Reversible MCAO without craniectomy in rats.

Stroke is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX 72514 1989;20;84-91 Stroke EZ Longa, PR Weinstein, S Carlson and R Cummins Reversible middle cerebral artery occlusion without craniectomy in rats 84 Reversible Middle Cerebral Artery Occlusion Without Craniectomy in Rats Enrique Zea Longa , MD, Philip R. Weinstein , MD , Sara Carlson, BS , and Rober t Cummins , MS To develop a simple, relatively noninvasive small-animal model of reversible regional cerebral ischemia, we tested various methods of inducing infarction in the territory of the right middle cerebral artery (MCA) by extracranjal vascular occlusion in rats. In preliminary studies, 60 rats were anesthetized with ketamine and different combinations of vessels were occluded; blood pressure and arterial blood gases were monitored. Neurologic deficit, mortality rate, gross pathology, and in some instances, electroencephalogram and histochemical staining results were evaluated in all surviving rats. The principal procedure consisted of introducing a 4-0 nylon intraluminal suture into the cervical internal carotid irtery (ICA) and advancing it intracra- nially to block blood flow into the MCA; collateral blood flow was reduced by interrupting all branches of the external carotid artery (EC A) and all extracranial branches of the ICA. In some groups of rats, bilateral vertebral or contralateral carotid artery occlusion was also performed. India ink perfusion studies in 20 rats documented blockage of MCA blood flow in 14 rats subjected to permanent occlusion and the restoration of blood flow to the MCA territory in six rats after withdrawal of the suture from the ICA. The best method of MCA occlusion was then selected for further confirmatory studies, including histologic examination, in five additional groups of rats anesthetized with halothane. Seven of eight rats that underwent permanent occlusion of the MCA had resolving moderately severe neurologic deficits (Grade 2 of 4) and unilateral infarcts averaging 37.6±5.5% of the coronal sectional area at 72 hours after the onset of occlusion. Five rats underwent the same procedure after bilateral vertebral artery occlusion was performed to reduce collateral blood flow. Only two of these five rats survived 72 hours; the neurologic deficits progressed from Grade 2.5 to 3, and the infarcts were larger than after MCA occlusion alone. In two groups of rats, the suture was withdrawn from the ICA to permit reperfusion after 2 or 4 hours of ischemia. Five of 10 rats subjected to 4-hour temporary MCA occlusion and one of six rats subjected to 2-hour temporary MCA occlusion did not survive 72 hours after the onset of occlusion. Infarct areas in surviving rats after 2-hour temporary MCA occlusion were 15.7% smaller than after permanent MCA occlusion, but the neurologic deficit was not significantly reduced by reperfusion. Fatal intracranial hemorrhage occurred in only two of 71 rats after occlusion of the MCA with an intraluminal suture. The results in the six sham-occluded rats showed that occlusion of the extracranial carotid branches, dissection of the cervical ICA, and placement of an intraluminal suture in the ECA did not produce stroke. This model provides a reliable method for studying reversible regional ischemia in rats without craniectomy. (Stroke 1989;20:84-91) The pathophysiology o f cerebral ischemia hasbeen studie d extensivel y i n rat s wit h vari -ous methods, including multiple vessel occlu- sion, hypotension , an d hypovolemia , t o produc e From the Department of Neurological Surgery , School of Med- icine, Universit y o f California , Sa n Francisco ; Sa n Francisc o Veteran's Administration Hospital ; and the Microsurgery Labora- tory, Ralph K. Davies Medical Center, San Francisco, California . Supported by the Veterans Administration Research Service . Address for correspondence: Philip R. Weinstein, MD, Depart- ment