Protective effects of brain hypothermia on behavior and histopathology following global cerebral ischemia in rats

Brain Research, 580 (1992) 197-204 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

197

BRES 17725

Protective effects of brain hypothermia on behavior and histopathology following global cerebral ischemia in rats E.J. Green a, W.D. Dietrich b, F. van Dijk a, R. Busto b, C.G. Markgraf ~, P.M. McCabe a, M.D. G i n s b e r g b a n d N. S c h n e i d e r m a n a Departments of aPsychology, bNeurology and Cerebral Vascular Disease Research Center, University of Miami, Coral Gables, FL 33124 (USA) (Accepted 24 December 1991)

Key words: Memory; Ischemia; Hippocampal formation; Hypothermia; Behavior

The present experiments were designed to assess whether brain hypothermia can reduce the behavioral and histopathological deficits associated with global forebrain ischemia. Animals were subjected to 12.5 min of four vessel occlusion (4VO) with moderate hypotension, and brain temperature maintained at either 37°C (4VO-37) or 30°C (4VO-30). Behavioral tests designed to assess forelimb reflexes and sensorimotor function were given on post-operative weeks 2 and 4. Beginning in week 5, the rats were trained on a variety of navigation problems in the Morris water maze. Histopathological examination of the tissue 2 months following reperfusion revealed that 4VO-37 animals sustained substantial cell death in hippocampal region CA1 and moderate damage to the dorsolateral neostriatum. 4VO-30 animals showed minimal cell death in CA1 and neostriatum. There were no group differences for any of the sensorimotor measures, or for acquisition performance on either the simple place task or visible platform version of the water maze. In contrast, during acquisition of the learning set task, the performance of 4VO-37 animals was impaired relative to either of the other groups, whereas the performance of 4VO-30 animals was not significantly different from the sham controls. These data suggest that moderate intra-ischemic brain hypothermia provides longlasting protection from behavioral deficits as well as neuronal injury following transient global ischemia. INTRODUCTION G l o b a l cerebral ischemia resulting from cardiac arrest is a p r o b l e m of increasing clinical significance. People who survive such insults are often h a m p e r e d by neurological impairments, which can range from transient sens o r y - m o t o r disturbances to persistent cognitive deficits, including m e m o r y loss 8'9. A n u m b e r of animal models of global ischemia have b e e n d e v e l o p e d in an a t t e m p t to d e t e r m i n e the mechanisms underlying ischemic-related neural injury, and to develop treatments that can attenuate the accompanying neurological deficits. In rats, one such m o d e l involves a transient occlusion of the vertebral and carotid arteries (four vessel occlusion; 4VO). This p r o c e d u r e is associated with a p a t t e r n of neural d a m a g e similar to that r e p o r t e d following global ischemia in humans, including substantial n e u r o n d e a t h in h i p p o c a m p a l subfield CA1 and variable amounts of neuron death in the c a u d o p u t a m e n and the cerebral cortex 1' 4,31,33

The cellular d a m a g e resulting from global ischemia evolves over the course of m a n y hours to days 21 and thus may be a m e n a b l e to therapeutic intervention. O n e fac-

tor known to positively influence ischemic o u t c o m e is hypothermia. Systemic h y p o t h e r m i a has long b e e n associated with an attenuation of ischemic injury 22'25'39, and recent histopathological studies involving animals indicate that a reduction in brain t e m p e r a t u r e mediates these protective effects 3'4'7'23'26'37. F o r example, rats subjected to a transient global ischemia at a brain t e m p e r a t u r e of 36-37°C exhibited extensive, bilateral necrosis in hippocampal area CA1 with m o d e r a t e necrosis in caudoputamen, whereas animals experiencing a similar ischemic insult at a brain t e m p e r a t u r e of 32°C showed no evidence of light microscopic p a t h o l o g y in these regions at 3 days after ischemia 4. A l t h o u g h histopathological measures, such as ischemic cell counts, are useful for providing a gross index of neurological state, they are incapable of revealing m o r e subtle changes which can significantly compromise brain function. F o r example, Jaspers et al. 19 have rep o r t e d significant place learning deficits 6 - 9 days following 2 V O , despite the absence of any obvious cellular death in CA1 hippocampus. A n analysis of the functional capacity of the affected brain regions, through the use of a p p r o p r i a t e behavioral assays, could provide a

