J Neurosurg 77:438-444, 1992
Reduction by delayed hypothermia of cerebral infarction following middle cerebral artery occlusion in the rat: a time-course study CHRISTOPHER J. BAKER, M.D., STEPHEN T. ONESTI, M.D., AND ROBERT A. SOLOMON, M.D.
Department of Neurological Surgery, Columbia-Presbyterian Medical Center, Columbia University College of Physicians and Surgeons, New York, New York t,, The effect of hypothermia on neuronal injury following permanent middle cerebral artery (MCA) occlusion in the rat was examined. Moderate hypothermia (body temperature 24"C) was induced before MCA occlusion (0-minute delay group) in six rats, at 30 minutes in eight rats, and at 1 (seven rats), 2 (seven rats), and 3 (nine rats) hours after occlusion. The rats were kept at a 24~ body temperature for 1 hour, then allowed to rewarm over 90 minutes. The animals were sacrificed 24 hours after MCA occlusion, and infarction was visualized by staining of coronal sections with 2,3,5-triphenyltetrazolium chloride. Infarct volumes were compared to matched normothermic control rats (body temperature 36'C). Additional groups of 0-minute delay hypothermic (10 rats) and control animals (nine rats) were sacrificed 72 hours after MCA occlusion to examine the effects of prolonged survival. A significant reduction in the percentage of infarcted fight hemisphere was seen in the animals sacrificed after 24 hours with 0-minute, 30-minute, and 1-hour delays in inducing hypothermia (mean _+standard error of the mean: 2.2% ___0.7%, 4.4% _+ 0.9%, and 3.6% - 1.1%, respectively) as compared to normothermic control rats (10.8% _+ 1.5%, p < 0.01 by Student's t-test). In the 2- and 3-hour delay groups, the percentage of infarcted fight hemisphere was 17.1% _+ 2.4% and 12.0% _+ 2.7%, respectively, and no decrease in infarct volume was observed. The 0-minute delay hypothermia group sacrificed after 72 hours also displayed a significant reduction in right hemisphere infarct compared to their respective controls (4.8% vs. 11.7%, p < 0.05). These findings indicate that, in the setting of permanent MCA occlusion, hypothermia markedly decreases brain injury even when its induction is delayed for up to 1 hour after the onset of ischemia. Ischemic damage does not appear to be merely retarded but permanently averted. KEY WORDS
'
hypothermia
9 ischemia
VER the last decade, there has been a resurgence in the use of hypothermic cerebral protection in neurosurgery. Systemic cooling and cardiopulmonary bypass have been increasingly used in the treatment of giant basilar and other complex aneurysms.3'16'25'39'41'61-63A number of centers have instituted new hypothermia-based protocols to re-examine the efficacy of systemic cooling in treating severe head trauma, t7 This renewed interest in hypothermia is both a product of, and a catalyst for, the expanding research devoted to understanding the cellular mechanisms of ischemic neuronal injury. In an effort to characterize the temporal nature of hypothermic protection in permanent focal ischemia, we studied the effects of immediate and delayed hypothermia on neuronal injury
O
438
9
middle cerebral artery occlusion
9 rat
following middle cerebral artery (MCA) occlusion in the rat. Materials and Methods
The protocol used in this study has previously been reported in detail and was approved by the Institutional Animal Care and Use Committee. TM Aseptic technique was employed during all experiments. Adult male Wistar rats, each weighing 350 to 400 gm, were anesthetized with intraperitoneal chloral hydrate (400 mg/kg). The animals subsequently underwent right femoral artery cannulation, ligation of the right common carotid artery, and tracheal intubation under direct visualization. The rats were artificially ventilated with a Harvard rodent ventilator and adjustments were made to main-
J. Neurosurg. / Volume 77/September, 1992
Delayed hypothermia for reduction of infarction TABLE 1 Comparison of temporalis muscle and brain temperature in rats*
Treatment
Temporalis Intraparenchymal Muscle Brain Group Temperature ('C) Temperature('C) normothermia(36*C) 36.0 _+0.1 35.4 _ 0.2 hypothermia(24"C) 24.2 _+0.1 25.0 _ 0.8 * Valuesare expressedas means + standard deviation. tain femoral artery blood gases within normal physiological limits. Blood gas and hematocrit levels were obtained preoperatively, after MCA occlusion, during hypothermia, and upon return to normothermia. Heart rate and blood pressure were recorded continuously. Contralateral temporalis muscle temperature was monitored throughout the experiment in all rats;* this appears to correlate closely with intraparenchymal brain temperature in our model (Table 1).45 With the animal in the left lateral decubitus position, an oblique incision was made between the inferior margin of the orbit and the tragus. The exposed temporalis muscle was dissected from the cranium to reveal the inferotemporal fossa. The coronoid process of the mandible was removed, and a craniectomy was performed with an electric drill.t The dura was opened and the MCA cauterized with a microbipolar unit.~ Interruption of the MCA took place midway between the olfactory tract and the inferior cerebral vein. The vessel was then divided, and the wound irrigated and closed. All hypothermic rats were cooled in a standard fashion to 24"C body temperature over 45 minutes by the application of ice packs and were maintained at 24"C for 1 hour. They were then rewarmed to 36"C over 90 minutes and maintained at this temperature until fully awake. The first group of six rats were cooled to 24"C immediately prior to MCA occlusion ("0-minute delay"); the MCA was not occluded in these animals until their body temperature reached 24~ In subsequent experimental groups, cooling was initiated at 30 minutes (eight rats), 1 hour (seven rats), 2 hours (seven rats), or 3 hours (nine rats) after MCA occlusion. Nine normothermic control rats were maintained at 36"C with the use of a heat lamp thermocouple system.w Femoral arterial catheters were removed immediately prior to the rats awakening. The animals were then extubated and returned to their cages with free access to food and water. Twenty-four hours after MCA occlusion, the rats * Telethermometer, Model 400, manufactured by Yellow Springs Instruments, Yellow Springs, Ohio. ~"Electric drill manufactured by Mototool Dremel, Denver, Colorado. ~tMalis bipolar coagulator manufactured by CodmanSchurtleff, Randolph, Massachusetts. w thermocouple system manufactured by Yellow Springs Instruments, Yellow Springs, Ohio. J. Neurosurg. / Volume 77/September, 1992
