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Different thresholds of HSP70 and HSC70 heat shock mRNA induction in post-ischemic gerbil brain
Brain Research, 599 (1992) 197-203 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
197
BRES 18369
Different thresholds of HSP70 and HSC70 heat shock mRNA induction in post-ischemic gerbil brain J. K a w a g o e ,
K. Abe and K. Kogure
Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan) (Accepted 21 July 1992)
Key words: Heat shock protein; Heat shock cognate protein; Cerebral ischemia; In situ hybridization; Gerbil
Thresholds of induction of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs after transient global ischemia in gerbil brain were investigated by in situ hybridization using cloned cDNA probes selective for each mRNA species. In sham control brain, HSP70 mRNA was little present, while HSC70 mRNA was present in most cell populations. A 0.5-min occlusion of bilateral common carotid arteries did not affect the amount of HSP70 and HSC70 mRNAs. The selective induction of HSC70 mRNA was observed in dentate granule cells at 1 h, and in most cells of hippocampus especially dentate gyrus at 3 h after 1 min of ischemia when induction of HSP70 mRNA was not evident in the identical brain. The selective induction diminished by 2 days. However, after 2 rain of ischemia, HSP70 and HSC70 mRNAs were induced together in hippocampal cells from 1 h of the reperfusion, and the co-induction prolonged in CA1 cells until 2 days. Body temperatures monitored at rectum increased after the reperfusion with a peak at 30 min. The degree of increase of the body temperature was significantly higher in the case after 2-min ischemia than in the cases after 0.5- and 1-min ischemia. Although HSP70 and HSC70 mRNAs are generally co-induced in stressful conditions, our results suggest the different thresholds of the induction between HSP70 and HSC70 mRNAs after transient brain ischemia. The selective induction of HSC70 mRNA which is not accompanied by the induction of HSP70 mRNA may relate to the differences of the duration of ischemia and the degree of the increase of body temperature after ischemia.
INTRODUCTION
different roles u n d e r n o r m a l c o n d i t i o n a n d the cooperative role of H S P 7 0 a n d HSC70 in the recovery process
V a r i o u s stressful c o n d i t i o n s of cells i n d u c e 'stress r e s p o n s e ' a n d s u b s e q u e n t l y p r o d u c e heat-shock proteins (HSPs) 6'11'13'21'27. B r a i n ischemia is o n e of such
from the ischemic injury. I n a d d i t i o n to the global ischemia m o d e l of gerbils, the c o - i n d u c t i o n of the
stresses to p r o d u c e HSPs i n c l u d i n g HSP7012'15'18'3° a n d the m R N m s 1'2'15'16'23-25'29. H S P 7 0 a n d heat-shock cogn a t e p r o t e i n ( H S C ) 70 are the m e m b e r s of so-called HSP70 family, a n d their protective roles in cells from the injury have b e e n suggested 2,4,tS'16AS'21,22,26-28. However, the precise role of the HSP70 a n d H S C 7 0 in ischemic b r a i n has n o t yet b e e n fully u n d e r s t o o d . W e have investigated the d i s t r i b u t i o n s of H S P 7 0 a n d HSC70 m R N A s after 10 m i n of global ischemia in gerbils with in situ h y b r i d i z a t i o n u s i n g c l o n e d c D N A s selective for the m R N A s 15. I n c o n t r a s t to the a b s e n c e of HSP70 m R N A , H S C 7 0 m R N A was p r e s e n t in the sham control brain. T h e m R N A s were c o - i n d u c e d after ischemia in b r a i n regions. T h e s e results suggested the
m R N A s was also d e t e c t e d in focal ischemia model of rats 16. However, HSC70 m R N A was selectively ind u c e d in some h i p p o c a m p a l cells of the rat m o d e l w h e r e obvious i n d u c t i o n of HSP70 m R N A was n e v e r found. Miller et al. 22 suggested that different i n d u c t i o n of HSP70 a n d HSC70 m R N A s occurred in amp h e t a m i n e - t r e a t e d h y p e r t h e r m i c b r a i n of rats by N o r t h e r n blot analysis, a n d that the elevation of HSC70 would be sufficient to protect cells from a relatively m i l d e r stress while the i n d u c t i o n of HSP70 would be r e q u i r e d in m o r e severe stress. Recently, W o n g et al. 32 also observed a selective i n d u c t i o n of HSP73 (HSC70) in the d e n t a t e gyrus by seizures in rats whereas n e i t h e r HSP70 n o r HSP84 genes were induced. However, no such dissociation in the i n d u c t i o n of HSP70 a n d HSC70
Correspondence: J.-i. Kawagoe, Tokyo Research Laboratories, Pharmaceutical Division, Kowa Company, LTD., 2-17-43, Noguchi-cho, Higashimurayama, Tokyo 189, Japan. Fax: (81) (423) 95 0312.
198
mRNAs
has been
reported
a f t e r t r a n s i e n t g l o b a l is-
c h e m i a in b r a i n .
or 2 min of ischemia at 30 and 60 min alter thu rcperfusion m~(i histopathological observation with Cres'd violet staining ~s was made at 7 days after the rcperfusion (n - 4 6).
