Neurological and Behavioral Outcomes of Focal Cerebral Ischemia in Rats

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1 67 Neurological and Behavioral Outcomes of Focal Cerebral Ischemia in Rats Florence Wahl, PharmD; Monique Allix, BS; Michel Plotkine, PhD; and Roger G. Boulu, PhD Downloaded from by on October, 8 Background and Purpose: The aim of this study was to investigate the neurobehavioral consequences of focal ischemia in rats. Methods: We induced permanent occlusion of the left middle cerebral artery in 4 Sprague- Dawley rats, and used 3 sham-operated rats as controls. During surgery, brain temperature and body temperature were kept at normothermia. Neurobehavioral studies (neurological examination, passive avoidance task, Y maze test, and modified open-field test) were carried out 4 days after ischemia before killing the rats to evaluate histological damage. Results: Ischemia induced large infarcts in the cortex (38.6±8.5 mm 3 ) and caudate-putamen (48.8±.6 mm 3 ) and, compared with sham-operated rats, produced a dramatic neurological deficit (p<.) characterized by sensorimotor dysfunctions and hemiplegia. Memory retention was significantly (p<.5) impaired in the passive avoidance task, but neither vigilance and exploratory behavior measured in the modified open-field test nor working memory evaluated in the Y maze test were disturbed. Infarct size was not correlated with the neurological or behavioral deficits. Conclusions: This lack of correlation indicates the necessity of carrying out parallel histological, neurological, and behavioral studies in any assays of new drugs using this model of focal ischemia. (Stroke 99;3:67-7) Occlusion of the left middle cerebral artery (MCA) in rats, first described by Tamura et al, is widely used to mimic focal cerebral ischemia in humans. Most studies on drug antiischemic activity using this model evaluate the drug's ability to reduce the volume of ischemic damage. 3 " 6 However, some reports have also described the functional consequences of focal ischemia using a simple, rapid neurological examination with grading scales for each symptom. 7-3 There have also been a few reports of memory dysfunction assessed using a step-through or passive avoidance test. 94 This report examines, using several neurobehavioral tests, the deficits present in the days following MCA occlusion. Our purpose was to evaluate more acutely the functional deficits induced by MCA occlusion, to determine whether these deficits correlate with histological damage, From the Laboratoire de Pharmacologie, Faculte des Sciences Pharmaceutiques et Biologiques, Universite Rene Descartes, Paris, France. Supported by grants from Rhone-Poulenc-Rorer, France, and the University Rene Descartes. Address for correspondence: Florence Wahl, Faculty des Sciences Pharmaceutiques et Biologiques, Laboratoire de Pharmacologie, 4, Avenue de 'Observatoire, 756 Paris, France. Received March 9, 99; accepted September 4, 99. and to indicate which neurobehavioral test(s) might be useful for assaying new anti-ischemic drugs. Materials and Methods The investigations were carried out in 7 male Sprague-Dawley rats weighing 8-3 g (Charles River, Cleon, France) divided into two groups: 4 MCA-occluded rats and 3 sham-operated rats. Each was anesthetized with 3 mg/kg i.p. chloral hydrate. Body temperature was maintained close to 37 C by a heating pad. Brain normothermia controlled by a thermocouple (Digi-Sense, Cole-Parmer Instrument Co., Chicago) inserted into the left temporalis muscle 5 was maintained by placing a 6-W lamp above the rat's head. The MCA was occluded using the technique of Tamura et al as modified by Shiraishi and Simon. 6 The scalp and temporalis muscle were reflected, and the zygomatic arch was removed. A subtemporal craniotomy beside the foramen ovale was performed using a dental drill. The proximal MCA was coagulated from its origin to its junction with the olfactory tract using microbipolar forceps. Virginiamycine (Smith Kline & French, Paris) was applied, the skin was sutured, and the rat was returned to its cage, which was warmed (4 C) for the night following surgery. The surgical procedure for

