176
Brain Research. 536 (1990) 176- 182 Elsevier
BRES 16148
Secondary hypotensive insults in a rat forebrain ischemia model David S. Warner, Daniel K. Reasoner, Michael M. Todd and Alice McAllister Neuroanesthesia Research Group, Department of Anesthesia, University of Iowa College of Medicine, Iowa City, IA 52242 (U.S.A.) (Accepted 10 July 1990) Key words: Rat; Brain; Ischemia; Hypoperfusion; Repeated ischemia
Previous studies have shown that the recently injured brain has an increased sensitivity to subsequent brief episodes of severe i~:.hemia. This investigation was designed to assess whether less severe secondary insults, which alone would be incapable of producing injury, exacerbate brain damage resulting from a primary episode of global ischemia. Rats were subjected to either 10 rain of 2-vessel forebrain isehemia (primary insult alone), 20 min of hypotension (mean arterial pressure, MAP = either 40 or 25 mmHg) without vessel occlusion (secondary insult alone), or 10 min isehemia followed 1 h later by the hypotensive challenge (primary+secondary insult). Seven days later, the animals were neurologically evaluated and the brains then prepared for histoiogic analysis. Neither magnitude of secondary insult alone was found to produce injury. In contrast, the primary insult alone caused moderate damage in the hippocampns, caudoputamen and neocortex. With the exception of increased neuronal necrosis in the hippocampal CA1 sector in rats receiving the primary + secondary insult (MAP = 25 mmHg), no worsening of outcome could be attributed to the secondary insults. These results indicate that the recovering brain may not be as sensitive to hypoperfusion as has previously been suggested. INTRODUCTION Investigations using rodents have demonstrated that the recently injured brain is uniquely susceptible to secondary ischemic insults 7-9'11'17. For example, Jenkins et al. have shown that when a 6 min episode of severe forebrai'n ischemia was produced 1 h after a fluid percussion injury, resultant histopathologic changes were worse than those produced by either insult in isolation 7. Similarly, Tomida et al. have demonstrated that three sequential 5 min episodes of cerebral ischemia, each separated from the next by 1 h, resulted in a worsened outcome compared to a single 15 min ischemic interval 17. Investigations concerning secondary insults have become even more interesting to the clinician given reports that such events, which in and of themselves were of an insufficient duration to injure the normal brain, caused a worsening of injury when administered to the recovering post-ischemic brain 8'9. However, such investigations have studied severe secondary insults, i.e. near-complete global ischemia, which are capable of producing both major electrophysiologic abnormalities (e.g. an isoelectric E E G ) as well as tissue damage. Secondary insults of this severity are not c o m m o n in clinical medicine, where moderate hypotension or hypoxia are far more likely to be superimposed on pre-existing disease states such as occlusive stroke, subarachnoid hemorrhage or cardiac arrest. To date there
have been no studies providing information on the sensitivity of the damaged brain to these less severe secondary insults. In other words, how severe must a secondary hypotensive insult be to worsen a pre-existent injury? To examine this question, we have employed the two-vessel forebrain ischemia model to produce a standardized 10 min primary insult in the rat. After one hour of recirculation, a transient (20 min) episode of either moderate (40 m m H g ) or severe (25 m m H g ) systemic hypotension was produced, followed by 7 days of recovery. The histopathologic and behavioral changes seen in these animals were then compared with those seen in animals subjected to either forebrain isehemia or hypotension alone. Both histologic and neurologic analyses indicate that the brain is surprisingly resistant to such secondary insults in this context.
MATERIALS AND METHODS Surgical preparation and experimental design With Institutional Animal Care and Use Committee approval, all animals underwent the following preparation. Male Spragu¢Dawley rats (age range 9-10 weeks) were fasted from food but allowed free access to water for 12-t6 h prior to the experiment. Following induction of anesthesia with 1-2% halothane in 30% Ozeoalance N2, each rat was intubated and the lungs were mechanically ventilated to maintain normocapnin (PoCO2 range = 38-42 mmHg) and normoxia (PaO2 range = 100--135mmHg). ~ e tail artery was cannulated to allow both blood pressure monitoring
Correspondence: D.S. Warner, Department of Anesthesia, University of Iowa College of Medicine, Iowa City, IA 52242, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
177 and sampling for blood gas/pH analysis. Via a ventral neck incision, both carotid arteries were isolated and loosely encircled with sutures. The right internal jugular vein was cannulated and 50 IU heparin were given intravenously. Muscle paralysis was obtained by a 1.0 mg i.v. bolus of succinylcholine repeated as necessary. Bipolar E E G activity was monitored from a pair of needle electrodes inserted into the temporalis muscle bilaterally and a reference electrode inserted into the animals snout. Finally, a 22 gauge needle thermistor was percutaneously placed adjacent to the skull and pericranial temperature was thenceforth servo-regulated at 37.0 + 0.1 °C. A total surgical interval of 30 min was allowed for each animal. The halothane concentration was then reduced to 0.5%, and a subsequent a 30 min interval was allowed for physiologic stabilization. Immediately thereafter, the animals were assigned to one of three groups in each of two separate experiments defined as follows.
Experiment 1 Primary insult only. Ten min forebrain ischemia (see below) followed by 0.5% halothane anesthesia for 80 min. Secondary insult only. Anesthesia only (sham ischemia) followed 1 h later by 20 min of systemic hypotension (MAP = 40 + 5 mmHg, see below). Primary+secondary insult. Ten min forebrain ischemia followed 1 h later by 20 min systemic hypotension (MAP = 40 + 5 mmHg, see below).
Experiment 2 When the results of Expt. 1 were analyzed, 3 more groups of rats were studied using precisely the same protocol as described above, except the severity of the secondary insult was enhanced by reducing MAP in these animals to 25 mmHg for 20 min in the appropriate groups. Note that in all groups (Expts. 1 and 2), the total duration of halothane anesthesia was identical. Primary insult. Forebrain ischemia (FBI) was produced as previously described 2, 14. Briefly, trimethaphan (3.5 mg) was given as an i.v. bolus and the carotid arteries were occluded bilaterally for 10 min using temporary aneurysm clips. During this 10 min interval, MAP was maintained at 50 + 5 mmHg by withdrawal of venous blood from the jugular catheter as necessary. E E G was continuously monitored and isoelectricity documented throughout the ischemic interval. Perfusion was restored by removal of the carotid artery clips and reinfusion of shed blood causing MAP to recover to at least 100 mmHg within 2 min. Secondary insult. In Expt. 1, systemic hypotension was produced by an intravenous bolus of 3.5 mg trimethaphan followed immediately by withdrawal of blood from the venous catheter as necessary to maintain MAP = 40 + 5 mmHg for 20 min. The blood was then reinfused and the animal recovered (see below). In these animals, resultant metabolic acidosis was of insufficient magnitude to warrant administration of NaHCO3. In Expt. 2 a similar protocol was employed with the following exceptions: (1) MAP was reduced to 25 + 5 mmHg for 20 min induced by 3.0 mg i.v. trimethaphan and maintained by bleeding from the jugular catheter; (2) a second i.v. bolus of trimethaphan (2.0 mg) was given 10 rain into the 20 min interval; and (3) metabolic acidosis occuring after reinfusion of shed blood was treated with i.v. NaHCO 3 as necessary to return arterial pH to the range of 7.38-7.40. Following completion of protocol in both experiments, the vascular catheters were removed and the wounds closed with suture. Anesthesia was discontinued, and when the rats were breathing spontaneously, the tracheas were extubated. The rats were then placed in an oxygen enriched environment for 45-60 min whereupon they were returned to their cages. A survival interval of 7 days with free access to food and water was allowed whereupon the rats were neurologically evaluated as follows.
