Journal of Cerebral Blood Flow and Metabolism 12:204--212 © 1992 The International Society of Cerebral Blood Flow and Metabolism Published by Raven Press, Ltd., New York
Regional Expression of Heat Shock Protein-70 mRNA and c-fos mRNA Following Focal Ischemia in Rat Brain
Frank A. Welsh, Donald J. Moyer, and Valerie A. Harris Division of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A.
Summary: In situ hybridization was used to estimate re gional levels of heat shock protein-70 (HSP-70) mRNA and c-fos mRNA in two related models of focal cerebral ischemia. In the first model, permanent occlusion of the distal middle cerebral artery (MCA) alone caused a patchy increase in HSP-70 mRNA by 1 h in the central zone of the MCA territory of the ipsilateral neocortex. Tissue levels of HSP-70 mRNA continued to increase for several hours and remained elevated at 24 h. In contrast to the focal expression of HSP-70, c-fos mRNA was in creased throughout the ipsilateral cerebral cortex by 15 min and remained elevated for least 3 h. The wide distri bution of c-fos expression suggests it may have been caused by spreading depression. In the second model, severe focal ischemia was produced with a combination of transient (l-h) bilateral carotid artery occlusion and permanent MCA occlusion. Combined occlusion for 1 h
without reperfusion caused expression of HSP-70 mRNA only in regions adjacent to the central zone of the MCA territory of the neocortex. However, reperfusion of the carotids for 2 h generated intense expression of HSP-70 mRNA throughout most of the ipsilateral cerebral cortex, white matter, striatum, and hippocampus. The wide spread increase in HSP-70 mRNA suggests that reperfu sion triggered expression in all previously ischemic re gions. However, at 24 h of reperfusion, increased levels of HSP-70 mRNA were restricted primarily to the isch
Cerebral ischemia induces increased synthesis of a number of specific proteins, despite an overall reduction in the rate of protein synthesis (Kleihues and Hossmann, 1971; Dienel et aI., 1980, 1986; Nowak, 1985). Several of the induced proteins are members of a group of "heat shock" or "stress" proteins that are expressed in the central nervous system following a number of stresses, including heat shock (Brown and Rush, 1990), trauma (Gower et aI., 1989), as well as ischemia. Although the func tion of the stress proteins is not well understood, their induction has been correlated with increased resistance to injury in many cell types (for review, see Lindquist and Craig, 1988). In brain, induction of stress proteins prior to an episode of cerebral
ischemia has been associated with decreased neu ronal necrosis in gerbil hippocampus (Kirino et aI., 1991). Thus, stress proteins may play an important role defending cells against ischemic injury. The 70-kDa heat shock protein (HSP-70) is one of the major stress proteins induced by cerebral isch emia (Nowak, 1985; Dienel et aI., 1986). Although the time course and regional induction of HSP-70 have been well characterized following transient global ischemia (Vass et aI., 1988; Kirino et aI., 1991; Simon et aI., 1991), expression of HSP-70 dur ing focal ischemia has not been extensively studied. Jacewicz et al. (1986) reported increased synthesis of a 70-kDa protein, presumed to be HSP-70, during focal ischemia in rat brain. Recently, HSP-70 was detected with immunocytochemical methods fol lowing occlusion of the middle cerebral artery (MCA) in rat brain (Gonzalez et aI., 1989; Sharp et aI., 1991). However, expression of HSP-70 in mod els of focal ischemia has not been further charac terized. Moreover, the effects of focal ischemia on transcription of the HSP-70 gene have not been pre-
emic core of the neocortex. These results suggest that expression of HSP-70 mRNA is prolonged in regions un dergoing injury, but is transient in surrounding regions that recover. Key Words: Heat shock protein-70--c-fos Focal ischemia-In situ hybridization-Messenger ribo nucleic acid-Middle cerebral artery.
Received April 5, 1991; revised July 19, 1991; accepted July 23, 1991. Address correspondence and reprint requests to Dr. F. A. Welsh at 313 Stemmler Hall, 36th and Hamilton Walk, Philadel phia, PA 19104-6070, U.S. A. Abbreviations used: HSP-70, heat shock protein-70; MCA, middle cerebral artery; SSC, sodium citrate saline.
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viously reported. Therefore, the objective of the present study was to determine the time course and regional expression of HSP-70 mRNA as a first step toward investigating the induction of stress proteins during focal ischemia and reperfusion. Regional levels of HSP-70 mRNA were estimated using in situ hybridization with a probe specific for the inducible form of HSP-70 mRNA (Miller et aI., 1987). To study the effects of increasing degrees of ischemia, two related models of focal ischemia were employed. First, mild focal ischemia was produced by permanent distal occlusion of the MeA. Second, severe focal ischemia was produced by combining MeA occlusion with transient bilateral carotid oc clusion for 1 h. Finally, since increased expression of the proto-oncogene c-fos has been reported fol lowing cerebral ischemia (Onodera et aI., 1989), re gional levels of c-fos mRNA were compared with those of HSP-70 mRNA following occlusion of the MeA alone. The results of this study have been presented previously in preliminary form (Welsh and Moyer, 1990; Moyer et aI., 1991).
METHODS Animal preparation Male Wistar rats, 35{}-550 g body wt, were given free access to food and water prior to surgery. The animals were anesthetized with pentobarbital (50 mg/kg i.p.),and core temperature was regulated at 37.5°C using a rectal thermistor connected to a heating lamp. The tail artery was cannulated to record arterial pressure and to obtain blood samples for measurement of P02, Pe02, pH, and hematocrit. Oxygen was delivered to the animals through a loose-fitting nose cone. In animals undergoing carotid occlusion,an incision was made in the neck,and the com mon carotid arteries were isolated and encircled loosely with surgical thread. The MCA on the right side was permanently occluded using the surgical approach described by Brint et al. (1988). After exposure of the MCA,the tip of a stainless steel wire,bent at 90° to form a hook,was inserted under the MCA just superior to the inferior cortical vein,using a micromanipulator. The MCA was lifted above the cor tical surface and permanently occluded by thermocoagu lation. A 24-gauge needle thermistor (YSI 524; Yellow Springs Instrument Co., Yellow Springs, OH, U.S.A.) was inserted through the craniotomy so that the tip rested in the subdural space over the MCA cortex. Subdural temperature was maintained at 3 7.SOC for 2 h using an overhead lamp. In animals undergoing transient carotid occlusion, miniature Mayfield clips were applied to both carotid arteries within 2 min of MCA occlusion. After occlusion for 1 h,the clips were removed to permit recir culation. Rectal temperature was maintained at 37.5°C until the animals awakened. In the first series (MCA only),animals were euthanized with a lethal injection of pentobarbital 3 h after sham operation (n 2) or after MCA occlusion for a period of 3),1 h (n 3), 3 h (n 3),or 24 h (n 2). 15 min (n
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Brains in the first series were analyzed for HSP-70 mRNA and c-fos mRNA using in situ hybridization. In the second series (combined MCA and carotid occlusion), animals were euthanized 3 h after sham operation (n 2), I h after combined occlusion without reperfusion (n 4), or after reperfusion for 15 min (n 3 ), 2 h (n 3), 24 h (n 3), or I week (n 2). Brains in the second series were analyzed for HSP-70 mRNA only. =
=
=
=
=
=
In situ hybridization Following euthanasia,the brain was removed from the cranium and frozen in a bed of dry ice. Brain sections were cut at a thickness of 15 fLm in a cryostat at - 11°C, dried on glass slides,fixed in 4% paraformaldehyde,and rinsed with 20x SSC buffer (0.3 M Na citrate,3 M NaC\). HSP-70 mRNA was detected using a 30-mer oligonucle otide probe with the sequence 3'-GG TAC CAC GAC TGG TTC TAC TTC CTC TAG C-5' (Nowak et aI., 1990b). This sequence, which is reported to be specific for the inducible form of HSP-70 mRNA (Miller et aI., 1987,1991),corresponds to a highly conserved region of the HSP-70 coding sequence,amino acids 122-129 of hu man HSP-70 (Hunt and Morimoto,1985). As a control for the HSP-70 probe,a 30-mer oligonucleotide with the com plementary sequence was also tested. c-fos mRNA was detected using a 30-mer oligonucleotide with sequence 3'-TGT GTC CTG AAA ACG CGT CTA GAC AGG CAG-5'. This sequence is complementary to base num bers 270-299 of rat c-fos mRNA (Curran et aI.,1987). The oligonucleotide probes were labeled on the 3'-end with eSS]dATP, using terminal transferase. The labeled probes were isolated and hybridized to tissue sections overnight at 3rC in 4x SSC. After rinsing for 2 h each in 1 x SSC and 0.5 x SSC at 37°C, the sections were dried and placed in contact with Kodak XAR film for 1-2 weeks. Expression of HSP-70 mRNA and c-fos mRNA was graded qualitatively by comparing optical density in several regions of the ipsilateral hemisphere to that in the homologous region of the contralateral hemisphere. The grading scale was as follows: 0 no difference, 1 moderate increase, 2 strong increase. =
=
=
RESULTS Arterial variables
Arterial pressure did not vary significantly during ischemia or reperfusion, relative to preocclusion levels (Table 1). Arterial pH and Pe02 remained within the normal range, while P02 was higher than TABLE 1. Arterial variables Stage of
MABP
Peo,
Po,
experiment
(mmHg)
(mm Hg)
(mm Hg)
pH
(%)
Preocclusion
113 ±16 (19)
38 ±6 (12)
230 ±63 (12)
7.33 ±0.06 (11)
41 ±4 (3)
112 ±21 (22)
35 ±8 (19)
216 ±71 (19)
7.36 ±0.1O (18)
38 ±4 (11)
119 ±13 (7)
34 ±4 (7)
225 ±72 (7)
7.38 ±0.04 (7)
35 ±3 (6)
Occlusion
(15
min)
Reperfusion
(30
min)
Hematocrit
=
=
=
=
=
Values are means
±
SD, (no. of animals).
