Journal of Cerebral Blood Flow and Metabolism 12:621--D28 © 1992 The International Society of Cerebral Blood Flow and Metabolism Published by Raven Press, Ltd" New York
The Effect of Hypothermia on Transient Middle Cerebral Artery Occlusion in the Rat
*Hua Chen, *:j:Michael Chopp, *:j:Zheng G. Zhang, and tJulio H. Garcia Departments of*Neurology and tPathology, Henry Ford Hospital, Detroit, Michigan, and fDepartment of Physics, Oakland University, Rochester, Michigan, U.S.A.
Summary: We investigated the effect of moderate whole body hypothermia (30°C) on transient middle cerebral ar tery occlusion (MCAO) in the rat. Male Wistar rats were subjected to 2 h of ischemia by inserting a suture into the lumen of the internal carotid artery and occluding the origin of the MCA. Experimental groups were (a) MCAO induced at 37°C body temperature (n 15); (b) 30°C body temperature induced prior to ischemia and maintained for 2 h of MCAO and I h of reperfusion (n 12); and (c) MCAO with regional brain and body temperatures mea sured in normothermic (n 3) and hypothermic MCAO rats (n 2). Histopathological evaluation was performed 96 h after reperfusion. All normothermic MCAO animals
exhibited ischemic infarct involving the ipsilateral cortex and basal ganglia with infiltration of neutrophils, macro phages, and microvascular proliferation. Hypothermic MCAO animals exhibited minor ischemic damage ranging from selective neuronal injury to small focal areas of in farct with minimal inflammatory response. Our data dem onstrate that transient ischemia induced by using the in tra-arterial suture method to occlude the MCA results in a reproducible brain lesion and that moderate hypother mia has a profound protective effect on the brain injury after transient MCAO. Key Words: Transient middle ce rebral artery occlusion-Hypothermia-Histopathol ogy-Rat.
Hypothermia has been widely investigated in an imal models of global cerebral ischemia, and mild to moderate hypothermia significantly reduces isch emic neuronal damage in these models (Busto et aI. , 1987; Boris-Moller et aI. , 1990; Welsh et aI. , 1990; Chopp et aI. , 1991). The effects of hypothermia on focal cerebral ischemia are uncertain. Data from hy pothermic intervention after permanent focal cere bral ischemia in subhuman primates suggest that severe hypothermia (25. 9°C) or prolonged hypo thermia (48 h) is detrimental to animals with focal brain ischemia (Michenfelder and Milde, 1977; Sim eone et aI. , 1979; Steen et aI. , 1979). To our knowl edge, there are no studies that examine the neuronal
protective effects of moderate hypothermia induced during and acutely after transient focal cerebral ischemia. Transient focal cerebral ischemia in the rat, in duced by advancing a suture into the internal ca rotid artery to occlude the middle cerebral artery (MCA), has provided a new model to investigate the development of cerebral tissue damage and thera peutic intervention (Koizumi et aI. , 1986; Nagasawa and Kogure, 1989; Zea Longa et aI. , 1989; Kawa mura et aI. , 1991). This model has the advantage of not requiring a craniotomy, thus eliminating the in vasive surgical procedure that itself may cause trauma to brain, as well as alter the intracranial pressure. In the present study, we employed the intraluminal model of transient middle cerebral ar tery occlusion (MCAO) in the rat to explore the pathological features of this model, and to test the effect of short duration (3 h), 30°C whole body hy pothermia on ischemic cell damage. Our data sug gest that reproducible brain injury is induced by 2 h of MCAO, and 30°C hypothermia significantly pro tects the brain from transient focal ischemia.
=
=
=
=
Received October 9, 1991; final revision received January 15, 1992; accepted January 15, 1992. Address correspondence and reprint requests to Dr. H. Chen at Department of Neurology, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202, U. S. A. Abbreviations used: ACA, anterior carotid artery; CCA, com mon carotid artery; ECA, external carotid artery; GFAP, glial fibrillary acid protein; HIE, hematoxylin and eosin; ICA, internal carotid artery; MCAO, middle cerebral artery occlusion; PA, pterygopalatine artery.
621
622
H. CHEN ET AL. MATERIALS AND METHODS
Male Wistar rats (weighing 270-JOO g, n 32) were used in the experiment. MCAO was induced by advanc ing a 4-0 surgical nylon suture into the internal carotid artery (ICA) to block the origin of the MCA (Koizumi et aI., 1986; Nagasawa and Kogure, 1989; Zea Longa et aI., 1989). Animals were anesthetized using a face mask de livering 1.0-2.0% halothane in 70% N20 and 30% O2, The right femoral artery was cannulated to measure blood gas and blood glucose before ischemia. Arterial blood pres sure was monitored prior to MCAO and continuously for 20 min after MCAO in both normothermic and hypother mic animals (groups 1 and 2, respectively; see below). In addition, in the hypothermic animals, arterial blood pres sure was continuously monitored throughout the period of MCAO and for 20 min after the onset of reperfusion. A 2 cm incision was made at the center of the neck, and the right common carotid artery (CCA), external carotid ar tery (ECA), and ICA were exposed under an operating microscope (Carl Zeiss, Inc., Thornwood, NY, U.S.A.). Care was taken to avoid injury to the vagus nerve. The ICA was further dissected to identify the pterygopalatine artery (PA) branch and the intracranial ICA branch (see Fig. 1). The CCA and ICA were temporarily clamped using microsurgical clips (Codman & Shurtleff, Inc., Ran dolf, MA, U.S.A.). A 5-0 silk suture was tied loosely at the origin of the ECA and ligated at the distal end of the ECA. A 4-0 surgical nylon suture, with its tip rounded by heating near a flame, was introduced into the ECA lumen through a small puncture. The silk suture around the ECA origin was tightened around the intraluminal nylon suture to prevent bleeding, and the microsurgical clips were re moved. A length of 18.5-19.5 mm of nylon suture, deter mined according to the animal's weight, was gently ad vanced from the ECA into the lumen of the ICA until the suture blocked the origin of the MCA. The incision was temporarily closed using skin clips. In group 1 (normo thermic animals, see below), anesthesia was then termi-
5
=
I
4
FIG. 2. Coronal section of rat brain illustrating the division of the rig ht hemisphere into eig ht regions for pathological eval uation. Stars represent the approximate locations of regional brain temperature measurements. IC, internal capsule; F, fornix; DB, diagonal band of Broca; AC, anterior commis sure; CPu, caudate putamen; GP, g lobus pallidus. Regions were as follows: (1) piriform cortex, (2) insular cortex, (3) parietal cortex, (4) hindlimb and forelimb area of cortex, (5) frontal and cingulate cortex, (6) caudate putamen, (7) g lobus putamen, and (8) preoptic area.
