(Stroke. 1995;26:1639-1648.)
© 1995 American Heart Association, Inc.
Articles |
Induction of c-fos and c-jun Gene Products and Heat Shock Protein After Brief and Prolonged Cerebral Ischemia in Gerbils
Presented at the 16th International Joint Conference on Stroke and Cerebral Circulation, San Francisco, Calif, February 21-23, 1991, and published in abstract form (Stroke. 1991;22:131).
From the Department of Neurology, Mayo Clinic and Mayo Foundation, Rochester, Minn.
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Abstract |
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Background and Purpose Proto-oncogene activation and induction of heat shock protein (HSP) occur in response to various stimuli to brain, but the role in neuronal survival after cerebral ischemia remains uncertain. We compared the extent of insults and induction of c-fos and c-jun gene products (c-FOS and c-JUN) as well as HSP in ischemic and postischemic gerbil brains immunohistochemically.
Methods Common carotid arteries of Mongolian gerbils were occluded for 5 or 15 minutes and recirculated for 0 minutes to 7 days. Antibodies for c-FOS, c-JUN, and HSP 70 were used for immunohistochemistry, and positive reactions were semiquantitatively analyzed. The presence of ischemic and postischemic lesions was ascertained with an antibody for microtubule-associated proteins.
Results After ischemia for 15 minutes and reperfusion, c-FOS was induced promptly after 1 to 6 hours in pyramidal cells of the CA3 and CA4 regions, while c-JUN became visible in the same areas after recirculation for 4 to 48 hours. HSP 70 was detected after recirculation for 24 hours in the CA3 region. In layers I and II of the cerebral cortex, c-FOS and c-JUN peaked at 3 hours and HSP 70 at 96 hours. Induction of these proteins was absent or negligible in the areas that developed ischemic or postischemic lesions, including the subiculum-CA1 and CA1 regions of the hippocampus and layers III/IV and Vb/VI of the cerebral cortex. After shorter ischemia for 5 minutes and reperfusion, c-FOS and c-JUN were rapidly induced at 15 minutes to 1 hour except for the subiculum-CA1 and CA1 regions of the hippocampus. Induction of HSP 70 did not occur for 24 hours and was noted only in the hippocampus.
Conclusions Induction of c-FOS and c-JUN occurred in the areas surviving after transient cerebral ischemia, but the extent of induction and the latent period varied depending on the duration of the insult and the location. In the areas with ischemic or postischemic damage detected by loss of the reaction for microtubule-associated proteins, the induction of c-FOS and c-JUN was either absent or minimal, suggesting that active induction of those immediate early gene products occurred early in surviving neurons. On the other hand, the induction of HSP 70 did not occur until reperfusion for 24 hours and actively occurred only in the areas with earlier induction of c-FOS and/or c-JUN, suggesting that the induction of HSP 70 occurred in neurons that survived to that point, but it did not participate in early responses for neuronal survival after global cerebral ischemia.
