Pal J, Buki A, Lengvari I, Povlishock JT, Doczi T. Effects of pituitary adenylate cyclase activating polypeptide in a rat model of traumatic brain injury. Regul Pept Dec 15;123(1-3):69-75.) A terápiás ablak meghatározása PACAP kezelés esetén, az kísérletben hatásosnak bizonyult dózis alkalmazásával (Tamas A, Zsombok A, Farkas O, Reglodi D, Pal J, Buki A, Lengvari I, Povlishock JT, Doczi T. Postinjury administration of pituitary adenylate cyclase activating polypeptide (PACAP) attenuates traumatically induced axonal injury in rats. J Neurotrauma May;23(5): ) 2. A diffúz idegsejt-károsodás vizsgálata A neuronális mechanoporáció, a potenciális membrán-helyreállítás (resealing) és következményeik, illetve az ezekhez kapcsolódó calpain-mediált strukturális fehérjebontás szerepének vizsgálata (Farkas O, Lifshitz J, Povlishock JT. Mechanoporation induced by diffuse traumatic brain injury: an irreversible or reversible response to injury? J Neurosci Mar 22;26(12): ) 22

23 3. A calpain- és caspase-aktiválódás szerepe a humán traumás agykárosodásban A calpain- és caspase-specifikus spektrin-degradációs termékek humán liquormintákból történő kimutatása Western blot segítségével súlyos koponyatraumát szenvedett betegek esetén (Farkas O, Polgar B, Szekeres-Bartho J, Doczi T, Povlishock JT, Buki A. Spectrin breakdown products in the cerebrospinal fluid in severe head injury--preliminary observations. Acta Neurochir (Wien) Aug;147(8): ) 23

24 4. ANYAGOK ÉS MÓDSZEREK A jelen fejezetben röviden ismertetjük azokat a módszereket, amelyeket több kísérletsorozatban is használtunk, míg részletes ismertetésük céljából utalunk a disszertáció tárgyát képező közleményekre. Az állatkísérletek mindegyikéhez felnőtt hím patkányokat használtunk; a Baranya Megyei Állategészségügyi Igazgatóság (illetve a 2. téma kísérletei során a Virginia Commonwealth University Institutional Animal Care and Use Committee, IACUC) engedélye alapján, a PTE ÁOK OEC Regionális Kutatási Etikai Bizottság által elfogadott normákat alkalmazva. A patkányok a kísérletek teljes ideje alatt mély intratracheális (30 % O 2 és 70 % N 2 O keverékéhez adott 4 % Isoflurane) vagy intraperitoneális (Thiopental és Seduxen 1:1 arányú keveréke) narkózisban voltak. Az egyes kísérletek során különböző vitális paraméterek hőmérséklet, artériás vérnyomás, artériás vérgázok, intrakraniális nyomás kerültek monitorozásra A diffúz agysérülés modellezése Az impact-accelerációs koponyatrauma modell A kísérletek egy része során a diffúz axonális és neuronális károsodás kiváltására a Marmarou által leírt, nemzetközileg elfogadott és széles körben használt impactaccelerációs koponyatrauma modell alkalmazásával került sor. A módszer lényege, hogy altatott állatok koponyacsontjára a bregma és a lambda varratok közé egy fém-korongot erősítünk, majd az állatokat egy speciális szivacs-ágyra helyezzük; a fejre erősített korongot egy plexicső alá centrálva. A trauma kiváltására a plexicsőn keresztül az állat fejére 450 g súlyt ejtünk. A szivacs-ágy trauma-utáni gyors eltávolításával akadályozzuk meg, hogy az állat fejét újabb ütés érje. E módszer koponyatöréstől és fokális agykárosodástól mentes koponyatraumát vált ki, ezáltal alkalmas a diffúz traumás agyi 24

25 elváltozások vizsgálatára [31, 72]. Az alkalmazott trauma középsúlyos/súlyos agysérülés kiváltását eredményezi, amely az állatok döntő többségénél átmeneti légzés-leállást okoz. Ezért a koponyatraumát követően minden esetben az állatot rövid ideig 100 % O 2 -nel lélegeztettük. Koponyacsont-törés esetén az állatot a további kísérletekből kizártuk Szelektív ultrastrukturális kompakciót kiváltó koponyatrauma modell Az ultrastrukturális kompakció szelektív kiváltására laboratóriumunkban kifejlesztettünk egy olyan modellt, amely a rugalmas koponyatetőt egy állítható mélységig pillanatszerűen benyomja, de nem töri be. A modell lényege, hogy az altatott állat fejét a készülékhez csatlakoztatható fej-tartóba rögzítjük, majd a fej-tartó mozgatásával a készülék impaktor részét kiválasztott koordinátáknak megfelelően a szabaddá tett koponyacsont fölé centráljuk. Az impaktort addig mozgatjuk lefelé, amíg az a koponyát éppen eléri, majd egy csavar segítségével a benyomódás kívánt mértékét beállítjuk. Ezután egy fémcsőn keresztül 200 g-os acél-súlyt ejtünk az impaktorra, amely a beállított mértékben benyomja a koponyacsontot. Ez a készülék alkalmas ultrastrukturális kompakció kiváltására úgy, hogy közben fokális agysérülés, illetve a diffúz axonkárosodás egyéb formái elsősorban AD/B nem következnek be, így segítségével a kompaktálódott axonok sorsa szelektíven vizsgálható Szövettani feldolgozás A kísérletek végén a kívánt az agysérülés kiváltásától eltelt túlélési idő elérésekor az állatok mély altatásban szíven keresztüli perfúziós fixálásra kerültek. A perfúzió aldehid-tartalmú fixáló oldatokkal történt, amelyek alkalmassá teszik az agyszövetet fény-mikroszkópos (formaldehid tartalmú fixáló) vagy elektronmikroszkópos (glutáraldehid tartalmú fixáló) feldolgozásra. A szíven keresztüli fixálás után az agyat az agytörzzsel és a gerincvelő felső nyaki szegmentumával együtt a koponyából eltávolítottuk, majd további immerziós fixáció céljából a perfúzióhoz használt oldatba tettük. Ezt követően az agyból a kívánt részt kivágtuk és vibratóm segítségével a nagyagyból koronális, az agytörzsből szagittális 25

26 metszeteket készítettünk Az argyrophil-iii típusú ezüstözési eljárás Ez az eljárás alkalmas a különféle behatások következtében a központi idegrendszerben kialakult bizonyos idegsejt- és axon-elváltozások érzékeny kimutatására [36], és ellentétben a hagyományos ezüstözési eljárások közismert megbízhatatlanságával jól-reprodukálható eredményeket biztosít [90]. A módszer lényege, hogy a metszeteket felszálló alkohol-sorban vízmentesítjük, majd 16 h-n át 56 C fokon 0.8 % kénsavat és 2 % vizet tartalmazó l-propanol oldatban inkubáljuk (észterifikáció). Ezután a metszeteket rehidráljuk, 10 percig 1 %-os ecetsavba helyezzük, majd egy speciális fizikai előhívó oldatba merítjük, amíg világos barnává válnak. Az előhívást 1 %-os ecetsavval állítjuk le Immunhisztokémiai technikák Az immunhisztokémia során alkalmazott antitestek a következők voltak: Target Elsődleges Antitest Másodlagos Kísérlet Antitest Intraaxonális transzportzavar Anti-APP Biotinilált anti-nyúl , 1.2. Neurofilament RMO-14 Biotinilált anti-egér , és kompakció Calpain-specifikus SBDP 150 Ab38 Biotinilált anti-nyúl 2. Calpain-specifikus Ab38 Alexa Fluor SBDP 150 jelölt anti-nyúl Alexa Fluor 488 Anti-Alexa Fluor Biotinilált anti-nyúl 2. jelölt dextrán 488 A fény-mikroszkópos technikák alkalmazása során az immunjelölések láthatóvá tételére avidin/biotin-peroxidáz komplex alkalmazását követően 0.05 % diaminobenzidint (DAB) és 0.01 % hidrogén-peroxidot tartalmazó 0.1 mólos foszfát puffert használtunk, 26

27 szemkontroll mellett. A 2. téma kísérletei során különböző fluorescens festékeket tartalmazó dextránokat juttattunk az agyszövetbe; ezek agyszöveti megoszlásának vizsgálata a metszést követően közvetlenül antitestek használata nélkül fluorescens mikroszkóppal történt Elektronmikroszkópos előkészítés Festetlen vagy elektrondenz terméket (DAB, az immunhisztokémia végterméke,) tartalmazó metszetekből a szomszédos ezüstözött, vagy egyéb módon festett metszeteken érdekesnek ítélt területeket kivágtuk, majd ozmium-tertoxiddal vagy ozmium-tetroxid és kálium-hexacyanoferrát(ii) keverékével tovább fixáltuk. (Az utóbbi vegyület a myelin szerkezetének megtartásában fontos; alkalmazása az axon-vizsgálatok esetén különösen indokolt volt.) A fixálás után a metszeteket Durcupan gyantába ágyaztuk. Félvékony metszetek toluidin-kékkel való festésével azonosítottuk a kívánt területeket, majd ultravékony szériákat metszettünk, amelyeket gridekre húztunk. A grideket az elektrondenzitás növelése céljából uranil-acetáttal és ólom-citráttal kontrasztosítottuk. Néhány esetben a már gyantába ágyazott metszeteken ezüstözést végeztünk, majd újrabeágyazás után ultra-metszettünk Western blot A 3. altéma kísérletei során kamrai katéteren keresztül vagy lumbál-punkcióval nyert liquor-mintákat a calpain- és caspase-specikifus SBDP-k kimutatására dolgoztuk fel. A mintákat 100 kd-s szűrőt használva Amicon ultrafiltrációs készüléken átszűrtük, majd a fehérje-koncentrációikat fotometriás módszerrel megmértük. 20 μg fehérjét tartalmazó mintákkal 6.5 %-os gélen nátrium-dodecil-szulfátos poliakrilamid gélelektroforézist végeztünk, majd a gélről a fehérjéket nitrocellúlóz membránra transzferáltuk. A membránokat az alfa-ii spektrint és lebontási termékeit kimutató egér antitesttel, majd biotinilált anti-egér másodlagos antitesttel, végül streptavidin-biotinilált peroxidáz komplex-szel inkubáltuk. Az immunjelölést kemiluminescens reagens segítségével Kodak filmeken tettük láthatóvá. Az eredményeket denzitometriás 27

28 módszerrel értékeltük Permeabilitás-vizsgálat Az intrakraniális nyomás monitorozására az oldalkamrába vezetett kanülön keresztül a membrán-permeabilitás változás kimutatására alkalmas (az ép sejtekbe az ép sejthártya által be-nem-engedett) nagy molekulasúlyú dextránokat infundáltunk az oldalkamrába. Az első dextrán infúzió Alexa Fluor 488 fluorescens festékkel jelölt 10 kda molsúlyú dextránnal 2 h-val a koponyatrauma előtt történt. A trauma kiváltása után 2 illetve 6 h-val a megnövekedett permabilitás elhúzódásának, illetve az esetleges sejthártya-helyreállításnak (resealing) kimutatására újabb dextrán-infúzió történt Texas Red fluorescens festékkel jelölt 10 kda mólsúlyú dextránnal. A második dextrán-infúziót követően 2 h-val az állatokat perfundáltuk. 28

29 5. EREDMÉNYEK ÉS KÖVETKEZTETÉSEK Az alábbiakban a Célkitűzések fejezetben vázoltaknak megfelelően kísérleteink hátterét, témáját, módszereit valamint legfontosabb eredményeit és következtetéseit foglaljuk össze. További részletek a disszertáció tárgyát képező közlemények megfelelő fejezeteiben találhatók A traumás axonkárosodás vizsgálata Az ultrastrukturális axon-kompakció vizsgálata Az ultrastrukturális axon-kompakció és az argyrophil axonkárosodás kapcsolata (Gallyas F, Farkas O, Mazlo M. Traumatic compaction of the axonal cytoskeleton induces argyrophilia: histological and theoretical importance. Acta Neuropathol (Berl) Jan;103(1):36-42.) Háttér: Különféle módon előidézett koponyatrauma után (i) az alkalmazott argyrophil-iii típusú speciális, és reprodukálható eredményű ezüstöző eljárás [32] hosszú axon-szakaszokat fest feketére, nem-festődő axonok közt sporadikus (diffúz) eloszlásban [33], (ii) valamint ultrastrukturális (neurofilament) kompakciót hoz létre számos elektronmikroszkópos axon-profilban, nem-kompaktálódott axon-profilok közt sporadikus (diffúz) eloszlásban [98]. Cél: Annak a tapasztalati úton nyert feltételezésnek igazolása, hogy az ezüstöződő axon-szakaszok azonosak az UC-t szenvedett axon-profilokkal. Módszer: A Marmarou-féle készülékkel végzett koponya-trauma után, a szíven keresztüli perfúzióval azonnal fixált patkányok agyából készített 150 µm-es vibratómos sorozat-metszetek közül minden ötödiket megezüstöztük. (A) vizsgálat: Az ezüstözött axon-szakaszt tartalmazó területeket elektronmikroszkópos megfigyelésre ozmifikáltuk, Durcupanba ágyaztuk, ultra-metszettük és kontrasztoztuk. (B) vizsgálat: Nemezüstözött vibratómos metszetek ugyanezen területeiből ozmifikálás és Durcupanba 29

30 ágyazás után 1 µm-es metszeteket készítettünk, ezeket megezüstöztük, majd újra beágyaztuk, ultrametszettük és kontrasztoztuk. Eredmények: (A) vizsgálat: Az ezüstözés erősen roncsolja az ultrastruktúrát; fellazítja a myelin-hüvelyek lemezeit, feloldja a normális axonokat, de - homogenizált formában - megőrzi a kompaktálódott axon-profilokat. Ezüst-szemcséket csak kompaktálódott axon-profilokban találtunk; másrészt nem találtunk olyan kompaktálódott axon-profilt, amely nem tartalmaztak ezüst-szemcséket. (B) vizsgálat: Minthogy az ezüstözést ozmifikálás és Durcupanba ágyazás előzte meg, az ultrastruktúra kiválóan megőrződött. Sok, és viszonylag nagy ezüst-szemcsét találtunk a kompakciót szenvedett axon-profilokban; másrészt nem találtunk olyan kompaktálódott axon-profilt, amely nem tartalmazott sok nagy ezüst-szemcsét. Következtetések: (1) Az alkalmazott ezüstöző módszer lehetővé teszi az UC-t szenvedett axon-szakaszok fénymikroszkópos feltüntetését vastag vibratómos metszeteken; ezáltal vizsgálhatóvá teszi ezen károsodást elszenvedett axonok térbeli elhelyezkedését, valamint az elektronmikroszkópos vizsgálatra érdemes területek kiválasztását. Továbbá, szemben a vonatkozó korábbi elképzelésekkel, (2) az UC a trauma pillanatában következik be, nem pedig 5-15 perces késéssel, (3) a citoszkeletális kompakció viszonylag hosszú, akár 1 mm-es axon-szakaszokra (de általában nem a teljes axon-hosszúságra) is kiterjedhet, azaz nem EM-szinten fokális elváltozás, (4) a neurotubulusok eltűnése, a neurofiament-oldalkarok részleges feloldódása, valamint a mitokondriumok duzzadása nem tartozik az UC elsődleges folyamatába. Megjegyzések: (I) A kompaktálódott axonok feltüntetésére más szerzők által alkalmazott RMO-14 immunfestéssel kapcsolatos megfigyelésünkről az alfejezetben lesz szó. (II) Ezüstöződést előidéző UC idegsejtek szóma-dendrit doménjében is végbe tud menni koponyatrauma hatására [25], ennek, és az axonális kompakciónak közös a korábbi nézeteknek részben ellentmondó mechanizmusáról az alfejezetben lesz szó. 30

31 Ultrastrukturális kompakció szelektív előidézésére alkalmas koponyatrauma modell kidolgozása (Pal J, Toth Z, Farkas O, Kellenyi L, Doczi T, Gallyas F. Selective induction of ultrastructural (neurofilament) compaction in axons by means of a new head-injury apparatus. J Neurosci Methods Jun 15;153(2):283-9.) Háttér: A diffúz axonkárosodás koponyatraumát szenvedett kísérleti állatokban két jól elkülöníthető morfológiai elváltozás formájában jelentkezik: (i) az előre-irányuló intraaxonális transzport-zavar által előidézett axonduzzadás/ballon-képződés (AD/B), (ii) az ezüstöződéssel járó ultrastrukturális kompakció (UC) ( lásd alfejezet) [135]. Míg az előbbi jól ismert humán vonatkozásban is, az utóbbi szerepe a DAI-ban kevésbé tisztázott. A meglévő koponyatrauma-modellek az említett axonális elváltozások és egyéb károsodások (1.2 alfejezet a Bevezetésben) együttes létrehozására, és ezáltal a TAI komplex vizsgálatára alkalmasak [40], az egyes morfológiai jellemzők szelektív vizsgálata azonban használatukkal nem lehetséges. Cél: Olyan koponyatrauma-modell kidolgozása, melynek segítségével az UC kialakulása és a kompaktálódott axonok sorsa más morfológiai elváltozások befolyásoló hatása nélkül vizsgálható. Módszer: Patkányok puha koponya-tetején kontrollálható-mélységű benyomódást hoztunk létre pillanat-szerűen, majd a patkányokat azonnal, illetve 1 vagy 4 órával később, formalinos vagy glutáraldehides fixálóval perfundáltuk. A koponyatrauma előtt, illetve a túlélés időtartamában monitoroztuk a légzés-számot, a pulzusszámot, a vérnyomást és a koponyaűri nyomást. Az 1 nap múlva kivett agyakból készített vibratómos metszetek közül minden harmadikat megezüstöztük; a formalinban fixáltaknál a közbeeső metszetek APP immun-hisztokémiára vagy RMO-14 immunhisztokémiára kerültek, míg a glutáraldehides közbeeső metszeteknek az ezüstözés alapján vizsgálni érdemesnek tartott területeit elektron-mikroszkópiára ágyaztuk be. Eredmények: 0.75 mm-es benyomódás esetén a vizsgált fiziológiai paraméterek a koponyatrauma után 1 perccel visszatértek a koponyatrauma előtti értékekre; ezüstözött illetve UC-t szenvedett axonok csak a benyomódás alatti agykéregben voltak megfigyelhetők, normális axonok között elszórtan, ép parenchimális környezetben. Egyéb morfológiai elváltozást nem találtunk. A károsodott axonok száma a koponyacsont-benyomódás mértékével párhuzamosan változott. Azonnal, illetve 1 órával 31

32 a trauma után a kompaktálódott axonok homogénen ezüstöződtek, míg 4 órával a traumát követően pontszerű ezüstöződés volt megfigyelhető. Intraaxonális transzportzavart mutató (anti-app-pozitív) axonok csak 1 mm-es, vagy nagyobb benyomódás esetén voltak láthatók; RMO-14 pozitivitást egyetlen esetben sem észleltünk. Következtetések: (1) A kidolgozott koponyatrauma-modell 0.75 mm-es koponyacsont-benyomódás esetén alkalmas az ultrastrukturális axon-kompakció szelektív előidézésére, ezáltal alkalmas a kompaktálódott axonok sorsának más morfológiai elváltozások vagy pathofiziológiai tényezők befolyásoló hatása nélküli vizsgálatára (lásd az alfejezetet), terápiás lehetőségek tesztelésére vagy akár ismételt enyhe koponyatrauma hatásának tanulmányozására. (2) Az APP-pozitivitás hiánya megerősíti azt az elképzelést, hogy az intraaxonális transzportzavar és az axon-kompakció két különböző axon-populácót érint. (3) A kompaktálódott axonok az agykéregben több óra elteltével sem festődnek az agytörzsi axon-kompakció kimutatására széles körben elterjedt markerrel, az RMO-14 antitesttel. A jelenség valószínűleg azzal magyarázható, hogy ellentétben az agytörzs nagy-kaliberű, myelinizált axonjaival az agykéreg vékony axonjaiban igen kevés az axon-kalibert meghatározó NF-M alegység [29], amelynek a calpain-aktiválódás hatására szabaddá vált rod doménjét mutatja ki az RMO-14 antitest [10]. Megjegyzések: (I) 1 mm-es koponyacsont-benyomódás, továbbá alacsonyabb sebességű koponyacsont benyomás, illetve egyéb koponya-trauma előidézési-módok [pl. 25] esetén nemcsak axonok, hanem idegsejtek és dendritfájuk is ezüstözhetővé válik, illetve kompaktálódik. Erről a jelenségről, illetve az ultrastrukturális axon- illetve idegsejt-kompakció háttérében feltételezett mechanizmusról az alfejezetben lesz szó. (II) Az ismertetett koponya-trauma módszer egy fontos alkalmazási területével az alfejezetben foglalkozunk A kompaktálódott axonok sorsának vizsgálata szelektív ultrastrukturális kompakciót kiváltó koponyatrauma modellben (Gallyas F, Pal J, Farkas O, Doczi T. The fate of axons subjected to traumatic ultrastructural (neurofilament) compaction: an electron-microscopic study. Acta Neuropathol (Berl) Mar;111(3): ) 32

33 Háttér: Az ultrastrukturális axon-kompakció a TAI konzekvens morfológiai velejárója, ennek ellenére eddig nem vizsgálták az érintett axonok sorsát. Ugyanakkor ismert, hogy UC-t szenvedett idegsejtek egy hányada néhány óra alatt spontán meggyógyul (recovery), másik hányada viszont elpusztul [25]. Minthogy az axonkompakció illetve az idegsejt-kompakció feltételezhetően azonos mechanizmussal megy végbe (lásd alfejezet), feltételezhető, hogy a kompaktálódott axonoknak is csak egy hányada pusztul el, a többi viszonylag gyorsan visszanyeri eredeti morfológiáját. Cél: Az alfejezetben ismertetett koponyatrauma módszer segítségével szelektíven kompakttá tett axonok sorsának nyomon követése. Módszer: 0.75 mm-es koponyacsont-benyomódást létrehozó koponyatraumát szenvedett patkányokat a trauma kiváltása után azonnal illetve 6 hónapig terjedő túlélési idő elteltével glutáraldehid-tartalmú fixálóval perfundáltunk, az agyakból készült metszetek egy hányadát ezüstöztük, a szomszédos metszetek EM feldolgozásra kerültek. Egy kiválasztott agykérgi területen a kompaktálódott, illetve a nem-kompaktálódott myelinizált axonok számát kvantitatív analízis céljából megállapítottuk. Eredmények: Az agykéreg vizsgált területén a kompaktálódott myelinizált axonok száma az azonnal-perfundált patkányokban több, mint kétszer annyi volt, mint az 1 napot vagy az 1 hetet túléltekben. Az azonnal, illetve a 4-órás túlélés után leölt patkányokban számos olyan myelinizált axon-profil volt megfigyelhető az és alfejezeteknek megfelelően, amelyekben a hosszanti ultrastrukturális elemek közötti távolságok drámai módon lecsökkentek (kompakció), míg az egyéb ultrastrukturális elemek épnek tűntek. A túlélési idő növekedésével egy sor egyéb ultrastrukturális elváltozás került megfigyelésre; így (i) membrán-örvények megjelenése az első két túlélési napban egyébként ép ultrastruktúrájú axon-profilokban, (ii) a kompaktálódott axon-profilok ultrastruktúrájának homogenizációja az első két túlélési napban, (iii) később a homogenizált ultrastrukturális területek fragmentációja, (iv) majd a fragmentumok felcserélődése oligodendroglia-citoplazmával, és (v) végül axolemmával körülvett, neurofilamenteket tartalmazó profilok megjelenése szokatlanul sok oligodendroglia-citoplazmát tartalmazó myelinizált axonokban. (vi) Kiemelendő, hogy makrofág infiltrációra, mikroglia proliferációra és a Waller-féle degeneráció késői, előrehaladott stádiumára utaló jeleket nem láttunk még 6 hónap elteltével sem. 33

34 Következtetések: (1) A kompaktálódott axonok >50%-a 1 napon belül, míg <10%- a 1 nap és 1 hét között visszanyeri eredeti ultrastruktúráját (meggyógyul); ennek jele hasonlóan az idegsejtek szómája és dendritjei esetében találtakhoz [25] a membránörvények megjelenése. (2) A többi kompaktálódott axon néhány hónap alatt regenerálódik (lásd az eredmények (ii)-(vi) pontjait). (3) Feltételezhető, hogy a fentiek szerepet játszanak a koponyatraumát szenvedett betegek fizikai és szellemi teljesítményeinek gyors (az első trauma-utáni nap folyamán végbemenő) illetve lassú (a néhány hónappal később végbemenő) javulásában. (4) A Waller-féle (irreverzibilis) degenerációval szembeni eltérés magyarázata abban keresendő, hogy a vizsgált esetben nem szakad meg a kapcsolat a kompaktálódott axon myelin-burka, és az azt tápláló oligodendroglia közt; ezért a myelin-burok nem pusztul el, és így pályát képez a regenerálódó axon számára. (5) Az alkalmazott modellről ismert, hogy nem vált ki tartós rosszabbodást a fiziológiai jellemezőkben (lásd alfejezet). Ennek tudható be a talált spontán-gyógyulási képesség, valamint a degeneráció reverzibilitása. Megjegyzés: Feltételezhető azonban, hogy tartósan-kedvezőtlen pathofiziológiai körülmények között pl. megnövekedett ICP, ischemia az axonoknak az általunk megfigyeltnél nagyobb hányada nem gyógyul meg, illetve a spontán nem-gyógyuló axonok egy hányada nem regenerálódik (elpusztul), amint ez a kompaktálódott idegsejtek esetében kimutatható volt [37] Az ultrastrukturális axon- illetve idegsejt-kompakció mechanizmusának tisztázása (Gallyas F, Farkas O, Mazlo M. Gel-to-gel phase transition may occur in mammalian cells: Mechanism of formation of "dark" (compacted) neurons. Biol Cell May;96(4): ) Háttér: Különböző patobiokémiai tényezők (köztük hypoglicaemia, ischemia és epilepsia), továbbá fizikai behatások (elektromos sokk, koponyatrauma) [25, 32] képesek azonos morfológiai elváltozások (ultrastrukturális kompakció) létrehozására. Fénymikroszkópos szinten ismert de elektron-mikroszkóppal korábban nem vizsgált, hogy ugyanilyen morfológiai elváltozás jön létre post mortem is idegsejtekben a nemfixált, vagy rosszul fixált agynak a koponyából való kivétele során elszenvedett mechanikai megterhelés hatására [14]. Az azonos morfológia a kiváltó károsító hatástól 34

35 független közös kialakulási mechanizmust feltételez; a post mortem kialakulás lehetősége pedig nem-enzim-közvetített folyamatra utal. A polimer gél-kémiából ismert egy, a kiváltó tényezőtől független mechanizmusú, és makromolekulák nem-kovalens kötései formájában tárolt energia hatására végbemenő, egy pontból kiindulóan tovaterjedő folyamat, a gélből gél fázisátalakulás [140], melynek szerepét az élő sejt egyes mechanizmusaiban már feltételezték [104, 141, 144]. Cél: (1) In vivo, illetve az enzim-közvetített folyamatok számára rendkívül kedvezőtlen körülmények között végzett, azonos típusú mechanikai behatás által kiváltott ultrastrukturális idegsejt- illetve axon-kompakció morfológiai jellemzőinek összehasonlítása annak bizonyítására, hogy a kompakció kialakulásának mechanizmusa nem enzim-közvetített folyamat. (2) Annak valószínűsítése, hogy ez a folyamat mind energetikai, mind strukturális szempontból gélből-gél fázisátalakulás. Módszerek: A Marmarou-féle készülékkel koponyatraumát hajtottunk végre egyrészt post mortem (glutáraldehides fixálóval végzett 30-perces, szíven-keresztüli perfúziót követően 0-fok közelébe hűtött patkányokon), másrészt in vivo (közvetlenül a perfúziós fixálás előtt a Marmarou-féle készülékkel traumatizált patkányokon). Az egy nappal később kivett agyakból készített vibratómos metszetek egy hányada ezüstözésre, másik hányada EM-feldolgozásra került. Kvantitatív analízis céljából meghatároztuk a hosszanti ultrastrukturális elemek közti átlag-távolságot mind kompaktálódott, mind pedig normál axonokban. Eredmények: (A) Az in vivo és a post mortem koponyatrauma egyforma morfológiai elváltozásokat eredményezett, mind a (i) a hyperbasophilia, (ii) a III-típusú argyrophilia, (iii) a hiper-elektrondenzitás, mind pedig (iv) az UC jellegzetességeinek tekintetében. (B) Az ultrastrukturális axon-kompakció foka a post mortem trauma esetén nem különbözött az in vivo trauma esetén meghatározottól. (C) A trauma-indukálta ezüstöződés (az UC fénymikroszkópos jele, lásd alfejezet) mindkét esetben minden vagy semmi jellegű volt, azaz az érintett szóma-dendrit domének teljes egészére, illetve hosszú axon-szakaszokra terjedt ki, de általában nem egyazon idegsejt szóma-dendrit doménjét és axonját érintette. Következtetések: Az ultrastrukturális idegsejt- és axon-kompakció mechanizmusában nem enzim-közvetített folyamatok játszanak szerepet, hiszen (1) 35

36 valószínűtlen, hogy ugyanolyan morfológiai eredménnyel folynak le biokémiai folyamatkaszkádok in vivo és post mortem (előzetes aldehides fixálás és 0 C fokra való lehűtés után), vagyis az enzimatikus folyamatoknak kedvező illetve rendkívül kedvezőtlen körülmények között. (2) A minden-vagy-semmi jelenség csak úgy magyarázható, hogy a kompakció egyetlen kezdeti pontból terjed ki az érintett idegsejt szóma-dendrit doménjének egészére, vagy egy hosszú axonszakaszra. Elképzelhetetlen ugyanis, hogy a kompakció az érintett idegsejt szóma-dendrit doménjének valamennyi pontján egyszerre iniciálódjon a néhány idegsejt környezetében kvázi-homogén mechanikai paraméterváltozásokat okozó pillanatszerű koponyatrauma hatására, a szomszédos idegsejtek szóma-dendrit doménjének pedig egyetlen pontján sem. Nagyon valószínűtlen az is, hogy egy enzim-közvetített reakció-kaszkád csillapodás nélkül végigterjedjen az érintett idegsejt szóma-dendrit doménjének egészén vagy hosszú axon-szakaszokon, különösen az enzimatikus reakcióknak rendkívül kedvezőtlen körülmények között. Az UC jelenségének magyarázatára feltételeztük, hogy az ismert ultrastrukturális elemek (organellumok, citoszkeleton) közti terekben egy összefüggő, fehérje-mátrixú gél-struktúra található, amely energiát tárol nem-kovalens kötések formájában. Ebben a gél-struktúrában a gél mátrixát felépítő fehérje-molekulák kooperatív (egyetlen pontból kiindulva tovaterjedő) konformáció-változása következtében fázisátalakulás megy végbe, miközben a megkötött vízmolekulák illetve K + -ionok egy jelentős hányada felszabadul a gél összehúzódását (térfogatcsökkenés) eredményezve. Az összehúzódó gél a hozzákötött ultrastrukturális elemeket magával vonja (kompakció). A polimer-kémiából ismert, hogy a gélből-gél fázisátalakulás iniciálható mind fizikai, mind kémiai behatásokkal, és reverzibilis lehet, összhangban a sötét idegsejtek és axonok ismert regenerációs képességével. Megjegyzések: (I) A spontán regenerációra képes idegsejtek valószínűleg megegyeznek azokkal, amelyek a trauma hatására plazmamembrán-permeabilitási zavart szenvednek, de nem mutatnak súlyos sejtkárosodásra utaló ultrastrukturális jeleket (lásd az 5.2. alfejezetet). (II) Ellentétben az axonokkal, az idegsejtek szómájában illetve dendritjeiben rendezetlen irányaik miatt nem vitelezhető ki az ultrastrukturális elemeik közti átlagtávolságok meghatározása. (III) Elektromos sokkal [56], valamint 0.1 mm SDS-t tartalmazó fiziológiás só szíven-keresztüli perfúziójával [59] is sikerült 36

37 ultrastrukturális kompakciót létrehozni post mortem körülmények közt. (IV) Az ultrastrukturális axon-kompakció mechanizmusában korábban feltételezett calpainmediált strukturfehérje-bontás feltételezhetően nem oka, hanem következménye az axonkompakciónak. Elképzelésünk szerint a gélből gélbe történő átalakulással létrejött ultrastrukturális kompakció során az axolemma vongálódása révén axolemmális permeabilitási zavar alakul ki, amely azután a jól ismert mechanizmussal a calpain aktiválódásához és ezáltal citoszkeletális fehérje-bontáshoz vezet (lásd alfejezet) Terápiás beavatkozások tesztelése Egy sejt-permeábilis calpain-inhibitor, az MDL-28170, hatásának vizsgálata a diffúz axonkárosodásra (Buki A, Farkas O, Doczi T, Povlishock JT. Preinjury administration of the calpain inhibitor MDL attenuates traumatically induced axonal injury. J Neurotrauma Mar;20(3):261-8.) Háttér: A koponyatrauma által kiváltott axolemma-permeabilitási zavar ( mechanoporáció a megnövekedett intracelluláris Ca 2+ -koncentráció révén calpain aktiválódáshoz, az axonális citoszkeleton részleges lebontásához és következményes NFC-hez vezet [10]. Kontúziós koponyatrauma-modellekben a calpain gátlása jótékony hatásúnak bizonyult az agykérgi citoszkeletális károsodások, és funkcionális károsodások csökkentésében [105, 123, 124], illetve a használni kívánt vegyület neuroprotektív hatású volt agyi ischemia során [68, 71]. Cél: Egy sejt-permeábilis calpain-inhibitor, az MDL-2817, hatásának vizsgálata a diffúz axonkárosodás két morfológiai jellemzőjére, a csökkent intraaxonális transzportra (IAT) és a neurofilament kompakcióra (NFC). Módszerek: A Marmarou-féle készülékkel kiváltott koponyatrauma előtt 30 perccel MDL t, vagy vivőanyagot (kontrol) iv-kapott patkányokat a trauma után 2 órával a szíven keresztül fixálószerrel perfundáltuk. A szagittális agytörzsi metszetek egy hányadán az IAT markerével (anti-app), a szomszédos metszeteken pedig az NFC markerével (RMO-14) immunhisztokémiát végeztünk. A károsodott axonok számát a kortikospinális pályában (CSpT) és a mediális hosszanti kötegben (MLF) 37

38 összehasonlítottuk a kezelt és a kontrol patkányok vonatkozásában. Eredmények: Az anti-app-pozitív és az RMO-14-pozitív axon-profilok morfológiai jellemzőiben nem volt különbség megfigyelhető a kezelt illetve a kontrol patkányokban. A kvantitatív analízis szerint a trauma előtt végzett MDL kezelés szignifikánsan csökkentette az RMO-14-pozitív axonok számát mind a CSpT (p<0.02), mind pedig az MLF területén (p<0.03). Az anti-app-pozitív axonok száma is statisztikailag szignifikáns csökkenést mutatott az MDL nel való kezelést követően mindkét vizsgált agytörzsi területen (p<0.03 a CSpT estén és p<0.01 az MLF esetén). Következtetések: A calpain-gátlásnak az NFC kialakulására kifejtett jótékony hatása magyarázható a calpain-mediált spektrin-bontás és az NFC ismert kapcsolatával. Bár a legújabb kísérleti eredmények azt mutatják, hogy az NFC és az IAT különböző axon-populációkat érint [73, 135], a calpain-gátlásnak az IAT-ra kifejtett pozitív hatása azt bizonyítja, hogy ha a kompakció nem is társul transzport-zavarral a calpain aktiválódása mindkét folyamatban jelentős szerepet játszik. Megjegyzés: Bár kísérleteink tovább erősítik a calpain és a CMSP szerepének az NFC-ben betöltött jelentőségét, ez nincs ellentmondásban az axon-kompakció általunk feltételezett mechanizmusával (lásd alfejezet) A trauma előtt illetve után iv- illetve icv-adott PACAP neuroprotektív hatásának vizsgálata; valamint a hatásos dózis megállapítása (Farkas O, Tamas A, Zsombok A, Reglodi D, Pal J, Buki A, Lengvari I, Povlishock JT, Doczi T. Effects of pituitary adenylate cyclase activating polypeptide in a rat model of traumatic brain injury. Regul Pept Dec 15;123(1-3):69-75.) Háttér: A PACAP számos neurotrófikus és neuroprotektív hatással rendelkezik in vitro. In vivo jótékony hatásúnak bizonyult mind a globális, mind a fokális agyi ischemia kezelésében [117, 118, 142], emellett traumás gerincvelő-sérülésekben [55], továbbá látóideg- illetve arcideg-átvágás esetén is [57, 127]. Az ischemiás és a traumás agykárosodás kialakulásában szerepet játszó részben hasonló mechanizmusokból kiindulva feltételezhető, hogy ez a vegyület traumás agykárosodás esetén is hatásos lehet. Cél: A PACAP esetleges neuroprotektív hatásának vizsgálata a traumás 38

39 agysérülések során kialakuló diffúz axonkárosodásra; illetve a vegyület hatásos dózisának megállapítása. Módszerek: (A) A Marmarou-féle készülékkel kiváltott koponyatrauma előtt PACAP-ot iv-kapott patkányokat 2 illetve 6 órával a koponyatrauma után perfúziósan fixáltuk, a szagittális agytörzsi metszeteket anti-app immunhisztokémiával vizsgáltuk, a károsodott axonok számát a kezelt illetve a kontrol patkányokban a CSpT és az MLF területén összehasonlítottuk. (B) A hatásos dózis megállapítása céljából 1 μg, 10 μg és 100 μg PACAP-ot adtunk be az (A) kísérlet eredményeiből kiindulva icv; a túlélési idő 2 óra volt. Eredmények: (A) Intravénás adagolás esetén más KIR betegségekben hatásosnak bizonyult dózisban a PACAP nem bizonyult neuroprotektívnek (sem a CSpT sem pedig az MLF területén nem csökkentette szignifikánsan az anti-app immunpozitív axonok számát). (B) A PACAP intracerebroventrikuláris adagolásban 1 μg-os és 10 μg-os dózis esetén szintén nem bizonyult hatásosnak, 100 μg-os dózis azonban szignifikánsan csökkentette a CSpT területén talált anti-app immunpozitív axonszakaszok számát (p<0.05). Következtetések: (1) A PACAP az apoptotikus és a gyulladásos mediátorok gátlása, illetve bizonyos mitokondriális enzimek befolyásolása révén fejthet ki neuroprotektív hatást. E folyamatok mind az elsődleges, mind a másodlagos (lásd az 1.2. alfejezetet a Bevezetésben) patológiás folyamatok következtében traumás agysérülések esetén is megfigyelhetők [11, 66, 143]. (2) Az intravénás adagolás sikertelensége abban keresendő, hogy bár a PACAP bizonyítottan átjut a vér-agy gáton transzportjának mértéke az agytörzsben sokkal alacsonyabb, mint más agyterületeken [91]. (3) Hasonlóan, az agytörzs területén a feltételezett alacsony mértékű liquor-agy transzport lehet a magyarázata a más kísérletekben tapasztaltabbaknál magasabb hatásos dózis igénynek icv alkalmazás esetén. Megjegyzés: A jelen kísérletek célja pusztán a PACAP traumás axonkárosodásra gyakorolt hatásának felmérése volt, ezért adása közvetlenül a trauma kiváltása után történt. A klinikai alkalmazhatóság feltételeként késleltetve adott PACAP hatékonyságának vizsgálatával az alfejezetben foglalkozunk. 39