Neurologic assessment The rats were first subjected to an open field test which was
intended to quantitate the potential for exploratory activity5. Each rat was placed in a wooden box having a square floor (90 x 90 era) painted with a grid, each square being 15 x 15 cm in area (i.e. a total of 36 squares). The rat was then observed over a 5 rain period and the total number of squares the animal's snout entered as well as the total number of times the animal raised up on its hind limbs or defecated was recorded. Motor function was then assessed as described by Combs and D'Alecy 4. Briefly, the rats were placed on a 29 x 30 cm screen (grid size 0.6 × 0.7 cm) that could be rotated from 0° (horizontal) to 90° (vertical). The duration of time that each rat was able to hold onto the vertically-oriented screen was recorded to a maximum of 15 secs (allowing a total of 3 points). Next, the rat was placed at the center of a horizontal wooden rod (1" diameter), and the duration that the animal remained balanced on the rod was recorded to a maximum of 30 secs (allowing a total of 3 points). Finally, a prehensile-traction test was administered. The duration the animal was able to cling to a horizontal rope was recorded to a maximum of 5 secs. From these 3 tests, a total motor score (9 possible points) was computed. All tests were performed by a single experimenter who was blinded to the experimental condition of the animal. In every case, evaluations were performed in the same darkened, quiet room after a brief period of acclimation.
Histologic evaluation Following neurologic evaluation, all rats were anesthetized with 3.0% halothane in 30% O2/balance N 2 and the brains were perfusion fixed with buffered formalin in situ. After being imbedded in paraffin, the brains were subserially sectioned (5 /~m in the coronal plane) and stained with Celestine blue-acid fuchsin. Damage was assessed as the presence of acidophilic (pink or red) neurons ~. At two anatomically standardized levels (bregma -3.3 mm and bregma -3.8 mm), hippocampal injury in the CA1 sector was quantified by direct visual counting of the acidophilic neurons. At the level where the septal nuclei are at their widest point, injury in the neocortex and caudate nucleus (in the lateral and dorsal aspects) was assessed using a crude damage index (CDI) as follows: 0, no damage; 1, rare to occasional acidophilic cells per field (approximately 10% damage); 2, moderate number of acidophilic cells per field (approximately 10-50% damage); or 3, frequent acidophilic cells per field (greater than 50% damage) 1. Finally, thalamic injury was quantitated in the posterior and ventromedial/lateral subregions at bregma -3.8 mm using the above scoring system. All histoiogic evaluations were performed at a magnification of 320x by an experimenter blinded to the treatment group.
Statistical analysis Because of lack of concurrence, results from the two experiments were analyzed separately. Within each experiment, physiologic values were compared by a one-way analysis of variance using the post hoc Newman-Keuls t-test for between group differences when indicated by significant F-ratio. Similar analysis was made of the open field test values. Percent acidophilic neurons in the CA1 sector of the hippocampus, crude damage index scores for the neocortex and caudate nucleus, and the total motor scores were compared using the Kruskal-Wallis H-statistic while pairwise comparisons were made with the Mann-Whitney rank sum test including a Bonferroni adjustment if indicated by a significant H-value. Statistical significance was assumed with P < 0.05 and values are reported as mean _+ S.D. where appropriate.
RESULTS
Physiologic values There were no between group differences for arterial blood gases/pH, head temperature,
plasma glucose, or
MAP throughout either experiment except where MAP
178 TABLE I Physiologic values at indicated experimental intervals for Expt. 1 No differences between groups were observed for any variable. Values for Expt. 2 were similar throughout without differences between roups. Values = mean + S.D. Body weight
Primary insult alone (n = 7) 338+14g
Secondary insult alone (n = 8) 333+14g
Primary + secondary insult (n = 7) 341++_17g
115 + 38.6 + 7.39 + 132 + 106 +
120 + 13 39.6 + 1.4 7.38 + 0.02 130 + 32 104 _+7
122 + 9 38.5 _+ 1.1 7.40 + 0.02 127 + 14 106 + 5
Baseline (0.5% halothane)
Forebraln Ischemia
10 min Pre-ischemia PaO2 (mmHg)
paCO2 (mmHg) Arterial pH Glucose (mg/dl) MAP (mmHg)
12 1.5 0.02 20 5
Pre-hypotension
10 min Pre-hypotension PaO2 (mmHg)
paCO 2 (mmHg) Arterial pH MAP (mmHg)
113 + 38.8 + 7.40 + 112 +
10 1.9 0.02 7
123 + 38.1 + 7.39 + 107 +
12 1.6 0.02 7
116 + 13 38.4 + 1.5 7.40 + 0.02 111 _+7
115 + 12 40.0 + 2.5 7.38 + 0.02 109+12
110 + 12 40.4 + 1.9 7.38 _+0.02 114+6
] 50~tV
10 min Post-hypotension PaO2 (mmHg)
paC02 (mmHg) Arterial pH MAP (mmHg)
116 + 8.6 39.2 + 1.0 7.40 + 0.01 118+11
was purposefully r e d u c e d in specific groups during either the p r i m a r y o r secondary insults p e r experimental design (Table I). Electrocortical activity Experiment 1. In rats receiving the secondary insult alone, no changes from baseline E E G were observed during the hypotensive challenge. In those rats receiving the p r i m a r y insult alone, an E E G p a t t e r n of isoelectricity was o b s e r v e d throughout the 10 min ischemic interval. A f t e r reperfusion, the E E G r e c o v e r e d over the subsequent h o u r to an a b n o r m a l pattern characterized by p r e d o m i n a n t slow wave activity and a reduction in high frequency activity. This p a t t e r n persisted throughout the s e c o n d a r y insult without change in 6 of 7 rats. In the remaining rat, increased slowing was o b s e r v e d during the s e c o n d a r y insult. Experiment 2. Again, 10 min of forebrain ischemia ( p r i m a r y insult alone) resulted in an isoelectric E E G which r e c o v e r e d over the subsequent 80 min. In those rats receiving the secondary insult alone, m o d e r a t e slowing with loss of high frequency activity was observed late in the hypotensive episode (15-20 min) which rapidly r e c o v e r e d to a n o r m a l p a t t e r n after return to n o r m o t e n sion. In those rats receiving the p r i m a r y + secondary insults, the secondary insult resulted in further decreases in a m p l i t u d e and reduction of high-frequency activity in 4 out of 5 rats relative to patterns o b s e r v e d i m m e d i a t e l y
10' hypotension ( MAP = 25 mmHg)
I
.I 1 sec
Fig. 1. Representative EEG recordings taken from a rat subjected to 10 min of forebrain ischemia followed 1 h later by an episode of systemic hypotension (25 mmHg for 20 min).