Key Words: cerebral ischemia • heat shock proteins • immunohistochemistry • gerbils
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nuclear proto-oncogenes have been recognized as a component of transmembrane signal transport and are activated by various physiological stimuli and noxious insults.1 Among them, c-fos and c-jun belong to immediate early genes (IEGs) known to have rapid but brief responses. In the central nervous system, these IEGs respond to various stimuli2 including epilepsy3 and motor and sensory cortical stimulation.4 Induction of c-fos and c-jun product proteins, c-FOS and c-JUN, has been investigated in focal ischemia in the cerebral cortex in rats,5 6 while c-FOS has been investigated after transient global ischemia in the hippocampus of gerbils.7 Induction of corresponding mRNA has also been investigated with an in situ hybridization technique after focal ischemia in the cerebral cortex of rats8 and after global ischemia in the hippocampus of rats9 10 and gerbils.11 12 These results indicated that induction of IEGs could occur in certain areas of the brain, but it was weak or absent in the part of the hippocampus known to develop delayed neuronal death. However, we have only limited information regarding the areas to have induction of IEGs and their products and the timing of induction in relation to the severity of cerebral ischemia and neuronal survival, despite the fact that there are many anatomic sites which could develop delayed neuronal death depending on the severity and duration of ischemic insults.13
Another group of cellular proteins that is known to emerge after various noxious stimuli is heat shock protein (HSP).14 Although identified after heat shock initially, HSP 70 has been found to be preferentially synthesized after transient global ischemia in gerbils,15 and subsequent immunohistochemical investigations, although primarily focused on the hippocampus, revealed the areas with and without induction of HSP 70 after global ischemia in gerbils16 and rats.17 18 While induction of HSP occurred more intensely in the areas less vulnerable to global ischemia, suggesting a protective effect of HSP,16 and was considered to be an early marker for neuronal injury,19 recent reports suggested that the presence of HSP 70 was only a marker of stress and not necessarily a marker for eventual neuronal survival.17 18 Thus, the relationship between HSP 70 and selective tissue vulnerability as well as delayed neuronal death remains uncertain, and its role in neuronal protection after ischemic insults is also uncertain, even though HSP 70 is believed to have such an effect.19
In the present investigation we therefore studied both the presence and absence of induction of c-FOS and c-JUN as well as HSP 70 not only in the hippocampus but also in other areas of the brain including the cerebral cortex, caudoputamen, and thalamus after global cerebral ischemia in gerbils, and we evaluated the extent and timing of induction of these proteins in vulnerable and tolerant areas after brief ischemia for 5 minutes, which would cause permanent damage only in small areas of the hippocampus, and prolonged ischemia for 15 minutes with extensive permanent damage.13 We also studied the hypothalamus to assess the response to ischemic stress. Since an absence of induction may indicate either the tolerant areas without ischemic damage or the vulnerable areas with irreversible ischemic or postischemic neuronal damage, we distinguished these areas in the adjacent tissue section by the immunohistochemical reaction for microtubule-associated proteins (MAPs), which has been shown to be very sensitive for detection of ischemic and postischemic damage regardless of the brain regions.20 21 The results of the present investigation have been reported in abstract form.22
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Materials and Methods |
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Mongolian gerbils weighing 60 to 80 g were used. The experimental protocol was in accordance with the Declaration of Helsinki of 1975 and has been approved by the Institutional Animal Research Committee. Each gerbil was anesthetized with ether inhalation, and both common carotid arteries were occluded in the neck by miniature aneurysmal clips. After occlusion for 5 or 15 minutes, the clips were removed under ether anesthesia. Each gerbil was allowed to live for 0, 15, or 30 minutes or 1, 3, 6, 24, 48, 72, or 96 hours if the ischemic period was 5 minutes; each gerbil was allowed to live for 0 or 30 minutes, 1, 3, 6, 12, 24, 48, or 96 hours, or 7 days if the ischemic period was 15 minutes. The gerbils had free access to food and water during reperfusion periods. Nonoperated as well as sham-operated gerbils were used as control. Sham-operated gerbils underwent the surgical procedure aimed at ischemia for 15 minutes and reperfusion for 3 hours but without actual clipping of the arteries. Four gerbils were used for each ischemic and postischemic period.
After a prescheduled reperfusion period, each gerbil was perfused through the ascending aorta with saline for 1 minute and then 4% paraformaldehyde and 0.2% picric acid in 0.1 mol/L phosphate buffer (pH 7.6) for 6 minutes. For gerbils without recirculation, perfusion fixation was performed just after removal of the clips. Each brain was then removed and divided into three coronal blocks and post-fixed with 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.6) for 6 hours at 4°C. After thorough washing in 15% sucrose in 0.1 mol/L phosphate buffer (pH 7.6), the tissue blocks were frozen in chilled isopentane and sliced into coronal sections (20 µm) in a cryostat at -20°C.
