Journal of Cerebral Blood Flow and Metabolism 12:794-S0I © 1992 The International Society of Cerebral Blood Flow and Metabolism Published by Raven Press, Ltd" New York
Distributions of Heat Shock Protein-70 mRNAs and Heat Shock Cognate Protein-70 mRNAs After Transient Global Ischemia in Gerbil Brain
J. Kawagoe, K. Abe, S. Sato, I. Nagano, S. Nakamura, and K. Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai, Japan
Summary: Distributions of heat shock protein (HSP)-70 mRN As and heat shock cognate protein (HSC)-70 mRNAs after 10 min of transient global ischemia were investigated in gerbil forebrain by in situ hybridization using cloned eDNA probes selective for the mRNAs. Ex pression of HSP70 immunoreactivity was also examined in the same brains. In hippocampal CAl neuronal cells, in which only a minimal induction of immunoreactive HSP70 protein was found, the strong hybridization for HSP70 mRNA disappeared at around 2 days before the death of CAl cells became evident. Furthermore, in hip pocampal CA3 cells, a striking induction of HSP70 mRNA was sustained even at 2 days along with a prom inent accumulation of HSP70 immunoreactivity. In con trast to the case of HSP70 mRNA, HSC70 mRNA was present in most neuronal cells, especially dense in CA3 cells, of the sham brain. A co-induction of HSP70 and HSC70 mRNAs was observed in several cell populations after the reperfusion with a peak at 8 h, although the
magnitude of HSC70 mRNA induction was lower than that of HSP70 mRNA, particularly in CAl cells. The ex pression of HSC70 mRNA in CAl cells also disappeared at around 2 days. All the induced signals of HSP70 and HSC70 mRNAs in other cell populations were diminished and returned to the sham level, respectively, by 7 days. These results are the first to show the time courses of distribution of HSP70 and HSC70 mRNAs and the immu noreactive HSP70 protein in the same gerbil brain after ischemia. The results suggest that the weak induction of HSP70 protein in CAl cells, which may relate to the vul nerability of this cell population, is due to both transla tional and transcriptional deficits. The different roles un der normal condition and the cooperative role in the re covery process from ischemic injury between HSP70 and HSC70, and the involvement of HSC70 in CAl cell death, are suggested. Key Words: Heat shock protein-Heat shock cognate protein-Cerebral ischemia-In situ hy bridization-Gerbil.
Selective neuronal vulnerability after global isch emia has been demonstrated in human (Petito et al., 1987) and rodent brains (Kirino, 1982; Pulsinelli et al., 1982; Smith et al., 1984; Hatakeyama et al. , 1988; Araki et al. , 1989; Araki et al. , 1991). In gerbil brain, hippocampal CAl pyramidal neurons are killed after transient ischemia while other neurons such as dentate gyrus granule cells and hippocam pal CA3 cells are much less vulnerable (Kirino, 1982; Hatakeyama et al., 1988; Araki et al., 1989;
Araki et al. , 1991). It is also demonstrated that tran sient global ischemia induces 70 kDa-heat shock protein (HSP70), which has been suggested to play a protective role under stressful conditions (Pel ham, 1984; Lindquist, 1986; Barbe et al. , 1988; Kirino et al. , 1991; Abe et al. , 1991). Recent immunohistochemical studies have re vealed a regional difference in the induction of im munoreactive HSP70 protein between the most vul nerable CAl cells and the more resistant CA3 and dentate granule cells after ischemia. Vass et al. (1988) reported a minimal induction of the HSP70 immunoreactivity in CAl cells of gerbil brain after ischemia, while CA3 and dentate granule cells showed pronounced immunoreactivity of HSP70. Magnusson and Wieloch (1989) demonstrated that the decreased immunostaining of ubiquitin, a stress inducible protein, in the CAl cells after ischemia
Received December II, 1991; final revision received March 5, 1992; accepted March 25, 1992. Address correspondence and reprint requests to Dr. 1. Kawagoe, Department of Neurology, Institute of Brain Dis eases, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan. Abbreviations used: ABC, avidin-biotin-peroxidase; HSP, heat shock protein; HSC, heat shock cognate protein; SSC, so dium chloride/sodium citrate; SDS, sodium dodecyl sulfate.