FIG. 1. Scatterplot showing the percentage of infarct volume of the right hemisphere (% IV) in experimental groups of control rats and rats with induced hypothermia before and after middle cerebral artery occlusion. Circles indicate individual rats and straight lines indicate the mean value for that group. were reanesthetized with chloral hydrate, perfused with 100 ml of normal saline, and decapitated. Additional groups of 10 0-minute delay rats and nine control animals were sacrificed 72 hours after MCA occlusion. The brains were removed and 3-mm coronal sections were made with the use of a rodent brain matrix.II Sections were immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) in 0.9% phosphate-buffered saline, incubated for 30 minutes at 37"C, then placed in formalin/ After TTC staining, infarcted brain was visualized as the area of unstained tissue; viable tissue stained red. Sections were photographed, and infarcted areas were computed at a blinded evaluation by planimetric analysis.45 Infarct volume was calculated by multiplying infarcted areas by slice thickness and integrating over all slices. Infarct volume was also expressed as a percentage of the right hemisphere volume to correct for any weight differences between the animals. Statistical comparisons between the two groups were performed using the Student two-sided unpaired t-test. Results Physiological variables in all rat groups are presented in Table 2. There was a transient, although significant, decline in heart rate and mean arterial blood pressure during systemic cooling. This most likely represents the cardiovascular depressive effect commonly seen with prolonged hypothermia.l~ The mean of the actual infarct volume and of the infarct volume expressed as a percentage of the right hemisphere volume are presented in Table 3; Fig. 1 represents a scatterplot of these data. In the rats cooled to 24~ before MCA occlusion (0-minute delay), 2.2% II Rodent brain matrix manufactured by Activational Systems, Inc., Warren, Michigan. 439
C. J. Baker, S. T. Onesti, and R. A. Solomon TABLE 2
Physiological parameters in rals sacrificed 24 or 72 hours after occlusion*
(mm Hg)
Hematocrit (%)
Mean Arterial Pressure (ram Hg)
(beats/rain)
39.7+1.2 35.3 _+ 1.9 35.7 4. 2.8
95__.3 105 + 6 102 _+ 8
43_+1 43 _+ 1 41 _ 2
104_+6 86 _+ 4 99 _+ 9
413+ 18 384 _+ 36 414 _+ 26
7.35 _+ 0.01 7.33 ___0.02 7.33 -+ 0.03
38.3 4. 1.4 32.6 4. 3.8 36,0 + 2.2
94 4. 3 107 4. 3 92 -+ 2
45 + 1 45 4. 1 43 __. 1
90 + 8 84 _+ 8 88 4. 3
450 + 26 118 _+ 40"~ 396 -+ 28
7.31 7.34 7.32 7.29
_+ 0.01 -+ 0.02 _+ 0.03 _+ 0.01
36.5 38.5 34.9 37.2
_+ 1.4 _+ 2.2 _+ 2.4 _ 1.1
92 _+ 2 97 -+ 2 113 _+ 2 100 _+ 5
41 43 43 42
_+ 2 4. 1 +_ 2 _ 1
96 95 72 90
_+ 3 _+ 5 _+ 6t --- 11
446 _+ 21 430 4. 16 104 _+ 12"~ 306 _+ 54
7.34 7.34 7,34 7.32
_+ 0.01 + 0.01 _+0.01 + 0.01
38.1 37.3 32.3 36.4
_+ 1.5 4. 2.0 4. 1.2 4. 0.9
85 96 97 101
_+ 4 -+ 10 _+ 2 4. 6
42 _+ 1 41 ___1 41 _ 1 41 _+ 1
91 83 63 89
+- 8 ___8 --- 6f 4. 6
437 463 84 463
Group & Time of Study
pH
pCO2 (ram Hg)
7.33+0.01 7.33 + 0.01 7.35 _+ 0.02
p02
Heart Rate
24-hour survival normothermic control (9 rats) pre-MCAO post-MCAO recovery 0-min delay hypothermia (6 rats) pre-MCAO post-MCAO/hypothermia recovery 30-rain delay hypothermia (8 rats) pre-MCAO post-MCAO hypothermia recovery 1-hr delay hypothermia (7 rats) pre-MCAO post-MCAO hypothermia recovery 2-hr delay hypothermia (7 rats) pre-MCAO post-MCAO hypothermia recovery 3-hr delay hypothermia (9 rats) pre-MCAO post-MCAO hypothermia recovery
_+ 17 -+ 11 4. 10t 4. 11
7,34 -+ 0.01 7,33 _+ 0.02 7,324.0.01 7,31 4. 0.01
38.l_+1.6 38.0 4. 2.2 37.1 _+ 1.9 39.0 _+ 1.6
103_+6 103 _+ 10 106_+6 105 _+ 6
43_+1 44 4. 1 40_+ 1 40 4. 1
89+__5 74 4. 2 50 -+ 5i" 76 _+ 8
433---24 369 --- 28 71 _+ 10i 360 _+ 32
7.33 4. 0.01 7.32 4. 0.01 7.30 +- 0.01 7,304.0.01
36.4 4. 1.4 36.6 + 1.5 38.7 _+ 2.4 38.34.1.2
107 _+ 6 92 4. 4 99 +- 4 I03___7
41 4. 1 42 _+ 1 41 -4- 1 4[_+1
85 ___6 71 ___5 48 _+ 6i" 734.5
428 4. 14 413 _+ 17 90 _+ 22t 368_+24
7.344.0.01 7.32 _+0.01 7,344.0.01
37.84.0.6 37,8 _ 0.8 37.7_+0.6
1134.6 101 _+ 7 1074. 12
424. 1 41 + 1 41 4. I
844.6 83 -+ 5 91 -+4
412_+ 19 400 + 28 3344.21
7.32 4. 0.01 7.34 4. 0.01 7.35-+0.01
38.6 4. 1.6 35.1 4. 1.5 38.1 -+ 1.0
101 -+ 6 99 4. 3 111 -+9
42 4. 1 41 _+ 1 41 _+ 1
84 4. 3 57 -4- 5i 73---2
418 + 15 73 4. 3~ 355_+24
72-hour survival normothermic control (9 rats) pre-MCAO post-MCAO recovery 0-min delay hypothermia (10 rats) pre-MCAO post-MCAO/hypothermia recovery
* Values expressed are means + standard error of the means. MCAO = middle control artery occlusion; recovery = immediately prior to extubation. i" Statistically significant difference (p < 0.01) from "pre-MCAO" values.
TABLE 3 Infarct volumes in rats in each study group* Group & Time of Study
No. Rats
Infarct Volume (cu mm)
% Infarct Volumet
9 6 8 7 7 9
99 __. 13 20 _+ 7 44 + 10 35 -+ 11 142 _+ 22 112 _+ 26
10.8 ___1.5 2.2 _ 0,7:~ 4.4 _ 0.9:~ 3.6 --. 1.1 $ 17.1 _+ 2.4~: 12.0 _+ 2.7
9 10
101 _+ 24 42 _+ 16
11.7 4. 2.8 4.8 __. 1.8w
24-hr survival normothermic control 0-rain delay hypothermia 30-min delay hypothermia 1-hr delay hypothermia 2-hr delay hypothermia 3-hr delay hylaothermia
72-hr survival normothermic control 0-rain delay hypothermia
* Values expressed are means 4. standard error of the means. l" Percentage of infarct volume of the right hemisphere, ~:Statistically significant differences (p < 0.01) from control rats. wStatistically significant difference (p < 0.05) from control rats. 440
-4- 0 . 7 % o f t h e r i g h t h e m i s p h e r e s u b s e q u e n t l y i n f a r c t e d a s c o m p a r e d to 10.8% _+ 1.5% i n t h e c o n t r o l g r o u p (p < 0.01). W h e n t h e i n d u c t i o n o f h y p o t h e r m i a w a s d e l a y e d 30 m i n u t e s o r 1 h o u r , t h e p e r c e n t a g e o f f i g h t h e m i s p h e r e i n f a r c t i o n w a s 4 . 4 % _+ 0 . 9 % a n d 3 . 6 % _+ 1.1%, r e s p e c t i v e l y ; i n f a r c t v o l u m e f o r t h e s e g r o u p s w a s also s i g n i f i c a n t l y l o w e r t h a n c o n t r o l v o l u m e (p < 0.01). T h e a n i m a l s in t h e 2 - h o u r d e l a y g r o u p h a d a significantly larger percentage of fight hemisphere infarction c o m p a r e d to c o n t r o l r a t s ( 1 7 . 1 % _+ 2 . 4 % vs. 10.8% + 1.5%, p < 0.01). H o w e v e r , t h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e 3 - h o u r d e l a y g r o u p ( 1 2 . 0 % _+ 2.7%) and the control group. The animals in the 0-minute delay group sacrificed at 7 2 h o u r s h a d a p e r c e n t a g e o f r i g h t h e m i s p h e r e i n f a r c t i o n o f 4 . 8 % + 1.8% c o m p a r e d to 11.7% + 2 . 8 % for t h e i r r e s p e c t i v e n o r r n o t h e r m i c c o n t r o l s (p < 0.05),
J. Neurosurg. / Volume 77/September, 1992
Delayed hypothermia for reduction of infarction There were no significant differences between the two 0-minute delay groups sacrificed at 24 or 72 hours (mean 2.2% vs. 4.8%) or between the two control groups sacrificed at 24 or 72 hours (mean 10.8% vs.