The immunohistochemical procedure was performed with the frontoparietal section including the caudoputamen and the parietal section including the hippocampus, thalamus, and hypothalamus. The avidin-biotin peroxidase method (Vectastain) was used for free-floating sections. Commercially available polyclonal antibodies for c-FOS (x1000) and c-JUN (x200) (Oncogene Science) and a monoclonal antibody for HSP 70 (x2000) (Amersham) were used as primary antibodies. Normal rabbit IgG was used as control for c-FOS and c-JUN antibodies and normal mouse IgG for HSP 70. Ischemic and postischemic lesions were visualized with a polyclonal antibody for MAP1 and MAP2 (x10 000) raised in a goat in our laboratory21 with normal goat serum as control. Each section was incubated with a primary antibody for overnight at 4°C. A 0.05 mol/L Tris-HCl buffer solution (pH 7.6) with 0.3% Triton-X was used throughout the entire immunohistochemical procedure. Diaminobenzidine tetrahydrochloride was used as a chromogen.
For semiquantitation of c-FOS and c-JUN as well as HSP 70,
neurons with positive reactions were counted under light microscopy and
scored from 0 to 5 (Table
) in the regions of interest.
Scores from eight hemispheres (four gerbils) were added to make a total
score. Thus, the total score of a given region of interest ranged from
a maximum of 40 to a minimum of 0.
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Results |
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Although no seizure was observed after ischemia for 5 minutes, more than 60% of gerbils experienced seizures after ischemia for 15 minutes. Under light microscopy, the positive reaction for c-FOS and c-JUN was observed mainly in neuronal nuclei, and the positive reaction for HSP 70 was detected in neuronal cytoplasm. After extended postischemic periods, small nuclei positive for c-FOS or c-JUN were observed within postischemic lesions, as depicted by loss of the immunohistochemical reaction for MAPs. These scattered cells with positive reactions were identified as astrocytes by the location and the shape and were eliminated from the semiquantitative evaluation. The distribution patterns of ischemic and postischemic lesions visualized by the reaction for MAPs were similar to those reported previously after ischemia for 5 and 10 minutes.13 While ischemic and postischemic lesions could be reversible in some areas of the brain, those seen after 96 hours following 5-minute ischemia and those seen after reperfusion for 7 days following 15-minute ischemia were considered irreversible because of coincidence with the corresponding lesions seen with hematoxylin-eosin staining after extended reperfusion.13 21 23
Hippocampus
Control and Sham Operation
Minimal c-FOS activities were noted in control gerbils, with a
total score of 4 in pyramidal cells of the CA3 and CA4
regions as well as in granular cells of the dentate gyrus; c-JUN
activities existed in granular cells of the dentate gyrus, with a total
score of 16, without notable activities in other areas. HSP 70 was not
detected in the hippocampus or any other areas of the brain.
In sham-operated gerbils, an increase of c-FOS was found in pyramidal cells of the CA3 and CA4 regions, with a total score of 6 and 4, respectively. However, no increase was found in c-JUN. HSP was not increased in the hippocampus or any other areas of the brain in sham-operated gerbils.
Ischemia for 5 Minutes
Mild induction of c-FOS and c-JUN occurred from 15 minutes
to 3 hours in pyramidal cells of the subiculum-CA1 (between
the subiculum and the medial CA1 region, which has been identified as
the paramedian part of the CA2 region24 in the gerbil
brain) to CA1 region of the hippocampus (Fig 1). There
was no difference in the pattern of induction of c-FOS and c-JUN in the
subiculum-CA1 and the medial as well as lateral part of the CA1 region.