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HSP70 AND HSC70 mRNAs IN POSTISCHEMIC GERBIL BRAIN
failed to recover although that in the other resistant cells of rat hippocampus recovered. Furthermore, a lower level of HSP70 induction in hippocampal CA1 cells was detected at the transcriptional level by Northern blot analysis in a to-min ischemia study in gerbils (Abe et aI. , 1991). In contrast to this result, the in situ hybridization technique revealed a strong and prolonged hybridization of HSP70 mRNA in CAl cells of gerbil brain after 5-min ischemia (Nowak, 1991). These results indicate the impor tance of investigating the regional distributions of immunoreactivity and mRNA of the HSP70 family in the same ischemic brain in order to consider mechanisms of ischemia-induced selective vulnera bility. Heat shock cognate protein (HSC)-70 is a mem ber of the HSP70 family, and is constitutively ex pressed in normal development and cell differenti ation (Flaherty et aI. , 1990). Northern blot analyses revealed an induction of HSC70 mRNA in the post ischemic gerbil brain (Nowak et aI. , 1990; Abe et aI., 1991). Abe et ai. (1991) also observed a partial regional difference in the amount of HSC70 mRNA under normal and postischemic conditions, and pro posed a possible protective role of HSC70 protein in CAl cell damage after ischemia. However, the spa tial distributions of HSC70 mRNA in normal and postischemic gerbil brains have not yet been deter mined. Therefore, it is of interest to investigate the distribution of HSC70 mRNA in subregions of nor mal and ischemic brains, especially in the selec tively vulnerable hippocampal formation. We recently isolated cDNA clones selective for HSP70 and HSC70 mRNAs from gerbil brain (Sato et aI., 1991). Using these cDNA clones as probes, we investigated the spatial distributions of HSP70 and HSC70 mRNAs by in situ hybridization in the same gerbil brain after to-min ischemia. The distri bution of HSP70 immunoreactivity was also deter mined. MATERIALS AND METHODS Animal model Male Mongolian gerbils (Meriones unguiculatus), aged 10--11 weeks and weighing 70--75 g, were lightly anesthe tized by inhalation of a nitrous oxide/oxygen/halothane (69%:30%:1%) mixture. Both common carotid arteries were exposed and anesthesia was then discontinued. When the animal began to regain consciousness, the ar teries were occluded for 10 min with surgical clips (Abe et aI., 1989). Body temperature was monitored in all animals with a rectal probe and was maintained at 37°C using a heating pad during surgical preparation and occlusion of the carotid arteries. After the restoration of blood flow, no attempt was made to maintain constant body temper ature. The animals recovered for 1,3,8 h,1,2,and 7 days
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3 for each time point) at ambient temperature (2123°C) and were then decapitated. Sham animals were killed 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 J.Lm) at the 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). Histological observation with cresyl violet staining, in situ hybridization for HSP70 and HSC70 mRNAs, and immunostaining against HSP70 protein were performed on the sections.