of Neurological Surgery , The Editorial Office , 136 0 Ninth Avenue, Suite 210, San Francisco, CA 94122 . Received Augus t 1 , 1987; accepted Jul y 7, 1988 . global alteratio n i n cerebra l bloo d flow an d metabolism.1-8 The searc h for a reliable, less inva - sive ra t strok e mode l o f temporar y regiona l isch - emia ha s bee n prompte d b y th e extensiv e neuro - chemical dat a alread y availabl e i n rats , th e rising cost o f experiment s wit h large r animals , an d th e limitations of other roden t models of focal cerebra l ischemia, suc h as occlusion of the common caroti d artery (CCA)9 or middle cerebral artery (MCA) 10 in gerbils. A direct microsurgical technique for perma- nently occludin g th e MC A throug h a craniec - tomy11-13 and an indirect method of producing hemi- Zea et al MCA Occlusion Without Craniectomy 85 spheric ischemia and reperfusion14 i n rats have also been described . We sought to develop a model of reversible regional cerebral ischemia in rats without craniectomy base d on advancing an intraluminal suture from the internal carotid arter y (ICA ) t o occlud e th e origi n o f th e MCA.13 We also tested the effects o f varying suture size, rat weight, and alternative methods of reducing collateral circulatio n t o supplemen t th e effec t o f MCA occlusion. We describe our preliminary exper- iments t o selec t th e optimal surgica l procedure an d present th e neurologic , electroencephalographi c (EEG), and pathologi c findings we obtained i n con- firmatory studies with this preparation . Materials and Method s Adult mal e Sprague-Dawle y rat s weighin g 400 - 500 g wer e anesthetize d wit h 80-10 0 mg/k g i.p . ketamine hydrochlorid e an d 5 mg/kg i.p . acepro - mazine maleate . A PE-50 cathete r wa s introduce d into the femoral artery for continuous monitoring of arterial bloo d pressur e an d samplin g o f bloo d fo r analysis of blood gases and hemoglobin concentra - tion. The rats' body temperature was maintained a t 37° C with an infrared hea t lamp and a heating pad. In on e grou p o f rats , monopola r EE G recording s were obtaine d wit h transcutaneou s needl e elec - trodes place d 3 mm lateral to the sagitta l sutur e in the parieta l an d fronta l regions ; a referenc e elec - trode wa s placed adjacen t t o the nasion . A Neuro- trac EEG recorder and spectral analyzer (Interspec, Conshohocken, Pennsylvania ) or a Grass Model 79 polygraph (Quincy, Massachusetts) with P511 ampli- fiers an d a n A.R . Vette r Co . C- 4 tap e recorde r (Rebersburg, Pennsylvania ) wer e used . Th e filter band pas s setting s wer e 0.3-300 Hz . The vascula r occlusiv e procedure s performe d i n each o f eigh t group s o f rat s i n th e preliminar y studies ar e show n i n Tabl e 1 . Groups 6 , 7 , an d 8 were included t o control for possible hemodynami - cally significan t effect s o f occludin g th e vertebra l artery o r external caroti d arter y (ECA) . The basic surgica l procedure consisted o f block- ing bloo d flow int o th e MC A wit h a n intralumina l suture introduce d throug h th e extracrania l ICA . Additional extracrania l vessels , includin g th e lef t ICA, right ECA , an d bot h vertebra l arteries , wer e occluded t o reduc e collatera l bloo d flow t o th e MCA territory . Under th e operatin g microscope , th e right CC A was expose d throug h a midlin e incision ; a self - retaining retracto r wa s positione d betwee n th e di - gastric and sternomastoid muscles , and the omohy- oid muscl e wa s divided . Th e occipita l arter y branches o f the EC A were then isolate d an d coag - ulated (Figur e 1 , left) . Next , th e superio r thyroi d and ascendin g pharyngea l arterie s wer e dissecte d and coagulated . Th e EC A wa s dissecte d furthe r distally an d coagulate d alon g wit h th e termina l lingual an d maxillar y arter y branches , whic h wer e then divided . Th e IC A wa s isolate d an d carefull y