Since verification of the CA2 region between the lateral CA1 region and
the CA3 region without any supplemental histological
procedure was difficult with the free-floating method, the CA2 region
was excluded from the evaluation. We expect that it would be the same
or very similar to the subiculum-CA1 region. Induction of c-FOS was
most prominent 1 to 3 hours after reperfusion in granular cells of the
dentate gyrus and diminished rapidly by 6 hours. Similarly, the peak of
c-FOS induction in pyramidal cells of the CA3 and CA4
regions occurred in 1 to 3 hours (Fig 1). Induction of c-JUN occurred
from 15 minutes to 6 hours in pyramidal cells of the CA3
and CA4 regions (Fig 1) and 15 minutes to 72 hours in granular cells of
the dentate gyrus. In scattered neurons of the intrahilar area of the
dentate gyrus, induction of c-FOS and c-JUN was observed in the same
manner as those in granular cells but to a lesser degree. HSP 70 was
only mildly to moderately induced in the CA3 and CA4 regions and
granular cells of the dentate gyrus and was only mildly induced in the
subiculum-CA1 to CA1 region after reperfusion for 24 hours (Fig 1).
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and 
indicate c-FOS after 5- and
15-minute ischemia, 
and 
indicate c-JUN after 5-
and 15-minute ischemia, and 
and 
indicate heat
shock protein 70 after 5- and 15-minute ischemia,
respectively.Ischemic lesions were identified immunohistochemically with the reaction for MAPs in the subiculum-CA1 region, and additionally postischemic lesions evolved in the intrahilar neurons in the dentate gyrus after reperfusion for 3 hours. Seventy-five percent of gerbils showed postischemic lesions in the subiculum-CA1 to CA1 region as well as in intrahilar neurons of the dentate gyrus after 96 hours, while no lesion was recognized at 96 hours in pyramidal cells of the CA3 and CA4 regions or granular cells of the dentate gyrus.
Ischemia for 15 Minutes
In the subiculum-CA1 to CA1 region, induction of c-FOS and c-JUN
as well as HSP 70 occurred in scattered neurons only faintly after
reperfusion for 6 hours (Figs 1
and 2A). Induction of
c-FOS was most prompt and marked in granular cells of the dentate gyrus
occurring in 1 to 3 hours but was already reduced at 6 hours. Induction
of c-FOS in pyramidal cells of the CA3 and CA4 regions
occurred after recirculation for 1 hour and was sustained for 96 hours
(Figs 1 and 2B). Induction of c-JUN was observed between 1 to 12 hours
in granular cells of the dentate gyrus and 6 to 48 hours in
pyramidal cells of the CA3 and CA4 regions (Fig 1). There
was a second but milder induction in granular cells of the dentate
gyrus between 48 hours and 7 days. In scattered neurons of the
intrahilar area of the dentate gyrus, mild induction of c-FOS was
observed at approximately 12 hours, while c-JUN was not visible in
those neurons. Induction of HSP 70 was observed mainly in
pyramidal cells of the CA3 and CA4 regions as well as in
neurons of the intrahilar area between 24 to 96 hours, and they were
more prominent than those after ischemia for 5 minutes (Fig 1).
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Ischemic lesions visualized by the reaction for MAPs already existed in the subiculum-CA1 to CA1 region as well as in the intrahilar area of the CA4 region at the end of ischemia even without recirculation. Additionally, postischemic lesions existed in all gerbils in these areas and in 75% of gerbils in the CA3 region at 7 days. However, no lesion was observed in granular cells of the dentate gyrus or pyramidal cells of the CA4 region even after 7 days.
Cerebral Cortex
Control and Sham Operation
In the frontoparietal cortex of control gerbils, c-FOS was
detected in layers I and II and lower layer VI, with a total score of 9
in each. In the parietal cortex, the entire depth of the cortex showed
c-FOS activities, with the total score ranging from 8 to 10. In the
frontoparietal cortex, c-JUN was detected in layers I and II and lower
layer VI, with a total score of 13, but no activity was present in
the parietal cortex. There was no detectable HSP activity in the
cerebral cortex of control gerbils.
In the frontoparietal cortex of sham-operated gerbils, no increase of c-FOS was found in layers I or II or lower layer VI, but minimal increases with a total score of 3 to 5 were detected in other layers. In the parietal cortex, further increases of c-FOS occurred in each layer, with a total score of 13 to 20. No increase in c-JUN activity was found in the cerebral cortex of sham-operated gerbils.