nated, and animals awoke 5-10 min thereafter. After 2 h of ischemia, anesthesia was reinstituted, and reperfusion was performed by withdrawal of the suture until the tip cleared the ICA lumen and reached the origin of ECA. By contrast, in group 2 (hypothermic animals, see below), halothane anesthesia was maintained throughout the 2 h ischemic period and 1 h of recirculation to allow accurate temperature control. Although CBF was not measured in the present study, the length of the suture required to block the MCA was confirmed in a preliminary study of 50 rats in which the suture was permanently placed to occlude the MCA and suture placement was verified after killing. Animals were fasted overnight before surgery but al lowed free access to water. Rectal temperature was con trolled with a feedback-regulated water heating system to maintain the body temperature. Animals were randomly divided into two experimental groups: In group 1, MCAO was induced at 37°C body temperature (n 15). In group 2, MCAO was induced at 30°C body temperature and maintained for 2 h of ischemia and 1 h of reperfusion (n 12). The 30°C whole body hypothermia was instituted 30 min prior to the onset of MCAO by spraying alcohol on the skin and fanning room air (22°C) toward the animal's body. The heating system was set to maintain the ani mal's body temperature at 30°C. Hypothermia was main tained throughout the 2 h period of ischemia and for an additional 1 h of recirculation. Animals were rewarmed to 37°C using the heating pad. Additional animals were used to measure body and re gional brain temperatures during ischemia and recircula tion in normothermic MCAO rats (n 3) and hypother mic MCAO rats (n 2), without the histopathology end point. Thirty minutes prior to MCAO, microthermocou pies (100 !-lm) placed into a 27 gauge needle were inserted into the right (lesion side) and left (control side) cortex, caudate putamen, and preoptic areas (see Fig. 2) through 1 mm burr holes in the skull. Brain and rectal tempera tures were recorded every 5 min throughout the experi ment using a digital thermometer (Physitemp, Clifton, NJ, U.S.A.). =
=
=
=
CCA FIG. 1. Schematic representation of rat extracranial and in
tracranial arteries (circled) and the suture position during middle cerebral artery occlusion (MCAO). CCA, common ca rotid artery; ECA, external carotid artery; ICA, internal carotid artery; PA, pterygopalatine artery; PCA, posterior cerebral artery; MCA, middle cerebral artery; ACA, anterior cerebral artery.
J
Cereb Blood Flow Metab, Vol.
12,
No.4,
1992
HYPOTHERMIA REDUCES FOCAL ISCHEMIC INFARCT TABLE 1.
Brain ischemic damage grading scales
Percentage of neuronal injury Neuron Acute damage Delayed damage Necrosis Cavitation Astrocyte
50%
I 4 7 10
2 5 8 11
3 6 9 12
0: normal 1: hypertrophic (increase in nuclear diameter, cellular processes, and GFAP intensity) 2: necrotic (no GFAP staining)
Inflammation Neutrophil Macrophage
Vasculature
not detectable polymorphonucleus detected not detectable peripheral nucleus with cytoplasmic granules detected 0: normal I: increased number of microvessels
0: 1: 0: 1:
Four days after MCAO, animals were anesthetized (i.m.) with ketamine (44 mg/kg) and xylazine (13 mg/kg), and transcardially perfused with heparinized saline and 10% neutral buffered formalin. The head was immersed in formalin solution for I h and then the brain was removed. The brain was cut into 2 mm thick coronal blocks, for a total of seven blocks per animal, using a rat brain matrix. The brain tissue was processed, embedded, and 6 /Lm thick paraffin sections from each block were cut and stained with hematoxylin and eosin (H/E). Brain ischemic damage was evaluated using light microscopy. A standard section, corresponding to the coronal sec tion at interaural 8.2 mm, bregma 0.8 mm (Paxinos and Watson, 1986), was selected from each animal. This sec tion is centered in the ischemic lesion, and the right hemi sphere was divided into eight anatomically distinct re gions for detailed histological analysis (Fig. 2). Paraffin embedded 6 /Lm thick sections adjacent to the H/E section were cut and mounted on gelatin-coated slides to evaluate the astrocytic responses. In order to identify the astrocytes, the avidin-biotin complex (ABC) method was performed using antibodies against glial fibrillary acid protein (OFAP, polyc1onal antibodies, purchased from DAKO, Carpinteria, CA, U.S.A.). Reactive/necrotic as trocytes were evaluated using OFAP staining. In the present study, detailed examination of the ICA lumen, by electron microscopy, was not performed. Neuronal dam age, astrocytic reaction/damage, and inflammatory re sponse (summation of infiltration by neutrophils, macro phages, and increased numbers of microvessels, identi fied using H/E staining) were evaluated in each distinct
TABLE 2.
region by means of grading scales described in Table I . The severity o f neuronal damage was divided into acute type (either shrunken or ballooned neurons), delayed type (eosinophilic neurons), tissue necrosis (no surviving tissue elements in an area), and cavitation. The percent age of ischemic neuronal damage (acute, delayed damage, necrosis, and cavitation) were factored into the numerical grading. For example, if < 10% of the neurons in region 1 were eosinophilic, the score of 4 was given; if> 10% but 50% eosino philic neurons in the region. Multiple histological changes within a region were averaged. With this grading system, the most severe injury would receive a score of 12 for neuronal damage and 2 for astrocytic and 3 for inflamma tory response.
Statistics Wilcoxon two-sample tests were performed to compare the response of neurons, astrocytes, and inflammation between the normothermic and hypothermic MCAO an imals. All data are presented as mean ± SD.
RESULTS
Blood gas values and arterial serum glucose lev els before MCAO were within normal physiological ranges (Table 2). The blood pressure fluctuated within 5-10 mm Hg during the MCAO surgery. There were no detectable differences in arterial blood pressure values prior to MCAO and 20 min after MCAO in both normothermic and hypother mic animals; likewise, there were no detectable dif ferences between groups at each time point of mea surement. In hypothermic animals, arterial blood pressure was stable during the 2 h of MCAO and 20 min of reperfusion (data not shown). Five animals developed subarachnoid hemorrhage (two in nor mothermic MCAO, three in hypothermic MCAO). Data from these animals were not included in the present study. No animals died after surgery. Figures 3A and 3B illustrate the brain (lesion side) and rectal temperature changes before, during, and after MCAO in a representative normother mic and hypothermic MCAO animal, respectively. Rectal temperatures were maintained constant at 37°C in normothermic and at 30°C in hypother mic MCAO animals. Prior to ischemia, brain tem perature was elevated above the rectal temper-
Serum arterial blood gas, glucose, and blood pressure (BP) values Pcoz (mm Hg)
pH Normothermia (n = 13) Hypothermia (n = 9)
623
7.43 7.35
± ±
0.04 0.08
37.3 39.0
± ±
2.1 5.0
Poz (mm Hg)
Glucose (mg/dl)
141 183
129 119
± ±
20 23
± ±
28 15
BP (mm Hg) Before
98 100
± ±
5 6
During
97 98
± ±
2 2
Data are obtained 20 min before MCAO and 20 min after MCAO (during).
J Cereb Blood Flow Metab, Vol. 12, No.4, 1992
H. CHEN ET AL.
624
NORMOTHERMIA (right hemisphere) 39.00 ,------,
a .......
38.50
o
o -
w a: :::> l e:{ a: w a.. ::::2: w I-
38.00
37.50
37.00
36.50
36.00
L-_...l._ ...--'---_--'--_----L_---L__L-_...l._ ...--'---_--'-- _--.l
-20
0
20
40
60
80
100
120
140
160
180
TIME (MIN)
HYPOTHERMIA (right hemisphere)
FIG. 3. Plot of rectal (+), cortical
(0),
caudate ([,), and preoptical (0) brain temperatures in a representative rat during normothermic MCAO (a) and hypothermic MCAO (b) and reperfu sion. Arrows indicate the initiation and termination of MCAO.