11.7%). Discussion Global Ischemia vs. Permanent Focal lschemia
The ability of hypothermia to minimize the ischemic susceptibility of the neuron has long been recognized. Rosomoff and coworkers,52-5s Raimondi, et al.? 1 and others 19 demonstrated 30 years ago that cooling the brain leads to a reduction in postischemic tissue damage. Hypothermia has since been shown in many studies to improve both neurological and histopathological outcome in ischemic neuronal tissue, y,io,35,56.65Investigators have recently focused on discovering the pathophysiological mechanisms underlying this protection. During the past few years, the effects of hypothermia on global and focal transient cerebral ischemia have been extensively examined. 7-11.14.~5,2~ Although the effects of hypothermia in these systems have been extensively studied, less attention has been given to models of permanent focal ischemia, Rat models of MCA occlusion are widely used paradigms of permanent focal ischemia, and more closely mimic thromboembolic stroke or intraoperative vascular occlusion than do models of global or transient ischemia. It is important to differentiate between global and focal ischemia because it appears that the mechanism of ischemic injury is not identical. In global ischemia, cessation of blood flow produces profound tissue hypoxia. This leads to depletion of cellular adenosine triphosphate and ensuing lactic acidosis due to glycolysis.s6 It is believed that this anoxic acidosis is largely responsible for glial and, subsequently, neuronal cell death. 49 The central portions of focally ischemic areas of brain behave in a similar fashion and undergo similar pathological changes. However, occlusion of a single vessel differs from global ischemia in that collateral blood flow partially perfuses the affected tissue. The ischemic penumbra is an area of incomplete ischemia surrounding a profoundly anoxic core of neuronal parenchyma. Astrup and colleagues~ have characterized neurons in this region as having sufficient blood flow to maintain cellular ion gradients but not electrical activity. Acidosis in this penumbral region is not as pronounced as it is in globally ischemic parenchyma. 14'5s Unfortunately, as cerebral blood flow (CBF) drops below a critical level in this region, the calciummediated release of glutamate and other excitotoxic mediators can lead to irreversible cell injury/4"6~ The collateral circulation in these areas appears to provide sufficient oxygen and high-energy metabolites to fuel calcium-mediated glutamate release, activate N-methylD-aspartate (NMDA) receptors, and allow arachidonic acid metabolism and free radical formation. ~4"~9'6~In contrast, globally ischemic regions only transiently go J. Neurosurg. / Volume 77/September, 1992
through this phase and little excitotoxic neurotransmitter damage occurs. In this study, we examined the effect of hypothermia on neuronal injury after permanent MCA occlusion in the rat. Hypothermia to 24~ significantly reduced cerebral infarction 24 hours after ischemia if rats were cooled before MCA occlusion or if the onset of cooling was delayed no longer than 1 hour after MCA occlusion. Delays of 2 hours or greater, however, resulted in no ischemic cerebral protection. We further demonstrated that, in rats subjected to hypothermia immediately prior to MCA occlusion, the degree of cerebral protection was not significantly different if the rats were sacrificed at 24 or 72 hours after MCA occlusion, This implies that the hypothermic protection is permanent and is not simply a temporary effect related to systemic cooling. We did not explore the relationship between cerebral protection and the degree of hypothermia. Moderate hypothermia (24"C) was chosen for our model because it recreates the conditions of intraoperative hypothermic circulatory arrest. However, many studies have proven that even minimal reductions in temperature can protect neurons from ischemia. J~~4,35,39,6?,68 M e c h a n i s m s o f Hypothermic Protection
Several theories have been proposed to explain the protection from cerebral ischemia afforded by hypothermia. It is well established that systemic cooling decreases the cellular metabolic rate. 2~ There is a linear decline in the cerebral metabolic rate for oxygen (CMRO2) down to 24~ and a log-linear decrease thereafter. Below a rat body temperature of 28"C, the reduction in CMRO2 is more profound than the decrease in CBF?' The decline in cellular glucose requirements follows a similar path. 2~'47The decrease in metabolic oxygen and glucose requirements as well as a myriad of other physiological effects have led to the view that, in part, hypothermia preserves neuronal viability by decreasing cellular energy expenditure. ~,6,1J,15, 20,28,34,38,43,66 By this mechanism, ischemic neuronal viability may be extended long enough for collateral blood flow to develop or until a transient period of ischemia subsides. 37'3s Hypothermia may also lower the critical threshold of CBF necessary to maintain neuronal viability, expanding the "ischemic penumbra" of potentially viable tissue surrounding an infarct.~'29 In addition, recent work has linked hypothermia with the suppression of glutamate excitotoxicity within the ischemic penumbra. The excessive release of glutamate and other neurotransmitters from ischemic neuronal tissue has been shown to contribute to cell injury and death. ~2'~3'59'6~It appears that this NMDA receptormediated injury occurs most convincingly in the penumbra surrounding focal areas of infarction. 12 A prolonged state of incomplete energy depletion persists which augments the excitotoxic process in these regions. 6~ Busto and coworkers ~~,2, and Duhaime and Ross 2z have independently examined the relationship between hypothermic cerebral protection and gluta441
C. J. Baker, S. T. Onesti, a n d R. A. S o l o m o n mate neurotoxicity. They have shown that part of the cerebral protection afforded by hypothermia can be linked to the presynaptic inhibition of glutamate release at the onset of neuronal ischemia. Thus, there appears to be an intimate relationship between hypothermic cerebral protection and suppression of glutamate-induced excitotoxicity in ischemic penumbral regions. In a model of transient cerebral ischemia, Moyer and Welsh (DJ Moyer and FA Welsh, unpublished data) and Busto, et al., ~ separately found no protection if hypothermia was induced 40 minutes after the onset of ischemia. Both groups were able to demonstrate neuronal protection only if cooling was started less than 20 minutes after ischemia began. Furthermore, Gill, et al., 23 have shown that glutamate excitotoxicity can be prevented by MK-801 only if it is given less than 30 minutes after the onset of ischemia. Other studies of transient global ischemia have shown that peak presynaptic release of glutamate occurs between 20 and 50 minutes after vessel occlusion. 5'11'22"26 These findings suggest that protection in transient ischemia models occurs only when hypothermic induction precedes glutamate release. In our model, however, significant protection was noted even when hypothermia was initiated 1 hour after the onset of ischemia, by which time peak glutamate release has already occurred. This may imply that in permanent focal ischemia, hypothermia acts on a different site of the glutamate excitotoxic cascade than that in transient ischemia. Alternatively, it may reflect a mechanism of protection other than suppression of excitotoxicity or cerebral metabolism. One possibility is that hypothermia alters the induction of the so-called "immediate-early genes" typically activated during the terminal events of cell injury. These genes, including the heat-shock genes and proto-oncogenes, are induced in dying cells and are considered to be markers of programmed cell death.18.3~176 Their resulting gene products are typically seen 1 to 2 hours after a cellular stress. Preliminary studies indicate that hypothermia can alter the expression of these genes and the expression of other genes intimately involved in cell death. 33 Delayed Neuronal Death
Pulsinelli and coworkers48'5~and Kirino 3~have shown in various animal systems that neuronal tissue exposed to transient ischemia may die in a delayed fashion over 72 hours or more. This progressive increase in irreversibly damaged cells includes cortical and striatal cells, as well as hippocampal neurons. A similar evolution in our model could lead the infarct volume in hypothermia-protected rats to approach the size of that in control animals after a number of days. However, our demonstration that differences in infarct volumes between the experimental and control groups are comparable at either 24 or 72 hours of survival suggests that substantial delayed neuronal death did not occur in this model. 442
Our finding that animals in the 2-hour delay group demonstrated significantly greater infarct than control animals is interesting. It is possible that the delayed induction of hypothermia resulted in an exacerbation of the infarct. However, since rats in the 3-hour delay group displayed a nearly identical infarct volume as the control rats, the apparent increase in infarct volume in the 2-hour delay group may simply reflect the relatively small sample size. Since rats in the 3-hour delay group had a lower average blood pressure during hypotherrnia than those in the 2-hour delay group, hypotension cannot explain this difference. Conclusions
In this rat model of permanent MCA occlusion, we were able to demonstrate that moderate hypothermia reduces infarct volume size when administered before or as much as I hour after the onset of ischemia. Furthermore, examination of the infarct in animals at 3 days suggests that this hypothermic protection is permanent in nature. Acknowledgments
The authors thank Daniel Batista for assistance in performing the experiments and Regina Ann Hartley for help in preparing the manuscript. References