(n
=
Hybridization experiments The inserts of cloned cDNAs used as probes in the present study were derived originally from cerebral cor tex of gerbils. The sizes of the inserts selective for HSP70 and HSC70 mRNAs were 1.0 (pGA3) and 1.4 kb (pGD 3), respectively (Sato et aI., 1991). The specificities of these probes for HSP70 and HSC70 mRNAs in gerbil brain were examined by Northern blot analysis. Total RNAs were extracted from cerebral cortices of the sham and the postischemic gerbil brains, electrophoresed, and trans ferred to a nylon membrane as in our previous report (Abe et aI., 1991). The probes were radiolabeled with et-[32p]_dATP (6000 Ci/mmol, Amersham, Tokyo, Japan) by random primer labeling and hybridized against North ern blots (15 J.Lg of total RNA/lane) at 42°C for 20 h in a hybridization solution containing formamide. After the hybridization, the filters were washed with 2 x SCC (1 x SSC 150 mM NaCI/15 mM sodium citrate), 1 x SSC, and 0.5 x SSC with 0.2% sodium dodecyl sulfate (SDS) at room temperature or 65°C. The filters were exposed to x-ray film for 20 h at -80°C. In situ hybridization was performed by a method of Yoshioka et al. (1990) with a slight modification. Briefly, the sections were fixed for 10 min in an ethanol/acetic acid (3:1) mixture, transferred to 0.2 M HCI for 20 min, and immersed for 20 min at 50°C in 2 x SSC (pH 7.0). They were then digested for 15 min at 37°C with 100 J.Lg/ml of proteinase K (Merck and Co. Rahway NJ) in 20 mM Tris-HCI buffer (pH 7.4) with 2 mM CaC!2 and dehy drated through graded ethanol. Slides were prehybridized for 2 h at room temperature in a solution containing 50% formamide, 600 mM NaC!, 10 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin, 100 J.Lg/ml sonicated, dena tured calf thymus DNA, and 0.5 mg/ml calf liver RNA. Hybridization was carried out for 20 h at 42°C using 35S_ labeled cDNA probe at a concentration of 0.1 ng/J.LI in prehybridization solution supplemented with 10% dex tran sulfate and 10 mM dithiothreitol. The cDNA inserts were radiolabeled with et-e5S]deoxycytidine triphosphate (1000 Ci/mmol, Amersham) by random primer labeling (Feinberg and Vogelstein, 1983) using a kit (Boehringer Mannheim Yamanouchi Co., Tokyo, Japan), resulting in specific activities of 5-6 x 108 dpm/J.Lg. The control probe was a similarly labeled plasmid vector (pHSG396,Takara Shuzo Co., Kyoto, Japan) fragment (Ll kb) digested by Hinfl (Takara). The sections were washed at 42°C for 2 h in 2 x SSC and for 1 h in 1 x SSC, and then dehydrated through a graded ethanol series containing 0.3 M ammo nium acetate. After visualization of the hybridization by exposure to x-ray film for 24 h at room temperature, the sections were dipped in a liquid emulsion (NR-M2, Kon ica Co., Tokyo, Japan) diluted (1:1) with 0.6 M ammo=
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nium acetate. They were then exposed at 4°C for approx imately 3 weeks, developed, and counterstained with he matoxylin. Several slides were treated with lOO j-Lg/ml RNase A and lO units/ml RNase Tl (Sigma, St. Louis, MO U.S.A.) in 2 x SSC at 37°C for 2 h prior to prehy bridization and were then hybridized with the probe for HSP70 or HSC70 mRNA.
ASP 70
HSC 70
S
S
8h 2d
8h
2d
Histochemical study Immunostaining against HSP70 in gerbil brain sections was performed by avidin-biotin-peroxidase (ABC) method using a kit (PK-6lO2, Vector Laboratories, Bur lingame CA). The sections were fixed for 10 min in ice cold acetone, air-dried, and rinsed in 0.01 M phosphate buffer containing 0.5 M NaCI (pH 7.4). After blocking with 10% normal horse serum for 2 h, the slides were washed and incubated for 12-15 h at 4°C with a mouse monoclonal antibody against the stress-inducible species of the HSP70 family (RPN1197, which was originally de signed as clone C92, Amersham) diluted in the buffer (11 200) containing 10% normal horse serum and 0.3% Triton X-I00. Specificity of the antibody has been described elsewhere (Welch and Suhan, 1986; Vass et aI., 1988; Kirino et ai., 1991). Endogenous peroxidase was blocked for 20 min with 0.3% H 202 and lO% methanol. Then the sections were washed and incubated for 3 h with biotiny lated anti-mouse IgG (11200) in the buffer, followed by incubation for 30 min with avidin-biotin-horseradish per oxidase complex. Staining was developed with 3,3' diaminobenzidine tetrahydrochloride (0.5 mg/ml in 100 mM Tris-HCI buffer, pH 7.2) in the presence of 0.02% H202•
Ischemia-induced cell damage was estimated by light microscopic observation of cresyl violet-stained brain sections (Pulsinelli et aI., 1982; Araki et aI., 1989; Araki et aI., 1991).