Correspondence: E.J. Green, Department of Psychology, University of Miami, PO Box 248185, Coral Gables, FL 33124, USA. Fax: (1) (305) 284-2522.

198 m o r e rigorous assessment of neural integrity. Accordingly, t h e goal o f t h e p r e s e n t e x p e r i m e n t s w a s to assess w h e t h e r neural h y p o t h e r m i a can attenuate the behavioral deficits a s s o c i a t e d w i t h i s c h e m i a i n d u c e d by t r a n s i e n t 4 V O in rats. H i p p o c a m p a l f u n c t i o n was a s s e s s e d by e v a l u a t i n g spatial n a v i g a t i o n in t h e M o r r i s w a t e r t a s k , which has b e e n shown to be sensitive to ischemic damage t o s u b f i e l d CA11'32. A series o f reflex a n d s e n s o r i m o t o r t e s t s t h a t a r e s e n s i t i v e t o d a m a g e in t h e c a u d o p u t a m e n a n d / o r c e r e b r a l c o r t e x was u s e d t o assess t h e functional integrity of those structures.

MATERIALS AND METHODS

Subjects Studies were carried out in 24 male Wistar rats, weighing between 250 and 300 g, obtained from Charles River Breeders. The rats were housed individually under 12 h light-dark conditions and allowed free access to food and water. The rats were fasted 24 h before the ischemic insult. Surgery The 4VO model used in these experiments is a variant of the model described previously4'12. Rats were initially anesthetized with 3% halothane, and subsequently maintained with 1-1.5% halothane, 70% nitrous oxide, and a balance of oxygen delivered via a closely fitting face mask. The percentage of halothane in the mixture was manipulated so as to produce an adequate depth of anesthesia without respiratory depression. Using aseptic surgical procedures, the vertebral arteries were bilaterally occluded using an electro-coagulating probe. The animals were then allowed to recover overnight. The following day, animals were intubated, connected to a respirator, and were ventilated with a mixture of 70% nitrous oxide, 1-1.5% halothane, and a balance of oxygen passed through a humidifier. Animals were then immobilized with pancuronium bromide (0.75 mg/kg i.v.) and were maintained with repeated doses of 0.35 mg/kg i.v. as needed. The common carotid arteries were exposed bilaterally, and the tail artery was cannulated to remove blood and to measure blood pressure. Arterial blood gases and blood pressure were measured throughout the procedure. Following a 45 min stabilization period, mean arterial pressure (MAP) was lowered to approximately 80 mm Hg by exsanguination into a heparinized syringe maintained at 36.4-37°C, and the ischemic insult was initiated by tightening the carotid ligature bilaterally for a period of 12.5 min. The ischemic insult was terminated by loosening the carotid ligatures and slowly re-infusing the shed blood to restore normotension. Brain temperature was monitored by means of a thermocouple implanted in the temporalis muscle, which can be used to estimate intracerebral temperature 4. Adjustments in brain temperature were accomplished by manipulating a small high-intensity lamp above the animals head, or by blowing cool air onto the surface of the head with a high speed fan as required. Brain temperature in the hypothermic (4VO-30; n = 9) and normothermic (4VO-37; n = 8) animals was maintained at 30 +_ 0.5°C and 36.4-37°C for the 12.5-min period, respectively. Rectal temperature in all of the animals was monitored and controlled at 37°C throughout the experimental protocol by a warming pad. The sham operated animals (n = 7) were subjected to all anesthetic and operative procedures described above, including preliminary bilateral vertebral artery electro-coagulation, but their common carotid arteries were not occluded. Four of the sham animals were conducted at a brain temperature of 37 _+ 0.5°C, and three were held at a brain temperature of 30 _+ 0.5°C for 12.5 min. All wounds were infiltrated with 1% lidocaine and cephazolin (Kefzo), 20 mg/kg i.m. Upon recovery of spontaneous ventilation, rats were placed in a chamber containing 100% oxygen humidified air for a period of 24

h prior to being returned to their cages.