TABLE 1 . Experimenta l Groups , Reversibl e Middl e Cerebra l Artery Occlusion Without Craniectomy in Rats Group Preliminary 1 2 3 4 5 6 7 8 n studies (N=60) 8 8 12 8 6 6 6 6 Confirmatory studies (N=34) A B C D E 5 8 6 10 5 Procedure RMCO R MCO+L ICO R MCO 24 hr after B VO 4-hr R MCO 24 hr after B VO 2-hr R MCO B VO ECO ECO 24 hr after B VO Suture in ECO (sham operation) Permanent R MCO 2-hr R MCO 4-hr R MCO Permanent R MCO immediately afte r B VO R MCO, right middle cerebral artery occlusion by intraluminal suture; L ICO , lef t interna l caroti d arter y occlusio n b y intralu - minal suture ; B VO , bilatera l vertebra l arter y occlusio n b y transection of terminal branches and placement of an intralumi- nal suture; ECO, external carotid artery occlusion by intralumi- nal suture. Group 5 was used for electroencephalographic stud - ies only; Groups 6, 7, and 8 were control groups. separated fro m th e adjacen t vagu s nerve . Furthe r dissection identifie d th e ans a o f th e glossopharyn - geal nerv e a t th e origi n o f th e pterygopalatin e artery; this posteriorly directed extracranial branc h of the ICA was ligated wit h 7-0 nylon sutur e clos e to it s origin . A t thi s point , th e IC A i s th e onl y remaining extracranial branc h of the CCA . Next, a 6-0 sil k sutur e wa s tie d loosel y aroun d the mobilized EC A stump , and a curved microvas - cular clip was placed acros s bot h the CCA and th e ICA adjacent t o the EC A origin . A 5-cm lengt h of 4-0 monofilamen t nylo n suture , it s ti p rounde d b y heating near a flame, was introduced int o the EC A lumen throug h a punctur e o r throug h on e o f th e terminal branches of the ECA. (In some smaller rats in the preliminary studies , a 5-0 intraluminal sutur e was use d whe n initia l attempt s t o advanc e a 4- 0 suture proved unsuccessful.) Th e silk suture around the ECA stump was tightened around the intralumi- nal nylon suture to prevent bleeding, and the micro- vascular cli p wa s removed . Th e nylo n sutur e wa s then gentl y advance d fro m th e EC A t o th e IC A lumen; th e positio n o f th e sutur e withi n th e IC A lumen coul d b e see n a s i t reache d th e bas e o f th e skull. Afte r a variabl e lengt h o f nylo n sutur e ha d been inserte d int o the EC A stump , resistanc e wa s felt and a slight curving of the suture or stretching of the IC A was observed , indicatin g tha t th e blunte d tip o f th e sutur e ha d passe d th e MC A origi n an d reached th e proximal segmen t of the anterio r cere- bral arter y (ACA) , whic h ha s a smalle r diamete r (Figure 1 , right). A t thi s point , th e intralumina l 86 Stroke Vol 20, No 1, January 1989 StamomaatoM mus. Ant. cefQbraJ •. MM. cerebral a. FIGURE 1 . Left : Diagram of cerebrovascular anatomy in rats illustrates extracranial and intracranial vascular relations exploited in our method of reversible occlusion of middle (Mid.) cerebral artery (a.). Vessel size is disproportionately enlarged for clarity. Cervical dissec- tion is illustrated on the left. Common (Com.), external (Ext.), and internal (Int.) carotid arteries and their branches are shown. Right : Photograph of rat brain at autopsy showing intravascular suture, introduced through cervical internal carotid artery, within lumen of right anterior cerebral artery. Suture occludes origin of middle cerebral artery from intracranial internal carotid artery. suture ha s blocked th e origin o f the MCA , occlud - ing al l source s o f blood flow from th e ICA , ACA , and posterio r cerebra l arter y (PCA) . Th e incisio n was closed , leavin g 1 cm o f th e nylo n sutur e pro - truding s o i t coul d b e withdraw n t o allo w reperfu - sion. Restoration of MCA blood flow did not require anesthesia. Th e sutur e wa s pulled bac k unti l resis- tance was felt, indicating that the tip had cleared the ACA-ICA lume n an d wa s i n the E C A stump , and then trimmed . Bilateral vertebra l arter y occlusio n (BVO ) wa s performed usin g a modificatio n o f th e techniqu e described b y Pulsinell i an d Brierley. 