Ischemia for 5 Minutes
Induction of c-FOS and c-JUN occurred markedly in all layers
after reperfusion for 15 minutes and was sustained for up to 6 hours in
both frontoparietal and parietal cortex (Fig 3).
Induction of HSP 70 was observed marginally at 96 hours only in layer
III/IV. Immunohistochemical ischemic lesions visualized by the
reaction for MAPs existed in layer III/IV in 25% of gerbils at the end
of ischemia without reperfusion. Postischemic
lesions persisted only in a small area of layer III/IV in 25% of
gerbils at 48 hours, and no postischemic lesions were
observed at 96 hours.
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Ischemia for 15 Minutes
In the frontoparietal cortex, induction of c-FOS was
observed moderately in layers I, II (Fig 3), and Va as well as in the
lower part of layer VI between 1 and 3 hours and persisted for up to 96
hours (Fig 3). Induction of c-JUN was observed after 6 hours in layers
I and II (Figs 3 and 4B) as well as in the lower part of
layer VI and, to a lesser degree, in layer Va. Induction of c-FOS and
c-JUN was mild or not present in layers III/IV or Vb/VI during 24
hours, but a second peak was noted from 48 to 96 hours (Figs 3 and 4A).
HSP 70 was moderately induced from 48 to 96 hours in layers I and II,
and it was more than after ischemia for 5 minutes (Figs 3 and 5B). HSP 70 was not induced in layer III/IV (Figs 3 and 5A). In the parietal cortex, the pattern of induction of c-FOS and
c-JUN as well as HSP 70 was almost identical to that in the
frontoparietal cortex.
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Immunohistochemical ischemic lesions visualized by the reaction for MAPs existed in layer III/IV in all gerbils at the end of ischemia without recirculation, and postischemic lesions persisted in layer III/IV in all gerbils and in layer Vb/VI in 50% of gerbils at 7 days.
Caudoputamen
Control and Sham Operation
No c-FOS activity was found in the caudoputamen of
control gerbils or sham-operated gerbils. The c-JUN activity was
detected in the medial and lateral caudoputamen, with a
total score of 6 and 5, respectively, without further increase in
sham-operated gerbils.
Ischemia for 5 Minutes
Induction of c-FOS and c-JUN was observed in the
medial and lateral parts of the caudoputamen to a
similar degree and between 15 minutes to 24 hours (Fig 6). Induction of HSP 70 was not apparent. No
immunohistochemical ischemic or postischemic lesion
was observed with the reaction for MAPs during ischemic and
postischemic periods.
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Ischemia for 15 Minutes
We found that c-FOS and c-JUN were induced mildly to
moderately both in the medial and lateral parts of the
caudoputamen from 1 to 48 hours and to a similar degree
(Fig 6). However, they were less active than those seen after
ischemia for 5 minutes for the first 60 minutes of reperfusion.
Induction of HSP 70 was not observed.
While there was no immunohistochemical ischemic lesion, postischemic lesions eventually evolved in the lateral part of the caudoputamen in 75% of gerbils and in the medial part in 50% of gerbils at 7 days.
Thalamus
Control and Sham Operation
No c-FOS activity was detected in the ventral or dorsomedial
nucleus in the thalamus of control gerbils. In sham-operated gerbils,
an increase was found in the dorsomedial nucleus, with a total score of
10. No c-JUN activity was found in the thalamus of either control or
sham-operated gerbils.
Ischemia for 5 Minutes
Induction of c-FOS occurred in the dorsomedial nucleus after 15
minutes to 96 hours and marginally in the ventral nucleus after 30
minutes to 6 hours (Fig 7), while induction of c-JUN
occurred after 15 minutes in both the dorsomedial and ventral nuclei
and continued for 48 to 72 hours (Fig 7). Induction of HSP 70 was not
observed. Immunohistochemical ischemic lesions were not
observed with the reaction for MAPs, but postischemic
lesions were transiently present in the ventral nucleus at 60
minutes and 48 hours.