40 A terápiás ablak meghatározása icv PACAP-kezelés esetén (Tamas A, Zsombok A, Farkas O, Reglodi D, Pal J, Buki A, Lengvari I, Povlishock JT, Doczi T. Postinjury administration of pituitary adenylate cyclase activating polypeptide (PACAP) attenuates traumatically induced axonal injury in rats. J Neurotrauma May;23(5): ) Háttér: Egy korábbi kísérlet szerint (lásd ) a koponyatrauma után azonnal icv-beadott PACAP csökkenti az axonális transzport fokális leállása által okozott axonkárosodást. Cél: (A) A trauma kiváltását követően késleltetve icv-adott PACAP hatékonyságának tesztelése diffúz axonkárosodásban az intraaxonális transzportzavar és a neurofilament kompakció vonatkozásában; (B) továbbá a terápiás ablak meghatározása. Módszerek: A Marmarou-féle készülékkel kiváltott koponyatrauma után 30 perccel illetve 1 órával 100 μg icv-beadott PACAP- vagy vivőanyag-kezelésben részesült patkányokat a traumát követően 2 órával perfundáltuk. A szagittális agytörzsi metszetek egy hányadán az IAT markerével (anti-app), a szomszédos metszeteken pedig az NFC markerével (RMO-14) immunhisztokémiát végeztünk. A károsodott axonok számát a kortikospinális pályában (CSpT) és a mediális hosszanti kötegben (MLF) összehasonlítottuk a kezelt illetve a kontrol patkányok vonatkozásában. Eredmények: A PACAP 100 μg-os dózisban (az alfejezet során megfigyelt hatásos dózis) mind 30 perccel (p<0.01), mind pedig 1 órával a koponyatrauma kiváltása után icv.-beadva szignifikánsan (p<0.001) csökkentette a CSpT területén az anti-app-pozitív axonok számát. Nem volt azonban hatással a fenti időpontokban egyikében sem az anti-app-pozitív axonok számára az MLF területén, illetve az RMO-14-pozitív axonok számára egyik vizsgált agytörzsi területen sem. Következtetések: (1) A koponyatrauma által okozott DAI progressziójának PACAP-kezeléssel való részleges gátlására jelentős nagyságú terápiás ablak áll rendelkezésre, ami a klinikai alkalmazhatóság alapfeltétele. (2) Az NFC-ra gyakorolt jótékony hatás elmaradása megerősíti azokat a megfigyeléseket, amelyek szerint az IAT és az NFC részben különböző axon-populációkat érint [73, 135], és kialakulásukban részben különböző mechanizmusok játszanak szerepet (lásd pl alfejezet). (3) Az IAT-ra gyakorolt PACAP-hatás elmaradása az MLF területén valószínűleg annak 40

41 köszönhető, hogy (i) az MLF területén sokkal kevesebb a károsodott axonok száma, mint a CSpT-ben, ami megnehezíti a statisztikai kimutathatóságot, illetve (ii) az MLF területén a traumát követően 2 órával az NFC-t mutató károsodott axon-profilok dominálnak A neuronális mechanoporáció, resealing és CMSP vizsgálata (Farkas O, Lifshitz J, Povlishock JT. Mechanoporation induced by diffuse traumatic brain injury: an irreversible or reversible response to injury? J Neurosci Mar 22;26(12): ) Háttér: A koponyatrauma hatására ébredő intracerebrális erők a plazmamembrán mechanoporációját válthatják ki [38, 133]. Ez lehetőséget nyit arra, hogy az érintett idegsejtek nagy mólsúlyú anyagokat tudjanak felvenni az extracelluláris térből, szemben más idegsejtekkel, amelyek ép plazma-membránja nem engedi át ezeket az anyagokat. A tartós permeabilitás-zavar az ion-homeosztázis felborulása elsősorban az intracelluláris Ca 2+ -koncentráció megnövekedése miatt, különböző enzimkaszkádok például a calpain aktiválódása révén sejthalálhoz vezethet. Cél: Annak a hipotézisnek a tesztelése, hogy (I) a mechanoporációt szenvedett neuronok egy része képes a sejtmembrán regenerációjára és a túlélésre, illetve, hogy (II) a permeabilitás-zavar calpain-aktiválódással és következményes spektrin-bontással társul a koponyatrauma által kiváltott diffúz idegsejt-károsodás esetén. Módszerek: A koponyatrauma előtt illetve különböző idő elteltével a trauma után adott fluorescens festékkel konjugált, nagy-mólsúlyú dextrán icv infúziójával teszteltük a tartós permeabilitási zavar fennállását és az esetleges membrán-helyreállítódást. (A) Formalinos perfúziós fixálás és vibratómos metszés után konfokális mikroszkóppal vizsgáltuk a metszeteket. (B) Dextránnal konjugált festék ellenes antitest segítségével végzett immunhisztokémia és EM-feldolgozás útján vizsgáltuk a permeabilitásváltozással asszociált ultrastrukturális elváltozásokat. (C) CMSP kimutatására képes antitesttel végzett fluorescens immunhisztokémia után konfokális mikroszkópiával vizsgáltuk a permeabilitás-változás és a calpain-aktiválódás ko-lokalizációját. A calpainasszociált ultrastrukturális elváltozások vizsgálatára a metszetek egy része EM feldolgozásra került. Kvantitatív analízist végeztünk a permeabilitás-változást és/vagy CMSP-t mutató idegsejtek arányának a vizsgált időpontokban való összehasonlítására. 41

42 Eredmények: A permeabilitás-változást szenvedett idegsejteknek kb. fele mutatott tartósan fennálló permeabilitási zavart a vizsgált időpontokban. E sejtek egy hányadánál súlyos sejtkárosodás jeleit észleltük, más hányaduk azonban csak minimális ultrastrukturális elváltozásokat mutatott. A tartós permeabilitási zavar csak kevesebb, mint 15 %-ban társult calpain aktiválódással. A sejtek egy hányadánál megtörtént a membrán helyreállítódása, ezek nem mutattak súlyos sejtkárosodás. Olyan idegsejtek is megfigyelhetőek voltak, melyek késői permeabilitási zavart mutattak. A CMSP-t mutató sejtek kb. harmada nem szenvedett permeabilitás-változást. E sejtek moderált ultrastrukturális károsodást mutattak, a nekrotikus sejtek nagy részében CMSP nem volt kimutatható. Következtetések: (1) A membrán-sérülés nem vezet feltétlenül gyors sejthalálhoz, szemben azzal, amit korábban feltételeztek. A membrán-regenerációt mutató sejtek mellett valószínűleg a tartós permeabilitási zavart mutató sejtek egy hányada is képes a regenerációra. (2) A késői permeabilitás-zavar hátterében a trauma után órákig tartó mérsékelten emelkedett intrakraniális nyomásnak lehet szerepe. (3) Kvantitatív elemzéseink azt mutatják, hogy a túlélési idő növekedésével a különféle membránsérülésben (elhúzódó permeabilitás-zavar, helyreállítódás, késői permeabilitási zavar) érintett neuronok között átrendeződés megy végbe, mely során a helyreállítódottmembránú neuronok egy hányadának újra kinyílik a membránja, míg a folyamatban korábban nem érintett neuronok egy hányada később permeabilitás-zavart szenvedhet. Ez a megfigyelés a késői permeabilitás-zavar jelentőségét hangsúlyozza, és felhívja a figyelmet a trauma által kiváltott másodlagos patológiás tényezők (lásd az 1.2. alfejezetet a Bevezetésben) szerepére. (4) A membrán-sérülés nem feltétlenül szükséges vagy elegendő triggere a calpain aktiválódásnak, a trauma indukálta sejthalál az adott időkereten belül nincs feltétlenül összefüggésben a calpain aktiválódással. Megjegyzés: Noha megfigyeléseink felhívják a figyelmet arra, hogy a CMSP nem döntő jelentőségű a diffúz traumás idegsejt-károsodásban, a calpain-aktiválódást a diffúz agysérülések fontos részfolyamatának tartjuk. Ennek bizonyítékai többek között a calpain-inhibitorokkal elért sikerek a TAI kezelésében (lásd alfejezetet), illetve a calpain-specifikus spektrin-lebontási termékek jelenléte a cerebrospinális folyadékban (lásd 5.3. fejezetet). 42

43 5.3. A calpain- és caspase-aktiválódás szerepe a humán traumás agykárosodásban (Farkas O, Polgar B, Szekeres-Bartho J, Doczi T, Povlishock JT, Buki A. Spectrin breakdown products in the cerebrospinal fluid in severe head injury-- preliminary observations. Acta Neurochir (Wien) Aug;147(8): ) Háttér: Habár a koponyatrauma által kiváltott fokális illetve diffúz agysérülések eltérő módon jönnek létre és klinikai manifesztációjuk is különböző, hasonló biokémiai folyamatok mindkét forma progressziójában szerepet játszanak. Ilyen folyamat például a calpain és a caspase aktiválódása [10, 11, 53, 54, 87, 100, 122]. Ezek következtében specifikus spektrin-degradációs termékek (SBDP) felszaporodása figyelhető meg az agyszövetben mind állatkísérletekben [10, 62, 63, 125], mind humán koponyatraumát követően [83], illetve kísérleti állatokban a cerebrospinális folyadékban (CSF) [102]. Cél: (I) A calpain- és caspase-mediált folyamatok részvételének igazolása a humán koponyatraumában specifikus spektrin-degradációs termékeknek a humán CSFből (liquorból) való kimutatásával; (II) továbbá patomechanizmus-specifikus biokémiai markerek azonosítása céljából annak vizsgálata, hogy ezen termékek liquorkoncentrációja mutat-e összefüggést az agysérülés klinikai képével, például súlyosságával és kimeneteléve Módszerek: 9 súlyos koponyatraumát (GCS<9), 3 subarachnoidális vérzést (SAV) illetve 3 intraventrikuláris vérzést (IVV) szenvedett, továbbá 3 agytumoros beteg ICP kontroll céljából behelyezett icv katéteren keresztül lebocsátott liquor-mintáin, illetve 5 diagnosztikus-célú lumbál-punkción átesett beteg liquor-mintáin a calpain- és caspasespecifikus SBDP-k kimutatására Western blottot végeztünk. A kapott eredményeket denzitometriás módszerrel hasonlítottuk össze. Néhány beteg esetén ezen kóros fehérjék jelenlétét a liquorban a traumától eltelt idő függvényében is meghatároztuk. Eredmények: (A) A 280 kd molsúlyú intakt, és a 120 kd súlyú caspase-specifikus spektrin-degradációs termék a koponyatraumát szenvedettek szignifikánsan nagyobb hányadában volt jelen, mint más betegek esetén. A diagnosztikus lumbál-punkción átesett betegek CSF-mintái nem tartalmaztak SBDP-t. (B) Az intakt spektrin, valamint a 150 kd és a 120 kd nagyságú SBDP-k szintjét szignifikánsan magasabbnak találtuk 43

44 koponyatrauma esetén, mint a vizsgált nem-traumás agysérülésekben. (C) A fehérjék liquor-szintje a trauma utáni 2-3. napon tetőzött, majd visszatért a kiindulási szintre, ezt követően egy újabb emelkedés volt tapasztalható. (D) A spektrin-szintek és a klinikai paraméterek összehasonítása során sem a súlyossági fokkal (GCS alapján), sem a kimenetellel (GOS alapján), sem pedig az ICP-emelkedés mértékével nem találtunk szignifikáns összefüggést. Következtetések: (1) A lumbál-punkción átesett betegek esetén tapasztalt negatív eredmény igazolja, hogy az SBDP-k megjelenése a CSF-ben agysérülés és/vagy emelkedett intrakraniális nyomás következménye. (2) A koponyatrauma során más ICP-emelkedéssel járó állapotokkal szemben tapasztalt magasabb SPDP-szint arra utal, hogy a megfigyelt jelenség a sérülés direkt következménye is, és nem csupán az emelkedett ICP eredménye. (3) A vizsgált fehérjék liquor-szintjének jellegzetes időösszefüggése alapján feltételezhető, hogy a calpain- és caspase-specifikus SBDP-k vizsgálata a jövőben hasznos pathomechanizmus-specifikus biomarker lehet a koponyatraumát szenvedettek állapotának nyomon követésében. Megjegyzés: (I) A kísérletbe bevont betegek viszonylag alacsony száma egyértelműen jelzi munkánk korlátait, a kísérletek pusztán tájékozódó jellegét. Valószínűleg a kis esetszám az oka annak is, hogy a megfigyelt tendenciák ellenére az SBDP-k liquor-szintje és a klinikai paraméterek között szignifikáns összefüggés nem volt megfigyelhető. (II) Klinikánkon megkezdődött a súlyos koponyatraumát szenvedett és ICP-monitorozás céljából behelyezett kamradrénes betegek CSF-mintáinak szisztematikus gyűjtése, ami a jövőben nagymértékben hozzájárulhat a különböző biomarkerek azonosítása és diagnosztikus/prognosztikus jelentőségük felmérése céljából indított vizsgálataink kiterjesztéséhez. 44

45 6. A TÉMÁBAN ELÉRT, NEMZETKÖZI-JELENTŐSÉGŰ EREDMÉNYEK 6.1. A traumás axonkárosodás Az ultrastrukturális axon-kompakció I. Elsőként igazoltuk, hogy a koponyatrauma következtében kialakuló, argyrophil- III típusú ezüstözési technikával festődő axonok azonosak a koponyatrauma következtében ultrastrukturális kompakciót szenvedett axonokkal. II. A traumás axon-kompakció mechanizmusának vizsgálata során elsőként igazoltuk, hogy (A) az ultrastrukturális axon-kompakció a trauma pillanatában következik be, nem pedig 5-15 perces késéssel, ahogy azt korábban feltételezték, (B) a citoszkeletális kompakció viszonylag hosszú, akár 1 mm-es axon-szakaszokra is kiterjedhet, azaz a korábbi feltételezésekkel ellentétben nem EM-szinten fokális elváltozás, (C) az in vivo és a post mortem koponyatrauma egyforma morfológiai elváltozásokat eredményez, mind a (i) a hyperbasophilia, (ii) a III-típusú argyrophilia, (iii) a hiperelektrondenzitás, mind pedig (iv) az ultrastrukturális kompakció tekintetében, (D) az ultrastrukturális axon-kompakció foka a post mortem trauma esetén nem különbözik az in vivo trauma esetén meghatározottól, (E) a trauma-indukálta ezüstöződés minkét esetben minden vagy semmi jellegű, azaz az érintett szóma-dendrit domének teljes egészére, illetve hosszú axon-szakaszokra terjed ki, de általában nem egyazon idegsejt szóma-dendrit doménjét és axonját érinti. III. Kísérleteink során kidolgoztunk egy olyan új koponyatrauma modellt, mely alkalmas az ultrastrukturális axon-kompakció szelektív előidézésére. IV. A koponyatrauma hatására kompaktálódott axonok sorsának vizsgálatával 45

46 elsőként írtuk le (A) a kompakciót szenvedett axonokban hónapok alatt lejátszódó morfológiai elváltozásokat, továbbá igazoltuk, hogy (B) az érintett axonok >50%-a 1 napon belül, míg <10%-a 1 nap és 1 hét között regenerálódik, azaz visszanyeri eredeti ultrastruktúráját. A többi kompaktálódott axon néhány hónap alatt regenerálódik, (C) a kompaktálódott axonok az agykéregben több óra elteltével sem festődnek az agytörzsi axon-kompakció kimutatására széles körben elterjedt markerrel, az RMO-14 antitesttel. (D) A kompaktálódott axonok nem mutatnak pozitivitást az IAT markerével, ami megerősíti azt az elképzelést, hogy az intraaxonális transzportzavar és az axonkompakció döntően két különböző axon-populácót érint Terápiás beavatkozások I. Kísérleteinkkel elsőként igazoltuk, hogy a sejt-permeábilis calpain-inhibitor, MDL szignifikánsan csökkenti az ultrastrukturális kompakciót szenvedett, illetve az intraaxonális transzport-zavart mutató axonok számát a TAI kialakulására jellegzetes agytörzsi régiókban, azaz a CSpT és az MLF területén. II. Elsőként igazoltuk, hogy a PACAP traumás axonkárosodás esetén szignifikánsan csökkenti az intraaxonális transzport-zavart mutató axonok számát az agytörzsben. III. Traumás axonkárosodás esetén kísérleteink során elsőként határoztuk meg PACAP-kezelés tekintetében (A) a hatásos dózist (B) a terápiás ablakot A diffúz idegsejt-károsodás 46

47 Kísérleteink során elsőként vizsgáltuk a koponyatrauma által kiváltott idegsejtmembrán permeabilitási zavar alakulását órákkal a traumát követően in vivo modellben. Elsőként igazoltuk, hogy (A) a traumás sejtmembrán-károsodás a korábbi nézetekkel ellentétben, az idegsejtek egy részében nem vezet súlyos ultrastrukturális elváltozásokhoz és gyors sejthalálhoz, (B) a tartós sejtmembrán permeabilitási zavar csak kevesebb, mint 15 %-ban társul calpain aktiválódással és következményes strukturális fehérjebontással, (C) a károsodott sejtmembrán a sejtek egy részénél helyreállítódik, ezek a sejtek nem szenvednek súlyos károsodást, (D) sejtmembrán permeabilitási zavar az idegsejtek egy részénél órákkal a traumát követően alakul ki, melynek hátterében feltételezhető a kísérleteink során észlelt, tartósan 20 Hgmm körüli ICP emelkedés, (E) a calpain-aktiválódást és következményes CMSP-t mutató sejtek kb. harmada nem szenved permeabilitás-változást. E sejtek közepes fokú ultrastrukturális károsodást mutatnak, a nekrotikus sejtek nagy részében nem igazolható CMSP. (F) a túlélési idő növekedésével a membrán-sérülésben érintett neuronok között (elhúzódó permeabilitás-zavar, helyreállítódás, késői permeabilitási zavar) átrendeződés megy végbe, mely során a helyreállítódott-membránú neuronok egy hányadának újra kinyílik a membránja, míg a folyamatban korábban nem érintett neuronok egy hányada később szintén permeabilitás-zavart szenvedhet A calpain- és caspase-aktiválódás szerepe a humán traumás agykárosodásban I. Kísérleteink során elsőként vizsgáltuk a calpain és caspase aktiváció és következményes strukturális fehérjebontás során létrejött spektrin degradációs termékek jelenlétét humán cerebrospinális folyadékban. Megerősítettük, hogy II. a calpain- és caspase-aktiváció fontos patobiokémiai tényező a traumás agykárosodás kialakulásában, továbbá elsőként igazoltuk, hogy 47

48 III. a specifikus SBDP-k megjelenése a CSF-ben agysérülés és/vagy emelkedett intrakraniális nyomás következménye, IV. az intakt spektrin, valamint a 150 kd és a120 kd nagyságú SBDP-k CSF-szintje szignifikánsan magasabb koponyatrauma esetén, mint a vizsgált nem-traumás agysérülésekben, V. a spektrin-degradációs termékek liquor-szintje jellegzetes idő-kinetikát mutat, azaz a trauma utáni 2-3. napon tetőzik, majd visszatér a kiindulási szintre, ezt követően egy újabb emelkedés tapasztalható. VI. Kísérleteink nagyban hozzájárultak egy nemzetközi, multicentrikus, a PTE ÁOK Idegsebészeti Klinika részvételével folyó vizsgálat indításához, amely különböző biomarkerek azonosítása és diagnosztikus/prognosztikus jelentőségük felmérése céljából több más fehérjével együtt a spektrin degradációs termékek vizsgálatát is célul tűzte. 48

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59 KÖSZÖNETNYILVÁNÍTÁS Mindenek előtt, hálás köszönettel tartozom témavezetőimnek, Gallyas Ferenc Professzor Úrnak és Dr. Büki Andrásnak a PhD éveim alatt nyújtott felbecsülhetetlen segítségükért, támogatásukért, bizalmukért, bíztatásukért, konstruktív kritikáikért. Köszönöm volt mentoromnak, Dr. Sándor Jánosnak, hogy elindított a tudomány rögös útján, és azóta is szemeit rajtam tartva, töretlen bizalommal és bíztatással kíséri végig tudományos életem alakulását. Külön köszönet illeti John T. Povlishock Professzor Urat, a Virginia Commonwealth University Anatómiai és Neurobiológiai Intézete vezetőjét, aki lehetővé tette, hogy 3 éven keresztül laboratóriumában dolgozhassam, és ott tartózkodásom ideje alatt illetve hazajövetelem óta is támogatásával és bizalmával tisztel meg. Köszönöm Dóczi Tamás Professzor Úrnak, hogy lehetővé tette, hogy a PTE ÁOK Idegsebészeti Klinika kutató laboratóriumában folytathassam munkáimat. Köszönet illeti a PTE ÁOK Idegsebészeti Klinikájának kutató laboratóriumában dolgozókat, név szerint: Nyirádi Józsefet, Andok Csabánét és Nádor Andrásnét, illetve a Virginia Commonwealth University Anatómiai és Neurobiológiai Intézetének laboránsait, Lynn Davist és Sue Walkert az évek alatt nyújtott felbecsülhetetlen technikai segítségükért. Köszönöm a PTE ÁOK Anatómiai Intézetéből Dr. Reglődi Dórának és Dr. Tamás Andreának közös munkáink során nyújtott együttműködését. Köszönöm a Pécsi Tudományegyetem Idegsebészeti Klinika intenzív osztályán dolgozóknak a súlyos koponyatraumás betegek liquor- és vér-mintagyűjtésében nyújtott segítségét. Továbbá köszönöm a PTE ÁOK Mikrobiológiai Intézetében dolgozóknak, elsősorban Dr. Szekeres Júlia Professzor Asszonynak, Dr. Polgár Beátának és Kiss Ágnesnek, e minták feldolgozásában való áldozatos közreműködését. Köszönetet mondok végül, de nem utolsó sorban minden kedves volt és jelenlegi kollégámnak, családomnak és barátaimnak a sok bíztatásért és lelki támaszért.

60 Acta Neuropathol (2002) 103 : DOI /s REGULAR PAPER F. Gallyas O. Farkas M. Mázló Traumatic compaction of the axonal cytoskeleton induces argyrophilia: histological and theoretical importance Received: 27 November 2000 / Revised: 26 February 2001 / Accepted: 25 May 2001 / Published online: 4 September 2001 Springer-Verlag 2001 Abstract It was earlier established that one of the primary morphopathological consequences of experimental traumatic brain injury is a dramatic reduction in the distances between the neurofilaments (cytoskeletal compaction) inside a number of axon segments that appear to be randomly distributed among normal axons in an otherwise undamaged parenchymal environment. The present results demonstrate that the cytoskeletal compaction instantly induces argyrophilia, thereby rendering possible selective visualisation of the affected axon segments for light microscopy through use of a special silver staining method. On combination of this method with electron microscopy, it was revealed that the cytoskeletal compaction is completed in much shorter times and extends to much longer axon segments than previously assumed. Keywords Head injury Axonal pathology Cytoskeletal compaction Silver staining Electron microscopy Introduction F. Gallyas ( ) O. Farkas Department of Neurosurgery, Faculty of Medicine, Pécs University, Rét utca 2, 7623 Pécs, Hungary ferenc.gallyas.sen@aok.pte.hu, Fax: M. Mázló Central Electron Microscopic Laboratory, Faculty of Medicine, Pécs University, Szigeti ùt 12, 7623 Pécs, Hungary In selected brain areas of experimental animals that survive for 5 min or longer following fluid percussion, impact acceleration or non-disruptive axonal injury to central myelinated nerve fibres, the following ultrastructural changes have recently been discovered in a number of axon profiles scattered among the normal-looking axon profiles: the mitochondria and myelin sheaths become swollen, a considerable proportion of the microtubules and the side-arms of the neurofilaments disappear, periaxonal water-filled spaces develop, and the distances between the neurofilaments are dramatically reduced [11, 12, 18, 19, 20]. In consequence of the latter feature, this morphological phenomenon has been designated as neurofilament/cytoskeletal/axonal compaction/condensation/ collapse. It has been hypothesised to precede axonal disconnection in experimental diffuse traumatic axonal injury (TAI) [14]. Using a special silver staining method [6, 8] that yields reproducible results [15], we have previously demonstrated [5, 7] that millimetre-long axon segments were stained with silver in a diffuse distribution within certain brain areas of rats that had been perfusion-fixed immediately after suffering either contusing or non-contusing concussive head injuries, whereas no axons were stained with silver in the control animals. This induced argyrophilia was suggested to be a manifestation of some, at that time unknown, primary morphological disturbance. Here we demonstrate that it is the axonal segments with a traumatically compacted cytoskeleton that are stained with silver by this method following a head injury. Furthermore, the histological and theoretical importance of this finding is discussed. Materials and methods Animal experiments Seven 400-g Sprague-Dawley rats were anaesthetised with a mixture of 4% isoflurane, 29% oxygen and 67% nitrous oxide using a SurgiVet respirator. The skull was exposed with a middle-line incision and a 4-mm-thick 10-mm-diameter brass disc was cemented with dental acrylic to the exposed skull vault, between the coronal suture and lambdoid. Thereafter, the animals were placed in a prone position on a 12-cm-thick foam bed under the impact-acceleration head-injury device of Marmarou et al. [13]. An intracerebral mechanical force was generated by dropping a metal rod with a mass of 450 g onto the brass disc from a height of 200 cm through a vertically positioned Plexiglas tube. Immediately after administration of the head injury, the rats were killed by transcardial perfusion with 50 ml physiological saline, followed by 500 ml of an aldehyde fixative (100 ml 20% paraformaldehyde, 100 ml 25% glutaraldehyde, 50 ml 0.1 mol/l calcium chloride, 500 ml 0.2 mol/l sodium cacodylate and 250 ml

61 37 distilled water were mixed, and the resulting solution was adjusted to ph 7.4 with a few drops of concentrated hydrochloric acid). Three additional animals, sham-operated and then perfusion-fixed without administration of the head injury, served as controls. Animal care and handling were carried out in accordance with order 243/1998 of the Hungarian Government, which is an adaptation of directive 86/609/EGK of the European Committee Council. Tissue processing All animals were left untouched at room temperature for 24 h after the start of fixation [3]. Thereafter the brains, together with a 5-mm-long stub of the spinal cord, were removed from the skull and then immersed in the fixative. Vibratome sections, 150 µm thick, were cut coronally from the caudal two-thirds of the cerebrum, and sagittally from the block including the cerebellum, pons, oblongata and spinal cord. Every fifth vibratome section was silver-stained. For electron microscopy, those areas of the subcortical white matter, caudate putamen and pons which contained numerous argyrophilic axon segments in the silver-stained sections were dissected from the nearby vibratome sections of head-injured animals. The corresponding areas were also cut from the vibratome sections of the control animals. These pieces of vibratome sections were post-fixed with a 1:1 mixture of 2% osmium tetroxide and 3% potassium ferrocyanide for 1 h at room temperature, and then flat-embedded in Durcupan ACM. Semithin sections were cut at 0.5 µm and air-dried onto microscopic slides previously coated with the Vectabond adhesive. Thin sections (50 nm) were contrasted with uranyl acetate and lead citrate. Silver staining Vibratome sections were dehydrated with graded 1-propanol (25%, 50%, 75% and 98%; 5 min in each), incubated for 16 h at 56 C in 1-propanol containing 0.8% sulphuric acid and 2% water (esterification), rehydrated with graded 1-propanol (75%, 50%, 25%) and distilled water (5 min in each), treated with 1% acetic acid for 10 min, and then immersed in a special physical developer until the background had become light-brown. The physical developer was prepared just before use by pipetting one part of stock solution B into one part of vigorously stirred stock solution A. Stock solution A consisted of 100 g anhydrous sodium carbonate dissolved in 1,000 ml of distilled water; stock solution B contained 2.5 g ammonium nitrate, 2.0 g silver nitrate, 100 ml 20% tungstosilicic acid, 100 ml 20% paraformaldehyde and 890 ml distilled water. Physical development was terminated by washing with 1% acetic acid for 30 min. Sections were dehydrated, cleared and covered with Canada balsam (for trouble shooting see [8]). Several silverstained sections were processed for electron microscopy. Durcupan-embedded semithin sections were silver-stained with the following modifications: (i) before esterification the sections were dehydrated by air-drying, and (ii) the treatment with acetic acid between the esterification and the physical development was replaced with a periodic acid treatment (1%, 10 min), followed by a short washing with distilled water (see discussion). Several silver-stained semithin sections were re-blocked, thin-sectioned and contrasted with uranyl acetate and lead citrate in the usual manner. Selected light microscopic areas of silver-stained semithin sections were compared with the electron microscopic pictures of the corresponding areas in nearby thin sections. Results Control animals Neither axons nor neurones were visualised by the silver method in the central nervous system of the control animals. However, hypertrophic astrocytic fibres were stained Fig. 1A, B Fibrous astrocytes in the pyramidal tract of the spinal cord of a control rat. A Silver-stained hypertrophic processes in a 150-µm-thick vibratome section; B hyperplastic glial filaments. The arrows in B point to dense bundles of glial filaments. Note that solitary long silver-stained astrocytic processes (arrow in A) can not be easily distinguished from silver-stained axon segments. A 225; bar B 500 nm with silver, mainly in the white matter of the spinal cord (Fig. 1a). The light microscopic distinction of solitary astrocytic fibres from argyrophilic axons in the silverstained sections of the head-injured animals can be difficult even for experienced observers. No axon profiles with cytoskeletal compaction were found under the electron microscope in the brain areas of the control animals that corresponded to the areas containing numerous silver-stained axon segments and compacted axon profiles in the head-injured animals. In the white matter of the spinal cord, hyperplastic bundles of glial filaments were found in several astrocytic processes (Fig. 1b).

62 38 Fig. 2 Silver-stained axon segments in 150-µm-thick vibratome sections cut from the subcortical white matter, corpus callosum and deep neocortex under the impact site (A), the reticular nucleus of the pons (B) and the caudate putamen (C) of a rat perfusionfixed immediately after suffering a head injury. The arrow points to a silver-stained interneurone, and the arrowhead to a neurone in the induseum griseum. Note that even millimetre-long axon segments are silver-stained. A, B 100; C 210 Head-injured animals Gross morphological observations Four of the seven head-injured animals suffered an impression fracture of the calvaria, with severe laceration and bleeding underneath. In all animals, either with or without a skull fracture, massive haemorrhage occupied the basal cisterns and extended to the subarachnoid spaces around the cerebral hemispheres, cerebellum and spinal cord. Light microscopic observations In all brains, long axon segments stained with silver in a scattered distribution or in dense bundles. The whole length of an axon was rarely stained. It was possible to follow individual axons for a maximum length of about 1 mm. The spatial distribution of silver-stained axon segments showed wide variations from one animal to another. The small number of animals involved in the study did not allow a statistical description of the distribution patterns of argyrophilic axon segments. For this reason, only those areas of the brain are demonstrated where silver-stained axon segments were consistently found at an appreciable distance from the focal parenchymal lesions, in a number that appeared sufficient for electron microscopic observation. Such areas in the animals in which the skull was not fractured were the deep neocortex with the subcortical white matter under the impact site (Fig. 2a), the caudate putamen (Fig. 2c) and the pons (Fig. 2b). The distribution of the silver-stained axon segments in the two cerebral hemispheres was not symmetrical. Their incidence displayed considerable differences in each of the above areas from animal to animal. The microphotos in Fig. 2 were taken from the most seriously affected animal. Pyramidal neurones were not demonstrated anywhere in the neocortex, but an insignificant number of interneurones were stained with silver (not shown). In addition to silver-stained axon segments, the brains of animals in which the skull was fractured contained numerous silver-stained neurones and dendrites, even in areas far from the impact site (not shown). The abundance of capillaries filled with erythrocytes indicated that these brains were imperfectly perfused with the fixative. For this reason, they were excluded from the following ultrastructural description, although they contained abundant axon profiles with compacted cytoskeleton (not shown).

63 39 Fig. 3 Normal-looking (A, C) and compacted (B, D) axon profiles in the reticular nucleus of the pons (A, B) and the subcortical white matter (C, D) of a rat perfusion-fixed immediately after suffering a head injury. A small periaxonal fluidfilled space is marked with an asterisk. Microtubuli in A and B are indicated by arrowheads. Note that the distances between individual neurofilaments are dramatically reduced in B and D. Bars A D 200 nm Fig. 4 Comparison of the light microscopic image of a selected area in a silver-stained semithin section (A) with the electron microscopic image of the corresponding area in a nearby ultrathin section (B) cut from the caudate putamen of a rat perfusion-fixed immediately after suffering a head injury. The short arrow points to a large fluid-filled periaxonal space, and the long arrow to a lamellar separation of the myelin sheath. Note that the pattern of the silver-stained axon profiles corresponds to that of the compacted ones. Bars A, B 2 µm Electron microscopic observations In those brain areas of head-injured animals where argyrophilic axon segments were present, a number of axon profiles displaying a dramatic decrease in the distances between the individual neurofilaments (Fig. 3) were scattered among normal-looking axon profiles (Fig. 4b). The mitochondria appeared to be non-swollen (Figs. 3, 4b). Lamellar separation of the myelin sheath was infrequent. Fluid-filled spaces of various shapes and volumes had developed between the axolemma and the myelin sheath (Fig. 4b). In compacted axon profiles with small periax-

64 40 Fig. 5 Axon profiles in the caudate putamen of a rat perfusion-fixed immediately after suffering a head injury. The specimen was osmicated, Durcupan-embedded, and semithin-sectioned, silver-stained, re-blocked and thin-sectioned before being contrasted with uranyl acetate and lead citrate. Note that the normal-looking axon profiles do not contain silver grains. Bar 500 nm Fig. 6 Axon profiles in the caudate putamen (A) and the subcortical white matter (B) of a rat perfusion-fixed immediately after suffering a head injury. The specimen was silverstained before being processed for electron microscopy. Note that the silver-containing axon profiles are homogeneously electron dense, while those without silver grains have disintegrated. Bars A, B 200 nm onal fluid-filled spaces, the myelin sheath appeared to be somewhat deformed (Fig. 4b). In silver-stained, re-blocked and then contrasted semithin sections, numerous silver grains were found in axon profiles with a compacted cytoskeleton, but there were hardly any in the normal-looking axons (Fig. 5). The myelin sheath, axolemma and mitochondria also contained a few silver grains, of somewhat smaller sizes. They rendered the myelin sheath brown in semithin sections, while the compacted axon segments turned black and the non-compacted axons remained unstained. A comparison of the light microscopic pictures of selected areas in the silver-stained semithin sections (Fig. 4a) with the electron micrographs of the corresponding areas in nearby ultrathin sections (Fig. 4b) showed that the pattern of the silver-stained axonal cross-sections corresponded to that of the compacted axon profiles. Minute differences between the two pictures in the distances between the individual axons and in their shapes were probably caused by the facts that their directions differed slightly from each other and, for technical reasons, not the next neighbouring sections were compared. In specimens silver-stained before being processed for electron microscopy, silver grains were found only in homogeneously electron-dense axon profiles. In contrast, the ultrastructure of axon profiles without silver grains was disintegrated and electron lucent (Fig. 6). Discussion Methodological considerations It is generally believed that silver staining methods are unreliable. However, several recent methods employing physical developers instead of the chemical developers

65 applied in the traditional methods, do give reproducible results [15]. The silver technique used in the present study, which employs a time-honoured physical developer [4], has been widely used to obtain information on neuronal death. An important step in this silver staining method, esterification with strongly acidic n-propanol at 56 C for 20 h, considerably impairs the ultrastructure in non-osmicated vibratome sections (Fig. 6). Nevertheless, traumatically compacted axon profiles can be identified under the electron microscope through their marking with silver grains. It appears that they are more resistant than the normal axons to the structure-disintegrating action of esterification (Fig. 6). In a separate study (Gallyas et al., submitted for publication), osmophilic structural elements were found to be more argyrophilic than compacted neurones, dendrites or axons. As a consequence, the microscopic images of semithin sections silver-stained according to the original procedure were dominated by silver-stained membranes and membranous structures such as myelin sheaths and mitochondria. Of the chemical agents proposed for the removal of bound osmium from Durcupan-embedded sections [10], periodic acid proved to be the most effective for suppression of the co-staining of myelin sheaths and mitochondria. Histological considerations Compacted axon segments have so far been selectively stained for light microscopic observation only in animals surviving for at least 5 min [horseradish peroxidase (HRP) uptake] or for 15 min (Ab38 or RMO14 antibody positivity) following a head injury. If injected in due time into the cerebrospinal fluid, HRP inundates the extracellular space; it remains excluded from the normal axons, but it penetrates into those which display cytoskeletal compaction [18, 19, 20]. Ab38 and RMO14 antibodies are effective because calcium-mediated processes linked to the proteolysis of spectrin and to alterations in the neurofilament-m subunit, respectively, take place in the compacted axon segments [1, 17, 20]. Our present findings clearly demonstrate that the silver method used here can also be utilised for demonstration of axon segments with a compacted cytoskeleton by light microscopy. This applies both to animals perfusion-fixed immediately following a head injury (Fig. 2) and to those surviving for several minutes or hours thereafter (not demonstrated). By virtue of the high selectivity and low background staining of this method, compacted axons can easily be located even in 150-µm-thick vibratome sections (Fig. 2). Similar findings in the brains of mice, rats and pigs traumatised in three different ways, and also in surgically removed human hippocampus [21], lead us to assume that this silver staining technique could be instrumental (i) in mapping the distribution of compacted axon segments throughout the brains of mammals, and (ii) in locating areas in mammalian brains that could be investigated in nearby vibratome sections by electron microscopy, immunocytochemistry or other techniques. The occurrence of hypertrophic fibrous astrocytes, which may hamper the identification of silver-stained axon segments, is not necessarily associated with any neurological disease. In the brains of control (unlesioned) rats weighing about 200 g, a few hypertrophic astrocytic fibres occur in subependymal and subpial positions (not demonstrated). Their number increases with age, especially in the spinal cord (Fig. 1a). Non-hypertrophic fibrous astrocytes are not stained with the silver method used here (Fig. 2). Neuronal somata and dendrites with a traumatically compacted cytoskeleton ( dark neurones) are also stained by this silver method [5, 7]. In specific cases, it is not easy to distinguish solitary compacted dendrites from solitary compacted axons. Theoretical considerations 41 The prevailing concept of the traumatic compaction of the axonal cytoskeleton [1, 2, 9, 11, 12, 14, 16, 17, 18, 19, 20] is based on the following experimental findings. In certain brain areas of animals perfusion-fixed at or after 5 min following a head injury, (i) HRP injected into the cerebrospinal fluid, (ii) calpain-mediated spectrin proteolysis, (iii) some kind of alteration in the neurofilament-m subunit, (iv) reduction of the extension of neurofilament sidearms, (v) the disappearance of neurotubuli and neurofilament side-arms, and (vi) the swelling of mitochondria are demonstrable in the axon segments with a compacted cytoskeleton, but not inside the normal axons. Accordingly, some intracerebral force generated by the head injury is assumed to stretch a number of axons to such an extent that a focal axolemmal perturbation allowing uncontrolled influx of ions including calcium occurs. The latter activates the proteolytic enzyme calpain. This mediates spectrin proteolysis and neurofilament-m subunit alteration, which gradually disturb the side-arms of the neurofilaments, resulting in the compaction of the axonal cytoskeleton. The present study throws light on two previously unconsidered features of the traumatic compaction of the axonal cytoskeleton: 1. To date, no data have been reported on either the presence or the absence of compacted axon profiles in animals perfusion-fixed immediately after a head injury. The reason for this is probably the lack of any light microscopic technique that could indicate where to look for them under the electron microscope. Our present data clearly demonstrate that the cytoskeletal compaction is already completed in numerous axons of animals surviving for less than 1 min after the head injury. Consequently, the pathometabolic cascade resulting in the cytoskeletal compaction must proceed at a much higher speed than previously assumed. 2. As demonstrated by the silver staining method used here, the cytoskeletal compaction extends to much