RESULTS The results obtained in the histochemical study and in situ hybridization were reproducible in all animals used at each time point. Hybridization experiments
Figure 1 shows the results of Northern blot anal ysis using cloned HSP70 and HSC70 cDNA probes against total RNA isolated from gerbil cerebral cor tex in the present ischemia model. Figure 1 shows the autoradiogram of the membrane filter finally washed with 0. 5 x SSC at 65°C. Final wash with 2 or 1 x SSC at room temperature yielded the same results (data not shown). In the sham control brain, there was no hybridization with the HSP70 mRNA while HSC70 mRNA was identified. At 8 h after the reperfusion, HSP70 mRNA was greatly induced and a slight increase of HSC70 mRNA level was also observed. Thus, HSP70 and HSC70 probes used in the present study selectively detected the HSP70 (2.8 kb) and the HSC70 (2.4 kb) mRNA spe cies, respectively, in the gerbil brain as reported by Sato et a1. (1991). The amount of HSP70 mRNA decreased, and that of HSC70 mRNA returned to
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FIG. 1. Northern blot analysis of HSP70 and HSC70 mRNAs in gerbil cerebral cortices of sham control (S) and postisch emie brains at B h (Bh) and 2 days (2d) after reperfusion. Note that HSP70 and HSC70 probes detected different mRNA spe cies in the gerbil brain and did not cross-react. Small and large arrowheads show positions of the detected HSP70 (2.B kb) and HSC70 (2.4 kb) mRNAs, respectively.
almost the sham control level by 2 days, respec tively. Neither the HSP70 probe nor the HSC70 probe cross-hybridized with any other mRNA spe cies. Distribution of HSP70 mRNA in postischemic gerbil brain is shown in the left column of Fig. 2. HSP70 mRNA was not abundantly present in sham brain; however, the mRNA was greatly induced in several cell popUlations even at 1 h after reperfu sion, with a significant induction in dentate gyrus granule cells, which continued until 1 day. In hip pocampal CAl neuronal cells, HSP70 mRNA in duction was gradually intensified with a peak at 8 h to 1 day, and was diminished at around 2 days. Maximum induction of HSP70 mRNA in the cere bral cortex, lateral geniculate body, medial habe nula, and ventral thalamic nuclei (especially the dorsomedial part) was also observed at 8 h. Lami nar pattern of hybridization in the cortex and less expression in medial ventral thalamic nuclei were noted at this time. In hippocampal CA3 pyramidal cells, the induction of HSP70 mRNA began at 1 h, reached maximum at 1 day, and was sustained even at 2 days. All of the induced signal of HSP70 mRNA disappeared by 7 days after the reperfusion. Localizations of HSC70 mRNA in sham and post-
HSP70 AND HSC70 mRNAs IN POSTISCHEMIC GERBIL BRAIN
HSP70
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HSC70
s
1h
3h FIG. 2. Distributions of HSP70
umn)
(left col
and HSC70 (right column) mRNAs in the sham control (S) and the postischemic gerbil brains. Note a strong hybridization of HSP70 mRNA in CA1 cells up to 1 day and the co-induction of HSP70 and HSC70 mRNAs in most cell populations of the brain after ischemia. Bar 2 mm.
8h
=
1d
2d
,-
7d
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ischemic gerbil brains are documented in the right column of Fig. 2. In contrast to the case of HSP70 mRNA, HSC70 mRNA was present in most cell populations of the sham brain. A relatively high level of HSC70 mRNA density was observed in CA3 cells, cells of medial habenula, medial amyg daloid, arcuatal nuclei, and ventromedial hypothal amus. Transient ischemia increased the amount of
HSC70 mRNA in several neuronal cell populations, including CAl cells, from 1 h with a peak at 8 h, although the magnitude of HSC70 mRNA induction was lower than that of HSP70 mRNA, particularly in CAl cells. At around 2 days, HSC70 mRNA in CAl cells was lost. The induction of HSC70 mRNA returned to the sham level by 7 days except for the CAl region.