Therefore, we have investigated possible thresholds of the induction of HSP70 and HSC70 mRNAs transient
global ischemia of gerbil brain
after
by in situ
hybridization technique. MATERIALS AND METHODS
Animal model Male Mongolian gerbils (Meriones unguiculatus), aged 10-11 weeks and weighing 70-75 g, were lightly anesthetized by inhalation of a nitrous oxide/oxygen/halothane (69% :30%: 1%) mixture. Both common carotid arteries were exposed and then the anesthesia was stopped. When the animal began to regain consciousness, the arteries were occluded for 0.5, 1, or 2 rain using surgical clips 2. Body temperatures were monitored in all animals with a rectal probe, and was maintained at 37°C using a heat pad during the surgical preparation and the occlusion of the carotid arteries. After the restoration of blood flow, no attempt was made to maintain constant body temperature of animals. The animals recovered for 1, 3 h and 2 days (n = 3 in each time point) at the ambient temperature (21-23°C), and then were decapitated. Sham animals were sacrificed just after exposing the carotid arteries without clamping the vessels. The dissected brains were frozen in powdered dry ice and stored at -80°C. Sections (10 p.m) at dorsal hippocampal level were cut on a cryostat at - 18°C and collected on the slides coated with Histostik (Accurate Chemical and Scientific Corp., Westbury, NY). In situ hybridization for HSP70 and HSC70 mRNAs was performed in the sections. In another experiment, rectal temperature was monitored after 0.5, 1,
HSP70
hz situ hybridization The inserts of cloned cDNAs used as probes in lhe present study were originally from cerebral cortex of gerbils. The sizes of the inserts selective for HSP70 and HSC70 mRNAs were 1.0 (pGA ~) and 1.4 kb (pGD3), respectively 29. In situ hybridization was performed by a method of Yoshioka et al. 3~ with a slight modification. Briefly, the sections were fixed for I(~ min in an ethanol/acetic acid (3:1) mixture, transferred to 0.2 M HCI for 20 rain, and immersed for 20 min at 50°C in 2×SSC (pH 7.0). They then were digested for 15 min at 37°C with 100/~g/ml of proteinase K (Merk & Co. Inc., Rahway, NJ) in 20 mM Tris-HCI buffer (pH 7.4) with 2 mM CaCl 2 and dehydrated through graded ethanol. Slides were prehybridized for 2 h at room temperature in a solution containing 50% formamide, 600 mM NaCI, l0 mM Tris-HC1 (pH 7.5), 1 mM EDTA, 0.02%, polyvinylpyrrolidone, 0.02% Ficoll. 0.02% bovine serum albumin, 100 p.g/ml sonicated, denatured calf thymus DNA, and 0.5 mg/ml calf liver RNA. Hybridization was done for 20 h at 42°C using SsS-labeled cDNA probe at a concentration of 0.1 ng//~l in prehybridization solution supplemented with 10% dextran sulfate and 10 mM dithiothreitol. The cDNA inserts were radiolabeled with [~-3sS]deoxycytidine triphosphate (1(100 Ci/mmol, Amersham) by random primer labeling s using a kit (Boehringer Mannheim Yamanouchi Co. Ltd., Tokyo, Japan), resulting in specific activities of 5-6 x 10 ~ dpm//sg. The control probe was a similarly-labeled plasmid vector (pHSG396, Takara Shuzo Co. Ltd., Kyoto, Japan) fragment (1.1 kb) digested by Hinfl (Takara). The sections were washed at 42°C for 2 h in 2xSSC and for I h in 1 x SSC, and then were dehydrated through graded ethanol series containing 0.3 M ammonium acetate. After visualization of the
HSC70
lh I I1!
Fig. 1. Distributions of HSP70 (left column) and HSC70 (right column) mRNAs at the level of dorsal hippocampus in the identical brain of sham control (S) and the post-ischemic gerbils at 1 h after 0.5 min of ischemia (lh). Note hybridization of HSC70 mRNA in most cell populations of the sham brain in contrast to little hybridization of HSP70 mRNA, and no significant effect of 0.5-min ischemia on the induction of HSP70 and HSC70 mRNAs. Bar = 3.1 ram.
199 hybridization by exposure against X-ray film for 24 h at room temperature, the sections were dipped in a liquid emulsion (NR-M2, Konica Co. Ltd., Tokyo, Japan) diluted (1 : 1) with 0.6 M ammonium acetate. They were then exposed at 4°C for approximately 3 weeks, developed, and counterstained with hematoxylin. Several slides were treated with 100 p,g/ml RNase A and 10 units/ml RNase T1 (Sigma, St. Louis. MO, USA) in 2×SSC at 37°C for 2 h prior to prehybridization and were hybridized with the probe for HSP70 or HSC70 mRNA.
d a n t l y p r e s e n t (Fig. l-S). A relatively high level of H S C 7 0 m R N A was o b s e r v e d in h i p p o c a m p a l C A 3 cells a n d t h e o r d e r o f h y b r i d i z a t i o n intensity o f H S C 7 0 m R N A in h i p p o c a m p a l cells was C A 3 > CA1 > d e n t a t e gyrus g r a n u l e . A 0.5 min o f t r a n s i e n t i s c h e m i a did not significantly affect t h e a m o u n t o f H S P 7 0 o r H S C 7 0 m R N A s at all e x a m i n e d time p o i n t s after the r e p e r f u s i o n (Fig. 1 - 1 h ) . H o w e v e r , o n e min o f i s c h e m i a a p p a r e n t l y i n d u c e d a f u r t h e r H S C 7 0 m R N A in d e n t a t e g r a n u l e cells, indic a t e d by a r r o w h e a d s in the figure, at 1 h as c o m p a r e d to t h e s h a m level (right c o l u m n in Fig. l-S), w h e r e a s no c h a n g e o f H S P 7 0 m R N A level o c c u r r e d again in the i d e n t i c a l b r a i n (Fig. 2 - 1 h ) . Such selective i n d u c t i o n o f H S C 7 0 m R N A was m o r e e v i d e n t in all cell types o f h i p p o c a m p u s especially d e n t a t e gyrus at 3 h a f t e r the r e p e r f u s i o n , w h e n t h e o r d e r o f t h e h y b r i d i z a t i o n intensity o f H S C 7 0 m R N A in h i p p o c a m p a l ceils c h a n g e d f r o m C A 3 > CA1 > d e n t a t e gyrus g r a n u l e in s h a m b r a i n (right c o l u m n in Fig. l - S ) to C A 3 >__d e n t a t e gyrus granule > CA1 (right c o l u m n in Fig. 2 - 3 h ) . Even at this t i m e point, 1 min of ischemia d i d not i n d u c e H S P 7 0 m R N A (left c o l u m n in Fig. 2 - 3 h ) . T h e level of H S C 7 0
Statistics The values obtained were expressed as means + S.E.M. Statistical comparisons were made by the Mann-Whitney U-test after the Kruskal-Wallis analysis. RESULTS
In situ hybridization T h e results o b t a i n e d in in situ h y b r i d i z a t i o n w e r e r e p r o d u c i b l e in all a n i m a l s u s e d in e a c h t i m e point. Figs. 1 to 3 show t h e a u t o r a d i o g r a m s o f the sections o f i d e n t i c a l b r a i n s u b j e c t e d to several d u r a t i o n s o f i s c h e m i a a n d r e p e r f u s i o n for the d e t e c t i o n o f H S P 7 0 a n d H S C 7 0 m R N A s by in situ h y b r i d i z a t i o n . In the s h a m c o n t r o l brain, H S C 7 0 m R N A was p r e s e n t in most cell p o p u l a t i o n s , while H S P 7 0 m R N A was n o t a b u n -
HSC70
HSP70 i
ii~
~, ili~iiii!~¸~ili~
1 h
r
3h
k
•
2d
Fig. 2. Chronological change of HSP70 (left column) and HSC70 (right column) mRNAs at 1 (lh), 3 h (3h) and 2 days (2d) after 1 min of ischemia. Note slight but significant induction of HSC70 mRNA in dentate gyrus granule cells (arrowheads) at 1 h and all cell types of hippocampus especially dentate gyrus at 3 h relative to the sham level (right column in Fig. l-S), while an induction of HSP70 mRNA was not evident. Bar = 3.1 mm.