66 42 greater distances in the affected axons (Fig. 2a, b) than previously estimated on the basis of pictures obtained using HRP histochemistry, Ab38 and RMO14 immunocytochemistry or electron microscopy. Thus, either the stretch-induced perturbation of the axolemma must extend to long axon segments (i.e. it is not focal) or, in case of focal axolemmal perturbation, the pathometabolic cascade resulting in the cytoskeletal compaction must spread over long distances during a relatively short period of time. Acknowledgements The authors express thanks to Andok Csabáné, Nyírádi József, Nádor Andrásné and Nagy Attila for their valuable help in the preparative work. This study was supported by Hungarian grants from OTKA (T ) and from FKFP (493/1999). References 1. Buki A, Siman R, Trojanowsky JQ, Povlishock JT (1999) The role of calpain mediated spectrin proteolysis in traumatically induced axonal injury. J Neuropathol Exp Neurol 58: Buki A, Onkowo DO, Wang KK, Povlishock JT (2000) Cytochrome c release and caspase activation in traumatic axonal injury. J Neurosci 20: Cammermeyer J (1961) The importance of avoiding dark neurons in experimental neuropthology. Acta Neuropathol (Berl) 1: Gallyas F (1971) A principle for silver staining of tissue elements. Acta Morphol Acad Sci Hung 19: Gallyas F, Zoltay G (1992) An immediate light microscopic response of neuronal somata, dendrites and axons to non-contusing concussive head injury. Acta Neuropathol 83: Gallyas F, Güldner FH, Zoltay G, Wolff JR (1990) Golgi-like demonstration of dark neurons with an argyrophil III method for experimental neuropathology. Acta Neuropathol 79: Gallyas F, Zoltay G, Balás I (1992) An immediate light microscopic response of neuronal somata, dendrites and axons to contusing concussive head injury. Acta Neuropathol 83: Gallyas F, Hsu M, Buzsáki G (1993) Four modified silver methods for thick sections of formaldehyde-fixed mammalian central nervous tissue: dark neurons, perikarya of all neurons, microglial cells and capillaries. J Neurosci Methods 50: Graham DI, McIntosh TK, Maxwell WL, Nicoll JAR (2000) Recent advances in neurotrauma. J Neuropathol Exp Neurol 59: Hayat MA (1981) Fixation for electron microscopy. Academic Press, New York, p Jafari SS, Maxwell WL, Neilson M, Graham DI (1997) Axonal cytoskeletal changes after nondisruptive axonal injury. J Neurocytol 26: Jafari SS, Neilson, M, Graham DI, Maxwell WL (1998) Axonal cytoskeletal changes after nondisruptive axonal injury. II. Intermediate sized axons. J Neurotrauma 15: Marmarou A, Foda MA, Brink W van den, Campbell J, Kita H, Demetriadou K (1994) A new model of diffuse brain injury in rats. Part I. Pathophysiology and biomechanics. J Neurosurg 80: Maxwell WL, Povlishock JT, Graham DL (1997) A mechanistic analysis of nondisruptive axonal injury: a review. J Neurotrauma 14: Newman GR, Jasani A B (1998) Silver development in microscopy and bioanalysis: past and present. J Pathol 186: Onkowo DO, Pettus EH, Moroi J, Povlishock JT (1998) Alteration of the neurofilament sidearm and its relation to neurofilament compaction. Brain Res 784: Onkowo DO, Büki A, Siman R, Povlishock JT (1999) Cyclosporin A limits calcium-induced axonal damage following traumatic brain injury. NeuroReport 10: Pettus EH, Povlishock JT (1996) Characterization of a distinct set of intra-axonal structural changes associated with traumatically induced alteration in axolemma permeability. Brain Res 772: Pettus EH, Christman CW, Giebel ML, Povlishock JT (1994) Traumatically induced altered membrane permeability: its relationship to traumatically induced reactive axonal change. J Neurotrauma 11: Povlishock JT, Marmarou A, McIntosh T, Trojanowsky JQ, Moroi J (1997) Impact acceleration injury in the rat: evidence for focal axolemmal change and related neurofilament sidearm alteration. J Neuropathol Exp Neurol 56: Van den Pol AN, Gallyas F (1990) Trauma-induced Golgi-like staining of neurons: a new approach to neuronal organization and response to injury. J Comp Neurol 296:

67 Journal of Neuroscience Methods 153 (2006) Selective induction of ultrastructural (neurofilament) compaction in axons by means of a new head-injury apparatus József Pál a,1, Zsolt Tóth b,2, Orsolya Farkas b,3,lóránd Kellényi b, Tamás Dóczi a,b, Ferenc Gallyas b, a Clinical Neuroscience Research Group of the Hungarian Academy of Sciences, Pécs University, Rét utca 2, H-7623 Pécs, Hungary b Department of Neurosurgery, Pécs University, Rét utca 2, H-7623 Pécs, Hungary Received 1 August 2005; received in revised form 10 November 2005; accepted 10 November 2005 Abstract A new weight-drop head-injury apparatus is described that can produce a momentary depression of predetermined depth at a predetermined site of the elastic calvaria of scalped young adult rats. In Wistar rats weighing about 200 g, a 0.75-mm deep calvaria depression immediately caused ultrastructural (neurofilament) compaction in many long axon segments, which were diffusely scattered among non-compacted axons in a well-defined area of cortical layers IV and V under the impact site. Apart from these morphological changes and swollen astrocytic processes in their vicinity, the brain tissue appeared non-impaired. The blood pressure, intracranial pressure, heart rate and respiration rate had returned to the normal range in 1 min. Diffuse axonal swelling caused by impaired axonal transport, ultrastructural compaction in neuronal soma-dendrite domains, impression fracture and subarachnoid or subdural hemorrhages were observed only in rats with a calvaria depression of 1 mm or more. All these features create favorable circumstances for study of various problems that are closely related to the ultrastructural (neurofilament) compaction in axons, such as the fate of the affected axons Elsevier B.V. All rights reserved. Keywords: Head injury; Calvaria depression; Axonal injury; Staining with silver; Electron microscopy; Ultrastructural compaction 1. Introduction The morphopathological responses of the human brain to blunt head injuries are rather complex (Graham et al., 2000). Certain responses set in rapidly (e.g. focal contusions or hemorrhages) whereas others are initiated primarily, but their final stages develop slowly (e.g. diffuse axonal injury or apoptotic neuronal death). Additionally, traumatically induced pathophysiological processes (e.g. ischemia or elevated intracranial pres- Corresponding author. Tel.: ; fax: addresses: jozsef.pal@aok.pte.hu (J. Pál), zsolt.toth@aok.pte.hu (Z. Tóth), ofarkas@mail1.vcu.edu (O. Farkas), kellenyi@ttk.pte.hu (L. Kellényi), tamas.doczi@aok.pte.hu (T. Dóczi), ferenc.gallyas.sen@aok.pte.hu (F. Gallyas). 1 Tel.: ; fax: Present address: Department of Heart Surgery, University of Pécs, H-7634 Pécs, Ifjuság út 13, Hungary. Tel.: ; fax.: Present address: Department of Anatomy, Medical College of Virginia, Campus of Virginia Commonwealth University, Richmond, VA, USA. sure) may secondarily cause their own morphological responses, or may influence the outcome of those elicited primarily. The term diffuse axonal injury (DAI) refers to the phenomenon of fatal damage to axons that are scattered among apparently normal axons in a widespread distribution in the human brain. DAI is a primary cause of death and neurological disability following head injuries (Adams et al., 1983). In human neuropathology, the pathomechanism of DAI cannot be elucidated mainly because of the complexity of traumatically induced morphopathological damages. Although the traumatic axonal injury observed in animal models (TAI) does not reproduce exactly the nature, extent and time course of axonal injury in DAI, the investigation of TAI provides good insight into the axonal changes occurring in DAI (Maxwell et al., 1997). By means of animal models, two distinct mechanisms of diffuse axonal damage were observed: focal impairment of the axonal transport and neurofilament (ultrastructural) compaction (Stone et al., 2001). In human neuropathology, focal axonal swellings are indicative of the impairment of axonal transport even for years post /$ see front matter 2005 Elsevier B.V. All rights reserved. doi: /j.jneumeth

68 284 J. Pál et al. / Journal of Neuroscience Methods 153 (2006) injury. Regarding the neurofilament (ultrastructural) compaction in axons, no persistent morphological change is known, which could facilitate the investigation of its participation in human cases. Since this kind of axonal damage was discovered only 10 years ago in animal experiments (Pettus et al., 1994), its morphological, neurological and behavioural consequences have not been cleared even in experimental neurotraumatology, probably because of the lack of a method capable of its selective production. The widely used experimental head-injury devises produce various sets of morphopathological responses such as those mentioned above (Gennarelli, 1994), thereby allowing the investigation of multiple aspects of traumatic brain injury in the complexity that can occur in various conditions in human neurotraumatology (Ellingson et al., 2005). However, another experimental approach, the investigation of a single morphopathological response without the influence of other responses, also holds out the promise of the acquisition of new information on this field. This paper describes a new weight-drop head-injury apparatus that can induce neurofilament (ultrastructural) compaction in long axon segments in the rat brain, but without inflicting other kinds of immediate or delayed morphological damage to axons and without pathophysiological processes ensuing in the postinjury period. Thus, various problems that are closely related to the neurofilament (ultrastructural) compaction in axons can be investigated by means of this apparatus without outside interference. Since microtubules, endoplasmic reticulum cisternae and the axolemma are also involved in neurofilament compaction we prefer the term ultrastructural compaction. As a further argument, head injuries can cause the same ultrastructural compaction in the neuronal soma-dendrite domain, where neurofilaments are sparse (Csordás et al., 2003; Gallyas et al., 2004). Fig. 2. (a) The head-injury apparatus in action. (b) The machinery allowing the precise positioning and fixation of the rat s head under the impactor. 2. Materials and methods 2.1. The new rat-head-injury apparatus and its use An earlier described weight-drop head-injury device designed to produce a depression of controlled depth on the exposed brain surface (Feeney et al., 1981) was modified substantially for the closed rat skull as follows (Figs. 1 and 2). A flanged steel cylinder 65 mm in length, 45 mm in outer diameter and 25 mm in inner diameter (impact-depth adjuster housing) is firmly fixed with a nut (fixing nut) into the bridge of a massive iron frame, which is welded to a heavy three-legged baseboard. Round the flange, a rotatable hollow disc (scale disc) is placed, which is marked every 36. A second steel cylinder 95 mm in length, 25 mm in outer diameter and 11 mm in inner diameter (impact-depth adjuster), the mantel of which is threaded at both ends (10 threads/cm), is fitted into its housing. The ver- Fig. 1. Schematic drawing of the calvaria-depression head-injury apparatus. (a c) Characteristic positions of the impactor relative to the rat s head.

69 J. Pál et al. / Journal of Neuroscience Methods 153 (2006) tical motion of the impact-depth adjuster is controlled with a nut (adjusting nut) screwed on its upper end. From the mantel of the impact-depth adjuster, a pin protrudes radially into a vertical slot milled in its housing, which allows it to move up or down, but not to rotate. The bottom end of a vertically positioned iron tube 1800 mm in length and 15 mm in inner diameter (guide tube) is soldered axially into the upper end of the impact-depth adjuster. Through the mantel of the guide tube, several vent holes are bored. An iron cylinder weighing about 5 kg (ballast weight) containing an axial hole 19 mm in diameter is fixed concentrically to the guide tube. This cylinder presses the impact-depth adjuster down, to prevent its possible axial play. A steel nose-piece containing an axial hole 5 mm in diameter is firmly screwed onto the bottom end of the impactdepth adjuster. The resulting sleeve (stopping sleeve) stops the downward movement of an 80-mm long, 11-mm thick steel piston (impactor). From its bottom (impactor shoulder), a 20-mm long, 4.0-mm thick steel rod with a lentiform bottom (impactor head) protrudes axially through the nose-piece. A bearing ball is brazed onto the upper end of the piston. Mechanical force is generated by dropping a 150-mm long, 13-mm thick steel bar weighing 200 g through the guide tube onto the impactor, which weighs 40 g. A head holder is fixed onto a dismounted microscope stage that is movable in two perpendicular directions by means of micrometer screws. The microscope stage is fastened to the baseboard under the impactor head at an adjustable angle, in order to permit the setting of a selected area on the rat s calvaria into the horizontal plane. The head injury is administered in the following sequence of steps: (1) the scalp of an anesthetized rat s head is incised longitudinally and stretched apart, and the soft tissue is then removed from the calvaria. (2) The rat s head is fixed in the head holder, and a selected area on the calvaria is then set in the horizontal plane and positioned under the impactor head (Fig. 1a). (3) The impact-depth adjuster, together with the impactor and the dropping weight on its top, is moved downwards by rotating the impact-depth adjusting nut until the face of the impactor head just reaches the exposed calvaria (Fig. 1b). (4) Thereafter, the adjusting nut is rotated again as far as the intended depth of the calvaria depression is adjusted, as measured on the scale disc (the angular displacement between any two neighboring marks means a depression depth of 0.1 mm). During this process, the rat s calvaria does not allow the impactor to move downwards. As a consequence, a distance equal to the intended depression depth results between the impactor shoulder and the stopping sleeve (Fig. 1c). (5) The dropping weight is pulled up to the selected height by means of a light string and than dropped onto the impactor. This latter suddenly moves downwards as far as allowed by the stopping sleeve, thereby producing a momentary depression of the intended depth in the calvaria. (6) Immediately thereafter, the rat is removed from the head holder Animal experiments A total of 36 Wistar rats weighing between 190 and 210 g were anesthetized by the intraperitoneal administration of 2 ml/kg of a 1:1 mixture of 25 mg/ml Thiopental (Biochemie GmbH, Austria) and 5 mg/ml Seduxen (Richter Gedeon Rt, Budapest). Nine groups of rats were studied, with four rats in each. The group 1 rats were not subjected to head injury, but were sham-operated and then perfusion-fixed transcardially (control rats). All other rats were subjected to the calvaria-depression head injury. The depression depth was set to 0.5 mm for the rats in group 2, 0.75 mm for those in groups 3 6, and 1.0 mm for those in groups 7 9. Thirty minutes before the administration of the head injury, the femoral artery in each rat in group 6 was exposed, channeled with polyethylene tubing 0.96 mm in outer diameter and 150 mm in length (Becton, Dickinson, Cat. no ) and connected to the pressure-measuring transducer (B. Braun, Melsungen, Germany) of a custom-made device for monitoring blood pressure (BP-Monitor, designed by L. Kellényi). Thereafter, their cisterna magna was channeled with polyethylene tubing of the above type, sealed hermetically with dental acrylate and connected to the pressure-measuring transducer (B. Braun, Melsungen, Germany) of a custom-made device for monitoring intracranial pressure (ICP-Monitor, designed by L. Kellényi). This device calculates the pulse rate and the respiration rate at intervals of 1 min by means of fast Fourier transformation of the oscillating waveform (Fig. 3a) of the monitored intracranial pressure. Before head injury, the head holder was tilted through 20 relative to the baseboard, and the axis of the impactor head was centered over the left hemisphere of each rat 3.5 mm caudal to the bregma and 2.5 mm lateral to the midline. Following the infliction of the head injury, the rats in groups 2, 3 and 7 were perfusion-fixed immediately, those in groups 4 and 8, 1 h later, and those in groups 5, 6 and 9, 4 h later. In each rat in group 6, anesthesia was maintained until the start of fixation, while the rats in groups 4, 5, 8 and 9 were allowed to recover, but re-anesthetized before fixation. Fig. 3. Respiration-caused and heart beat-caused oscillations of the intracranial pressure before and after the production of a 0.75-mm deep calvaria depression. (a and b) Intracranial pressure, (c) heart rate, (d) respiration rate and (e) blood pressure. Arrows point to the time of the head injury.

70 286 J. Pál et al. / Journal of Neuroscience Methods 153 (2006) From each group, two rats were perfused transcardially with 500 ml of 4% paraformaldehyde in PBS buffer of ph 7.4 for immunohistochemistry, while the remaining two rats were perfused with an electron-microscopic fixative. This was prepared by mixing 250 ml of 0.2 mol/l sodium cacodylate, 50 ml of 20% paraformaldehyde, 50 ml of 25% glutaraldehyde, 25 ml of 0.1 mol/l calcium chloride and 125 ml of 10% polyvinylpyrrolidone K25, followed by adjustment of the mixture to ph 7.5 with hydrochloric acid. Before fixation, the vascular system was rinsed with physiological saline for 30 s. After fixation, the rats were left untouched at room temperature for 24 h before removal of the brain from the skull (Cammermeyer, 1961). Animal care and handling were carried out in strict accordance with directive 86/609/EGK of the European Committee Council Tissue processing Brains were cut into three blocks close to the coronal planes 1 and 7 mm caudal to the bregma. The middle blocks were serial-sectioned on a vibratome in the coronal plane, while the caudal blocks, which included a part of the brain stem, the cerebellum, the pons, the oblongata and the spinal cord were serial-sectioned in the sagittal plane. From the glutaraldehydefixed blocks, sections were cut on a vibratome at 150 m. The formaldehyde-fixed blocks were embedded in 5% agarose and cut at 30 m. For each block, every sixth of the serial section was placed in the same collecting well. Sections from wells 1 and 4 were stained with a special silver method (Gallyas et al., 1993) that selectively demonstrates compacted axons (Gallyas et al., 2002) and also compacted neuronal soma-dendrite domains (Csordás et al., 2003). All formaldehyde-fixed sections from wells 2 and 5 were processed for RMO14 immunohistochemistry while those from wells 3 and 6 for APP immunohistochemistry. From the glutaraldehyde-fixed sections collected in wells 2, 3, 5 and 6, 2 mm 2 mm areas corresponding to those which contained many silver-stained axons in adjacent sections, were cut, postfixed with a 1:1 mixture of 2% osmium tetroxide and 3% potassium hexocyanoferrate(ii) for 1 h at room temperature, and then flat-embedded in Durcupan ACM. Thin (50 nm) sections were contrasted with uranyl acetate and lead citrate in the usual manner Staining procedures Silver staining The protocol described in a previous paper (Gallyas et al., 1993) was strictly observed. Briefly, following dehydration with graded 1-propanol, vibratome sections were incubated for 16 h at 56 C in 1-propanol containing 0.8% sulfuric acid and 2% water for glutaraldehyde-fixed sections, or 1.0% sulfuric acid for formaldehyde-fixed sections (esterification), then rehydrated with graded 1-propanol, treated with 1% acetic acid for 10 min and finally immersed in a special physical developer until the background had become light-brown. Thereafter, the sections were washed with three changes of 1% acetic acid, dehydrated with graded 1-propanol, cleared with clove oil and covered with Canada balsam APP immunohistochemistry After the suppression of endogenous peroxidase activity by a 30-min treatment with 0.5% hydrogen peroxide in PBS followed by washing with PBS for three 10-min periods, the sections were exposed to controlled-temperature microwave retrieval (Stone et al., 1999) and then incubated for 20 min in 0.2% Triton X-100 (Sigma, Cat. no. T-8532) dissolved in PBS. Next, the sections were rinsed with PBS, immersed for 40 min in 1% normal horse serum (NHS, Vectastain Universal Elite ABC Kit, Vector PK- 6200) diluted with PBS (henceforth 1% NHS/PBS), incubated overnight in rabbit anti- -APP (Zymed Laboratories Inc., Cat. no ) diluted with 1% NHS/PBS at 1:250, and then washed with 1% NHS/PBS three times for 10 min each. Thereafter, the sections were subjected to the staining protocol of the Vectastain Universal Elite ABC Kit (Vector, Cat. no. PK- 6200). Finally, the product of the immunological reaction was visualized with a diaminobenzidine (DAB) peroxidase substrate kit (Vector, Cat. no. SK-4100) and then washed with PBS for 10 min RMO14 immunohistochemistry The procedure was similar to that detailed above for - APP, except that the primary antiserum was mouse monoclonal RMO14 antibody (Zymed Laboratories Inc., Cat. no ) diluted with 1% NHS/PBS at 1: Results 3.1. Control rats In the brains of the sham-operated rats, and also in the nontraumatized hemispheres of the head-injured rats, anomalous staining properties or morphological features were not found Head-injured rats, gross clinical and pathological observations None of the head-injured rats developed apnea or convulsions either immediately after the head injury or later. In the rats that suffered a 0.75-mm deep calvaria depression, the intracranial pressure displayed a sudden increase (14.3 ± 2.1 mmhg), which was immediately followed by a sudden decrease (2.3 ± 0.4 mmhg) and then a gradually fading. The intracranial pressure returned to the normal range in 1 min (Fig. 3a). No other change was monitored thereafter (Fig. 3b). Neither the pulse rate nor the respiration rate could be measured in the first minute post-insult due to the absence of an oscillating waveform of the intracranial pressure. Otherwise, the post-insult values were similar to those which were measured pre-insult (Fig. 3c and d). Immediately after the head injury, the blood pressure decreased slightly then returned to the normal range in 1 min (Fig. 3e). No other pathological event was monitored in the 4-h survival period. Impression fracture of the skull was observed only in one rat with a 1-mm depression depth. Except for this rat, hemorrhages were not present in the basal cisternae, the ventricles, the cisterna magna or over the surface of the affected hemisphere. In the rats

71 J. Pál et al. / Journal of Neuroscience Methods 153 (2006) with a 1-mm deep calvaria depression, but without fractured calvaria, small subarachnoid hemorrhages were observed just under the impact site Head-injured rats, immediate sacrifice, silver staining In the rats subjected to a 0.5-mm deep calvaria depression, a few long (even about 1 mm) axon segments were evenly silverstained in the cortical layers IV and V under the impact site (Fig. 4b), but nowhere else. In each rat that received a 0.75-mm deep calvaria depression, many more silver-stained axon segments were present in the above location (Fig. 4c). These were diffusely distributed in a crescent-shaped area in the coronal plane under the center of the impact site (Fig. 4a). At increasing distances from this plane, the density of the silver-stained axons in the neocortex gradually decreased, reaching zero at about 1.5 mm in either direction. Hardly any neuronal soma-dendrite domains were silver-stained either under the impact site or anywhere else. No intra- or extracellular edema, or any other morphological damage was observed at all. When the calvaria depressions were 1 mm deep there were higher numbers of affected axons, predominantly in cortical layers V and VI and the subcortical white matter (Fig. 4d and e) under the impact site. A few neuronal soma-dendrite domains were also demonstrated, mainly just above the subcortical white matter, but occasionally in cortical layers II V, and also in the hilus and the granule cell layers of the hippocampal dentate gyrus (not demonstrated). Neither silver-stained axons nor silver-stained soma-dendrite domains were present in other brain areas Head-injured rats, 1 or 4-h survival, silver staining In the rats with 1.0-mm deep calvaria depressions, evenly silver-stained axons were scarce. However, many axons were Fig. 4. Light-microscopic (a h) and electron-microscopic (i) damages in rats sacrificed immediately (a e and i) or at 4 h (f h) following the production of a 0.5-mm deep (b), a 0.75-mm deep (a, c and i), a 1.0-mm deep (d h) momentary calvaria depression. Thirty micrometer (f h) or 150- m (a e) vibratome sections were stained with silver (a e and g), anti-app (f) or RMO14 antibody (h). Because of overcrowding, individual silver-stained axons or neuronal soma-dendrite domains cannot be seen in (a and e), and can be hardly discerned in (d and g). (a and e) Coronal sections, 3 mm caudal to the bregma. (b d) Cortical areas in the same position as that framed in (a). (f h) Cortical areas in the same position as that framed in (e). The border between the neocortex and the subcortical white matter is marked with dashed lines. (i) Cross-sectioned compacted (C) and normal (N) myelinated axons in normal neuropil. Black arrowheads point to a long, curving compacted axon segment in (b), to a subarachnoidal bleeding in (e) and an APP-positive axonal swelling in (f). White arrowheads point to compacted neuronal soma-dendrite domains in d and to an axon traced out with silver-stained dots in (g). Scale bars: (a and e) 1 mm; (b d and f g) 50 m; (i) 500 nm.

72 288 J. Pál et al. / Journal of Neuroscience Methods 153 (2006) traced out with silver-stained dots (Fig. 4g; see also Fig. 4c in Gallyas and Zoltay, 1992, and Fig. 2d in Csordás et al., 2003). With depressions that were less deep, there were fewer such axons Head-injured rats, immunohistochemical staining Anti-APP stained several axonal swellings in the subcortical white matter under the impact site, but only a few in the neocortex of the rats that survived for 1 or 4 h following the production of calvaria depressions of 1 mm in depth (Fig. 4f). However, hardly any anti-app-positive axon swellings were found in the rats with depressions that were less deep. RMO14 antibody stained no axons in the affected cortical area or the subcortical white matter of any of the head-injured rats, but cell bodies of normal neurons were faintly stained (Fig. 4h) Head-injured rats, electron microscopy In the affected cortical area of each rat sacrificed either immediately or 1 4 h after the production of 0.50 or mm deep calvaria depressions, numerous myelinated (but no non-myelinated) axons (Fig. 4i) displayed a marked reduction of the distances between any two neighboring parts of apparently intact ultrastructural elements, mainly neurofilaments. The spaces among the compacted ultrastructural elements were hyper-electrondense, which gave the affected axon profiles a dark appearance. The axolemma did not exhibit any abnormality at an intermediate magnification of electron microscopy, despite the fact that a relatively large volume of fluid must have passed through it. Some of this fluid was found between the myelin sheath and the axolemma, and some in nearby astrocytic processes. The compacted axonal profiles were randomly scattered in an otherwise intact environment. There was no indication of axonal profiles characteristic of impaired intra-axonal transport (the focal accumulation of mitochondria, vesicles and endoplasmic reticulum intermingled with disorganized neurofilament bundles; Stone et al., 2001). In the rats with a 1-mm deep calvaria depression, in addition to the above morphological changes, a few axonal profiles displayed the characteristics of impaired intra-axonal transport, and a few somal and/or dendritic profiles displayed the characteristics of somato-dendritic ultrastructural compaction described elsewhere (Csordás et al., 2003; Gallyas et al., 2004). 4. Discussion 4.1. Methodological comments Immediately after the administration of head injury and also 1 h later, the silver method used here evenly blackens the axons possessing compacted ultrastructure in a selective (Gallyas et al., 2002) and reproducible (Newman and Jasani, 1998) manner. Later, the affected axons are traced out with silver-stained dots (Gallyas and Zoltay, 1992). The dotted axonal staining disappears gradually in 2 days (Zsombok et al., 2005). RMO14 immunostaining is a widely used tool for the lightmicroscopic identification of axons that have suffered traumatic neurofilament (ultrastructural) compaction (Lee et al., 1987; Stone et al., 2001). However, it does not work in animals that survive for less than 15 min post-injury (Buki et al., 1999), nor in thin (less than a few m in diameter) compacted axons in the rat neocortex, even after a survival period of 4 h (Fig. 4h). The target of the RMO14 antibody is the rod domain of the mid-sized neurofilament protein, which is masked in normal axons, but becomes unmasked as a result of calcium-initiated and calpain-mediated spectrin proteolysis in the axons that display neurofilament (ultrastructural) compaction (Buki et al., 1999). Mid-sized neurofilament protein is known to be the main regulator of axon thickness; its absence decreases the axon caliber, while its presence is required to achieve the maximal axon diameter (Elder et al., 1998). This is probably the reason for the lack of RMO-14 staining in thin axons in the cortex. The difference in location between the anti-app-stained axonal swellings and the silver-stained axonal segments in the rats that survived for 4 h following the production of a 1.0-mm deep calvaria depression confirms the observation of Stone et al. (2001) that the neurofilament (ultrastructural) compaction and the impairment of axonal transport are independent phenomena Nature of the mechanical load delivered by the present head-injury apparatus With the present head-injury apparatus, the mechanical load to the brain depends only on two reproducible, adjustable and quantifiable parameters: the depth of the calvaria depression and the velocity of its production. The latter can be adjusted via the height from which the weight is dropped. A height of 1.5 m, which gives a final velocity of 5.44 m/s to the dropped weight, proved to be suitable for the selective induction of axonal compaction in the neocortex. Lower velocities tended to favor the initiation of ultrastructural compaction in the neuronal somadendrite domains (our unpublished observation), whereas a very low velocity (0.5 mm/s), produces ultrastructural compaction mainly in astrocytes, much less in neurons, but not at all in axons (Tóth et al., 1997). All these suggest that it may be the velocity of the calvaria depression, i.e. a dynamic factor that determines which of the tissue elements mentioned will be damaged. A further factor that should be considered as the cause of the axonal damage in our case is a strain in the cortical tissue under the impact site, which is evoked by the lentiform shape of depression in its superficial layers. This possibility is supported by the fact that most affected axons in our experiments run parallel to the outline of the depressed cortical surface. Consequently, a sudden stretch in the cortical tissue may contribute to the development of the axonal damage in our case. A third factor that should also be considered consists in the fact that, in our case, the sudden increase in intracranial pressure is immediately followed by a sudden decrease in intracranial pressure, which is also able to cause morphological damage to the rat brain (Shreiber et al., 1999). Since the experiments with our apparatus are not qualified to decide among these factors, we share the non-committed opinion of Shreiber et al. (1999) that

73 J. Pál et al. / Journal of Neuroscience Methods 153 (2006) some dynamic deformation in the cortical tissue is responsible for causing the axonal damage in our case. Above a critical value (150 g, our unpublished observation) the mass of the dropping weight does not appreciably influence the numbers of affected axons in the rats that suffered calvaria depressions of 0.75 mm in depth, probably because the energy in excess to that needed for the production of the calvaria depression is absorbed by the stopping sleeve when the dropping weight strikes against it Possible fields of application of the head-injury apparatus presented In case of a depression depths of 0.75 mm, the head-injury apparatus presented is unparalleled in four senses: (1) it produces ultrastructural compaction in diffusely distributed axon segments without co-production of other kinds of primary or secondary axon damage, (2) the affected axon segments are present in an apparently intact environment, (3) in a well-defined area of the neocortex, but nowhere else and (4) the blood pressure, intracranial pressure, heart rate and respiration rate return to the normal ranges in 1 min. These qualities make this apparatus suitable for the electron-microscopic investigation of the fate of the compacted axon segments during a long post-injury period, without the interference of other kinds of morphological damages or of unfavorable post-injury circumstances. Furthermore, the effect of experimentally induced post-injury pathological circumstances (such as elevated intracranial pressure or impaired blood circulation) or the effect of future therapeutic experiments on the fate of the compacted axons, or else the cumulative consequences of recurrent minor head injuries can also be investigated by means of this apparatus. Acknowledgments The authors thank Andok Csabáné, Nyirádi József and Dr. Nádor Andrásné for their valuable help in the light- and electronmicroscopic, and the photographic preparative work, respectively. This study was supported by the Hungarian grants NKFP 1/A 00026/2002 and ALK-00126/2002, and the Hungarian Academy of Sciences. References Adams JH, Graham DI, Gennarelli TA. Head injury in man and experimental animals. Acta Neurochir 1983;32(Suppl.): Buki A, Siman R, Trijanovsky JQ, Povlishock JR. The role of calpain mediated spectrin proteolysis in traumatically induced axonal injury. J Neuropathol Exp Neurol 1999;58: Cammermeyer J. The importance of avoiding dark neurons in experimental neuropathology. Acta Neuropathol 1961;1: Csordás A, Mázló M, Gallyas F. Recovery versus death of dark (compacted) neurons in non-impaired parenchymal environment. Acta Neuropathol 2003;106: Elder GA, Friedrich Jr VL, Bosco P, Kang C, Gourov A, Tu PH, et al. Absence of mid-sized neurofilament subunit decreases axonal calibers, levels of light neurofilament (NF-L), and neurofilament content. J Cell Biol 1998;141: Ellingson BM, Fijalkowski LJ, Pintar FA, Yoganandan N, Gennarelli TA. New mechanism for inducing closed head injury in the rat. Biomed Sci Instrum 2005;41: Feeney DM, Boyeson MG, Linn RT, Murray HM, Dail WG. Responses to cortical injury. I. Methodology and local effects of contusion in the rat. Brain Res 1981;11: Gallyas F, Zoltay G. An immediate light microscopic response of neuronal somata, dendrites and axons to non-contusing consussive head injury. Acta Neuropathol 1992;83: Gallyas F, Hsu M, Buzsáki G. Four modified silver methods for thick sections of formaldehyde-fixed mammalian central nervous tissue: dark neurons, perikarya of all neurons, microglial cells and capillaries. J Neurosci Method 1993;50: Gallyas F, Farkas O, Mázló M. Traumatic compaction of the axonal cytoskeleton induces argyrophilia: histological and theoretical importance. Acta Neuropathol 2002;103: Gallyas F, Farkas O, Mázló M. Whole-cell ultrastructural compaction ( dark cell formation) in mammalian tissues may consist in gel-to-gel phase transition. Biol Cell 2004;96: Gennarelli TA. Animate models of human head injury. J Neurotrauma 1994;11: Graham DI, McIntosh TK, Maxwell WL, Nicoll JAR. Recent advances in neurotrauma. J Neuropathol Exp Neurol 2000;59: Lee VM, Carden MJ, Schlaepfer WW, Trojanowsky JQ. Monoclonal antibodies distinguish several differentially phosphorylated states of the two largest rat neurofilament subunits (NF-H and NF-M) and demonstrate their existence in the normal nervous system of adult rats. J Neurosci 1987;7: Maxwell WL, Povlishock JT, Graham DL. A mechanistic analysis of nondisruptive axonal injury: a review. J Neurotrauma 1997;14: Newman GR, Jasani AB. Silver development in microscopy and bioanalysis: past and present. J Pathol 1998;186: Pettus EH, Christman CW, Giebel ML, Povlishock JT. Traumatically induced altered membrane permeability; its relationship to traumatically induced reactive axonal change. J Neurotrauma 1994;11: Shreiber DI, Bain AC, Ross DT, Smith DH, Gennarelli TA, McIntosh TK, et al. Experimental investigation of cerebral contusion: histopathological and immunohistochemical evaluation of dynamic cortical deformation. J Neuropathol Exp Neurol 1999;58: Stone JR, Walker SA, Povlishock JT. The visualization of a new class of traumatically injured axons through the use of a modified method of microwave antigen retrieval. Acta Neuropathol 1999;97: Stone JR, Singleton RH, Povlishock JT. Intraaxonal neurofilament compaction does not evoke local axonal swelling in all traumatically injured axons. Exp Neurol 2001;172: Tóth Zs, Seress L, Tóth P, Ribak CE, Gallyas F. A common morphological response of astrocytes to various injuries: dark astrocytes. A light and electron microscopic analysis. J Brain Res 1997;38: Zsombok A, Tóth Z, Gallyas F. Basophilia, acidophilia and argyrophilia of dark (compacted) neurons in the course of their formation, recovery or death in an otherwise undamaged environment. J Neurosci Method 2005;142:

74 Acta Neuropathol (2006) 111: DOI /s ORIGINAL PAPER Ferenc Gallyas Æ Jo zsef Pa l Æ Orsolya Farkas Tama s Do czi The fate of axons subjected to traumatic ultrastructural (neurofilament) compaction: an electron-microscopic study Received: 14 November 2005 / Revised: 7 December 2005 / Accepted: 9 December 2005 / Published online: 17 February 2006 Ó Springer-Verlag 2006 Abstract By means of a new head-injury apparatus, a 0.75-mm-deep depression was produced momentarily at a predetermined site of the rat calvaria. This immediately evoked ultrastructural (neurofilament) compaction in many myelinated axon segments in layers IV and V of the neocortex under the impact site. The affected axon segments run quasi-parallel to the brain surface in a diffuse distribution among normal axons. Other kinds of damage to the brain tissue were insignificant; the conditions were therefore favorable for investigation of the fate of the compacted axons. Quantitative analysis of the findings on groups of ten rats that were sacrificed either immediately after the head injury or following a 1 day or a 1 week survival period showed that around 50% of the compacted axons recovered in 1 day, and a further less than 10% did so in 1 week. Electron microscopy revealed that the non-recovering compacted axons underwent a sequence of degenerative morphological changes including homogenization, fragmentation and resorption of the fragments. However, the myelin sheaths around these degenerating axons remained apparently unchanged even in the long-surviving rats, and hardly any phagocytotic cells were encountered. On the other hand, many such myelin sheaths contained axolemma-bound, normal-looking axoplasm besides the above morphological signs of axon-degeneration. It is concluded that the non-recovering compacted axons undergo an uncommon (non-wallerian) kind of degeneration, which is mostly reversible. Keywords Recovery Æ Degeneration Æ Regeneration Æ Electron microscopy F. Gallyas (&) Æ O. Farkas Æ T. Do czi Department of Neurosurgery, Faculty of Medicine, Pe cs University, Re t utca 2, 7623, Pécs, Hungary ferenc.gallyas.sen@aok.pte.hu Tel.: Fax: J. Pál Æ T. Do czi Clinical Neuroscience Research Group of the Hungarian Academy of Sciences, Pe cs University, 7623, Pe cs, Hungary Introduction In human neurology, diffuse axonal injury (DAI) is a primary cause of disability or death following head injuries [1]. However, mainly because of the complexity of primary and secondary morphological damages to the brain tissue [12], human neuropathology is not qualified for the elucidation of its patho-mechanism. Although its equivalent in experimental animals, traumatic axonal injury (TAI), does not parallel exactly its nature, distribution and time course, the investigation of TAI is considered [14] to improve our knowledge on DAI. Two distinct mechanisms of TAI have been established: focal impairment of the intra-axonal transport leading to delayed axon disconnection, and neurofilament (ultrastructural) compaction in axons [21]. The latter phenomenon consists in a dramatic decrease of the distances between adjacent ultrastructural elements, mainly neurofilaments. The affected axons are scattered among apparently normal axons, frequently in an apparently intact environment. Although this phenomenon was discovered 10 years ago [19], its contribution to the morphological, neurological and behavioural consequences of head injury in human neuropathology has not been investigated at all. Even the fate (recovery or death) of the affected axons in animal experiments has not been cleared. A newly elaborated rat head injury apparatus [17] is able to immediately produce ultrastructural (neurofilament) compaction in myelinated axons in a well-defined area of the neocortex under the impact site, without giving rise to any appreciable extent of other kinds of morphological damage to the brain. Thus, the ultrastructural appearance of the compacted axons, which presumably changes during a long post-injury period, cannot be confused with the ultrastructural formations originating from the axonal degeneration initiated by direct tearing or delayed disconnection, or death of the parent neuron. Further, under the circumstances used in the present study, the blood pressure, intracranial pres-