prior to onset of the s e c o n d a r y insult (Fig. 1). These changes b e c a m e obvious early in the secondary insult interval and in two of these rats occasional episodes of burst suppression ( 2 - 5 s duration) were evident. In the remaining rat, the E E G b e c a m e isoelectric early in the hypotensive e p i s o d e which persisted until n o r m o t e n s i o n was restored. Histology Experiment 1. T h e secondary insult alone ( M A P = 40 m m H g for 20 min; n = 7), resulted in no detectable histologic injury in the h i p p o c a m p a l CA1 sector. In contrast, the p r i m a r y insult alone (10 min of forebrain ischemia; n = 7) resulted in 54 + 19% d e a d cells in CA1. A combination of the p r i m a r y and s e c o n d a r y insults (n = 7) caused a d e a t h rate of 59 + 27% for CA1 neurons which was not different from those animals receiving the p r i m a r y insult alone (Fig. 2). In the n e o c o r t e x (Fig. 3), the secondary insult alone p r o d u c e d no histologic injury with the exception of one rat wherein a few acidophilic neurons were observed. In contrast, in the p r i m a r y insult alone and the p r i m a r y + s e c o n d a r y insult groups, consistent neocortical injury was o b s e r v e d although there was no statistical difference b e t w e e n these two groups. A similar p a t t e r n of injury for the different groups was observed in the caudate nucleus (Fig. 3) and thalamic nuclei. Experiment 2. Ten min of forebrain ischemia alone (n
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= 7) again resulted in moderate CA1 injury (55 + 23%). The secondary insult alone failed to result in CA1 neuronal necrosis (n = 5). In contrast, the combination of primary + secondary insults (n = 6) caused a significant worsening (P < 0.05) of histologic injury with 73 + 20% of CA1 neurons staining acidophilic. In the neocortex and caudate nucleus, the secondary insult
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alone caused no histologic injury (Fig. 3). In contrast to the hippocampus, however, consistent but similar neocortical, caudate and thalamic injuries were observed in both the primary insult alone and primary + secondary insult groups. The one rat in the latter group sustaining E E G isoelectricity during the secondary insult had the worst histologic outcome in the study (CA1 dead cells = 91%, cortex CDI -- 3, caudate CDI = 3). This rat also displayed a relative hyperactivity in the open field test although it also achieved a total motor score of 9.
Neurology Neurologic outcome was similar for all groups in both experiments. Evaluation of motor function on day 7 revealed a maximal score of 9 in all Expt. 1 rats except for one rat from the primary + secondary insult group which scored an 8 and one rat from the primary insult alone group scoring a 7. In Expt. 2, again all rats scored a 9 except for one rat in the secondary insult alone group which scored a 6 and one rat in the primary + secondary insult group which scored an 8. Values from the open field test (mean total squares entered, rearings or defecations during a 5 min interval) were not different amongst treatment groups in either experiment.
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180 DISCUSSION The rationale for our initial experimental design was two-fold. First we sought to establish a secondary insult which the recovering post-ischemic brain might find injurious but also that the normal brain would readily tolerate. Pilot studies were performed which indicated that a 60-70% reduction of MAP from baseline values taken during anesthesia produced no histologic changes. Thus a secondary insult consisting of hypotension (MAP = 40 mmHg) for 20 min was adopted. Second, recent investigations exploring the role of secondary insults in affecting outcome from primary ischemia have identified the brain to be most vulnerable during the interval of post-ischemic delayed hypoperfusion 9'17. In the gerbil, where the pioneering investigations of secondary insults were performed, delayed hypoperfusion is clearly evident after one hour of recirculation 17. Because work utilizing the two-vessel rat forebrain ischemia model has also documented delayed hypoperfusion after 1 h of recirculation 3, we chose to examine the influence of the secondary insult at this interval. Nevertheless, when a secondary hypotensive insult consisting of MAP = 40 m m H g for 20 min was administered 1 h after reperfusion from 10 min forebrain ischemia, no worsening of histologic injury was observed nor was there worsening of neurologic outcome. Because 10 min of forebrain ischemia alone resulted in an intermediate severity of histologic injury in the selectively vulnerable hippocampal CA1 sector, there clearly was opportunity for outcome to be altered as has previously been demonstrated 2,6. Due to the failure of that secondary insult to worsen outcome from ischemia we hypothesized that a more severe challenge would be necessary to elicit such an effect. We therefore extended our work in Expt. 2, using a more severe hypotensive insult (MAP = 25 m m H g for 20 min), again administered during the interval of presumed maximal post-ischemic sensitivity. In pilot studies, rats (not having undergone the primary insult) were subjected to even more severe hypotensive challenges (e.g. MAP 15-20 mmHg), but exhibited an exceedingly high mortality rate which was associated with profound systemic acidosis. This suggests that animals in Expt. 2 were subjected to the most severe hypotensive insult that could practically be achieved without incorporating other systemic pathophysiology such as vascular occlusion or hypoxia. It should be noted, however, that Yamauchi et al. have recently reported a hemorrhagic shock model (MAP = 25 mm Hg) which produces a reproducible and rapid onset of E E G isoelectricity in spontaneously breathing rats 23. When blood pressure was reduced to 25 m m H g in our mechanically ventilated rats