200
HSP70
HSC70
lh
3h
2d
Fig. 3. Chronological change of HSP70 (left column) and HSC70 (right column) m R N A s at 1 (lh), 3 h (3h) and 2 days (2d) after 2 min of ischemia. Note a co-induction of HSP70 and HSC70 m R N A s from 1 h to 2 days and the long-lasting hybridization of the m R N A s in CA1 cells even at 2 days. Bar = 3.1 mm.
mRNA returned to the sham level with little change in HSP70 mRNA at 2 days after 1-min ischemia (Fig. 2-2d). After 2 min of ischemia, both HSP70 and HSC70 mRNAs were markedly induced in a cooperative manner in dentate granule cells at 1 h (Fig. 3-1h) and in all cell types of hippocampus especially dentate gyrus at 3 h (Fig. 3-3h). Strong hybridization of HSP70 and HSC70 mRNAs remained in CA1 cells even at 2 days after the reperfusion (Fig. 3-2d) although the hybridization of the mRNAs in cells of dentate gyrus and CA3 diminished or returned to the sham level. Observation of sections dipped in the liquid emulsion revealed that grains for HSP70 and HSC70 probes were predominantly located in the neuronal cell body (data not shown). Neither the sections hybridized with the probe from plasmid vector nor the sections treated with RNases before incubation with the probe for HSP70 or HSC70 mRNA exhibited any specific hybridization (data not shown).
Histopathological study Cresyl violet staining revealed that 2 min of ischemia resulted in little morphological change even at 7 days after the reperfusion (data not shown) as reported previously TM.
Body temperature Fig. 4 shows the changes of body temperature after different durations of ischemia (0.5-2 min). Transient ischemia definitely increased the body temperature at 30 and 60 min after the reperfusion with the peak time at 30 min. The peak of the increased body temperature in case of 2 min of ischemia (39.8 + 0.1°C, n = 5) was
40.0
"
0
39.0
~
38.0
E ~" "0 0 rn
37.0
36.0
Pre
Ischemia
30 min
60 min
Fig. 4. C h a n g e s of body temperature monitored at rectum before (Pre) and during ischemia (Ischemia), and at 30 and 60 rain after the reperfusion in gerbil. The peak body temperature at g0 ~ after the reperfusion was significantly higher in the case With 2 min of ischemia (e) than in those with 1 ( D ) and 0.5 (©) min of ischemia. * P < 0.01 vs. 0.5 and 1 min of ischemia groups (n = 4-6).
201 significantly higher than those of 1 min (38.7 _+0.1°C, n = 6) and 0.5 min (38.5 + 0.1°C, n = 4) of ischemia (P < 0.01). There was no statistical difference between the body temperature in the different durations of ischemia at 60 min after the reperfusion. DISCUSSION Northern blot analysis revealed that the HSP70 and HSC70 probes employed in the present study selectively detected HSP70 and HSC70 mRNAs, respectively, in the gerbil brain and did not cross-react 15,29. In in situ hybridization analysis, grains for HSP70 and HSC70 probes were located predominantly in the cell body. The specificities of the probes were further supported by the negative signal after a hybridization with the irrelevant plasmid vector probe and by the eliminated signal after pretreatment with RNases prior to incubation with the probe for HSP70 or HSC70 mRNA. These results provide the evidence that the HSP70 and HSC70 cDNA probes used here selectively recognize HSP70 and HSC70 mRNAs of gerbil brain in situ, respectively. HSP70 mRNA was induced by 2 min of forebrain ischemia from 1 h to 2 days after the reperfusion (left column in Fig. 3). However, 1 or 0.5 min of ischemia did not induce HSP70 mRNA (left column in Fig. 1-1h and Fig. 2). These results are in good accordance with that by Nowak and Osborne 25. They reported that 2 min of ischemia induced HSP70 mRNA while 1 min of ischemia did not. Further induction of HSC70 mRNA relative to the sham level was evident after 2 (right column in Fig. 3) and 1 (right column in Fig. 2-1h and 3h) min of ischemia. However, after 0.5 min of ischemia, further induction of HSC70 mRNA was not detected (right column in Fig. 1-1h). These results indicate the different thresholds of the induction of HSP70 (2 min) and HSC70 (1 min) mRNAs in transient ischemia model of gerbils and the selective induction of HSC70 mRNA which is not accompanied by the induction of HSP70 mRNA after 1-min ischemia. The results with strong induction of both HSP70 and HSC70 mRNAs after 2 min of ischemia demonstrate the occurrence of the co-induction of HSP70 and HSC70 mRNAs in hippocampal cells (Fig. 3) and the long-lasting co-induction in CA1 cells at 2 days (Fig. 3-2d). The co-induction of HSP70 and HSC70 mRNAs has generally been found after 10 min of transient global ischemia in gerbils ~5 and after 30 min of transient focal ischemia of rats ~6. From the suggested roles of HSP70 and HSC70 under stressful conditions 5'9A°33'2°'26'27, the co-induction of HSP70 and HSC70 mRNAs may indicate a cooperative role of these proteins for an active
folding of damaged a n d / o r newly synthesized proteins in hippocampal cells of post-ischemic brain. Two min of ischemia produced little morphological damage in brain cells even at 7 days (data not shown, ref. 14) in contrast to an evident damage in CA1 cells after 5-min ischemia337. However, the cooperative induction of HSP70 and HSC70 mRNAs after 2 min of ischemia was similar to that after 5-min ischemia although the extent and the intensity of hybridization were more severe in the latter (Kawagoe et al., unpublished data). Therefore, the ischemia of 2 to 5 min induced the same pattern of stress response in vulnerable CA1 cells independently of the lethality. The involvement of HSP70 and HSC70 in CA1 cell death after ischemia has been suggested 2'1538'33. Recently, Kirino et alJ 8 reported that 2 min of ischemia successfully produced HSP70 immunoreactivity in CA1 cells but that 5 min of ischemia failed. Combined with our results in 2 rain of ischemia, the transcriptional and the translational activities in producing HSP70 and the transcriptional activity in producing HSC70 are well maintained in CA1 ceils after 2 min of ischemia. Although immunoreactivity for HSC70 after lethal and non-lethal ischemia has not been examined, it is speculated that translational failure in HSPs after 5-min ischemia may relate to the lethality of the CA1 ceils after ischemia. Transcriptional failure in producing HSP70 and HSC70 in CA1 cells has been reported at around 2 days after 10 min of ischemia of gerbils ~5. One min of ischemia selectively induced HSC70 mRNA in cells of hippocampus especially dentate gyrus after the reperfusion, while an induction of HSP70 mRNA was not evident (Fig. 2-1h and 3h). Recently, such selective induction of HSC70 mRNA without HSP70 mRNA induction has been suggested in some hippocampal cells in transient focal ischemia 16 and seizure 32 models of rats by in situ hybridization and in amphetamine-treated hyperthermic brain of rats by Northern blot analysis22. Miller et al. 22 suggested that the elevation in HSC70 mRNA levels would be sufficient to protect cells from lower levels of stress and HSP70 would be induced only in higher levels of stress. Therefore, the selective induction of HSC70 mRNA in the present study may indicate that 1 min of ischemia produced a minimum stress to cells which may be protected by only further induction of constitutive HSC70, and that the induction of HSP70 may be required in more stressful conditions such as 2 or more than 2 min of ischemia. The present study demonstrated that the selective induction of HSC70 mRNA was pronounced in granule cells of dentate gyrus at 1 and 3 h after the reperfusion and diminished by 2 days (right column in Fig. 2). In