75 230 sure, heart-pulse rate and respiration rate return to the normal range in 1 min. [17], which creates exogenous circumstances favorable for a spontaneous recovery. It has long been known that a proportion of neurons that have undergone somato-dendritic ultrastructural compaction, caused either by a pathometabolic condition (such as hypoglycemia [2]) or by a physical insult (including head injury [6]) recover spontaneously. Since the mechanism of ultrastructural compaction in the soma-dendrite domain is probably the same as that in the axons [11], it is reasonable to assume that a proportion of traumatically compacted axons may also recover spontaneously. By means of the head-injury apparatus mentioned, the present study follows for 6 months the fate of axon segments subjected to traumatic ultrastructural compaction. As no light-microscopic marker is available that could demonstrate the structural changes in the compacted axons for longer than a few days (see later), electron microscopy is used throughout. Materials and methods Animal experiments A total of 48 Wistar rats weighing between 190 and 210 g were anesthetized by the intraperitoneal administration of 2 ml/kg of a 1:1 mixture of 25 mg/ml Thiopental (Biochemie GmbH, Austria) and 5 mg/ml Seduxen (Richter Gedeon Rt, Budapest). Nine groups of rats were studied, with three rats in each of groups 1, 3, and 6 9, and 10 rats in each of groups 2, 4 and 5. The group 1 rats were not subjected to head injury, but were sham-operated and then perfusion-fixed transcardially (control rats). In all the other rats, a 0.75-mm-deep momentary calvaria depression was produced with a new rat head-injury apparatus [17]. Its head holder was tilted at 20 relative to the baseboard, and the axis of the impactor head was centered over the left side of the calvaria, 2.0 mm caudal to the bregma and 2.0 mm lateral to the midline. After the infliction of the head injury, the rats in group 2 were perfusion-fixed immediately, whereas the rats in groups 3 9 were perfusionfixed after survival for 4 h, 1 day, 1 week 1 month, or 2, 4 and 6 months, respectively. The rats in groups 3 9 were allowed to recover, but re-anesthetized before fixation. Each rat was perfused transcardially with 500 ml of an electron-microscopic fixative, prepared by mixing 250 ml of 0.2 mol/l sodium cacodylate, 50 ml of 20% paraformaldehyde, 50 ml of 25% glutaraldehyde, 25 ml of 0.1 mol/l calcium chloride and 125 ml of 10% polyvinylpyrrolidone K25, followed by adjustment of the mixture to ph 7.5 with a few drops of 0.1 mol/l hydrochloric acid. Before fixation, the vascular system was rinsed with physiological saline for 30 s. After fixation, the rats were left untouched at room temperature for 24 h before removal of the brain from the skull [4]. Animal care and handling were carried out in accordance with order 243/1998 of the Hungarian Government, which is an adaptation of directive 86/609/EGK of the European Committee Council. Tissue processing and staining The middle thirds of the brains were serial-sectioned at 150 lm on a vibratome. The sections corresponding to the coronal plane 2 mm caudal to the bregma (according to the atlas of Paxinos and Watson [16]) were selected. From each of them, the parts of the neocortex between 1.5 and 2.5 mm, both right and left from the midline, together with the underlying white matter, were cut out. Additionally, from three rats in each traumatized group, three further parts of the left (traumatized) neocortex were cut out, together with the subcortical white matter (0 1.5 mm, mm and mm with respect to the midline). All these specimens were postfixed with a 1:1 mixture of 2% osmium tetroxide and 3% potassium hexocyanoferrate(ii) for 1 h at room temperature, and then flat-embedded in Durcupan ACM. The specimens from the right (non-traumatized) hemisphere of the head-injured rats served as internal control. From each specimen, semithin (1 lm) sections were cut and stained for 1 min. at 90 C in a solution containing 0.05% toluidine blue, 0.05% sodium tetraborate and 0.1% saccharose (ph 9.5). Thereafter, the cortical layers I and II were trimmed out from the specimens from the head-injured rats. Thin (50 nm) sections were mounted on G300 square mesh grids (TAAB GG007/C) in such a manner that the direction perpendicular to the cortical surface should be parallel to either of the grid bars. Sections were contrasted with uranyl acetate and lead citrate in the usual manner. From the remaining vibratome sections, every fifth one was stained with a silver method that selectively demonstrates freshly-compacted axons and neuronal soma-dendrite domains [9]. Briefly, following dehydration with graded 1-propanol, vibratome sections were incubated for 16 h at 56 C in 1-propanol containing 0.8% sulfuric acid and 2% water (esterification) then rehydrated with graded 1-propanol, treated with 1% acetic acid for 10 min. and finally immersed in a special physical developer until the background had become light-brown. Thereafter, the sections were washed with three changes of 1% acetic acid, dehydrated with graded 1-propanol, cleared with clove oil and covered with Canada balsam. Quantitative analysis In the control rats, the myelinated axon profiles were counted in 240 grid-squares belonging to ten adjacent columns arranged perpendicularly to the cortical surface. At a magnification of 3,000, the electron-microscopic image in the grid-square just examined was

76 231 moved at a right angle to a straight line drawn vertically through the center of the picture screen of the electron microscope, and the myelinated axons just reaching this straight line were counted. The counts belonging to the rows of grid-squares at the same distance from the cortical surface were averaged for the three control rats and plotted, together with the standard deviation, against the serial number of the grid-squares as counted from the cortical surface. In the head-injured rats, all the myelinated axon profiles and the compacted axon profiles were counted in those grid-squares aligned in eight adjacent columns perpendicular to the cortical surface that did not contain more than 150 or less than 40 myelinated axon profiles (for an explanation, see the Results). The resulting counts were then totaled for each specimen examined. Thereafter, the totals for the rats that survived for 1 day (group 4) or 1 week (group 5) following the infliction of the head injury were compared with those for the rats that were sacrificed immediately (group 2), using the Student s t test. Results Control specimens In all brain areas of the sham-operated rats and in the brain areas outside the neocortex under the impact site of the head-injured rats, the silver staining method did not demonstrate either axons or neuronal soma-dendrite domains. Electron microscopically, none of the ultrastructural abnormalities that were found in the traumatically damaged cortical areas were observed either in the specimens obtained from the control rats or in the internal-control specimens obtained from the headinjured rats. Head-injured rats, light-microscopic observations In the rats sacrificed immediately after the infliction of the head injury, as described and illustrated in a previous paper [17], the silver method stained evenly numerous long axon segments (Fig. 1a) in the neocortex under the impact site, but nowhere else. Most affected axons run quasi-parallel to the brain surface, but no one into the subcortical white matter. The frequency of silver-stained axons decreased with the distance from the center of the impact site. In the rats that survived for 4 h, evenly silver-stained axons were scarce. However, many axons (Fig. 1b) were traced out by silver-stained dots. At 1 day post-injury, the incidence of axons displaying dotted silver staining was much lower (Fig. 1c). In the silver-stained sections of the rats with longer survival times, nothing was indicative of a previous axonal disturbance. Head-injured rats, electron-microscopic observations In the rats that were sacrificed immediately, as described and illustrated in a previous paper [17], many myelinated axons, but no non-myelinated axons or boutons displayed a marked reduction of the distances between any two neighboring parts of apparently intact ultrastructural elements, mainly neurofilaments, in the neocortex under the impact site, but nowhere else (compare Fig. 2a, b). However, the interior of membrane-bound organelles, such as the mitochondria, remained non-compacted. In parallel with the compaction, the spaces surrounding the ultrastructural elements visible in the transmission electron microscope became more electrondense, which gave the affected axon profiles a dark appearance. The axolemma did not exhibit any abnormality at an intermediate magnification of transmission electron microscopy, despite the fact that a relatively large volume of fluid must have passed through it. A proportion of this fluid was found between the myelin sheath and the axolemma, and another proportion appeared to swell nearby astrocytic processes. As a result, the myelin sheaths of the affected axons became deformed and reduced in caliber. The compacted axon profiles were randomly scattered among normal axon profiles in an otherwise intact environment. Fig. 1 Light-microscopic appearance of compacted axons in 150-lm-thick silver-stained vibratome sections of layers IV and V of the neocortex in rats sacrificed immediately (a), or 4h(b) or 1 day (c) after the production of a 0.75-mm-deep momentary calvaria depression. Arrowheads point to continuously silver-stained axons, and arrows to axons that are outlined with silver-stained puncta. Scale bars: a c 50 lm

77 232 Fig. 2 Electron-microscopic appearance of normal (a), compacted (b), homogenized (c), presumably recovering (d, e), watery (f) and ballooning (g) axons in layer IV or V of the neocortex in rats sacrificed 4 h (a, b, g) or 1 day (c f) after the production of a 0.75-mm-deep momentary calvaria depression. White triangles denote mitochondria, white asterisks membranous whorls and white circles dense bodies. Scale bars: a c, e and f 200 nm; d 500 nm; g 1 lm In the rats that survived for 4 h, the ultrastructural abnormalities in the affected cortical area were very similar to those found in the rats that were sacrificed immediately. As regards the compacted axons, all their individual ultrastructural elements could be easily discerned (Fig. 2b). Very infrequently, misaligned neurofilaments running through loosely piled-up mitochondria, dense bodies, endoplasmic reticulum sacs and vesicles were found to fill slightly distended myelin sheath profiles (Fig. 2g). In a few compacted axons of the rats that survived for 1 day, individual ultrastructural elements could still be discerned, whereas the overwhelming majority of the compacted axons had become homogenized (Fig. 2c). In the latter, fluid-filled spaces were not found. Mitochondrion-sized membranous whorls present in axon profiles of otherwise normal appearance were frequently observed (Fig. 2d, e). Very infrequently, considerably distended myelin sheath profiles filled with some fluid (henceforth: watery axons; Fig. 2f) or with densely piledup mitochondria, mitochondrion-sized dense bodies, endoplasmic reticulum sacs and misaligned neurofilaments (henceforth: ballooned axons; similar to those demonstrated in Fig. 2g) were observed. The astrocytic profiles in the vicinity of the compacted axons appeared normal, but many astrocytic foot processes around the blood vessels in the affected cortical area were extremely swollen. In other respects, the tissue structure was normal. In the rats that survived for 1 week or 1 month, in addition to the ultrastructural features found in the 1 day rats, four other types of morphological changes were found. (1) In a large proportion of the compacted axons, the membrane-bound, homogenous interior had become convoluted or even fragmented (Fig. 3a, b). The spaces around such formations appeared frequently (but not always) to be extensions of the space present between the axolemma and the myelin sheath in the non-compacted axons. Thus, the ultrastructural elements in such spaces might be of oligodendrocytic origin. (2) Ultrastructural elements of such origin filled a large part (Fig. 3c, d) or the whole

78 233 Fig. 3 Electron-microscopic appearance of compacted axons in various stages of degeneration (a e) and regeneration (f i) in layer IV or V of the neocortex in rats sacrificed 1 week (a, b) or 1 month (c i) after the production of a 0.75-mm-deep momentary calvaria depression. White asterisks denote changes in the compacted axonal ultrastructure, black asterisks axolemma-bound axoplasm, white squares some structure of either oligodendrocytic or neuronal origin, and white triangles mitochondria. In i, an arrowhead points to an apparently normal myelin sheath in an unusual position. Scale bars: a, c and h 500 nm; b, d g and i 200 nm (Fig. 3e) of the interior of several myelin sheath profiles. (3) In a few cases, apparently normal, axolemmabound, normal-looking axoplasm shared the interior of the myelin sheath with one of the above-described ultrastructural formations (Fig. 3f i). (4) Very infrequently, myelin sheath profiles engulfed by astrocytes (Fig. 4a), lipoid-containing phagolysosomes in pericytes (Fig. 4b), and anomalous myelin profiles (Figs. 3i, 4c e) were present. In the rats that survived for 2, 4 or 6 months, all the morphological changes in the compacted axons demonstrated in Fig. 3a i were still present, but far less frequent than in the 1 week or 1 month surviving rats. The myelin sheaths around each of such axon profiles were apparently normal even in the 6 month surviving rats (Fig. 4f). In the astrocytes, debris of engulfed myelin sheaths, phagolysosomes, tight bundles of glial filaments, or other abnormalities were not present. Nothing indicated macrophage infiltration or microglial proliferation. Fat-containing phagolysosomes were observed in a negligible number of pericytes, but in none of the resting microglial cells. It should be emphasized that compacted or degenerated boutons, and degenerated myelin sheath formations similar to those in the advanced or chronic phases of Wallerian degeneration, were never observed in any of the cortical and subcortical areas examined, even in the long-surviving rats. Quantitative analysis The thin sections of the neocortex of the control rats covered about 28 rows of grid-squares counted from the

79 234 Fig. 4 Electron-microscopic pictures in layer IV or V of the neocortex in rats sacrificed 1 month (a e) or 6 months (f) after the production of a mm-deep momentary calvaria depression. The pictures in a and b indicate the phagocytosis of myelinated axons, in c e a malfunction of oligodendrocytes and in f the possibility that apparently normal myelin sheaths may exist around compacted and homogenized axons even after a 6 months survival period. In a, an arrow points to an engulfed myelin sheath; A denotes astrocytic cytoplasm and N astrocytic nucleus. In b, white arrowheads point to basal laminae; black squares denote fat-containing phagolysosomes, white squares other lysosomes and A an extremely swollen astrocytic foot process. In c e, arrows point to abnormal myelin sheath figures. In d, O denotes oligodendroglia cytoplasm and N an oligodendroglia nucleus. In e, an asterisk denotes compacted and homogenized axoplasm. In f, arrowheads point to normal myelin sheaths; the insert is a magnification of the compacted axon denoted by an asterisk. Scale bars: a and b 500 nm; c 200 nm; d f 1 lm brain surface. Cortical layers IV and V, where a majority of the compacted axon segments resided in the rats that were sacrificed immediately, corresponded approximately to the grid-squares in rows 9 22 (Fig. 5). In these, the numbers of myelinated axon profiles per gridsquare were on average between 40 and 150. Thus, in each head-injured rat, about 100 such grid squares were present in the eight columns examined. As regards the head-injured rats involved in the quantitative analysis, the average and the standard deviation of the numbers of all myelinated axon profiles were 8,451±908 in group 2, 8,494±960 in group 4 and 8,549±924 in group 5. The differences between any two of them were insignificant: t 18 =0.1028, P>90% between groups 2 and 4; t 18 =0.2282, P>80% between groups 2 and 5; and t 18 =0.1331; P>80% between groups 4 and 5. On the other hand, the average and standard deviation of the numbers of compacted axon profiles were 542±270 in group 2, 240±181 in group 4 and 191±137 in group 5. The differences between the rats that were sacrificed immediately (group 2) and those which survived for 1 day (group 4) or 1 week (group 5) were significant: t 18 =2.917, P<1% and t 18 =3.905, P<1%, respectively. The average number of compacted axon profiles was less in the 1 week rats than in the 1 day rats, but the difference between them was not significant (t 18 =0.684, P50%). In the rats that were sacrificed immediately, no ballooning axon was counted, whereas in the 1 day and the 1 week rats, there were 3 and 9 ballooned axons, respectively. The incidences of these damaged structures as compared to the total numbers of compacted axons in

80 235 Fig. 5 The average number and standard deviation of myelinated axons per grid-square in relation to the serial numbers of the gridsquares counted from the cortical surface the same rat groups (henceforth: relative incidence) were less than 0.5%. Discussion Methodological comments One of our observations (discussed below) was the presence of membranous whorls inside several myelinated axon profiles displaying an otherwise normal ultrastructure. In some respects, these whorls resembled the myelin bodies produced by inadequate brain fixation. In order to avoid the artificial formation of myelin bodies, calcium chloride and polyvinylpyrrolidone were added to the fixative, and potassium hexacyanoferrat(ii) was added to the osmicating solution [13]. It should be noted that the quality of the ultrastructural preservation in the surroundings of each electron-microscopic area in Figs. 2 and 3 was as good as that demonstrated in Fig. 4f. This photo also demonstrates that both the compacted and the non-compacted myelinated axon profiles are clearly visible in the electron microscope at a magnification of 3,000, where they were counted for a quantitative analysis. The number of axon segments stained either evenly or dotted with silver decreased to zero within a few days. This could be assumed to be a result of the spontaneous recovery of all compacted axons or a decrease in time of their stainability with silver [22]. A comparison with the electron microscopic observations and the quantitative data suggests that both assumptions are partly valid. Furthermore, the compacted axons in the rat neocortex cannot be stained with the RMO14 antibody [17], which antibody is widely used for their light-microscopic demonstration in the brain stem, pons and oblongata [21]. This inconsistency may be explained by the difference in thickness of the axons studied. Specifically, the target of the RMO14 antibody is the rod domain of the mid-sized neurofilament protein, which is masked in normal axons, but becomes unmasked as a result of calcium-initiated and calpain-mediated spectrin proteolysis in the axons that display neurofilament (ultrastructural) compaction [3]. Mid-sized neurofilament protein is known to be the main regulator of axon thickness; its absence decreases the axon caliber, while its presence is required to achieve the maximal axon diameter [7]. This is probably the reason for the lack of RMO-14 staining in thin axons in the cortex. For all these, only electron microscopy is qualified to furnish a reliable answer regarding the fate of traumatically compacted axons. In this respect it should be noted that the decrease in distance between adjacent neurofilaments in thin compacted axons in the neocortex immediately after the head injury (58.4%, [11]) is similar to that found in thick axons in the brain stem (62.6%, [18]). Besides the well-known ultrastructural features of crushed or dissected axons in the early phase of Wallerian degeneration (the accumulation of mitochondria, endoplasmic reticulum cisternae, vesicles and mitochondrion-sized dense bodies), two other types of axon degeneration occur in the central nervous system: dark degeneration and watery degeneration [15]. The electron-microscopic features of dark degeneration are very similar to those demonstrated in our Figs. 2c and 3a, b, whereas the appearance of watery degeneration resembles that demonstrated in our Fig. 2f. This is the reason why head-injury apparatuses that also produce an immediate or a delayed disconnection in axons are not suitable for clarification of the fate of the compacted axons. Conclusions drawn from the quantitative analysis The fact that the differences between any two of the three rat groups concerning the numbers (mean ± - E.S.M.) of all (normal + compacted) axon profiles were insignificant indicates that similar populations of axons were examined, and that the method of data sampling used is suitable for quantitative comparisons. As regards the number of compacted axons, there was some diversity among the individual rats in any of the three groups examined. In addition to chance error attributable to the head-injury apparatus, this could be caused by variation in the thickness and elasticity of the rat s calvaria, which certainly depends on the rat s age and breed. However, the degree of this diversity does not contradict the use of three rat groups for qualitative purposes. In Wistar rats weighing about 200 g, approximately 6% of all the axons in the cortical area examined became compacted. The significantly lower numbers of compacted axon profiles in the rats that survived for 1 day as compared with those in the rats sacrificed immediately clearly showed that a large proportion (more than 50%) of the compacted axons recover in less than 1 day while a small proportion (less than 10%) between 1 day and 1 week.

81 236 The ability of the compacted axons to recover spontaneously indirectly supports our assumption [11] that their mechanism of formation is the same as that of the compacted ( dark ) neurons, which are also able to recover spontaneously [6]. The low relative incidence of ballooned axons allows the supposition that neither the somato-dendritic ultrastructural compaction nor the focal impairment of intra-axonal transport (which leads to the formation of axon balloons, [20]) influences the conclusions drawn from the present quantitative and electron-microscopic findings. Moreover, the low relative incidence of ballooned axons confirms the observation [21] that axonal compaction and the focal impairment of axonal transport are not directly related. Conclusions drawn from the electron-microscopic observations Despite the momentary nature of the traumatic ultrastructural compaction in axons [11], the onset time of the subsequent pathological changes (convolution, fragmentation, resorption of the fragments) in the affected axons segments varied considerably. This made it meaningless to attempt an exact correlation of the sequence of morphological changes with the duration of survival. For the same reason, the interpretation of the ultrastructural findings could be composed only of a mixed set of mosaics. Overall, however, three pathways appear to be available for the compacted thin axons in the neocortex as concerns their fate: (1) spontaneous recovery, (2) irreversible degeneration and (3) reversible degeneration. It should be noticed that the axon caliber, the anatomical region and the animal species may influence considerably the extent of involvement of these pathways. The only electron-microscopic sign of spontaneous recovery, in which more than 50% of the compacted axons is involved, is the presence of membranous whorls in several axons displaying an otherwise normal-looking ultrastructure. Such membranous whorls were found in the affected cortical areas of the rats that survived for 1 day or 1 week, but not in their corresponding internal-control areas or in the electron-microscopic specimens of the other head-injured rats. Similar membranous whorls were found in otherwise normallooking neuronal somata and dendrites in otherwise normal-looking granule neurons in the hippocampal dentate gyrus where electrically compacted neurons had recovered [6]. Lamellated myelin sheaths invaginated into axons, which occasionally resemble to our membranous whorls, had been found by other authors and interpreted as the response of myelin to the application of tensile strain to nerve fibers (reviewed in [14]). However, since our membranous whorls are present in axons surrounded by normal-looking myelin sheaths we assume that they are not myelin-derived abnormalities. The chance for recovery probably exists as long as the ultrastructural formations produced immediately after the head injury persists, i.e., for several hours after the head injury. This affords a good opportunity for future therapeutic attempts to increase the probability of recovery. On the other hand, pathophysiological conditions such as an increased intracranial pressure or ischemia, if they exist in this period, may decrease the probability of recovery. The persistence for hours of the primary ultrastructure in traumatically compacted axons in an animal brain was first observed by other authors [18]. The phenomenon of the irreversible degeneration of the compacted axons is indicated directly by the presence of myelin debris in astrocytes, and indirectly by the presence of fat-containing phagolysosomes in pericytes. The incidences of these abnormalities were estimated to be very low as compared with those of the ultrastructural changes of the compacted axons in long-surviving rats. The absence of microglial proliferation and macrophage infiltration indicates that, at least under nonimpaired circumstances, appreciable proportions of compacted axons are not involved in this pathway. However, long-lasting pathophysiological conditions such as an increased intracranial pressure or ischemia may increase the probability of irreversible degeneration. The abnormal myelin sheath configurations depicted in Figs. 3i and 4c e are probably related to some malfunction of oligodendrocytes rather than to myelin degeneration concomitant to the Wallerian axon degeneration. As regards reversible degeneration, a comparison of the message of the previous paragraph with the relevant quantitative data suggests that a considerable proportion of the compacted axons regain their normal ultrastructure within a few months following a degeneration process. The latter begins with the homogenization of the axonal ultrastructure, which is completed during the first day post-injury in most compacted axons. In the meantime, the fluid expressed from the axoplasm flows somehow out of the myelin sheath, or becomes accumulated inside the myelin sheath, filling most part or even the whole of the interior of a cross-sectioned myelin profile. Thereafter, the homogenized axon structure undergoes convolution and fragmentation, which can start at any time between 1 day and 6 months post-injury. The next process is the gradual resorption of these fragments, accompanied by their replacement probably with oligodendrocyte cytoplasm. In parallel with all these processes, axolemma-bound normal axoplasm is assumed to grow into the compacted segment, setting out from its proximal end, and can be expected to establish a functioning connection between the parent cell body and its boutons. The myelin sheaths around the degenerating compacted axons remain non-impaired for several months and ensure guide tubes for the regenerating axons. The reason why the axon degeneration following ultrastructural compaction does not lead to myelin degeneration, which is characteristic of the Wallerian degeneration, is probably that the affected axon segments remain in physical contact with the

82 237 parent neurons. Concerning the effort of mammalian brain neurons to attain axon regeneration, it is known that an attempt at regeneration takes place in the distal end of the proximal part even of the disconnected axons [5]. As revealed by the silver method used here, extensive axonal compaction in various brain areas of the rat, including the brain stem, cerebellum and pons, is a frequent concomitant of various head injuries [8, 10]. If this were the case in human head injuries too, the potential of the compacted axons for a spontaneous recovery as well as a reversible degeneration could be one of the causes leading to an hours-long as well as a months-long improvement of physical or mental capabilities that were lost immediately. Acknowledgments The authors thank Andok Csaba ne, Nyira di Jo zsef, and Dr. Nádor Andra sne for their valuable help in the lightmicroscopic, electron-microscopic and photographic preparative work, respectively. This study was supported by Hungarian grants NKFP 1/A 00026/2002 and ALK-00126/2002, and by the Hungarian Academy of Sciences. References 1. Adams JH, Graham DI, Gennarelli TA (1983) Head injury in man and experimental animals. Acta Neurochir 32(Suppl): Auer RN, Kalimo H, Olsson Y, Siesjo BK (1985) The temporal evolution of hypoglycemic brain damage. I. Light- and electron-microscopic findings in the rat cerebral cortex. Acta Neuropathol 67: Buki A, Siman R, Trojanovsky JQ, Povlishock JR (1999) The role of calpain mediated spectrin proteolysis in traumatically induced axonal injury. J Neuropathol Exp Neurol 58: Cammermeyer J (1961) The importance of avoiding dark neurons in experimental neuropathology. Acta Neuropathol 1: Christman CW, Salivan JB Jr, Walker SA, Povlishock JT (1997) Characterization of a prolonged regenerative attempt by diffusely injured axons following traumatic brain injury in adult cat: a light and electron microscopic immunocytochemical study. Acta Neuropathol 94: Csorda s A, Mázlo M, Gallyas F (2003) Recovery versus death of dark (compacted ) neurons in non-impaired parenchymal environment. Acta Neuropathol 106: Elder GA, Friedrich VL Jr, Bosco P, Kang C, Gourov A, Tu PH, Lee VM, Lazzarini RA (1998) Absence of mid-sized neurofilament subunit decreases axonal calibers, levels of light neurofilament (NF-L), and neurofilament content. J Cell Biol 141: Gallyas F, Zoltay G (1992) An immediate light microscopic response of neuronal somata, dendrites and axons to noncontusing consussive head injury. Acta Neuropathol 83: Gallyas F, Hsu M, Buzsaki G (1993) Four modified silver methods for thick sections of formaldehyde-fixed mammalian central nervous tissue: dark neurons, perikarya of all neurons, microglial cells and capillaries. J Neurosci Meth 50: Gallyas F, Farkas O, Ma zlo M (2002) Traumatic compaction of the axonal cytoskeleton induces argyrophilia: histological and theoretical importance. Acta Neuropathol 103: Gallyas F, Farkas O, Mázlo M (2004) Whole-cell ultrastructural compaction ( dark cell formation) in mammalian tissues may consist in gel-to-gel phase transition. Biol Cell 96: Graham DI, McIntosh TK, Maxwell WL, Nicoll JAR (2000) Recent advances in neurotrauma. J Neuropathol Exp Neurol 59: Hayat MA (1981) Fixation for electron microscopy Academic, New York, pp 88, 123, Maxwell WL, Povlishock JT, Graham DL (1997) A mechanistic analysis of nondisruptive axonal injury: A review. J Neurotrauma 14: Narciso MS, Hokoc JN, Martinez AMB (2001) Watery and dark axons in Wallerian degeneration of the opossum s optic nerve: different patterns of cytoskeletal breakdown? Ann Acad Bras Cienc 73: Paxinos G, Watson C (1982) The rat brain in stereotaxic coordinates Academic, Sydney, New York, plates 18 and Pál J, To th Zs, Farkas O, Kelle nyi L, Do czi T, Gallyas F Selective induction of ultrastructural (neurofilament) compaction in axons by means of a new head injury apparatus. J Neurosci Meth (in press) 18. Pettus EH, Povlishock JT (1996) Characterization of a distinct set of intraaxonal ultrastructural changes associated with traumatically induced alteration in axolemmal permeability. Brain Res 722: Pettus EH, Christman CW, Giebel ML, Povlishock JT (1994) Traumatically induced altered membrane permeability: its relationship to traumatically induced reactive axonal change. J Neurotrauma 11: Povlishock JT, Jenkins LW (1995) Are the pathobiological changes evoked by traumatic brain injury immediate and irreversible? Brain Pathol 5: Stone JR, Singleton RH, Povlishock JT (2001) Intraaxonal neurofilament compaction does not evoke local axonal swelling in all traumatically injured axons. Exp Neurol 172: Zsombok A, To th Zs, Gallyas F (2005) Basophilia, acidophilia and argyrophilia of dark (compacted) neurons in the course of their formation, recovery or death in an otherwise undamaged environment. J Neurosi Meth 142:

83 Biology of the Cell 96 (2004) Gel-to-gel phase transition may occur in mammalian cells: Mechanism of formation of dark (compacted) neurons Ferenc Gallyas a, *, Orsolya Farkas a, Mária Mázló b a Department of Neurosurgery, Pécs University, H-7623, Rét utca 2, Pécs, Hungary b Central Electron Microscopic Laboratory, Faculty of Medicine, Pécs University, H-7623, Rét utca 2, Pécs, Hungary. Received 2 October 2003; accepted 23 January 2004 Available online 12 April 2004 Abstract In the course of many diseases, individual non-apoptotic cells that are randomly distributed among undamaged cells in various mammalian tissues become shrunken and hyperbasophilic ( dark ). The light microscopic shrinkage is caused by a potentially reversible, dramatic compaction of all ultrastructural elements inside the affected cells, and escape of the excess water through apparently intact plasma membrane. In the case of neurons, the ultrastructural compaction rapidly involves the soma-dendrite domains in an all-or-nothing manner, and also mm-long axon segments. The present paper demonstrates that such ultrastructural compaction in neurons, which affects the whole soma-dendrite domain or long axon segments, can take place both immediately after an in vivo head injury and in rat brains perfusion-fixed for 30 min., and then chilled to just above the freezing point before the same kind of head injury was inflicted. This argues strongly against any enzyme-mediated compaction mechanism. On the analogy of gel-to-gel phase transitions in polymer chemistry, we hypothesize a pure physico-chemical compaction mechanism. Specifically, after initiation at a single site in each affected cell, the ultrastructural compaction is propelled throughout the whole cell on the domino principle by the free energy stored in the form of non-covalent interactions among the constituents of some cytoplasmic gel structure Elsevier SAS. All rights reserved. Keywords: Head injury; Post mortem; Cytoplasmic gel; Mechanical energy; Energy storing 1. Introduction Head injuries, electric shock and many diseases or pathometabolic conditions can cause a common type of morphological damage to a number of cells randomly scattered among morphologically intact cells of the same kind in mammalian tissues. The morphological features of the affected cells, which are traditionally called dark, are (a) hyperbasophilia of the cytoplasm, (b) nuclear pyknosis, (c) massive shrinkage of the whole cell, (d) increased electron density, (e) compaction of the ultrastructural elements, and (f) aggregation of the nuclear chromatin with a non-apoptotic pattern. Dark cell formation has been reported to occur in the somatotrophic cells of the adenohypophysis, the reticular cells of the thymus and lymph nodes, the chief cells of the carotid body, the cortical cells of the adrenal gland, the * Corresponding author: Ferenc Gallyas, Ph.D., Department of Neurosurgery, University of Pécs, H-7623 Pécs, Rét utca 2, Hungary; Tel.: +36/72/535900, Fax: +36/72/ address: ferenc.gallyas.sen@aok.pte.hu (F. Gallyas) Elsevier SAS. All rights reserved. doi: /j.biolcel epithelial cells of the cornea and the mammary gland, the tubule cells of the kidney, the keratinocytes of the epidermis, mastocytoma cells, hepatic cells and neurons (reviewed by Harmon, 1987), and also in astrocytes (Tóth et al., 1997). As a possible mechanism of their formation, compression resulting from overcrowding, i.e. a physical injury, has been suggested. In the neurons, dark cell formation is initiated in many human diseases, such as hypoglycemia, ischemia, status epilepticus and head injury, and it can be initiated in experimental animals by excessive stimulation, deafferentation, hyperosmotic shock, poisoning with folic acid, tunicamycin, methoxypyridoxine, 6-hy droxydopamine, 3-acetylpyridine, colchicine, 3-aminopyridine, kainic acid or 3-nitropropionic acid (reviewed by Gallyas et al., 1990), and also by electric shock (Csordás et al., 2003). Neurons are particularly suitable objects for elucidation of the mechanism of dark cell formation, because they possess long, ramified dendrites that are always involved in the ultrastructural compaction. This paper compares the morphological changes caused in neuronal somata, dendrites and axons of the rat brain by a

84 314 F. Gallyas et al. / Biology of the Cell 96 (2004) head injury under in vivo conditions with those caused under conditions extremely unfavorable for enzyme-mediated processes, and concludes from their similarity that the formation of dark neurons may not be a result of a cascade of (patho)- biochemical processes. It is hypothesized that the compaction of apparently intact ultrastructural elements, which is the essence of dark cell formation, consists in a gel-to-gel phase transition that extends to the whole of the cell. As regards gel-to-gel phase transitions, polymer chemistry has invented synthetic gels that can assume two or more metastable phases, each having a distinct free-energy minimum, a distinct set of macromolecular conformations, a distinct degree of water content (Annaka and Tanaka, 1992), and therefore a distinct volume. Initiated at a single point, transition from one gel phase to another can spread throughout the gel, propelled on the domino principle by the difference in free energy (Tanaka et al., 1992). The initiation can be performed by a subtle change around a critical value of chemical composition, ph or temperature of the surrounding medium, and also by illumination, an electric field or a mechanical stress (reviewed by Pollack, 2001). Such gel-togel phase transitions restricted to special intracellular compartments have already been suggested to play essential roles in certain activities of the living cell, such as muscle contraction (Pollack, 1996), action potential (Tasaki, 1999) and mucin secretion (Verdugo et al., 1992). 2. Results 2.1. Control rats Neither the traumatically induced anomalous lightmicroscopic staining properties nor the ultrastructural anomalies described below were observed in the control rats or in the rats that underwent head-injury 1 day after perfusion fixation. Special attention was paid to the neocortex under the impact site In vivo experiments Fig. 1. Light-microscopic appearance of compacted neuronal somata (thick arrows), dendrites (thick arrowheads) and axons (medium arrows) in the brain cortex under the impact site following an in vivo head injury. (A) Silver-stained, 150-µm vibratome section. The nucleoli (thin arrows) of normal neurons are darker, while the nuclei lighter than the background. (B) Toluidine blue-stained, 0.5-µm Durcupan-embedded section. The nuclei (N) and main dendrites (asterisks) of normal neurons are much lighter, whereas the nucleoli (thin arrow) are darker than the background. Scale bars: A=50µm,B=10µm. Intracerebral mechanical force was generated by dropping a brass rod with a mass of 450 g from a height of 100 cm through a vertically positioned Plexiglas tube onto a metal disc cemented to the exposed calvaria of anesthetized rats (impact acceleration head injury, Marmarou et al., 1994). Light microscopy: In the neocortex under the impact site in the rats transcardially perfused with a glutaraldehyde fixative immediately after the infliction of the impactacceleration head injury, several neuronal somata together with their dendritic trees (henceforth: soma-dendrite domains) and numerous long axon segments were stained black with silver in a randomly scattered distribution among unstained neuronal cell bodies, dendrites and axons (Fig. 1a). The nucleoli of normal neurons also became black, whereas the nuclei stained less intensely than the light brown background. The numbers of affected axon segments and somadendrite domains (henceforth: affected neuronal parts) varied widely from rat to rat. The microphoto in Fig. 1a was taken from the most seriously affected rat. On revolution of the micrometer screw, individual axons could be followed in 150-µm-thick vibratome sections for a maximum length of 1.4 mm. Interestingly, the axons relating to the silver-stained soma-dendrite domains usually remained unstained. Not only the neocortex, but also the caudate putamen, brainstem, pons and oblongata contained silver-stained neuronal parts in a scattered distribution (for a detailed description of the morphological damage caused by the head injury paradigm utilized, see Foda and Marmarou, 1994). In Durcupanembedded semithin sections, toluidine blue stained homogenously and intensely the somata and dendrites of several considerably shrunken neurons in the same locations as did the silver method. Even their thin dendrites could be discerned from the background under a high-power microscopic lens (Fig. 1b). On the other hand, only the nucleolus and the rim of the nucleus were visualized in the normal neurons, while their dendrites were much lighter than the background. Several axons were also stained with toluidine blue (not shown), but, in consequence of the co-staining of the myelin sheaths, this could be discerned only under a high-power microscopic lens (similar to that demonstrated in Fig. 5b). Electron microscopy: In those brain areas where silverstained neuronal parts were present in adjacent vibratome

85 F. Gallyas et al. / Biology of the Cell 96 (2004) Fig. 2. (A) Electron-microscopic appearance of normal (N) and compacted (D) neuronal cell bodies in the brain cortex under the impact site following an in vivo head injury. White, thick arrowheads point to dilated Golgi cisternae; n denotes nucleolus, black circles markedly swollen astrocytic processes, and small white squares staining artifacts. In (B), which is a magnification of the area framed in (A), black or white arrowheads point to endoplasmic reticulum cisternae, and black or white arrows to ribosome rosettes; m denotes a mitochondrion. Scale bars: A = 2 µm, B = 500 nm. sections, a number of neuronal cell bodies (Fig. 2), dendrites (Fig. 3) and axons (Fig. 4) displayed a dramatic compaction of apparently intact ultrastructural elements and a considerable volume decrease. The degree of compaction appeared to be similar throughout each affected axonal, somatic and dendritic profile including the dendritic spines. In the compacted axons, the cross-section area relating to a single longitudinal ultrastructural element was 880±59.4 nm 2, whereas in the normal-looking axons 2116±245 nm 2. Their quotient, i.e. the degree of compaction, was 0.416± The plasma membrane did not exhibit any abnormality, despite the fact that a relatively large volume of water must have passed through it. Interestingly, the excess water was taken up by nearby astrocytic processes (but not by the extracellular space). However, in several compacted axons, some excess water remained between the axolemma and the myelin sheath (Fig. 4b). The affected neuronal parts were randomly scattered among normal neuronal parts in an otherwise morphologically intact environment. The compaction involved a marked reduction of the distances between any two neighboring parts of the ultrastructural elements that were visible in the conventional transmission electron microscope (nuclear and plasma membranes, free ribosomes, ribosome rosettes, the outer surfaces of the endoplasmic reticulum cisternae, Golgi cisternae, mitochondria, lysosomes and multivesicular bodies, and also the components of the filamentous cytoskeleton). The distances between the inner surfaces of the endoplasmic reticulum cisternae were also reduced. All the ultrastructural elements involved in the compaction appeared to be interconnected with fine side-arms. In parallel with the compaction, these became shorter and more electron-dense, which gave a dark appearance to the affected neuronal parts. The nuclei of the neurons with a compacted cytoplasm were also compacted. The nuclear chromatin aggregated to numerous small clumps with irregular outlines and a myriad of minute granules, which is a non-apoptotic pattern. No changes were apparent in the volumes of the mitochondria, multivesicular bodies and lysosomes, whereas the interior of the Golgi cisternae was always dilated Post mortem experiments Rats were transcardially perfused with a glutaraldehyde fixative and then chilled to just above the freezing point by being immersed into icy water. Thereafter, a head injury was inflicted by means of the same impact-acceleration paradigm as that used in the in vivo experiment. Brains were removed 1 day later. Light microscopy: In the neocortex under the impact site, similar results were found as in the in vivo experiment. Specifically, the traumatically induced stainability with silver was an all-or-nothing (whole-cell) phenomenon, with the following restrictions: (i) if the soma of a neuron was affected, a continuous network of its dendrites was also affected, whereas its axon was rarely; (ii) the affected axon segments were always long (up to 1.2 mm); and (iii) the neuronal soma-dendrite domains relating to affected axon segments remained predominantly unaffected (Fig. 5a).