after 1 h of reperfusion, histologic changes in the cortex and caudate nucleus, as well as neurologic outcomes, were not worsened. In contrast, there was a worsening of hippocampal CA1 injury, indicating that the hypoperfusion threshold necessary for the secondary insult to cause injury had been achieved for that structure. Because the hippocampus has repeatedly been demonstrated to be the most vulnerable of those cerebral structures evaluated in this experiment 1°'15, the hierarchy of selective vulnerability may persist in the context of secondary hypotensive insults. It could thus be postulated that a more prolonged secondary hypotensive challenge would have recruited further injury in either the neocortex or caudoputamen. In contrast, Kato et al. have observed that the thalamus exhibits a unique hypersensitivity to secondary 'sublethal' isoelectric ischemic challenges presenting an enigma as this structure is not normally considered to be selectively vulnerable to ischemia 9. Of interest is the fact that we failed to detect neurologic worsening in the ischemia groups relative to those which received hypotension only after 7 days recovery. This discrepancy between histologic and neurologic outcome from global brain ischemia in the rat has been encountered previously. For example, Poignet et al., while able to document early post-ischemic neurologic deficits, found these changes to be essentially normalized after 72 h of recovery such that in the final analysis neurology and histology did not correlate j2. Our use of the total motor score developed by Combs and D'Alecy was adopted because of their reported ability to discriminate changes in motor function following forebrain ischemia 4. However, review of the Combs and D'Alecy data suggests a progressive improvement of function until 48 h when the final evaluation was made. Given the relatively modest injury in the neocortex and basal ganglia observed in our study, it seems likely that functional plasticity would prevail over the 7 day recovery interval and obscure deficits characteristic of injury in these regions. We did not assess hippocampal function where persistent deficits (e.g. performance in the radial maze) following forebrain ischemia have been observed 20,21. We are left to conclude that the value of assaying motor function as late as 7 days post-ischemia is not specific for histologic injury in this model. Our results do indicate that the animals were not neurologically devastated by the addition of the secondary insult. This is significant in revealing the robustness of the brain to secondary hypotensive insults relative to other potential challenges. For example, moderate pre-ischemic hyperglycemia in the same rat model leads to severe neurologic changes (i.e. audiogenic status epilepticus) and high mortality in the early recovery interval (i.e. within 24 h) 22. It is tempting to speculate that abolition of electrical
181 activity by the secondary insult is a prerequisite for causing substantial worsening of outcome. Other work evaluating secondary insults has been performed primarily in the Mongolian gerbil where the carotid arteries are occluded 8' 9,17. In that model, the E E G becomes isoelectric within 60 s in both the cortex and hippocampus 16. Similarly, the remaining study which demonstrated worsening of outcome from primary brain injury (after a fluid percussion injury in the rat) as a result of a secondary insult incorporated as the secondary insult the two-vessel occlusion ischemia model which we used for the primary insult. That insult by definition 14, also invokes E E G isoelectricity. Since the ischemic threshold for E E G isoelectridty is generally considered to be greater than that which produces ion pump failure 13, it would follow that irreversible events are unlikely to be initiated in the presence of an active E E G . This is supported by recent work which has demonstrated that in vivo fluorometrically determined influx of calcium occurs only in the presence of severe attenuation of regional E E G activity TM. To the extent that the recorded electrocortical activity in Expt. 2 reflects electrical activity in the hippocampus (i.e. hippocampal activity was not directly assessed), the marked attenuation of E E G during the secondary insult can be correlated with increased CA1 neuronal necrosis. Similarly, in Expt. 1, where little or no E E G changes were associated with hypotension administered after forebrain ischemia, no histologic injury was observed.
On the other hand, in the neocortex where we are able to compare E E G changes with histology directly, the substantial attenuation of electrical activity observed during the secondary insult was not associated with increased neocortical injury. Therefore, further investigation utilizing more regionally specific electrical monitoring will be necessary to determine critical electrographic thresholds for secondary hypotensive challenges to worsen histologic outcome. In summary, this investigation explored the effect of secondary hypotensive insults, which were insufficiently severe to independently cause histologic injury, on outcome from global ischemia. The secondary insults were applied during the post-ischemic interval when maximal sensitivity to such a challenge would be expected (i.e. during delayed hypoperfusion). Despite the fact that severe hypotensive challenges were applied, such secondary insults failed to worsen neurologic outcome. In contrast, the hippocampus exhibited an enhanced injury within the CA1 sector resulting from the more severe secondary hypotensive challenge. Otherwise, histologic outcome was unaffected by the secondary insult. Within this model, the recovering post-ischemic brain therefore demonstrates a surprising robustness to subsequent hypotensive challenges.
REFERENCES
Neuropathol., 79 (1990) 494-500. 10 Kirino, T.,Sano, K., Selective vulnerability in the gerbil hippocampus following transient ischemia, Acta NeuropathoL, 62 (1984) 201-208 11 Pluta, R., Tomida, S., Ikeda, J., Nowak, J. T. and Klatzo, I., Cerebral vascular volume after repeated isehemie insults in the gerbil: comparison with changes in CBF and brain edema, Z Cereb. Blood Flow Metab., 9 (1989) 163-170. 12 Poignet, H., Beaughard, M., Lecoin, G. and Massingham, R., Functional, behavioral, and histologic changes induced by transient global cerebral ischemia in rats: effects of cinnarizine and flunarizine, J. Cereb. Blood Flow Meta~ol., 9 (1989) 646-654. 13 Siesjo, B., Cerebral circulation and metabolism, J. Neurosurg., 60 (1984) 883-908. 14 Smith, M., Bendek, G., Dahlgren, N., Rosen, I. and Wieloeh, T. and Siesjo, B., Models for studying long-term recovery following forebrain isehemia in the rat. 2: A 2-vessel occlusion model, Acta Neurol. Scand., 69 (1984) 385-401. 15 Smith, M.-L., Auer, R. and Siesjo, B., The density and distribution of ischemic brain injury in the rat following 2-10 rain of forebrain ischemia, Acta Neuropathol., 64 (1984) 319-332. 16 Suzuki, R., Yamaguchi, T., Li, C. and Klatzo, I., The effects of 5-minute ischemia in Mongolian gerbils: II. Changes of spontaneous neuronal activity in cerebral cortex and CA1 sector of hippocampus, A cta Neuropathol. , 60 (1983) 217-222. 17 Tomida, S., Nowak, J. T., Vass, K., Lohr, J. and Klatzo, I., Experimental model for repetitive ischemic attacks in the gerbil: the cumulative effect of repeated ischemie insults, J. Cereb. Blood Flow Metab., 7 (1987) 773-782. 18 Uematsu, D., Greenberg, J., Reivich, M. and Hiekey, W., Direct evidence for calcium-induced ischemic and reperfusion injury, Ann. Neurol., 26 (1989) 280-283.