202 the previous experiments, such selective HSC70 mRNA induction was again predominant in the dentate granule cells 16'32. We reported the occurrence of the selective induction from 1 h to 1 day after transient focal ischemia t6. Wong et al. 32 revealed that the increased levels of the mRNA was present at 2 h but returned to baseline within 24 h after seizure, and suggested that HSC70 may play a role in the adaptation a n d / o r in the maintenance of the dentate gyrus cell integrity following seizures. These results suggest the selective induction of HSC70 mRNA occurs relatively selective in dentate granule cells within relatively early phase after transient ischemia or seizure. In addition to the suggestion of Wong et al. 32, we postulate that the cells of dentate gyrus may be sensitive to ischemia a n d / o r postischemic stresses such as glutamate release and changes in blood flow and body temperature. The increase in body temperature after transient ischemia has been demonstrated to play a role in neuronal i n j u r y 19'31 and the different increase in body temperature has been reported to produce different mRNA induction of HSP70 and HSC7022. In the drug-induced hyperthermic rat, relative levels of HSC70 mRNA increased at body temperatures greater than 39°C, whereas HSP70 mRNA synthesis was induced at temperatures greater than 40°C 22. Thus, the change in body temperature may relate to the different thresholds of the induction of HSP70 and HSC70 mRNAs observed in the present study. Based on the fact that the peak of body temperature at 30 min after 2 min of ischemia was significantly higher than those after 0.5 and 1 min of ischemia (Fig. 4), there may be a relation between the marked increase in body temperature and the threshold of induction of HSP70 mRNA. However, the same change of the body temperature after 0.5 or 1 min of ischemia (Fig. 4) made a difference in the induction of HSC70 mRNA. Therefore, both the duration of ischemia and body temperature may be important factors to determine thresholds of the induction of HSP70 and HSC70 mRNAs. Acknowledgments. This work was partly supported by Monbusho Grants 01044018 and 03404028, a grant of the Ministry of Welfare and Health of Japan, and the Sasakawa medical research foundation. The authors would like to express their appreciation to Drs. S. Sato and M. Aoki, and Mrs. M. Matsumoto for their excellent technical assistance.
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3 Araki, T., Kato, H. and Kogure, K.. Selective neuronal vulnerability following transient cerebral ischemia in the gerbil: Distribu tion and time course, Acta Neurol, Stand., 8tl ( 19891 548-553. 4 Barbe, M.F., Tytell, M., Gower, D.J. and Welch. W..l.. ltyperthermia protects against light damage in the rat retina. Science, 241 (1988) 1817 1820. 5 Beckmann, R.P., Mizzen. L.A. and Welch, W.J.. lnleractio~ o[ hsp 70 with newly synthesized proteins: Implications for protein folding and assembly, Science, 248 (19901 850 ~54. 6 Brown, I.R., Rush, S. and Ivy, G.O.. Induction of a heat shock gene at the site of tissue injury, in the rat brain. Neuron, 2 (19891 1559-1564. 7 Collins, R.C., Selective vulnerability of brain: new insights from the excitatory synapse, Metab. Brain Dis., 1 (19861 231-240. 8 Feinberg, A.P. and Vogelstein, B., A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity, Anal, Biochem., 132 (1983) 6-13. 9 Flaherty, K.M., DeLuca-Flaherty, C. and McKay, D.B., Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein, Nature, 346 (1990) 623-628. 10 Gething, M.J. and Sambrook, J., Protein folding in the cell, Nature, 355 (19921 33-45. 11 Gonzalez, M.F., Shiraishi, K., Hisanaga, K., Sagar, S.M., Mandabach, M. and Sharp, F.R., Heat shock proteins as markers of neuronal injury, Mot. Brain Res., 6 (1989) 93-10(I. 12 Gonzalez, M.F., Lowenstein, D., Fernyak, S., Hisanaga, K., Simon, R. and Sharp, F.R., Induction of heat shock protein 72-like immunoreactivity in the hippocampal formation following transient global ischemia, Brain Res. Bull., 26 (199l) 241-250. 13 Hightower, L.E., Heat shock, stress proteins, chaperones, and proteotoxicity, Cell, 66 (1991) 191-197. 14 Kato, H., Liu, Y., Araki, T. and Kogure, K., Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects. Brain Res., 553 (1991) 238-242. 15 Kawagoe, J., Abe, K., Sato, S., Nagano, 1., Nakamura, S. and Kogure, K., Distributions of heat shock protein-70 mRNAs and heat shock cognate protein-70 mRNAs after transient global ischemia in gerbil brain, Z Cereb. Blood Flow Metab., 12 (1992) 794-801. 16 Kawagoe, J., Abe, K., Sato, S., Nagano, I., Nakamura, S. and Kogure, K., Distributions of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs after transient focal ischemia in rat brain, Brain Res., 587 (1992) 195-202. 17 Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57-69. 18 Kirino, T., Tsujita, Y. and Tamura, A., Induced tolerance to ischemia in gerbil hippocampal neurons, Z Cereb. Blood Flow Metab., 11 (19911 299-307. 19 Kuroiwa, T., Bonnekoh, P. and Hossmann, K.-A., Prevention of postischemic hyperthermia prevents ischemic injury of CA l neurons in gerbils, J. Cereb. Blood Flow Metab,, 10 (19901 550-556. 20 Lewis, M.T. and Pelham, H.R.B., Involvement of ATP in the nuclear and nucleolar functions of the 70kd heat shock protein. EMBO J., 4 (1985) 3137-3143. 21 Lindquist, S., The heat-shock response, Annu. Ret. Biochem.. 55 (1986) 1151-1191. 22 Miller, E.K., Raese, J.D. and Morrison-Bogorad, M., Expression of heat shock protein 70 and heat shock cognate 70 messenger RNAs in rat cortex and cerebellum after heat shock or amphetamine treatment, J. Neurochem., 56 (1991) 2060-2071. 23 Nowak Jr., T.S., Bond, U. and Schlesinger, M.J., Heat shock RNA levels in brain and other tissues after hyperthermia and transient ischemia, J. Neurochem., 54 (19901 451-458. 24 Nowak Jr., T.S., Localization of 70 kDa stress protein mRNA induction in gerbil brain after ischemia, J. Cereb. Blood Flow Metab., 11 (1991) 432-439. 25 Nowak Jr., T.S. and Osborne, O.C., Threshold ischemic duration for stress protein induction in gerbil brain, Stroke, 9_2 (1991) 131. 26 Pelham, H.R.B., HSP70 accelerates the recovery of nucleolar morphology after heat shock, EMBO J., 3 (10841 3005-310(t.