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Ischemia for 15 Minutes
Induction of c-FOS occurred after 1 hour and remained
activated for 96 hours in the dorsomedial nucleus (Fig 7),
while c-JUN was induced mildly after 3 hours, with a second peak at 48
to 96 hours. In the ventral nucleus, mild induction of c-FOS was
observed after 3 to 12 hours and of c-JUN after 12 to 96 hours (Fig 7).
HSP 70 was not induced.
While no immunohistochemical ischemic lesion was observed in the thalamus at the end of ischemia without reperfusion by the reaction for MAPs, postischemic lesions were observed in the ventral nucleus in 50% of gerbils after 1 hour and 100% at 3 hours and persisted in 50% of gerbils at 7 days. Postischemic lesions were also observed transiently in the dorsomedial nucleus in 50% of gerbils after 1 hour but disappeared thereafter.
Hypothalamus
Control and Sham Operation
The c-FOS activity was minimal in control gerbils but increased in
sham-operated gerbils to a total score of 14. No reaction or increase
of c-JUN was found in control or sham-operated gerbils.
Ischemia for 5 Minutes
Induction of c-FOS occurred at the end of ischemia without
reperfusion, reaching a total score of 30 after 3 hours, and persisted
for up to 48 hours, while induction of c-JUN occurred after 15 minutes,
reaching a total score of 20, and persisted for up to 48 hours. HSP 70
was not induced in the hypothalamus at any reperfusion period (Fig 8). Neither ischemic nor
postischemic lesions were observed throughout the entire
observation period with the immunohistochemical reaction for MAPs.
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Ischemia for 15 Minutes
Marked induction of c-FOS occurred after 60 minutes, reaching a
total score of 35, and remained active for up to 96 hours, while c-JUN
was induced mildly after 3 to 48 hours, with a maximal total score of
14. HSP 70 was not induced in this area. Neither ischemic nor
postischemic lesions were recognized with the
immunohistochemical reaction for MAPs (Fig 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present investigation demonstrated the spatial and temporal distribution of the induction of c-FOS and c-JUN as well as HSP 70 throughout the forebrain. Although a number of studies described induction of IEGs after transient ischemia9 10 11 25 26 and c-FOS in the hippocampus,7 26 no report has focused attention on the spatial and temporal pattern of the induction of IEG proteins throughout the whole brain and compared brief ischemia with mostly reversible insults and prolonged ischemia with irreversible damages. A comparison of induction of c-FOS and c-JUN in various areas of the brain with different ischemic vulnerabilities in the present study indicated that induction of IEG proteins occurred promptly and vigorously in areas of the brain that did not develop irreversible ischemic or postischemic damage, particularly after brief ischemia with milder insults. In these areas, the responding neurons promptly produced IEG proteins more rigorously than they would respond to acute stress caused by sham operation. After prolonged ischemia with more severe insults, however, the responses to produce IEG proteins were delayed and often much less rigorous compared with their responses after ischemia for 5 minutes, and in some areas IEG responses did not occur. Neurons in the most vulnerable areas promptly ceased to respond or showed only faint responses and developed irreversible ischemic or postischemic damage promptly. The hypothalamus responded to ischemic insults rigorously without having irreversible ischemic or postischemic damage.