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Observation of sections dipped in liquid emulsion revealed that grains for HSP70 and HSC70 probes were predominantly located in the neuronal cell body (data not shown). Neither the sections hybrid ized with the probe from the plasmid vector nor the sections treated with RNases before incubation with the probe for HSP70 or HSC70 mRNA exhib ited any specific hybridization (data not shown). Histochemical study
Results obtained in the immunostaining against HSP70 protein were almost consistent with those of Vass et al. ( 1988). Sham brains did not express HSP70 immunoreactivity in the hippocampal for mation (Fig. 3A). After transient ischemia, CA t cells showed a weak immunoreactivity of HSP70 from t to 2 days (Fig. 3B), while a prominent im munoreactivity of HSP70 in the cell body and den drites of CA3 neurons was found (Fig. 3B and C).
FIG. 4. High-magnification photographs of cresyl violet stained hippocampal CA1 cells of sham (A) and postischemic brains at 2 days (8) and 7 days (C) after reperfusion. Note that the majority of CA1 cells is still present at 2 days (8) although the staining begins to decrease (B) as compared with sham control (A). Delayed neuronal cell death in CA1 is apparent at 7 days (C). Bar 1 0 f.Lm. =
FIG. 3. Representative photographs of immunostaining against HSP70 protein in the hippocampal formation of sham (A) or 1 0-min ischemia plus 2-day reflow (8 and C) gerbil brain. Note the minimal immunostaining of CA1 cells as com pared with pronounced staining in CA3 and dentate gyrus granule cells at 2 days. Bar 500 f.Lm in A and B, and 25 f.Lm inC. =
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Staining of dentate gyrus granule cells was also ob served (Fig. 3B). Cresyl violet staining of sections from identical ischemic brain showed that the majority of hippo campal CAl cells was still preserved at 2 days (Fig. 4B), when HSP70 and HSC70 mRNAs were almost lost (in 2d of Fig. 2). At this time, however, the staining of CAl cells began to decrease (Fig. 4B) as compared with sham brains (Fig. 4A). Hippocampal CA t neuronal cells completely disappeared at 7 days after reperfusion (Fig. 4C).
HSP70 AND HSC70 mRNAs IN POSTISCHEMIC GERBIL BRAIN
DISCUSSION
Northern blot analysis demonstrated 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 (Fig. 1). Furthermore, the temporal and quantitative changes of HSP70 and HSC70 mRNA levels in Northern blot analysis (Fig. 1) were consistent with those of the in situ hybridiza tion analysis (Fig. 2). In the in situ hybridization analysis, grains for the HSP70 and HSC70 probes were located predominantly in the cell body. The specificities of these probes were further supported by the negative signal after hybridization with the irrelevant plasmid vector probe and by the elimina tion of the signal after pretreatment with RNases prior to incubation with the probe for HSP70 or HSC70 mRNA. These results prove that the HSP70 and HSC70 cDNA probes used here selectively rec ognize HSP70 and HSC70 mRNAs of gerbil brain in situ, respectively. HSP70 mRNA was induced with regional differ ences among the subregions of ischemic gerbil brain (left column in Fig. 2). However, the temporal pro file of the induction of HSP70 mRNA detected with cDNA probe in the present study using a JO-min ischemic gerbil brain differed somewhat from the results reported by Nowak (1991) using a 5-min ischemic gerbil brain and a synthetic oligonucle otide probe selective for HSP70 mRNA. The present study revealed that significant HSP70 mRNA induction occurred in hippocampal CAl cells and disappeared at around 2 days, and that the more resistant hippocampal CA3 and dentate gran ule cells showed a sustained, striking induction of HSP70 mRNA to at least 2 days and 1 day, respec tively (left column in Fig. 2). On the other hand, in the 5-min ischemic gerbil forebrain, a more sus tained induction of HSP70 mRNA in CAl cells up to 2 days and a great reduction of hybridization