203 27 Pelham, H.R.B., Functions of the hsp70 protein family: An overview. In R.I. Morimoto, A. Tissi6res and C. Georgopoulos (Eds.), Stress Proteins in Biology and Medicine, Cold Spring Harbor Laboratory Press, New York, 1990, pp. 287-299. 28 Riabowol, K.T., Mizzen, L.A. and Welch, W.J., Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70, Science, 242 (1988) 433-436. 29 Sato, S., Abe, K., Kawagoe, J., Aoki, M. and Kogure, K., Isolation of complementary DNAs for heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 genes and expressions in post ischemic gerbil brain, Neurol. Res., (1992) in press. 30 Vass, K., Welch, W.J. and Nowak Jr., T.S., Localization of 70-kDa stress protein induction in gerbil brain after ischemia, Aeta Neuropathol. (Berl.), 77 (1988) 128-135. 31 Welsh, F.A., Sims, R.E. and Harris, V.A., Mild hypothermia
prevents ischemic injury in gerbil hippocampus, Z Cereb. Blood Flow Metab., 10 (1990) 557-563. 32 Wong, M.-L., Weiss, S.R.B., Gold, P.W., Doi, S.Q., Banerjee, S, Licinio, J., Lad R., Post, R.M. and Smith, M.A., Induction of constitutive heat shock protein 73 mRNA in the dentate gyrus by seizures, Mol. Brain Res., 13 (1992) 19-25. 33 Yoshimi, K., Takeda, M., Nisbimura, T., Kudo, T., Nakamura, Y., Tada, K. and Iwata, N., An immunobistochemical study of MAP2 and clathrin in gerbil hippocampus after cerebral ischemia, Brain Res., 560 (1991) 149-158. 34 Yoshioka, M., Nagano, I., Nakamura, S., Imaizumi, M. and Kimura, N., Detection of vasoactive intestinal polypeptide messenger RNA in ganglioneuroblastoma by in situ hybridization, Endocr. Pathol., 1 (1990) 51-57.
197
BRES 18369
Different thresholds of HSP70 and HSC70 heat shock mRNA induction in post-ischemic gerbil brain J. K a w a g o e ,
K. Abe and K. Kogure
Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan) (Accepted 21 July 1992)
Key words: Heat shock protein; Heat shock cognate protein; Cerebral ischemia; In situ hybridization; Gerbil
Thresholds of induction of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs after transient global ischemia in gerbil brain were investigated by in situ hybridization using cloned cDNA probes selective for each mRNA species. In sham control brain, HSP70 mRNA was little present, while HSC70 mRNA was present in most cell populations. A 0.5-min occlusion of bilateral common carotid arteries did not affect the amount of HSP70 and HSC70 mRNAs. The selective induction of HSC70 mRNA was observed in dentate granule cells at 1 h, and in most cells of hippocampus especially dentate gyrus at 3 h after 1 min of ischemia when induction of HSP70 mRNA was not evident in the identical brain. The selective induction diminished by 2 days. However, after 2 rain of ischemia, HSP70 and HSC70 mRNAs were induced together in hippocampal cells from 1 h of the reperfusion, and the co-induction prolonged in CA1 cells until 2 days. Body temperatures monitored at rectum increased after the reperfusion with a peak at 30 min. The degree of increase of the body temperature was significantly higher in the case after 2-min ischemia than in the cases after 0.5- and 1-min ischemia. Although HSP70 and HSC70 mRNAs are generally co-induced in stressful conditions, our results suggest the different thresholds of the induction between HSP70 and HSC70 mRNAs after transient brain ischemia. The selective induction of HSC70 mRNA which is not accompanied by the induction of HSP70 mRNA may relate to the differences of the duration of ischemia and the degree of the increase of body temperature after ischemia.
INTRODUCTION
different roles u n d e r n o r m a l c o n d i t i o n a n d the cooperative role of H S P 7 0 a n d HSC70 in the recovery process
V a r i o u s stressful c o n d i t i o n s of cells i n d u c e 'stress r e s p o n s e ' a n d s u b s e q u e n t l y p r o d u c e heat-shock proteins (HSPs) 6'11'13'21'27. B r a i n ischemia is o n e of such
from the ischemic injury. I n a d d i t i o n to the global ischemia m o d e l of gerbils, the c o - i n d u c t i o n of the
stresses to p r o d u c e HSPs i n c l u d i n g HSP7012'15'18'3° a n d the m R N m s 1'2'15'16'23-25'29. H S P 7 0 a n d heat-shock cogn a t e p r o t e i n ( H S C ) 70 are the m e m b e r s of so-called HSP70 family, a n d their protective roles in cells from the injury have b e e n suggested 2,4,tS'16AS'21,22,26-28. However, the precise role of the HSP70 a n d H S C 7 0 in ischemic b r a i n has n o t yet b e e n fully u n d e r s t o o d . W e have investigated the d i s t r i b u t i o n s of H S P 7 0 a n d HSC70 m R N A s after 10 m i n of global ischemia in gerbils with in situ h y b r i d i z a t i o n u s i n g c l o n e d c D N A s selective for the m R N A s 15. I n c o n t r a s t to the a b s e n c e of HSP70 m R N A , H S C 7 0 m R N A was p r e s e n t in the sham control brain. T h e m R N A s were c o - i n d u c e d after ischemia in b r a i n regions. T h e s e results suggested the
m R N A s was also d e t e c t e d in focal ischemia model of rats 16. However, HSC70 m R N A was selectively ind u c e d in some h i p p o c a m p a l cells of the rat m o d e l w h e r e obvious i n d u c t i o n of HSP70 m R N A was n e v e r found. Miller et al. 22 suggested that different i n d u c t i o n of HSP70 a n d HSC70 m R N A s occurred in amp h e t a m i n e - t r e a t e d h y p e r t h e r m i c b r a i n of rats by N o r t h e r n blot analysis, a n d that the elevation of HSC70 would be sufficient to protect cells from a relatively m i l d e r stress while the i n d u c t i o n of HSP70 would be r e q u i r e d in m o r e severe stress. Recently, W o n g et al. 32 also observed a selective i n d u c t i o n of HSP73 (HSC70) in the d e n t a t e gyrus by seizures in rats whereas n e i t h e r HSP70 n o r HSP84 genes were induced. However, no such dissociation in the i n d u c t i o n of HSP70 a n d HSC70
Correspondence: J.-i. Kawagoe, Tokyo Research Laboratories, Pharmaceutical Division, Kowa Company, LTD., 2-17-43, Noguchi-cho, Higashimurayama, Tokyo 189, Japan. Fax: (81) (423) 95 0312.