Behavioral assessment Each rat was subjected to a series of behavioral tests by experimenters who were blind to which group the rats had been assigned. During post-operative weeks 2 and 4 the animals were evaluated using a battery of sensorimotor tests, which has been previously shown to be sensitive to a combination of unilateral striatal and neocortical damage observed following focal middle cerebral artery occlusionz4. Beginning during the fifth post-operative week, the rats were trained to perform 3 distinct problems in the Morris water maze 27. The simple place test assessed the rat's ability to locate a platform concealed in opaque water, using distal spatial cues present in the room. The visible platform test required the rat to navigate to a distinctly marked platform that protruded above the surface of the water. Performance on both of these tasks has been demonstrated to be sensitive to striatal damage in rodents38, and the simple place task has been shown to be sensitive to damage of the hippocampuszT. The learning set task is similar to the simple place task, except that the location of the submerged platform is changed from day to day. This task has been proven to be sensitive to ischemic damage to hippocampal field CAI 1"32, whereas the simple place task was not H6. Sensorimotor battery Postural reflex test. This test consisted of holding the rat by the tail and suspending it 1 m above the floor. Normal rats extend both forelimbs toward the floor, and receive a score of 0. Abnormal behavior consists of either flexion of a forelimb, or rotation of the body. If either behavior occurred, the animal was held so that its forelimbs were in contact with a sheet of plasticbacked paper that could easily be gripped. The animal was then slid back and forth while its claws remained in contact with the paper. If it resisted sliding by gripping the paper, it received a score of 1. If it did not resist sliding, it received a score of 2. Limb placing test. This test quantified forelimb placing reactions to visual, tactile, and proprioceptive stimuli. The rat was gripped in such a way that twisting was prevented, while leaving the forelimbs free to move. Forward visual placing was elicited by bringing the rat to within approximately 10 cm of a tabletop, while holding it perpendicular to the tabletop edge; sideways placing was elicited following the same procedures, but holding the rat parallel to the edge. Normal rats reach for the tabletop with both forelimbs when forward visual placing is tested, and the appropriate limb when sideways placing is tested. Tactile placing was measured by touching the dorsal and lateral surface of each paw to the tabletop. Normal rats immediately place their paw upon the tabletop. Proprioceptive placing was measured by pushing the entire forelimb against the table. Normal rats either immediately place their paw upon the tabletop, or do not allow their leg to be pushed completely against the table. On all tests rats received scores for each forelimb. Normal rats received a score of 0; rats which showed slow or incomplete placing received a score of 1, and rats which showed no sign of placing after 3 s received a score of 2. A total score was obtained by adding the individual scores. Foot fault test. This test consisted of putting the animal for 2 rain on a elevated grid floor of approximately 30 cmz with grid openings of approximately 3 cm2. The animals walked on the wire grid, occasionally failing through with one foot, scoring a 'foot fault'. The total number of foot faults, and total number of forelimb steps taken during the interval were recorded. The number of foot faults was expressed as a percentage of the total number of forelimb steps. Vertical screen test. Rats were placed on a horizontal wire mesh screen measuring 50 x 22 cm with grid openings of 1.5 cm2. The screen was then slowly tilted to the vertical and the rat was observed for 5 s. Normal rats grip the screen for the entire interval, and receive a score of 0; if the rat slips but does not fall off the screen, it receives a score of 0.5; rats that grasp the screen but fall off within the 5 s period receive a score of 1; rats that fall off im-