16 Unde r a surgical microscope , th e ala r forame n wa s locate d at the lateral edg e of Cl an d enlarged wit h a dental drill. Each vertebra l arter y an d it s occipital branc h were coagulated wit h microbipola r cauter y forcep s and divided . In a subsequen t serie s o f experiment s t o confir m the optimum surgical technique, 34 younger rats weigh- ing 300-400 g were anesthetized b y inhalation of 1% halothane an d subjecte d t o on e o f five procedure s (Groups A-E, Table 1) . The occlusion technique was standardized by advancing a 4-0 suture, which had a rounded, slightl y large r tip , exactl y 1 7 mm int o th e ICA from th e origin of the ECA in each rat . Neurologic examination s wer e performe d ever y 12 hours in Groups 1-8 . I n Groups A-E, neurologi c examinations were performed 2,4 , and 8 hours afte r the onset of occlusion and then daily until sacrifice . The neurologic findings were scored on a five-point scale: a score of 0 indicated no neurologic deficit , a score o f 1 (failure t o exten d lef t forepa w fully ) a mild focal neurologic deficit, a score of 2 (circling to the left ) a moderat e foca l neurologi c deficit , an d a score of 3 (falling t o the left ) a severe foca l deficit ; rats wit h a scor e o f 4 did no t wal k spontaneousl y and had a depressed leve l o f consciousness . All survivin g rat s wer e kille d 7 2 hours afte r th e onset of occlusion, and the brains were removed and inspected to determine the position of the ICA suture. In 1 4 rats selected arbitrarily from Group s 1 , 2, and 3, an India ink solution was injected int o the ascend- ing aort a befor e sacrific e t o verif y obstructio n o f anterograde blood flow to the territory of the MCA; the same technique was used to verify reperfusion of the MCA afte r reversa l o f the occlusio n i n si x rat s from Group 4. The absence or presence of staining of the MCA and it s branches wa s considered proo f of Zea et al MCA Occlusion Without Craniectomy 87 TABLE 2. Neurologic Deficit Score and Infarct Size After Intrahunlnal Suture Occlusion of Middle Cerebral Artery in 15 Rats That Survived 72 Hours Neurologic score 2 h r 4 h r 8 h r 24hr 48hr 72 hr 2 2 0 1 1 1 Area ofischemic injury Each rat Group mean±S D 38. 1 1 1 1 1 1 Group B 2 2 2 2 2 1 2 2 1 1 1 1 1 2 2 1 1 1 on single coronal section 3 32. 6 34.3 37.6±5 36.2 .5 46.6 2 at optic 31.6 2 2 2 2 2 2 jroup 2 1 1 0 0 0 chiasm (%) 34.0 21 7.0 .9+14 C 2 1 1 2 1 1 5.1 .5 2 2 2 1 1 1 32.0 2 2 1 2 1 1 32.1 Group D 1 0 0 0 0 0 8.1 25 2 2 1 1 1 1 1 1 1 1 1 1 14.6 36. 7 7±13.4 2 2 1 1 1 1 37.0 Five rats fro m eac h group. Group B , permanent right middle cerebral arter y occlusion ; Group C, 2-hou r right middle cerebral artery occlusion; Group D, 4-hour right middle cerebral artery occlusion. Two rats in Group B were excluded fro m pathologi c analysis because of inadequate brai n fixation . MCA occlusio n o r reperfusion . Som e o f the brain s from th e rat s i n th e preliminar y studie s wer e sec - tioned coronally, incubate d fo r 6 0 minutes i n a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) at 37 ° C fo r vita l staining, 17-19 photographed , an d fixed b y immersio n i n 10 % formalin solution . Th e remaining brains wer e placed in 10 % formalin solu - tion for late r sectioning . In Group s A-E , th e brain s wer e fixed b y intra - cardiac perfusio n o f heparinize d 0.9 % salin e fol - lowed by 10 % formalin i n a 0.1 M phosphate buffe r (pH 7.4) , removed , an d store d i n fixative. Block s containing tissu e from th e anterio r to the posterio r edges o f th e corpu s callosu m wer e embedde d i n paraffin, cu t int o 6-/x m sections , an d staine d wit h hematoxylin an d eosin . Section s o f interes t wer e selected at 1-mm intervals and evaluated microscop- ically for ischemic tissue