198
mRNAs
has been
reported
a f t e r t r a n s i e n t g l o b a l is-
c h e m i a in b r a i n .
or 2 min of ischemia at 30 and 60 min alter thu rcperfusion m~(i histopathological observation with Cres'd violet staining ~s was made at 7 days after the rcperfusion (n - 4 6).
Therefore, we have investigated possible thresholds of the induction of HSP70 and HSC70 mRNAs transient
global ischemia of gerbil brain
after
by in situ
hybridization technique. MATERIALS AND METHODS
Animal model Male Mongolian gerbils (Meriones unguiculatus), aged 10-11 weeks and weighing 70-75 g, were lightly anesthetized by inhalation of a nitrous oxide/oxygen/halothane (69% :30%: 1%) mixture. Both common carotid arteries were exposed and then the anesthesia was stopped. When the animal began to regain consciousness, the arteries were occluded for 0.5, 1, or 2 rain using surgical clips 2. Body temperatures were monitored in all animals with a rectal probe, and was maintained at 37°C using a heat pad during the surgical preparation and the occlusion of the carotid arteries. After the restoration of blood flow, no attempt was made to maintain constant body temperature of animals. The animals recovered for 1, 3 h and 2 days (n = 3 in each time point) at the ambient temperature (21-23°C), and then were decapitated. Sham animals were sacrificed just after exposing the carotid arteries without clamping the vessels. The dissected brains were frozen in powdered dry ice and stored at -80°C. Sections (10 p.m) at dorsal hippocampal level were cut on a cryostat at - 18°C and collected on the slides coated with Histostik (Accurate Chemical and Scientific Corp., Westbury, NY). In situ hybridization for HSP70 and HSC70 mRNAs was performed in the sections. In another experiment, rectal temperature was monitored after 0.5, 1,
HSP70
hz situ hybridization The inserts of cloned cDNAs used as probes in lhe present study were originally from cerebral cortex of gerbils. The sizes of the inserts selective for HSP70 and HSC70 mRNAs were 1.0 (pGA ~) and 1.4 kb (pGD3), respectively 29. In situ hybridization was performed by a method of Yoshioka et al. 3~ with a slight modification. Briefly, the sections were fixed for I(~ min in an ethanol/acetic acid (3:1) mixture, transferred to 0.2 M HCI for 20 rain, and immersed for 20 min at 50°C in 2×SSC (pH 7.0). They then were digested for 15 min at 37°C with 100/~g/ml of proteinase K (Merk & Co. Inc., Rahway, NJ) in 20 mM Tris-HCI buffer (pH 7.4) with 2 mM CaCl 2 and dehydrated through graded ethanol. Slides were prehybridized for 2 h at room temperature in a solution containing 50% formamide, 600 mM NaCI, l0 mM Tris-HC1 (pH 7.5), 1 mM EDTA, 0.02%, polyvinylpyrrolidone, 0.02% Ficoll. 0.02% bovine serum albumin, 100 p.g/ml sonicated, denatured calf thymus DNA, and 0.5 mg/ml calf liver RNA. Hybridization was done for 20 h at 42°C using SsS-labeled cDNA probe at a concentration of 0.1 ng//~l in prehybridization solution supplemented with 10% dextran sulfate and 10 mM dithiothreitol. The cDNA inserts were radiolabeled with [~-3sS]deoxycytidine triphosphate (1(100 Ci/mmol, Amersham) by random primer labeling s using a kit (Boehringer Mannheim Yamanouchi Co. Ltd., Tokyo, Japan), resulting in specific activities of 5-6 x 10 ~ dpm//sg. The control probe was a similarly-labeled plasmid vector (pHSG396, Takara Shuzo Co. Ltd., Kyoto, Japan) fragment (1.1 kb) digested by Hinfl (Takara). The sections were washed at 42°C for 2 h in 2xSSC and for I h in 1 x SSC, and then were dehydrated through graded ethanol series containing 0.3 M ammonium acetate. After visualization of the
HSC70
lh I I1!
Fig. 1. Distributions of HSP70 (left column) and HSC70 (right column) mRNAs at the level of dorsal hippocampus in the identical brain of sham control (S) and the post-ischemic gerbils at 1 h after 0.5 min of ischemia (lh). Note hybridization of HSC70 mRNA in most cell populations of the sham brain in contrast to little hybridization of HSP70 mRNA, and no significant effect of 0.5-min ischemia on the induction of HSP70 and HSC70 mRNAs. Bar = 3.1 ram.
199 hybridization by exposure against X-ray film for 24 h at room temperature, the sections were dipped in a liquid emulsion (NR-M2, Konica Co. Ltd., Tokyo, Japan) diluted (1 : 1) with 0.6 M ammonium acetate. They were then exposed at 4°C for approximately 3 weeks, developed, and counterstained with hematoxylin. Several slides were treated with 100 p,g/ml RNase A and 10 units/ml RNase T1 (Sigma, St. Louis. MO, USA) in 2×SSC at 37°C for 2 h prior to prehybridization and were hybridized with the probe for HSP70 or HSC70 mRNA.