1. Astrup J, Siesj6 BK, Symon L: Thresholds in cerebral ischemia - - the ischemic penumbra. Stroke 12:723-725, 1981 2. Baker CJ, Onesti ST, Barth KNM, et al: Hypothermic protection following middle cerebral artery occlusion in the rat. Surg Neural 36:175-180, 1991 3. Baumgartner WA, Silverberg GD, Ream AK, et al: Reappraisal of cardiopulmonary bypass with deep hypothermia and circulatory arrest for complex neurosurgical operations. Surgery 94:242-249, 1983 4. Bederson JB, Pitts LH, Germano IM, et al: Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 17:1304-1308, 1986 5. Benveniste H, Drejer J, Schousboe A, et al: Elevation of the extracellular concentrations of glutamate and aspartale in rat hippocampus during transient cerebralischemia monitored by intracerebral rnicrodialysis. J Neurochem 43:1369-1374, 1984 6. Berntman L, Welsh FA, Harp JR: Cerebral protective effect of low-grade hypothermia. Anesthesiology 55: 495-498, 1981 7. Buchan A, PulsineUi WA: Hypothermia but not the Nmethyl-D-aspartate antagonist, MK-801, attenuates neuronal damage in gerbils subjected to transient global ischemia. J Neurosci 10:311-316, 1990 8. Busto R, Dietrich WD, Globus MYT, et al: The importance of brain temperature in cerebral ischemic injury. Stroke 20:1113-1114, 1989 9. Busto R, Dietrich WD, Globus MYT, et al: Postischemic moderate hypothermia inhibits CAI hippocampal ischemic neuronal injury. Neuroscl Lett 101:299-304, 1989 10. Busto R, Dietrich WD, Globus MYT, et al: Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury, d Cereb J. Neurosurg. / Volume 77/September, 1992
Delayed hypothermia for reduction of infarction Blood Flow Metab 7:729-738, 1987 11. Busto R, Globus MYT, Dietrich WD, et al: Effect of mild hypothermia on ischemia-induced release of neurotransmitters and free fatty acids in rat brain. Stroke 20: 904-910, 1989 12. Choi DW: Cerebral hypoxia: some new approaches and unanswered questions. J Neurosci 10:2493-2501, 1990 13. Choi DW, Rothman SM: The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu Roy Neurosci 13:171-182, 1990 14. Chopp M, Chen H, Dereski MO, et al: Mild hypothermic intervention after graded ischemic stress in rats. Stroke 22:37-43, 1991 15. Chopp M, Knight R, Tidwell CD, et al: The metabolic effects of mild hypothermia on global cerebral ischemia and recirculation in the cat: comparison to normothermia and hyperthermia. J Cereb Blood Flow Metab 9: 141-148, 1989 16. Chyatte D, Elefteriades J, Kim B: Profound hypothermia and circulatory arrest for aneurysm surgery. Case report. J Neurosurg 70:489-491, 1989 17. Cold GE: Cerebral blood flow in acute head injury. The regulation of cerebral blood flow and metabolism during the acute phase of head injury, and its significance for therapy. Acta Neurochir Suppl 49:1-64, 1990 18. Cole AJ, Saffen DW, Baraban JM, et al: Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation. Nature 340:474-476, 1989 (Letter) 19. Connolly JE, Boyd RJ, Calvin JW: The protective effect of hypothermia in cerebral ischemia: experimental and clinical application by selective brain cooling in the human. Surgery 52:15-24, 1962 20. Dempsey RJ, Combs DJ, Maley ME, et al: Moderate hypothermia reduces postischemic edema development and leukotriene production. Neurosurgery 21:177-181, 1987 21. Drummond JC, Shapiro HM: Cerebral physiology, in Miller RD (ed): Anesthesia. New York: Churchill Livingstone, 1990, Vol 1, pp 642-648 22. Duhaime AC, Ross DT: Degeneration of hippocampal CA 1 neurons following transient ischemia due to raised intracranial pressure: evidence for a temperature-dependent excitotoxic process. Brain Res 512:169-174, 1990 23. Gill R, Foster AC, Woodruff GN: MK-801 is neuroprotective in gerbils when administered during the postischaemic period. Neuroscience 25:847-855, 1988 24. Globus MYT, Busto R, Dietrich WD, et al: Intra-ischemic extracellular release of dopamine and glutamate is assodated with striatal vulnerability to ischemia. Neurosci Lett 91:36-40, 1988 25. Gonski A, Acedillo AT, Stacey RB: Profound hypothermia in the treatment of intracraniai aneurysms. Aust NZ J Surg 56:639-643, 1986 26. Hagberg H, Lehmann A, Sandberg M, et al: Ischemiainduced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments. J Cereb Blood Flow Metab 5:413-419, 1985 27. Ikonomidou C, Mosinger JL, Olney JW: Hypothermia enhances protective effect of MK-801 against hypoxic/ ischemic brain damage in infant rats. Brain Res 487: 184-187, 1989 28. Inuzuka T, Tamura A, Sato S, et al: Changes in the concentrations of cerebral proteins following occlusion of the middle cerebral artery in the rat. Stroke 21:917-922, 1990 29. Jones TH, Morawetz RB, Crowell RM, et al: Thresholds of focal cerebral ischemia in awake monkeys. J Neurosnrg 54:773-782, 1981
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30. J0rgensen MB, Deckert J, Wright DC, et al: Delayed cfos proto-oncogene expression in the rat hippocampus induced by transient global cerebral ischemia: an in situ hybridization study. Brain Res 484:393-398, 1989 31. Kirino T: Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res 239:57-69, 1982 32. Lafferty J J, Keykhah MM, Shapiro HM, et al: Cerebral hypometabolism obtained with deep pentobarbital anesthesia and hypothermia (30 C). Anesthesiology 49: 159-164, 1978 33. Lanks KW: Inhibition of glucose-regulated and heat shock protein induction by low temperature. Biochlm Biophys Acta 1034:62-66, 1990 34. Lantos J, Temes G, Tbr6k B: Changes during ischaemia in extracellular potassium ion concentration of the brain under nitrous oxide or hexobarbital-sodium anaesthesia and moderate hypothermia. Acta Physiol Hung 67: 141-153, 1986 35. Leonov Y, Sterz F, Safar P, et al: Mild cerebral hypothermia during and after cardiac arrest improves neurologic outcome in dogs. J Cereb Blood Flow Metab 10:57-70, 1990 36. Ljunggren B, Schutz H, Siesj6 BK: Changes in energy state and acid-base parameters of the rat brain during complete ischemia. Brain Res 73:277-289, 1974 37. Michenfelder JD: Cerebral preservation for intraoperative focal ischemia. Clin Neurosurg 32:105-113, 1985 38. Michenfelder JD, Theye RA: The effects of anesthesia and hypothermia on canine cerebral ATP and lactate during anoxia produced by decapitation. Anesthesiology 33:430-439, 1970 39. Minamisawa H, Smith ML, Siesj6 BK: The effect of mild hypertherrnia and hypotherrnia on brain damage following 5, 10, and 15 minutes of forebrain ischemia. Ann Neurol 28:26-33, 1990 40. Mizzen LA, Welch WJ: Characterization of the therrnotolerant cell. I. Effects of protein synthesis activity and the regulation of heat-shock protein 70 expression. J Cell Biol 106:1105-1116, 1988 41. Mooij JJA, Buchthal A, Belopavlovic M: Somatosensory evoked potential monitoring of temporary middle cerebral artery occlusion during aneurysm operation. Neurosurgery 21:492-496, 1987 42. Morgan JI, Curran T: Stimulus-transcription coupling in neurons: role of cellular immediate-early genes. Trends Neurosci 12:459-462, 1989 43. Natale JE, D'Alecy LG: Protection from cerebral ischemia by brain cooling without reduced lactate accumulation in dogs. Stroke 20:770-777, 1989 44. Nowak TS Jr, Ikeda J, Nakajima T, et al: 70 kDa heat shock protein and c-los gone expression after transient ischemia. Stroke 21 (Suppl):107-111, 1990 45. Onesti ST, Baker CJ, Sun PP, et al: Transient hypothermia reduces focal ischemic brain injury in the rat. Neurosurgery 29:369-373, 1991 46. Onodera H, Kogure K, Ono Y, et al: Proto-oncogene clos is transiently induced in the rat cerebral cortex after forebrain ischemia. Neurosci Lett 98:101-104, 1989 47. Palmer C, Vannucci RC, Christensen MA, et at: Regional cerebral blood flow and glucose utilization during hypothermia in newborn dogs. Anesthesiology 71:730-737, 1989 48. Petito CK, Feldmann E, Pulsinelli WA, et al: Delayed hippocampal damage in humans following cardiorespiratory arrest. Neurology 37:1281-1286, 1987 49. Plum F: The clinical problem: how much anoxia-ischemia damages the brain? Arch Neurol 29:359-360, 1973 50. Pulsinelli W, Brierley JB, Plum F: Temporal profile of neuronal damage in a model of transient forebrain ische443
C. J. Baker, S. T. Onesti, and R. A. Solomon mia. Ann Neurol 11:491-498, 1982 51. Raimondi AJ, Clasen RA, Beattie EJ, el al: The effect of hypothermia and steroid therapy on experimental cerebral injury. Surg Gynecol Obstet 108:333-338, 1959 52. Rosomoff HL: Experimental brain injury during hypothermia. J Neurosurg 16:177-187, 1959 53. Rosomoff HL: Hypothermia and cerebral vascular lesions. I. Experimental interruption of the middle cerebral artery during hypothermia. J Neurosurg 13:332-343, 1956 54. Rosomoff HL: Hypothermia and cerebral vascular lesions. II. Experimental middle cerebral artery interruption followed by induction of hypothermia. Arch Neurol Psychiatry 78:454-464, 1957 55. RosomoffHL, Shulman K, Raynor R, etal: Experimental brain injury and delayed hypothermia. Surg Gynecol Obstet 110:27-32, 1960 56. Rothman SM, Olney JW: Glutamate and the pathophysiology of hypoxic-ischemic brain damage. Ann Neuro119: 105-111, 1986 57. Schlesinger MJ: Heat shock proteins. J Biol Chem 265: 12111-12114, 1990 58. Siesj6 BK: Acidosis and ischemic brain damage. Neuroehem Pathol 9:31-88, 1988 59. Siesjo BK: Mechanisms of ischemic brain damage. Crit Care Med 16:954-963, 1988 60. Siesj6 BK, Bengtsson F: Calcium fluxes, calcium antagonists, and calcium-related pathology in brain ischemia, hypoglycemia, and spreading depression: a unifying hypothesis. J Cereb Blood Flow Metab 9:127-140, 1989 61. Silverberg GD, Reitz BA, Ream AK: Hypothermia and cardiac arrest in the treatment of giant aneurysms of the cerebral circulation and hemangioblastoma of the medulla. J Neurosurg 55:337-346, 1981
444
62. Solomon RA, Smith CR, Raps EC, et al: Deep hypothermic circulatory arrest for the management of complex anterior and posterior circulation aneurysms. Neurosurgery 29:732-738, 1991 63. Spetzler RF, Hadley MN, Rigamonti D, et al: Aneurysms of the basilar artery treated with circulatory arrest, hypothermia, and barbiturate cerebral protection. J Neurosurg 68:868-879, 1988 64. Szekely AM, Barbaccia ML, Alho H, et al: In primary cultures of cerebellar granule cells the activation of Nmethyl-D-aspartate-sensitive giutamate receptors induces c-fosmRNA expression.MolPharmaco135:401-408, 1989 65. Tyson GW, Teasdale GM, Graham DI, et al: Focal cerebral ischemia in the rat: topography of hemodynamic and histopathological changes. Ann Neuro115:559-567, 1984 66. Vaagenes P: Effects of therapeutic hypothermia on activity of some enzymes in cerebrospinal fluid of patients with anoxic-ischemic brain injury. Clin Chem 32: 1336-1340, 1986 67. VanBogelen RA, Neidhardt FC: Ribosomes as sensors of heat and cold shock in Escherichia coli. Proc Natl Acad Sci USA 87:5589-5593, 1990 68. Welsh FA, Sims RE, Harris VA: Mild hypothermia prevents ischemic injury in gerbil hippocampus. J Cereb Blood Flow Metab 10:557-563, 1990
Manuscript received July 3, 1991. Accepted in final form January 22, 1992. Address reprint requests to: Robert A. Solomon, M.D., The Neurological Institute, Room 212, 710 West 168th Street, New York, New York 10032.