32.50 ,------,
.......
b
32.00
o
o
-
w a: :::> l e:{ a: w a.. ::::2: w I-
31.50
31.00
30.50
30.00
+ ..... ++++++++++++++++++++++++++++++++++++
t
t
29.50 L-_-'--_-'---_...L.-_-'--_-'-_--'--_--'-_---'-_---' -20 20 40 60 80 100 120 140 160 180 0
TIME (MIN) ature by OSC in normothermic and lSC in hypo thermic MCAO rats. After the onset of MCAO, brain temperatures declined approximately 0. 5°C and fluctuated at 37. 2 ± 0. 3 and 31. 2 ± 0. 3°C in normothermic and hypothermic MCAO rats, re spectively. Brain temperatures in the nonlesioned left side declined and fluctuated slightly, as in the lesioned side, during the MCAO in both normo thermic and hypothermic MCAO rats (data not shown). In both groups of animals, after withdrawal J
Cereb Blood Flow Metab, Vol.
12,
No.4,
1992
of the suture, brain temperatures in both sides of the brain fluctuated slightly, similar to the temper ature fluctuation found during ischemia. Figures 4A-D show coronal sections of brain from a representative normothermic MCAO animal exhibiting ischemic infarct and from a hypothermic MCAO animal exhibiting minor ischemic injury. All normothermic MCAO animals revealed a consistent and homogeneous pattern of ischemic infarct in re gions 1, 2, 3, 6, and 8 (Fig. 4A). The size of the
HYPOTHERMIA REDUCES FOCAL ISCHEMIC INFARCT
625 (b)
(a)
(e)
FIG. 4. HIE staining of 6 fJ-m par
aff in-embedded coronal sec tions from a representative rat brain subjected to normother mic MCAO (a,c) and hypother mic MCAO (b,d). a: A well demarcated paled lesion is noted in the right frontoparietal cortex and basal ganglia,region 1,2,3,6,7,and 8 (x6). b: Small pale areas are noted in the right lateral caudate putamen and preoptic area, region 6 and 8 (x6). c: An enlargement (x570) of the box in a demonstrating eosinophilic neurons (arrows) and inf iltrating inflammatory cells (neutrophil, arrowhead; macrophage, open arrow). d: An enlargement (x 570) of the box in b, demonstrating selec tive individual ischemic neu rons (large arrows) and intact neurons (small arrows).
(d)
J cerea
Blood Flow Metab, Vol.
n. *f. JIlf1'I
626
H. CHEN ET AL.
lesion in regions 4 and 7, located at the boundary of infarct, varied from animal to animal. Region 5, which is primarily supplied by the anterior cerebral artery (ACA), exhibited only minor acute ischemic neuronal change. However, an increased reactivity of GFAP was present (increase of nuclear diameter, cytoplasmic processes, and GFAP intensity). Within an infarct, neurons were eosinophilic or ex hibited ghost-like appearance, and no astrocytes were detected by GFAP staining. In addition to the neuronal and astrocytic injury, massive infiltration by neutrophils and macrophages as well as in creased numbers of microvessels (compared to the contralateral side) were detected in the lesioned re gions 1, 2, 3, 6, and 8 (Fig. 4C). In the hypothermic MCAO group, two-thirds of the animals exhibited only selective neuronal damage in regions 6, 8, and/ or 2 and 3 (Fig. 4D), accompanied by an increased GFAP reactivity, without any evidence of infarct. One-third of the animals demonstrated a small in farct, straddling regions 6, 8, and/or 3, along with a loss of GFAP reactivity and only minor inflamma tory response (Fig. 4B). Figure 5 a, b, and c sum marize the neuronal damage and astrocytic and in flammatory responses in each of the eight regions in both the normothermic and hypothermic MCAO animals. Significant differences were detected be tween the normothermic and hypothermic animals for neuronal, astrocytic, and inflammatory re sponse in all the regions (p < 0. 01), except in region 5, which demonstrated significance between the two groups only in the astrocytic response (p < 0.01).
Ischemic Neuronal Damage
a
12 10 Q)
8
0
0 (/J
4 2 0 3
6
4
7
Regions
Astrocytic reaction
b 2
�
0 0 (/J
�
2
3
6
4
8
Regions
Inflammatory response
DISCUSSION
Our data demonstrate that moderate whole-body hypothermia eliminates or significantly reduces the size of the ischemic infarct and alters the type of ischemic neuronal damage after transient focal ce rebral ischemia in the rat. Focal cerebral ischemia in rat has been previ ously studied using a model of intracranial exposure and coagulation of the origin of the MCA (Tamura et al., 1981; Brint et al., 1988; Chen et aI. , 1991). An extracranial approach to occlude the MCA, by in troducing a suture into the ICA, has been recently developed (Koizumi et al. , 1986; Nagasawa and Kogure, 1989; Zea Longa et al. , 1989). Reperfusion can be easily induced by withdrawing the suture. The degree of tissue damage and mortality rate is a function of duration of ischemia and reperfusion time (Nagasawa and Kogure, 1989; Zea Longa et al., 1989). We chose a 2 h duration of ischemia to avoid mortality and a 96 h reperfusion time to allow J
Cereb Blood Flow Metab.
Vol. 12,
No.4.
1992
c
3
�
o o (/J
2
2
3
4
5
8
Regions ± SO of neuronal damage (a), astrocytic response (b), and inflammatory responses (c) in normothermic MCAO (open bar) and hypothermic MeAO (hatched bar) in the eig ht different brain regions. All regions except region 5 for neurons and inflammation exhibited a sig nificant difference (p < 0.01) in neuronal, astrocytic, and inflammatory response between the normothermic and hy pothermic g roups.