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normal owing to administration of 02' The hemat ocrit decreased moderately during reperfusion. MCA In sham-operated animals, levels of HSP-70 mRNA were low in cerebral cortex, white matter, and striatum (Fig. 1). By contrast, c-fos mRNA was moderately increased throughout the ipsilateral ce rebral cortex in sham-operated animals (Fig. 1; Ta ble 2). Occlusion of the distal MCA caused increased expression of HSP-70 mRNA in the central zone of the MCA neocortex by I h but not 15 min post occlusion (Fig. 1; Table 2). Expression continued to increase for 3 h, although the distribution of HSP-70 mRNA was somewhat patchy and was accentuated in the outer layers of cortex. By 3 h post occlusion, increased levels of HSP-70 mRNA extended ante rior to the level of the caudate and posterior to the level of the hippocampus, with strongest expression occurring in layer 4 of the neocortex (Fig. 2). At 24 h, levels of HSP-70 mRNA remained elevated in the MCA territory of the neocortex, sometimes distrib uted in a columnar orientation (Fig. 1). HSP-70 mRNA was not detectably increased in subcortical structures of the ipsilateral hemisphere or in the contralateral hemisphere. Further, the antisense control probe for HSP-70 did not exhibit increased hybridization in regions with elevated HSP-70 mRNA. In contrast to the focal induction of HSP-70 mRNA, a widespread increase of c-fos mRNA was apparent throughout the ipsilateral cerebral cortex at all times studied (Fig. 1; Table 2). Expression of c-fos mRNA was increased by 15 min post occlu sion and continued to increase at 1 and 3 h. Within the cerebral cortex, highest levels of c-fos mRNA occurred in the outer layers of parietal cortex, in layer 2 of paramedian (cingulate) cortex, and in the pyramidal cell layer of entorhinal cortex (Fig. 1). Permanent occlusion of distal
MCA and carotid arteries In sham-operated animals, similar to those in the first series, expression of HSP-70 mRNA was low throughout the ipsilateral hemisphere (Fig. 3; Table 3). Combined occlusion of the MCA and carotid arteries for 1 h without reperfusion produced a moderate increase in HSP-70 mRNA in the parame dian cortex, entorhinal cortex, striatum, and hippo campus in two of four animals. Within the hippo campus, expression was strongest in the upper blade of the dentate gyrus. There was no detectable increase in HSP-70 mRNA in the central zone of the MCA territory of the neocortex. Reperfusion of the carotids for 15 min following 1 h of combined occlusion caused a regional expresCombined occlusion of
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sion of HSP-70 mRNA similar to that observed without reperfusion (Table 3). Thus, there was no marked change in HSP-70 mRNA levels during the first 15 min of reperfusion. However, by 2 h reper fusion, a profound increase in HSP-70 mRNA oc curred throughout the ipsilateral cerebral cortex, white matter, striatum, and hippocampus (Fig. 3; Table 3). Within the cortex, expression was most intense in the ischemic core. However, HSP-70 mRNA was also elevated in paramedian and ento rhinal cortex. Subcortical white matter tracts also exhibited increased expression in the ipsilateral hemisphere, especially at the level of the striatum. Within the striatum, increased expression of HSP70 mRNA occurred rather evenly, with slightly higher levels along the medial edge. In hippocam pus, increased expression was evident in the pyra midal cell layer of all subfields (most intense in CA4) and in the dentate granule cell layer, particularly in the upper blade. However, in one of three animals at this time, hippocampal expression of HSP-70 mRNA was not elevated (Table 3). Increased levels of HSP-70 mRNA were not detected in thalamus. Reperfusion for 24 h was associated with a reduc tion of HSP-70 mRNA levels in most regions of the ipsilateral hemisphere (Fig. 3; Table 3). While strong expression persisted in the ischemic core of the neocortex, especially layer 4, levels of HSP-70 mRNA had returned nearly to background in para median and entorhinal cortex, striatum, and hippo campus. Interestingly, in one of three animals at 24 h, increased levels of HSP-70 mRNA were present in subiculum and CAl pyramidal cell layer of the hippocampus (Fig. 3). Reperfusion for 1 week showed only a faint increase in HSP-70 mRNA along the margins of a presumed cortical infarct. DISCUSSION The present results describe the time course and regional expression of HSP-70 mRNA during focal ischemia and reperfusion of rat brain. Three distinct regional patterns of expression were detected. First, permanent occlusion of distal MCA alone caused an accumulation of HSP-70 mRNA that was restricted to the central zone of the MCA territory of the neocortex. Second, combined occlusion of the MCA and carotid arteries without reperfusion caused increased expression only in regions adja cent to the MCA territory of the neocortex. Third, reperfusion of the carotids following combined oc clusion of the MCA and carotid arteries produced a robust expression of HSP-70 mRNA that encom passed both the ischemic core and adjacent regions. These regional patterns of expression were deter mined, presumably, by the level of regional blood
HSP-70 mRNA EXPRESSION IN FOCAL ISCHEMIA
HSP-70
207
c-fos
Sham
(3 h)
Occlusior 15 min
1
hour
3 hours
24
hours
FIG. 1. Heat shock protein-70 (HSP-70) mRNA and c-fos mRNA levels during permanent occlu sion of the distal middle cerebral artery (MCA). Following sham operation or MCA occlusion for 15 min or 1,3, or 24 h, the brain was sectioned, and adjacent brain sections were hybridized with 35S-labeled probes specific for HSP-70 mRNA or c-fos mRNA and exposed to x-ray film. Increased levels of mRNA are indicated by in creased optical density. Hybridization for c-fos mRNA was not performed in animals 24 h post occlusion.
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TABLE 2. Heat shock protein-70 (HSP-70) mRNA and c-fos mRNA during middle cerebral artery (MeA) occlusion Duration of occlusion Sham (3 h) 15 min
1 h
3 h
24 h
c-fos
HSP-70 MC
PM
ER
CP
MC
PM
ER
CP
0 1 0 0 0 2 2 0 1 2 2 1 2
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 1 0 0 0 0 0
2 2 0 1 0 1 2 2 0 0 0 0 0
1 1 1 1 2 2 1 2 2
I 1 2 1 1 2 1 2 2
0 1 2 1 1 2 1 2 1
0 0 0 0 0 0 0 0 0
the neocortex. Although blood flow was not mea sured in the present study, occlusion of the distal MCA alone in rats of the Long-Evans strain was reported to decrease cortical flow to 62% of pre ischemic levels (Chen et al., 1986). These results suggest that rather modest reductions of blood flow for 1-3 h may be sufficient to activate transcription of the HSP-70 gene. Combined MCA and bilateral carotid occlusion for 1 h without reperfusion permitted accumulation of HSP-70 mRNA in regions adjacent to, but not within, the central zone of the MCA territory of the neocortex. Presumably, blood flow in the ischemic core was too low to permit synthesis of the mRNA.
MC, MCA neocortex; PM, paramedian cortex; ER, entorhinal cortex; CP, choroid plexus; 0 = no increase; 1 = moderate increase; 2 = strong increase.
flow during vessel occlusion, although intrinsic re gional differences in expression cannot be ruled out. Further, within a severely ischemic zone, it is apparent that reperfusion is required before HSP-70 mRNA is strongly expressed. The mechanism by which ischemia triggers ex pression of the HSP-70 gene is not well understood. In many cells, hyperthermia activates a preexisting cytoplasmic protein that then migrates to the nu cleus and increases transcription of the HSP-70 gene (Wu et aI., 1987). Whether cerebral ischemia activates the same protein has not been determined. Indeed, the degree and duration of ischemia re quired to induce expression in brain remain poorly defined. In models of global ischemia, 2 min of isch emia was reported to trigger hippocampal expres sion of HSP-70 (Simon and Cho, 1990; Kirino et aI., 199 1; Nowak and Osborne, 199 1). Since ischemia for 2 min in normothermic gerbil brain is sufficient to deplete levels of ATP in most regions of hippo campus (Welsh et aI., 1990), it is apparent that even a brief period of energy failure is associated with increased expression of HSP-70. Induction of 70-kDa proteins during focal isch emia in rat brain was demonstrated in regions of neocortex in which blood flow was reduced to 40 ml/lOO g/min and below (Jacewicz et al., 1986). Since this level of flow is considered well above that required to cause energy failure (Astrup et al., 1981), increased expression of HSP-70 may be as sociated with moderate alterations of energy metab olites when the duration of ischemia is prolonged. In the present study, occlusion of the distal MCA alone triggered expression of HSP-70 mRNA by 1-3 h within the central zone of the MCA territory of J Cereb Blood Flow Metab, Vol. 12. No.2, 1992
FIG. 2. Heat shock protein-70 (HSP-70) mRNA in three levels of brain after 3-h permanent occlusion of the distal middle cerebral artery. Sections from the level of striatum, fornix, and hippocampus were hybridized with a 35S-labeled probe specific for HSP-70 mRNA and exposed to x-ray film. In creased levels of HSP-70 mRNA are indicated by increased optical density. Note strong expression in layer 4 of neocor tex.