199 mediately receive a score of 2. Water maze Following sensorimotor testing, rats were tested using a water maze. The maze consisted of a round pool (122 cm diameter; 60 cm deep) filled with water at 21°C, and rendered opaque by adding two pounds of white, powdered non-toxic paint. The maze was located in a quiet, well-lit, windowless room, with a variety of large, distinct, extra-maze cues. Four points on the rim designated as north (N), east (E), south (S) and west (W), served as starting positions and divided the maze into four quadrants. A round platform (13 cm diameter) was located 1.5 cm beneath the surface of the water. A microcomputer-based tracking system (Prototype Systems Ltd., Boulder, CO) was used to record and analyze the rat's swim paths. This system recorded the position of the rat by tracking a piece of black tape on the rat's head, using a digital camera suspended above the pool. Path length (cm) and latency to reach the platform (s) were automatically determined by the computer and recorded. Average swim speed (path length/latency) was calculated as well. Water maze testing was accomplished in 3 phases, including a simple place task, a visible platform task and a learning set task, conducted in succession. Simple place task. Testing occurred on ten consecutive days following sensorimotor testing, commencing approximately 4 weeks following the surgical procedure. The hidden platform was placed in the center of the NW quadrant throughout the 10 days of testing. Animals were given four trials daily in a random order, one from each starting location. Each trial consisted of releasing the animal into the water, facing the side of the pool. If the platform was found within 60 s, the animal was allowed to remain on it for 10 s; if the 60 s elapsed without the animal finding the platform, the animal was taken out of the water and placed on the platform for 10 s. Inter-trial intervals lasted between 1 and 2 min, during which the rats were placed under a heat lamp. Visible platform task. Testing occurred on two consecutive days following completion of the simple place task. The submerged platform was placed in the NE quadrant, and all procedures used were identical to the simple place task except that the platform was raised approximately 1.5 cm out of the water, making it visible to the animals. Learning set task. Testing occurred on six consecutive days following completion of the visible platform task. The platform was moved each day to one of the following six locations: location 1 was at the center of the NE quadrant; location 2 was at the pool center; location 3 was at the center of the SE quadrant; location 4 was 18 cm from the N point on the pool rim; location 5 was 18 cm from the W point on the pool rim; and location 6 was at the center of the SW quadrant. Each animal received sixteen 60 s trials, which occurred in random pairs, with two pairs from each starting location each day. If the rat successfully located the platform it was allowed to remain for 5 s; otherwise, it was put on the platform for

a period of 5 s. After the first trial of the pair, the second trial was given immediately, using the same starting location. Inter-trial intervals lasted 2-4 min, during which rats were placed under a warming lamp. Histology Following completion of the learning set task (approximately 8 weeks following brain ischemia or sham procedures), rats were deeply anesthetized with 2% halothane and perfusion-fixed with a mixture of 40% formaldehyde-glacial acetic acid-methanol (FAM) (1:1:8 by volume). The brains were held in FAM and removed from the skull 24 h later and processed for paraffin histopathological analysis by methods previously described 12. 10/zm sections were taken, stained with hematoxylin and eosin, and examined by light microscopy. Quantification of CA1 damage was performed by one of the experimenters (WDD) who was blind to the experimental group assignments. Direct counts were made of the remaining viable neurons at 400 x magnification in a rectangular area (200 × 50 ~m) of the dorsal hippocampal CA1 region at 5 rostrocaudal levels of the hippocampus (after Rod et al.32). These five levels corresponded roughly to 1.6, 2.2, 2.6, 3.0 and 3.4 mm posterior to bregma 2s. Viable neurons were defined as those cells showing a distinct nucleus and nucleolus. Pathology in the neostriatum and cerebral cortex was evaluated by means of the following semi-quantitative scale: 0 = normal; 1 -- few affected neurons; 2 = many affected neurons; 3 = most neurons affected. Statistical analyses Non-parametric statistics (Kruskal-Wallis tests) were used to evaluate data from the sensorimotor battery. Repeated-measures analyses of variance (ANOVA) were performed on the data from the water maze tests, followed by simple effects comparisons where appropriate. One way ANOVAs were performed on physiological and histological data. Scheffe tests were used to evaluate statistically post-hoc comparisons.