FIG. 5. Bar g raphs of mean
HYPOTHERMIA REDUCES FOCAL ISCHEMIC INFARCT
for maturation and clear demarcation of the lesion. Our data from normothermic animals show a sharply demarcated, reproducible ischemic lesion localized in the frontoparietal cortex and basal gan glia. This model of ischemia in our hands is repro ducible and allows for detailed histopathological evaluation of ischemic cell damage and therapeutic interventions. The transient MCAO rat model used in our ex periment differs slightly from that employed by Zea Longa and Nagasawa (Koizumi et aI. , 1986; Naga sawa and Kogure, 1989; Zea Longa et aI. , 1989). In our study, the CCA was left open and the branches of the ECA were left intact to allow complete reper fusion after withdrawal of the suture, and to avoid undue stress to the animal. Because of the minimal lesion, and consequently the absence of a clearly demarcated infarct in the hypothermic animals, we developed a scoring system for differential cellular evaluation in eight anatomically distinct brain sub regions. Measuring only the area or volume of the lesion fails to demonstrate the anatomical sensitiv ity and distribution of an ischemic lesion. The present scoring system provides a detailed evalua tion of the anatomical distribution of the cellular response and confirms the presence of a reproduc ible lesion localized at the neocortex and basal gan glia after MCAO. Hypothermia has been studied extensively for its effect on selective/delayed neuronal damage after transient global cerebral ischemia (Busto et aI. , 1989; Boris-Moller et aI. , 1990; Welsh et aI. , 1990; Chopp et aI. , 1991). Hypothermia reduces ischemic neuronal death, possibly by inhibiting the release of excitatory neurotransmitters (Busto et aI. , 1987). However, the exact mechanism of hypothermic protection in cerebral ischemia remains unknown. Normothermic MCAO induces a regional reduction of CBF and development of a localized cerebral in farction (Nagasawa and Kogure, 1989). Brain tissue undergoes cell death (including neurons and glia) in conjunction with infiltration of neutrophils and macrophages and microvascular proliferation (Gar cia and Kamijyo, 1974). The effect of hypothermia on MCAO may be different from that on global ce rebral ischemia. In the latter, hypothermia primar ily reduces the number of necrotic neurons and ac tivated astrocytes, without altering the distribution of the ischemic lesion (Busto et aI. , 1987; Boris Moller et aI. , 1990; Petito et aI. , 1990; Welsh et aI. , 1990; Chopp et aI. , 1991). In contrast, in transient focal ischemia induced by MCAO, the pattern of the lesion is also altered by hypothermia, from focal infarct to primarily selective neuronal ischemic in jury. In addition, there is a pronounced reduction in
627
the infiltration of inflammatory cells, which are ab sent in the global ischemia. Our finding, in part, may reflect differences in anesthesia during the ischemic period. Halothane anesthesia was maintained during ischemia in the animals subjected to hypothermia, while anesthesia was terminated during ischemia in the normother mic animals. Anesthesia may have a protective ef fect on ischemic brain cell damage (Baughman et aI. , 1988). Kuroiwa et ai. (1990) found that postisch emic halothane anesthesia lessens ischemic cell damage in the rat after global cerebral ischemia by preventing postischemic hyperthermia. However, in a study of 2 h of MCAO in the rat, maintenance of halothane anesthesia during the ischemic period still resulted in a large cerebral infarct (Warner et aI. , 1991). Our present study cannot exclude the possibility that halothane administered during hy pothermic ischemia may have contributed to pro tecting tissue from ischemic cell damage. Transient global ischemia causes a significant re duction in brain temperature (4-5°C) and an abrupt return to normal temperature after restoration of blood flow (Minamisawa et aI. , 1990a,b; Chopp et aI. , 1991). Focal cerebral ischemia, however, both in normothermic and hypothermic animals, causes only minor fluctuations of brain temperature. The maintenance of brain temperatures during focal ischemia may be attributed to the reduction in blood flow in a segment of tissue embedded in normal perfused surrounding brain and extracerebral tis sue. In summary, our data demonstrate that (a) hypo thermia during and after transient MCAO signifi cantly alleviates ischemic cell damage and (b) tran sient MCAO induced by the intracarotid suture method is a reliable model of ischemia with which to study the tissue response to therapeutic interven tion. Acknowledgment: The authors wish to thank Dr. Yasuji Yoshida for stimulating discussions, Patricia Ruffin for manuscript preparation, and Lisa Pietrantoni and Cecylia Zaloga for technical assistance. This work is supported by NINDS 23393 and NS29463.
REFERENCES Baughman VL, Hoffman WE, Miletich DJ, Albrecht RF, Thom as C (1988) Neurologic outcome in rats following incomplete cerebral ischemia during halothane, isoflurane, or N20. A n
esthesiology 69:192-198 Boris-Moller F, Smith ML, Siesj6 BK (1990) Effects of hypo thermia on ischemic brain damage: A comparison between preischemic and postischemic cooling. Neurosci Res Com
mun 5:87-94 Brint S, Jacewicz M, Kiessling M, Tanabe J, Pulsinelli W (1988) Focal brain ischemia in the rat: Methods for reproducible
J
Cereb Blood Flow Metab, Vol.
12,
No.4,
1992
628
H. CHEN ET AL. neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries. J
Cereb Blood Flow Metab 8:474-485 Busto R, Dietrich WD, Globus MYT, Valdes I, Scheinberg P, Ginsberg MD (1987) Small differences in intraischemic brain temperature critically determine the extent of ischemic neu ronal injury. J Cereb Blood Flow Metab 7:729-738 Busto R, Globus MYT, Dietrich WD, Martinez E, Valdes I, Ginsberg MD (1989) Effect of mild hypothermia on isch emia-induced release of neurotransmitters and free fatty ac ids in the rat brain. Stroke 20:904-910 Chen H, Chopp M, Welch KMA (1991) Effect of mild hyperther mia on the ischemic infarct volume after middle cerebral artery occlusion in the rat. Neurology 41: 1133-1135 Chopp M, Chen H, Dereski MO, Garcia JH (1991) Mild hypo thermic intervention after graded ischemic stress in rats.
Stroke 22:37-43 Garcia JH, Kamijyo Y (1974) Cerebral infarction. Evolution of histopathological changes after occlusion of a middle cere bral artery in primates. J Neuropathol Exp Neurol 33:409-
421 Kawamura S, Shirasawa M, Fukasawa H, Yasui N (1991) At tenuated neuropathology by nilvadipine after middle cere bral artery occlusion in rats. Stroke 22:51-55 Koizumi J, Yoshida Y, Nakazawa T, Ooneda G (1986) Experi mental studies of ischemic brain edema. I. A new experi mental model of cerebral embolism in rats in which recircu lation can be introduced in the ischemic area. Jpn J Stroke
8:1-8 Kuroiwa T, Bonnekoh P, Hossmann KA (1990) Prevention of postischemic hyperthermia prevents ischemic injury of CAl neurons in gerbils. J Cereb Blood Flow Metab 10:550--556 Michenfelder JD, Milde JH (1977) Failure of prolonged hypo capnia, hypothermia, or hypertension to favorably alter acute stroke in primates. Stroke 8:87-91
J
Cereb Blood Flow Metab, Vol. 12, No.4, 1992
Minamisawa H, Nordstrom CH, Smith ML, Siesj6 BK (1990a) The influence of mild body and brain hypothermia on isch emic brain damage. J Cereb Blood Flow Metab 10:365-374 Minamisawa H, Mellergard P, Smith ML, Bengtsson F, Thean der S, Boris-Moller F, Siesj6 BK (1990b) Preservation of brain temperature during ischemia in rats. Stroke 21:758-764 Nagasawa H, Kogure K (1989) Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion. Stroke 20:1037-1043 Paxinos G, Watson C (1986) The Rat Brain in Stereotaxic Coor dinates, 2nd edition. Orlando, FL, Academic Press Petito C, Morgello S, Felix JC, Lesser ML (1990) The two pat terns of reactive astrocytosis in postischemic rat brain. J
Cereb Blood Flow Metab 10:850--859 Simeone FA, Frazer G, Lawner P (1979) Ischemic brain edema: Comparative effects of barbiturates and hypothermia.
Stroke 10:8-12 Steen PA, Edward HS, Michenfelder JD (1979) Detrimental ef fect of prolonged hypothermia in cats and monkeys with and without regional cerebral ischemia. Stroke 10:522-528 Tamura A, Graham Dl, McCulloch J, Teasdale GM (1981) Focal cerebral ischemia in the rat: 1. Description of technique and early neuropathological consequences following middle ce rebral artery occlusion. J Cereb Blood Flow Metab 1:53-60 Warner DS, Zhou J, Ramani R, Todd MM (1991) Reversible focal ischemia in the rat: Effects of halothane, isoflurane, and methohexital anesthesia. J Cereb Blood Flow Metab
11:794-802 Welsh FA, Sims RE, Harris VA (1990) Mild hypothermia pre vents ischemic injury in gerbil hippocampus. J Cereb Blood
Flow Metab 10:557-563 Zea Longa E, Weinstein PR, Carlson S, Cummins R (1989) Re versible middle cerebral artery occlusion without craniec tomy in rats. Stroke 20:84-91
The Effect of Hypothermia on Transient Middle Cerebral Artery Occlusion in the Rat
*Hua Chen, *:j:Michael Chopp, *:j:Zheng G. Zhang, and tJulio H. Garcia Departments of*Neurology and tPathology, Henry Ford Hospital, Detroit, Michigan, and fDepartment of Physics, Oakland University, Rochester, Michigan, U.S.A.