2 68 Stroke Vol 3, No February 99 TABLE. Neurological Examination Grading Scale for Rats Item Normal score Equilibrium Horizontal bar Inclined plane (time spent face up) Grasping reflex Righting reflex Head tilted On back Dropped Placing reactions Visual Leg hanging Motility Spontaneous Circling behavior Forelimb flexion Thorax twisting Global neurological score for each paw (-5 seconds) for each paw for each side for each paw (absent) (absent) (absent) Total Deficit (5-3 seconds) or (>3 seconds) (walk on digits) or (no walking) (present) (present) (present) Downloaded from by on October, 8 sham-operated rats was similar except for MCA cauterization. The neurological deficit was evaluated on day 3 after MCA occlusion or sham operation using the protocol of Tupper and Wallace 7 as modified by Capdeville et al. 8 Neurological status was rated for the following items: equilibrium: on a horizontal bar and an inclined plane; grasping reflex in which observation of the four paws allowed for separate scoring of the contralateral and ipsilateral (i.e., occlusion) sides; righting reflexes: "head tilted," "on back," and "dropped"; placing reactions: visual and "leg hanging" in which observation of the four paws allowed for separate scoring of the contralateral and ipsilateral sides; and motility: spontaneous and circling behavior. As proposed by Bederson et al, 3 we observed each rat for forelimb flexion and rated the rat's thorax twisting when suspended by its tail. A grading scale (Table ) was used to assess the effects of ischemia, and the sum of the partial scores gave the global neurological score, which is 4 in normal nonischemic rats. The passive avoidance task described by Bures et al 9 has been slightly modified as follows: the training was carried out with two acquisition tests on days and 3 after surgery and one retention test on day 4. The apparatus was an illuminated safe compartment with a Plexiglas door in the wall through which the rat could enter a dark compartment that had a grid on the floor. In the first acquisition test, the rat was placed in the illuminated safe compartment for a 6-second habituation period. The door was then opened, and the response latency (time to enter the dark compartment) was measured. Once all four paws of the rat were on the grid, the door was closed and a foot shock was delivered ( ma, 5 seconds). The rat was immediately returned to its home cage. For the second acquisition test, the rat was placed in the illuminated compartment for 3 seconds before the door was opened. The response latency was recorded, and any entry into the dark compartment was followed by a similar foot shock. For the retention test, the rat was placed in the illuminated compartment for 3 seconds, and the response latency was measured. The response latency of rats that did not enter the dark compartment during the observation period was taken to be 3 seconds. A Y maze test was performed on day 3 after surgery. Normal rats visit the arms of the maze one after the other. This behavior, spontaneous alternation, was used to evaluate the working memory of rats placed in a new environment. The apparatus comprised three (numbered,, or 3) black wooden arms (4x5x35 cm) placed in a dark room and illuminated with a 6-W lamp placed.5 m above it. The rat was placed in one arm (no. ); three possibilities were offered to the rat for its first choice: staying in arm, moving into arm, or moving into arm 3. An alternation was considered as correct if the rat visited a new arm and did not return to the two previously visited arms. The ratio of correct alternations to the number of visits during an 8-minute observation period was calculated to give the frequency of alternation. A frequency of >5% indicated spontaneous alternation. Spontaneous exploratory behavior was studied with an open field test on day 3 after surgery. The apparatus (9x7 cm), with x cm squares painted on its floor, was illuminated by a 6-W lamp placed m above it. Four wooden parallelepipeds