1 Auer, R., Olsson, Y. and Siesjo, B., Hypoglycemic brain injury in the rat. Correlation of density of brain damage with the EEG isoelectric time: a quantitative study, Diabetes, 33 (1984) 1090--1098. 2 Blair, J., Warner, D. and Todd, M., Effects of elevated plasma magnesium versus calcium on cerebral ischemic injury in rats, Stroke, 20 (1989) 507-512. 3 Blomqvist, P., Lindvall, O. and Wieloch, T., Delayed postischemic hypoperfusion: evidence against involvement of the noradrenergic locus ceruleus system, J. Cereb. Blood Flow Metab., 4 (1984) 425-429. 4 Combs, D. and D'Alecy, L., Motor performance in rats exposed to severe forebrain ischemia: effect of fasting and 1,3-butanediol, Stroke, 18 (1987) 503-511. 5 Denenberg, V., Open field test: what does it mean?, Ann. N.Y. Acad. Sci., 159 (1969) 852-863. 6 Deshpande, J. and Wieloch, T., Flunarizine, a calcium entry blocker, ameliorates ischemic brain damage in the rat, Anesthesiology, 64 (1986) 215-224. 7 Jenkins, L., Moszynski, K., Lyeth, B., Lewelt, W., DeWitt, D., Allen, A., Dixon, C., Povlishock, J., Majewski, T., Clifton, G., Young, H., Becker, D. and Hayes, R., Increased vulnerability of the mildly traumatized rat brain to cerebral ischemia: the use of controlled secondary ischemia as a research tool to identify common or different mechanisms contributing to mechanical and ischemic injury, Brain Research, 477 (1989) 211-224. 8 Kato, H., Kogure, K. and Nakano, S., Neuronal damage following repeated brief ischemia in the gerbil, Brain Research, 479 (1989) 366-370. 9 Kato, H. and Kogure, K., Neuronal damage following nonlethal but repeated cerebral ischemia in the gerbil, Acta
Acknowledgements. This work was supported in part by NIH Grants R29-GM39771 (D.S.W.) and RO1-NS24517 (M.M.T.).
182 19 Vass, K., Tomida, S., Hossmann, K., Nowak, J.T. and Klatzo, I., Microvascular disturbances and edema formation after repetitive ischemia of gerbil brain, Acta Neuropathol., 75 (1988) 288-294. 20 Volpe, B., Davis, H. and Colombo, P., Preoperative training modifies radial maze performance in rats with ischemic hippocampal injury, Stroke, 20 (1989) 1700-1706. 21 Volpe, B., Pulsinelli, W., Tribuna, J. and Davis, H., Behavioral
performance of rats following transient forebrain ischemia,
Stroke, 15 (1984) 558-562. 22 Warner, D., Smith, M. and Siesjo, B., Ischemia in normo- and hyperglycemic rats: Effects on brain water and electrolytes, Stroke, 18 (1987) 464-471. 23 Yamauchi, Y., Kato, H. and Kogure, K., Brain damage in a new hemorrhagic shock model in the rat using long-term recovery, J. Cereb. Blood Flow Metab., 10 (1990) 207-212.
Brain Research. 536 (1990) 176- 182 Elsevier
BRES 16148
Secondary hypotensive insults in a rat forebrain ischemia model David S. Warner, Daniel K. Reasoner, Michael M. Todd and Alice McAllister Neuroanesthesia Research Group, Department of Anesthesia, University of Iowa College of Medicine, Iowa City, IA 52242 (U.S.A.) (Accepted 10 July 1990) Key words: Rat; Brain; Ischemia; Hypoperfusion; Repeated ischemia
Previous studies have shown that the recently injured brain has an increased sensitivity to subsequent brief episodes of severe i~:.hemia. This investigation was designed to assess whether less severe secondary insults, which alone would be incapable of producing injury, exacerbate brain damage resulting from a primary episode of global ischemia. Rats were subjected to either 10 rain of 2-vessel forebrain isehemia (primary insult alone), 20 min of hypotension (mean arterial pressure, MAP = either 40 or 25 mmHg) without vessel occlusion (secondary insult alone), or 10 min isehemia followed 1 h later by the hypotensive challenge (primary+secondary insult). Seven days later, the animals were neurologically evaluated and the brains then prepared for histoiogic analysis. Neither magnitude of secondary insult alone was found to produce injury. In contrast, the primary insult alone caused moderate damage in the hippocampns, caudoputamen and neocortex. With the exception of increased neuronal necrosis in the hippocampal CA1 sector in rats receiving the primary + secondary insult (MAP = 25 mmHg), no worsening of outcome could be attributed to the secondary insults. These results indicate that the recovering brain may not be as sensitive to hypoperfusion as has previously been suggested. INTRODUCTION Investigations using rodents have demonstrated that the recently injured brain is uniquely susceptible to secondary ischemic insults 7-9'11'17. For example, Jenkins et al. have shown that when a 6 min episode of severe forebrai'n ischemia was produced 1 h after a fluid percussion injury, resultant histopathologic changes were worse than those produced by either insult in isolation 7. Similarly, Tomida et al. have demonstrated that three sequential 5 min episodes of cerebral ischemia, each separated from the next by 1 h, resulted in a worsened outcome compared to a single 15 min ischemic interval 17. Investigations concerning secondary insults have become even more interesting to the clinician given reports that such events, which in and of themselves were of an insufficient duration to injure the normal brain, caused a worsening of injury when administered to the recovering post-ischemic brain 8'9. However, such investigations have studied severe secondary insults, i.e. near-complete global ischemia, which are capable of producing both major electrophysiologic abnormalities (e.g. an isoelectric E E G ) as well as tissue damage. Secondary insults of this severity are not c o m m o n in clinical medicine, where moderate hypotension or hypoxia are far more likely to be superimposed on pre-existing disease states such as occlusive stroke, subarachnoid hemorrhage or cardiac arrest. To date there
have been no studies providing information on the sensitivity of the damaged brain to these less severe secondary insults. In other words, how severe must a secondary hypotensive insult be to worsen a pre-existent injury? To examine this question, we have employed the two-vessel forebrain ischemia model to produce a standardized 10 min primary insult in the rat. After one hour of recirculation, a transient (20 min) episode of either moderate (40 m m H g ) or severe (25 m m H g ) systemic hypotension was produced, followed by 7 days of recovery. The histopathologic and behavioral changes seen in these animals were then compared with those seen in animals subjected to either forebrain isehemia or hypotension alone. Both histologic and neurologic analyses indicate that the brain is surprisingly resistant to such secondary insults in this context.