HSP-70 mRNA EXPRESSION IN FOCAL ISCHEMIA
Anterior
209
Posterior
Sham (3 h)
1
h occlusion no reflow
1
h occlusion 2 h reflow
1
h occlusion 24 h reflow
1
h occlusion 1 wk reflow
FIG. 3. Heat shock protein-70 (HSP-70) mRNA at the level of striatum and hippocampus following combined occlusion of the middle cerebral artery and carotid arteries without reperfusion and following reperfusion for 2 h, 24 h, and 1 week. Brain sections were hybridized with a 35S-labeled probe specific for HSP-70 mRNA and exposed to x-ray film. Increased levels of HSP-70 mRNA are indicated by increased optical density. Note that persistent expression at 24 h in hippocampus occurred in only one of three animals.
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TABLE 3. Heat shock protein-70 mRNA during combined middle cerebral artery (MeA) and carotid occlusion and reperjusion Cortex Group Sham (3 h) I-h occlusion, no reperfusion
I-h occlusion, IS-min reperfusion I-h occlusion, 2-h reperfusion I-h occlusion, 24-h reperfusion I-h occlusion, I-wk reperfusion
Hippocampus
MC
PM
ER
STR
CAl
CA3
DEN
0 I 0 0 0 0 0 0 0 2 2 2 2 2 2 I 0
0 0 I 0 I 0 I 0 I I 2 2 1 I 0 0 0
0 0 I 0 I 0 I 0 I I 2 I 0 0 0 0 0
0 0 I 0 I 0 I 0 I 2 2 I 0 0 0 0 0
0 0 0 0 I 0 I 0 I 2 0 I 2 0 0 0 0
0 0 0 0 I 0 I 0 I 2 0 I 0 0 0 0 0
0 0 2 0 2 0 0 2 2 2 0 I 0 0 0 0 0
MC, MCA neocortex; PM, paramedian cortex; ER, entorhinal cortex; STR, striatum; DEN, dentate; 0 = no expression; 1 � moderate; 2 = strong.
Previous work with this model of combined occlu sion has demonstrated a profound increase in tissue NADH fluorescence and depletion of ATP in the central zone of the MCA territory of the neocortex by 1 h (Welsh et al., 199 1). In the absence of an adequate supply of ATP, synthesis of mRNA and other macromolecules will be negligible. By con trast, in regions adjacent to the ischemic core, en ergy metabolites may be sufficiently preserved to support synthesis of mRNA. Although metabolite levels were not measured in the present study, pre vious results revealed only minor changes in ATP and lactate in regions adjacent to the ischemic core (Welsh et al., 199 1). Thus, similar to the correlation with modest reductions of blood flow, expression of HSP-70 mRNA may be associated with minor per turbations in tissue metabolite levels. Recirculation for 2 h following a I-h period of combined occlusion produced intense, widespread expression of HSP-70 mRNA. Presumably, restora tion of blood flow to the ischemic core permitted sufficient recovery of energy metabolites to synthe size mRNA. Previous work has demonstrated that even after combined occlusion for 2.5 h, tissue lev els of ATP were restored to nearly 50% of control during reperfusion for 1 h (Welsh et al., 199 1). Al though metabolite levels have not been measured during reperfusion following occlusion for 1 h in the present model, substantial restoration of high energy phosphates has been demonstrated in other models of focal ischemia in rat brain, provided the duration of ischemia was �2 h (Selman et al., 1990). J Cereb Blood Flow Metab, Vol. 12, No.2, 1992
Whatever the degree of metabolic recovery in the present study, it was apparently adequate to sup port strong expression of HSP-70 mRNA in all pre viously ischemic regions. It should be noted, how ever, that reperfusion for 15 min was not long enough to permit accumulation of HSP-70 mRNA. Whether the difference in expression between 15 min and 2 h represents a delay in reperfusion, re generation of ATP, or synthesis of mRNA itself was not addressed in the present investigation. After reperfusion for 24 h, high levels of HSP-70 mRNA persisted in the central zone of the MCA territory of the neocortex, but not in most adjacent regions. With the present methods, it is difficult to distinguish whether HSP-70 mRNA was expressed within the ischemic core continuously or was ex pressed early and not degraded. It is conceivable that inhibition of ribonuclease within the ischemic core might prolong the half-life of HSP-70 mRNA. Whatever the explanation, levels of HSP-70 mRNA had returned nearly to background by 1 week, even in the central zone of the MCA neocortex. Loss of hybridization after the acute phase of recirculation may have occurred by two distinct processes. First, in regions adjacent to the ischemic core, expression of the HSP-70 gene may be terminated as cells re cover from the ischemic stress. Second, within the ischemic core, mRNA will eventually be degraded during the process of infarction. The relationship between the ischemic induction of stress proteins and cellular injury remains poorly defined. The present investigation indicates that in both models of focal ischemia employed, HSP-70 mRNA was expressed in a wider distribution than the expected extent of ischemic infarction. In pre vious studies of distal MCA occlusion, neocortical infarction was limited to a few cubic millimeters in several strains of rats (Coyle, 1982, 1986; Coyle et al., 1984; Bederson et al., 1986). In our own labo ratory, the volume of neocortical infarction in Wistar rats was 33 ± 26 mm3 (mean ± SD, n 5) following distal MCA occlusion alone (unpublished experiments). While the volume of infarction ap pears to be smaller than that expressing HSP-70 mRNA, it will be necessary to evaluate histologic change and hybridization in adjacent sections of the same brain to compare the topography of the two endpoints directly. Following combined occlusion of the MCA and carotid arteries, HSP-70 mRNA was expressed in paramedian cortex, striatum, and hippocampus, re gions not known to undergo infarction. Thus, in previous work with this model, infarction was de tected in the MCA territory of the neocortex, but not in subcortical structures (Chen et al., 1986). =
HSP-70 mRNA EXPRESSION IN FOCAL ISCHEMIA
Similarly, in preliminary studies from our own lab oratory, combined occlusion for 1 h produced in farction in the neocortex (93 ± 30 mm3 ; mean ± SD, n 4), but not in paramedian cortex, striatum, or hippocampus (Moyer and Welsh, 1990). Thus, the transient expression of HSP-70 mRNA in parame dian and entorhinal cortex, striatum, and hippocam pus in the present study is apparently not associated with tissue infarction. However, the occurrence of selective neuronal necrosis in these regions cannot be ruled out. In particular, the expression of HSP70 mRNA in the hippocampal CAl pyramidal cell layer at 24 h in one of the animals may be indicative of delayed neuronal necrosis. Finally, it should be cautioned that the present experiments were directed at levels of mRNA only. Thus, the efficiency of translation of mRNA into protein cannot be inferred from these results. In deed, it is possible that within the ischemic core there are regions able to synthesize mRNA, but un able to synthesize protein. Prolonged inhibition of protein synthesis has been well documented follow ing cerebral ischemia (Kleihues and Hossmann, 197 1; Dienel et aI., 1980). Further, an uncoupling between expression of HSP-70 mRNA and protein in gerbil hippocampus has recently been reported (Nowak, 199 1). Thus, elevation of HSP-70 mRNA implies only that transcription of the HSP-70 gene has been activated. However, work from other lab oratories has demonstrated expression of HSP-70 protein in models of focal ischemia. Thus, the in duction of immunoreactive HSP-70 in cortex and striatum following permanent occlusion of the prox imal MCA in rats indicates that synthesis of the protein occurs within the center of the ischemic core (Gonzalez et aI., 1989). Further, transient oc clusion of the proximal MCA has recently been shown to cause expression of HSP-70 protein within multiple foci (Sharp et aI., 199 1). Expression of c-fos mRNA following MCA oc clusion differed markedly from that of HSP-70 mRNA both in time course and in regional distribu tion. The proto-oncogene c-fos encodes a nuclear protein that is one component of a system that ac tivates transcription of genes having an AP-l bind ing site (Curran and Franza, 1988). While c-fos and HSP-70 have been reported to be induced in parallel under several conditions (Andrews et aI. , 1987; Gu bits and Fairhurst, 1988), the HSP-70 gene is not known to contain an AP-l binding site. Thus, it is unlikely that induction of c-fos has a direct role in activating the expression of the HSP-70 gene. In deed, the difference in regional expression of c-fos and HSP-70 mRNAs in the present study is consis tent with this conclusion. =
211
Increased expression of the c-fos gene has been associated with a variety of external stimuli, includ ing neuronal activation (Morgan et aI., 1986; Sagar et aI., 1988), trauma (Dragunow and Robertson, 1988), ischemia (Onodera et aI., 1989), and even sham procedures (Daval et aI., 1989). In the present study, increased expression of c-fos mRNA was de tected in the ipsilateral cortex of animals killed 3 h after sham manipulation of the MCA. Thus, even a minor degree of localized trauma appears to be suf ficient to trigger expression of c-fos over a large area of cortex. However, more intense expression of c-fos mRNA was produced following MCA oc clusion throughout the ipsilateral cerebral cortex. The widespread distribution of c-fos expression suggests that the underlying cause may be spread ing depression, which has previously been demon strated during focal ischemia in rat brain (Neder gaard and Astrup, 1986). Spreading depression is characterized by recurrent waves of neuronal depo larization, accompanied by a transient influx of cal cium (Kraig and Nicholson, 1978). Expression of c-fos is regulated, in part, by calcium influx through voltage-sensitive channels (Morgan and Curran, 1986). Thus, spreading depression-induced calcium influx could easily explain the widespread expres sion of c-fos, in both sham-operated and MCA occluded animals. Indeed, spreading depression has previously been associated with accumulation of c-fos protein in rat brain following removal of pial vessels (Herrera and Robertson, 1989) and follow ing direct application of KCI (Herrera and Robert son, 1990). In both cases, induction of c-fos protein was inhibited by N-methyl-D-aspartate antagonists, suggesting that N-methyl-D-aspartate-linked cal cium entry may mediate increased expression of c-fos. Finally, the presence of c-fos expression for at least 3 h post occlusion in the present study in dicates that the stimulus for expression may have continued for this duration, since expression of c-fos mRNA is short-lived in models of reversible ischemia (Onodera et aI. , 1989; Nowak et aI. , 1990a). Acknowledgment: This work was supported, in part, by the Research Foundation of the University of Pennsylva nia and by NIH grant NS-29331. The authors thank L. Davis, R. Krause, and D. Tyrrell at E.1. du Pont de Ne mours & Co. for their help with in situ hybridization.