RESULTS Physiological measures Physiological measures taken prior and subsequent to s u r g e r y a r e p r e s e n t e d in T a b l e I. T h e p a r t i a l p r e s s u r e s o f c a r b o n d i o x i d e ( p C O 2 ) , o x y g e n (pOE) , b l o o d p H a n d mean

arterial pressure

were

generally within normal

p h y s i o l o g i c a l limits. T h e t w o i s c h e m i c g r o u p s e x h i b i t e d a m i l d e l e v a t i o n in p C O 2 f o l l o w i n g t h e i s c h e m i c insult.

TABLE I Physiological measures o f rats subjected to normothermic, hypothermic or sham forebrain ischemia Group

Pre-Ischemiaa pO 2

pCO 2

Sham (n = 7) Ischemia 4VO-37 (n = 8) 4VO-30 (n = 9)

.

38+1 38+1

Post-Ischemia b

.

pH .

136+7 142_+11

MAP

.

7.424+0.01 7.406+0.01

110+3 113+4

almmediately prior to onset of ischemia

b30 min followitTg ischemia pCO2, pO 2 and MAP (mean arterial blood pressure) are expressed in mm Hg * Significantly different from pre-ischemic value by one-way ANOVA (P < 0.05)

pCO 2

pO 2

pH

MAP

37+1

136-+9

7.406+0.02

118+2

43+1" 44-+2*

141_+12 142+6

7.342+0.01 7.351+0.01

112+2 111-+3

200

Histology There was no detectable histopathology in sham animals maintained at either 30 or 37°C. Thus the data from these animals were pooled for statistical purposes. A typical example of the pattern of hippocampal injury is shown in Fig. 1. The 4VO-37 (normothermicischemic) animals exhibited substantial CA1 cell loss throughout the hippocampal CA1 region. Quantitative counts of remaining viable neurons indicated that each of the animals had fewer than 50% of the number of neurons present in sham animals (group mean = 11.5% of sham). Relative to shams, normothermic animals had significantly fewer viable cells at each of the 5 rostrocaudal levels (Fig. 2). Reactive astrocytes and macrophages around necrotic CA1 perikaryia were routinely observed (Fig. 1). There was no obvious indication of damage to the CA3 region or to the fascia dentata. Semi-quantitative data indicated that significant damage (i.e. a histopathological grade 2 or 3) to the caudate nu-

cleus occurred in 5 out of the 8 normothermic rats. These animals exhibited evidence of glial and macrophage proliferation, and a decreased density of striatal neurons. In 1 of the 8 hormothermic rats, large numbers of dark, shrunken neurons were detected throughout the cerebral cortex; the remaining 7 animals exhibited little or no damage in this structure (histopathological grades of 0 or 1). In contrast to the normothermic-ischemic results, large numbers of viable C A I neurons were detected in the hypothermic (4VO-30)-ischemic group. In 6 of the 8 animals, the CA1 layer appeared essentially intact, whereas in the other 2 animals a thinning of the CA1 pyramidal cell layer, suggesting a drop-out of neurons, was evident. The number of viable cells counted in this group averaged 81.3% of that found in the sham control group (Fig. 2); this was not statistically different from sham values at any rostrocaudal level (all comparisons, P < 0.05). Light microscopic indicators of significant ischemic damage in other brain regions, including the caudate nucleus

Fig. 1. Paraffin-embedded coronal rat brain sections stained with hematoxylin and eosin from normothermic- (a,b) and hypothermic- (c,d) ischemic rats. a: two months after 12.5 min of normothermic (37°C) global ischemia, severe necrosis of CA1 hippocampus is apparent (× 120). b: higher magnification of injured CA1 sector illustrates only 2 viable neurons (arrowheads) among reactive astrocytes and microglia (x 1100). c: in contrast to normothermic results, a relatively intact CA1 sector is present in a rat which underwent hypothermic (30°C) global ischemia (x120). d: higher magnification of CA1 demonstrates many viable neurons containing a distinct nucleus and nucleolus

(× 1100).