Summary: We investigated the effect of moderate whole body hypothermia (30°C) on transient middle cerebral ar tery occlusion (MCAO) in the rat. Male Wistar rats were subjected to 2 h of ischemia by inserting a suture into the lumen of the internal carotid artery and occluding the origin of the MCA. Experimental groups were (a) MCAO induced at 37°C body temperature (n 15); (b) 30°C body temperature induced prior to ischemia and maintained for 2 h of MCAO and I h of reperfusion (n 12); and (c) MCAO with regional brain and body temperatures mea sured in normothermic (n 3) and hypothermic MCAO rats (n 2). Histopathological evaluation was performed 96 h after reperfusion. All normothermic MCAO animals
exhibited ischemic infarct involving the ipsilateral cortex and basal ganglia with infiltration of neutrophils, macro phages, and microvascular proliferation. Hypothermic MCAO animals exhibited minor ischemic damage ranging from selective neuronal injury to small focal areas of in farct with minimal inflammatory response. Our data dem onstrate that transient ischemia induced by using the in tra-arterial suture method to occlude the MCA results in a reproducible brain lesion and that moderate hypother mia has a profound protective effect on the brain injury after transient MCAO. Key Words: Transient middle ce rebral artery occlusion-Hypothermia-Histopathol ogy-Rat.
Hypothermia has been widely investigated in an imal models of global cerebral ischemia, and mild to moderate hypothermia significantly reduces isch emic neuronal damage in these models (Busto et aI. , 1987; Boris-Moller et aI. , 1990; Welsh et aI. , 1990; Chopp et aI. , 1991). The effects of hypothermia on focal cerebral ischemia are uncertain. Data from hy pothermic intervention after permanent focal cere bral ischemia in subhuman primates suggest that severe hypothermia (25. 9°C) or prolonged hypo thermia (48 h) is detrimental to animals with focal brain ischemia (Michenfelder and Milde, 1977; Sim eone et aI. , 1979; Steen et aI. , 1979). To our knowl edge, there are no studies that examine the neuronal
protective effects of moderate hypothermia induced during and acutely after transient focal cerebral ischemia. Transient focal cerebral ischemia in the rat, in duced by advancing a suture into the internal ca rotid artery to occlude the middle cerebral artery (MCA), has provided a new model to investigate the development of cerebral tissue damage and thera peutic intervention (Koizumi et aI. , 1986; Nagasawa and Kogure, 1989; Zea Longa et aI. , 1989; Kawa mura et aI. , 1991). This model has the advantage of not requiring a craniotomy, thus eliminating the in vasive surgical procedure that itself may cause trauma to brain, as well as alter the intracranial pressure. In the present study, we employed the intraluminal model of transient middle cerebral ar tery occlusion (MCAO) in the rat to explore the pathological features of this model, and to test the effect of short duration (3 h), 30°C whole body hy pothermia on ischemic cell damage. Our data sug gest that reproducible brain injury is induced by 2 h of MCAO, and 30°C hypothermia significantly pro tects the brain from transient focal ischemia.
=
=
=
=
Received October 9, 1991; final revision received January 15, 1992; accepted January 15, 1992. Address correspondence and reprint requests to Dr. H. Chen at Department of Neurology, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202, U. S. A. Abbreviations used: ACA, anterior carotid artery; CCA, com mon carotid artery; ECA, external carotid artery; GFAP, glial fibrillary acid protein; HIE, hematoxylin and eosin; ICA, internal carotid artery; MCAO, middle cerebral artery occlusion; PA, pterygopalatine artery.
621
622
H. CHEN ET AL. MATERIALS AND METHODS
Male Wistar rats (weighing 270-JOO g, n 32) were used in the experiment. MCAO was induced by advanc ing a 4-0 surgical nylon suture into the internal carotid artery (ICA) to block the origin of the MCA (Koizumi et aI., 1986; Nagasawa and Kogure, 1989; Zea Longa et aI., 1989). Animals were anesthetized using a face mask de livering 1.0-2.0% halothane in 70% N20 and 30% O2, The right femoral artery was cannulated to measure blood gas and blood glucose before ischemia. Arterial blood pres sure was monitored prior to MCAO and continuously for 20 min after MCAO in both normothermic and hypother mic animals (groups 1 and 2, respectively; see below). In addition, in the hypothermic animals, arterial blood pres sure was continuously monitored throughout the period of MCAO and for 20 min after the onset of reperfusion. A 2 cm incision was made at the center of the neck, and the right common carotid artery (CCA), external carotid ar tery (ECA), and ICA were exposed under an operating microscope (Carl Zeiss, Inc., Thornwood, NY, U.S.A.). Care was taken to avoid injury to the vagus nerve. The ICA was further dissected to identify the pterygopalatine artery (PA) branch and the intracranial ICA branch (see Fig. 1). The CCA and ICA were temporarily clamped using microsurgical clips (Codman & Shurtleff, Inc., Ran dolf, MA, U.S.A.). A 5-0 silk suture was tied loosely at the origin of the ECA and ligated at the distal end of the ECA. A 4-0 surgical nylon suture, with its tip rounded by heating near a flame, was introduced into the ECA lumen through a small puncture. The silk suture around the ECA origin was tightened around the intraluminal nylon suture to prevent bleeding, and the microsurgical clips were re moved. A length of 18.5-19.5 mm of nylon suture, deter mined according to the animal's weight, was gently ad vanced from the ECA into the lumen of the ICA until the suture blocked the origin of the MCA. The incision was temporarily closed using skin clips. In group 1 (normo thermic animals, see below), anesthesia was then termi-
5
=
I
4
FIG. 2. Coronal section of rat brain illustrating the division of the rig ht hemisphere into eig ht regions for pathological eval uation. Stars represent the approximate locations of regional brain temperature measurements. IC, internal capsule; F, fornix; DB, diagonal band of Broca; AC, anterior commis sure; CPu, caudate putamen; GP, g lobus pallidus. Regions were as follows: (1) piriform cortex, (2) insular cortex, (3) parietal cortex, (4) hindlimb and forelimb area of cortex, (5) frontal and cingulate cortex, (6) caudate putamen, (7) g lobus putamen, and (8) preoptic area.