REFERENCES Andrews OK, Harding MA, Calvet JP, Adamson ED (1987) The heat shock response in HeLa cells is accompanied by ele vated expression of the c-fos proto-oncogene. Mol Cell Bioi 7:3452-3458 Astrup J, Siesj6 BK, Symon L (1981) Thresholds in cerebral ischemia-the ischemic penumbra. Stroke 12:723-725
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Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H (1986) Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17:472-476 Brint S, Jacewicz M, Kiessling M, Tanabe J, Pulsinelli W (1988) Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries. J Cereb Blood Flow Metab 8:474-485 Brown IR, Rush SJ (1990) Expression of heat shock genes (hsp70) in the mammalian brain: distinguishing constitu tively expressed and hyperthermia-inducible mRNA spe cies. J Neurosci Res 25:14-19 Chen ST, Hsu CY, Hogan EL, Maricq H, Balentine JD (1986) A model of focal ischemic stroke in the rat: reproducible ex tensive cortical infarction. Stroke 17:738-743 Coyle P (1982) Middle cerebral artery occlusion in the young rat. Stroke 13:855-859 Coyle P (1986) Different susceptibilities to cerebral infarction in spontaneously hypertensive (SHR) and normotensive Sprague-Dawley rats. Stroke 17:520--525 Coyle P, Odenheimer DJ, Sing CF (1984) Cerebral infarction after middle cerebral artery occlusion in progenies of spon taneously stroke-prone and normal rats. Stroke 15:711-716 Curran T, Franza BR (1988) Fos and Jun: the AP-I connection. Cell 55:395-397 Curran T, Gordon MB, Rubino KL, Sambucetti LC (1987) Iso lation and characterization of the c-fos (rat) cDNA and anal ysis of posttranslational modification in vitro. Oncogene 2:79-84 Daval J-L, Nakajima T, Gleiter CH, Post RM, Marangos PJ (1989) Mouse brain c-fos mRNA distribution following a sin gle electroconvulsive shock. J Neurochem 52:1954-1957 Dienel GA, Pulsinelli WA, Duffy TE (1980) Regional protein synthesis in rat brain following acute hemispheric ischemia. J Neurochem 35:1216-1226 Dienel GA, Kiessling M, Jacewicz M, Pulsinelli WA (1986) Syn thesis of heat shock proteins in rat brain cortex after tran sient ischemia. J Cereb Blood Flow Metab 6:505-510 Dragunow M, Robertson HA (1988) Brain injury induces c-fos protein(s) in nerve and glial-like cells in the adult mammalian brain. Brain Res 455:295-299 Gonzalez MF, Shiraishi K, Hisanaga K, Sagar SM, Mandabach M, Sharp FR (1989) Heat shock proteins as markers of neu ral injury. Mol Brain Res 6:93-100 Gower DJ, Hollman C, Lee KS, Tytell M (1989) Spinal cord injury and the stress protein response. J Neurosurg 70:605611 Gubits RM, Fairhurst JL (1988) c-fos mRNA levels are increased by the cellular stressors, heat shock and sodium arsenite. Oncogene 3:163-168 Herrera DG, Robertson HA (1989) Unilateral induction of c-fos protein in cortex following cortical devascularization. Brain Res 503:205-213 Herrera DG, Robertson HA (1990) Application of potassium chloride to the brain surface induces the c-fos proto oncogene: reversal by MK-801. Brain Res 510:166-170 Hunt C, Morimoto RI (1985) Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide se quence of human hsp70. Proc Natl Acad Sci USA 82:64556459 Jacewicz M, Kiessling M, Pulsinelli WA (1986) Selective gene expression in focal cerebral ischemia. J Cereb Blood Flow Metab 6:263-272 Kirino T, Tsujita Y, Tamura A (1991) Induced tolerance to isch emia in gerbil hippocampal neurons. J Cereb Blood Flow Metab 11:299-307 Kleihues P, Hossmann K-A (1971) Protein synthesis in the cat brain after prolonged cerebral ischemia. Brain Res 35:409418 Kraig RP, Nicholson C (1978) Extracellular ionic variations dur ing spreading depression. Neuroscience 3:1045-1059
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Lindquist S, Craig EA (1988) The heat shock proteins. Annu Rev Genet 22:631-677 Miller EK, Trulson ME, Mues GI, Munn TZ, Goggans ML, Morrison MR, Raese JD (1987) The differential induction of two hsp70 mRNAs in several regions of rat brain by D-meth amphetamine. Soc Neurosci Abstr 13:1709 Miller EK, Raese JD, Morrison-Bogorad M (1991) Expression of heat shock protein 70 and heat shock cognate 70 messenger RNAs in rat cortex and cerebellum after heat shock or am phetamine treatment. J Neurochem 56:2060--2071 Morgan JI, Curran T (1986) Role of ion flux in the control of c-fos expression. Nature 322:552-555 Morgan JI, Cohen DR, Hempstead JL, Curran T (1987) Mapping patterns of c-fos expression in the central nervous system after seizure. Science 237:192-197 Moyer DJ, Welsh FA (1990) Delayed hypothermia reduces in farct volume following transient focal ischemia in rat brain. Soc Neurosci Abstr 16:938 Moyer DJ, Welsh FA, Harris VA (1991) Regional expression of HSP-70 mRNA: comparison between two models of focal ischemia in rat brain. Stroke 22:131 Nedergaard M, Astrup J (1986) Infarct rim: effect of hypergly cemia on direct current potential and [14C]2-deoxyglucose phosphorylation. J Cereb Blood Flow Metab 6:607-615 Nowak TS Jr (1985) Synthesis of a stress protein following tran sient ischemia in the gerbil. J Neurochem 45:1635-1641 Nowak TS Jr (1991) Localization of 70 kDa stress protein mRNA induction in gerbil brain after ischemia. J Cereb Blood Flow Metab 11:432-439 Nowak TS Jr, Osborne OC (1991) Threshold ischemic duration for stress protein induction in gerbil brain. Stroke 22:131 Nowak TS Jr, Ikeda J, Nakajima T (l990a) 70 kilodalton heat shock protein and c-fos gene expression following transient ischemia. Stroke 21 (suppl 3):107-111 Nowak TS Jr, Bond U, Schlesinger MJ (l990b) Heat shock RNA levels in brain and other tissues after hyperthermia and tran sient ischemia. J Neurochem 54:451-458 Onodera H, Kogure K, Ono Y, Igarashi K, Kiyota Y, Nagaoka A (1989) Proto-oncogene c-fos is transiently induced in the rat cerebral cortex after forebrain ischemia. Neurosci Lett 98:101-104 Sagar SM, Sharp FR, Curran T (1988) Expression of c-fos pro tein in brain: metabolic mapping at the cellular level. Science 240:1328-1331 Selman WR, Crumrine RC, Ricci AJ, LaManna JC, Ratcheson RA, Lust WD (1990) Impairment of metabolic recovery with increasing periods of middle cerebral artery occlusion in rats. Stroke 21:467-471 Sharp FR, Lowenstein D, Simon R, Hisanaga K (1991) Heat shock protein hsp72 induction in cortical and striatal astro cytes and neurons following infarction. J Cereb Blood Flow Metab 11:621-627 Simon RP, Cho H (1990) The temporal profile of stress protein production following global ischemia. Neurology 40 (suppl 1):383 Simon RP, Cho H, Gwinn R, Lowenstein DH (1991) The tem poral profile of 72-kDa heat-shock protein expression fol lowing global ischemia. J Neurosci 11:881-889 Vass K, Welch WJ, Nowak TS Jr (1988) Localization of 70 kDa stress protein induction in gerbil brain after ischemia. Acta Neuropathol (Berl) 77:128-135 Welsh FA, Moyer DJ (1990) Expression of heat shock protein-70 mRNA following reversible focal ischemia in rat brain. Soc Neurosci Abstr 16:938 Welsh FA, Sims RE, Harris VA (1990) Mild hypothermia pre vents ischemic injury in gerbil hippocampus. J Cereb Blood Flow Metab 10:557-563 Welsh FA, Marcy VR, Sims RE (1991) NADH fluorescence and regional energy metabolites during focal ischemia and reper fusion of rat brain. J Cereb Blood Flow Metab II :459-465 Wu C, Wilson S, Walker B, Dawid I, Paisley T, Zimarino V, Veda H (1987) Purification and properties of Drosophila heat shock activator protein. Science 238: 1247-1253
Regional Expression of Heat Shock Protein-70 mRNA and c-fos mRNA Following Focal Ischemia in Rat Brain
Frank A. Welsh, Donald J. Moyer, and Valerie A. Harris Division of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, U.S.A.