86 316 F. Gallyas et al. / Biology of the Cell 96 (2004) Fig. 3. Electron-microscopic appearance of longitudinally sectioned normal (d) and compacted (black arrows) neuronal dendrites in the brain cortex under the impact site following an in vivo head injury. Black or white arrowheads point to microtubules; m denotes mitochondria, a black asterisk glial filaments, and small white squares staining artifacts. Scale bar = 500 nm. However, all the brain areas other than the neocortex did not demonstrate such effects, and the numbers of affected neuronal parts in the neocortex were much smaller. The microphoto in Fig. 5a was taken from the most seriously affected rat. Similarly as found in the in vivo case, the head injury induced stainability with toluidine blue in injured neuronal somata, dendrites and axons (Fig. 5b). Electron microscopy: Several neuronal somata, dendrites and axons revealed the same ultrastructural features (Figs 6-8) as those observed in the in vivo head-injured rats. No exceptions were found. In the compacted axons, the crosssection area relating to a single longitudinal ultrastructural element was 867±63.6 nm 2, whereas in the normal-looking axons 2066±246 nm 2. Their quotient, i.e. the degree of compaction, was 0.420± Discussion 3.1. Comments on the methods used and the results obtained A special silver staining method was utilized to find brain areas worthy of electron-microscopic investigation, i.e. in which compacted neuronal parts were present in sufficient numbers (Figs 1a and 5a). Because of the poor reputation of the traditional silver impregnation methods, it should be stressed that various recent methods that employ physical developers, including the present method, are reliable (Newman and Jasani, 1998). In the rat brain, this method demonstrates ultrastructural compaction selectively and reproducibly both in axons (Gallyas et al., 2002) and in neuronal soma-dendrite domains (Csordás et al., 2003). No other method is available for the demonstration of freshly compacted neuronal dendrites and axons in thick vibratome sections. In thin frozen or paraffin sections, in addition to the usual basophilic substances (nucleic and ribonucleic acids and polyanions) neurons with compacted somata can also be stained at about ph 4 with cationic dyes such as toluidine blue. In osmicated and Durcupan embedded 1-µm-thick sections, nothing can be stained with cationic dyes at about ph 4, but both the usual basophilic substances and the compacted neuronal somata, dendrites and axon segments are selectively stained at ph 9.5 (see Figs. 1b and 5b). In surviving animals, from the 5 th min. post injury, compacted axons can be demonstrated by the uptake of intraventricularly administered horseradish peroxidase (Pettus et al., 1994), and from the 15 th min. on by Ab-38 and RMO-14 immunohis-

87 F. Gallyas et al. / Biology of the Cell 96 (2004) Fig. 4. Electron-microscopic appearance of cross-sectioned (A) and longitudinally sectioned (B) normal (white arrowheads) and compacted (white arrows) myelinated axons in the brain cortex under the impact site following an in vivo head injury. Black arrows point to neurotubulí in a normal dendrite (d); white m denotes myelin sheaths, and black circles fluid-filled spaces between the axolemma and myelin sheath. Neurofilaments and microtubules appear as fine dots inside the cross-sectioned axons, while as fine lines inside the longitudinally sectioned axons. Scale bars: A and B = 1 µm. tochemistry (Büki et al., 1999). Freshly-produced dark neurons are both strongly basophilic and slightly acidophilic, thereby stainable with many dyes (Brown, 1977). The acidophilia of the dark neurons that die gradually increases, while their basophilia decreases (Auer et al., 1984; Kiernan et al., 1998). For the investigation of the ultrastructural compaction in neuronal somata, dendrites and axons without the influence of any other kinds of traumatic damage to the brain tissue, it is of fundamental importance that the affected area should remain otherwise intact. This is, why the rats with a calvaria fracture (which results in serious laceration of the brain cortex) were discarded from the present investigation (see Materials and Methods). The differences, between the normal and the affected axons, in the average cross-section area relating to a single longitudinal ultrastructural element were highly significant both after an in vivo (t 18 = 15.5; P < 0.1%), and a post mortem (t 18 = 14.9; P < 0.1%.) head injury. On the supposition that the axonal length remains unchanged during the compaction, the degree of compaction is directly proportional to the degree of reduction in the axon volume. Accordingly, the average volume of the compacted axons following an in vivo head injury is 41.6±3.7% of the average volume of the normal-looking axons, while this parameter is 42.0±3.9% for the post-mortem case. These values are very similar (the difference between them is not significant: t 18 = 0.235; 10% < P <20%), indicating that the compaction may proceed with the same mechanism both in vivo and post mortem. (The volume decrease in neuronal somata and dendrites could not be analyzed quantitatively due to problems in sampling comparable data.) The essence of the morphological damage caused to neuronal somata, dendrites and axons by a head injury in the present post mortem experiment is the same as that caused in the present in vivo experiment. Specifically, in both cases, (i) a high-grade compaction of apparently intact ultrastructural elements occurred in the affected neuronal somata (Figs 2 and 6) dendrites (Figs 3 and 7) and axons (Figs 4 and 8), (ii) the degree of volume reduction in the axons was similar (see above;), (iii) the interior of the membrane-bound ultrastructural elements (mitochondria, lysosomes, vesicles and multivesicular bodies) remained non-compacted, (iv) the Golgi cisternae became dilated, (v) the compacted neuronal parts were scattered among normal neuronal parts in an otherwise intact environment, (vi) whole soma-dendrite domains and long axon segments were always affected, and the compacted neuronal parts immediately acquired (vii) basophilia, (viii) argyrophilia and (ix) hyper-electrondensity. Dissimilarities between the in vivo and the post mortem cases were found only in the number of affected neuronal parts and in the extent of the affected areas. Both were smaller under the post mortem circumstances, probably because of a fixation-derived decrease in the elasticity of the brain tissue. However, these dissimilarities are not sufficient to negate the message of the present investigation, specified in the first sentence of the previous paragraph.

88 318 F. Gallyas et al. / Biology of the Cell 96 (2004) The sporadic formation of dark (strikingly shrunken and hyperbasophilic) neurons as a result of post mortem mechanical injuries to unfixed or improperly fixed brains, and its avoidance by transcardial perfusion fixation and a 1-day delay of the brain autopsy, have long been known (Cammermeyer, 1960 and 1961). Since these dark neurons have been regarded as artifacts, their ultrastructure has not been investigated so far. The in vivo formation of compacted axons scattered among normal axons was discovered a decade ago in experimental animals that survived for 5 min. or longer following a head injury (Pettus et al., 1994). Shorter survival periods were not investigated. It was demonstrated only recently that the process of ultrastructural compaction both in axon segments (Gallyas et al., 2002) and in neuronal soma-dendrite domains (Csordás et al., 2003) is completed even in rats that are perfused transcardially with an electronmicroscopic fixative immediately after the infliction of a head injury. As regards the soma-dendrite compaction caused by an in vivo head injury, no biochemical mechanism has been postulated so far. For axons, traumatic ultrastructural (neurofilament) compaction has been assumed by other authors (e.g. Buki et al., 1999) to be a morphological manifestation of calpain-mediated spectrin proteolysis, initiated by an uncontrolled influx of Ca ++ through the axolemma, perturbed focally by some traumatically generated intracerebral shearing force Two arguments against any enzyme-mediated compaction mechanism Fig. 5. Light-microscopic appearance of compacted neuronal somata (thick arrows), dendrites (thick arrowheads) and axons (medium arrows) in the brain cortex under the impact site following a post mortem head injury. (A) Silver-stained, 150-µm vibratome section. The nuclei of normal neurons are much lighter, whereas the nucleoli (thin arrows) are darker than the background. (B) Toluidine blue-stained, 0.5-µm Durcupan-embedded section. A compacted neuron (thick arrow), the compacted myelinated axons (medium arrows), and also all myelin sheaths are intensely stained, whereas the normal neurons (N) and the non-compacted myelinated axons (medium arrowheads) and are unstained. Open circles denote nuclei of astrocytes, and a black square a capillary lumen. Scale bars: A = 25 µm, B = 10 µm. The first argument is based on the high degree of similarity between the pathomorphologic features found in our in vivo and post mortem experiments. This suggests that the mechanism of compaction is the same in both cases. However, it appears improbable that any complex enzymemediated process should result in the same morphological features under both optimum and extremely unfavorable conditions. Namely, the individual biochemical reactions that run in cascade and/or in parallel in any complex enzymemediated process would be slowed down to different degrees by the partial fixation and the marked temperature drop applied in our post mortem experiment, which would substantially disturb the in vivo harmony of their action. The second argument is based on the all-or-nothing nature of the compaction. It is impossible that a head injury should simultaneously produce ultrastructural compaction at each point of a number of randomly distributed neuronal somadendrite domains and axons and, at the same time, should spare all points of the neighboring soma-dendrite domains and axons. The all-or-nothing nature can be explained only by the assumption that the compaction spreads throughout each affected soma-dendrite domain or axon from a single initiation point. It appears highly unlikely that any complex enzyme-mediated process should spread over long intracellular distances without fading under the extremely unfavorable conditions applied in our post mortem experiment. If the above train of thoughts is true, any post-traumatic enzyme-mediated pathological process should be assumed to be a consequence, and not the cause of the in vivo ultrastructural compaction. In accordance with this assumption, calpain-mediated spectrin proteolysis in compacted axons could be detected only in rats that survived for at least 15 min. after an in vivo head injury (Buki et al., 1999) The hypothesized non-enzymatic mechanism of the whole-cell ultrastructural compaction We propose here a non-enzymatic mechanism, first in terms of the energy available to propel the whole-cell ultrastructural compaction both in vivo and post mortem, and then in terms of the structure capable of executing the compaction at the expense of the available energy. The free energy of this structure comprises that of the covalent bonds and that of the non-covalent interactions (ion-ion, dipole-dipole, van der Waals, hydrophobic and hydrogen bonding). In contrast with the release of the free energy of the covalent bonds, the release of the free energy of the non-covalent interactions does not necessarily need enzyme mediation. If the free energy of the non-covalent inter-

89 F. Gallyas et al. / Biology of the Cell 96 (2004) Fig. 6. (A) Electron-microscopic appearance of normal (N) and compacted (D) neuronal cell bodies in the brain cortex under the impact site following a post mortem head injury. White, thick arrows point to dilated Golgi cisternae; black circles denote markedly swollen astrocytic processes. In (B), which is a magnification of the area framed in (A), black or white thin arrows point to endoplasmic reticulum cisternae, black or white thin arrowheads to ribosome rosettes, and a thick arrow to a multivesicular body; white asterisks denote mitochondria, and black circles swollen astrocytic processes. Scale bars: A = 2 µm, B=500nm. actions in the non-compacted state of this structure is higher than that in the compacted state, the difference between these energy levels (henceforth: stored non-covalent free energy) can propel the compaction both in vivo and post mortem, provided that the following two energetic requirements are met: First, the non-compacted state of the structure responsible for the compaction must be metastable. Thus, enzymemediated energy is needed only for its building up, but not for its maintenance. Furthermore, some exogenous energy, called activation energy, is needed for the release of the stored energy. The fulfillment of this energetic requirement is plausible: without exogenous intervention, the neurons never become compacted post mortem, even in the unfixed brain. Second, in the non-compacted state, the structure responsible for the compaction must be built up in such a way that the stored non-covalent free energy, if released somehow at any point, inevitably serves as activation energy at the neighboring points (domino principle). If this requirement is fulfilled, the all-or-nothing nature of the compaction can be explained as follows: Some intracerebral mechanical force induced by the head injury transmits activation energy to only a single point in each somatodendritic domain or axon that will become compacted (initiation phase). Thereafter, the structural transformation concomitant with the energy release spreads over long intracellular distances, propagated by the stored non-covalent free energy (spreading phase). The scattered occurrence of the affected neuronal somadendrite domains or axon segments indicates that the probability of initiation is very low, while the fact that whole soma-dendrite domains or long axon segments are generally affected indicates that the process of spreading is relatively rapid. We suggested earlier (Gallyas et al., 1992) that the neurofilament network, which had been assumed to store torsional energy (Metuzals and Izzard, 1969; Gilbert, 1975), is responsible for the compaction. However, the morphological substratum of the compaction cannot be any of the ultrastructural elements visible in the conventional transmission electron microscope (such as the neurofilaments), since intracellular spaces devoid of them (the dendritic spines, the interior of the endoplasmic reticulum cisternae, or the cell nucleus) are also involved in the compaction. Conversely, this structure must be present and continuous in all intracellular spaces involved in the compaction. We propose here that this structure consists in a gel built up as outlined below. Numerous non-covalent binding sites in numerous protein molecules that are present in the aqueous cytoplasm (all cytoplasmic spaces not occupied by the ultrastructural components visible in the conventional transmission electron

90 320 F. Gallyas et al. / Biology of the Cell 96 (2004) Fig. 7. (A) Electron-microscopic appearance of a longitudinally sectioned compacted neuronal dendrite in the brain cortex under the impact site following a post mortem head injury. Black arrows point to dendritic spines; black circles denote swollen astrocytic processes. (B) A normal (left side) and a compacted (right side) dendrite in juxtaposition. Black or white arrowheads point to microtubules; black circles denote swollen astrocytic processes. Scale bars: A = 5 µm, B=200nm. microscope; Clegg, 1984) are engaged not in maintaining the secondary and tertiary structures of their native (in-vitroactive) state, but in mutually chaperoning their in vivo conformation, anchoring the resulting protein matrix to the ultrastructural elements visible in the conventional transmission electron microscope, binding K + (Ling and Ochsenfeld 1973) and adsorbing water molecules in multiple ordered layers (Ling and Walton, 1975). In this case, the whole-cell ultrastructural compaction ( dark cell formation) could consist in a gel-to-gel phase transition, i.e. in a co-operative conformational change in the protein molecules building up the matrix of this gel, which conformational change is accompanied by the release of bound water molecules (compaction) and K + (hyperbasophilia). Namely, the gel-to-gel phase transition (for its relevant qualities see the Introduction) can (i) fulfil the two energetic requirements discussed earlier, (ii) cause a dramatic volume reduction, (iii) be initiated by both chemical and physical injuries and (iv) be reversible (see in section 3.6.), like the whole-cell compaction. In aldehyde-fixed specimens, the microtrabecular lattice observable in the high-voltage stereo electron microscope (Porter, 1989) might be the post-fixation morphological equivalent of the matrix of this gel. The absence of somatodendritic or axonal compaction from the brains of rats headinjured 24 h after transcardial perfusion with an aldehyde fixative can be explained as follows. By gradually forming chemical bridges between protein molecules, the aldehyde fixation stabilizes the matrix of the presumed intracellular gel in 1 day at room temperature, but not in much shorter periods of time Supporting arguments drawn from the post mortem development of hyperbasophilia and argyrophilia Two all-or-nothing phenomena accompany the whole-cell compaction both in vivo and post mortem: hyperbasophilia (Figs 1b and 5 b) and argyrophilia (Figs 1a and 5a). Basophilia is the histochemical manifestation of structure-bound anionic groups that are capable of binding positively charged dye molecules. In the normal neurons, only the Nissl granules, the nucleoli and the rim of the nucleus (nucleic acids) are basophilic (see Fig. 1a and 5a). As a consequence of the ultrastructural compaction, the affected neuronal somata, dendrites and axons homogeneously acquire excess basophilia (become hyperbasophilic). Enzymatic digestion of

91 F. Gallyas et al. / Biology of the Cell 96 (2004) Fig. 8. (A) Electron-microscopic appearance of cross-sectioned normal (arrowhead) and compacted (arrow) myelinated (white m) axons in the brain cortex under the impact site following a post mortem head injury. A black circle denotes a swollen astrocytic process. (B) and (C) are enlargements of the normal and the compacted axons, respectively, demonstrated in (A). Black or white arrows point to microtubules, and black or white arrowheads to endoplasmic reticulum cisternae; white m denotes myelin sheaths. Neurofilaments appear as small dots. Scale bars: A = 500 nm, B and C = 200 nm. nucleic acids cannot remove the excess basophilia, whereas it removes completely the basophilia of the normal neurons (our unpublished observation). Consequently, hyperbasophilia of the dark (compacted) neurons can not be the simple consequence of the condensation of preexisting basophilic substances. Furthermore, it appears improbable that any enzyme-mediated process could form negatively charged substances in excess under extremely unfavorable conditions. Conversely, as a result of compaction-derived conformational changes in the affected protein molecules, preexisting structure-bound anionic groups can become detached from the counter-ions (K +?), escape from a hydrophobic environment or become more electronegative. The silver staining method utilized demonstrates type-iii argyrophilia. In its case, the formation of silver grains is mediated by tissue-related catalytic sites, which consist of a few side-groups of protein molecules in a favorable spatial arrangement (Gallyas, 1982). It appears improbable that any enzyme-mediated process could form new proteins or protein side-groups in excess under extremely unfavorable conditions. Conversely, catalytic sites may result from compaction-derived conformational changes in the affected

92 322 F. Gallyas et al. / Biology of the Cell 96 (2004) protein molecules that lead to a favorable change in the spatial arrangement of their pre-existing side-groups Two previous conceptions of phase transition in the aqueous cytoplasm The association-induction theory of Ling (1962) postulates the existence of a physicochemical process, called cardinal adsorption, which is in fact micro-compartmentalized gel-to-gel phase transition. Ling assumes that the binding of cardinal adsorbents, such as ATP or Ca ++, donates additional free energy to an association of fully extended proteins, inorganic ions and water molecules. This induces a spreading change in protein conformation accompanied by the selective adsorption of K + and the large-scale polarization of water molecules into multilayers. The reverse of this process supplies free energy to various physiological processes. The Pollack (2001) conception considers that gel-to-gel phase transition may be a central mediator of physiological cell functions, especially those in which chemical energy is transformed to physical work. The different physiological needs are met by various types of phase transition, which are confined to intracellular compartments of diverse size and gel composition. Mammalian cells could undergo both the above physiological types and the currently reported pathological type of gel-to-gel phase transition, provided that the hypothesized cytoplasmic gel has at least three metastable phases representing different levels of molecular organization. It should be stressed that complex synthetic gels that possess such a property do exist (Annaka and Tanaka, 1992) Presumed function of whole-cell gel-to-gel phase transition in pathology Dark cells are widely believed to be fated inevitably to die. However, as concerns dark neurons, strong evidence has been presented in the past twenty years that, under favorable circumstances, a varying proportion of them can spontaneously recover (reviewed by Csordás et al., 2003). Since the dying dark neurons are removed from an excitotoxic or a necrotic environment through a necrotic-like sequence of ultrastructural changes (gross swelling and disintegration of the organelles and the gradual disappearance of the plasma and nuclear membranes), it has generally been concluded that their death is caused by necrosis (e.g. Ingvar et al., 1988). However, a recent publication (Csordás et al., 2003) demonstrates that, from an intact environment, dark neurons are removed through a sequence of ultrastructural changes similar to that for apoptotic neurons. Specifically: marked convolution of the nuclear and cellular outlines, separation of the pedunculated protuberances forming membrane-bound bodies of compacted ultrastructure, which are later engulfed by phagocytotic cells. After completion of the programmed biochemical cascade and segregation of the nuclear chromatin into sharply delineated masses, apoptotic cells are known to undergo a sudden cytoplasmic shrinkage (e.g. Wyllie, 1987). Ultrastructural similarities between the shrunken cytoplasm of apoptotic neurons and that of the dark neurons produced in a noncompromised environment (our unpublished observation) suggest that their mechanisms of formation are the same. If this is true, the formation of dark cells might be a malfunction of the currently hypothesized metastable intracellular gel structure, the genuine function of which is to execute whole-cell ultrastructural compaction in an advanced morphological phase of apoptotic cell death. The capacity of dark neurons to recover indicates that at least the neurons are prepared to attempt to remedy the damage caused by this malfunction. It should be stressed that no idea of the function of dark -cell formation (the ultrastructural compaction in non-apoptotic cells) has been suggested so far Concluding remarks It remains the task of further investigations to establish how this conception of the whole-cell ultrastructural compaction can be harmonized with the prevailing conceptions of the physical state of the living cell and concomitant features such as membrane pumps and ion channels. Even within the realm of the current conception, tremendous complementary problems demanding a solution will emerge, such as the very low probability of initiation of whole-cell compaction or its stop at the axon hillock in the case of neurons. Regardless of whether or not the present conception holds true, some overall cell-biological phenomenon is lying hidden behind whole-cell ultrastructural compaction, and this must not be passed over in silence. 4. Materials and methods 4.1. Animal experiments A total of 29 Sprague-Dawley rats weighing about 200 g were anaesthetized with a mixture of 4% isoflurane, 29% oxygen and 67% nitrous oxide, using a SurgiVet respirator (Waukesha, WI, USA). Each of them was scalped and a 4-mm-thick metal disc 10 mm in diameter was cemented to the exposed skull vault between the coronal suture and lambdoid. Seven rats were placed in a prone position on a 12-cmthick foam bed under an impact-acceleration head injury device (Marmarou et al., 1994). An intracerebral mechanical force was generated by dropping a brass rod with a mass of 450 g onto the metal disc from a height of 100 cm through a vertically positioned Plexiglas tube. Two of these rats suffered impression fracture of the calvaria and were excluded from further investigation (for the reason, see the Discussion). Immediately after the head injury, the 5 rats with a non-fractured calvaria were perfused transcardially at room temperature, first with 50 ml of physiological saline and then, for 30 min. with 500 ml of an aldehyde fixative (100 ml of

93 F. Gallyas et al. / Biology of the Cell 96 (2004) % paraformaldehyde, 100 ml of 25% glutaraldehyde, 500 ml of 0.2 mol/l sodium cacodylate, and 300 ml of distilled water were mixed, and the resulting solution was adjusted to ph 7.4 with a few drops of concentrated hydrochloric acid). Without any previous manipulation, each of the remaining 22 rats was perfused with the above fixative for 30 min. Thereafter, the bead probe of a digital thermometer was positioned deep in the rectum and the skin over the abdomen and thorax was tightly sutured. Finally, such rats were immersed into icy water until the rectal temperature fell to just above the freezing point (about 90 min.). Twelve of them were head-injured as described above. Seven of the headinjured rats suffered impression fracture of the calvaria, and were excluded from the further investigation (for the reason, see the Discussion). The remaining 5 head-injured rats and 10 rats without head injury were allowed to warm up to room temperature. In 5 of the latter rat group, the above head injury was provoked 24 h later. None of them suffered calvaria fracture. The non-traumatized 5 rats served as controls. Apart from the above manipulations, all rats were left untouched at room temperature for 24 h after the start of fixation. Thereafter, the brains were removed from the skull and immersed in the fixative. Animal care and handling were carried out in accordance with order 243/1998 of the Hungarian Government, which is an adaptation of directive 86/609/EGK of the European Committee Council Tissue processing and staining Vibratome sections 150 µm in thickness were cut coronally from the caudal two-thirds of the cerebrum. Every fifth vibratome section was stained by a special silver technique, as described previously (Gallyas et al., 1990). Briefly, following dehydration with graded 1-propanol, vibratome sections were incubated for 16 h at 56 C in 1-propanol containing 0.8% sulfuric acid and 2% water (esterification), rehydrated with graded 1-propanol, treated with 1% acetic acid for 10 min. and then immersed in a special physical developer until the background had become light-brown. For electron microscopy, 2x2-mm 2 areas of the neocortex and the subcortical white matter were cut from the sections adjacent to those displaying the highest number of silverstained neuronal parts. These specimens were postfixed with a 1:1 mixture of 2% osmium tetroxide and 3% potassium hexocyanoferrate(ii) for 1 h at room temperature, and flatembedded in Durcupan ACM. Semithin (1-µm) sections were stained in a solution containing 0.05% toluidine blue, 0.05 % sodium tetraborate and 0.1% saccharose (ph 9.5) for 1 min. at 90 C. Thin (50-nm) sections were contrasted with 5% uranyl acetate in 50% methanol for 2 min. and then 0.5% lead citrate for 1 min. Ultrastructural investigations were carried out with a Jeol JEM 1200EX transmission electron microscope Quantitative analysis From each of the 5 in vivo and the 5 post mortem headinjured rats, cross-sections of 10 normal-looking and 10 compacted axons were photographed at a magnification of 25000, digitized at 1200 dpi with an Epson GT 9600 scanner and magnified electronically to times their actual size. Thereafter, the longitudinal ultrastructural elements (neurofilaments and microtubules) were counted in them. From each 10-member group, 2 axons that contained longitudinal elements in similar numbers (between 195 and 216) were selected. In these axons, the area inside the axolemma but outside the mitochondria and endoplasmic reticulum cisternae was measured, and then divided by , and finally by the relevant number of longitudinal elements. The quotients relating to each of the 4 axon groups of interest (normal axons, in vivo head injury; compacted axons, in vivo head injury, normal axons, post mortem head injury; compacted axons, post mortem head injury) were next averaged. These averages, which represent the cross section areas that relate to a single longitudinal ultrastructural element, were compared using the Student t-test. Acknowledgements We thank Andok Csabáné, Nyírádi József and Nádor Andrásné for technical assistance. Supported by Hungarian grants from OTKA (T ) and from NKFP (1/A 00026/2002). References Annaka, M., Tanaka, T., Multiple phases of polymer gels. Nature 355, Auer, R.N., Wieloch, T., Olsson, Y., Siesjö, B.K., The distribution of hypoglycemic brain damage. Acta Neuropathol. 64, Brown, A.W., Structural abnormalities in neurons. J. Clin. Pathol. 30 (Suppl), (Royal College of Pathologists) 11. Buki, A., Siman, R., Trojanowsky, J.Q., Povlishock, J.T., The role of calpain mediated spectrin proteolysis in traumatically induced axonal injury. J. Neuropathol. Exp. Neurol. 58, Cammermeyer, J., The post mortem origin and mechanism of neuronal hyperchromatosis and nuclear pycnosis. Exp. Neurol. 2, Cammermeyer, J., The importance of avoiding dark neurones in experimental neuropathology. Acta Neuropathol. 1, Clegg, J.S., Properties and metabolism of the aqueous cytoplasm and its boundaries. Am. J. Physiol. 246, R133 R151. Csordás, A., Mázló, M., Gallyas, F., Recovery versus death of dark (compacted) neurones in non-impaired parenchimal environment. Light and electron microscopic observations. Acta Neuropathol. 106, Foda, M.M.A., Marmarou, A., A new model of diffuse brain injury in rats. Part II: Morphological characterization. J. Neurosurg 80, Gallyas, F., Physico-chemical mechanism of the argyrophil III reaction. Histochemistry 74, Gallyas, F., Güldner, F.H., Zoltay, G., Wolff, J.R., Golgi-like demonstration of dark neurones with an argyrophil III method for experimental neuropathology. Acta Neuropathol. 79,

94 324 F. Gallyas et al. / Biology of the Cell 96 (2004) Gallyas, F., Zoltay, G., Dames, W., Formation of dark (argyrophilic) neurones of various origin proceeds with a common mechanism of biophysical nature. Acta Neuropathol. 83, Gallyas, F., Farkas, O., Mázló, M., Traumatic compaction of the axonal cytoskeleton induces argyrophilia: histological and theoretical importance. Acta Neuropathol. 103, Gilbert, D., Axoplasm architecture as seen in the Myxicola giant axon. J. Physiol 253, Harmon, B.V., An ultrastructural study of spontaneous cell death in mouse mastocytoma with particular reference to dark cells. J. Pathol 153, Ingvar, M., Morgan, P.F., Auer, R.N., The nature and timing of excitotoxic neuronal necrosis in the cerebral cortex, hippocampus and thalamus due to flurothyl-induced status epilepticus. Acta Neuropathol. 75, Kiernan, J.A., Macpherson, C.M., Price, A., Sun, T., A histochemical examination of the staining of kainate-induced neuronal degeneration by anionic dyes. Biotechnic & Histochemistry 73, Ling, G.N., A Physical Theory of the Living State: The Association- Induction Hypothesis. Blaisdell, Waltham, pp Ling, G.N., Ochsenfeld, M.M., Mobility of potassium ion in frog muscle cells, both living and dead. Science 181, Ling, G.N., Walton, C.L., What retains water in living cells. Science 191, Marmarou, A., Foda, M.A., Brink, W., Chambell, J., Khita, H., Demetriadu, K., A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics. J. Neurosurg 80, Metuzals, J., Izzard, C.S., Spatial patterns of threadlike elements in the axoplasm of the giant nerve fibre of the squid (Loligo pealei L) as disclosed by differential interference microscopy and by electron microscopy. J. Cell Biol. 43, Newman, G.R., Jasani, A.B., Silver development in microscopy and bioanalysis: past and present. J. Pathol 186, Pettus, E.H., Christman, C.W., Giebel, M.L., Povlishock, J.T., Traumatically induced altered membrane permeability: its relationship to traumatically induced reactive axonal change. J. Neurotrauma 11, Pollack, G.H., Phase transitions and the molecular mechanism of contraction. Biophys. Chem 59, Pollack, G.H., Cells, Gels and the Engines of Life. Ebner & Sons, Seattle, pp Porter, K.R., The cytoplasm and its matrix. Prog. Clin. Biol. Res. 295, Tanaka, T., Annaka, M., Ilmain, F., Ishii, K., Kokufuta, E., Suzuki, A., Tokita, M., Phase transitions of gels. In: Karalis, T.K. (Ed.), Mechanics of Swelling, H64. Springer, Berlin, pp NATO ASI Series. Tasaki, I., Rapid structural changes in nerve fibers and cells associated with their excitation processes. (Review). Jpn. J. Physiol. 49, Tóth, Z.S., Seress, L., Tóth, P., Ribak, C.E., Gallyas, F., A common morphopathological response of astrocytes to various injuries: dark astrocytes: A light and electron microscopic analysis. J. Brain Res. 38, Verdugo, P., Deyrup-Olsen, I., Martin, A.W., Luchtel, D.L., Polymer gel phase transition: the molecular mechanism of product release in mucin secretion. In: Karalis, T.K. (Ed.), Mechanics of Swelling, H64. Springer, Berlin, pp NATO ASI Series. Wyllie, A.H., Apoptosis: Cell death in tissue regulation. J. Pathol. 153,

95 JOURNAL OF NEUROTRAUMA Volume 20, Number 3, 2003 Mary Ann Liebert, Inc. Preinjury Administration of the Calpain Inhibitor MDL Attenuates Traumatically Induced Axonal Injury A. BUKI, 1 O. FARKAS, 1 T. DOCZI, 1 and J.T. POVLISHOCK 2 ABSTRACT Traumatic brain injury (TBI) evokes diffuse (traumatic) axonal injury (TAI), which contributes to morbidity and mortality. Damaged axons display progressive alterations gradually evolving to axonal disconnection. In severe TAI, the tensile forces of injury lead to a focal influx of Ca 21, initiating a series of proteolytic processes wherein the cysteine proteases, calpain and caspase modify the axonal cytoskeleton, causing irreversible damage over time postinjury. Although several studies have demonstrated that the systemic administration of calpain inhibitors reduces the extent of ischemic and traumatic contusional injury a direct beneficial effect on TAI has not been established to date. The current study was initiated to address this issue in an impact acceleration rat-tbi model in order to provide further evidence on the contribution of calpain-mediated proteolytic processes in the pathogenesis of TAI, while further supporting the utility of calpain-inhibitors. A single tail vein bolus injection of 30 mg/kg MDL was administered to Wistar rats 30 min preinjury. After injury the rats were allowed to survive 120 min when they were perfused with aldehydes. Brains were processed for immunohistochemical localization of damaged axonal profiles displaying either amyloid precursor protein (APP) or RMO-14 immunoreactivity (IR), both considered markers of specific features of TAI. Digital data acquisition and statistical analysis demonstrated that preinjury administration of MDL significantly reduced the mean number of damaged RMO-14 as well as APP-IR axonal profiles in the brainstem fiber tracts analyzed. These results further underscore the role of calpain-mediated proteolytic processes in the pathogenesis of DAI and support the potential use of cell permeable calpain-inhibitors as a rational therapeutic approach in TBI. Key words: axonal injury; calpain; MDL-28170; spectrin; traumatic brain injury INTRODUCTION TRAUMATIC BRAIN INJURY (TBI) has long been associated with the generation of traumatic axonal injury (TAI) both in animals and humans (Gennarelli et al., 1982; Blumbergs et al., 1989; Adams et al., 1977, 1980; Strich, 1956). In the last two decades, detailed investigation of the pathobiology of traumatic axonal injury has revealed that the majority of traumatically injured axons are not mechanically severed at the time of impact, instead showing progressive changes gradually evolving to axonal disconnection (Povlishock et al., 1983, 1992; Povlishock, 1992; Maxwell et al., 1997; Meaney et al., 2000). Typically, this process of progressive axonal 1 Department of Neurosurgery, Medical Faculty of Pécs University, Pécs, Hungary. 2 Department of Anatomy and Neurobiology, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, Virginia. 261

96 BUKI ET AL. change evolves over a several hour period, leading to an upstream swelling due to an impairment of axonal transport (Povlishock et al., 1983), although it is now recognized that not all injured axons go on to swell (Stone et al., 2000). The precise subcellular mechanisms responsible for initiating some, if not all of these progressive changes, have also been unmasked over the last several years. In vitro studies by Wolf and colleagues (2001) have shown that the traumatic event is associated with intraaxonal calcium influx, which was linked to traumatically induced depolarization, activation of voltage sensitive calcium channels and the concomitant activation of sodium/calcium exchangers. In the in vivo setting, the involvement of calcium in this progressive pathobiology has also been confirmed through several indirect approaches. Maxwell and colleagues (1995) showed significant intraaxonal calcium increases through the use of pyroantimonate in the injured optic nerve. Our own group demonstrated, in foci of traumatic axonal injury, alterations in axolemmal permeability that were directly correlated with cytoskeletal and mitochondrial damage, a finding consistent with local calcium overloading, occurring as a result of alterated axolemmal permeability (Pettus et al., 1994; Pettus and Povlishock, 1996). When first observed, it was assumed that this altered axolemmal permeability and presumed calcium influx would cause catastrophic and ultra-rapid degradation of the intraaxonal cytoskeleton. However, continued work did not confirm this. Detailed studies conducted by Büki and colleagues (1999a) examining calcium-mediated pathology in terms of calcium-mediated protease activation demonstrated a compartmentalized and progressive series of changes that led to axonal disconnection. Specifically, through the use of antibodies targeting calpain-mediated spectrin-proteolysis Büki and colleagues (1999a) recognized, in the early post-traumatic period, that the calpainmediated processes were confined to the sub-axolemmal domain, progressing over time to involve the entire extent of the axon diameter, thereby causing the progressive degradation of the axon cylinder. These studies confirmed and extended earlier studies by Saatman and colleagues (1996b), Newcomb and collaborators (1997) and McCracken et al. (1999) which also had alluded, in part, to the occurrence of calpain-mediated axonal damage occurring together with other forms of TBI-induced pathology. Because of these observed progressive pathological changes involving calpain-mediated damage, it appeared that therapeutic strategies targeting calcium and its downstream targets such as calpain would constitute rational approaches to blunt the progression of axonal damage, thereby translating into improved outcome. The correctness of this assumption was supported, in part, by findings employing hypothermia, wherein the use of posttraumatic hypothermia followed by slow rewarming was shown to translate into significant axonal protection which was directly correlated with the attenuation of markers targeting calpain- mediated proteolysis. (Koizumi and Povlishock, 1998; Buki et al., 1999b). The potential usefulness of inhibitors of calpain-mediated proteolysis is not a new concept and, in fact, this approach has been used in the study of ischemic brain injury as well as traumatic brain injury paradigms focusing primarily upon contusional change (Saatman et al., 1996a, 2000; Posmantur, 1997). In studies of cerebral ischemia, Bartus and colleagues (1994) have reported that the systemic administration of calpain inhibitors reduced the overall degree of the ischemic alteration, with local cytoskeletal protection also reported in a contusional model of TBI (Posmanter et al., 1997). Saatman and colleagues (1996a) also demonstrated, in the context of traumatic brain injury, that the use of calpain inhibitors translated into improved behavioral outcome. However, this occurred without concomitant neuroprotection in terms of reduced contusional size or a blunting of apoptotic neuronal cell death (Saatman et al., 2000). Unfortunately, as Saatman and colleagues did not examine any potential reduction in the degree of axonal damage, the possibility remained that the observed behavioral protection resided in concomitant axonal sparing. In that we have already confirmed the presence of calpain-mediated axonal damage in well-characterized model of traumatic brain injury, it seemed reasonable and rational to revisit our animal models, now using inhibitors of calpain-mediated damage to determine if they significantly attenuate axonal damage. To this end, we used a rodent model of traumatic brain injury in concert with the administration of a cell-permeable peptidyl-aldehyde calpain inhibitor (MDL-28170) to determine whether this drug attenuates traumatically induced axonal damage. This approach was also initiated to provide further evidence of the physical role of calpainmediated damage in the pathogenesis of TAI while setting the stage for additional therapeutic studies on the utility of calpain inhibitors for the treatment of TAI. MATERIALS AND METHODS Drug Administration and Induction of Traumatic Brain Injury In this study we utilized the well-characterized rodent model of impact acceleration head injury described in detail elsewhere (Marmarou et al., 1994; Foda and Marmarou, 1994). In all, 12 Wistar rats (Charles River Hungary) weighing g were used for the experiments; three animals served as sham-injured controls. For the induction of anesthesia, the animals were ex- 262

97 CALPAIN INHIBITOR PREVENTS TRAUMATIC AXONAL INJURY posed to 4% isoflurane (Forane, Abbott, Hungary) in a bell jar for 5 min, and then intubated and ventilated with a mixture of 1 2% isoflurane in 30% O 2 and 70% N 2 O. A single tail vein bolus injection of 30 mg/kg MDL (courtesy of Aventis Pharmaceuticals) dissolved in 1 ml of the vehicle (PEG300/EtOH, 9:1) was administered 30 min prior to injury (n 5 4). The dosage and route of administration were selected based on pharmacokinetic studies described by Markgraf and colleagues (1998). Other rats were treated with a single tail vein bolus injection of the vehicle alone (n 5 5). Next, the skull between the coronal and lambdoid sutures was exposed with a midline incision. A metallic disc-shaped helmet of 10mm diameter was firmly glued to this point on the skull. The animal was placed in a prone position on a foam bed with the metallic helmet centered under the edge of a Plexiglas tube. The rat was prevented from falling by two belts secured to the foam pad. Brass weights weighing 450 g were allowed to fall from a height of 200 cm through the Plexiglas tube directly onto the metallic disc fixed to the animal s skull a setting that precludes cerebral contusion or subdural hemorrhage. Immediately after injury the animal was ventilated with 100% O 2. The helmet was removed and the skull was investigated for any sign of fracture which, if found, disqualified the animal from further evaluation. The scalp wound was sutured, with the animal remaining on artificial ventilation until the pre-determined survival of 120 min after injury. This relatively brief survival time was employed to optimize the usefulness of the antibodies described below, particularly those targeting the altered rod domains of the NFM subunits which yield the potential for false positives with increased survival (Povlishock et al. 1997). Physiological Assessments The respiratory status was monitored through pulse oximetry via the footpad and/or the ear. Additionally, brain temperature was monitored by a temporalis muscle probe, while core temperature was determined by a rectal probe. Immunohistochemistry At the designated survival time, the rats were re-anesthetized with an overdose of sodium pentobarbital and transcardially perfused with 4% paraformaldehyde in Millonig s buffer. Brains were removed and immersed in the same fixative overnight (16 18 h). On the basis of previously published observations concerning the topography of diffusely injured axons in rat brain (Koizumi and Povlishock, 1998; Okonkwo et al., 1999; Okonkwo and Povlishock, 1999), a midline, 5-min-wide block of the brain was removed using a sagittal brain blocking device to include the region extending from the interpeduncular fossa to the first cervical segment (World Precision Instruments, Sarasota, FL). The tissue was sectioned with Vibratome Series 1000 (Polysciences Inc., Warrington, PA) at a thickness of 40 mm and collected in 0.1 M phosphate buffer. The sections were collected in two groups in a semi-serial fashion, rinsed three times ten minutes in phosphate buffered saline (PBS), and processed for immunohistochemical localization of damaged axonal profiles. Half of the sections were single labeled for the detection of RMO-14 immunoreactivity. This antibody is known to exclusively target an epitope on the rod domains of altered NFM subunits, that are exposed upon modification of the NF sidearms, an assumed consequence of calcium induced enzymatic processes (Lee et al., 1987; Povlishock et al., 1997; Buki et al., 1999a; Okonkwo et al., 1998). Every second section was processed for immunohistochemical detection of the amyloid precursor protein (APP). This classical marker of TAI is carried by fast axonal transport and pools at foci affected by TAI (Gentleman et al., 1993; Sherriff et al., 1994; Stone et al., 1999). In our experiments we utilized a polyclonal antibody targeted to the C-terminus of the b-amyloid precursor protein. Characterization and detailed description of the advantages of this antibody were recently published by Stone et al. (2000). After washing in PBS, sections from both groups were incubated for 35 min with 0.2% Triton X (Sigma Chemical Co., St. Louis, MO [products of Sigma Chemical Co. were purchased via their Hungarian representative, Sigma Aldrich, Hungary, Budapest]) in 10% normal goat or horse serum, respectively (NGS or NHS) (Sigma) in PBS. After two quick rinses in PBS containing 1% NGS (or NHS in the case of RMO14) the above-defined groups of sections were incubated overnight in rabbit anti-app antibody (Zymed Laboratories, CT) at a dilution of 1:3000 or in mouse monoclonal RMO14 antibody at a dilution of 1:500 (kindly provided by Dr. John Q. Trojanowski, University of Pennsylvania Department of Pathology). After min washes in PBS containing 1% NGS (or NHS) sections were incubated in biotinylated anti-rabbit immunoglobulin derived from goat (diluted 1:200 in 1% NGS/PBS; Vector, Burlingame, CA [products of Vector were purchased via their Hungarian representative, Biomarker Ltd., Godollo]) or in 1:400 dilution of biotinylated, rat adsorbed anti-mouse immunoglobulin derived from horse (Vector) for 60 min followed by min rinses in PBS. After incubation in an avidin biotin peroxidase complex (ABC standard Elite kit, Vector, Burlingame, CA, at a dilution of 1:100) and rinsing in PBS and 0.1 M phosphate buffer min and min, respectively, sections were processed for visualiza- 263