MATERIALS AND METHODS Surgical preparation and experimental design With Institutional Animal Care and Use Committee approval, all animals underwent the following preparation. Male Spragu¢Dawley rats (age range 9-10 weeks) were fasted from food but allowed free access to water for 12-t6 h prior to the experiment. Following induction of anesthesia with 1-2% halothane in 30% Ozeoalance N2, each rat was intubated and the lungs were mechanically ventilated to maintain normocapnin (PoCO2 range = 38-42 mmHg) and normoxia (PaO2 range = 100--135mmHg). ~ e tail artery was cannulated to allow both blood pressure monitoring
Correspondence: D.S. Warner, Department of Anesthesia, University of Iowa College of Medicine, Iowa City, IA 52242, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
177 and sampling for blood gas/pH analysis. Via a ventral neck incision, both carotid arteries were isolated and loosely encircled with sutures. The right internal jugular vein was cannulated and 50 IU heparin were given intravenously. Muscle paralysis was obtained by a 1.0 mg i.v. bolus of succinylcholine repeated as necessary. Bipolar E E G activity was monitored from a pair of needle electrodes inserted into the temporalis muscle bilaterally and a reference electrode inserted into the animals snout. Finally, a 22 gauge needle thermistor was percutaneously placed adjacent to the skull and pericranial temperature was thenceforth servo-regulated at 37.0 + 0.1 °C. A total surgical interval of 30 min was allowed for each animal. The halothane concentration was then reduced to 0.5%, and a subsequent a 30 min interval was allowed for physiologic stabilization. Immediately thereafter, the animals were assigned to one of three groups in each of two separate experiments defined as follows.
Experiment 1 Primary insult only. Ten min forebrain ischemia (see below) followed by 0.5% halothane anesthesia for 80 min. Secondary insult only. Anesthesia only (sham ischemia) followed 1 h later by 20 min of systemic hypotension (MAP = 40 + 5 mmHg, see below). Primary+secondary insult. Ten min forebrain ischemia followed 1 h later by 20 min systemic hypotension (MAP = 40 + 5 mmHg, see below).
Experiment 2 When the results of Expt. 1 were analyzed, 3 more groups of rats were studied using precisely the same protocol as described above, except the severity of the secondary insult was enhanced by reducing MAP in these animals to 25 mmHg for 20 min in the appropriate groups. Note that in all groups (Expts. 1 and 2), the total duration of halothane anesthesia was identical. Primary insult. Forebrain ischemia (FBI) was produced as previously described 2, 14. Briefly, trimethaphan (3.5 mg) was given as an i.v. bolus and the carotid arteries were occluded bilaterally for 10 min using temporary aneurysm clips. During this 10 min interval, MAP was maintained at 50 + 5 mmHg by withdrawal of venous blood from the jugular catheter as necessary. E E G was continuously monitored and isoelectricity documented throughout the ischemic interval. Perfusion was restored by removal of the carotid artery clips and reinfusion of shed blood causing MAP to recover to at least 100 mmHg within 2 min. Secondary insult. In Expt. 1, systemic hypotension was produced by an intravenous bolus of 3.5 mg trimethaphan followed immediately by withdrawal of blood from the venous catheter as necessary to maintain MAP = 40 + 5 mmHg for 20 min. The blood was then reinfused and the animal recovered (see below). In these animals, resultant metabolic acidosis was of insufficient magnitude to warrant administration of NaHCO3. In Expt. 2 a similar protocol was employed with the following exceptions: (1) MAP was reduced to 25 + 5 mmHg for 20 min induced by 3.0 mg i.v. trimethaphan and maintained by bleeding from the jugular catheter; (2) a second i.v. bolus of trimethaphan (2.0 mg) was given 10 rain into the 20 min interval; and (3) metabolic acidosis occuring after reinfusion of shed blood was treated with i.v. NaHCO 3 as necessary to return arterial pH to the range of 7.38-7.40. Following completion of protocol in both experiments, the vascular catheters were removed and the wounds closed with suture. Anesthesia was discontinued, and when the rats were breathing spontaneously, the tracheas were extubated. The rats were then placed in an oxygen enriched environment for 45-60 min whereupon they were returned to their cages. A survival interval of 7 days with free access to food and water was allowed whereupon the rats were neurologically evaluated as follows.
Neurologic assessment The rats were first subjected to an open field test which was
intended to quantitate the potential for exploratory activity5. Each rat was placed in a wooden box having a square floor (90 x 90 era) painted with a grid, each square being 15 x 15 cm in area (i.e. a total of 36 squares). The rat was then observed over a 5 rain period and the total number of squares the animal's snout entered as well as the total number of times the animal raised up on its hind limbs or defecated was recorded. Motor function was then assessed as described by Combs and D'Alecy 4. Briefly, the rats were placed on a 29 x 30 cm screen (grid size 0.6 × 0.7 cm) that could be rotated from 0° (horizontal) to 90° (vertical). The duration of time that each rat was able to hold onto the vertically-oriented screen was recorded to a maximum of 15 secs (allowing a total of 3 points). Next, the rat was placed at the center of a horizontal wooden rod (1" diameter), and the duration that the animal remained balanced on the rod was recorded to a maximum of 30 secs (allowing a total of 3 points). Finally, a prehensile-traction test was administered. The duration the animal was able to cling to a horizontal rope was recorded to a maximum of 5 secs. From these 3 tests, a total motor score (9 possible points) was computed. All tests were performed by a single experimenter who was blinded to the experimental condition of the animal. In every case, evaluations were performed in the same darkened, quiet room after a brief period of acclimation.
Histologic evaluation Following neurologic evaluation, all rats were anesthetized with 3.0% halothane in 30% O2/balance N 2 and the brains were perfusion fixed with buffered formalin in situ. After being imbedded in paraffin, the brains were subserially sectioned (5 /~m in the coronal plane) and stained with Celestine blue-acid fuchsin. Damage was assessed as the presence of acidophilic (pink or red) neurons ~. At two anatomically standardized levels (bregma -3.3 mm and bregma -3.8 mm), hippocampal injury in the CA1 sector was quantified by direct visual counting of the acidophilic neurons. At the level where the septal nuclei are at their widest point, injury in the neocortex and caudate nucleus (in the lateral and dorsal aspects) was assessed using a crude damage index (CDI) as follows: 0, no damage; 1, rare to occasional acidophilic cells per field (approximately 10% damage); 2, moderate number of acidophilic cells per field (approximately 10-50% damage); or 3, frequent acidophilic cells per field (greater than 50% damage) 1. Finally, thalamic injury was quantitated in the posterior and ventromedial/lateral subregions at bregma -3.8 mm using the above scoring system. All histoiogic evaluations were performed at a magnification of 320x by an experimenter blinded to the treatment group.
Statistical analysis Because of lack of concurrence, results from the two experiments were analyzed separately. Within each experiment, physiologic values were compared by a one-way analysis of variance using the post hoc Newman-Keuls t-test for between group differences when indicated by significant F-ratio. Similar analysis was made of the open field test values. Percent acidophilic neurons in the CA1 sector of the hippocampus, crude damage index scores for the neocortex and caudate nucleus, and the total motor scores were compared using the Kruskal-Wallis H-statistic while pairwise comparisons were made with the Mann-Whitney rank sum test including a Bonferroni adjustment if indicated by a significant H-value. Statistical significance was assumed with P < 0.05 and values are reported as mean _+ S.D. where appropriate.