nated, and animals awoke 5-10 min thereafter. After 2 h of ischemia, anesthesia was reinstituted, and reperfusion was performed by withdrawal of the suture until the tip cleared the ICA lumen and reached the origin of ECA. By contrast, in group 2 (hypothermic animals, see below), halothane anesthesia was maintained throughout the 2 h ischemic period and 1 h of recirculation to allow accurate temperature control. Although CBF was not measured in the present study, the length of the suture required to block the MCA was confirmed in a preliminary study of 50 rats in which the suture was permanently placed to occlude the MCA and suture placement was verified after killing. Animals were fasted overnight before surgery but al lowed free access to water. Rectal temperature was con trolled with a feedback-regulated water heating system to maintain the body temperature. Animals were randomly divided into two experimental groups: In group 1, MCAO was induced at 37°C body temperature (n 15). In group 2, MCAO was induced at 30°C body temperature and maintained for 2 h of ischemia and 1 h of reperfusion (n 12). The 30°C whole body hypothermia was instituted 30 min prior to the onset of MCAO by spraying alcohol on the skin and fanning room air (22°C) toward the animal's body. The heating system was set to maintain the ani mal's body temperature at 30°C. Hypothermia was main tained throughout the 2 h period of ischemia and for an additional 1 h of recirculation. Animals were rewarmed to 37°C using the heating pad. Additional animals were used to measure body and re gional brain temperatures during ischemia and recircula tion in normothermic MCAO rats (n 3) and hypother mic MCAO rats (n 2), without the histopathology end point. Thirty minutes prior to MCAO, microthermocou pies (100 !-lm) placed into a 27 gauge needle were inserted into the right (lesion side) and left (control side) cortex, caudate putamen, and preoptic areas (see Fig. 2) through 1 mm burr holes in the skull. Brain and rectal tempera tures were recorded every 5 min throughout the experi ment using a digital thermometer (Physitemp, Clifton, NJ, U.S.A.). =
=
=
=
CCA FIG. 1. Schematic representation of rat extracranial and in
tracranial arteries (circled) and the suture position during middle cerebral artery occlusion (MCAO). CCA, common ca rotid artery; ECA, external carotid artery; ICA, internal carotid artery; PA, pterygopalatine artery; PCA, posterior cerebral artery; MCA, middle cerebral artery; ACA, anterior cerebral artery.
J
Cereb Blood Flow Metab, Vol.
12,
No.4,
1992
HYPOTHERMIA REDUCES FOCAL ISCHEMIC INFARCT TABLE 1.
Brain ischemic damage grading scales
Percentage of neuronal injury Neuron Acute damage Delayed damage Necrosis Cavitation Astrocyte
50%
I 4 7 10
2 5 8 11
3 6 9 12
0: normal 1: hypertrophic (increase in nuclear diameter, cellular processes, and GFAP intensity) 2: necrotic (no GFAP staining)
Inflammation Neutrophil Macrophage
Vasculature
not detectable polymorphonucleus detected not detectable peripheral nucleus with cytoplasmic granules detected 0: normal I: increased number of microvessels
0: 1: 0: 1:
Four days after MCAO, animals were anesthetized (i.m.) with ketamine (44 mg/kg) and xylazine (13 mg/kg), and transcardially perfused with heparinized saline and 10% neutral buffered formalin. The head was immersed in formalin solution for I h and then the brain was removed. The brain was cut into 2 mm thick coronal blocks, for a total of seven blocks per animal, using a rat brain matrix. The brain tissue was processed, embedded, and 6 /Lm thick paraffin sections from each block were cut and stained with hematoxylin and eosin (H/E). Brain ischemic damage was evaluated using light microscopy. A standard section, corresponding to the coronal sec tion at interaural 8.2 mm, bregma 0.8 mm (Paxinos and Watson, 1986), was selected from each animal. This sec tion is centered in the ischemic lesion, and the right hemi sphere was divided into eight anatomically distinct re gions for detailed histological analysis (Fig. 2). Paraffin embedded 6 /Lm thick sections adjacent to the H/E section were cut and mounted on gelatin-coated slides to evaluate the astrocytic responses. In order to identify the astrocytes, the avidin-biotin complex (ABC) method was performed using antibodies against glial fibrillary acid protein (OFAP, polyc1onal antibodies, purchased from DAKO, Carpinteria, CA, U.S.A.). Reactive/necrotic as trocytes were evaluated using OFAP staining. In the present study, detailed examination of the ICA lumen, by electron microscopy, was not performed. Neuronal dam age, astrocytic reaction/damage, and inflammatory re sponse (summation of infiltration by neutrophils, macro phages, and increased numbers of microvessels, identi fied using H/E staining) were evaluated in each distinct
TABLE 2.
region by means of grading scales described in Table I . The severity o f neuronal damage was divided into acute type (either shrunken or ballooned neurons), delayed type (eosinophilic neurons), tissue necrosis (no surviving tissue elements in an area), and cavitation. The percent age of ischemic neuronal damage (acute, delayed damage, necrosis, and cavitation) were factored into the numerical grading. For example, if < 10% of the neurons in region 1 were eosinophilic, the score of 4 was given; if> 10% but 50% eosino philic neurons in the region. Multiple histological changes within a region were averaged. With this grading system, the most severe injury would receive a score of 12 for neuronal damage and 2 for astrocytic and 3 for inflamma tory response.
Statistics Wilcoxon two-sample tests were performed to compare the response of neurons, astrocytes, and inflammation between the normothermic and hypothermic MCAO an imals. All data are presented as mean ± SD.
RESULTS
Blood gas values and arterial serum glucose lev els before MCAO were within normal physiological ranges (Table 2). The blood pressure fluctuated within 5-10 mm Hg during the MCAO surgery. There were no detectable differences in arterial blood pressure values prior to MCAO and 20 min after MCAO in both normothermic and hypother mic animals; likewise, there were no detectable dif ferences between groups at each time point of mea surement. In hypothermic animals, arterial blood pressure was stable during the 2 h of MCAO and 20 min of reperfusion (data not shown). Five animals developed subarachnoid hemorrhage (two in nor mothermic MCAO, three in hypothermic MCAO). Data from these animals were not included in the present study. No animals died after surgery. Figures 3A and 3B illustrate the brain (lesion side) and rectal temperature changes before, during, and after MCAO in a representative normother mic and hypothermic MCAO animal, respectively. Rectal temperatures were maintained constant at 37°C in normothermic and at 30°C in hypother mic MCAO animals. Prior to ischemia, brain tem perature was elevated above the rectal temper-
Serum arterial blood gas, glucose, and blood pressure (BP) values Pcoz (mm Hg)
pH Normothermia (n = 13) Hypothermia (n = 9)
623
7.43 7.35
± ±
0.04 0.08
37.3 39.0
± ±
2.1 5.0
Poz (mm Hg)
Glucose (mg/dl)
141 183
129 119
± ±
20 23
± ±
28 15
BP (mm Hg) Before
98 100
± ±
5 6
During
97 98
± ±
2 2
Data are obtained 20 min before MCAO and 20 min after MCAO (during).
J Cereb Blood Flow Metab, Vol. 12, No.4, 1992
H. CHEN ET AL.
624
NORMOTHERMIA (right hemisphere) 39.00 ,------,
a .......
38.50
o
o -
w a: :::> l e:{ a: w a.. ::::2: w I-
38.00
37.50
37.00
36.50
36.00
L-_...l._ ...--'---_--'--_----L_---L__L-_...l._ ...--'---_--'-- _--.l
-20
0
20
40
60
80
100
120
140
160
180
TIME (MIN)
HYPOTHERMIA (right hemisphere)
FIG. 3. Plot of rectal (+), cortical
(0),
caudate ([,), and preoptical (0) brain temperatures in a representative rat during normothermic MCAO (a) and hypothermic MCAO (b) and reperfu sion. Arrows indicate the initiation and termination of MCAO.