201

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100

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40

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ROSTRO-CAUDAL L (DISTANCE FROM BREGMA IN MM)

Fig. 2. Effect of intra-ischemic brain temperature on the number of viable CA1 cells found 8 weeks following transient forebrain ischemia. This figure illustrates the mean (_+ S.E.M.) number of viable cells counted within a prescribed zone (depicted by the rectangular box) at each of 5 rostrocaudal levels of the hippocampus. CA1 cell counts from animals subjected to 12.5 min. of ischemia at a brain temperature of 37°C (n = 8) averaged only 11.5% of those from sham operated controls (*P < 0.01 compared with sham). The number of cells counted in animals subjected to ischemia at a brain temperature of 30°C (n = 9) did not differ significantly from values obtained from sham animals (n -- 8).

and frontoparietal cortex, were not seen in the hypothermic animals (all rats had histopathological grades of 0 or

Visible platform task. A n analysis of path length data for the visible platform failed to indicate a significant ef-

1).

fect of group (F2,20 = 1.47, P = 0.25), days (F1,20 = 3.35, P = 0.08), or a group by day interaction (F2.2o = 0.25, P = 0.78). Learning set task. The results of the learning set task are presented in Fig. 3. Analyses of variance revealed a

Behavioral assessment Sensorimotor battery Data from the four tests comprising the sensorimotor battery administered during post-operative week 2 are presented in Table II. A n i m a l s from all three groups appeared normal in these measures. Analyses of variance revealed no significant effect of t r e a t m e n t for either postural reflex (H E = 1.09), limb placing (H E = 1.90), foot faults (H 2 = 2.54), or on the vertical screen test (H 2 = 2.0); P > 0.05 for all tests. Very similar results were obtained on the sensorimotor tests administered during post-operative week 4. Water maze Simple place task. There was n o significant difference in the acquisition of this task by the three groups. There was a significant decrease in m e a n path length scores over days (F9,t89 = 11.16, P = 0.0001), indicating that all groups became more proficient at finding the hidden platform as training progressed. However, no significant effects of group (F2,189 = 0.54, P = 0.59), or interaction of group and days (F18,189 = 0.63, P = 0.87) were seen.

significant effect of group on path length (F2,21 = 5.22, P < 0.02), and a significant decrease in path length over trials (F15,315 = 15.07, P = 0.0001). However, there was

TABLE II Means and standard error scores for the sensoriraotor battery conducted during post-operative week two Ischemic rats appeared similar to sham animals in all respects. These results suggest that there were no permanent deficits in reflex or sensorimotor integration produced by the ischemic insult, a result which is consistent with the lack of cortical pathology observed 2 months following reperfusion. Surgical group Sham (n = 7) 4VO-37 (n -- 8) 4VO-30 (n = 9)

Postural reflex

Placing

Foot-faults Vertical (%) screen

0.14+0.14

0

3.92+1.06

0

0.43+0.3

6.23_+1.78 0.14_+0.14

0.14_+0.14 0.43+0.3

5.39-+1.04

0

0

202

~; (J

Ischemlc-30

Sham /#12186

1400 1200

o----

Ischemlc-37 (n=8)

~-

Ischemlc-30 (n=9)

# 12062

v

-r

1000

<3 Z

8O0

I-

uJ .J .,-

Isehemlc-37 #12042

600

I--

< I1.

400 200

• ~

0

2

•

I

4

•

I

6

!•

!

8

•

!

10

•

I

12

•

!

14

•

!