98 BUKI ET AL. tion of the immunohistochemical complex using 0.05% diaminobenzidine (DAB; Sigma) and 0.01% hydrogen peroxide in 0.1 M phosphate buffer. Next the sections were mounted and cleared for routine light microscopic examination. Immunohistochemical Controls The employed antibodies have been well characterized and extensively used. However, control studies were performed to further assure specificity. To this end all immunocytochemical incubations included sections incubated without the addition of the primary or secondary antibodies. Image Analysis Semi-serial brainstem sections (six from each animal) were examined with a NIKON light microscope interfaced with a computer-assisted image analysis system (NIH 1.55) in a blinded fashion. At the pontomedullary junction, two adjacent grids of 40,000 mm 2 were superimposed over the corticospinal tracts (CSpT), images were captured and digitized at a magnification of 503 and the total number of damaged APP or RMO14 immunopositive axonal profiles within this area were marked and counted. In the same brainstem sections an adjacent image of the medial longitudinal fasciculus (MLF) was captured and digitized at 253, a 160,000 mm 2 grid was superimposed over the region of the MLF and the number of damaged axonal profiles within the grid were counted and expressed as density of APP or RMO- 14 immunopositive axons. For the purpose of image analyses each immunoreactive section was captured in a blinded fashion, with each section scanned to identify the plane of maximal immunoreactivity for counting. Only strongly positive immunoreactive profiles were included in the data analyses. Statistical Analysis For each antibody and region twelve fields of the CSpT, and six of the MLF where counted in each animal and the mean number of immunopositive axons was computed as number of immunopositive axons per mm 2. The mean number of immunopositive axons in both regions in vehicle treated animals (n 5 5) was compared with mean density in MDL treated rats (n 5 4) with an independent samples t-test. Differences were assumed significant at a level of p # RESULTS In accordance with previous observations from our laboratories and others the use of the above-described experimental protocols did not result in any significant alteration in the physiological parameters monitored. Light microscopic examination of vehicle and drug treated animals subjected to TBI and reacted for the visualization of the APP and RMO-14 antibodies showed discrete immunocytochemical reaction products within FIG. 1. Traumatically injured axons displaying APP immunoreactivity indicating axonal injury (disturbed axonal transport) in light micrographs of thecorticospinal tract (A,B) and medial longitudinal fasciculus (C,D) from injured animals. Note that the number of immunoreactive damaged axons appears dramatically reduced in the MDL treated (B,D) compared to the vehicle-treated (A,C) sections. (Bar indicates 50 mm.) 264

99 CALPAIN INHIBITOR PREVENTS TRAUMATIC AXONAL INJURY scattered axons localized in the CSPT and MLF. Morphological characteristics of the swollen, occasionally disconnected APP-IR-axonal segments and the lobulated/vacuolated, partially or totally disconnected RMO- 14 IR-axonal segments were entirely consistent with previous descriptions of damaged axonal profiles 2 h post-injury (Stone et al., 1999; Buki et al., 1999a,c). The sham-injured animals displayed no immunoreactive axonal profiles. In the MDL treated group (Figs. 1B,D and 2B,D), both the localization and the appearance of APP and the RMO-14 immunopositive axonal profiles were similar to that observed in the vehicle treated group (Figs. 1A,C and 2A,C); however, now the number of the damaged, immunopositive axonal profiles appeared dramatically decreased. Digital acquisition followed by statistical analysis confirmed that preinjury administration of MDL significantly reduced the mean number of damaged RMO- 14-IR axonal profiles in both the corticospinal tract (CSpT) (from RMO-IR (mean 6 SEM, /mm 2 ) to RMO-IR (t , df 5 7, p, 0.02) and the medial longitudinal fasciculus (MLF; from RMO-IR to RMO-IR (t , df 5 7, p, 0.03); Figs. 3 and 4). Similarly, pre-injury administration of MDL significantly reduced the mean numbers of damaged APP-IR axonal profiles both in the brainstem in the CSpT (from APP- IR to APP-IR (t , df 5 7, p, 0.03)) and at the MLF (from APP-IR to APP- IR (t , df 5 7, p, 0.01); Figs. 3 and 4). DISCUSSION In the present study, we have demonstrated that preinjury administration of cell permeable calpain-inhibitor MDL is capable of significantly reducing traumatically evoked axonal injury as assessed by the immunocytochemical markers APP and RMO-14, both of which target specific features of TAI. Not only do these studies strongly support the potential utility of calpaininhibitors in blunting the progression of traumatically induced axonal injury, but also they provide further evidence of the role of calcium-induced, calpain-mediated structural proteolysis in traumatic brain injury as previously suggested through multiple lines of evidence in animals (Pike et al., 2001; Kampfl et al., 1997; Saatman et al., 1996b), and in man (McCracken et al., 1999). While the present studies did not utilize parallel ultrastructural analyses to define the precise sub-cellular targets of the chosen calpain inhibitor, the study s use of two different antibodies targeting specific features of traumatic brain injury provides insight to its potential mode of action. As it is recognized that RMO-14 immunoreactivity is associated with neurofilament compaction secondary to cleavage of the neurofilament sidearm (Povlishock et al., 1997), the observed reduction in the immunreactive prod- FIG. 2. Traumatically injured axons displaying RMO-14 immunoreactivity indicating axonal injury (neurofilament-compaction) in light micrographs of corticospinal tract (A,B) and medial longitudinal fasciculus (C,D) from injured animals. Note that the number of immunoreactive damaged axons appears dramatically reduced in the MDL treated (B,D) compared to the vehicle-treated (A,C) sections. (Bar indicates 50 mm.) 265

100 BUKI ET AL. FIG. 3. Bar graph of the mean numbers of immunopositive axons displaying APP and RMO-14 immunoreactivity in the corticospinal tracts (CSpT) of vehicle and MDL treated animals subjected to impact acceleration injury. Black lines represent standard error of mean, asterisks indicate a statistically significant difference. uct clearly speaks to the utility of the chosen calpain inhibitor in blunting the calpain-mediated proteolytic cleavage of the neurofilament sidearms. Similarly, the overall reduction in the APP immunreactive axonal swellings most likely speaks to a further attenuation of the calpainmediated processes. As alluded to previously, the traumatically induced intraaxonal rise in intraaxonal calcium, coupled with the increase in calpain proteolytic activity, contributes to microtubular loss and dispersion, disrupting axonal transport kinetics, with the net effect of axonal transport impairment (Pettus and Povlishock, 1996). This leads to upstream intraaxonal swelling with the accumulation of anterogradely transported products such as APP. The attenuation of this APP accumulation via use of a calpain inhibitor speaks to protective effect that this agent has in preventing calpain-linked microtubular disassembly and alterations in axonal transport. Until recently, it was assumed that the above described markers APP and RMO-14, were co-localized in the same axonal population. However, it is now understood that, in some cases, these markers can co-localize whereas in other cases, they may identify different populations of axons, with the RMO-14 fibers identifying more severely injured axons, with the APP immunreactivity confined to those axons sustaining less severe insult (Stone et al., 2001). While in the current communication, we have made no direct RMO-14/APP colocalization, their change parallels that described in our recent publication (Stone et al., 2001), thereby confirming the utility of the calpain-inhibitors against a broad spectrum of traumatic axonal injury. The results of the current communication are novel in the context of traumatically induced axonal injury, yet, they do join a relatively large volume of literature suggesting the utility of calpain inhibitors in various central nervous system disorders. Calpain-inhibitors have proven efficacious in reducing brain ischemic injury (Markgraf et al., 1998; Bartus et al., 1994; Hong et al., 1994; Lee et al., 1997) and spinal cord injury (Banik et al., 1998). The studies reported herein also extend other work conducted in traumatic brain injury wherein the use of calpain-inhibitors was observed to improve behavioral outcome although it did not exert neuroprotection in terms of contusional volume reduction and/or apoptotic change. As noted in the introduction to this manuscript, the currently observed axonal protection may constitute a major substrate of behavioral protection described in these original studies of Saatman and colleagues (1996a; 2000), a point which requires further study. The major limitation of the current investigation resides in the fact that the results rely on the use of a single pre-injury injection strategy, which was mandated by the limited availability of the chosen drug. Ideally, to demonstrate potential pre-clinical relevance, it would have been of interest to observe the axonal protection afforded by calpain-inhibitor delivered in a delayed, postinjury injection schedule. Based upon our published work FIG. 4. Bar graph of the mean numbers of immunopositive axons displaying APP and RMO-14 immunoreactivity in the medial longitudinal fasciculus (MLF) of vehicle and MDL treated animals subjected to impact acceleration injury. Black lines represent standard error of mean, asterisks indicate statistically significant difference. 266

101 CALPAIN INHIBITOR PREVENTS TRAUMATIC AXONAL INJURY looking at calpain-mediated spectrin proteolysis (Buki et al., 1999a), we would assume that the window of opportunity for maximal protection would reside within the first hour or two postinjury, however, these issues require further study. Another issue that must be addressed concerns the fact that while compelling, the present study does not provide direct evidence that calpain inhibition attenuates TAI. It is known that MDL is not solely calpain specific. Further, as the antibodies used did not specifically recognize axonal breakdown products generated by calpain activation, some caution must be exercised to preclude over interpretation of the data. ACKNOWLEDGMENTS We thank Aventis Pharmaceuticals for the kind donation of the MDL-28170, John Trojanowski for the RMO- 14 antibody, and Carrie G. Markgraf for her excellent technical advices. We also thank Csabáné Andok, Andrásné Nádor, and József Nyirádi for their technical assistance as well as Carolyn Davis and Susan Walker for their continuous support. Grants of the Hungarian Science Foundation (OTKA T , ETT), Bólyai Scholarship of the Hungarian Academy of Sciences and the Fogarty International Research Collaboration Award (1- RO3-TW A1) and NIH Grant NS20193 supported this work. REFERENCES ADAMS, H., MITCHELL, D.E., GRAHAM, D.I., et al. (1977). Diffuse brain damage of immediate impact type. Its relationship to primary brain-stem damage in head injury. Brain 100, ADAMS, J.H., GRAHAM, D.I., SCOTT, G., et al. (1980). Brain damage in fatal non-missile head injury. J. Clin. Pathol. 33, BANIK, N.L., SHIELDS, D.C., RAY, S., et al. (1998). Role of calpain in spinal cord injury: effects of calpain and free radical inhibitors. Ann. N.Y. Acad. Sci. 844, BARTUS, R.T., HAYWARD, N.J., ELLIOTT, P.J., et al. (1994). Calpain inhibitor AK295 protects neurons from focal brain ischemia. Effects of postocclusion intra-arterial administration. Stroke 25, BLUMBERGS, P.C., JONES, N.R., and NORTH, J.B. (1989). Diffuse axonal injury in head trauma. J. Neurol. Neurosurg. Psychiatry 52, BUKI, A., KOIZUMI, H., and POVLISHOCK, J.T. (1999b). Moderate posttraumatic hypothermia decreases early calpainmediated proteolysis and concomitant cytoskeletal compromise in traumatic axonal injury. Exp. Neurol. 159, BUKI, A., OKONKWO, D.O., and POVLISHOCK, J.T. (1999c). Postinjury cyclosporin A administration limits axonal damage and disconnection in traumatic brain injury. J. Neurotrauma 16, BUKI, A., OKONKWO, D.O., WANG, K.K., et al. (2000). Cytochrome c release and caspase activation in traumatic axonal injury. J. Neurosci. 20, BUKI, A., SIMAN, R., TROJANOWSKI, J.Q., et al. (1999a). The role of calpain-mediated spectrin proteolysis in traumatically induced axonal injury. J. Neuropathol. Exp. Neurol. 58, FODA, M.A., and MARMAROU, A. (1994). A new model of diffuse brain injury in rats. Part II: Morphological characterization. J. Neurosurg. 80, GENNARELLI, T.A., THIBAULT, L.E., ADAMS, J.H., et al. (1982). Diffuse axonal injury and traumatic coma in the primate. Ann. Neurol. 12, GENTLEMAN, S.M., NASH, M.J., SWEETING, C.J., et al. (1993). Beta amyloid precursor protein (beta-app) as a marker for axonal injury after head injury. Neurosci. Lett. 160, HONG, S.C., GOTO, Y., LANZINO, G., et al. (1994). Neuroprotection with a calpain inhibitor in a model of focal cerebral ischemia. Stroke 25, KAMPFL, A., POSMANTUR, R.M., ZHAO, X., et al. (1997). Mechanisms of calpain proteolysis following traumatic brain injury: implications for pathology and therapy: implications for pathology and therapy: a review and update. J. Neurotrauma 14, KOIZUMI, H., and POVLISHOCK, J.T. (1998). Posttraumatic hypothermia in the treatment of axonal damage in an animal model of traumatic axonal injury. J. Neurosurg. 89, LEE, K.S., YANAMOTO, H., FERGUS, A., et al. (1997). Calcium-activated proteolysis as a therapeutic target in cerebrovascular disease. Ann. N.Y. Acad. Sci. 825, LEE, V.M., CARDEN, M.J., SCHLAEPFER, W.W., et al. (1987). Monoclonal antibodies distinguish several differentially phosphorylated states of the two largest rat neurofilament subunits (NF-H and NF-M) and demonstrate their existence in the normal nervous system of adult rats. J. Neurosci. 7, MARKGRAF, C.G., VELAYO, N.L., JOHNSON, M.P., et al. (1998). Six-hour window of opportunity for calpain inhibition in focal cerebral ischemia in rats. Stroke 29, MARMAROU, A., FODA, M.A., VAN DEN BRINK, W., et al. (1994). A new model of diffuse brain injury in rats. Part I: Pathophysiology and biomechanics. J. Neurosurg. 80,

102 BUKI ET AL. MAXWELL, W.L., POVLISHOCK, J.T., and GRAHAM, D.L. (1997). A mechanistic analysis of nondisruptive axonal injury: a review. J. Neurotrauma 14, MCCRACKEN, E., HUNTER, A.J., PATEL, S., et al. (1999). Calpain activation and cytoskeletal protein breakdown in the corpus callosum of head-injured patients. J. Neurotrauma 16, MEANEY, D. F., MARGULIES, S.S., and SMITH, D. H. (2000). Diffuse axonal injury. J. Neurosurg. 95, OKONKWO, D.O., BUKI, A., SIMAN, R., et al. (1999). Cyclosporin A limits calcium-induced axonal damage following traumatic brain injury. Neuroreport 10, OKONKWO, D.O., PETTUS, E.H., MOROI, J., et al. (1998). Alteration of the neurofilament sidearm and its relation to neurofilament compaction occurring with traumatic axonal injury. Brain Res. 784, 1 6. OKONKWO, D.O., and POVLISHOCK, J.T. (1999). An intrathecal bolus of cyclosporin A before injury preserves mitochondrial integrity and attenuates axonal disruption in traumatic brain injury. J. Cereb. Blood Flow Metab. 19, PETTUS, E.H., CHRISTMAN, C.W., GIEBEL, M.L., et al. (1994). Traumatically induced altered membrane permeability: its relationship to traumatically induced reactive axonal change. J. Neurotrauma 11, PETTUS, E.H., and POVLISHOCK, J.T. (1996). Characterization of a distinct set of intra-axonal ultrastructural changes associated with traumatically induced alteration in axolemmal permeability. Brain Res. 722, PIKE, B.R., FLINT, J., DUTTA, S., et al. (2001). Accumulation of non-erythroid alpha II-spectrin and calpain-cleaved alpha II-spectrin breakdown products in cerebrospinal fluid after traumatic brain injury in rats. J. Neurochem. 78, PIKE, B.R., ZHAO, X., NEWCOMB, J.K., et al. (1998). Regional calpain and caspase-3 proteolysis of alpha-spectrin after traumatic brain injury. Neuroreport 9, POSMANTUR, R., KAMPFL, A., SIMAN, R., et al. (1997). A calpain inhibitor attenuates cortical cytoskeletal protein loss after experimental traumatic brain injury in the rat. Neuroscience 77, POVLISHOCK, J.T. (1992). Traumatically induced axonal injury: pathogenesis and pathobiological implications. Brain Pathol. 2, POVLISHOCK, J.T., BECKER, D.P., CHENG, C.L., et al. (1983). Axonal change in minor head injury. J. Neuropathol. Exp. Neurol. 42, POVLISHOCK, J.T., ERB, D.E., and ASTRUC, J. (1992). Axonal response to traumatic brain injury: reactive axonal change, deafferentation, and neuroplasticity. J. Neurotrauma 9, S189 S200. POVLISHOCK, J.T., MARMAROU, A., MCINTOSH, T., et al. (1997). Impact acceleration injury in the rat: evidence for focal axolemmal change and related neurofilament sidearm alteration. J. Neuropathol. Exp. Neurol. 56, SAATMAN, K.E., BOZYCZKO-COYNE, D., MARCY, V., et al. (1996b). Prolonged calpain-mediated spectrin breakdown occurs regionally following experimental brain injury in the rat. J. Neuropathol. Exp. Neurol. 55, SAATMAN, K.E., MURAI, H., BARTUS, R.T., et al. (1996a). Calpain inhibitor AK295 attenuates motor and cognitive deficits following experimental brain injury in the rat. Proc. Natl. Acad. Sci. U.S.A. 93, SAATMAN, K.E., ZHANG, C., BARTUS, R.T., et al. (2000). Behavioral efficacy of posttraumatic calpain inhibition is not accompanied by reduced spectrin proteolysis, cortical lesion, or apoptosis. J. Cereb. Blood Flow Metab. 20, SHERRIFF, F.E., BRIDGES, L.R., GENTLEMAN, S.M., et al. (1994). Markers of axonal injury in post mortem human brain. Acta Neuropathol. (Berl.) 88, STONE, J.R., SINGLETON, R.H., and POVLISHOCK, J.T. (2000). Antibodies to the C-terminus of the beta-amyloid precursor protein (APP): a site specific marker for the detection of traumatic axonal injury. Brain Res. 871, STONE, J.R., SINGLETON, R.H., and POVLISHOCK, J.T. (2001). Intra-axonal neurofilament compaction does not evoke local axonal swelling in all traumatically injured axons. Exp. Neurol. 172, STONE, J.R., WALKER, S.A., and POVLISHOCK, J.T. (1999). The visualization of a new class of traumatically injured axons through the use of a modified method of microwave antigen retrieval. Acta Neuropathol. (Berl.) 97, STRICH, S.J. (1956). Diffuse degeneration of the cerebral white matter in severe dementia following head injury. J. Neurol. Neurosurg. Psychiatry 19, WOLF, J.A., STYS, P. K., LUSARDI T., et al. (2001). Traumatic Axonal Injury induces calcium influx modulated by tetrodotoxin-sensitive sodium channels. J. Neurosci. 21, Address reprint requests to: John T. Povlishock, Ph.D. VCU Neuroscience Center Medical College of Virginia Campus Virginia Commonwealth University P.O. Box E. Marshall St. Richmond, VA jpovlish@hsc.vcu.edu 268

103 Regulatory Peptides 123 (2004) Effects of pituitary adenylate cyclase activating polypeptide in a rat model of traumatic brain injury Orsolya Farkas a, Andrea Tamás b, *, Andrea Zsombok a,c,dóra Reglo}di b,józsef Pál a, Andras Büki a, István Lengvári b, John T. Povlishock d, Tamás Dóczi a a Department of Neurosurgery, University of Pécs, Medical Faculty, Hungary b Department of Anatomy (Neurohumoral Regulations Research Group of the Hungarian Academy of Sciences), University of Pécs, Medical Faculty, Szigeti u 12, Pécs 7624, Hungary c Department of Central Laboratory of Animal Research, University of Pécs, Medical Faculty, Hungary d Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, VA, USA Available online 28 July 2004 Abstract Pituitary adenylate cyclase activating polypeptide (PACAP) is a widely distributed neuropeptide that has numerous different actions. Recent studies have shown that PACAP exerts neuroprotective effects not only in vitro but also in vivo, in animal models of global and focal cerebral ischemia, Parkinson s disease and axonal injuries. Traumatic brain injury has an increasing mortality and morbidity and it evokes diffuse axonal injury which further contributes to its damaging effects. The aim of the present study was to examine the possible neuroprotective effect of PACAP in a rat model of diffuse axonal injury induced by impact acceleration. Axonal damage was assessed by immunohistochemistry using an antiserum against beta-amyloid precursor protein, a marker of altered axoplasmic transport considered as key feature in axonal injury. In these experiments, we have established the dose response curves for PACAP administration in traumatic axonal injury, demonstrating that a single post-injury intracerebroventricular injection of 100 Ag PACAP significantly reduced the density of damaged, beta-amyloid precursor protein-immunoreactive axons in the corticospinal tract. D 2004 Elsevier B.V. All rights reserved. Keywords: Neuroprotection; Traumatic brain injury; Beta-amyloid precursor protein; Corticospinal tract; Traumatic axonal injury 1. Introduction Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the vasoactive intestinal peptide (VIP)/secretin/glucagon peptide family [1]. PACAP was discovered as a hypothalamic peptide on its potential of increasing adenylate cyclase activity in the pituitary gland. Since its discovery, various distinct effects in the central and peripheral nervous systems have been described [2 4]. Among them, PACAP has neurotrophic and neuroprotective actions [2 4]. Numerous studies have proven its neuroprotective effects in vitro, where PACAP protects neurons against different neurotoxic agents [5 10]. A * Corresponding author. Tel.: /5398; fax: address: andrea.tamas@aok.pte.hu (A. Tamás). few studies have shown that this effect is also present in vivo, in optic nerve axotomy, spinal cord injury, facial nerve transection, and 6-hydroxydopamine-induced lesion of the substantia nigra [11 14]. Also, PACAP has neuroprotective effects in animal models of global and focal ischemia [15 17]. Although cerebral ischemia and traumatic brain injury are triggered by different events, the majority of cellular events leading eventually to death of neurons are shared by both ischemic and traumatic brain injuries [18 21]. These include excitotoxicity, nitric oxide, inflammation, reactive oxygen species, elevated Ca levels and apoptosis [18 21]. The presence of these common pathways suggests that those interventions that target the consensus pathways of these two major diseases of the central nervous system should be effective in both types of brain injury. Based on the several lines of evidence on the neuroprotective effects of PACAP in cerebral ischemia, /$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi: /j.regpep

104 70 O. Farkas et al. / Regulatory Peptides 123 (2004) the aim of the present study was to investigate the possible protective effect of this neuropeptide in a rat model of traumatic brain injury. 2. Methods 2.1. Induction of traumatic brain injury Adult Wistar rats ( g) were subjected to impact acceleration traumatic brain injury in accordance with previous descriptions [22,23]. Animals were anesthetized in a bell jar for 5 min under 4% isoflurane and a 2:1 mixture of N 2 O/O 2, endotracheally intubated and maintained under 1.5 2% isoflurane. Rectal and temporal temperatures were monitored and maintained at 37 jc with a ventral heating pad. A midline scalp incision was made and the skull was exposed with blunt dissection. Parietal bones were exposed, cleaned and dried with bone wax. A 10-mm stainless steel disc helmet was secured with dental acrylic to the skull between bregma and lambda. Rats were then fastened prone to a foam bed beneath the injury tower and injured from 2 m with a 450 g weight. After injury, animals were immediately ventilated with 100% O 2, the helmet was removed, and the skull examined for sign of fracture, which, if found, disqualified the rat from further evaluation. Selected animals underwent monitoring of other physiological parameters including invasive blood pressure, pulse oxymetry and arterial blood gas analysis Drug administration In the first study, a single bolus of 125 Ag/kg PACAP dissolved in physiological saline (n =11) or the same volume of vehicle in control animals (n = 13) was given intravenously before the induction of traumatic brain injury. The dose of PACAP was selected based on previous studies [16]. These animals were sacrificed 2 h (n = 5 and 7 in PACAP-treated and control groups, respectively), or 6 h(n = 6 in both groups) after brain injury. Since intravenous PACAP pre-treatment did not prove to be effective (see Results), intravenous administration as a post-treatment did not seem reasonable. Due to the failure of the intravenous treatment, PACAP was administered intracerebroventricularly in the second study. This model of traumatic brain injury requires intact skull; therefore, administration was started post-injury. Animals received 1 Ag (n = 8), 10 Ag (n = 11) 100 Ag (n = 13) PACAP, or vehicle (n = 16) intracerebroventricularly. Sham-injured rats (n = 4) were anesthetized, intubated and the parietal bones were exposed without performing the impact acceleration injury. In the first experiment, no significant increase of immunopositive axonal profiles could be observed 6 h after injury compared to the results at 2 h; therefore, histological assessment was done only 2 h after the induction of traumatic brain injury or sham operation in the second study Immunohistochemistry At the designed survival time, animals were reanesthetized and transcardially perfused with Zamboni s fixative [24]. The brains were immersed in the same fixative overnight (16 18 h). Based upon previously published observations concerning the topography of diffusely injured axons in the rat brainstem [25], a median 5 mm wide block of the brain was removed using a sagittal brain blocking device (Braintree Scientific) to include the region extending from the interpeduncular fossa to the first cervical segment. The brainstem was blocked just lateral to the pyramids to encompass known vulnerable fiber tracts in this model of traumatic brain injury: the corticospinal tract and the medial longitudinal fascicle. Immunohistochemistry was used to detect axonal damage. In the present study, we utilized immunostaining with a polyclonal antiserum targeting the C-terminus of betaamyloid precursor protein, a marker of altered axoplasmic transport [26,27]. Vibratome sections were cut serially at 40 Am and microwaved for optimizing antigenicity with the retention of excellent ultrastructural detail according to former publications [28]. Sections were then washed in phosphate-buffered saline (PBS) containing 1% normal goat serum (Fluca), and were incubated overnight in rabbit anti-beta-amyloid precursor protein antibody at a dilution of 1:3000 (Zymed). Having been rinsed in PBS, sections were incubated in biotinylated anti-rabbit immunoglobulin (Sigma) for 60 min. After incubation in avidin biotin peroxidase complex (ABC kit, Vector) and rinsing, sections were processed for visualization of the immunohistochemical complex using 0.05% diaminobenzidine and 0.01% hydrogen peroxide in 0.1 M PBS. The sections were then mounted and cleared for routine light microscopic examination Image analysis Damaged immunopositive axon profiles in the area of the corticospinal tract and the medial longitudinal fascicle were captured and digitized at a magnification of 50. The total number of damaged, immunoreactive axons were counted using an NIH Image J software in a blinded fashion. Student s t-test was used to compare the density of immunopositive profiles (expressed as number/mm 2 ) between the control and PACAP-treated animals. 3. Results In accordance with previous observations from our laboratories and others, the use of the above-described experimental protocols did not result in any significant

105 O. Farkas et al. / Regulatory Peptides 123 (2004) alteration in the physiological parameters monitored during the experiment. Light microscopic examination of the animals subjected to traumatic brain injury and reacted for the visualization of beta-amyloid precursor protein antibody revealed discrete focal immunoreactivity within scattered axons in the corticospinal tract and medial longitudinal fascicle. Sham-injured animals did not display immunoreactive axonal profiles (Fig. 1A). Morphological characteristics of swollen, occasionally disconnected immunoreactive axonal segments appeared entirely consistent with previous descriptions of damaged axonal profiles 2 6 h post-injury (Fig. 1B) [21,26,29]. There was no significant difference between the number of immunopositive axonal profiles 2 or 6 h after the injury. Results of pre-injury intravenous PACAP administration revealed that the mean densities of beta-amyloid precursor protein-immunopositive axon profiles in PACAP-treated animals in the corticospinal tract and medial longitudinal fascicle were not significantly different from those in control animals at either 2 or 6 h post-injury (Fig. 1C). When PACAP was administered intracerebroventricularly after the induction of traumatic brain injury, treatment with 100 Ag PACAP resulted in a significant reduction of beta-amyloid precursor protein-immunopositive axon profiles in the corticospinal tract when compared to control animals 2 h postinjury (Figs. 1F and 2A). Treatment with lower doses of PACAP (1 and 10 Ag) did not result in significant reduction of damaged axons (Figs. 1D,E and 2A). In animals treated with 1 Ag PACAP, even an elevation in immunopositive axon profiles could be observed, which was not significantly different from control animals, but significantly higher when compared to animals treated with 100 Ag PACAP. In contrast to the corticospinal tract, no statistical significance A B C D E F Fig. 1. Representative beta-amyloid precursor protein-immunostained sections of the corticospinal tract from a sham-operated animal (A), a control rat (B), an animal receiving pre-injury intravenous PACAP (C), and animals treated with 1 Ag (D), 10 Ag (E) and 100 Ag (F) PACAP. The number of arrows (indicating some of the immunopositive axon profiles) is proportional to the mean densities of immunostained axon profiles.

106 72 O. Farkas et al. / Regulatory Peptides 123 (2004) Fig. 2. Mean densities of beta-amyloid precursor protein (APP)-immunoreactive axon profiles in the corticospinal tract (A) and medial longitudinal fascicle (B) of control animals and those treated with different doses of PACAP. *P < 0.05 vs. control group. could be established between the various treatment groups in the medial longitudinal fascicle (Fig. 2B). 4. Discussion In the present study we demonstrated that post-traumatic treatment with high dose of PACAP significantly reduced the density of damaged axons in the most vulnerable corticospinal tract. Our results showing that PACAP is neuroprotective in vivo are in accordance with numerous other studies that prove protective effects of this pleiotropic neuropeptide. VIP, which shows close homology to PACAP, has also been shown to exert neuroprotective effects in various in vitro and in vivo systems [30 33]. Soon after its discovery of PACAP, several studies demonstrated that PACAP also has neurotrophic actions in vitro, enhancing survival and neurite outgrowth of cultured neuroblasts [4,34,35]. Later, it was demonstrated that PACAP is able to protect neurons against various neurotoxic agents. Its protective effects have been shown against glutamate-induced neurotoxicity in cortical and retinal neurons [5,6,8], in PC12 cells against betaamyloid-induced toxicity [7], against lipopolysaccharideinduced toxicity in cortical neurons [9] and against HIV envelope protein in PC12 cells [4]. A few studies have demonstrated that PACAP is able to exert neuroprotection in vivo, in animal models of various brain pathologies. PACAP shows neuroprotection in rat global cerebral ischemia [15] and focal ischemia in rats and mice [16,17], in facial nerve injury [12] and in optic nerve transection [13]. Considering traumatic injuries, there are only a few studies that indicate the potentially protective effect of PACAP. In a rat model of spinal cord injury, induced by extradural static weight compression, post-injury PACAP treatment significantly reduced the number of apoptotic cells assessed by TUNEL staining in the damaged spinal cord [11]. Pro-inflammatory cytokines are expressed in

107 O. Farkas et al. / Regulatory Peptides 123 (2004) spinal cord within 1 2 h after traumatic injury, and this temporal profile of cytokine production is mimicked in spinal cord slices, where PACAP has been shown to eliminate the increase of tumor necrosis factor after spinal cord transection [36]. In a model of traumatic brain injury, similar to the one used in our study, PACAP mrna was induced after injury, and the temporal profile of its upregulation paralleled the decrease in the number of apoptotic cells [37]. These studies indicate that PACAP is a promising therapeutic agent in traumatic brain and spinal cord injuries which is further supported by our observations. The exact mechanism underlying the in vivo neuroprotective effect of PACAP has not been clarified so far. In vitro studies however indicate that various effects can play a role, including antiapoptotic and anti-inflammatory actions. The antiapoptotic effects of PACAP have been proven in different neuronal cultures in control conditions and against several apoptosis-inducing agents [10,38 40]. In cerebellar granule cells, PACAP efficiently reduces caspase-3 activation, the final effector of apoptotic cell death, which is activated also in traumatic brain injury [21,39]. Inflammation accompanies and further aggravates neuronal injury in most brain pathologies, including traumatic brain injury [18]. PACAP is a potent inactivator of induced microglial release of pro-inflammatory cytokines and nitric oxide [9,36,41]. Mitochondrial damage has long been associated with the pathogenesis of traumatic brain injury [20]. The mitochondrial failure caused by calciuminduced opening of the permeability transition pore is a key phenomenon in multiple neurological insults, including axonal injury [21,42]. Aconitase is a key mitochondrial enzyme influencing the viability of neurons, and it is inactivated upon mitochondrial calcium influx. Recently, it has been shown that the inactivation of aconitase is attenuated by PACAP [43]. We attempted to show the possible protective effect of PACAP as an intravenous injection because this route of administration is clinically more relevant. As far as the preinjury treatment is concerned, we followed the generally used routine to test candidate neuroprotective drugs first as pre-treatment. Several examples show that pre- and posttreatments can be equally effective [29,44] in models of brain injuries. Due to the failure of intravenous PACAP treatment given pre-injury, giving PACAP as intravenous post-treatment did not seem reasonable. The failure may be due to the low concentration reaching the brainstem. PACAP crosses the blood brain barrier relatively well in comparison with other neuropeptides both in the normal and injured brain [45,46]. However, results of the regional differences in brain transport revealed that the transport rate of PACAP is lower or not measurable in the area examined in our study (corticospinal tract and medial longitudinal fascicle), in contrast to other brain areas [47]. Furthermore, recent studies have demonstrated that intravenous efficacy of PACAP requires continuous infusion an observation explained by the presence of a serum-binding protein [48,49]. To this end, in a model of focal cerebral ischemia, a single injection was not effective; nevertheless, continuous intravenous infusion significantly attenuated the ischemic brain damage [16]. Due to the failure of intravenous administration of PACAP, we chose to administer PACAP intracerebroventricularly, where significant neuroprotection could be achieved by high concentrations of PACAP. Several studies have proven the dose dependence of the neuroprotective effect of PACAP [6,7]. It has also been postulated that different doses of PACAP act through different signal transduction pathways [50,51]. Our results indicate that relatively high concentration is required to achieve significant neuroprotection in this rodent model of diffuse traumatic brain injury. Although most of the in vivo studies have shown protective effects of PACAP at lower doses [14 16,38], our results are in accordance with some other studies where neuroprotection by PACAP was reached by relatively high concentrations, such as in spinal cord injury [11], in optic nerve transection [13] and in facial nerve injury [12]. According to these few in vivo studies, it seems that protective effects can be reached by only higher doses of PACAP in traumatic injuries. No data are available on the passage of PACAP from the cerebrospinal fluid to the brain; therefore, the exact tissue concentration reached in our study is not known. It is possible, however, that the passage of PACAP to the brainstem is relatively low similarly to the low transport rate across the blood brain barrier in this area [46]. This may also explain why higher concentrations were required to reach significant neuroprotection in the corticospinal tract. Further examination of the exact dose dependence and therapeutic window is reasonable in models of traumatic brain injury, where our observations suggest a promising therapeutic value for PACAP. Acknowledgements This work was supported by the National Science Research Fund (OTKA T034491/046589, T035195, ETT), Fogarty IRCA (1-RO3-TW A1), NIH Grant NS20193, National Science Projects NFKP1A/00026/2002 and ALK /2002, the Hungarian Academy of Sciences, Bolyai and Bekesy Scholarships. References [1] Miyata A, Arimura A, Dahl RR, Minamino N, Uehara A, Jiang L, et al. Isolation of a novel 38-residue hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells. 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109 O. Farkas et al. / Regulatory Peptides 123 (2004) [43] Tabuchi A, Funaji K, Nakatsubo J, Fukuchi M, Tsuchiya T, Tsuda M. Inactivation of aconitase during apoptosis of mouse cerebellar granule neurons induced by a deprivation of membrane depolarization. J Neurosci Res 2003;71: [44] Okonkwo DO, Buki A, Siman R, Povlishock JT. Cyclosporin A limits calcium-induced axonal damage following traumatic brain injury. NeuroReport 1999;10: [45] Banks WA, Kastin AJ, Komaki G, Arimura A. Passage of pituitary adenylate cyclase activating polypeptide 1 27 and pituitary adenylate cyclase activating polypeptide 1 38 across the blood brain barrier. J Pharmacol Exp Ther 1993;267: [46] Somogyvári-Vigh A, Pan W, Reglo}di D, Vigh S, Kastin AJ, Arimura A. The passage of pituitary adenylate cyclase activating polypeptide across the blood brain barrier during focal cerebral ischemia. Regul Pept 2000;91: [47] Nonaka N, Banks WA, Mizushima H, Shioda S, Morley JE. Regional differences in PACAP transport across the blood brain barrier in mice: a possible influence of strain, amyloid h protein and age. Peptides 2002;23: [48] Somogyvári-Vigh A, Svoboda-Teet J, Vigh S, Arimura A. Is an intravenous bolus injection required prior to initiating slow intravenous infusion of PACAP38 for prevention of neuronal death induced by global ischemia? The possible presence of a binding protein for PACAP38 in blood. Ann N Y Acad Sci 1998;865: [49] Tams JW, Johnsen AH, Fahrenkrug J. Identification of pituitary adenylate cyclase activating polypeptide 1 38-binding factor in human plasma, as ceruloplasmin. Biochem J 1999;341: [50] Arimura A. Perspectives on the development of a neuroprotective drug based on PACAP. Regul Pept 2003;115:40 [abstract]. [51] Zhou CJ, Shioda S, Yada T, Inagaki N, Pleasure SJ, Kikuyama S. PACAP and its receptors exert pleiotropic effects in the nervous system by activating multiple signaling pathways. Curr Prot Pept Sci 2002;3:

110 JOURNAL OF NEUROTRAUMA Volume 23, Number 5, 2006 Mary Ann Liebert, Inc. Pp Postinjury Administration of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Attenuates Traumatically Induced Axonal Injury in Rats ANDREA TAMÁS, 1 ANDREA ZSOMBOK, 2,3 ORSOLYA FARKAS, 2 DÓRA REGLÖDI, 1 JÓZSEF PÁL, 2,5 ANDRÁS BÜKI, 2 ISTVÁN LENGVÁRI, 1 JOHN T. POVLISHOCK, 4 and TAMÁS DÓCZI 2,5 ABSTRACT Pituitary adenylate cyclase activating polypeptide (PACAP) has several different actions in the nervous system. Numerous studies have shown its neuroprotective effects both in vitro and in vivo. Previously, it has been demonstrated that PACAP reduces brain damage in rat models of global and focal cerebral ischemia. Based on the protective effects of PACAP in cerebral ischemia and the presence of common pathogenic mechanisms in cerebral ischemia and traumatic brain injury (TBI), the aim of the present study was to investigate the possible protective effect of PACAP administered 30 min or 1 h postinjury in a rat model of diffuse axonal injury. Adult Wistar male rats were subjected to impact acceleration, and PACAP was administered intracerebroventricularly 30 min (n 4), and 1 h after the injury (n 5). Control animals received the same volume of vehicle at both time-points (n 5). Two hours after the injury, brains were processed for immunhistochemical localization of damaged axonal profiles displaying either amyloid precursor protein ( -APP) or RMO-14 immunoreactivity, both considered markers of specific features of traumatic axonal injury. Our results show that treatment with PACAP (100 g) 30 min or 1 h after the induction of TBI resulted in a significant reduction of the density of -APP immunopositive axon profiles in the corticospinal tract (CSpT). There was no significant difference between the density of -APP immunopositive axons in the medial longitudinal fascicle (MLF). PACAP treatment did not result in significantly different number of RMO-14 immunopositive axonal profiles in either brain areas 2 hours post-injury compared to normal animals. While the results of this study highlighted the complexity of the pathogenesis and manifestation of diffuse axonal injury, they also indicate that PACAP should be considered a potential therapeutic agent in TBI. Key words: -amyloid precursor protein; corticospinal tract; neuroprotection; PACAP; traumatic brain injury Departments of 1 Anatomy (Neurohumoral Regulations Research Group of the Hungarian Academy of Sciences), 2 Neurosurgery and 3 Central Laboratory of Animal Research, University of Pécs, Medical Faculty, Pécs, Hungary. 4 Department of Anatomy and Neurobiology, Virginia Commonwealth University, Richmond, Virginia. 5 Clinical Neuroscience Research Group of the Hungarian Academy of Sciences, Department of Neurosurgery, University of Pécs, Medical Faculty, Pécs, Hungary. 686