J. Neurosurg. / Volume 77/September, 1992
Reduction by delayed hypothermia of cerebral infarction following middle cerebral artery occlusion in the rat: a time-course study CHRISTOPHER J. BAKER, M.D., STEPHEN T. ONESTI, M.D., AND ROBERT A. SOLOMON, M.D.
Department of Neurological Surgery, Columbia-Presbyterian Medical Center, Columbia University College of Physicians and Surgeons, New York, New York t,, The effect of hypothermia on neuronal injury following permanent middle cerebral artery (MCA) occlusion in the rat was examined. Moderate hypothermia (body temperature 24"C) was induced before MCA occlusion (0-minute delay group) in six rats, at 30 minutes in eight rats, and at 1 (seven rats), 2 (seven rats), and 3 (nine rats) hours after occlusion. The rats were kept at a 24~ body temperature for 1 hour, then allowed to rewarm over 90 minutes. The animals were sacrificed 24 hours after MCA occlusion, and infarction was visualized by staining of coronal sections with 2,3,5-triphenyltetrazolium chloride. Infarct volumes were compared to matched normothermic control rats (body temperature 36'C). Additional groups of 0-minute delay hypothermic (10 rats) and control animals (nine rats) were sacrificed 72 hours after MCA occlusion to examine the effects of prolonged survival. A significant reduction in the percentage of infarcted fight hemisphere was seen in the animals sacrificed after 24 hours with 0-minute, 30-minute, and 1-hour delays in inducing hypothermia (mean _+standard error of the mean: 2.2% ___0.7%, 4.4% _+ 0.9%, and 3.6% - 1.1%, respectively) as compared to normothermic control rats (10.8% _+ 1.5%, p < 0.01 by Student's t-test). In the 2- and 3-hour delay groups, the percentage of infarcted fight hemisphere was 17.1% _+ 2.4% and 12.0% _+ 2.7%, respectively, and no decrease in infarct volume was observed. The 0-minute delay hypothermia group sacrificed after 72 hours also displayed a significant reduction in right hemisphere infarct compared to their respective controls (4.8% vs. 11.7%, p < 0.05). These findings indicate that, in the setting of permanent MCA occlusion, hypothermia markedly decreases brain injury even when its induction is delayed for up to 1 hour after the onset of ischemia. Ischemic damage does not appear to be merely retarded but permanently averted. KEY WORDS
'
hypothermia
9 ischemia
VER the last decade, there has been a resurgence in the use of hypothermic cerebral protection in neurosurgery. Systemic cooling and cardiopulmonary bypass have been increasingly used in the treatment of giant basilar and other complex aneurysms.3'16'25'39'41'61-63A number of centers have instituted new hypothermia-based protocols to re-examine the efficacy of systemic cooling in treating severe head trauma, t7 This renewed interest in hypothermia is both a product of, and a catalyst for, the expanding research devoted to understanding the cellular mechanisms of ischemic neuronal injury. In an effort to characterize the temporal nature of hypothermic protection in permanent focal ischemia, we studied the effects of immediate and delayed hypothermia on neuronal injury
O
438
9
middle cerebral artery occlusion
9 rat
following middle cerebral artery (MCA) occlusion in the rat. Materials and Methods
The protocol used in this study has previously been reported in detail and was approved by the Institutional Animal Care and Use Committee. TM Aseptic technique was employed during all experiments. Adult male Wistar rats, each weighing 350 to 400 gm, were anesthetized with intraperitoneal chloral hydrate (400 mg/kg). The animals subsequently underwent right femoral artery cannulation, ligation of the right common carotid artery, and tracheal intubation under direct visualization. The rats were artificially ventilated with a Harvard rodent ventilator and adjustments were made to main-
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Delayed hypothermia for reduction of infarction TABLE 1 Comparison of temporalis muscle and brain temperature in rats*
Treatment
Temporalis Intraparenchymal Muscle Brain Group Temperature ('C) Temperature('C) normothermia(36*C) 36.0 _+0.1 35.4 _ 0.2 hypothermia(24"C) 24.2 _+0.1 25.0 _ 0.8 * Valuesare expressedas means + standard deviation. tain femoral artery blood gases within normal physiological limits. Blood gas and hematocrit levels were obtained preoperatively, after MCA occlusion, during hypothermia, and upon return to normothermia. Heart rate and blood pressure were recorded continuously. Contralateral temporalis muscle temperature was monitored throughout the experiment in all rats;* this appears to correlate closely with intraparenchymal brain temperature in our model (Table 1).45 With the animal in the left lateral decubitus position, an oblique incision was made between the inferior margin of the orbit and the tragus. The exposed temporalis muscle was dissected from the cranium to reveal the inferotemporal fossa. The coronoid process of the mandible was removed, and a craniectomy was performed with an electric drill.t The dura was opened and the MCA cauterized with a microbipolar unit.~ Interruption of the MCA took place midway between the olfactory tract and the inferior cerebral vein. The vessel was then divided, and the wound irrigated and closed. All hypothermic rats were cooled in a standard fashion to 24"C body temperature over 45 minutes by the application of ice packs and were maintained at 24"C for 1 hour. They were then rewarmed to 36"C over 90 minutes and maintained at this temperature until fully awake. The first group of six rats were cooled to 24"C immediately prior to MCA occlusion ("0-minute delay"); the MCA was not occluded in these animals until their body temperature reached 24~ In subsequent experimental groups, cooling was initiated at 30 minutes (eight rats), 1 hour (seven rats), 2 hours (seven rats), or 3 hours (nine rats) after MCA occlusion. Nine normothermic control rats were maintained at 36"C with the use of a heat lamp thermocouple system.w Femoral arterial catheters were removed immediately prior to the rats awakening. The animals were then extubated and returned to their cages with free access to food and water. Twenty-four hours after MCA occlusion, the rats * Telethermometer, Model 400, manufactured by Yellow Springs Instruments, Yellow Springs, Ohio. ~"Electric drill manufactured by Mototool Dremel, Denver, Colorado. ~tMalis bipolar coagulator manufactured by CodmanSchurtleff, Randolph, Massachusetts. w thermocouple system manufactured by Yellow Springs Instruments, Yellow Springs, Ohio. J. Neurosurg. / Volume 77/September, 1992
FIG. 1. Scatterplot showing the percentage of infarct volume of the right hemisphere (% IV) in experimental groups of control rats and rats with induced hypothermia before and after middle cerebral artery occlusion. Circles indicate individual rats and straight lines indicate the mean value for that group. were reanesthetized with chloral hydrate, perfused with 100 ml of normal saline, and decapitated. Additional groups of 10 0-minute delay rats and nine control animals were sacrificed 72 hours after MCA occlusion. The brains were removed and 3-mm coronal sections were made with the use of a rodent brain matrix.II Sections were immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) in 0.9% phosphate-buffered saline, incubated for 30 minutes at 37"C, then placed in formalin/ After TTC staining, infarcted brain was visualized as the area of unstained tissue; viable tissue stained red. Sections were photographed, and infarcted areas were computed at a blinded evaluation by planimetric analysis.45 Infarct volume was calculated by multiplying infarcted areas by slice thickness and integrating over all slices. Infarct volume was also expressed as a percentage of the right hemisphere volume to correct for any weight differences between the animals. Statistical comparisons between the two groups were performed using the Student two-sided unpaired t-test. Results Physiological variables in all rat groups are presented in Table 2. There was a transient, although significant, decline in heart rate and mean arterial blood pressure during systemic cooling. This most likely represents the cardiovascular depressive effect commonly seen with prolonged hypothermia.l~ The mean of the actual infarct volume and of the infarct volume expressed as a percentage of the right hemisphere volume are presented in Table 3; Fig. 1 represents a scatterplot of these data. In the rats cooled to 24~ before MCA occlusion (0-minute delay), 2.2% II Rodent brain matrix manufactured by Activational Systems, Inc., Warren, Michigan. 439
C. J. Baker, S. T. Onesti, and R. A. Solomon TABLE 2
Physiological parameters in rals sacrificed 24 or 72 hours after occlusion*