•

16

TRIAL

Fig. 3. Mean (+ S.E.M.) distance taken to the platform during the learning set task. The animals were required to locate a platform hidden at a new location on each of 6 days. Data are collapsed across days. The performance of animals subjected to transient global ischemia at a brain temperature of 37°C was significantly different from that of sham ischemic animals (P < 0.05); the performance of 30°C animals did not differ from that of sham animals (P > 0.3). The right column shows swim paths taken by an individual animal from each of the treatment groups on the sixth day of testing using the learning set paradigm. These trials illustrate the relative difference in path lengths taken by sham, normothermic and hypothermic-ischemic animals at this final stage of testing.

no interaction between group and trials (F15,315 = 0.77, P = 0.80). Simple main effects tests revealed that 4VO-37 animals had longer path lengths than either the sham (P < 0.05) or the 4VO-30 animals (P < 0.05). The scores of sham and 4VO-30 animals were not statistically different (P > 0.3). A n analysis of the latency data failed to indicate a statistically significant group effect (F2,21 = 2.93, P = 0.08), and there was no interaction between group and trials (F15,315 = 0.71, P = 0.87). Average swim speeds across the 6 days of the learning set testing were, in cm/s: 4VO-37, 29.2 + 0.72; 4VO-30, 26.8 + 1.3; sham, 27 + 0.66. There were no significant effects of either group (P = 0.16) or trial (P = 0.40) on this measure. The relationship between behavioral performance on the learning set and histopathological damage was quantitatively assessed by computing Spearman correlation coefficients. Behavioral ranks were determined by calculating a mean path length for each animal, collapsing the learning set data across both trials and days. The behavioral ranks and histopathological grades of damage to the caudoputamen in the 8 normothermic-ischemic rats were then used to calculate the correlation coefficient (hypothermic and sham animals were not considered, as none exhibited any evidence of caudoputamen damage). There was no evidence of a significant relationship between behavioral performance and damage in the caudoputamen (r s = 0.04, P > 0.05). A similar test was conducted of the relationship between the number of surviving CA1 cells and learning set performance for the 13 animals showing evidence of cell loss. Again, there was no significant correlation between the two measures (rs = 0.24, P > 0.05), although the range of variation in cell counts may not have been large enough to assess the

relationship adequately. DISCUSSION The data from this study provides direct evidence that intra-ischemic hypothermia can confer marked behavioral, as well as histopathological, protection from global ischemia at an extended survival interval. Animals subjected to transient 4VO at a brain temperature of 37°C exhibited severe cell loss in the hippocampal region CA1, and moderate cell loss in striatum. These animals were significantly impaired in their performance on the learning set task in which they were required to find a submerged platform hidden at a distinct location each day. In contrast, animals subjected to global ischemia at a brain temperature of 30°C exhibited a general sparing of cell loss in region CA1 and striatum, and no significant deficits on the learning set task. The pattern of behavioral deficits exhibited by ischemic animals indicates that those deficits were due primarily to ischemic cell loss in region CA1. Although the ischemic insult used in this study resulted in moderate amounts of damage in the caudoputamen, there was no evidence of a relationship between caudoputamen damage and the animals' performance on the learning set. Furthermore, rats with lesion-induced hippocampal damage typically show no deficits in acquiring simple cued tasks such as the visible platform test used in the present study (e.g. refs. 27,34), whereas rats with medial caudoputamen lesions exhibit impaired acquisition on those tasks 38. The 4VO-37 animals showed no deficits during the visible platform trials. Finally, deficits in acquisition of the learning set have been reported by other investigators examining animals with ischemic neuronal loss