3 Wahl et al Neurobehavioral Outcome in Focal Ischemia 69 Downloaded from by on October, 8 (x7x5 cm) were placed symetrically in the center of the floor. Each rat was placed in a corner of the apparatus and observed for 6 minutes. The latency (time before leaving the start point), motility (number of squares crossed), number of visits to the objects (i.e., stops with sniffing and/or rearing), and number of rightings against the apparatus walls were noted. The rats were decapitated under ether anesthesia on day 4 after surgery, and their brains were rapidly removed and frozen in isopentane at -4 C. Coronal cryostat sections 3 /im thick were cut at levels throughout the rostrocaudal extent of the brain, from 3. to 3. mm anterior to the interaural line. 3 The sections were stained with cresyl violet, and areas of infarction were delineated and measured with an image analyzer (Imstar, Paris, France). Total infarct volume was determined by integrating the areas of infarction (with correction for edema) at each level and the distances between them. Edema was assessed as the ratio between the area of the right cerebral hemisphere and that of the left cerebral hemisphere at each coronal level. Results are expressed as mean±sem. Differences between groups were evaluated using Student's t test. Spearman's test was used to calculate correlations. Results In the MCA-occluded rats total infarct volume was 87.4±9.8 mm 3 and edema was.8±.6%. Histological damage (Figure ) occurred in both the cortex (38.6±8.5 mm 3 ) and the caudate-putamen (48.8±.6 mm 3 ). Ischemia induced lesions in the motor and sensorimotor frontoparietal cortex. The dorsolateral caudate-putamen was always infarcted, with maximal infarction being 8.7 mm from the interaural line (Figure ). The MCA-occluded rats had a much lower global neurological score (.8±.5) than the sham-operated rats (.7±.7; df=75, f=.384,/><.). Ischemia disturbed several items of the neurological examination (Table ): the contralateral grasping reflex (f=3.38), the contralateral leg hanging reaction (f=5.98), and the head tilted righting reflex (/=5.76) (df=5 andp<. for each item). Disturbance of the visual placing reaction (df =5, f=.59,/?<.) may be associated with increased thorax twisting (df=5, f=.53,/j<.). Ischemia disturbed spontaneous motility (df=5, f=8.498, /><.) and induced forelimb flexion. The other items were not modified. In the passive avoidance task the response latencies of the two groups did not differ for the first and second acquisition tests (Table 3). However, MCAoccluded rats showed a shorter response latency than the sham-operated rats (df=5, t=.48, /?<.5) in the retention test. In the Y maze test the spontaneous alternation percentage was not modified by ischemia (67. ±4.7% in the MCA-occluded group versus 76.±3.9% in the sham-operated group). In the open field test (Table 4), focal ischemia did not alter the FIGURE. Representative distribution of ischemic damage (black areas) at different coronal levels after proximal middle cerebral artery occlusion in rats. latency, motility, or number of visits to the objects. Only the number of rightings against the apparatus walls was reduced in the MCA-occluded rats (df=5, t=.79, p<.). There was no significant correlation between these neurological or behavioral deficits and the size of the infarct (Figure ) (total infarct volume, cortex infarct volume, or caudate-putamen infarct volume). Discussion We evaluated the consequences of MCA occlusion-induced histological damage on neurological status and behavior. The cerebral infarct was larger than that obtained by others 34 in the same rat strain in both the cortex and the caudate-putamen, perhaps because we maintained both body and brain temperature to avoid the protective effect of hypothermia. 5 The neurological status of MCA-occluded rats was evaluated,, and 3 days after ischemia in preliminary studies (unpublished). The neurological deficit was maximum on day, and no further change occurred during the following days. Hence, we allowed the rats to recover from the surgical and occlusion stresses for 4 hours, began the tests 48 hours after ischemia, and continued the neurobehav-