32.50 ,------,
.......
b
32.00
o
o
-
w a: :::> l e:{ a: w a.. ::::2: w I-
31.50
31.00
30.50
30.00
+ ..... ++++++++++++++++++++++++++++++++++++
t
t
29.50 L-_-'--_-'---_...L.-_-'--_-'-_--'--_--'-_---'-_---' -20 20 40 60 80 100 120 140 160 180 0
TIME (MIN) ature by OSC in normothermic and lSC in hypo thermic MCAO rats. After the onset of MCAO, brain temperatures declined approximately 0. 5°C and fluctuated at 37. 2 ± 0. 3 and 31. 2 ± 0. 3°C in normothermic and hypothermic MCAO rats, re spectively. Brain temperatures in the nonlesioned left side declined and fluctuated slightly, as in the lesioned side, during the MCAO in both normo thermic and hypothermic MCAO rats (data not shown). In both groups of animals, after withdrawal J
Cereb Blood Flow Metab, Vol.
12,
No.4,
1992
of the suture, brain temperatures in both sides of the brain fluctuated slightly, similar to the temper ature fluctuation found during ischemia. Figures 4A-D show coronal sections of brain from a representative normothermic MCAO animal exhibiting ischemic infarct and from a hypothermic MCAO animal exhibiting minor ischemic injury. All normothermic MCAO animals revealed a consistent and homogeneous pattern of ischemic infarct in re gions 1, 2, 3, 6, and 8 (Fig. 4A). The size of the
HYPOTHERMIA REDUCES FOCAL ISCHEMIC INFARCT
625 (b)
(a)
(e)
FIG. 4. HIE staining of 6 fJ-m par
aff in-embedded coronal sec tions from a representative rat brain subjected to normother mic MCAO (a,c) and hypother mic MCAO (b,d). a: A well demarcated paled lesion is noted in the right frontoparietal cortex and basal ganglia,region 1,2,3,6,7,and 8 (x6). b: Small pale areas are noted in the right lateral caudate putamen and preoptic area, region 6 and 8 (x6). c: An enlargement (x570) of the box in a demonstrating eosinophilic neurons (arrows) and inf iltrating inflammatory cells (neutrophil, arrowhead; macrophage, open arrow). d: An enlargement (x 570) of the box in b, demonstrating selec tive individual ischemic neu rons (large arrows) and intact neurons (small arrows).
(d)
J cerea
Blood Flow Metab, Vol.
n. *f. JIlf1'I
626
H. CHEN ET AL.
lesion in regions 4 and 7, located at the boundary of infarct, varied from animal to animal. Region 5, which is primarily supplied by the anterior cerebral artery (ACA), exhibited only minor acute ischemic neuronal change. However, an increased reactivity of GFAP was present (increase of nuclear diameter, cytoplasmic processes, and GFAP intensity). Within an infarct, neurons were eosinophilic or ex hibited ghost-like appearance, and no astrocytes were detected by GFAP staining. In addition to the neuronal and astrocytic injury, massive infiltration by neutrophils and macrophages as well as in creased numbers of microvessels (compared to the contralateral side) were detected in the lesioned re gions 1, 2, 3, 6, and 8 (Fig. 4C). In the hypothermic MCAO group, two-thirds of the animals exhibited only selective neuronal damage in regions 6, 8, and/ or 2 and 3 (Fig. 4D), accompanied by an increased GFAP reactivity, without any evidence of infarct. One-third of the animals demonstrated a small in farct, straddling regions 6, 8, and/or 3, along with a loss of GFAP reactivity and only minor inflamma tory response (Fig. 4B). Figure 5 a, b, and c sum marize the neuronal damage and astrocytic and in flammatory responses in each of the eight regions in both the normothermic and hypothermic MCAO animals. Significant differences were detected be tween the normothermic and hypothermic animals for neuronal, astrocytic, and inflammatory re sponse in all the regions (p < 0. 01), except in region 5, which demonstrated significance between the two groups only in the astrocytic response (p < 0.01).
Ischemic Neuronal Damage
a
12 10 Q)
8
0
0 (/J
4 2 0 3
6
4
7
Regions
Astrocytic reaction
b 2
�
0 0 (/J
�
2
3
6
4
8
Regions
Inflammatory response
DISCUSSION
Our data demonstrate that moderate whole-body hypothermia eliminates or significantly reduces the size of the ischemic infarct and alters the type of ischemic neuronal damage after transient focal ce rebral ischemia in the rat. Focal cerebral ischemia in rat has been previ ously studied using a model of intracranial exposure and coagulation of the origin of the MCA (Tamura et al., 1981; Brint et al., 1988; Chen et aI. , 1991). An extracranial approach to occlude the MCA, by in troducing a suture into the ICA, has been recently developed (Koizumi et al. , 1986; Nagasawa and Kogure, 1989; Zea Longa et al. , 1989). Reperfusion can be easily induced by withdrawing the suture. The degree of tissue damage and mortality rate is a function of duration of ischemia and reperfusion time (Nagasawa and Kogure, 1989; Zea Longa et al., 1989). We chose a 2 h duration of ischemia to avoid mortality and a 96 h reperfusion time to allow J
Cereb Blood Flow Metab.
Vol. 12,
No.4.
1992
c
3
�
o o (/J
2
2
3
4
5
8
Regions ± SO of neuronal damage (a), astrocytic response (b), and inflammatory responses (c) in normothermic MCAO (open bar) and hypothermic MeAO (hatched bar) in the eig ht different brain regions. All regions except region 5 for neurons and inflammation exhibited a sig nificant difference (p < 0.01) in neuronal, astrocytic, and inflammatory response between the normothermic and hy pothermic g roups.