(mm Hg)
Hematocrit (%)
Mean Arterial Pressure (ram Hg)
(beats/rain)
39.7+1.2 35.3 _+ 1.9 35.7 4. 2.8
95__.3 105 + 6 102 _+ 8
43_+1 43 _+ 1 41 _ 2
104_+6 86 _+ 4 99 _+ 9
413+ 18 384 _+ 36 414 _+ 26
7.35 _+ 0.01 7.33 ___0.02 7.33 -+ 0.03
38.3 4. 1.4 32.6 4. 3.8 36,0 + 2.2
94 4. 3 107 4. 3 92 -+ 2
45 + 1 45 4. 1 43 __. 1
90 + 8 84 _+ 8 88 4. 3
450 + 26 118 _+ 40"~ 396 -+ 28
7.31 7.34 7.32 7.29
_+ 0.01 -+ 0.02 _+ 0.03 _+ 0.01
36.5 38.5 34.9 37.2
_+ 1.4 _+ 2.2 _+ 2.4 _ 1.1
92 _+ 2 97 -+ 2 113 _+ 2 100 _+ 5
41 43 43 42
_+ 2 4. 1 +_ 2 _ 1
96 95 72 90
_+ 3 _+ 5 _+ 6t --- 11
446 _+ 21 430 4. 16 104 _+ 12"~ 306 _+ 54
7.34 7.34 7,34 7.32
_+ 0.01 + 0.01 _+0.01 + 0.01
38.1 37.3 32.3 36.4
_+ 1.5 4. 2.0 4. 1.2 4. 0.9
85 96 97 101
_+ 4 -+ 10 _+ 2 4. 6
42 _+ 1 41 ___1 41 _ 1 41 _+ 1
91 83 63 89
+- 8 ___8 --- 6f 4. 6
437 463 84 463
Group & Time of Study
pH
pCO2 (ram Hg)
7.33+0.01 7.33 + 0.01 7.35 _+ 0.02
p02
Heart Rate
24-hour survival normothermic control (9 rats) pre-MCAO post-MCAO recovery 0-min delay hypothermia (6 rats) pre-MCAO post-MCAO/hypothermia recovery 30-rain delay hypothermia (8 rats) pre-MCAO post-MCAO hypothermia recovery 1-hr delay hypothermia (7 rats) pre-MCAO post-MCAO hypothermia recovery 2-hr delay hypothermia (7 rats) pre-MCAO post-MCAO hypothermia recovery 3-hr delay hypothermia (9 rats) pre-MCAO post-MCAO hypothermia recovery
_+ 17 -+ 11 4. 10t 4. 11
7,34 -+ 0.01 7,33 _+ 0.02 7,324.0.01 7,31 4. 0.01
38.l_+1.6 38.0 4. 2.2 37.1 _+ 1.9 39.0 _+ 1.6
103_+6 103 _+ 10 106_+6 105 _+ 6
43_+1 44 4. 1 40_+ 1 40 4. 1
89+__5 74 4. 2 50 -+ 5i" 76 _+ 8
433---24 369 --- 28 71 _+ 10i 360 _+ 32
7.33 4. 0.01 7.32 4. 0.01 7.30 +- 0.01 7,304.0.01
36.4 4. 1.4 36.6 + 1.5 38.7 _+ 2.4 38.34.1.2
107 _+ 6 92 4. 4 99 +- 4 I03___7
41 4. 1 42 _+ 1 41 -4- 1 4[_+1
85 ___6 71 ___5 48 _+ 6i" 734.5
428 4. 14 413 _+ 17 90 _+ 22t 368_+24
7.344.0.01 7.32 _+0.01 7,344.0.01
37.84.0.6 37,8 _ 0.8 37.7_+0.6
1134.6 101 _+ 7 1074. 12
424. 1 41 + 1 41 4. I
844.6 83 -+ 5 91 -+4
412_+ 19 400 + 28 3344.21
7.32 4. 0.01 7.34 4. 0.01 7.35-+0.01
38.6 4. 1.6 35.1 4. 1.5 38.1 -+ 1.0
101 -+ 6 99 4. 3 111 -+9
42 4. 1 41 _+ 1 41 _+ 1
84 4. 3 57 -4- 5i 73---2
418 + 15 73 4. 3~ 355_+24
72-hour survival normothermic control (9 rats) pre-MCAO post-MCAO recovery 0-min delay hypothermia (10 rats) pre-MCAO post-MCAO/hypothermia recovery
* Values expressed are means + standard error of the means. MCAO = middle control artery occlusion; recovery = immediately prior to extubation. i" Statistically significant difference (p < 0.01) from "pre-MCAO" values.
TABLE 3 Infarct volumes in rats in each study group* Group & Time of Study
No. Rats
Infarct Volume (cu mm)
% Infarct Volumet
9 6 8 7 7 9
99 __. 13 20 _+ 7 44 + 10 35 -+ 11 142 _+ 22 112 _+ 26
10.8 ___1.5 2.2 _ 0,7:~ 4.4 _ 0.9:~ 3.6 --. 1.1 $ 17.1 _+ 2.4~: 12.0 _+ 2.7
9 10
101 _+ 24 42 _+ 16
11.7 4. 2.8 4.8 __. 1.8w
24-hr survival normothermic control 0-rain delay hypothermia 30-min delay hypothermia 1-hr delay hypothermia 2-hr delay hypothermia 3-hr delay hylaothermia
72-hr survival normothermic control 0-rain delay hypothermia
* Values expressed are means 4. standard error of the means. l" Percentage of infarct volume of the right hemisphere, ~:Statistically significant differences (p < 0.01) from control rats. wStatistically significant difference (p < 0.05) from control rats. 440
-4- 0 . 7 % o f t h e r i g h t h e m i s p h e r e s u b s e q u e n t l y i n f a r c t e d a s c o m p a r e d to 10.8% _+ 1.5% i n t h e c o n t r o l g r o u p (p < 0.01). W h e n t h e i n d u c t i o n o f h y p o t h e r m i a w a s d e l a y e d 30 m i n u t e s o r 1 h o u r , t h e p e r c e n t a g e o f f i g h t h e m i s p h e r e i n f a r c t i o n w a s 4 . 4 % _+ 0 . 9 % a n d 3 . 6 % _+ 1.1%, r e s p e c t i v e l y ; i n f a r c t v o l u m e f o r t h e s e g r o u p s w a s also s i g n i f i c a n t l y l o w e r t h a n c o n t r o l v o l u m e (p < 0.01). T h e a n i m a l s in t h e 2 - h o u r d e l a y g r o u p h a d a significantly larger percentage of fight hemisphere infarction c o m p a r e d to c o n t r o l r a t s ( 1 7 . 1 % _+ 2 . 4 % vs. 10.8% + 1.5%, p < 0.01). H o w e v e r , t h e r e w a s n o s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e 3 - h o u r d e l a y g r o u p ( 1 2 . 0 % _+ 2.7%) and the control group. The animals in the 0-minute delay group sacrificed at 7 2 h o u r s h a d a p e r c e n t a g e o f r i g h t h e m i s p h e r e i n f a r c t i o n o f 4 . 8 % + 1.8% c o m p a r e d to 11.7% + 2 . 8 % for t h e i r r e s p e c t i v e n o r r n o t h e r m i c c o n t r o l s (p < 0.05),
J. Neurosurg. / Volume 77/September, 1992
Delayed hypothermia for reduction of infarction There were no significant differences between the two 0-minute delay groups sacrificed at 24 or 72 hours (mean 2.2% vs. 4.8%) or between the two control groups sacrificed at 24 or 72 hours (mean 10.8% vs.
11.7%). Discussion Global Ischemia vs. Permanent Focal lschemia
The ability of hypothermia to minimize the ischemic susceptibility of the neuron has long been recognized. Rosomoff and coworkers,52-5s Raimondi, et al.? 1 and others 19 demonstrated 30 years ago that cooling the brain leads to a reduction in postischemic tissue damage. Hypothermia has since been shown in many studies to improve both neurological and histopathological outcome in ischemic neuronal tissue, y,io,35,56.65Investigators have recently focused on discovering the pathophysiological mechanisms underlying this protection. During the past few years, the effects of hypothermia on global and focal transient cerebral ischemia have been extensively examined. 7-11.14.~5,2~ Although the effects of hypothermia in these systems have been extensively studied, less attention has been given to models of permanent focal ischemia, Rat models of MCA occlusion are widely used paradigms of permanent focal ischemia, and more closely mimic thromboembolic stroke or intraoperative vascular occlusion than do models of global or transient ischemia. It is important to differentiate between global and focal ischemia because it appears that the mechanism of ischemic injury is not identical. In global ischemia, cessation of blood flow produces profound tissue hypoxia. This leads to depletion of cellular adenosine triphosphate and ensuing lactic acidosis due to glycolysis.s6 It is believed that this anoxic acidosis is largely responsible for glial and, subsequently, neuronal cell death. 49 The central portions of focally ischemic areas of brain behave in a similar fashion and undergo similar pathological changes. However, occlusion of a single vessel differs from global ischemia in that collateral blood flow partially perfuses the affected tissue. The ischemic penumbra is an area of incomplete ischemia surrounding a profoundly anoxic core of neuronal parenchyma. Astrup and colleagues~ have characterized neurons in this region as having sufficient blood flow to maintain cellular ion gradients but not electrical activity. Acidosis in this penumbral region is not as pronounced as it is in globally ischemic parenchyma. 14'5s Unfortunately, as cerebral blood flow (CBF) drops below a critical level in this region, the calciummediated release of glutamate and other excitotoxic mediators can lead to irreversible cell injury/4"6~ The collateral circulation in these areas appears to provide sufficient oxygen and high-energy metabolites to fuel calcium-mediated glutamate release, activate N-methylD-aspartate (NMDA) receptors, and allow arachidonic acid metabolism and free radical formation. ~4"~9'6~In contrast, globally ischemic regions only transiently go J. Neurosurg. / Volume 77/September, 1992
through this phase and little excitotoxic neurotransmitter damage occurs. In this study, we examined the effect of hypothermia on neuronal injury after permanent MCA occlusion in the rat. Hypothermia to 24~ significantly reduced cerebral infarction 24 hours after ischemia if rats were cooled before MCA occlusion or if the onset of cooling was delayed no longer than 1 hour after MCA occlusion. Delays of 2 hours or greater, however, resulted in no ischemic cerebral protection. We further demonstrated that, in rats subjected to hypothermia immediately prior to MCA occlusion, the degree of cerebral protection was not significantly different if the rats were sacrificed at 24 or 72 hours after MCA occlusion, This implies that the hypothermic protection is permanent and is not simply a temporary effect related to systemic cooling. We did not explore the relationship between cerebral protection and the degree of hypothermia. Moderate hypothermia (24"C) was chosen for our model because it recreates the conditions of intraoperative hypothermic circulatory arrest. However, many studies have proven that even minimal reductions in temperature can protect neurons from ischemia. J~~4,35,39,6?,68 M e c h a n i s m s o f Hypothermic Protection