Summary: In situ hybridization was used to estimate re gional levels of heat shock protein-70 (HSP-70) mRNA and c-fos mRNA in two related models of focal cerebral ischemia. In the first model, permanent occlusion of the distal middle cerebral artery (MCA) alone caused a patchy increase in HSP-70 mRNA by 1 h in the central zone of the MCA territory of the ipsilateral neocortex. Tissue levels of HSP-70 mRNA continued to increase for several hours and remained elevated at 24 h. In contrast to the focal expression of HSP-70, c-fos mRNA was in creased throughout the ipsilateral cerebral cortex by 15 min and remained elevated for least 3 h. The wide distri bution of c-fos expression suggests it may have been caused by spreading depression. In the second model, severe focal ischemia was produced with a combination of transient (l-h) bilateral carotid artery occlusion and permanent MCA occlusion. Combined occlusion for 1 h
without reperfusion caused expression of HSP-70 mRNA only in regions adjacent to the central zone of the MCA territory of the neocortex. However, reperfusion of the carotids for 2 h generated intense expression of HSP-70 mRNA throughout most of the ipsilateral cerebral cortex, white matter, striatum, and hippocampus. The wide spread increase in HSP-70 mRNA suggests that reperfu sion triggered expression in all previously ischemic re gions. However, at 24 h of reperfusion, increased levels of HSP-70 mRNA were restricted primarily to the isch
Cerebral ischemia induces increased synthesis of a number of specific proteins, despite an overall reduction in the rate of protein synthesis (Kleihues and Hossmann, 1971; Dienel et aI., 1980, 1986; Nowak, 1985). Several of the induced proteins are members of a group of "heat shock" or "stress" proteins that are expressed in the central nervous system following a number of stresses, including heat shock (Brown and Rush, 1990), trauma (Gower et aI., 1989), as well as ischemia. Although the func tion of the stress proteins is not well understood, their induction has been correlated with increased resistance to injury in many cell types (for review, see Lindquist and Craig, 1988). In brain, induction of stress proteins prior to an episode of cerebral
ischemia has been associated with decreased neu ronal necrosis in gerbil hippocampus (Kirino et aI., 1991). Thus, stress proteins may play an important role defending cells against ischemic injury. The 70-kDa heat shock protein (HSP-70) is one of the major stress proteins induced by cerebral isch emia (Nowak, 1985; Dienel et aI., 1986). Although the time course and regional induction of HSP-70 have been well characterized following transient global ischemia (Vass et aI., 1988; Kirino et aI., 1991; Simon et aI., 1991), expression of HSP-70 dur ing focal ischemia has not been extensively studied. Jacewicz et al. (1986) reported increased synthesis of a 70-kDa protein, presumed to be HSP-70, during focal ischemia in rat brain. Recently, HSP-70 was detected with immunocytochemical methods fol lowing occlusion of the middle cerebral artery (MCA) in rat brain (Gonzalez et aI., 1989; Sharp et aI., 1991). However, expression of HSP-70 in mod els of focal ischemia has not been further charac terized. Moreover, the effects of focal ischemia on transcription of the HSP-70 gene have not been pre-
emic core of the neocortex. These results suggest that expression of HSP-70 mRNA is prolonged in regions un dergoing injury, but is transient in surrounding regions that recover. Key Words: Heat shock protein-70--c-fos Focal ischemia-In situ hybridization-Messenger ribo nucleic acid-Middle cerebral artery.
Received April 5, 1991; revised July 19, 1991; accepted July 23, 1991. Address correspondence and reprint requests to Dr. F. A. Welsh at 313 Stemmler Hall, 36th and Hamilton Walk, Philadel phia, PA 19104-6070, U.S. A. Abbreviations used: HSP-70, heat shock protein-70; MCA, middle cerebral artery; SSC, sodium citrate saline.
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viously reported. Therefore, the objective of the present study was to determine the time course and regional expression of HSP-70 mRNA as a first step toward investigating the induction of stress proteins during focal ischemia and reperfusion. Regional levels of HSP-70 mRNA were estimated using in situ hybridization with a probe specific for the inducible form of HSP-70 mRNA (Miller et aI., 1987). To study the effects of increasing degrees of ischemia, two related models of focal ischemia were employed. First, mild focal ischemia was produced by permanent distal occlusion of the MeA. Second, severe focal ischemia was produced by combining MeA occlusion with transient bilateral carotid oc clusion for 1 h. Finally, since increased expression of the proto-oncogene c-fos has been reported fol lowing cerebral ischemia (Onodera et aI., 1989), re gional levels of c-fos mRNA were compared with those of HSP-70 mRNA following occlusion of the MeA alone. The results of this study have been presented previously in preliminary form (Welsh and Moyer, 1990; Moyer et aI., 1991).
METHODS Animal preparation Male Wistar rats, 35{}-550 g body wt, were given free access to food and water prior to surgery. The animals were anesthetized with pentobarbital (50 mg/kg i.p.),and core temperature was regulated at 37.5°C using a rectal thermistor connected to a heating lamp. The tail artery was cannulated to record arterial pressure and to obtain blood samples for measurement of P02, Pe02, pH, and hematocrit. Oxygen was delivered to the animals through a loose-fitting nose cone. In animals undergoing carotid occlusion,an incision was made in the neck,and the com mon carotid arteries were isolated and encircled loosely with surgical thread. The MCA on the right side was permanently occluded using the surgical approach described by Brint et al. (1988). After exposure of the MCA,the tip of a stainless steel wire,bent at 90° to form a hook,was inserted under the MCA just superior to the inferior cortical vein,using a micromanipulator. The MCA was lifted above the cor tical surface and permanently occluded by thermocoagu lation. A 24-gauge needle thermistor (YSI 524; Yellow Springs Instrument Co., Yellow Springs, OH, U.S.A.) was inserted through the craniotomy so that the tip rested in the subdural space over the MCA cortex. Subdural temperature was maintained at 3 7.SOC for 2 h using an overhead lamp. In animals undergoing transient carotid occlusion, miniature Mayfield clips were applied to both carotid arteries within 2 min of MCA occlusion. After occlusion for 1 h,the clips were removed to permit recir culation. Rectal temperature was maintained at 37.5°C until the animals awakened. In the first series (MCA only),animals were euthanized with a lethal injection of pentobarbital 3 h after sham operation (n 2) or after MCA occlusion for a period of 3),1 h (n 3), 3 h (n 3),or 24 h (n 2). 15 min (n
205
Brains in the first series were analyzed for HSP-70 mRNA and c-fos mRNA using in situ hybridization. In the second series (combined MCA and carotid occlusion), animals were euthanized 3 h after sham operation (n 2), I h after combined occlusion without reperfusion (n 4), or after reperfusion for 15 min (n 3 ), 2 h (n 3), 24 h (n 3), or I week (n 2). Brains in the second series were analyzed for HSP-70 mRNA only. =
=
=
=
=
=
In situ hybridization Following euthanasia,the brain was removed from the cranium and frozen in a bed of dry ice. Brain sections were cut at a thickness of 15 fLm in a cryostat at - 11°C, dried on glass slides,fixed in 4% paraformaldehyde,and rinsed with 20x SSC buffer (0.3 M Na citrate,3 M NaC\). HSP-70 mRNA was detected using a 30-mer oligonucle otide probe with the sequence 3'-GG TAC CAC GAC TGG TTC TAC TTC CTC TAG C-5' (Nowak et aI., 1990b). This sequence, which is reported to be specific for the inducible form of HSP-70 mRNA (Miller et aI., 1987,1991),corresponds to a highly conserved region of the HSP-70 coding sequence,amino acids 122-129 of hu man HSP-70 (Hunt and Morimoto,1985). As a control for the HSP-70 probe,a 30-mer oligonucleotide with the com plementary sequence was also tested. c-fos mRNA was detected using a 30-mer oligonucleotide with sequence 3'-TGT GTC CTG AAA ACG CGT CTA GAC AGG CAG-5'. This sequence is complementary to base num bers 270-299 of rat c-fos mRNA (Curran et aI.,1987). The oligonucleotide probes were labeled on the 3'-end with eSS]dATP, using terminal transferase. The labeled probes were isolated and hybridized to tissue sections overnight at 3rC in 4x SSC. After rinsing for 2 h each in 1 x SSC and 0.5 x SSC at 37°C, the sections were dried and placed in contact with Kodak XAR film for 1-2 weeks. Expression of HSP-70 mRNA and c-fos mRNA was graded qualitatively by comparing optical density in several regions of the ipsilateral hemisphere to that in the homologous region of the contralateral hemisphere. The grading scale was as follows: 0 no difference, 1 moderate increase, 2 strong increase. =
=
=
RESULTS Arterial variables
Arterial pressure did not vary significantly during ischemia or reperfusion, relative to preocclusion levels (Table 1). Arterial pH and Pe02 remained within the normal range, while P02 was higher than TABLE 1. Arterial variables Stage of
MABP
Peo,
Po,
experiment
(mmHg)
(mm Hg)
(mm Hg)
pH
(%)
Preocclusion
113 ±16 (19)
38 ±6 (12)
230 ±63 (12)
7.33 ±0.06 (11)
41 ±4 (3)
112 ±21 (22)
35 ±8 (19)
216 ±71 (19)
7.36 ±0.1O (18)
38 ±4 (11)
119 ±13 (7)
34 ±4 (7)
225 ±72 (7)
7.38 ±0.04 (7)
35 ±3 (6)
Occlusion
(15
min)
Reperfusion
(30
min)
Hematocrit
=
=
=
=
=
Values are means
±
SD, (no. of animals).