d a n t l y p r e s e n t (Fig. l-S). A relatively high level of H S C 7 0 m R N A was o b s e r v e d in h i p p o c a m p a l C A 3 cells a n d t h e o r d e r o f h y b r i d i z a t i o n intensity o f H S C 7 0 m R N A in h i p p o c a m p a l cells was C A 3 > CA1 > d e n t a t e gyrus g r a n u l e . A 0.5 min o f t r a n s i e n t i s c h e m i a did not significantly affect t h e a m o u n t o f H S P 7 0 o r H S C 7 0 m R N A s at all e x a m i n e d time p o i n t s after the r e p e r f u s i o n (Fig. 1 - 1 h ) . H o w e v e r , o n e min o f i s c h e m i a a p p a r e n t l y i n d u c e d a f u r t h e r H S C 7 0 m R N A in d e n t a t e g r a n u l e cells, indic a t e d by a r r o w h e a d s in the figure, at 1 h as c o m p a r e d to t h e s h a m level (right c o l u m n in Fig. l-S), w h e r e a s no c h a n g e o f H S P 7 0 m R N A level o c c u r r e d again in the i d e n t i c a l b r a i n (Fig. 2 - 1 h ) . Such selective i n d u c t i o n o f H S C 7 0 m R N A was m o r e e v i d e n t in all cell types o f h i p p o c a m p u s especially d e n t a t e gyrus at 3 h a f t e r the r e p e r f u s i o n , w h e n t h e o r d e r o f t h e h y b r i d i z a t i o n intensity o f H S C 7 0 m R N A in h i p p o c a m p a l ceils c h a n g e d f r o m C A 3 > CA1 > d e n t a t e gyrus g r a n u l e in s h a m b r a i n (right c o l u m n in Fig. l - S ) to C A 3 >__d e n t a t e gyrus granule > CA1 (right c o l u m n in Fig. 2 - 3 h ) . Even at this t i m e point, 1 min of ischemia d i d not i n d u c e H S P 7 0 m R N A (left c o l u m n in Fig. 2 - 3 h ) . T h e level of H S C 7 0
Statistics The values obtained were expressed as means + S.E.M. Statistical comparisons were made by the Mann-Whitney U-test after the Kruskal-Wallis analysis. RESULTS
In situ hybridization T h e results o b t a i n e d in in situ h y b r i d i z a t i o n w e r e r e p r o d u c i b l e in all a n i m a l s u s e d in e a c h t i m e point. Figs. 1 to 3 show t h e a u t o r a d i o g r a m s o f the sections o f i d e n t i c a l b r a i n s u b j e c t e d to several d u r a t i o n s o f i s c h e m i a a n d r e p e r f u s i o n for the d e t e c t i o n o f H S P 7 0 a n d H S C 7 0 m R N A s by in situ h y b r i d i z a t i o n . In the s h a m c o n t r o l brain, H S C 7 0 m R N A was p r e s e n t in most cell p o p u l a t i o n s , while H S P 7 0 m R N A was n o t a b u n -
HSC70
HSP70 i
ii~
~, ili~iiii!~¸~ili~
1 h
r
3h
k
•
2d
Fig. 2. Chronological change of HSP70 (left column) and HSC70 (right column) mRNAs at 1 (lh), 3 h (3h) and 2 days (2d) after 1 min of ischemia. Note slight but significant induction of HSC70 mRNA in dentate gyrus granule cells (arrowheads) at 1 h and all cell types of hippocampus especially dentate gyrus at 3 h relative to the sham level (right column in Fig. l-S), while an induction of HSP70 mRNA was not evident. Bar = 3.1 mm.
200
HSP70
HSC70
lh
3h
2d
Fig. 3. Chronological change of HSP70 (left column) and HSC70 (right column) m R N A s at 1 (lh), 3 h (3h) and 2 days (2d) after 2 min of ischemia. Note a co-induction of HSP70 and HSC70 m R N A s from 1 h to 2 days and the long-lasting hybridization of the m R N A s in CA1 cells even at 2 days. Bar = 3.1 mm.
mRNA returned to the sham level with little change in HSP70 mRNA at 2 days after 1-min ischemia (Fig. 2-2d). After 2 min of ischemia, both HSP70 and HSC70 mRNAs were markedly induced in a cooperative manner in dentate granule cells at 1 h (Fig. 3-1h) and in all cell types of hippocampus especially dentate gyrus at 3 h (Fig. 3-3h). Strong hybridization of HSP70 and HSC70 mRNAs remained in CA1 cells even at 2 days after the reperfusion (Fig. 3-2d) although the hybridization of the mRNAs in cells of dentate gyrus and CA3 diminished or returned to the sham level. Observation of sections dipped in the liquid emulsion revealed that grains for HSP70 and HSC70 probes were predominantly located in the neuronal cell body (data not shown). Neither the sections hybridized with the probe from plasmid vector nor the sections treated with RNases before incubation with the probe for HSP70 or HSC70 mRNA exhibited any specific hybridization (data not shown).
Histopathological study Cresyl violet staining revealed that 2 min of ischemia resulted in little morphological change even at 7 days after the reperfusion (data not shown) as reported previously TM.
Body temperature Fig. 4 shows the changes of body temperature after different durations of ischemia (0.5-2 min). Transient ischemia definitely increased the body temperature at 30 and 60 min after the reperfusion with the peak time at 30 min. The peak of the increased body temperature in case of 2 min of ischemia (39.8 + 0.1°C, n = 5) was
40.0
"
0
39.0
~
38.0
E ~" "0 0 rn
37.0
36.0
Pre
Ischemia
30 min
60 min
Fig. 4. C h a n g e s of body temperature monitored at rectum before (Pre) and during ischemia (Ischemia), and at 30 and 60 rain after the reperfusion in gerbil. The peak body temperature at g0 ~ after the reperfusion was significantly higher in the case With 2 min of ischemia (e) than in those with 1 ( D ) and 0.5 (©) min of ischemia. * P < 0.01 vs. 0.5 and 1 min of ischemia groups (n = 4-6).