203 which was essentially limited to the CA1 region, with little extra-hippocampal involvement 1. The lack of sensorimotor deficits in ischemic animals is consistent with the relatively sparse neocortical damage exhibited by these animals. The tactile and proprioceptive limb placing task is extremely sensitive to damage in the frontal and parietal forelimb areas of the neocortex t°'ll, and performance on the foot-fault test is sensitive to lesions of the sensorimotor cortex 17. The postural reflex test has been used to assess ischemic damage in the cortex and basal ganglia, but appears to be most sensitive to combined damage to the two structures 2. The present results indicate that there were no permanent sensorimotor disturbances in t h e s e animals. This conclusion is supported by data from the water maze, showing normal swim speeds and performance on the visible platform task, suggesting that there was no general motor impairment or simple sensory deficit during the testing period. Previous experiments evaluating the beneficial effects of intra-ischemic hypothermia on histopathological outcome have involved relatively short (3-7 days) post-ischemic periods 4A5'26'37. These studies left open the question of whether intra-ischemic hypothermia provided permanent protection or only delayed ischemic cell injury, and raised the possibility that hypothermia attenuates cellular mortality yet produces some functional anomaly which is undetectable at the light microscopic level. The current findings indicate that intra-ischemic hypothermia protects against behavioral deficits and CA1 necrosis for at least 2 months following the ischemic insult, suggesting that the protective effects of intraischemic hypothermia are permanent. Experimental work with animals 2°'3°'31 as well as studies involving cardiac arrest in humans 29 suggest that ischemic neuronal death can take days to develop fully. However, clinical observations indicate that behavioral deficits resulting from an ischemic insult occur immediately, and are often transient in nature. Thus, it is apparent that some behavioral consequences of brain ischemia are not directly associated with neuronal death, and it is likely that these deficits arise as a consequence of transient circuit dysfunction. Indeed, recent experiments indicate that the metabolic response to activation of a sensory circuit is significantly attenuated immediREFERENCES 1 Auer, R.N., Jensen, M.L. and Whishaw, I.Q., Neurobehavioral deficit due to ischemic brain damage limited to half of the CA1 sector of the hippocampus, J. Neurosci., 9 (1989) 16411647. 2 Bederson, J.B., Pitts, L.H., Tsuji, M., Nishimura, M.C., Davis, R.L. and Bartkowski, H., Rat middle cerebral artery occlusion:

ately following global ischemia, despite a lack of histopathological evidence for ischemic neuronal injury 14'18' 35. Interestingly, moderate intra-ischemic hypothermia has been shown to prevent the transient metabolic depression commonly detected following global ischemia 13. In the present study, behavioral testing began more than 2 weeks following ischemia, and thus it is not known if there were transient behavioral deficits at earlier postischemic time points. Future studies will be necessary to assess early behavioral deficits following global ischemia, and whether these behavioral changes can be modulated by hypothermia. The clinical usefulness of intra-ischemic brain hypothermia is somewhat limited because of the necessity of anticipating an ischemic episode. However, recent histopathological studies indicate that post-ischemic brain hypothermia can also attenuate cell death following global ischemia 5'6. While the therapeutic window for such postischemic effects is rather short ( < 30 min), and extension of this window, perhaps by combining hypothermia with pharmacotherapy, might be clinically useful for limiting the neurological consequences of stroke in humans. Ongoing studies are currently evaluating the effects of post-ischemic hypothermia on behavior. The present data suggest that histopathological measures can be reasonable predictors of chronic behavioral outcome, though they may not accurately indicate functional integrity at all survival times and with all treatments. Thus, although morphological endpoints are extremely useful for screening potential therapeutic treatments, behavioral endpoints will continue to be advantageous to confirm the efficacy of potential treatment strategies. Of the several behavioral procedures that have been proven useful for detecting behavioral deficits associated with ischemic-related hippocampal damage (e.g. refs. 1,16,36), the sensitivity of the learning set task to partial hippocampal damage, along with the lack of need for extended training and food deprivation, make it particularly well suited for screening potential therapeutic treatments directed at attenuating behavioral deficits associated with cerebral ischemia.

Acknowledgements. This work was supported by NS-05820. We thank Ofelia Alonso and Susan Kraydieh for assistance with surgical and histological procedures.

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