RESULTS
Physiologic values There were no between group differences for arterial blood gases/pH, head temperature,
plasma glucose, or
MAP throughout either experiment except where MAP
178 TABLE I Physiologic values at indicated experimental intervals for Expt. 1 No differences between groups were observed for any variable. Values for Expt. 2 were similar throughout without differences between roups. Values = mean + S.D. Body weight
Primary insult alone (n = 7) 338+14g
Secondary insult alone (n = 8) 333+14g
Primary + secondary insult (n = 7) 341++_17g
115 + 38.6 + 7.39 + 132 + 106 +
120 + 13 39.6 + 1.4 7.38 + 0.02 130 + 32 104 _+7
122 + 9 38.5 _+ 1.1 7.40 + 0.02 127 + 14 106 + 5
Baseline (0.5% halothane)
Forebraln Ischemia
10 min Pre-ischemia PaO2 (mmHg)
paCO2 (mmHg) Arterial pH Glucose (mg/dl) MAP (mmHg)
12 1.5 0.02 20 5
Pre-hypotension
10 min Pre-hypotension PaO2 (mmHg)
paCO 2 (mmHg) Arterial pH MAP (mmHg)
113 + 38.8 + 7.40 + 112 +
10 1.9 0.02 7
123 + 38.1 + 7.39 + 107 +
12 1.6 0.02 7
116 + 13 38.4 + 1.5 7.40 + 0.02 111 _+7
115 + 12 40.0 + 2.5 7.38 + 0.02 109+12
110 + 12 40.4 + 1.9 7.38 _+0.02 114+6
] 50~tV
10 min Post-hypotension PaO2 (mmHg)
paC02 (mmHg) Arterial pH MAP (mmHg)
116 + 8.6 39.2 + 1.0 7.40 + 0.01 118+11
was purposefully r e d u c e d in specific groups during either the p r i m a r y o r secondary insults p e r experimental design (Table I). Electrocortical activity Experiment 1. In rats receiving the secondary insult alone, no changes from baseline E E G were observed during the hypotensive challenge. In those rats receiving the p r i m a r y insult alone, an E E G p a t t e r n of isoelectricity was o b s e r v e d throughout the 10 min ischemic interval. A f t e r reperfusion, the E E G r e c o v e r e d over the subsequent h o u r to an a b n o r m a l pattern characterized by p r e d o m i n a n t slow wave activity and a reduction in high frequency activity. This p a t t e r n persisted throughout the s e c o n d a r y insult without change in 6 of 7 rats. In the remaining rat, increased slowing was o b s e r v e d during the s e c o n d a r y insult. Experiment 2. Again, 10 min of forebrain ischemia ( p r i m a r y insult alone) resulted in an isoelectric E E G which r e c o v e r e d over the subsequent 80 min. In those rats receiving the secondary insult alone, m o d e r a t e slowing with loss of high frequency activity was observed late in the hypotensive episode (15-20 min) which rapidly r e c o v e r e d to a n o r m a l p a t t e r n after return to n o r m o t e n sion. In those rats receiving the p r i m a r y + secondary insults, the secondary insult resulted in further decreases in a m p l i t u d e and reduction of high-frequency activity in 4 out of 5 rats relative to patterns o b s e r v e d i m m e d i a t e l y
10' hypotension ( MAP = 25 mmHg)
I
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Fig. 1. Representative EEG recordings taken from a rat subjected to 10 min of forebrain ischemia followed 1 h later by an episode of systemic hypotension (25 mmHg for 20 min).
prior to onset of the s e c o n d a r y insult (Fig. 1). These changes b e c a m e obvious early in the secondary insult interval and in two of these rats occasional episodes of burst suppression ( 2 - 5 s duration) were evident. In the remaining rat, the E E G b e c a m e isoelectric early in the hypotensive e p i s o d e which persisted until n o r m o t e n s i o n was restored. Histology Experiment 1. T h e secondary insult alone ( M A P = 40 m m H g for 20 min; n = 7), resulted in no detectable histologic injury in the h i p p o c a m p a l CA1 sector. In contrast, the p r i m a r y insult alone (10 min of forebrain ischemia; n = 7) resulted in 54 + 19% d e a d cells in CA1. A combination of the p r i m a r y and s e c o n d a r y insults (n = 7) caused a d e a t h rate of 59 + 27% for CA1 neurons which was not different from those animals receiving the p r i m a r y insult alone (Fig. 2). In the n e o c o r t e x (Fig. 3), the secondary insult alone p r o d u c e d no histologic injury with the exception of one rat wherein a few acidophilic neurons were observed. In contrast, in the p r i m a r y insult alone and the p r i m a r y + s e c o n d a r y insult groups, consistent neocortical injury was o b s e r v e d although there was no statistical difference b e t w e e n these two groups. A similar p a t t e r n of injury for the different groups was observed in the caudate nucleus (Fig. 3) and thalamic nuclei. Experiment 2. Ten min of forebrain ischemia alone (n
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= 7) again resulted in moderate CA1 injury (55 + 23%). The secondary insult alone failed to result in CA1 neuronal necrosis (n = 5). In contrast, the combination of primary + secondary insults (n = 6) caused a significant worsening (P < 0.05) of histologic injury with 73 + 20% of CA1 neurons staining acidophilic. In the neocortex and caudate nucleus, the secondary insult
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alone caused no histologic injury (Fig. 3). In contrast to the hippocampus, however, consistent but similar neocortical, caudate and thalamic injuries were observed in both the primary insult alone and primary + secondary insult groups. The one rat in the latter group sustaining E E G isoelectricity during the secondary insult had the worst histologic outcome in the study (CA1 dead cells = 91%, cortex CDI -- 3, caudate CDI = 3). This rat also displayed a relative hyperactivity in the open field test although it also achieved a total motor score of 9.
Neurology Neurologic outcome was similar for all groups in both experiments. Evaluation of motor function on day 7 revealed a maximal score of 9 in all Expt. 1 rats except for one rat from the primary + secondary insult group which scored an 8 and one rat from the primary insult alone group scoring a 7. In Expt. 2, again all rats scored a 9 except for one rat in the secondary insult alone group which scored a 6 and one rat in the primary + secondary insult group which scored an 8. Values from the open field test (mean total squares entered, rearings or defecations during a 5 min interval) were not different amongst treatment groups in either experiment.