111 POSTINJURY ADMINISTRATION OF PACAP IN TBI INTRODUCTION NEUROPATHOLOGICAL STUDIES, clinical observations, and other data derived from animal models have significantly increased the understanding of both primary and secondary brain damage over the past decades. In up to 45% of severely head-injured patients, the neuropathological changes after head injury may be due to the effects of secondary mechanisms. The early insults, such as transient global ischemia, haematoma, or diffuse axonal injury, are followed and complicated by secondary mechanisms, which may be mediated by complex cascades of biochemical processes. Many of these secondary posttraumatic events have been targeted as potential sites for pharmacological interventions to prevent or to significantly reduce secondary brain damage (Bullock, 1993). Numerous neuroprotective drugs and therapeutic interventions have been tested in different animal models of traumatic brain injury (TBI). Despite the obvious differences in their pathogenesis, TBI and ischemic brain damage do share some common pathways providing a solid basis for the application of at least partially similar treatment strategies. Such mechanisms include excitotoxicity, overproduction of free radicals, inflammation, and apoptosis (Bramlett and Dietrich, 2004; Leker and Shohami, 2002). One of the most widely used therapeutic approaches is hypothermia. Postinjury hypothermia is extensively studied and utilized for neuroprotection in several models of ischemia and traumatic injuries of the nervous system (Bethea and Dietrich, 2002; Bramlett et al., 1995; 1997; Koizumi and Povlishock, 1998; Lyeth et al., 1993). Hypothermia prevents free radical production (Globus et al., 1995), nitric oxide synthesis (Sakamoto et al., 1997), intracellular calcium accumulation (Mitani et al., 1991), and the rise in excitatory amino acid secretion (Koizumi et al., 1997). There are several other therapeutic interventions effective both in cerebral ischemia and TBI. The efficacy of posttraumatic Mg 2 as a neuroprotective agent has been shown in both diffuse axonal injury and brain ischemia (Heath and Vink, 1999; Tsuda et al., 1991). Similarly, treatment with NMDA channel blockers limits excitotoxin-induced secondary neuronal damage in models of TBI (Goda et al., 2002) and cerebral ischemia (Dirnagl et al., 1990). Inhibition of inflammatory mediators and calpain has also been proven effective in both models (Buki et al., 1999b, 2003; Cernak et al., 2002; Hong et al., 1994; Kadoya et al., 1995; Nakayama et al, 1998; Sanderson et al., 1999; Yoshimoto and Siesjo, 1999). Several neurotrophic factors, such as brain-derived neurotrophic factor, nerve growth factor and fibroblast growth factor, have been shown to play an important role in the endogenous response following both cerebral ischemia and traumatic brain injury, and to be effective in both brain pathologies when given exogenously (Dietrich et al., 1996; Kawamata et al., 1997; Leker and Shohami, 2002; Truettner et al., 1999). Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide structurally belonging to the secretin/glucagon/ VIP family. PACAP was isolated from ovine hypothalamus based on its ability to activate adenylate cyclase in the pituitary gland. PACAP is a bioactive peptide with diverse activities in the nervous system. In addition to its more classic role as a neurotransmitter, PACAP functions as a neurotrophic factor (Somogyvari-Vigh and Reglodi, 2004). Numerous studies have shown its neuroprotective effect in vitro and in vivo (Arimura, 1998; Somogyvari- Vigh and Reglodi, 2004; Vaudry et al., 2000a). Previously, it has been demonstrated that PACAP has neuroprotective effects in a rat model of global and focal cerebral ischemia treated before and after the injury (Uchida et al., 1996; Reglodi et al., 2000, 2002). Based on the findings with PACAP in cerebral ischemia and the presence of common pathogenetic mechanisms in cerebral ischemia and TBI, we applied PACAP therapy in a rat model of TBI in a previous study. It was found that PACAP had neuroprotective effects when administered immediately after the injury (Farkas et al., 2004). In acute cerebral injuries, it is of utmost importance to determine the therapeutic time window of candidate neuroprotective agents, since the immediate therapy is not possible in most cases. Of similar importance is the identification of therapeutic interventions that could be applied at the scene of the accident in an attempt to prolong the time window for further neuroprotective interventions and measures to inhibit progression to irreversible structural changes. The aim of the present study was therefore, to investigate the possible protective effect of PACAP in a rat model of TBI given 30 min and 1 h postinjury in order to examine the possibility of delaying the therapeutic window. MATERIALS AND METHODS Induction of Traumatic Brain Injury Adult Wistar male rats ( g) were subjected to impact acceleration TBI in accordance with previous descriptions (Marmarou et al., 1994; Foda and Marmarou, 1994). Animals were anesthetized in a bell jar for 5 min under 4% isoflurane (Forane, Abott, Hungary) and a 2:1 mixture of N 2 O:O 2, endotracheally intubated, and maintained under 1.5 2% isoflurane. Rectal and temporal temperature were monitored and maintained at 37 C with a ventral heating pad (FHC BOWDOINHAMME

112 TAMÁS ET AL. USA Temperature Control). A midline scalp incision was made and the skull was exposed with blunt dissection. Parietal bones were exposed, cleaned, and dried with bone wax. A 10-mm stainless steel disc helmet was secured with dental acrylic to the skull between bregma and lambda. Rats were then fastened prone to a foam bed beneath the injury tower and injured from 2 m with a 450-g weight. After injury, animals were immediately ventilated with 100% O 2, the helmet was removed, and the skull examined for sign of fracture, which, if found, disqualified the rat from further evaluation. Selected animals underwent monitoring of other physiological parameters including invasive blood pressure, pulse oxymetry and arterial blood gas analysis. All procedures were performed in accordance with the ethical guidelines of NIH and guidelines approved by the University of Pécs (no. BA02/ /2001) to minimize pain and suffering of the animals. The minimal number of animals required to achieve statistically meaningful results was used in all cases. Drug Administration A single bolus of 100 g PACAP (Sigma, Hungary) dissolved in 5 L of physiological saline was administered intracerebroventriculary 30 min after the injury in one group of rats (n 4), and 1 h after the injury in another group (n 5). Control animals received the same volume of vehicle at both time-points (n 5). The dose of PACAP was selected based on previous studies describing dose-dependent characteristics of PACAP administration (Farkas et al., 2004). Sham-injured rats (n 4) were anesthetized, intubated and the parietal bones were exposed without performing the impact acceleration injury. The animals were sacrificed for the histological assessment 2 h after brain injury. Immunohistochemistry At the designed survival time, animals were reanesthetized with an overdose of sodium pentobarbital and transcardially perfused with Zamboni s fixative (Zamboni and Martino, 1967). The brains were removed and immersed in the same fixative overnight (16 18 h). Based upon previously published observations concerning the topography of diffusely injured axons in the rat brainstem (Povlishock et al., 1997), a median 5-mm-wide block of the brain was removed using a sagittal brain blocking device (Braintree Scientific Inc.) to include the region extending from the interpeduncular fossa to the first cervical segment. The brainstem was blocked just lateral to the pyramids to encompass known vulnerable fiber tracts in this model of TBI: the corticospinal tract (CSpT) and the medial longitudinal fascicle (MLF). The tissue was sectioned with Vibrotome Series 1000 (Polysciences Inc., Warrington, PA) at a thickness of 40 m and collected in 0.1 M phosphate buffer. The sections were collected in two groups in a semi-serial fashion, rinsed three times for 10 min in phosphate-buffered saline (PBS), and processed for immunhistochemical localization of damaged axonal profiles. Half of the sections were single labeled for the detection of RMO-14 immunoreactivity. This antibody in known to exclusively target an epitope on the rod domains of altered NF-M subunits, which are exposed upon modification of the NF sidearms, and assumed consequence of calcium induced enzymatic processes (Lee et al., 1987; Okonkwo et al., 1998; Povlishock et al., 1997). Every second section was processed for immunohistochemical detection of the amyloid precursor protein ( -APP). This classical marker of traumatic axonal injury (TAI) is carried by fast axoplasmic transport and will pool at foci affected by TAI. In the present study, we utilized a polyclonal antiserum targeting the C-terminus of -APP (Gentleman et al., 1993; Stone et al., 2000). Vibratome sections microwaved for optimizing antigenicity with the retention of excellent ultrastructural detail according to former publications (Stone et al., 1999). Sections from both groups were then washed in PBS containing 1% normal goat serum (Fluca), and were incubated for 35 min with 0.2% Triton X (Sigma-Aldrich, Hungary) in 10% normal goat or horse serum, respectively (NGS or NHS; Sigma Chemical Co., St. Louis, MO) in PBS. After two quick rinses in PBS containing 1% NGS (or NHS in the case of RMO-14) the above defined groups of sections were incubated overnight in rabbit anti-app antibody at a dilution of 1:3000 (Zymed Laboratories, Hartford, CT) or in mouse monoclonal RMO-14 antibody (kindly provided by Dr. John Q. Trojanowski, University of Pennsylvania Department of Pathology) at the dilution of 1:500. Having been rinsed 3 10 min in PBS containing 1% NGS (or NHS), sections were incubated in biotinylated anti-rabbit immunoglobuline (Sigma) derived from goat or in 1:400 dilution of biotinylated, rat adsorbed anti-mouse immunoglobulin derived from horse (Vector) for 60 min. After incubation in avidin biotin-peroxidase complex (ABC kit, Vector) and rinsing in PBS and 0.1 M phosphate buffer 3 10 min and 2 10 min respectively, sections were processed for visualization of the immunohistochemical complex using 0.05% diaminobenzidine (Sigma) and 0.01% hydrogen peroxide in 0.1 M PBS. The sections were then mounted and cleared for routine light microscopic examination. Image Analysis Semi-serial brainstem sections were examined with a Nikon light microscope interfaced with a computer-as- 688

113 POSTINJURY ADMINISTRATION OF PACAP IN TBI sisted image analysis system (NIH Image J software) in a blinded fashion. At the pontomedullary junction, two adjacent grids of 40,000 m 2 were superimposed over the CSpT and the MLF, images were captured and digitized at a magnification of 50 and the total number of damaged APP and RMO-14 immunopositive axonal profiles within this area were marked and counted. The mean density of immunopositive axons was computed as a number of immunopositive axons per mm 2. Statistical Analysis Student t-test was used to compare the density of immunopositive profiles (expressed as mean number/mm 2 ) between the control and PACAP-treated animals. RESULTS Neither the use of the above-described experimental TBI protocol nor the administration of PACAP resulted in any significant alteration in the physiological parameters monitored during the experiment (temporal and rectal temperature, blood pressure, and blood gases), consistent with previous observations from our laboratories and others (Buki et al., 1999a, 2003; Otani et al., 2002; Reglodi et al., 2000, 2002; Singleton et al., 2001). Previous observations with PACAP treatment found a transient, slight drop in blood pressure only when the peptide was administered intravenously: fall in the blood pressure was observed 5 min after intravenous injection, but it soon returned to normal (Reglodi et al., 2000). Consistent with the current study, no such alteration was found in previous studies when PACAP was given icv (Reglodi et al., 2002). Light microscopic examination of vehicle- and drugtreated animals subjected to TBI and reacted for the visualization of -APP and RMO-14 antibodies, revealed discrete focal immunoreactivity within scattered axons in the CSpT and the MLF. Sham-injured animals did not display immunoreactive axonal profiles. Morphological characteristics of swollen, occasionally disconnected - APP immunoreactive axonal segments and the lobulated/vacuolated, partially or totally disconnected RMO- 14 immunoreactive axonal segments appeared entirely consistent with previous descriptions of damaged axonal profiles 2 h post-injury (Buki et al., 1999a,b, 2000; Stone et al., 2000). Treatment with PACAP (100 g) at 30 min or 1 h after the induction of TBI resulted in a significant reduction of -APP immunopositive axon profiles in the CSpT when compared to control animals 2 h postinjury. There was no significant difference between the number of -APP immunopositive axons in the MLF and RMO-14 immunopositive axonal profiles in both brain areas at 2 h after the injury compared to normal animals (Figs. 1,2,3). Quantitative findings followed by statistical analysis confirmed that postinjury administration of 100 g of PACAP significantly reduced the mean density of damaged -APP immunoreactive axonal profiles in the CSpT from to (p 0.001) or to (p ) 30 min or 1 h after injury, respectively (Figs. 1,3). There was no significant change in the MLF, where the mean number of -APP immunoreactive axon profiles varied from in the control group to and to in groups treated with PACAP at 30 min and 1 h postinjury, respectively (Fig. 1). FIG. 1. Mean densities of amyloid precursor protein (APP) immunopositive axons in the corticospinal tract (CSpT) and the medial longitudinal fascicle (MLF) in control and pituitary adenylate cyclase activating polypeptide (PACAP) treated animals. **p 0.01, ***p versus control group. 689

114 TAMÁS ET AL. FIG. 2. Mean densities of RMO-14 immunopositive axons in the corticospinal tract (CSpT) and the medial longitudinal fascicle (MLF) in control and pituitary adenylate cyclase activating polypeptide (PACAP) treated animals. In contrast, the mean density of RMO-14 immunorective axonal profiles showed no significant differences in either brain areas. In the CSpT, the mean density of RMO14 immunorective profiles varied from in the control group to and to after administration of PACAP at 30 min and 1 h postinjury. The mean number of RMO-14 immunoreactive profiles in the MLF varied from in the control group to and to in groups treated with PACAP at 30 min and 1 h after trauma (Fig. 2). DISCUSSION In the present study, we demonstrated that administration of PACAP 30 as late as 60 min post-injury sig- FIG. 3. Traumatically injured axons displaying amyloid precursor protein (APP) immunreactivity in the corticospinal tract (CSpT) from injured animals. (A) Control animals. (B) Animals treated with pituitary adenylate cyclase activating polypeptide (PACAP) at 30 min after the injury. (C) Animals treated with PACAP at 1 h postinjury. Arrows indicate some immunostained axons, and the number of arrows is proportional to the mean densities of immunostained axon profiles. Higher magnification of the same areas is seen in the inserts. 690

115 POSTINJURY ADMINISTRATION OF PACAP IN TBI nificantly reduced traumatically evoked axonal injury in the most vulnerable CSpT in the brainstem. Thus, our study provides further evidence for the previously described neuroprotective effects of PACAP. Recently, many therapeutic agents have been used in models of TBI, such as antiinflammatory and antiapoptotic drugs, as well as substances protective against excitotoxicity, to attenuate the main pathogenetic mechanisms present in brain injuries (Leker and Shohami, 2002). Most recent results indicate that PACAP may also be a promising therapeutic agent in injuries of the nervous system. PACAP is thought to interact with growth factors during development and following injury (Somogyvari-Vigh and Reglodi, 2004). In a model of TBI, increase of PACAP mrna has been observed in the vulnerable cortex and dentate gyrus concomitant with the decrease of TUNEL-positive apoptotic cells, which may indicate a role for PACAP in reducing the number of apoptotic neurons (Skoglosa et al., 1999). Since its discovery, numerous in vitro studies have demonstrated that PACAP has neurotrophic and neuroprotective effects. The first reports of the in vivo efficacy of PACAP showed that the peptide exerts neuroprotection in a rat model of global and focal cerebral ischemia (Ohtaki et al. 2003; Reglodi et al., 2000, 2002; Uchida et al., 1996). Subsequently, it has been shown that local injections of PACAP enhance facial nerve recovery after transection, and is neuroprotective in models of optic nerve transection, spinal cord injury, and 6-OHDA induced lesion (Katahira et al., 2003; Kimura et al., 2003; Reglodi et al., 2004; Seki et al., 2003) Based on the findings with PACAP in nervous injuries and the presence of common features of pathogenetic mechanisms in cerebral ischemia and TBI, we administered PACAP in a rat model of TBI. The application of PACAP was considered on the basis of widespread mechanisms of action. Glutamate excitotoxicity plays a role in the damage caused by TBI, and NMDA-antagonists have been shown to reduce the neuronal damage after impact-acceleration brain injury (Goda et al., 2002). The survival promoting effect of PACAP has been demonstrated against glutamate-induced toxicity in cultured cortical and retina neurons (Frechilla et al., 2001; Morio et al., 1996; Shoge et al., 1999). A recent study has shown that PACAP upregulates several genes that increase resistance to neurotoxic agents, including cytochrome P450 and FGF regulated protein (Vaudry et al., 2002b). PACAP also increases the velocity of glutamate uptake by cortical astroglial cells by promoting the expression of glutamate transporters and by inducing the expression of glutamine synthetase (Figiel and Engele, 2000). Inflammation is not restricted to infectious or autoimmune disorders of the nervous system, but occurs in cerebral ischemia, trauma and neurodegenerative disorders (Leker and Shohami, 2002). Inhibition of COX-2, an enyzme responsible for the production of prostaglandins at sites of inflammation, improves cognitive and motor outcome following diffuse TBI in rats (Cernak et al., 2002). Interleukin-1 receptor antagonists attenuate regional neuronal cell death and cognitive dysfunction after experimental brain injury (Sanderson et al., 1999). In addition, postinjury hypothermia, which is widely utilized for neuroprotection in TBI, also has anti-inflammatory effect (Deng et al., 2003). PACAP has been proven to be a potent inactivator of induced microglial release of proinflammatory cytokines and chemokines, such as TNF, IL-1, IL-6, IL-12, and NO (Delgado et al., 2002, 2003; Kim et al., 2000). Moreover, PACAP stimulates the anti-inflammatory cytokine IL-10 (Ganea and Delgado, 2002). Recently, it has also been shown that PACAP has inhibitory action on chemotaxis and neutrophil migration which may play a role in its neuroprotective effect (Kinhult et al., 2001). A few studies have demonstrated that the anti-inflammatory actions of PACAP exist also in vivo (Delgado et al., 1999). Apoptotic cell death plays a role in tissue injury following brain trauma and can be prevented by manipulation of the steps that lead to activation of the caspase cascade (Leker and Shohami, 2002). Recently, it has been demonstrated that inhibition of the caspase cascade reduces post-tbi (Fink et al., 1999). PACAP decreases the naturally occurring apoptotic cell death as well as apoptosis induced by various agents in neuronal cell lines (Canonico et al., 1996; Shioda et al., 1998; Somogyvari- Vigh and Reglodi, 2004; Tanaka et al., 1997; Vaudry et al., 2000b, 2002a). In cerebellar granule cells, PACAP reduces caspase-3 activation, which is also activated in TBI (Buki et al., 2000; Vaudry et al., 2000b). Mitochondrial damage has long been associated with the pathogenesis of TBI (Maxwell et al., 1997; Okonkwo et al., 1999). The mitochondrial failure purportedly caused by calcium-induced opening of the permeability transition pore is a key phenomenon in multiple neurological insults, including axonal injury (Buki et al., 2000; Pettus et al., 1994). Cyclosporin A, a potent inhibitor of Ca 2 -induced mitochondrial permeability transition, blunts the progression of calcium-mediated cytoskeletal change and reduces the number of disconnected/dysfunctional axons in rat TBI (Buki et al., 1999b; Okonkwo et al., 1999). Aconitase, a key mitochondrial enzyme influencing the viability of neurons in response to oxidative stress, is inactivated by a deprivation of Ca 2 influx into neurons and PACAP attenuates this inactivation (Tabuchi et al., 2003). In the present study we demonstrated that administration of PACAP at 30 min and 1 h after the injury reduced 691

116 TAMÁS ET AL. traumatically evoked axonal injury as assessed by immunocytochemical marker -APP in the CSpT, while there was no significant changes in the MLF. Similar surprising finding of this study was the lack of therapeutic effect in the case of damaged RMO-14 immunoreactive fibers. These findings might be explained by several reasons: first, the design of this study may not have allowed enough statistical power to establish significant difference in the MLF where the density of damaged axonal profiles was found in previous studies about 10% of that of the CSpT. Further, recent observations by Stone et al. (2000) indicated that APP and RMO-14 purportedly label at least partially different axon populations; 2 h post-injury, the former is more frequently localized to the CSpT while the latter is prevalent in the MLF at this time point. Similar results have been obtained by Suehiro et al. (2001) where the immunophilin ligand FK506 was administered following posttraumatic hypothermia associated with rapid rewarming. Recently, it has been demonstrated that the ability to protect mitochondria from undergoing mitochondrial permeability transition is very important in the neuroprotection following posthypothermic rewarming (Okonkwo and Povlishock, 1999), and RMO-14 immunoreactivity correlates with the percentage of calpain-mediated spectrin proteolysis and mitochondrial damage (Buki et al., 1999a; Povlishock et al., 1997; Povlishock and Stone, 2001). Based on the observation that FK506 did not decrease the number of RMO- 14 positive fibers, the authors confirmed that FK506 has no known action on mitochondrial permeability transition (Liu et al., 1991; Suehiro et al., 2001). Presently, there are no data available on the effects of PACAP on calpain-mediated spectrin proteolysis, which should correlate RMO-14 immunoreactivity. The differences between the structure of fibers situated in the CSpT and the MLF, and the different pathogenesis of axonal damage in these structures could also be the reason for different protective effects (Povlishock and Stone, 2001; Suehiro et al., 2001). A major finding in our study is that PACAP was effective even when given delayed after the trauma. Numerous investigations have shown the protective effect of different treatments applied before injury (Buki et al., 2003; Singleton et al., 2001) and immediately after the injury (Koizumi and Povlishock, 1998). Clinical therapies on the other hand focus on delayed administration strategies post-injury. It has been shown that cyclosporin A has neuroprotective effect when given 30 min after TBI (Buki et al., 1999b), while MgSO 4 improves motor outcome when administered up to 24 h after injury in rat TBI (Heath et al., 1999). 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120 3130 The Journal of Neuroscience, March 22, (12): Neurobiology of Disease Mechanoporation Induced by Diffuse Traumatic Brain Injury: An Irreversible or Reversible Response to Injury? Orsolya Farkas, 1,2 Jonathan Lifshitz, 1 and John T. Povlishock 1 1 Department of Anatomy and Neurobiology, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, Virginia 23298, and 2 Department of Neurosurgery, Medical Faculty of Pecs University, H-7623 Pecs, Hungary Diffuse traumatic brain injury (DTBI) is associated with neuronal plasmalemmal disruption, leading to either necrosis or reactive change without cell death. This study examined whether enduring membrane perturbation consistently occurs, leading to cell death, or if there is the potential for transient perturbation followed by resealing/recovery. We also examined the relationship of these events to calpainmediated spectrin proteolysis (CMSP). To assess plasmalemmal disruption, rats (n 21) received intracerebroventricular infusion 2 h before DTBI of a normally excluded 10 kda fluorophore-labeled dextran. To reveal plasmalemmal resealing or enduring disruption, rats were infused with another labeled dextran2h(n 10)or6h(n 11) after injury. Immunohistochemistry for the 150 kda spectrin breakdown product evaluated the concomitant role of CMSP. Neocortical neurons were followed with confocal and electron microscopy. After DTBI at 4 and 8 h, 55% of all tracer-flooded neurons contained both dextrans, demonstrating enduring plasmalemmal leakage, with many demonstrating necrosis. At 4 h, 12.0% and at 8 h, 15.7% of the dual tracer-flooded neurons showed CMSP, yet, these demonstrated less advanced cellular change. At 4 h, 39.0% and at 8 h, 24.4% of all tracer-flooded neurons revealed only preinjury dextran uptake, consistent with membrane resealing, whereas 7.6 and 11.1%, respectively, showed CMSP. At 4 h, 35% and at 8 h, 33% of neurons demonstrated CMSP without dextran flooding. At 4 h, 5.5% and at 8 h, 20.9% of tracer-flooded neurons revealed only postinjury dextran uptake, consistent with delayed membrane perturbation, with 55.0 and 35.4%, respectively, showing CMSP. These studies illustrate that DTBI evokes evolving plasmalemmal changes that highlight mechanical and potential secondary events in membrane poration. Key words: diffuse traumatic brain injury; neuron; dextrans; membrane disruption; membrane resealing; calpain Received Aug. 18, 2005; revised Jan. 16, 2006; accepted Feb. 8, This work was supported by National Institutes of Health Grants R01 NS , F32 HD-49343, and NS andbythecommonwealthcenterfortraumaticbraininjury. WethankSueWalker, LynnDavis, andtomcoburnfor their technical assistance. We also thank for Dr. Robert Hamm for his advice on statistical analysis and Dr. Scott Henderson for his help with confocal microscopy. Correspondence should be addressed to Dr. John T. Povlishock, Department of Anatomy and Neurobiology, Medical College of Virginia Campus of Virginia Commonwealth University, P.O. Box , Richmond, VA jtpovlis@vcu.edu. DOI: /JNEUROSCI Copyright 2006 Society for Neuroscience /06/ $15.00/0 Introduction Our understanding of diffuse traumatic brain injury (DTBI) has long moved on the assumption that the forces of injury evoke focal as well as diffuse neuronal death, contributing to morbidity and mortality. However, little work exists to support this premise. In animals and humans, contusions are associated with regional neuronal death, together with the finding of scattered neuronal death in the neocortex, hippocampus, and diencephalon (Cortez et al., 1989; Dietrich et al., 1994a,b; Colicos et al., 1996; Hicks et al., 1996; Saatman et al., 1996). In these cases, both apoptotic and necrotic cell death have been identified, with the assumption that the observed neuronal death proceeds from traumatically induced neuroexcitation, oxygen radical-mediated damage and/or secondary insults. Few have considered the potential that the forces of injury directly cause mechanically induced neuronal perturbation or death. Recently, after DTBI, we identified axotomy-mediated change in the neuronal somata associated with impaired, yet transient protein translation (Singleton et al., 2002). We also observed progressive necrosis in neurons adjacent to the axotomized neurons. Studies using extracellular tracers normally excluded by intact neuronal membranes revealed that these necrotic neurons suffered altered tracer permeability at the moment of injury most likely allowing for influx of damaging ions (Singleton and Povlishock, 2004). Other neurons in the same field, however, also took up these normally excluded tracers, yet reorganized them over time, manifesting no evidence of cell death. These observations led to the premise that the DTBI evoked direct mechanical poration of the neuronal cell membrane, which, if enduring, contributed to cell death. Conversely, it was posited that those neurons that contained tracers, yet remained viable, resealed their mechanically damaged membranes. In this communication, we tested this hypothesis by reexamining the potential for traumatically induced mechanical poration of neuronal membranes while evaluating whether continuing membrane perturbation leads to cell death. We also explored the potential that some neuronal membranes may reseal and recover. Fluorescently labeled tracers were administered both before and after injury to evaluate poration and its continuance. Parallel immunocytochemical investigations examined calpain-mediated spectrin proteolysis (CMSP) long associated with the damaging proteolytic death cascades activated in DTBI (Kampfl et al., 1996, 1997; Saatman et al., 1996; Newcomb et al., 1997; Pike et al., 1998; Buki et al., 1999; McCracken et al.,

121 Farkas et al. The Consequences of Mechanoporation in DTBI J. Neurosci., March 22, (12): ). In fact, the significance of CMSP is highlighted by experimental and clinical studies that have suggested that CSF CMSP breakdown products are surrogate markers of morbidity and mortality (Pike et al., 2001; Farkas et al., 2005). Confocal microscopy and concomitant ultrastructural analyses using antibodies targeting the fluorophore-tagged tracers confirmed that TBI caused direct mechanical poration in numerous neuronal somata scattered throughout the neocortex. Over 50% of the tracercontaining neurons were labeled with both tracers with many of these revealing necrotic cell death. Other flooded neurons contained only one tracer, consistent either with membrane resealing or delayed opening. Surprisingly, CMSP did not frequently colocalize with neurons, demonstrating tracer influx and/or necrosis. Collectively, these studies illustrate the complex and evolving plasmalemmal changes ongoing with DTBI. Materials and Methods In all, 21 adult male Sprague Dawley rats weighing g were used for the experiments (n 10 in 4 h survival group; n 11 in 8 h survival group). Four animals served as tracer-infused, sham-injured controls to demonstrate that infusion into the lateral ventricle itself does not lead to neuronal cellular membrane disruption. Ten noninjected, injured animals were used as intracranial pressure (ICP) controls to obviate the concern that any tracer infusion-induced rise in ICP could constitute a confound. Surgical preparation and injury induction. For the induction of anesthesia, animals were exposed to 4% isoflurane in a mixture of 30% O 2 and 70% N 2 O for 5 min, then intubated and ventilated with 1 2% isoflurane in the mixture of 30% O 2 and 70% N 2 O. Anesthesia was maintained throughout the duration of the experiment until transcardial perfusion. Animals were placed on a feedback-controlled heating pad (Harvard Apparatus, Holliston, MA) to maintain body temperature at 37 C during surgery. A PE50 polyethylene tube (Becton Dickinson, Sparks, MD) was placed into the left femoral artery to monitor the mean arterial blood pressure (MABP) and blood gases. After cannulation animals were placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). The skull between the coronal and lambdoidal sutures was exposed with a midline incision. A 2 mm burr hole was drilled in the right parietal bone. A 26 gauge needle connected to a pressure transducer and a microinfusion pump via a PE50 polyethylene tube was placed into the lateral ventricle 0.5 mm posterior and 1.3 mm lateral from bregma through the burr hole. During the needle placement, sterile saline was infused with a 3 l/min rate within a closed fluid-pressure system, while intracranial pressure monitoring was obtained via a PowerLab system (ADInstruments, Colorado Springs, CO). With the advancement of the needle, the detection of a significant pressure drop indicated the moment that the needle had broached the ventricular wall (Amorini et al., 2003). This controlled and monitored needle placement assured consistent needle placement within the ventricular compartment without the potential confound of brain placement and its potentially damaging consequences on tracer infusion. After placing the needle into the ventricle, 75 l of Alexa Fluor 488-labeled 10 kda molecular weight dextran (Invitrogen, San Diego, CA) was infused into the ventricle at 2 l/min rate using a CMA/100 microinjection pump (Carnegie Medicin, Stockholm, Sweden), with continuous intracranial pressure monitoring. The dextran was diluted in sterile saline in mg/ml concentration; the final amount infused was 5 mg/kg. The needle was allowed to remain in the ventricle for 10 min after completion of the infusion, after which the needle was slowly removed. Gelfoam and bone wax were used to restore skull integrity before the induction of closed head injury. Next, the animal was prepared for injury as described in previous protocols (Foda and Marmarou, 1994; Marmarou et al., 1994). Briefly, a metallic disc-shaped helmet of 10 mm diameter was firmly glued between the bregma and lambdoid sutures of the skull. The animal was disconnected from the ventilator and placed in a prone position on a foam bed with the metallic helmet centered under the edge of a Plexiglas tube. The rat was prevented from falling by two belts secured to a foam bed of known spring constant (Type E bed; Foam to Size, Ashland, VA). Brass weights weighing 450 g were allowed to fall from a height of 2 m through the Plexiglas tube directly to the metallic disc fixed to the animal s skull, a setting that precluded cerebral contusion or subdural hemorrhage. Rebound impact was prevented by sliding the foam bed containing the animal away from the tube immediately after the initial impact. Each animal sustained injury 2 h after preinjury tracer administration. Noninjured controls were prepared for injury in the same manner but were not injured. Immediately after the injury, the animal was reconnected to the ventilator. The helmet was removed and the skull was studied for any sign of fracture, which, if found, disqualified the animal from further evaluation. The scalp wound was then sutured while the animal remained on artificial ventilation. Delayed tracer infusion. Before the second tracer infusion, animals were returned to the stereotaxic device, the skull was exposed, and another hole was drilled through the left parietal bone. Texas Red-labeled 10 kda dextran (Invitrogen) was then infused at the same concentration as the preinjury dextran. Once again, this was accomplished via a needle placed in the left ventricle 0.5 mm posterior and 1.3 mm lateral to bregma with 2 l/min infusion rate with continuous ICP monitoring. This tracer was infused either at 2 h after injury (n 10) or 6 h after injury (n 11). The tracer was then allowed to diffuse for additional 2 h before perfusion with fixative, resulting in the respective 4 and 8 h postinjury survival groups. Sham-injured animals were infused either 4 or 8 h after the first infusion. Non-injected-injured animals (n 10) were prepared in the same manner for ICP monitoring and injury, but no tracers were infused in their ventricles. Rather, ICP was monitored through a needle placed in the ventricle at the same time points used in injected animals. Five minutes before transcardial perfusion, the animals were injected with 150 mg/kg euthasol. At either 4 or 8 h after injury or at either 6 or 10 h after the first infusion (sham injury), the animals underwent transcardial perfusion with saline and then 4% paraformaldehyde in Millonig s buffer. Physiological assessment. MABP was monitored via a femoral line in each animal for the duration of the surgical procedures. Blood gases were analyzed before the tracer infusions as well as before and after injury. As noted above, ICP was also monitored before, during, and after tracer infusion or throughout the duration of all procedures in noninjected animals. Body temperature was monitored via a rectal probe and maintained at 37 C using a feedback control heating pad. PowerLab was used to monitor all physiological parameters (MABP, ICP, temperature). Tissue processing. After perfusion, the brains were removed and stored in 4% paraformaldehyde in Millonig s buffer at 4 C for 24 h, after which they were coronally blocked at the optic chiasm and midbrain and sectioned in 0.1 M phosphate buffer with a vibratome (Leica, Banockburn, IL) at a thickness of 40 m. Sections were collected serially in 24-well culture plates and stored in Millonig s buffer at 4 C until further processing. Multiple fluorescent analysis. Because dextrans infused to the brain were labeled with fluorophores (Alexa Fluor 488 for the preinjury dextran and Texas Red for postinjury dextran), no additional steps were needed to visualize the tracer or any tracer-containing neurons. In addition to the identification of tracer-containing neurons, antibody against the 150 kda calpain-specific spectrin breakdown product was used to examine the link between any observed membrane perturbation and the ensuing induction of calpain-mediated spectrin proteolysis, previously linked to TBI-induced proteolytic cascades. The antibody Ab 38 was used for this approach, because it is well characterized, targeting the N-terminal fragment of the 150 kda calpain-specific breakdown of IIspectrin, a cytoskeletal protein known to be cleaved after traumatic brain injury (Siman et al., 1989; Roberts-Lewis et al., 1994). In this procedure, some sections were washed in PBS three times for 10 min and then subjected to temperature-controlled microwave antigen retrieval as described previously (Stone et al., 1999). After retrieval, sections were again rinsed in PBS three times for 10 min and treated for 60 min in 10% normal goat serum (NGS) with 0.2% Triton X-100 in PBS and 5% BSA. The tissue was then incubated in a 1:5000 dilution of Ab 38 (generous gift from Dr. R. Siman, University of Pennsylvania, Philadelphia, PA) in 1% NGS/PBS with 1% BSA overnight. The following day, sections were rinsed three times for 10 min 1% NGS/PBS and then incubated for2hin a 1:1500 dilution of Alexa Fluor 647-labeled anti-rabbit IgG (Invitrogen)

122 3132 J. Neurosci., March 22, (12): Farkas et al. The Consequences of Mechanoporation in DTBI in 1% NGS/PBS. After rinsing three times for 10 min in PBS and twice for 10 min in 0.1 M phosphate buffer, sections were mounted on nontreated slides and coverslipped (ProLong; Invitrogen). Visualization of fluorophores at ultrastructural level. Because the fluorescent dyes that were used are not detectable at electron microscopic level, for parallel assessments of the ultrastructural change, conversion of fluorophores to a stable, electron-dense product was performed. This was achieved through the use of an antibody against the fluorophores themselves. The same sections previously scanned with confocal microscope for tracer uptake or individually captured with a Spot RT camera attached to an epifluorescent Nikon (Tokyo, Japan) Eclipse E800 microscope were used to provide a visual fluorescent map to which the subsequently prepared chromagen-based sections could be directly compared at the LM and EM levels. After capture, the tissues were soaked off the slides, and brain regions demonstrating preinjury and/or postinjury tracer uptake were removed and processed to investigate any ultrastructural change related to membrane disruption by using antibodies targeted to Alexa Fluor 488. This method allowed the identification of the same cells that previously revealed evidence of the uptake of either one or both dextran tracers, allowing the ultrastructural assessment of any alteration associated with enduring membrane change and/or membrane resealing. In this approach, sections were postfixed with 0.1% glutaraldehyde in 4% paraformaldehyde, then rinsed five times for 10 min in Millonig s buffer and three times for 10 min in PBS. Endogenous peroxidase activity within the tissue was blocked with 0.5% H 2 O 2 in PBS for 30 min. Sections were processed using the temperature-controlled microwave antigen retrieval approach described above. After microwave antigen retrieval, sections were preincubated for 60 min in 10% NGS with 0.2% Triton X-100 in PBS. The tissue was incubated overnight in a 1:5000 dilution of rabbit anti-alexa Fluor 488 antibody (Invitrogen) in 1% NGS in PBS. Sections were then incubated for 1 h in biotinylated anti-rabbit antibody (IgG; Vector Laboratories, Burlingame, CA) diluted 1:2000 in 1% NGS in PBS and then for 1hina1:200 dilution of an avidin horseradish peroxidase complex (ABC Standard Elite kit; Vector Laboratories). The reaction product was visualized with 0.05% diaminobenzidine, 0.01% hydrogen peroxide, and 0.3% imidazole in 0.1 M phosphate buffer for min. Visualization of calpain-mediated spectrin proteolysis at the ultrastructural level. Brain regions demonstrating evidence for tracer uptake with confocal microscopy were removed and processed to investigate CMSP using the previously described Ab 38 antibody. In this approach, after postfixation tissues were processed for EM analysis as described above, using Ab 38 as primary antibody in 1:8000 dilution and biotinylated anti-rabbit antibody in 1:2000 dilution. Electron microscopy. Using confocal and routine fluorescent images as described above, the once-fluorescent cells, which now contained a peroxidase reaction product, were prepared for EM. In this approach, the previously prepared fluorescent maps were used to identify specific sites of interest. These were dissected out of the tissue sections, and then osmicated, dehydrated, and embedded in epoxy resins on plastic slides with plastic coverslips. After resin curing, the plastic slides were studied with routine LM to identify the precise neurons of interest. Once identified, these sites were removed, mounted on plastic studs, and thick sectioned to the depth of the immunoreactive sites of interest. Serial 70 nm sections were cut and picked up on to Formvar-coated slotted grids. The grids then were stained in 5% uranyl acetate in 50% methanol for 2 min and 0.5% lead citrate for 1 min. Ultrastructural analysis was performed using a JEM 1230 electron microscope (JEOL-USA, Peabody, MA). Specifically, at 1000, the grid field was scanned to identify the cell(s) of interest. Once identified, these cells were digitally acquired at 5000 as a montage. These digital montages were copied to a digital video disk and transferred to a Dell Dimension XPS Gen 4 computer (Dell Computer Company, Round Rock, TX), on which the images were further enlarged and assessed for any evidence of cellular/subcellular change. Confocal fluorescent microscopy. Zeiss (Oberkochen, Germany) 510 Meta and Leica TCS SP2 AOBS laser scanning confocal microscope systems were used to analyze double- and/or triple-labeled sections. Appropriate laser lines (argon 488 nm for Alexa Fluor 488, He-Ne 594 nm for Texas Red, and He-Ne 633 nm for Alexa Fluor 647) and emission filters (Zeiss) or emission bands (Leica) were used for exciting and detecting fluorescent signals. Semiquantification of fluorescent images. The TBI model used in the current study evokes diffuse injury throughout the neocortex, resulting in scattered neuronal injury with heterogeneous pathologies. These modest numbers of damaged neurons, particularly those revealing only one tracer, would violate a basic tenant of stereology and preclude further Figure 1. Bar charts representing the mean levels of intracranial pressure measured 1 and 2hbeforeinjuryaswellas2,3,6,and7hafterinjury(blackcolumn,noninfusedinjured,n 10; gray column, infused injured, 4 h survival, n 10; white column, infused injured, 8 h survival, n 11). No elevation in ICP was found in injected-non-injured sham animals, indicating that controlled infusion into the lateral ventricle does not increase ICP (data not shown). No significant difference in ICP was observed between injected-injured and non-injected-injured animalsat4or8hafterinjury, indicatingthatinfusionintothelateralventricledoesnotcomplicate any ICP change associated with closed head injury. Note, however, that in this injury model, the traumatic episode was associated with a significant elevation of ICP. The ICP was elevated at each time point measured postinjury, particularly at 2, 3, 6, and 7 h after injury. Data are shown as mean SEM. The asterisk indicates statistical significance ( p 0.001). Figure 2. This double-labeled confocal image from a sham-injured animal receiving fluorophore-labeleddextraninfusionsdemonstratesthediffusionofthetracersthroughoutthe interstices of the neocortex without any evidence of neuronal uptake of any of the administrated dextrans. Note that in addition to their passage through the interstices of the brain parenchyma, the labeled dextrans can be easily visualized in the perivascular regions, consistent with a control pattern of dextran distribution (arrows). Scale bar, 100 m.