201 significantly higher than those of 1 min (38.7 _+0.1°C, n = 6) and 0.5 min (38.5 + 0.1°C, n = 4) of ischemia (P < 0.01). There was no statistical difference between the body temperature in the different durations of ischemia at 60 min after the reperfusion. DISCUSSION Northern blot analysis revealed that the HSP70 and HSC70 probes employed in the present study selectively detected HSP70 and HSC70 mRNAs, respectively, in the gerbil brain and did not cross-react 15,29. In in situ hybridization analysis, grains for HSP70 and HSC70 probes were located predominantly in the cell body. The specificities of the probes were further supported by the negative signal after a hybridization with the irrelevant plasmid vector probe and by the eliminated signal after pretreatment with RNases prior to incubation with the probe for HSP70 or HSC70 mRNA. These results provide the evidence that the HSP70 and HSC70 cDNA probes used here selectively recognize HSP70 and HSC70 mRNAs of gerbil brain in situ, respectively. HSP70 mRNA was induced by 2 min of forebrain ischemia from 1 h to 2 days after the reperfusion (left column in Fig. 3). However, 1 or 0.5 min of ischemia did not induce HSP70 mRNA (left column in Fig. 1-1h and Fig. 2). These results are in good accordance with that by Nowak and Osborne 25. They reported that 2 min of ischemia induced HSP70 mRNA while 1 min of ischemia did not. Further induction of HSC70 mRNA relative to the sham level was evident after 2 (right column in Fig. 3) and 1 (right column in Fig. 2-1h and 3h) min of ischemia. However, after 0.5 min of ischemia, further induction of HSC70 mRNA was not detected (right column in Fig. 1-1h). These results indicate the different thresholds of the induction of HSP70 (2 min) and HSC70 (1 min) mRNAs in transient ischemia model of gerbils and the selective induction of HSC70 mRNA which is not accompanied by the induction of HSP70 mRNA after 1-min ischemia. The results with strong induction of both HSP70 and HSC70 mRNAs after 2 min of ischemia demonstrate the occurrence of the co-induction of HSP70 and HSC70 mRNAs in hippocampal cells (Fig. 3) and the long-lasting co-induction in CA1 cells at 2 days (Fig. 3-2d). The co-induction of HSP70 and HSC70 mRNAs has generally been found after 10 min of transient global ischemia in gerbils ~5 and after 30 min of transient focal ischemia of rats ~6. From the suggested roles of HSP70 and HSC70 under stressful conditions 5'9A°33'2°'26'27, the co-induction of HSP70 and HSC70 mRNAs may indicate a cooperative role of these proteins for an active
folding of damaged a n d / o r newly synthesized proteins in hippocampal cells of post-ischemic brain. Two min of ischemia produced little morphological damage in brain cells even at 7 days (data not shown, ref. 14) in contrast to an evident damage in CA1 cells after 5-min ischemia337. However, the cooperative induction of HSP70 and HSC70 mRNAs after 2 min of ischemia was similar to that after 5-min ischemia although the extent and the intensity of hybridization were more severe in the latter (Kawagoe et al., unpublished data). Therefore, the ischemia of 2 to 5 min induced the same pattern of stress response in vulnerable CA1 cells independently of the lethality. The involvement of HSP70 and HSC70 in CA1 cell death after ischemia has been suggested 2'1538'33. Recently, Kirino et alJ 8 reported that 2 min of ischemia successfully produced HSP70 immunoreactivity in CA1 cells but that 5 min of ischemia failed. Combined with our results in 2 rain of ischemia, the transcriptional and the translational activities in producing HSP70 and the transcriptional activity in producing HSC70 are well maintained in CA1 ceils after 2 min of ischemia. Although immunoreactivity for HSC70 after lethal and non-lethal ischemia has not been examined, it is speculated that translational failure in HSPs after 5-min ischemia may relate to the lethality of the CA1 ceils after ischemia. Transcriptional failure in producing HSP70 and HSC70 in CA1 cells has been reported at around 2 days after 10 min of ischemia of gerbils ~5. One min of ischemia selectively induced HSC70 mRNA in cells of hippocampus especially dentate gyrus after the reperfusion, while an induction of HSP70 mRNA was not evident (Fig. 2-1h and 3h). Recently, such selective induction of HSC70 mRNA without HSP70 mRNA induction has been suggested in some hippocampal cells in transient focal ischemia 16 and seizure 32 models of rats by in situ hybridization and in amphetamine-treated hyperthermic brain of rats by Northern blot analysis22. Miller et al. 22 suggested that the elevation in HSC70 mRNA levels would be sufficient to protect cells from lower levels of stress and HSP70 would be induced only in higher levels of stress. Therefore, the selective induction of HSC70 mRNA in the present study may indicate that 1 min of ischemia produced a minimum stress to cells which may be protected by only further induction of constitutive HSC70, and that the induction of HSP70 may be required in more stressful conditions such as 2 or more than 2 min of ischemia. The present study demonstrated that the selective induction of HSC70 mRNA was pronounced in granule cells of dentate gyrus at 1 and 3 h after the reperfusion and diminished by 2 days (right column in Fig. 2). In
202 the previous experiments, such selective HSC70 mRNA induction was again predominant in the dentate granule cells 16'32. We reported the occurrence of the selective induction from 1 h to 1 day after transient focal ischemia t6. Wong et al. 32 revealed that the increased levels of the mRNA was present at 2 h but returned to baseline within 24 h after seizure, and suggested that HSC70 may play a role in the adaptation a n d / o r in the maintenance of the dentate gyrus cell integrity following seizures. These results suggest the selective induction of HSC70 mRNA occurs relatively selective in dentate granule cells within relatively early phase after transient ischemia or seizure. In addition to the suggestion of Wong et al. 32, we postulate that the cells of dentate gyrus may be sensitive to ischemia a n d / o r postischemic stresses such as glutamate release and changes in blood flow and body temperature. The increase in body temperature after transient ischemia has been demonstrated to play a role in neuronal i n j u r y 19'31 and the different increase in body temperature has been reported to produce different mRNA induction of HSP70 and HSC7022. In the drug-induced hyperthermic rat, relative levels of HSC70 mRNA increased at body temperatures greater than 39°C, whereas HSP70 mRNA synthesis was induced at temperatures greater than 40°C 22. Thus, the change in body temperature may relate to the different thresholds of the induction of HSP70 and HSC70 mRNAs observed in the present study. Based on the fact that the peak of body temperature at 30 min after 2 min of ischemia was significantly higher than those after 0.5 and 1 min of ischemia (Fig. 4), there may be a relation between the marked increase in body temperature and the threshold of induction of HSP70 mRNA. However, the same change of the body temperature after 0.5 or 1 min of ischemia (Fig. 4) made a difference in the induction of HSC70 mRNA. Therefore, both the duration of ischemia and body temperature may be important factors to determine thresholds of the induction of HSP70 and HSC70 mRNAs. Acknowledgments. This work was partly supported by Monbusho Grants 01044018 and 03404028, a grant of the Ministry of Welfare and Health of Japan, and the Sasakawa medical research foundation. The authors would like to express their appreciation to Drs. S. Sato and M. Aoki, and Mrs. M. Matsumoto for their excellent technical assistance.
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prevents ischemic injury in gerbil hippocampus, Z Cereb. Blood Flow Metab., 10 (1990) 557-563. 32 Wong, M.-L., Weiss, S.R.B., Gold, P.W., Doi, S.Q., Banerjee, S, Licinio, J., Lad R., Post, R.M. and Smith, M.A., Induction of constitutive heat shock protein 73 mRNA in the dentate gyrus by seizures, Mol. Brain Res., 13 (1992) 19-25. 33 Yoshimi, K., Takeda, M., Nisbimura, T., Kudo, T., Nakamura, Y., Tada, K. and Iwata, N., An immunobistochemical study of MAP2 and clathrin in gerbil hippocampus after cerebral ischemia, Brain Res., 560 (1991) 149-158. 34 Yoshioka, M., Nagano, I., Nakamura, S., Imaizumi, M. and Kimura, N., Detection of vasoactive intestinal polypeptide messenger RNA in ganglioneuroblastoma by in situ hybridization, Endocr. Pathol., 1 (1990) 51-57.