4 7 Stroke Vol 3, No February 99 Downloaded from by on October, 8 TABLE. Scores on Items of Neurological Examination in Rats Item Equilibrium Horizontal bar Inclined plane Grasping reflex Contralateral Ipsilateral Righting reflex Head tilted On back Dropped Placing reactions Visual Leg hanging Contralateral Ipsilateral Motility Spontaneous Circling behavior Forelimb flexion Thorax twisting Values are mean±sem. *p<. different from Sham-operated (n-3).8±.5.4±..8±..9±..8±. sham-operated Occluded (n=4).9±.4.9±..8±.*.9±. l±.*.±.*.+.* l.l±.*.±.* ioral studies on the third day to carry out all the tests in parallel. In this model Bederson et al, 3 Persson et al, and Obana et al 8 all performed simple neurological examinations evaluating forelimb flexion, resistance to a lateral push, and circling behavior. Germano et al 7 performed a neurological examination with the same items except circling behavior. Yamamoto et al 9 - evaluated the degree of hemiplegia and abnormal posture. Tominaga and Ohnishi 4 examined motor performance using an inclined plane test, a balance beam test, and a prehensile test. These neurological examination protocols detect hemiplegia, motor performance, and abnormal postures. The neurological examination that we used provides a more detailed description of ischemia-induced sensorimotor dysfunctions. The contralateral leg hanging placing reaction was disturbed in the same way as the grasping reflex and the head tilted righting reflex and might be due to hemiplegia. Thorax twisting may be responsible for the incorrect visual placing reaction. Forelimb flexion induced by ischemia, as observed by others, 73 was always present. Spontaneous motil INFARCT SIZE (mm 3 ) FIGURE. Scatterplot of correlation between global neurological score and total infarct volume for rats (r=.96, n=4, p>.5). ity was also affected; the rats walked on their digits instead of on their foot pads. This feature occurred mostly with the right hind paw and generated a crawling walk. All of these deficits can probably be attributed to neuronal lesions affecting sensorimotor areas. The cortical motor area of rats, the anterior dorsal cortex, 6 includes areas corresponding to the hind limb and the forepaw that are both almost always damaged by ischemia. Circling behavior never occurred, but rolling behavior occurred 3-4 hours after MCA occlusion. This might be attributed to the striatal lesion because a similar rolling was observed after injecting quinolinic acid (a neurotoxic glutamate receptor agonist) into the striatum to induce local tissue necrosis (personal observations). This rolling might also be due to released dopamine because Globus et al 7 demonstrated postischemic hyperactivity of the striatal dopaminergic system after ischemia that persisted for at least 4 hours. This transient effect might explain why the rats showed no circling behavior 3 days after MCA occlusion. In the passive avoidance test, focal ischemia induced a memory impairment during the acute phase. Our results are in agreement with those of others 94 showing a mnemic disturbance in MCA-occluded rats in a one-trial passive avoidance test 94 or a step-through procedure. The hippocampus, the temporal lobe, and the cholinergic system are generally considered to be the main centers of memory processes. 8 Our results suggest that the cerebral cortex or striatum might also be involved in learning memory. There was no evidence of hippocampal TABLE 3. Response Latencies of Passive Avoidance Task Acquisition test Group Sham-operated Occluded n 3 4 First 35.5±.8 7.±5.3 Second 55.9± ±37.5 Values are mean±sem seconds. *p<.5 different from sham-operated. Retention test 3 7.±36.4*