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normal owing to administration of 02' The hemat ocrit decreased moderately during reperfusion. MCA In sham-operated animals, levels of HSP-70 mRNA were low in cerebral cortex, white matter, and striatum (Fig. 1). By contrast, c-fos mRNA was moderately increased throughout the ipsilateral ce rebral cortex in sham-operated animals (Fig. 1; Ta ble 2). Occlusion of the distal MCA caused increased expression of HSP-70 mRNA in the central zone of the MCA neocortex by I h but not 15 min post occlusion (Fig. 1; Table 2). Expression continued to increase for 3 h, although the distribution of HSP-70 mRNA was somewhat patchy and was accentuated in the outer layers of cortex. By 3 h post occlusion, increased levels of HSP-70 mRNA extended ante rior to the level of the caudate and posterior to the level of the hippocampus, with strongest expression occurring in layer 4 of the neocortex (Fig. 2). At 24 h, levels of HSP-70 mRNA remained elevated in the MCA territory of the neocortex, sometimes distrib uted in a columnar orientation (Fig. 1). HSP-70 mRNA was not detectably increased in subcortical structures of the ipsilateral hemisphere or in the contralateral hemisphere. Further, the antisense control probe for HSP-70 did not exhibit increased hybridization in regions with elevated HSP-70 mRNA. In contrast to the focal induction of HSP-70 mRNA, a widespread increase of c-fos mRNA was apparent throughout the ipsilateral cerebral cortex at all times studied (Fig. 1; Table 2). Expression of c-fos mRNA was increased by 15 min post occlu sion and continued to increase at 1 and 3 h. Within the cerebral cortex, highest levels of c-fos mRNA occurred in the outer layers of parietal cortex, in layer 2 of paramedian (cingulate) cortex, and in the pyramidal cell layer of entorhinal cortex (Fig. 1). Permanent occlusion of distal
MCA and carotid arteries In sham-operated animals, similar to those in the first series, expression of HSP-70 mRNA was low throughout the ipsilateral hemisphere (Fig. 3; Table 3). Combined occlusion of the MCA and carotid arteries for 1 h without reperfusion produced a moderate increase in HSP-70 mRNA in the parame dian cortex, entorhinal cortex, striatum, and hippo campus in two of four animals. Within the hippo campus, expression was strongest in the upper blade of the dentate gyrus. There was no detectable increase in HSP-70 mRNA in the central zone of the MCA territory of the neocortex. Reperfusion of the carotids for 15 min following 1 h of combined occlusion caused a regional expresCombined occlusion of
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sion of HSP-70 mRNA similar to that observed without reperfusion (Table 3). Thus, there was no marked change in HSP-70 mRNA levels during the first 15 min of reperfusion. However, by 2 h reper fusion, a profound increase in HSP-70 mRNA oc curred throughout the ipsilateral cerebral cortex, white matter, striatum, and hippocampus (Fig. 3; Table 3). Within the cortex, expression was most intense in the ischemic core. However, HSP-70 mRNA was also elevated in paramedian and ento rhinal cortex. Subcortical white matter tracts also exhibited increased expression in the ipsilateral hemisphere, especially at the level of the striatum. Within the striatum, increased expression of HSP70 mRNA occurred rather evenly, with slightly higher levels along the medial edge. In hippocam pus, increased expression was evident in the pyra midal cell layer of all subfields (most intense in CA4) and in the dentate granule cell layer, particularly in the upper blade. However, in one of three animals at this time, hippocampal expression of HSP-70 mRNA was not elevated (Table 3). Increased levels of HSP-70 mRNA were not detected in thalamus. Reperfusion for 24 h was associated with a reduc tion of HSP-70 mRNA levels in most regions of the ipsilateral hemisphere (Fig. 3; Table 3). While strong expression persisted in the ischemic core of the neocortex, especially layer 4, levels of HSP-70 mRNA had returned nearly to background in para median and entorhinal cortex, striatum, and hippo campus. Interestingly, in one of three animals at 24 h, increased levels of HSP-70 mRNA were present in subiculum and CAl pyramidal cell layer of the hippocampus (Fig. 3). Reperfusion for 1 week showed only a faint increase in HSP-70 mRNA along the margins of a presumed cortical infarct. DISCUSSION The present results describe the time course and regional expression of HSP-70 mRNA during focal ischemia and reperfusion of rat brain. Three distinct regional patterns of expression were detected. First, permanent occlusion of distal MCA alone caused an accumulation of HSP-70 mRNA that was restricted to the central zone of the MCA territory of the neocortex. Second, combined occlusion of the MCA and carotid arteries without reperfusion caused increased expression only in regions adja cent to the MCA territory of the neocortex. Third, reperfusion of the carotids following combined oc clusion of the MCA and carotid arteries produced a robust expression of HSP-70 mRNA that encom passed both the ischemic core and adjacent regions. These regional patterns of expression were deter mined, presumably, by the level of regional blood
HSP-70 mRNA EXPRESSION IN FOCAL ISCHEMIA
HSP-70
207
c-fos
Sham
(3 h)
Occlusior 15 min
1
hour
3 hours
24
hours
FIG. 1. Heat shock protein-70 (HSP-70) mRNA and c-fos mRNA levels during permanent occlu sion of the distal middle cerebral artery (MCA). Following sham operation or MCA occlusion for 15 min or 1,3, or 24 h, the brain was sectioned, and adjacent brain sections were hybridized with 35S-labeled probes specific for HSP-70 mRNA or c-fos mRNA and exposed to x-ray film. Increased levels of mRNA are indicated by in creased optical density. Hybridization for c-fos mRNA was not performed in animals 24 h post occlusion.
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TABLE 2. Heat shock protein-70 (HSP-70) mRNA and c-fos mRNA during middle cerebral artery (MeA) occlusion Duration of occlusion Sham (3 h) 15 min
1 h
3 h
24 h
c-fos
HSP-70 MC
PM
ER
CP
MC
PM
ER
CP
0 1 0 0 0 2 2 0 1 2 2 1 2
0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 1 0 0 0 0 0
2 2 0 1 0 1 2 2 0 0 0 0 0
1 1 1 1 2 2 1 2 2
I 1 2 1 1 2 1 2 2
0 1 2 1 1 2 1 2 1
0 0 0 0 0 0 0 0 0
the neocortex. Although blood flow was not mea sured in the present study, occlusion of the distal MCA alone in rats of the Long-Evans strain was reported to decrease cortical flow to 62% of pre ischemic levels (Chen et al., 1986). These results suggest that rather modest reductions of blood flow for 1-3 h may be sufficient to activate transcription of the HSP-70 gene. Combined MCA and bilateral carotid occlusion for 1 h without reperfusion permitted accumulation of HSP-70 mRNA in regions adjacent to, but not within, the central zone of the MCA territory of the neocortex. Presumably, blood flow in the ischemic core was too low to permit synthesis of the mRNA.
MC, MCA neocortex; PM, paramedian cortex; ER, entorhinal cortex; CP, choroid plexus; 0 = no increase; 1 = moderate increase; 2 = strong increase.
flow during vessel occlusion, although intrinsic re gional differences in expression cannot be ruled out. Further, within a severely ischemic zone, it is apparent that reperfusion is required before HSP-70 mRNA is strongly expressed. The mechanism by which ischemia triggers ex pression of the HSP-70 gene is not well understood. In many cells, hyperthermia activates a preexisting cytoplasmic protein that then migrates to the nu cleus and increases transcription of the HSP-70 gene (Wu et aI., 1987). Whether cerebral ischemia activates the same protein has not been determined. Indeed, the degree and duration of ischemia re quired to induce expression in brain remain poorly defined. In models of global ischemia, 2 min of isch emia was reported to trigger hippocampal expres sion of HSP-70 (Simon and Cho, 1990; Kirino et aI., 199 1; Nowak and Osborne, 199 1). Since ischemia for 2 min in normothermic gerbil brain is sufficient to deplete levels of ATP in most regions of hippo campus (Welsh et aI., 1990), it is apparent that even a brief period of energy failure is associated with increased expression of HSP-70. Induction of 70-kDa proteins during focal isch emia in rat brain was demonstrated in regions of neocortex in which blood flow was reduced to 40 ml/lOO g/min and below (Jacewicz et al., 1986). Since this level of flow is considered well above that required to cause energy failure (Astrup et al., 1981), increased expression of HSP-70 may be as sociated with moderate alterations of energy metab olites when the duration of ischemia is prolonged. In the present study, occlusion of the distal MCA alone triggered expression of HSP-70 mRNA by 1-3 h within the central zone of the MCA territory of J Cereb Blood Flow Metab, Vol. 12. No.2, 1992
FIG. 2. Heat shock protein-70 (HSP-70) mRNA in three levels of brain after 3-h permanent occlusion of the distal middle cerebral artery. Sections from the level of striatum, fornix, and hippocampus were hybridized with a 35S-labeled probe specific for HSP-70 mRNA and exposed to x-ray film. In creased levels of HSP-70 mRNA are indicated by increased optical density. Note strong expression in layer 4 of neocor tex.