123 Farkas et al. The Consequences of Mechanoporation in DTBI J. Neurosci., March 22, (12): stereological analysis of the number of neurons with membrane perturbation at 4 or 8 h after injury. Densitometric evaluation of injured neurons showing different dextran uptake profiles could not be performed, considering the high and variable background level of the fluorophores associated with the intraventricular administration of the labeled dextrans, making the necessary standardization difficult. Based on these deficiencies, we conducted traditional cell counting using images captured by confocal microscopy with rigorous inclusion criteria to semiquantify neurons showing primary and/or secondary dextran uptake both at 4 and 8 h after injury. Sections captured at 40 magnification and containing at least three fluorescent labeled neurons were included (number of animals at 4 h, n 8; at 8 h, n 7; mean number of sections Figure 3. Confocal images of tracer-infused-injured animals. A, This double-labeled image demonstrates numerous cortical neurons (arrows) revealing intracellular tracer flooding with both the preinjury and postinjury infused dextrans. Scale bar, 100 m. B D, These confocal images, revealing the initially administered dextran flooding (B), the postinjury infused dextran flooding (C), and their overlay (D), demonstrate that the majority of the neurons sustaining membrane disruption and tracer uptake are double labeled both with preinjury- and postinjury-infused dextrans (arrows). Such double labeling was observed at both 4 and 8 h after injury. Note that some neurons flooding with the preinjury dextran alone can also be observed (arrowheads). Scale bar, 100 m. per animal, 7.8). The definition for positive labeled neurons was all neurons showing any dextran flooding. Neurons showing preinjury dextran uptake, postinjury dextran uptake, or both preinjury and postinjury dextran uptake were quantified as a percentage of the total number of neurons demonstrating any tracer uptake in each section and averaged for each animal. Similar methods were used to quantify calpain mediated spectrin proteolysis and its relationship to membrane disruption. Statistical analysis. Quantitative data are presented as mean SEM. One-way ANOVA was used to compare the physiological parameters (MABP, po 2, pco 2, body temperature, and ICP) between the different groups or between different time points. For further analysis, Duncan post hoc tests were performed, and a p value 0.05 was considered statistically significant. To detect a redistribution of tracer-filled neurons over time after injury, serial 2 tests between proportions of neurons with specific membrane perturbations were performed. The three 2 tests (resealed vs enduring, enduring vs delayed, resealed vs delayed) were evaluated at p to correct for multiple analyses. Results Physiological assessments Mean arterial blood pressure, pco 2,pO 2, and ph were monitored and maintained at physiological levels in each animal for the duration of the experiments. There were no differences in these physiological parameters between injected and noninjected animals, demonstrating no systemic effects of the intracerebroventricular injections (data not shown). ICP was also monitored to reveal the effects of preinjury and postinjury infusions on ICP as well as any ICP change related to diffuse traumatic brain injury. No elevation in ICP was found in the injected, noninjured sham animals. This indicated that the intraventricular infusion into the lateral ventricle did not increase ICP itself (data not shown). No significant differences in ICP over time were observed between injected-injured and noninjected-injured animals at any time point, indicating that intracerebroventricular infusion had no effect on ICP change related to DTBI. However, it is important to note that in this injury model, like the human condition, the traumatic episode itself was associated with ICP elevation. Specifically, ICP was elevated 2, 3, 6, and 7 h after injury compared with preinjury levels both in non-injected-injured and injected-injured animals (Fig. 1). In some cases, the ICP approached 20 mmhg, a value consistent with that seen in brain-injured humans (Miller et al., 1977; Marshall et al., 1979). Figure 4. A D, Double-labeled confocal images. In A C, neurons flooding with both dextrans reveal evidence of concomitant cellular injury, reflected in their irregular, distorted profiles and vacuolization (arrows). In those cells showing the most severe damage, dextrans are also typically found within the nucleus (arrowhead). Note that other double-labeled neurons (D) demonstrate little or no pathological damage and that despite homogenous tracer uptake, no nuclear accumulation or vacuolization occurs (arrows). Scale bars, 100 m. Evidence for altered neuronal membrane permeability after DTBI Administration of high-molecular-weight dextrans normally excluded from the neuronal and glial cytoplasm by intact cell membranes was used to investigate altered membrane permeability related to diffuse

124 3134 J. Neurosci., March 22, (12): brain injury. Based on our previous observation that the infusion of 10 kda dextran into the lateral ventricle is a reliable approach for the detection of plasmalemmal alteration related to closed head injury (Singleton and Povlishock, 2004), Alexa Fluor 488-conjugated 10 kda dextran was infused 2 h before injury. Additionally, Texas Red-conjugated 10 kda dextran was used either 2 or 6 h after TBI to examine potential membrane resealing and/or enduring membrane disruption. The rationale here was that neurons containing both fluorophores sustained enduring membrane damage over the experimental time course, whereas the presence of one fluorophore alone would argue for either membrane resealing in the case of the preinjury-administered fluorophore or delayed opening in the case of the postinjury-administered fluorophorelabeled dextran. Sham-injured animals receiving fluorophore-labeled dextran infusions demonstrated interstitial or perivascular diffusion of the tracers throughout the neocortex without any evidence of cellular uptake. This indicated that controlled dextran infusion into the lateral ventricles did not lead to cellular uptake of the tracers (Fig. 2). This pattern of tracer distribution was consistent with that described with the use of comparable molecular weight tracers described by our laboratory and others (Singleton and Povlishock, 2004). In contrast, the injured animals revealed tracer-containing neurons indicative of membrane disruption scattered bilaterally throughout the rostrocaudal extent of the neocortex, primarily within layers IV and V (Fig. 3A). The relatively large number of dextran-flooded neurons in injured tissue indicated that the membrane disruption was not an isolated phenomenon but rather occurred throughout the extent of the neocortex scattered among other apparently unaltered neurons. In all cases, based on at both light and electron microscopy evaluation, there was no evidence of dextranflooded glia. At 4 and 8 h after injury, respectively, 55.4% 7.4 and 54.7% 6.4 of neurons sustaining any membrane disruption and tracer flooding were double labeled both with the preinjury- and postinjury-infused dextrans (Alexa Fluor 488 and Texas Red conjugated). These findings suggest that neuronal cell membranes were opened initially after injury and maintained continued membrane disruption detected by the administration of the second tracer (Fig. 3B D). With confocal microscopy, many of the neurons flooded with both dextrans demonstrated Farkas et al. The Consequences of Mechanoporation in DTBI Figure 5. Ultrastructural analysis of the dual tracer-containing neurons reveals various forms of pathologic change. A, The fluorescent image obtained via routine fluorescent microscopy. B, The anti-alexa Fluor 488-immunostained image of the same region. C, D, An electron micrograph of the same neuron. Note that this neuron demonstrates moderately increased electron density, cytoplasmic and nuclear tracer flooding (arrows), and perinuclear organelle vacuolization (arrowheads) without overt mitochondrial damage (double arrowheads), best shown in the enlarged panel D. Scale bar, 2 m. E, Three tracer-flooded neurons, again confirmed by routine fluorescent microscopy and followed via EM. The most severely damaged neuron (asterisk) demonstrates increased electron density, organelle vacuolization, and perisomatic glial ensheathment best illustrated in enlarged panel F. The two other cells (double and triple asterisks) demonstrate little or no pathological change. Note that the surrounding neuropil demonstrates little overt pathologic change consistent with the confocal observations. Scale bar, 5 m

125 Farkas et al. The Consequences of Mechanoporation in DTBI J. Neurosci., March 22, (12): Figure 6. A, Triple-labeled confocal image demonstrating dextran flooded neurons as well as CMSP-immunopositive neurons. Note that at both 4 and 8 h after injury, CMSP-immunoreactive neurons were observed throughout the neocortex (arrows). Scale bar, 100 m. B E, Confocal images of preinjury dextran flooding (B), postinjury dextran flooding (C), CMSP immunopositivity (D), and their overlay (E) demonstrate neurons showing enduring membrane disruption reflected in their content of both tracers. Note that some neurons colocalize with CMSP immunopositivity (arrow), whereas other neurons demonstrate tracer flooding without CMSP (arrowheads). Also note that at these same time points, several CMSP-immunoreactive neurons also could be identifiedwithoutconcomitanttracerflooding(doublearrowhead). Finally, notethatinthesameregion, aneurondemonstrating onlytheinitialtracerflooding(bigarrow), aswellasaneuronrevealingonlysecondarytracerflooding(triplearrowhead), canalso be seen. Scale bar, 50 m. evidence of concomitant cell injury reflected in their irregular profiles and vacuolization, all of which were suggestive of necrotic alteration leading to cell death (Fig. 4A C). In those neurons showing the most dramatic structural change, the dextrans typically were found within the nucleus. In contrast to these findings, a limited number of double-labeled neurons at both time points demonstrated little or no pathological damage. These neurons revealed homogenous tracer flooding without nuclear accumulation or vacuolization (Fig. 4D). Concomitant ultrastructural analyses confirmed and supplemented the morphological changes observed with confocal microscopy. With electron microscopy, many of the doubleflooded neurons appeared damaged with evidence of necrosis. Such necrotic change was reflected in occasional nuclear pyknosis, dilation of the Golgi apparatus and the smooth endoplasmic reticulum (SER), together with occasional vesicular swelling, isolated mitochondrial change, increased nuclear and cytoplasmic electron density, and perisomatic glial swelling in the most severely necrotic neurons (Fig. 5E,F). However, as noted above, a limited number of double-flooded cells demonstrated only modest ultrastructural change, consistent with a less severe form of cellular perturbation (Fig. 5C E). Within these injury foci, CMSP revealed a complex and unanticipated repertoire of responses in relation to the above-described pathologies. No CMSPimmunopositive neurons were observed in sham-injured animals. However, at both 4 and 8 h after injury, CMSPimmunoreactive neurons were observed throughout the neocortex (Fig. 6a). At 4 h after injury, 12.0% 8.1 of neurons demonstrating enduring membrane disruption, evidenced by their content of both tracers, were also found to be Ab 38 positive. By 8 h after injury, 15.7% 4.3 of the double-flooded neurons revealed evidence of CMSP. Furthermore, at 4 and 8hrespectively, 34.3% 3.1 and 35.4% 6.3 of all neurons analyzed revealed CMSP immunoreactivity without concomitant tracer flooding (Fig. 6B D). Ultrastructural analysis of these Ab 38-positive neurons revealed only moderate subcellular damage (Fig. 7A C). Typically, the CMSP reaction product was found scattered throughout the cytoplasm, with local concentrations around dilated mitochondria dispersed throughout the neuronal cell body. In contrast, neurons demonstrating severe ultrastructural alterations failed to show CMSP immunoreactivity (Fig. 7A,B). Evidence for membrane resealing after DTBI In contrast to those neurons demonstrating enduring membrane alteration as evidenced by their content of both dextrans, at 4 h, 39.1% 6.4 and at 8 h, 24.3% 9.4 of cortical neurons, showing any evidence of tracer uptake, flooded with the preinjury infused dextran alone, thereby suggesting membrane resealing (Fig. 8A C). With confocal microscopy, these neurons displayed normal cellular detail, an observation consistent with post-tbi membrane

126 3136 J. Neurosci., March 22, (12): Farkas et al. The Consequences of Mechanoporation in DTBI Figure 7. A C, A toluidine blue-stained 1- m-thick section reacted for the visualization of CMSP is shown in A, whereas B and C reveal an EM of the same neurons. Note that in B, one CMSP-immunopositive neuron demonstrates moderate subcellular damage. Note that C, taken fromthatareablockedoffinb, revealsthecmspreactionproductaroundswollenmitochondria (arrows) and dispersed throughout the cytoplasm (double arrowheads). A neuron demonstrating overt necrosis with no evidence of CMSP is also shown (arrowhead). Scale bar, 5 m. resealing and cell survival. Parallel ultrastructural analyses failed to reveal significant pathological change within these neurons (Fig. 8F,G). Furthermore, at 4 and 8 h respectively, 7.6% 5.5 and 11.1% 2.9 of these resealed neurons showed evidence of calpain-mediated spectrin proteolysis. Evidence for delayed membrane perturbation after DTBI Despite the consistent finding of neurons containing the initially infused tracer without any delayed tracer flooding, at 4 h, 5.5% 2.7 and at 8 h, 20.9% 9.1 of all tracer-containing neurons were found to flood with the postinjury infused dextran alone (Fig. 9A C). At 4 h, 55.1% 12.9 and at 8 h, 32.5% 11.8 of neurons demonstrating delayed membrane perturbation also showed CMSP. Importantly, all of the above-described populations of neurons could be found in the same neocortical loci, excluding the possibility that different regions exposed to different rates of dextran diffusion could have sustained different degrees of dextran flooding. Redistribution of neuronal membrane perturbation after DTBI As noted above, neurons scattered throughout the neocortex exhibited varying degrees of membrane perturbation and cellular pathology after DTBI. In the present study design, three categories of neurons with membrane perturbation were detected: (1) resealed neurons, (2) neurons with enduring membrane perturbation, (3) neurons with delayed membrane damage. Similarly, the experimental design separated each survival group (4 and 8 h after injury) into periods before and after the availability of the postinjury tracer. In this way, the relative proportions of neurons with resealed, enduring, and delayed membrane perturbations could be analyzed in relationship to time periods of tracer availability, to provide a model of membrane perturbation pathology after DTBI (Fig. 10). Of all the neurons that suffer membrane perturbation by 4 h after injury, 95% are damaged within the first 2 h. However, 40% of the neurons reseal by 2 h, leaving 60% with enduring or delayed membrane perturbations at 4 h after injury (Fig. 10). From the 8 h experimental group, 80% of all neurons with membrane perturbation suffer damage within the first 6 h, with 25% of the neurons resealing before the postinjury tracer administration at 6 h, and 75% of the population manifests enduring or delayed membrane perturbation (Fig. 10). Because the percentages of the population are normalized and derived from the same data set, statistical tests cannot be performed within a time point. Between time points, the proportion of neurons with delayed membrane perturbation differs significantly from the proportion of resealed neurons and the proportion of enduring membrane perturbation ( 2, p 0.016). The change in the proportion of neurons with resealed and enduring membrane perturbation over time was not significant ( 2, p 0.19). Together, these tests confirm a significant redistribution of the neuronal population with membrane perturbations between 4 and 8 h. These analyses were based on fractions of all neurons that demonstrate perturbation, but the total populations were not necessarily equal. Discussion Using tracer exclusion and immunohistochemical techniques, together with confocal and electron microscopy, the current study provides new evidence for the diversity of the neuronal cellular responses to DTBI, emphasizing the significance of neuronal membrane mechanoporation in DTBI pathobiology. Membrane disruption and enduring membrane permeability The present study is the first to demonstrate, in an in vivo model of DTBI, the potential for either enduring membrane perturbation progressing to cell death or neuronal membrane resealing compatible with recovery. These neuronal changes were both widespread and diffusely distributed, interspersed with apparently uninjured neurons, a finding consistent with the pathobiology of DTBI in humans (Povlishock and Katz, 2005). These data emphasize that the mechanical forces initiate the observed pathology. The early, diffuse damage identified is not consistent with focal ischemia that would have precipitated a more delayed and highly focal neuronal response (Dietrich et al., 1987; Dereski et al., 1993; Dietrich, 1998; Mennel et al., 2000; Bramlett and Dietrich, 2004). Although in this study, DTBI evoked elevated ICP, this elevation never exceeded 20 mmhg, suggesting no significant alteration in cerebral perfusion pressure with the normal arterial blood pressure described in this study. This does not preclude, however, the potential that over time the elevated ICP evoked secondary neuronal damage (vide infra). Using lactate dehydrogenase (LDH) release or highmolecular-weight tracer uptake to evaluate membrane damage in various TBI models, our laboratory and others have established that membrane disruption can occur at the moment of injury, suggesting that the traumatic event evokes direct mechanical poration of the neuronal cell membrane (Pettus et al., 1994; LaPlaca et al., 1997; Geddes and Cargill, 2001; Geddes et al., 2003; Prado and LaPlaca, 2004; Prado et al., 2004; Singleton and Povlishock, 2004). In these studies, it was suggested that the altered neuronal membrane permeability generated ionic disturbances to precipitate cell death. This premise, however, was neither directly addressed nor followed comprehensively via multiple endpoints (LaPlaca et al., 1997; Geddes et al., 2003; Prado et al., 2004). The

127 Farkas et al. The Consequences of Mechanoporation in DTBI J. Neurosci., March 22, (12): Figure 8. A C, Confocal images of preinjury-infused dextrans (A), postinjury-infused dextrans (B), and their overlay (C) demonstrate some cortical neurons flooding with the preinjury-infused dextran alone without concomitant flooding with the postinjury-administrated tracer (arrows). Note that one double-flooded neuron demonstrating severe damage (arrowhead) and one double-labeled neuron demonstrating less severe pathology (double arrowhead) are also shown. Scale bar, 50 m. D,A routine fluorescent image that reveals a single labeled cell. E, The same neuron as in D is visualized through the use of antibodies tothefluorophoreandthencarriedtotheemlevel(f, G). Notethatneuronsfloodingwiththepreinjurydextranalonedonotshow overt pathological damage. Immunoreactive products (anti-alexa Fluor IR) are labeled with arrows in G, which is an enlargement of the area, blocked out in F. Scale bar, 2 m. Figure 9. A C, Confocal images of preinjury-infused dextran (A), postinjury-infused dextran (B), and their overlay (C) demonstrate scattered neurons flooding with the postinjury-infused dextran alone (arrows), among with double-flooded neurons (arrowheads). Scale bar, 100 m. current study significantly extends previous observations, demonstrating that membrane perturbation persists for, at least, several hours after injury in many injured neurons. Significance is also attached to the LM observation that these neurons revealed morphological features consistent with irreversible damage, including cellular distortion, vacuolization, and pyknotic nuclei. The parallel EM findings of increased electron density, together with the dilation of the Golgi and SER and increased numbers of dilated vesicles, were also consistent with a necrotic cascade triggered by the translocation of lysosomal cathepsins and other hydrolytic enzymes (White et al., 1993; Chan, 1996; Yamashima et al., 1998; Yamashima, 2004). However, we cannot rule out that this organelle and vesicular dilation are linked to an albeit abortive attempt at membrane repair mediated by the lysosomes and the synaptotagmin found on their surfaces (Andrews, 2005). These issues will require highly targeted investigations for their resolution. Although our finding of isolated neurons demonstrating enduring membrane permeability without severe ultrastructural damage at both 4 and 8 h after injury suggests that membrane perturbation may not always lead to cell death, we cannot preclude the possibility that with increased survival these same neurons would die. Membrane resealing In addition to enduring membrane alteration, other neurons displayed evidence of membrane resealing via their uptake of only the preinjury tracer. Previous studies in nonneuronal cells demonstrated that, after mechanical injury, membrane resealing can occur within seconds to minutes (McNeil and Ito, 1989; McNeil and Khakee, 1992; Yu and McNeil, 1992; Steinhardt et al., 1994; Terasaki et al., 1997; McNeil et al., 2000). These studies investigated cardiac muscle (Clarke et al., 1995), skeletal muscle (McNeil and Khakee, 1992), skin (McNeil and Ito, 1990), vascular endothelium (Yu and McNeil, 1992), or gastrointestinal cells (McNeil and Ito, 1989), which frequently experience mechanical stress/strain under normal conditions. To date, only limited studies have investigated membrane resealing in the CNS. In vitro or nonmammalian models have focused primarily on resealing after axonal transection (Yawo and Kuno, 1983, 1985; Fishman et al., 1990; Xie and Barrett, 1991; Godell et al., 1997; Shi and Pryor, 2000; Shi et al., 2001; Geddes et al., 2003). In contrast to non-neuronal cells, these studies suggested that neuronal cell membrane resealing takes minutes to hours rather than seconds described in non-neuronal cells. Our finding of neurons labeled with the preinjury tracer alone is consistent with membrane repair; however, the sampling period occurred over several hours after injury precluding the determination of whether this resealing was immediate or rather occurred at a later time point. Delayed membrane perturbation Our observation of neurons flooded with the postinjury infused dextran alone suggests the potential for delayed membrane disruption occurring several hours after injury. The precise mechanism(s) for this delayed opening is (are) unknown; however, the possibility exists that the sustained, elevated ICP occurring after injury could alter the neuronal cell membrane. This issue is of more than mere academic interest, because sustained elevated ICP is one of the major adverse prognostic factors for recovery after TBI and obviously this issue will merit continued evaluation (The Brain Trauma Foundation et al., 2000).

128 3138 J. Neurosci., March 22, (12): Farkas et al. The Consequences of Mechanoporation in DTBI Figure 10. Distribution of neurons with resealed, enduring, or delayed membrane perturbation within the population of neurons with DTBI-induced membrane perturbation. As membranes remain closed, open, or reseal in response to injury, tracer availability will determine the type of membrane perturbation categorically assigned to each neuron (table within figure legend). The preinjury administration of Alexa Fluor 488 and postinjury administration of Texas Red-conjugated dextrans permit the evaluation of neuronal membrane perturbation distribution at 4 and 8 h after injury. Across time points, the proportion of neurons with delayed membrane perturbation is significantly different from the proportion of resealed neurons and the proportion of enduring membrane perturbation ( 2, p 0.016). The change in the proportion of neurons with resealed and enduring membrane perturbation over time was not significant ( 2, p 0.19). The redistribution of the types of membrane perturbation between 4 and 8 h after injury may result from resealed neurons reopening to become neurons with enduring damage and/or additional neurons suffering delayed membrane perturbation. See Results for more details. The coincidence of CMSP with altered membrane permeability The present study also examined the potential role of calpain activation and its relation to membrane permeability and/or neuronal damage. Calpain activation and the subsequent CMSP have long been associated with the pathogenesis of different brain injuries and TBI cell death (Kampfl et al., 1996, 1997; Saatman et al., 1996; Newcomb et al., 1997; Pike et al., 1998, 2000, 2001; Buki et al., 1999; McCracken et al., 1999; Kupina et al., 2002, 2003). Calpain activation occurs at elevated intracellular calcium levels. Membrane perturbation can lead to excessive calcium influx from the extracellular space, resulting in calpain activation (Povlishock et al., 1997; Buki et al., 1999), that then causes direct subcellular damage or lysosome membrane rupture with the release of damaging enzymes such as cathepsins (Yamashima, 2004). The cysteine proteases, such as calpain and caspase, can also maintain increased membrane permeability, theoretically permitting additional pathology (Liu and Schnellmann, 2003; Liu et al., 2004). Furthermore, it has been demonstrated, in vitro, that CMSP correlates with LDH release and propidium iodide uptake, markers of increased membrane permeability after stretch injury (Pike et al., 2000). Other studies, however, have also suggested that calpain might have an important role in membrane resealing (Godell et al., 1997; Howard et al., 1999; Shi et al., 2000). In the present study, the majority of cells demonstrating CMSP did not reveal tracer flooding either at 4 or 8 h after injury, suggesting that calpain activation occurs independent of cell membrane disruption. Furthermore, the majority of CMSPpositive neurons revealed only limited ultrastructural change, suggesting that calpain activation is not necessarily associated with cell death after DTBI, at least within the time frames assessed. Alternatively, the initiation of neuronal death may await the activation of a secondary calpain cascade, a premise supported by several studies (Saido et al., 1993; Yokota et al., 1995; Neumar et al., 2001; Saatman et al., 2003; Czogalla and Sikorski, 2005). Equally surprising was the finding that the majority of cells flooding with both dextrans and manifesting necrotic change did not show CMSP. Conceivably, the calpain-specific spectrin breakdown products in the necrotic neurons were cleaved before the assessment, yet, this argument is countered by the fact that spectrin fragments are highly stable in vivo and in vitro (Wang et al., 1989; Czogalla and Sikorski, 2005). Neurons triple labeled with both dextrans and Ab 38 were observed, yet they represented the smallest population of injured neurons. Collectively, these results suggest that the observed membrane disruption is not necessary or sufficient for calpain-mediated spectrin proteolysis. Together with other studies (Saatman et al., 1996; Brana et al., 1999), these findings emphasize the need for caution in interpreting the occurrence of CMSP and its overall implications for neuronal injury and death. Redistribution of neuronal membrane perturbation after TBI In addition to the above provocative information regarding membrane perturbation, resealing, and delayed opening, the shifts over time in the percentages of neuronal populations showing membrane damage provide compelling evidence for a redistribution of the membrane damaged population over time. Specifically, the fraction of neurons that reseal early postinjury may suffer secondary membrane reopening, converting these neurons to those linked with enduring membrane perturbation. Furthermore, with increased survival, proportionally more neurons suffer delayed membrane perturbation. Theoretically to achieve such a distribution from 4 8 h after injury, 25% of the resealed neurons would be required to reopen, with an additional 20% of the previously uninjured neurons now suffering delayed membrane perturbation. 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131 Acta Neurochir (Wien) (2005) 147: DOI /s Clinical Article Spectrin breakdown products in the cerebrospinal fluid in severe head injury preliminary observations O. Farkas 1;3, B. Polgár 2, J. Szekeres-Bartho 2,T.Doczi 1, J. T. Povlishock 3, and A. Büki 1 1 Department of Neurosurgery, Center for Medical and Health Sciences, Pecs University, Pecs, Hungary 2 Clinical Microbiology and Immunology, Center for Medical and Health Sciences, Pecs University, Pecs, Hungary 3 Department of Anatomy and Neurobiology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA Received May 19, 2004; accepted April 28, 2005; published online June 9, 2005 # Springer-Verlag 2005 Summary Background. Calcium-induced proteolytic processes are considered key players in the progressive pathobiology of traumatic brain injury (TBI). Activation of calpain and caspases after TBI leads to the cleavage of cytoskeletal proteins such as non-erythroid alpha II-spectrin. Recent reports demonstrate that the levels of spectrin and spectrin breakdown products (SBDPs) are elevated in vitro after mechanical injury, in the cerebrospinal fluid (CSF) and brain tissue following experimental TBI, and in human brain tissue after TBI. Methods. This study was initiated to detect spectrin and SBDP accumulation in the ventricular CSF of 12 severe TBI-patients with raised intracranial pressure (ICP). Nine patients with non-traumatically elevated ICP and 5 undergoing diagnostic lumbar puncture (LP) served as controls. Intact spectrin and calpain and caspase specific SBDPs in CSF collected once a day over a several day period were assessed via Western blot analysis. Parameters of severity and outcome such as ICP, Glasgow Coma Scale and Glasgow Outcome Scale were also monitored in order to reveal a potential correlation between these CSF markers and clinical parameters. Results. In control patients undergone LP no immunoreactivity was detected. Non-erythroid alpha-ii-spectrin and SBDP occurred more frequently and their level was significantly higher in the CSF of TBI patients than in other pathological conditions associated with raised ICP. Those TBI patients followed for several days post-injury revealed a consistent temporal pattern for protein accumulation with the highest level achieved on the 2 nd 3 rd days after TBI. Conclusion. Elevation of calpain and caspase specific SBDPs is a significant finding in TBI patients indicating that intact brain spectrin- and SBDP-levels are closely associated with the specific neurochemical processes evoked by TBI. The results strongly support the potential utility of these surrogate markers in the clinical monitoring of patients with severe TBI and provide further evidence of the role of calcium-induced, calpain- and caspase-mediated structural proteolysis in TBI. Keywords: Traumatic brain injury; calpain; caspase; spectrin; human; cerebrospinal fluid. Introduction Traumatic brain injury (TBI) is the primary cause of death in the population under the age of 40 representing an extreme challenge for individuals and families affected as well as the society at large [10]. Thus, understanding the pathobiochemical processes evoked by or operant in TBI is integral to developing new therapeutic strategies while exploring potential biochemical markers that can help assess the severity of the injury and predict outcome. Traumatic brain injuries are typically classified on whether the primary damage evokes more localized brain damage and=or mass lesion formation (focal injury), or more scattered, non-focal injuries typically involving scattered axonal and=or microvascular damage (diffuse injury). Although focal and diffuse injuries can be evoked by different mechanism and their clinical manifestations can be different, several studies to date have shown that similar biochemical cascades take part in the pathobiology of these different injury types. These studies have demonstrated increased pathological activation of calpain and=or caspase-3 both after focal TBI [8, 9, 12, 16] and diffuse injuries [5, 6, 18]. Calpain and caspase-3, both members of cysteine protease family, have also been shown to play an important role in the proteolytic cascades associated with several other central nervous system disorders such as stroke, hypoxiaischemia [3, 13], experimental hydrocephalus [7] and spinal cord injury [1]. Non-erythroid alpha II-spectrin, major component of the cytoskeleton, constitutes a

132 856 O. Farkas et al. substrate for both calpain and caspase-3 [21]. Calpainmediated cleavage of intact spectrin (280 kda) results in 150 and 145 kda-fragments specific for calpain, whereas the caspase-3-specific products are linked to 150 and 120-fragments [20]. Using antibodies targeting alpha II-spectrin breakdown products numerous recent reports have demonstrated that non-erythroid alpha II-spectrin and spectrin breakdown products (SBDPs) are elevated in vitro after mechanical injury [15, 4], also being found in the cerebrospinal fluid and brain following experimental TBI [16, 12, 8, 18], as well as human brain tissue post TBI [11]. The goal of the current study was to further define the role of calcium-induced, calpain- and caspase-mediated structural proteolysis in the pathobiology of TBI, identifying potential biomarkers specifically associated with the pathological processes evoked by TBI and thereby purportedly capable of predicting the severity of the initial injury and outcome to help in the better clinical management of severely brain-injured patients. In this study, the levels of non-erythroid alpha IIspectrin and SBDPs in the CSF of TBI patients sustaining severe injury were compared to two control groups: one with other disorders associated with elevated intracranial pressure (ICP) and the other with normal ICP. Clinical parameters of severity and outcome such as intracranial pressure, Glasgow Coma Scale and Glasgow Outcome Scale were monitored. Materials and methods The study included twelve patients with severe TBI (GCS< 9). The first control group consisted of patients with comparably raised ICP not associated with TBI and included 3 patients with subarachnoid haemorrhage (SAH), 3 with intraventricular haemorrhage (IVH), and 3 with brain tumours. The second control group consisted of 5 patients undergoing diagnostic LP that subsequently proved negative for SAH and=or meningitis (clinical data of all patients are presented in Table 1). All CSF harvesting was part of routine patient management, in accordance with institutional guidelines; the study protocol was approved by the Regional Ethics Committee (NIH-approved Institutional Review Board) of the Centre of Medical and Health Sciences of Pecs University. In the traumatically brain injured group all patients were equipped with intraventricular catheters for the control of ICP. Via these catheters ventricular CSF samples were collected once a day, in some patients for several days following ventriculostomy. They were centrifuged at 6000 g for 6 min and stored at 80 C. After defrosting samples were washed in fivefold PBS on Amicon ultrafiltration cell using polyethersulfone ultrafiltration membranes with 100 kda nominal molecular weight limit. During this procedure the samples were purified from serum albumin and other proteins with less than 100 kda molecular weight and concentrated to about five-fold. Protein concentrations were determined using Bio-Rad Protein Assay. Samples containing less than 1mg=ml protein were dried by Heto dry Winner. The dehydrated substances were dissolved in distilled water to get a protein concentration of 1mg=ml. Sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli using a mini-gel apparatus (Bio-Rad). Samples containing 20 micrograms of Table 1. Group Age Sex Diagnosis CT GCS o.a. ICP or OP Date GOS Date T-1 41 M SDH þ Cont 4 35 Hgmm D T-2 79 FM Cont IVH 4 20 Hgmm D T-3 28 M Cont IVH 5 20 Hgmm D T-4 48 M SDH 4 25 Hgmm D T-5 51 M EDH þ Cont 6 10 Hgmm D T-6 21 M Cont 7 20 Hgmm D1 4 9 T-7 51 M SDH þ Cont 5 40 Hgmm D T-8 68 M SDH 6 20 Hgmm D T-9 51 FM Cont IVH 4 40 Hgmm D1 1 3 T M EDH 4 35 Hgmm D T M SDH þ Cont 6 35 Hgmm D T FM EDH þ Cont 5 10 Hgmm D C FM SAH HC 9 20 Hgmm D C M SAH HC 8 15 Hgmm D C FM SAH OE 4 30 Hgmm D C FM SAH IVH Hgmm D C M SAH IVH þ HC 6 20 Hgmm D C M IVH HC 7 40 Hgmm D2 1 7 C FM Tu. V-3 HC 9 25 Hgmm D C FM Tu. V-4 HC 8 10 Hgmm D C M Tu. V-4 HC Hgmm D C M MGITIS? Neg Hgmm NA NA NA C FM SAH? Neg Hgmm NA NA NA C M MGITIS? Neg Hgmm NA NA NA C M MGITIS? Neg Hgmm NA NA NA C FM SAH? Neg Hgmm NA NA NA

133 Spectrin breakdown products in the CSF in severe head injury preliminary observations 857 protein (equal loading from all samples) were electrophoresed in 6.5% Tris=glycine gels. Next, separated proteins were transferred to nitrocellulose membranes. Blots were blocked for 1 hour in 1% non-fat milk in TBS with 0.1% Tween-20 (TBST). The presence of non-erythroid alpha II-spectrin and its breakdown products were examined with monoclonal mouse anti-spectrin antibody (Chemicon) capable of detecting intact non-erythroid alpha II-spectrin (280 kd) and 150, 145 and 120 kd cleavage fragments. (These proteins in the CSF detected by the antibody are exclusively derived from the central nervous system as erythroid spectrin is not recognized by the antibody applied. This premise excludes the possibility of false positive reactions due to intraventricular haemorrhages and focal lesions leading to the accumulation of erythroid spectrin in the CSF.) Following 1 hour incubation at room temperature with this primary antibody (1:500 in 1% non-fat milk TBST) blots were incubated with biotinylated anti-mouse Ig (Amersham) in 1:400 dilution in 1% non-fat milk TBST for 1 hour at room temperature, next were incubated with Streptavidin-Biotinylated Horseradish Peroxidase Complex (Amersham, 1:1000) for half an hour at room temperature. Western Lighting TM Chemiluminescence reagents were used to visualize immunolabelling on Kodak films. Semi-quantitative evaluation of detected proteins was performed by computer-assisted densitometric scanning (Bio-Profile Bio-1Dþþ, Vilber Lourmat). Data were normalized to background density as well as negative control reacted without the presence of the antigens. Densitometric data gained from CSF samples of TBI-patients and controls was compared with an independent samples t-test. Differences were assumed significant at a level of p Correlation between protein levels and clinical parameters was tested by linear regression. Results Thirty-four CSF samples from the 12 brain-injured patients in addition to 15 samples from 9 control patients were evaluated in the current investigation (Table 1). Analysis of all protein bands examined demonstrated that intact (280 kda) spectrin as well as the 120 kda breakdown product was present in significantly higher percentage of TBI-patients than in patients with raised ICP of different etiologies (Table 2). The CSF samples from patients undergoing routine, diagnostic LP (second control group) contained neither intact spectrin nor SBDP. Selected patients (on Table 1 these patients could be identified as T-1 and T-2) with severe TBI were followed for several days to explore the time course of spectrin release in CSF. One patient (GCS ¼ 4, GOS ¼ 2, ICP ¼ 35 post admission) revealed a peak of both intact spectrin and SBDPs on the third day (Table 3 and Fig. 1a and c). The density of all proteins decreased to the baseline levels by the day 7 8 followed by another increase in the density of 150 kda spectrin at the 8 th day post-injury. Similarly, another patient (GCS ¼ 4, GOS ¼ 2, ICP ¼ 20 at admission) had a peak of SBDPs at the second day followed by a density decrease, however by the fourth day postinjury another increase was observed in the 150 kda spectrin level as well as in the density of intact and 145 kda spectrin (Table 3 and Fig. 1b and c). Independent samples t-test was used to compare the density of protein bands taken from head injured patients versus those suffering from other central nervous system disorders (clinical data of all patients are summarized in Table 1). One sample from each patient, taken on the first Table 2. Band (kda) Occurrence in trauma 95% CI value Occurrence in control 95% CI value Significance % 26.7% 11% 20.4% % 88% 21.2% % 24.5% 44% 32% % 24.5% 22% 27% Table 3. Date Band 280 kda 280 kda 150 kda 150 kda 145 kda 145 kda 120 kda 120 kda patient 1 patient 2 patient 1 patient 2 patient 1 patient 2 patient 1 patient 2 12 hrs hrs days days days days days days days days

134 858 O. Farkas et al. Fig. 1. Western blots ((a) patient 1, (b) patient 2) and charts (c) indicating time course in densitometric units of the accumulation of nonerythroid alpha II-spectrin and its breakdown products in ventricular CSF of two severe head injured patients. (solid line: patient 1, dotted line: patient 2, &: 280 kda intact spectrin, ^: 150 kda SBDP, ~: 145 kda SBDP, : 120 kda SBDP) to third day post-injury, was analyzed based upon the observations described above regarding the time course of SBDP levels in ventricular CSF. In those TBI patients, where multiple samples were available, maximal levels have been considered. In the first control group, where ventriculostomy was performed to decrease ICP primarily evoked by occlusive or malresorptive hydrocephalus and oedema, maximal levels detected at the insertion of the ventricular drainage were considered. The result of the densitometric analysis and statistical comparison is summarized in detail in Table 4. In sum, Table 4. Band Diagnosis Mean density SEM P Significance 280 kda TBI Control-I kda TBI Control-I kda TBI Control-I kda TBI Control-I Fig. 2. Characteristic Western blots (a) and bar charts (b) indicating ventricular CSF level of non-erythroid alpha II-spectrin and its breakdown products in TBI and non-injured control patients with other pathological conditions associated with raised ICP. (Blots from patients underwent diagnostic lumbar punction are not shown.) Asterisks indicate significance of difference, error bars represent standard error of means, data derive from TBI and non-injured control patients, 34 versus 15 samples, respectively). P < 0.05