5 Wahl et al Neurobehavioral Outcome in Focal Ischemia 7 TABLE 4. Results of Modified Group Sham-operated Occluded n 3 4 Open-Field Test Latency (seconds) 5.3±.7 8.±.8 Motility (number of squares) ±5 3 ±8 Visits (number) 7.7±.5 4.3±.4 Rightings (number).4± * Values are mean±sem. different from sham-operated. Downloaded from by on October, 8 damage although the histological study protocol provided no fine microscopic observation of the neurons. This learning memory impairment may also be due to brain edema because memory disturbance is correlated with brain edema. 4 Memory function assessed with the Y maze did not reveal any disturbance of working memory. However, the Y maze is a simple and rapidly performed test, and using a radial-arm maze 3 months following focal ischemia in rats, Kanemitsu et al 9 observed an impairment of working memory that was attributed to a decrement of acetylcholine levels in the cortex. This test is more rigorous but takes longer, which is not suitable for our purpose. The decrease in the number of rightings against the apparatus walls in the open field test might be due to slight hemiplegia-induced motor frailty preventing the rats from remaining stable on their hind paws. In rats with forebrain ischemia Le Peillet et al reported a decrease in exploratory behavior but no hypermotility using the same modified open field test. We found no disturbance of exploratory behavior. A hypermotility phase (in a conventional open field test without objects) was observed 3 in right MCA-occluded but not left MCA-occluded rats and was attributed to anatomic or physiological asymmetries in the brain. Hence, focal ischemia does not disturb exploratory behavior or induce any motor incapacity. Because the latency was unchanged, our data suggest that vigilance remained unchanged in MCAoccluded rats. None of these neurobehavioral outcomes correlate with the cortical, striatal, or total infarct volume. Bederson et al 3 studied the influence of site and length of the MCA occlusion on neurological deficits. Their occlusions induced infarcts of highly variable size, resulting in different neurological outcomes. Similarly, Obana et al 8 found a correlation between the neurological grade and size of the infarct. Both results disagree with our findings. However, in our conditions, the MCA was always occluded in the same proximal portion, leading to a homogeneous volume of infarction. Hence, this limited range of infarct volume was associated with reproducible neurological deficits, so there was no correlation between these measurements. Although staining with cresyl violet allowed us to delineate clearly the boundaries of necrotic tissues, this staining did not allow fine evaluation of the morphological status of the neurons. Thus, it cannot be excluded that some damaged areas of the brain might be nonfunctional without being necrotic. Hence, the neurobehavioral deficits we describe might be related to transient dysfunction of the neurons, without any alteration detectable with cresyl violet staining. The lack of correlation between infarct size and neurological status indicates that a parallel examination of neurological deficits and behavioral disturbances (particularly passive avoidance reactions) should be performed to evaluate the cerebral antiischemic profile of new compounds. References. Tamura A, Graham DI, McCulloch J, Teasdale GM: Focal cerebral ischemia in the rat:. Description of technique and early neuropathological consequences following middle cerebral artery occlusion. / Cereb Blood Flow Metab 98;l:53-6. Nedeergard M: Neuronal injury in the infarct border: A neuropathological study in the rat. Ada Neuropathol 987;73: Park CK, Nehls DG, Graham DI, Teasdale GM, McCulloch J: The glutamate antagonist MIC-8 reduces focal ischemic brain damage in the rat. Ann Neurol 988;4: Gotti B, Duverger D, Bertin J, Carter C, Dupont R, Frost J, Gaudilliere B, MacKenzie E, Rousseau J, Scatton B, Wick A: Ifenprodil and SL 8.75 as cerebral anti-ischemic agents. I: Evidence for efficacy in models of focal cerebral ischemia. J Pharmacol Exp Ther 988;47:- 5. Mohamed AA, Gotoh O, Graham DI, Osborne KA, McCulloch J, Mendelow AD, Teasdale GM, Harper AM: Effect of pretreatment with the calcium antagonist nimodipine on local cerebral blood flow and histopathology after middle cerebral artery occlusion. Ann Neurol 985;8: Nakayama H, Ginsberg MD, Dietrich WD: (SJ-Emopamil, a novel calcium channel blocker