HSP-70 mRNA EXPRESSION IN FOCAL ISCHEMIA
Anterior
209
Posterior
Sham (3 h)
1
h occlusion no reflow
1
h occlusion 2 h reflow
1
h occlusion 24 h reflow
1
h occlusion 1 wk reflow
FIG. 3. Heat shock protein-70 (HSP-70) mRNA at the level of striatum and hippocampus following combined occlusion of the middle cerebral artery and carotid arteries without reperfusion and following reperfusion for 2 h, 24 h, and 1 week. Brain sections were hybridized with a 35S-labeled probe specific for HSP-70 mRNA and exposed to x-ray film. Increased levels of HSP-70 mRNA are indicated by increased optical density. Note that persistent expression at 24 h in hippocampus occurred in only one of three animals.
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TABLE 3. Heat shock protein-70 mRNA during combined middle cerebral artery (MeA) and carotid occlusion and reperjusion Cortex Group Sham (3 h) I-h occlusion, no reperfusion
I-h occlusion, IS-min reperfusion I-h occlusion, 2-h reperfusion I-h occlusion, 24-h reperfusion I-h occlusion, I-wk reperfusion
Hippocampus
MC
PM
ER
STR
CAl
CA3
DEN
0 I 0 0 0 0 0 0 0 2 2 2 2 2 2 I 0
0 0 I 0 I 0 I 0 I I 2 2 1 I 0 0 0
0 0 I 0 I 0 I 0 I I 2 I 0 0 0 0 0
0 0 I 0 I 0 I 0 I 2 2 I 0 0 0 0 0
0 0 0 0 I 0 I 0 I 2 0 I 2 0 0 0 0
0 0 0 0 I 0 I 0 I 2 0 I 0 0 0 0 0
0 0 2 0 2 0 0 2 2 2 0 I 0 0 0 0 0
MC, MCA neocortex; PM, paramedian cortex; ER, entorhinal cortex; STR, striatum; DEN, dentate; 0 = no expression; 1 � moderate; 2 = strong.
Previous work with this model of combined occlu sion has demonstrated a profound increase in tissue NADH fluorescence and depletion of ATP in the central zone of the MCA territory of the neocortex by 1 h (Welsh et al., 199 1). In the absence of an adequate supply of ATP, synthesis of mRNA and other macromolecules will be negligible. By con trast, in regions adjacent to the ischemic core, en ergy metabolites may be sufficiently preserved to support synthesis of mRNA. Although metabolite levels were not measured in the present study, pre vious results revealed only minor changes in ATP and lactate in regions adjacent to the ischemic core (Welsh et al., 199 1). Thus, similar to the correlation with modest reductions of blood flow, expression of HSP-70 mRNA may be associated with minor per turbations in tissue metabolite levels. Recirculation for 2 h following a I-h period of combined occlusion produced intense, widespread expression of HSP-70 mRNA. Presumably, restora tion of blood flow to the ischemic core permitted sufficient recovery of energy metabolites to synthe size mRNA. Previous work has demonstrated that even after combined occlusion for 2.5 h, tissue lev els of ATP were restored to nearly 50% of control during reperfusion for 1 h (Welsh et al., 199 1). Al though metabolite levels have not been measured during reperfusion following occlusion for 1 h in the present model, substantial restoration of high energy phosphates has been demonstrated in other models of focal ischemia in rat brain, provided the duration of ischemia was �2 h (Selman et al., 1990). J Cereb Blood Flow Metab, Vol. 12, No.2, 1992
Whatever the degree of metabolic recovery in the present study, it was apparently adequate to sup port strong expression of HSP-70 mRNA in all pre viously ischemic regions. It should be noted, how ever, that reperfusion for 15 min was not long enough to permit accumulation of HSP-70 mRNA. Whether the difference in expression between 15 min and 2 h represents a delay in reperfusion, re generation of ATP, or synthesis of mRNA itself was not addressed in the present investigation. After reperfusion for 24 h, high levels of HSP-70 mRNA persisted in the central zone of the MCA territory of the neocortex, but not in most adjacent regions. With the present methods, it is difficult to distinguish whether HSP-70 mRNA was expressed within the ischemic core continuously or was ex pressed early and not degraded. It is conceivable that inhibition of ribonuclease within the ischemic core might prolong the half-life of HSP-70 mRNA. Whatever the explanation, levels of HSP-70 mRNA had returned nearly to background by 1 week, even in the central zone of the MCA neocortex. Loss of hybridization after the acute phase of recirculation may have occurred by two distinct processes. First, in regions adjacent to the ischemic core, expression of the HSP-70 gene may be terminated as cells re cover from the ischemic stress. Second, within the ischemic core, mRNA will eventually be degraded during the process of infarction. The relationship between the ischemic induction of stress proteins and cellular injury remains poorly defined. The present investigation indicates that in both models of focal ischemia employed, HSP-70 mRNA was expressed in a wider distribution than the expected extent of ischemic infarction. In pre vious studies of distal MCA occlusion, neocortical infarction was limited to a few cubic millimeters in several strains of rats (Coyle, 1982, 1986; Coyle et al., 1984; Bederson et al., 1986). In our own labo ratory, the volume of neocortical infarction in Wistar rats was 33 ± 26 mm3 (mean ± SD, n 5) following distal MCA occlusion alone (unpublished experiments). While the volume of infarction ap pears to be smaller than that expressing HSP-70 mRNA, it will be necessary to evaluate histologic change and hybridization in adjacent sections of the same brain to compare the topography of the two endpoints directly. Following combined occlusion of the MCA and carotid arteries, HSP-70 mRNA was expressed in paramedian cortex, striatum, and hippocampus, re gions not known to undergo infarction. Thus, in previous work with this model, infarction was de tected in the MCA territory of the neocortex, but not in subcortical structures (Chen et al., 1986). =
HSP-70 mRNA EXPRESSION IN FOCAL ISCHEMIA
Similarly, in preliminary studies from our own lab oratory, combined occlusion for 1 h produced in farction in the neocortex (93 ± 30 mm3 ; mean ± SD, n 4), but not in paramedian cortex, striatum, or hippocampus (Moyer and Welsh, 1990). Thus, the transient expression of HSP-70 mRNA in parame dian and entorhinal cortex, striatum, and hippocam pus in the present study is apparently not associated with tissue infarction. However, the occurrence of selective neuronal necrosis in these regions cannot be ruled out. In particular, the expression of HSP70 mRNA in the hippocampal CAl pyramidal cell layer at 24 h in one of the animals may be indicative of delayed neuronal necrosis. Finally, it should be cautioned that the present experiments were directed at levels of mRNA only. Thus, the efficiency of translation of mRNA into protein cannot be inferred from these results. In deed, it is possible that within the ischemic core there are regions able to synthesize mRNA, but un able to synthesize protein. Prolonged inhibition of protein synthesis has been well documented follow ing cerebral ischemia (Kleihues and Hossmann, 197 1; Dienel et aI., 1980). Further, an uncoupling between expression of HSP-70 mRNA and protein in gerbil hippocampus has recently been reported (Nowak, 199 1). Thus, elevation of HSP-70 mRNA implies only that transcription of the HSP-70 gene has been activated. However, work from other lab oratories has demonstrated expression of HSP-70 protein in models of focal ischemia. Thus, the in duction of immunoreactive HSP-70 in cortex and striatum following permanent occlusion of the prox imal MCA in rats indicates that synthesis of the protein occurs within the center of the ischemic core (Gonzalez et aI., 1989). Further, transient oc clusion of the proximal MCA has recently been shown to cause expression of HSP-70 protein within multiple foci (Sharp et aI., 199 1). Expression of c-fos mRNA following MCA oc clusion differed markedly from that of HSP-70 mRNA both in time course and in regional distribu tion. The proto-oncogene c-fos encodes a nuclear protein that is one component of a system that ac tivates transcription of genes having an AP-l bind ing site (Curran and Franza, 1988). While c-fos and HSP-70 have been reported to be induced in parallel under several conditions (Andrews et aI. , 1987; Gu bits and Fairhurst, 1988), the HSP-70 gene is not known to contain an AP-l binding site. Thus, it is unlikely that induction of c-fos has a direct role in activating the expression of the HSP-70 gene. In deed, the difference in regional expression of c-fos and HSP-70 mRNAs in the present study is consis tent with this conclusion. =
211
Increased expression of the c-fos gene has been associated with a variety of external stimuli, includ ing neuronal activation (Morgan et aI., 1986; Sagar et aI., 1988), trauma (Dragunow and Robertson, 1988), ischemia (Onodera et aI., 1989), and even sham procedures (Daval et aI., 1989). In the present study, increased expression of c-fos mRNA was de tected in the ipsilateral cortex of animals killed 3 h after sham manipulation of the MCA. Thus, even a minor degree of localized trauma appears to be suf ficient to trigger expression of c-fos over a large area of cortex. However, more intense expression of c-fos mRNA was produced following MCA oc clusion throughout the ipsilateral cerebral cortex. The widespread distribution of c-fos expression suggests that the underlying cause may be spread ing depression, which has previously been demon strated during focal ischemia in rat brain (Neder gaard and Astrup, 1986). Spreading depression is characterized by recurrent waves of neuronal depo larization, accompanied by a transient influx of cal cium (Kraig and Nicholson, 1978). Expression of c-fos is regulated, in part, by calcium influx through voltage-sensitive channels (Morgan and Curran, 1986). Thus, spreading depression-induced calcium influx could easily explain the widespread expres sion of c-fos, in both sham-operated and MCA occluded animals. Indeed, spreading depression has previously been associated with accumulation of c-fos protein in rat brain following removal of pial vessels (Herrera and Robertson, 1989) and follow ing direct application of KCI (Herrera and Robert son, 1990). In both cases, induction of c-fos protein was inhibited by N-methyl-D-aspartate antagonists, suggesting that N-methyl-D-aspartate-linked cal cium entry may mediate increased expression of c-fos. Finally, the presence of c-fos expression for at least 3 h post occlusion in the present study in dicates that the stimulus for expression may have continued for this duration, since expression of c-fos mRNA is short-lived in models of reversible ischemia (Onodera et aI. , 1989; Nowak et aI. , 1990a). Acknowledgment: This work was supported, in part, by the Research Foundation of the University of Pennsylva nia and by NIH grant NS-29331. The authors thank L. Davis, R. Krause, and D. Tyrrell at E.1. du Pont de Ne mours & Co. for their help with in situ hybridization.