Several theories have been proposed to explain the protection from cerebral ischemia afforded by hypothermia. It is well established that systemic cooling decreases the cellular metabolic rate. 2~ There is a linear decline in the cerebral metabolic rate for oxygen (CMRO2) down to 24~ and a log-linear decrease thereafter. Below a rat body temperature of 28"C, the reduction in CMRO2 is more profound than the decrease in CBF?' The decline in cellular glucose requirements follows a similar path. 2~'47The decrease in metabolic oxygen and glucose requirements as well as a myriad of other physiological effects have led to the view that, in part, hypothermia preserves neuronal viability by decreasing cellular energy expenditure. ~,6,1J,15, 20,28,34,38,43,66 By this mechanism, ischemic neuronal viability may be extended long enough for collateral blood flow to develop or until a transient period of ischemia subsides. 37'3s Hypothermia may also lower the critical threshold of CBF necessary to maintain neuronal viability, expanding the "ischemic penumbra" of potentially viable tissue surrounding an infarct.~'29 In addition, recent work has linked hypothermia with the suppression of glutamate excitotoxicity within the ischemic penumbra. The excessive release of glutamate and other neurotransmitters from ischemic neuronal tissue has been shown to contribute to cell injury and death. ~2'~3'59'6~It appears that this NMDA receptormediated injury occurs most convincingly in the penumbra surrounding focal areas of infarction. 12 A prolonged state of incomplete energy depletion persists which augments the excitotoxic process in these regions. 6~ Busto and coworkers ~~,2, and Duhaime and Ross 2z have independently examined the relationship between hypothermic cerebral protection and gluta441
C. J. Baker, S. T. Onesti, a n d R. A. S o l o m o n mate neurotoxicity. They have shown that part of the cerebral protection afforded by hypothermia can be linked to the presynaptic inhibition of glutamate release at the onset of neuronal ischemia. Thus, there appears to be an intimate relationship between hypothermic cerebral protection and suppression of glutamate-induced excitotoxicity in ischemic penumbral regions. In a model of transient cerebral ischemia, Moyer and Welsh (DJ Moyer and FA Welsh, unpublished data) and Busto, et al., ~ separately found no protection if hypothermia was induced 40 minutes after the onset of ischemia. Both groups were able to demonstrate neuronal protection only if cooling was started less than 20 minutes after ischemia began. Furthermore, Gill, et al., 23 have shown that glutamate excitotoxicity can be prevented by MK-801 only if it is given less than 30 minutes after the onset of ischemia. Other studies of transient global ischemia have shown that peak presynaptic release of glutamate occurs between 20 and 50 minutes after vessel occlusion. 5'11'22"26 These findings suggest that protection in transient ischemia models occurs only when hypothermic induction precedes glutamate release. In our model, however, significant protection was noted even when hypothermia was initiated 1 hour after the onset of ischemia, by which time peak glutamate release has already occurred. This may imply that in permanent focal ischemia, hypothermia acts on a different site of the glutamate excitotoxic cascade than that in transient ischemia. Alternatively, it may reflect a mechanism of protection other than suppression of excitotoxicity or cerebral metabolism. One possibility is that hypothermia alters the induction of the so-called "immediate-early genes" typically activated during the terminal events of cell injury. These genes, including the heat-shock genes and proto-oncogenes, are induced in dying cells and are considered to be markers of programmed cell death.18.3~176 Their resulting gene products are typically seen 1 to 2 hours after a cellular stress. Preliminary studies indicate that hypothermia can alter the expression of these genes and the expression of other genes intimately involved in cell death. 33 Delayed Neuronal Death
Pulsinelli and coworkers48'5~and Kirino 3~have shown in various animal systems that neuronal tissue exposed to transient ischemia may die in a delayed fashion over 72 hours or more. This progressive increase in irreversibly damaged cells includes cortical and striatal cells, as well as hippocampal neurons. A similar evolution in our model could lead the infarct volume in hypothermia-protected rats to approach the size of that in control animals after a number of days. However, our demonstration that differences in infarct volumes between the experimental and control groups are comparable at either 24 or 72 hours of survival suggests that substantial delayed neuronal death did not occur in this model. 442
Our finding that animals in the 2-hour delay group demonstrated significantly greater infarct than control animals is interesting. It is possible that the delayed induction of hypothermia resulted in an exacerbation of the infarct. However, since rats in the 3-hour delay group displayed a nearly identical infarct volume as the control rats, the apparent increase in infarct volume in the 2-hour delay group may simply reflect the relatively small sample size. Since rats in the 3-hour delay group had a lower average blood pressure during hypotherrnia than those in the 2-hour delay group, hypotension cannot explain this difference. Conclusions
In this rat model of permanent MCA occlusion, we were able to demonstrate that moderate hypothermia reduces infarct volume size when administered before or as much as I hour after the onset of ischemia. Furthermore, examination of the infarct in animals at 3 days suggests that this hypothermic protection is permanent in nature. Acknowledgments
The authors thank Daniel Batista for assistance in performing the experiments and Regina Ann Hartley for help in preparing the manuscript. References
1. Astrup J, Siesj6 BK, Symon L: Thresholds in cerebral ischemia - - the ischemic penumbra. Stroke 12:723-725, 1981 2. Baker CJ, Onesti ST, Barth KNM, et al: Hypothermic protection following middle cerebral artery occlusion in the rat. Surg Neural 36:175-180, 1991 3. Baumgartner WA, Silverberg GD, Ream AK, et al: Reappraisal of cardiopulmonary bypass with deep hypothermia and circulatory arrest for complex neurosurgical operations. Surgery 94:242-249, 1983 4. Bederson JB, Pitts LH, Germano IM, et al: Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 17:1304-1308, 1986 5. Benveniste H, Drejer J, Schousboe A, et al: Elevation of the extracellular concentrations of glutamate and aspartale in rat hippocampus during transient cerebralischemia monitored by intracerebral rnicrodialysis. J Neurochem 43:1369-1374, 1984 6. Berntman L, Welsh FA, Harp JR: Cerebral protective effect of low-grade hypothermia. Anesthesiology 55: 495-498, 1981 7. Buchan A, PulsineUi WA: Hypothermia but not the Nmethyl-D-aspartate antagonist, MK-801, attenuates neuronal damage in gerbils subjected to transient global ischemia. J Neurosci 10:311-316, 1990 8. Busto R, Dietrich WD, Globus MYT, et al: The importance of brain temperature in cerebral ischemic injury. Stroke 20:1113-1114, 1989 9. Busto R, Dietrich WD, Globus MYT, et al: Postischemic moderate hypothermia inhibits CAI hippocampal ischemic neuronal injury. Neuroscl Lett 101:299-304, 1989 10. Busto R, Dietrich WD, Globus MYT, et al: Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury, d Cereb J. Neurosurg. / Volume 77/September, 1992