and serotonin S antagonist, markedly reduces infarct size following middle cerebral artery occlusion in the rat. Neurology 988;38: Germano IM, Bartowsky HM, Cassel M, Pitts E: The therapeutic value of nimodipine in experimental focal ischemia. Neurosurgery 987;67: Obana WG, Pitts LH, Nishimura MC: Effect of opiate antagonists on middle cerebral artery occlusion infarct in the rat. JNeurosurg 988;69: Yamamoto M, Tamura A, Kirino T, Shimizu M, Sano K: Behavioral changes after focal cerebral ischemia by left middle cerebral artery occlusion in rats. Brain Res 988;45: Yamamoto M, Tamura A, Kirino T, Hirakawa M, Shimizu M, Sano K: Effects of a new thyrotropin-releasing hormone analogue administered in rats week after middle cerebral artery occlusion. Stroke 989;:89-9. Yamamoto M, Tamura A, Kirino T, Shimizu M, Sano K: Effects of a new thyrotropin-releasing hormone derivative on behavioral changes after focal cerebral ischemia in rats. Stroke 989;: Persson L, Hardemark H-G, Bolander HG, Hillered L, Olsson Y: Neurologic and neuropathologic outcome after middle cerebral artery occlusion in rats. Stroke 989;: Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H: Rat middle cerebral artery occlusion: Evaluation of the model and development of a neurologic examination. Stroke 986;7:47-476

6 7 Stroke Vol 3, No February Tominaga T, Ohnishi ST: Interrelationship of brain edema, motor deficits, and memory impairment in rats exposed to focal ischemia. Stroke 989;: Busto R, Dietrich WD, Globus MYT, Valdes I, Scheinberg P, Ginsberg MD: Small differences in intra-ischemic brain temperature critically determine the extent of ischemic neuronal injury. J Cereb Blood Flow Metab 987;7: Shiraishi K, Simon RP: A model of proximal middle cerebral artery occlusion in rat. / Neurosci Methods 989;3: Tupper DE, Wallace RB: Utility of the neurological examination in rats. Ada Neurobiol Exp 98;4: Capdeville C, Pruneau D, Allix M, Plotkine M, Boulu RG: Naloxone effect on the neurological deficit induced by forebrain ischemia in rats. Life Sci 986;38: Bures J, Bures O, Huston J: Techniques and Basic Experiments for the Study of Brain and Behavior. Amsterdam/New York, Elsevier Science Publishers BV, 983. Oades R, Taghzouti K, Simon H, Le Moal M: Dopaminesensitive alternation and collateral behavior in a Y-maze: Effects of d-amphetamine and haloperidol. Psychopharmacology 985;85:3-8. Soubrie P, Boissier JR: Psychophysiologie: Redressements et comportements chez le rat. CRAcad Sci 97;74: Le Peillet E, Lekieffre D, Allix M, Plotkine M, Boulu RG: Modified open-field test to study the protective activity of drugs in cerebral ischemia in rats ("4 vessel model"), in Krieglstein J (ed): Pharmacology of Cerebral Ischemia. Elsevier Science Publishing Co, Inc, 988, pp Paxinos G, Watson C: The Rat Brain in Stereotaxic Coordinates. New York, Academic Press, Inc, Duverger D, McKenzie ET: The quantification of cerebral infarction following focal ischemia in the rat: Influence of strain, arterial pressure, blood glucose concentration and age. J Cereb Blood Flow Metab 988;8: Boris-Moller F, Smith MJ, Siesjo BK: Effects of hypothermia on ischemic brain damage: A comparison between preischemic and postischemic cooling. Neurosci Res Commun 989;5: Hall RD, Lindholm EP: Organization of motor and somatosensory neocortex in the albino rat. Brain Res 974;66: Globus MYT, Ginsberg MD, Harik SI, Busto R, Dietrich WD: Role of dopamine in ischemic striatal injury: Metabolic evidence. Neurology 987;37: Olton DS: Interventional approaches to memory: Lesions, in Martinez JL Jr, Kesner RP (eds): Learning and Memory. New York, Academic Press, Inc, 986, pp Kanemitsu H, Tamura A, Nagashima H, Tahira Y, Kirino T, Sano K, Iwasaki K, Fujiwara M: Disturbance of spatial cognition and ACh level in rat following middle cerebral artery occlusion (abstract). J Cereb Blood Flow Metab 99;ll(suppl ) 3. Robinson RG: Differential behavioral and biochemical effects of right and left hemispheric cerebral infarction in the rat. Science 979;5:77-7 KEY WORDS behavior, animal cerebral ischemia rats Downloaded from by on October, 8