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180 DISCUSSION The rationale for our initial experimental design was two-fold. First we sought to establish a secondary insult which the recovering post-ischemic brain might find injurious but also that the normal brain would readily tolerate. Pilot studies were performed which indicated that a 60-70% reduction of MAP from baseline values taken during anesthesia produced no histologic changes. Thus a secondary insult consisting of hypotension (MAP = 40 mmHg) for 20 min was adopted. Second, recent investigations exploring the role of secondary insults in affecting outcome from primary ischemia have identified the brain to be most vulnerable during the interval of post-ischemic delayed hypoperfusion 9'17. In the gerbil, where the pioneering investigations of secondary insults were performed, delayed hypoperfusion is clearly evident after one hour of recirculation 17. Because work utilizing the two-vessel rat forebrain ischemia model has also documented delayed hypoperfusion after 1 h of recirculation 3, we chose to examine the influence of the secondary insult at this interval. Nevertheless, when a secondary hypotensive insult consisting of MAP = 40 m m H g for 20 min was administered 1 h after reperfusion from 10 min forebrain ischemia, no worsening of histologic injury was observed nor was there worsening of neurologic outcome. Because 10 min of forebrain ischemia alone resulted in an intermediate severity of histologic injury in the selectively vulnerable hippocampal CA1 sector, there clearly was opportunity for outcome to be altered as has previously been demonstrated 2,6. Due to the failure of that secondary insult to worsen outcome from ischemia we hypothesized that a more severe challenge would be necessary to elicit such an effect. We therefore extended our work in Expt. 2, using a more severe hypotensive insult (MAP = 25 m m H g for 20 min), again administered during the interval of presumed maximal post-ischemic sensitivity. In pilot studies, rats (not having undergone the primary insult) were subjected to even more severe hypotensive challenges (e.g. MAP 15-20 mmHg), but exhibited an exceedingly high mortality rate which was associated with profound systemic acidosis. This suggests that animals in Expt. 2 were subjected to the most severe hypotensive insult that could practically be achieved without incorporating other systemic pathophysiology such as vascular occlusion or hypoxia. It should be noted, however, that Yamauchi et al. have recently reported a hemorrhagic shock model (MAP = 25 mm Hg) which produces a reproducible and rapid onset of E E G isoelectricity in spontaneously breathing rats 23. When blood pressure was reduced to 25 m m H g in our mechanically ventilated rats
after 1 h of reperfusion, histologic changes in the cortex and caudate nucleus, as well as neurologic outcomes, were not worsened. In contrast, there was a worsening of hippocampal CA1 injury, indicating that the hypoperfusion threshold necessary for the secondary insult to cause injury had been achieved for that structure. Because the hippocampus has repeatedly been demonstrated to be the most vulnerable of those cerebral structures evaluated in this experiment 1°'15, the hierarchy of selective vulnerability may persist in the context of secondary hypotensive insults. It could thus be postulated that a more prolonged secondary hypotensive challenge would have recruited further injury in either the neocortex or caudoputamen. In contrast, Kato et al. have observed that the thalamus exhibits a unique hypersensitivity to secondary 'sublethal' isoelectric ischemic challenges presenting an enigma as this structure is not normally considered to be selectively vulnerable to ischemia 9. Of interest is the fact that we failed to detect neurologic worsening in the ischemia groups relative to those which received hypotension only after 7 days recovery. This discrepancy between histologic and neurologic outcome from global brain ischemia in the rat has been encountered previously. For example, Poignet et al., while able to document early post-ischemic neurologic deficits, found these changes to be essentially normalized after 72 h of recovery such that in the final analysis neurology and histology did not correlate j2. Our use of the total motor score developed by Combs and D'Alecy was adopted because of their reported ability to discriminate changes in motor function following forebrain ischemia 4. However, review of the Combs and D'Alecy data suggests a progressive improvement of function until 48 h when the final evaluation was made. Given the relatively modest injury in the neocortex and basal ganglia observed in our study, it seems likely that functional plasticity would prevail over the 7 day recovery interval and obscure deficits characteristic of injury in these regions. We did not assess hippocampal function where persistent deficits (e.g. performance in the radial maze) following forebrain ischemia have been observed 20,21. We are left to conclude that the value of assaying motor function as late as 7 days post-ischemia is not specific for histologic injury in this model. Our results do indicate that the animals were not neurologically devastated by the addition of the secondary insult. This is significant in revealing the robustness of the brain to secondary hypotensive insults relative to other potential challenges. For example, moderate pre-ischemic hyperglycemia in the same rat model leads to severe neurologic changes (i.e. audiogenic status epilepticus) and high mortality in the early recovery interval (i.e. within 24 h) 22. It is tempting to speculate that abolition of electrical
181 activity by the secondary insult is a prerequisite for causing substantial worsening of outcome. Other work evaluating secondary insults has been performed primarily in the Mongolian gerbil where the carotid arteries are occluded 8' 9,17. In that model, the E E G becomes isoelectric within 60 s in both the cortex and hippocampus 16. Similarly, the remaining study which demonstrated worsening of outcome from primary brain injury (after a fluid percussion injury in the rat) as a result of a secondary insult incorporated as the secondary insult the two-vessel occlusion ischemia model which we used for the primary insult. That insult by definition 14, also invokes E E G isoelectricity. Since the ischemic threshold for E E G isoelectridty is generally considered to be greater than that which produces ion pump failure 13, it would follow that irreversible events are unlikely to be initiated in the presence of an active E E G . This is supported by recent work which has demonstrated that in vivo fluorometrically determined influx of calcium occurs only in the presence of severe attenuation of regional E E G activity TM. To the extent that the recorded electrocortical activity in Expt. 2 reflects electrical activity in the hippocampus (i.e. hippocampal activity was not directly assessed), the marked attenuation of E E G during the secondary insult can be correlated with increased CA1 neuronal necrosis. Similarly, in Expt. 1, where little or no E E G changes were associated with hypotension administered after forebrain ischemia, no histologic injury was observed.
On the other hand, in the neocortex where we are able to compare E E G changes with histology directly, the substantial attenuation of electrical activity observed during the secondary insult was not associated with increased neocortical injury. Therefore, further investigation utilizing more regionally specific electrical monitoring will be necessary to determine critical electrographic thresholds for secondary hypotensive challenges to worsen histologic outcome. In summary, this investigation explored the effect of secondary hypotensive insults, which were insufficiently severe to independently cause histologic injury, on outcome from global ischemia. The secondary insults were applied during the post-ischemic interval when maximal sensitivity to such a challenge would be expected (i.e. during delayed hypoperfusion). Despite the fact that severe hypotensive challenges were applied, such secondary insults failed to worsen neurologic outcome. In contrast, the hippocampus exhibited an enhanced injury within the CA1 sector resulting from the more severe secondary hypotensive challenge. Otherwise, histologic outcome was unaffected by the secondary insult. Within this model, the recovering post-ischemic brain therefore demonstrates a surprising robustness to subsequent hypotensive challenges.
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Acknowledgements. This work was supported in part by NIH Grants R29-GM39771 (D.S.W.) and RO1-NS24517 (M.M.T.).
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