FIG. 5. Bar g raphs of mean
HYPOTHERMIA REDUCES FOCAL ISCHEMIC INFARCT
for maturation and clear demarcation of the lesion. Our data from normothermic animals show a sharply demarcated, reproducible ischemic lesion localized in the frontoparietal cortex and basal gan glia. This model of ischemia in our hands is repro ducible and allows for detailed histopathological evaluation of ischemic cell damage and therapeutic interventions. The transient MCAO rat model used in our ex periment differs slightly from that employed by Zea Longa and Nagasawa (Koizumi et aI. , 1986; Naga sawa and Kogure, 1989; Zea Longa et aI. , 1989). In our study, the CCA was left open and the branches of the ECA were left intact to allow complete reper fusion after withdrawal of the suture, and to avoid undue stress to the animal. Because of the minimal lesion, and consequently the absence of a clearly demarcated infarct in the hypothermic animals, we developed a scoring system for differential cellular evaluation in eight anatomically distinct brain sub regions. Measuring only the area or volume of the lesion fails to demonstrate the anatomical sensitiv ity and distribution of an ischemic lesion. The present scoring system provides a detailed evalua tion of the anatomical distribution of the cellular response and confirms the presence of a reproduc ible lesion localized at the neocortex and basal gan glia after MCAO. Hypothermia has been studied extensively for its effect on selective/delayed neuronal damage after transient global cerebral ischemia (Busto et aI. , 1989; Boris-Moller et aI. , 1990; Welsh et aI. , 1990; Chopp et aI. , 1991). Hypothermia reduces ischemic neuronal death, possibly by inhibiting the release of excitatory neurotransmitters (Busto et aI. , 1987). However, the exact mechanism of hypothermic protection in cerebral ischemia remains unknown. Normothermic MCAO induces a regional reduction of CBF and development of a localized cerebral in farction (Nagasawa and Kogure, 1989). Brain tissue undergoes cell death (including neurons and glia) in conjunction with infiltration of neutrophils and macrophages and microvascular proliferation (Gar cia and Kamijyo, 1974). The effect of hypothermia on MCAO may be different from that on global ce rebral ischemia. In the latter, hypothermia primar ily reduces the number of necrotic neurons and ac tivated astrocytes, without altering the distribution of the ischemic lesion (Busto et aI. , 1987; Boris Moller et aI. , 1990; Petito et aI. , 1990; Welsh et aI. , 1990; Chopp et aI. , 1991). In contrast, in transient focal ischemia induced by MCAO, the pattern of the lesion is also altered by hypothermia, from focal infarct to primarily selective neuronal ischemic in jury. In addition, there is a pronounced reduction in
627
the infiltration of inflammatory cells, which are ab sent in the global ischemia. Our finding, in part, may reflect differences in anesthesia during the ischemic period. Halothane anesthesia was maintained during ischemia in the animals subjected to hypothermia, while anesthesia was terminated during ischemia in the normother mic animals. Anesthesia may have a protective ef fect on ischemic brain cell damage (Baughman et aI. , 1988). Kuroiwa et ai. (1990) found that postisch emic halothane anesthesia lessens ischemic cell damage in the rat after global cerebral ischemia by preventing postischemic hyperthermia. However, in a study of 2 h of MCAO in the rat, maintenance of halothane anesthesia during the ischemic period still resulted in a large cerebral infarct (Warner et aI. , 1991). Our present study cannot exclude the possibility that halothane administered during hy pothermic ischemia may have contributed to pro tecting tissue from ischemic cell damage. Transient global ischemia causes a significant re duction in brain temperature (4-5°C) and an abrupt return to normal temperature after restoration of blood flow (Minamisawa et aI. , 1990a,b; Chopp et aI. , 1991). Focal cerebral ischemia, however, both in normothermic and hypothermic animals, causes only minor fluctuations of brain temperature. The maintenance of brain temperatures during focal ischemia may be attributed to the reduction in blood flow in a segment of tissue embedded in normal perfused surrounding brain and extracerebral tis sue. In summary, our data demonstrate that (a) hypo thermia during and after transient MCAO signifi cantly alleviates ischemic cell damage and (b) tran sient MCAO induced by the intracarotid suture method is a reliable model of ischemia with which to study the tissue response to therapeutic interven tion. Acknowledgment: The authors wish to thank Dr. Yasuji Yoshida for stimulating discussions, Patricia Ruffin for manuscript preparation, and Lisa Pietrantoni and Cecylia Zaloga for technical assistance. This work is supported by NINDS 23393 and NS29463.
REFERENCES Baughman VL, Hoffman WE, Miletich DJ, Albrecht RF, Thom as C (1988) Neurologic outcome in rats following incomplete cerebral ischemia during halothane, isoflurane, or N20. A n
esthesiology 69:192-198 Boris-Moller F, Smith ML, Siesj6 BK (1990) Effects of hypo thermia on ischemic brain damage: A comparison between preischemic and postischemic cooling. Neurosci Res Com
mun 5:87-94 Brint S, Jacewicz M, Kiessling M, Tanabe J, Pulsinelli W (1988) Focal brain ischemia in the rat: Methods for reproducible
J
Cereb Blood Flow Metab, Vol.
12,
No.4,
1992
628
H. CHEN ET AL. neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries. J
Cereb Blood Flow Metab 8:474-485 Busto R, Dietrich WD, Globus MYT, Valdes I, Scheinberg P, Ginsberg MD (1987) Small differences in intraischemic brain temperature critically determine the extent of ischemic neu ronal injury. J Cereb Blood Flow Metab 7:729-738 Busto R, Globus MYT, Dietrich WD, Martinez E, Valdes I, Ginsberg MD (1989) Effect of mild hypothermia on isch emia-induced release of neurotransmitters and free fatty ac ids in the rat brain. Stroke 20:904-910 Chen H, Chopp M, Welch KMA (1991) Effect of mild hyperther mia on the ischemic infarct volume after middle cerebral artery occlusion in the rat. Neurology 41: 1133-1135 Chopp M, Chen H, Dereski MO, Garcia JH (1991) Mild hypo thermic intervention after graded ischemic stress in rats.
Stroke 22:37-43 Garcia JH, Kamijyo Y (1974) Cerebral infarction. Evolution of histopathological changes after occlusion of a middle cere bral artery in primates. J Neuropathol Exp Neurol 33:409-
421 Kawamura S, Shirasawa M, Fukasawa H, Yasui N (1991) At tenuated neuropathology by nilvadipine after middle cere bral artery occlusion in rats. Stroke 22:51-55 Koizumi J, Yoshida Y, Nakazawa T, Ooneda G (1986) Experi mental studies of ischemic brain edema. I. A new experi mental model of cerebral embolism in rats in which recircu lation can be introduced in the ischemic area. Jpn J Stroke
8:1-8 Kuroiwa T, Bonnekoh P, Hossmann KA (1990) Prevention of postischemic hyperthermia prevents ischemic injury of CAl neurons in gerbils. J Cereb Blood Flow Metab 10:550--556 Michenfelder JD, Milde JH (1977) Failure of prolonged hypo capnia, hypothermia, or hypertension to favorably alter acute stroke in primates. Stroke 8:87-91
J
Cereb Blood Flow Metab, Vol. 12, No.4, 1992
Minamisawa H, Nordstrom CH, Smith ML, Siesj6 BK (1990a) The influence of mild body and brain hypothermia on isch emic brain damage. J Cereb Blood Flow Metab 10:365-374 Minamisawa H, Mellergard P, Smith ML, Bengtsson F, Thean der S, Boris-Moller F, Siesj6 BK (1990b) Preservation of brain temperature during ischemia in rats. Stroke 21:758-764 Nagasawa H, Kogure K (1989) Correlation between cerebral blood flow and histologic changes in a new rat model of middle cerebral artery occlusion. Stroke 20:1037-1043 Paxinos G, Watson C (1986) The Rat Brain in Stereotaxic Coor dinates, 2nd edition. Orlando, FL, Academic Press Petito C, Morgello S, Felix JC, Lesser ML (1990) The two pat terns of reactive astrocytosis in postischemic rat brain. J
Cereb Blood Flow Metab 10:850--859 Simeone FA, Frazer G, Lawner P (1979) Ischemic brain edema: Comparative effects of barbiturates and hypothermia.
Stroke 10:8-12 Steen PA, Edward HS, Michenfelder JD (1979) Detrimental ef fect of prolonged hypothermia in cats and monkeys with and without regional cerebral ischemia. Stroke 10:522-528 Tamura A, Graham Dl, McCulloch J, Teasdale GM (1981) Focal cerebral ischemia in the rat: 1. Description of technique and early neuropathological consequences following middle ce rebral artery occlusion. J Cereb Blood Flow Metab 1:53-60 Warner DS, Zhou J, Ramani R, Todd MM (1991) Reversible focal ischemia in the rat: Effects of halothane, isoflurane, and methohexital anesthesia. J Cereb Blood Flow Metab
11:794-802 Welsh FA, Sims RE, Harris VA (1990) Mild hypothermia pre vents ischemic injury in gerbil hippocampus. J Cereb Blood
Flow Metab 10:557-563 Zea Longa E, Weinstein PR, Carlson S, Cummins R (1989) Re versible middle cerebral artery occlusion without craniec tomy in rats. Stroke 20:84-91