Delayed hypothermia for reduction of infarction Blood Flow Metab 7:729-738, 1987 11. Busto R, Globus MYT, Dietrich WD, et al: Effect of mild hypothermia on ischemia-induced release of neurotransmitters and free fatty acids in rat brain. Stroke 20: 904-910, 1989 12. Choi DW: Cerebral hypoxia: some new approaches and unanswered questions. J Neurosci 10:2493-2501, 1990 13. Choi DW, Rothman SM: The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu Roy Neurosci 13:171-182, 1990 14. Chopp M, Chen H, Dereski MO, et al: Mild hypothermic intervention after graded ischemic stress in rats. Stroke 22:37-43, 1991 15. Chopp M, Knight R, Tidwell CD, et al: The metabolic effects of mild hypothermia on global cerebral ischemia and recirculation in the cat: comparison to normothermia and hyperthermia. J Cereb Blood Flow Metab 9: 141-148, 1989 16. Chyatte D, Elefteriades J, Kim B: Profound hypothermia and circulatory arrest for aneurysm surgery. Case report. J Neurosurg 70:489-491, 1989 17. Cold GE: Cerebral blood flow in acute head injury. The regulation of cerebral blood flow and metabolism during the acute phase of head injury, and its significance for therapy. Acta Neurochir Suppl 49:1-64, 1990 18. Cole AJ, Saffen DW, Baraban JM, et al: Rapid increase of an immediate early gene messenger RNA in hippocampal neurons by synaptic NMDA receptor activation. Nature 340:474-476, 1989 (Letter) 19. Connolly JE, Boyd RJ, Calvin JW: The protective effect of hypothermia in cerebral ischemia: experimental and clinical application by selective brain cooling in the human. Surgery 52:15-24, 1962 20. Dempsey RJ, Combs DJ, Maley ME, et al: Moderate hypothermia reduces postischemic edema development and leukotriene production. Neurosurgery 21:177-181, 1987 21. Drummond JC, Shapiro HM: Cerebral physiology, in Miller RD (ed): Anesthesia. New York: Churchill Livingstone, 1990, Vol 1, pp 642-648 22. Duhaime AC, Ross DT: Degeneration of hippocampal CA 1 neurons following transient ischemia due to raised intracranial pressure: evidence for a temperature-dependent excitotoxic process. Brain Res 512:169-174, 1990 23. Gill R, Foster AC, Woodruff GN: MK-801 is neuroprotective in gerbils when administered during the postischaemic period. Neuroscience 25:847-855, 1988 24. Globus MYT, Busto R, Dietrich WD, et al: Intra-ischemic extracellular release of dopamine and glutamate is assodated with striatal vulnerability to ischemia. Neurosci Lett 91:36-40, 1988 25. Gonski A, Acedillo AT, Stacey RB: Profound hypothermia in the treatment of intracraniai aneurysms. Aust NZ J Surg 56:639-643, 1986 26. Hagberg H, Lehmann A, Sandberg M, et al: Ischemiainduced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments. J Cereb Blood Flow Metab 5:413-419, 1985 27. Ikonomidou C, Mosinger JL, Olney JW: Hypothermia enhances protective effect of MK-801 against hypoxic/ ischemic brain damage in infant rats. Brain Res 487: 184-187, 1989 28. Inuzuka T, Tamura A, Sato S, et al: Changes in the concentrations of cerebral proteins following occlusion of the middle cerebral artery in the rat. Stroke 21:917-922, 1990 29. Jones TH, Morawetz RB, Crowell RM, et al: Thresholds of focal cerebral ischemia in awake monkeys. J Neurosnrg 54:773-782, 1981
J. Neurosurg. / Volume 77/September, 1992
30. J0rgensen MB, Deckert J, Wright DC, et al: Delayed cfos proto-oncogene expression in the rat hippocampus induced by transient global cerebral ischemia: an in situ hybridization study. Brain Res 484:393-398, 1989 31. Kirino T: Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res 239:57-69, 1982 32. Lafferty J J, Keykhah MM, Shapiro HM, et al: Cerebral hypometabolism obtained with deep pentobarbital anesthesia and hypothermia (30 C). Anesthesiology 49: 159-164, 1978 33. Lanks KW: Inhibition of glucose-regulated and heat shock protein induction by low temperature. Biochlm Biophys Acta 1034:62-66, 1990 34. Lantos J, Temes G, Tbr6k B: Changes during ischaemia in extracellular potassium ion concentration of the brain under nitrous oxide or hexobarbital-sodium anaesthesia and moderate hypothermia. Acta Physiol Hung 67: 141-153, 1986 35. Leonov Y, Sterz F, Safar P, et al: Mild cerebral hypothermia during and after cardiac arrest improves neurologic outcome in dogs. J Cereb Blood Flow Metab 10:57-70, 1990 36. Ljunggren B, Schutz H, Siesj6 BK: Changes in energy state and acid-base parameters of the rat brain during complete ischemia. Brain Res 73:277-289, 1974 37. Michenfelder JD: Cerebral preservation for intraoperative focal ischemia. Clin Neurosurg 32:105-113, 1985 38. Michenfelder JD, Theye RA: The effects of anesthesia and hypothermia on canine cerebral ATP and lactate during anoxia produced by decapitation. Anesthesiology 33:430-439, 1970 39. Minamisawa H, Smith ML, Siesj6 BK: The effect of mild hypertherrnia and hypotherrnia on brain damage following 5, 10, and 15 minutes of forebrain ischemia. Ann Neurol 28:26-33, 1990 40. Mizzen LA, Welch WJ: Characterization of the therrnotolerant cell. I. Effects of protein synthesis activity and the regulation of heat-shock protein 70 expression. J Cell Biol 106:1105-1116, 1988 41. Mooij JJA, Buchthal A, Belopavlovic M: Somatosensory evoked potential monitoring of temporary middle cerebral artery occlusion during aneurysm operation. Neurosurgery 21:492-496, 1987 42. Morgan JI, Curran T: Stimulus-transcription coupling in neurons: role of cellular immediate-early genes. Trends Neurosci 12:459-462, 1989 43. Natale JE, D'Alecy LG: Protection from cerebral ischemia by brain cooling without reduced lactate accumulation in dogs. Stroke 20:770-777, 1989 44. Nowak TS Jr, Ikeda J, Nakajima T, et al: 70 kDa heat shock protein and c-los gone expression after transient ischemia. Stroke 21 (Suppl):107-111, 1990 45. Onesti ST, Baker CJ, Sun PP, et al: Transient hypothermia reduces focal ischemic brain injury in the rat. Neurosurgery 29:369-373, 1991 46. Onodera H, Kogure K, Ono Y, et al: Proto-oncogene clos is transiently induced in the rat cerebral cortex after forebrain ischemia. Neurosci Lett 98:101-104, 1989 47. Palmer C, Vannucci RC, Christensen MA, et at: Regional cerebral blood flow and glucose utilization during hypothermia in newborn dogs. Anesthesiology 71:730-737, 1989 48. Petito CK, Feldmann E, Pulsinelli WA, et al: Delayed hippocampal damage in humans following cardiorespiratory arrest. Neurology 37:1281-1286, 1987 49. Plum F: The clinical problem: how much anoxia-ischemia damages the brain? Arch Neurol 29:359-360, 1973 50. Pulsinelli W, Brierley JB, Plum F: Temporal profile of neuronal damage in a model of transient forebrain ische443
C. J. Baker, S. T. Onesti, and R. A. Solomon mia. Ann Neurol 11:491-498, 1982 51. Raimondi AJ, Clasen RA, Beattie EJ, el al: The effect of hypothermia and steroid therapy on experimental cerebral injury. Surg Gynecol Obstet 108:333-338, 1959 52. Rosomoff HL: Experimental brain injury during hypothermia. J Neurosurg 16:177-187, 1959 53. Rosomoff HL: Hypothermia and cerebral vascular lesions. I. Experimental interruption of the middle cerebral artery during hypothermia. J Neurosurg 13:332-343, 1956 54. Rosomoff HL: Hypothermia and cerebral vascular lesions. II. Experimental middle cerebral artery interruption followed by induction of hypothermia. Arch Neurol Psychiatry 78:454-464, 1957 55. RosomoffHL, Shulman K, Raynor R, etal: Experimental brain injury and delayed hypothermia. Surg Gynecol Obstet 110:27-32, 1960 56. Rothman SM, Olney JW: Glutamate and the pathophysiology of hypoxic-ischemic brain damage. Ann Neuro119: 105-111, 1986 57. Schlesinger MJ: Heat shock proteins. J Biol Chem 265: 12111-12114, 1990 58. Siesj6 BK: Acidosis and ischemic brain damage. Neuroehem Pathol 9:31-88, 1988 59. Siesjo BK: Mechanisms of ischemic brain damage. Crit Care Med 16:954-963, 1988 60. Siesj6 BK, Bengtsson F: Calcium fluxes, calcium antagonists, and calcium-related pathology in brain ischemia, hypoglycemia, and spreading depression: a unifying hypothesis. J Cereb Blood Flow Metab 9:127-140, 1989 61. Silverberg GD, Reitz BA, Ream AK: Hypothermia and cardiac arrest in the treatment of giant aneurysms of the cerebral circulation and hemangioblastoma of the medulla. J Neurosurg 55:337-346, 1981
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62. Solomon RA, Smith CR, Raps EC, et al: Deep hypothermic circulatory arrest for the management of complex anterior and posterior circulation aneurysms. Neurosurgery 29:732-738, 1991 63. Spetzler RF, Hadley MN, Rigamonti D, et al: Aneurysms of the basilar artery treated with circulatory arrest, hypothermia, and barbiturate cerebral protection. J Neurosurg 68:868-879, 1988 64. Szekely AM, Barbaccia ML, Alho H, et al: In primary cultures of cerebellar granule cells the activation of Nmethyl-D-aspartate-sensitive giutamate receptors induces c-fosmRNA expression.MolPharmaco135:401-408, 1989 65. Tyson GW, Teasdale GM, Graham DI, et al: Focal cerebral ischemia in the rat: topography of hemodynamic and histopathological changes. Ann Neuro115:559-567, 1984 66. Vaagenes P: Effects of therapeutic hypothermia on activity of some enzymes in cerebrospinal fluid of patients with anoxic-ischemic brain injury. Clin Chem 32: 1336-1340, 1986 67. VanBogelen RA, Neidhardt FC: Ribosomes as sensors of heat and cold shock in Escherichia coli. Proc Natl Acad Sci USA 87:5589-5593, 1990 68. Welsh FA, Sims RE, Harris VA: Mild hypothermia prevents ischemic injury in gerbil hippocampus. J Cereb Blood Flow Metab 10:557-563, 1990
Manuscript received July 3, 1991. Accepted in final form January 22, 1992. Address reprint requests to: Robert A. Solomon, M.D., The Neurological Institute, Room 212, 710 West 168th Street, New York, New York 10032.
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