REFERENCES Andrews OK, Harding MA, Calvet JP, Adamson ED (1987) The heat shock response in HeLa cells is accompanied by ele vated expression of the c-fos proto-oncogene. Mol Cell Bioi 7:3452-3458 Astrup J, Siesj6 BK, Symon L (1981) Thresholds in cerebral ischemia-the ischemic penumbra. Stroke 12:723-725
J
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F. A. WELSH ET AL .
Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL, Bartkowski H (1986) Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17:472-476 Brint S, Jacewicz M, Kiessling M, Tanabe J, Pulsinelli W (1988) Focal brain ischemia in the rat: methods for reproducible neocortical infarction using tandem occlusion of the distal middle cerebral and ipsilateral common carotid arteries. J Cereb Blood Flow Metab 8:474-485 Brown IR, Rush SJ (1990) Expression of heat shock genes (hsp70) in the mammalian brain: distinguishing constitu tively expressed and hyperthermia-inducible mRNA spe cies. J Neurosci Res 25:14-19 Chen ST, Hsu CY, Hogan EL, Maricq H, Balentine JD (1986) A model of focal ischemic stroke in the rat: reproducible ex tensive cortical infarction. Stroke 17:738-743 Coyle P (1982) Middle cerebral artery occlusion in the young rat. Stroke 13:855-859 Coyle P (1986) Different susceptibilities to cerebral infarction in spontaneously hypertensive (SHR) and normotensive Sprague-Dawley rats. Stroke 17:520--525 Coyle P, Odenheimer DJ, Sing CF (1984) Cerebral infarction after middle cerebral artery occlusion in progenies of spon taneously stroke-prone and normal rats. Stroke 15:711-716 Curran T, Franza BR (1988) Fos and Jun: the AP-I connection. Cell 55:395-397 Curran T, Gordon MB, Rubino KL, Sambucetti LC (1987) Iso lation and characterization of the c-fos (rat) cDNA and anal ysis of posttranslational modification in vitro. Oncogene 2:79-84 Daval J-L, Nakajima T, Gleiter CH, Post RM, Marangos PJ (1989) Mouse brain c-fos mRNA distribution following a sin gle electroconvulsive shock. J Neurochem 52:1954-1957 Dienel GA, Pulsinelli WA, Duffy TE (1980) Regional protein synthesis in rat brain following acute hemispheric ischemia. J Neurochem 35:1216-1226 Dienel GA, Kiessling M, Jacewicz M, Pulsinelli WA (1986) Syn thesis of heat shock proteins in rat brain cortex after tran sient ischemia. J Cereb Blood Flow Metab 6:505-510 Dragunow M, Robertson HA (1988) Brain injury induces c-fos protein(s) in nerve and glial-like cells in the adult mammalian brain. Brain Res 455:295-299 Gonzalez MF, Shiraishi K, Hisanaga K, Sagar SM, Mandabach M, Sharp FR (1989) Heat shock proteins as markers of neu ral injury. Mol Brain Res 6:93-100 Gower DJ, Hollman C, Lee KS, Tytell M (1989) Spinal cord injury and the stress protein response. J Neurosurg 70:605611 Gubits RM, Fairhurst JL (1988) c-fos mRNA levels are increased by the cellular stressors, heat shock and sodium arsenite. Oncogene 3:163-168 Herrera DG, Robertson HA (1989) Unilateral induction of c-fos protein in cortex following cortical devascularization. Brain Res 503:205-213 Herrera DG, Robertson HA (1990) Application of potassium chloride to the brain surface induces the c-fos proto oncogene: reversal by MK-801. Brain Res 510:166-170 Hunt C, Morimoto RI (1985) Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide se quence of human hsp70. Proc Natl Acad Sci USA 82:64556459 Jacewicz M, Kiessling M, Pulsinelli WA (1986) Selective gene expression in focal cerebral ischemia. J Cereb Blood Flow Metab 6:263-272 Kirino T, Tsujita Y, Tamura A (1991) Induced tolerance to isch emia in gerbil hippocampal neurons. J Cereb Blood Flow Metab 11:299-307 Kleihues P, Hossmann K-A (1971) Protein synthesis in the cat brain after prolonged cerebral ischemia. Brain Res 35:409418 Kraig RP, Nicholson C (1978) Extracellular ionic variations dur ing spreading depression. Neuroscience 3:1045-1059
J Cereb Blood Flow Metab, Vol. 12, No.2, 1992
Lindquist S, Craig EA (1988) The heat shock proteins. Annu Rev Genet 22:631-677 Miller EK, Trulson ME, Mues GI, Munn TZ, Goggans ML, Morrison MR, Raese JD (1987) The differential induction of two hsp70 mRNAs in several regions of rat brain by D-meth amphetamine. Soc Neurosci Abstr 13:1709 Miller EK, Raese JD, Morrison-Bogorad M (1991) Expression of heat shock protein 70 and heat shock cognate 70 messenger RNAs in rat cortex and cerebellum after heat shock or am phetamine treatment. J Neurochem 56:2060--2071 Morgan JI, Curran T (1986) Role of ion flux in the control of c-fos expression. Nature 322:552-555 Morgan JI, Cohen DR, Hempstead JL, Curran T (1987) Mapping patterns of c-fos expression in the central nervous system after seizure. Science 237:192-197 Moyer DJ, Welsh FA (1990) Delayed hypothermia reduces in farct volume following transient focal ischemia in rat brain. Soc Neurosci Abstr 16:938 Moyer DJ, Welsh FA, Harris VA (1991) Regional expression of HSP-70 mRNA: comparison between two models of focal ischemia in rat brain. Stroke 22:131 Nedergaard M, Astrup J (1986) Infarct rim: effect of hypergly cemia on direct current potential and [14C]2-deoxyglucose phosphorylation. J Cereb Blood Flow Metab 6:607-615 Nowak TS Jr (1985) Synthesis of a stress protein following tran sient ischemia in the gerbil. J Neurochem 45:1635-1641 Nowak TS Jr (1991) Localization of 70 kDa stress protein mRNA induction in gerbil brain after ischemia. J Cereb Blood Flow Metab 11:432-439 Nowak TS Jr, Osborne OC (1991) Threshold ischemic duration for stress protein induction in gerbil brain. Stroke 22:131 Nowak TS Jr, Ikeda J, Nakajima T (l990a) 70 kilodalton heat shock protein and c-fos gene expression following transient ischemia. Stroke 21 (suppl 3):107-111 Nowak TS Jr, Bond U, Schlesinger MJ (l990b) Heat shock RNA levels in brain and other tissues after hyperthermia and tran sient ischemia. J Neurochem 54:451-458 Onodera H, Kogure K, Ono Y, Igarashi K, Kiyota Y, Nagaoka A (1989) Proto-oncogene c-fos is transiently induced in the rat cerebral cortex after forebrain ischemia. Neurosci Lett 98:101-104 Sagar SM, Sharp FR, Curran T (1988) Expression of c-fos pro tein in brain: metabolic mapping at the cellular level. Science 240:1328-1331 Selman WR, Crumrine RC, Ricci AJ, LaManna JC, Ratcheson RA, Lust WD (1990) Impairment of metabolic recovery with increasing periods of middle cerebral artery occlusion in rats. Stroke 21:467-471 Sharp FR, Lowenstein D, Simon R, Hisanaga K (1991) Heat shock protein hsp72 induction in cortical and striatal astro cytes and neurons following infarction. J Cereb Blood Flow Metab 11:621-627 Simon RP, Cho H (1990) The temporal profile of stress protein production following global ischemia. Neurology 40 (suppl 1):383 Simon RP, Cho H, Gwinn R, Lowenstein DH (1991) The tem poral profile of 72-kDa heat-shock protein expression fol lowing global ischemia. J Neurosci 11:881-889 Vass K, Welch WJ, Nowak TS Jr (1988) Localization of 70 kDa stress protein induction in gerbil brain after ischemia. Acta Neuropathol (Berl) 77:128-135 Welsh FA, Moyer DJ (1990) Expression of heat shock protein-70 mRNA following reversible focal ischemia in rat brain. Soc Neurosci Abstr 16:938 Welsh FA, Sims RE, Harris VA (1990) Mild hypothermia pre vents ischemic injury in gerbil hippocampus. J Cereb Blood Flow Metab 10:557-563 Welsh FA, Marcy VR, Sims RE (1991) NADH fluorescence and regional energy metabolites during focal ischemia and reper fusion of rat brain. J Cereb Blood Flow Metab II :459-465 Wu C, Wilson S, Walker B, Dawid I, Paisley T, Zimarino V, Veda H (1987) Purification and properties of Drosophila heat shock activator protein. Science 238: 1247-1253