damage. Areas of neuronal injury o r infarctio n wer e plotte d o n tracing s fro m projections o f th e corona l sections . Th e are a o f neuronal injur y o r tissu e necrosi s wa s divide d b y the total area of the whole-brain corona l sectio n a s assessed b y pola r planimetr y t o obtain th e percen t infarcted are a i n eac h section . Averag e infarcte d areas fo r eac h ra t wer e calculate d usin g three sec - tions fro m eac h brain . Thi s analysi s showe d n o significant differenc e i n mean infarct area s between Groups B , C, an d D . Infarcte d area s take n fro m a single section of each brain at the level of the rostral edge of the opti c chiasm were then used to calculate the mea n an d standar d deviation s fo r eac h group . Nonparametric analysi s (Kruskal-Walli s test ) wa s used t o determin e significan t difference s i n infarc t size between groups . Results Cardiorespiratory functio n remaine d stabl e i n all rats i n the preliminar y experiments . Ther e wa s n o significant alteratio n in Pc^ (90-120 torr), Pco2 (28- 45 torr) , arteria l p H (7.30-7.35) , o r systoli c bloo d pressure (90-11 3 torr) durin g anesthesia; th e aver - age values were similar in all groups. The hemoglo- bin concentrations wer e 11-1 5 g% and varie d littl e within or between groups . EEG monitoring in the six rats in Group 5 showed a consisten t bilatera l decreas e i n amplitud e afte r 2-hour temporary right MCA occlusion. After occlu - sion, the mean±SD EEG amplitude had declined to 25.4±28.5% and 69.2±16.0% of the baseline value s in the ischemic and nonischemic hemispheres, respec- tively. Afte r 2 hours o f reperfusion , th e mean±S D TABLE 3. Pathologic Findings in 36 Rats in Preliminary Studies of Reversible Middle Cerebral Artery Occlusion Without Craniectomy in Rats Group 1 2 3 4 n 8 8 12 8 Procedure RMCO R MCO+L ICO B VO+R MC O 4-hr temporar y MC O after B VO Surviving Suture positio n or vessel patenc y 7 in ACA, 1 i n intracranial ICA 8 in ACA 10 in ACA No thrombosis in ICA rats TTC stain results 6 of 8 infarcte d 6 of 8 infarcte d 10 of 1 0 infarcted 1 of 7 infarcte d Dying rats Suture position — 1 in ICA, 1 perforated IC A 1 perforated AC A Two rats died of hemorrhage and one of pulmonary insufficiency. R MCO, right middle cerebral artery occlusion; L ICO , lef t interna l caroti d arter y occlusion ; B VO , bilatera l vertebra l arter y occlusion ; TTC , 2,3,5 - triphenyltetrazolium chloride ; ACA, anterior cerebra l artery ; ICA, internal carotid artery . 88 Strok e Vol 20, No 1, January 1989 i1Era HiEM B 0 P Zea et al MCA Occlusion Without Craniectomy V * • , ,' ' ''" * '• •'• f ' • .•'• » »r »; FIGURE 3. Left : Low-power photomicrograph (x.3.7) of infarct border zone in parasagittal region (arrow) shown in Figure 2, top right. Right : High-power photomicrograph (X18.5) of parasagittal infarct border (arrow) shown in Figure 2, bottom left. Note edema, nuclear pleomorphism, and pyknosis. EEG amplitudes were 36.4±25.9% of baseline in the ischemic hemisphere and 58.8± 16.3% in the nonisch- emic hemisphere . Thus , EE G amplitud e di d no t recover significantl y afte r reperfusion . I n on e rat , EEG amplitud e decrease d i n th e ischemi c hemi - sphere but was unchanged in the nonischemic hemi- sphere throughout occlusio n and reperfusion . Rats i n Group s 1 an d 2 ha d mil d neurologi c deficits a t 1 2 hours after th e onset of occlusion tha t resolved completel y b y 7 2 hours . I n Grou p 3 , moderate foca l deficit s (averag e scor e 2.3 ) wer e observed afte r ful l recover y fro m anesthesi a (1 2 FIGURE 2. Photomicrographs of coronal sections of rat brain removed 72 hours after onset of 4-hour temporary occlusion of middle cerebral artery with intraluminal suture. Neurologic deficit score of 2 was observed after 2 hours of reperfusion; at sacrifice, deficit had improved to score of 1. Infarct area in right hemisphere (arrows) measured