Suppression of the production of IEG proteins in the areas known to have irreversible ischemic or postischemic damage could be from suppression of IEG gene activation and/or damage to the subsequent steps for production of IEG proteins. Production of mRNA for IEGs and the HSP gene has been investigated after transient global cerebral ischemia by the use of an in situ hybridization technique.9 10 11 12 26 These studies revealed much more prompt induction of IEGs than the HSP gene. This may be due to superinduction of IEGs as the result of inhibition in general protein synthesis.27
A comparison of our results showing induction of IEG proteins and others showing activation of IEGs (ie, induction of mRNAs) is difficult, since the areas of investigations by others have often been confined to the hippocampus and the severity of global cerebral ischemia is different between rats and gerbils. Although a gross parallelism can be observed between activation of IEGs and induction of IEG proteins, some discrepancies exist between them. While induction of c-FOS and c-JUN occurred to lesser degrees in vulnerable regions compared with more resistant regions, the mRNA levels for IEGs were similar between the vulnerable CA1 region and the resistant CA3 region.9 10 11 12 25 26 Although the impairment of both IEG activation and production of IEG proteins may be responsible for ischemic and postischemic neuronal death, the status of IEG proteins would reflect the eventual outcome of affected neurons better than the status of IEG activation. Indeed, our results demonstrated close correlation between prompt induction of IEG proteins and neuronal survival after ischemic stress. Thus, we suspect that induction of IEG proteins may be closely related to the molecular process determining neuronal survival after global cerebral ischemia, although there is no direct proof at the present time and the exact mechanism is still uncertain. We previously suggested that ischemic neuronal damage was a dynamic process, with neurons in a given area having potentials for acute death, progressive deterioration, delayed neuronal death, and recovery depending on the severity and the duration of ischemia.13 The present investigation also demonstrated that induction of IEG products was a dynamic process, with variation in the extent and time course depending on the severity and the duration of ischemia.
HSPs are also known to be induced in a variety of conditions including brain injury,14 status epilepticus,28 29 cerebral ischemia,15 16 17 18 30 31 32 and hyperthermia.19 33 34 HSPs are selectively expressed in neurons subjected to sublethal stress,31 and lethal ischemic damage could be ameliorated by preceding hyperthermia,33 34 brief ischemia,35 36 37 or oxidative stress,38 which could induce HSPs. While these findings support the neuroprotective role of preexisting HSPs, induction of HSP 72 has been observed in neurons in the CA1 region of the rat hippocampus that were destined to die,17 and the neuroprotective role of HSPs induced after the onset of cerebral ischemia remains uncertain.
In the present investigation, HSP 70 was detected in the hippocampus and cerebral cortex but not in the caudoputamen, thalamus, or hypothalamus. In the areas vulnerable to ischemia, including the subiculum-CA1 region of the hippocampus and layer III/IV of the cerebral cortex, HSP 70 was induced after reperfusion for 24 to 96 hours, but it was modest compared with induction in the areas relatively tolerant of ischemia, including the CA3 and CA4 regions of the hippocampus and layers I and II of the cerebral cortex. It was particularly so after ischemia for 15 minutes, in which case HSP 70 induction was very prominent in the areas relatively tolerant of ischemia, as pointed out previously.18 HSP 70 is known to be preferentially synthesized when general protein synthesis recovers after transient cerebral ischemia.15 The timing of induction of HSP 70 in the present investigation corresponded to the timing of polyribosomal reaggregation and recovery of polypeptide synthesis shown electron microscopically, biochemically,39 40 and by autoradiography.41 42
From our findings and the above information on IEG proteins and HSP 70, we can find the following correlations. First, IEG proteins were induced during the early reperfusion period before induction of HSP 70 even before recovery of general protein synthesis was expected. Second, there was a close correlation between prompt induction of c-FOS/c-JUN and the absence of permanent ischemic or postischemic damage. Third, no area of the brain showed induction of HSP 70 in the absence of preceding c-FOS or c-JUN induction. These findings have led us to speculate that induction of IEG proteins was closely associated with the molecular process determining neuronal survival after global cerebral ischemia and that the role of HSP 70 in neuronal survival may be limited after global cerebral ischemia, unless HSP 70 is already present at the onset of the ischemic insult.
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Acknowledgments |
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This study was supported by grant NS-06663 from the National Institutes of Health, US Public Health Service.
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Footnotes |
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Reprint requests to T. Yanagihara, MD, Department of Neurology, Osaka University Medical School, 2-2, Yamada-oka, Suita, Osaka, 565, Japan.
Received October 10, 1994; revision received February 27, 1995; accepted May 5, 1995.
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