in cells of the dentate granule and hippocampal CA3 by 1 day were detected in the study of Nowak (1991). Although ischemia was induced during anes thesia (Nowak, 1991) or after the recovery from an esthesia (present study), the difference in HSP70 mRNA induction may mainly be due to the differ ence in the duration of ischemia. In cells of the dentate granule and hippocampal CA3, it is reason able to infer that 10-min ischemia induced stronger and longer-lasting hybridization of HSP70 mRNA than 5-min ischemia, since the greater stress condi tion may produce greater stress response in general. However, in CAl cells, HSP70 mRNA induction disappeared at around 2 days after 10-min ischemia
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(left column in Fig. 2), in contrast to the "superin duction" of HSP70 mRNA after 5-min ischemia (Nowak, 1991). The majority of CAl cells was still spared at 2 days in the present experiment (Fig. 4B), and calcium accumulation was not yet ob served at this time point in the same 10-min gerbil ischemia model (Araki et aI. , 1989). Therefore, these results suggest that a transcriptional inhibition of HSP70 production may begin at around 2 days in CAl cells after JO-min ischemia before the death of cells becomes evident. Abe et al. (1991) showed less expression of HSP70 mRNA in CAl cells than in the cerebral cortex at 8 h after 10-min transient isch emia by Northern blot analysis using a human ge nomic DNA probe which detected both HSP70 and HSC70 mRNAs. However, it is difficult to compare directly these results because of the differences in the technique of detecting mRNA and the probes used. HSP70 has been demonstrated to be induced by transient ischemia in rodent brain (Vass et aI., 1988; Gonzalez et aI. , 1991; Kirino et aI. , 1991) and to play a protective role under various stressful con ditions (Pelham, 1984; Lindquist, 1986; Barbe et aI., 1988; Abe et aI. , 1991; Kirino et aI., 1991). It is interesting that CAl cells produce only a minimal HSP70 immunoreactivity from 1 to 2 days post ischemia (Fig. 3B and Vass et aI., 1988) in spite of the prolonged, striking induction of HSP70 mRNA at least by 1 day (left column in Fig. 2). On the other hand, more resistant cells, such as dentate granule and hippocampal CA3, successfully produced both HSP70 mRNA and protein even at 1 day (left col umn in Fig. 2 and Vass et aI. , 1988). These results suggest that a translational deficit in HSP70 produc tion may begin earlier than the beginning of tran scriptional failure. Our results are the first to demonstrate the spatial distribution of HSC70 mRNA in normal and post ischemic gerbil brains (right column in Fig. 2). In contrast to the case of HSP70 mRNA, HSC70 mRNA was present in most cell populations of the gerbil brain under sham control conditions. These results suggest different roles for HSP70 and HSC70 under normal conditions. HSC70 has been suggested to be associated with nascent polypeptide chains (Beckmann et aI. , 1990; Hightower, 1991). It is interesting that the distribution of total protein synthesis in the sham gerbil brain (Thilmann et aI. , 1986; Yoshidomi et al. , 1989) is similar to that of HSC70 mRNA. A possibility might be that the rel ative densities of cells were higher in those regions. However, in cresyl violet-stained sham control brains, the neuronal densities among the cell popu lations such as the CA3 area and the dorsomedial
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thalamic nuclei were not significantly different from those in the CAl area and the ventromedial tha lamic nuclei, respectively (data not shown). There fore, the relatively high level of expression of HSC70 mRNA in some cell populations including CA3 cells may relate to active protein synthesis in these regions rather than to the density of cell pop ulations. The obvious co-induction of HSP70 and HSC70 mRNAs was observed in several cell populations of the postischemic brain although the magnitude of induction of these mRNAs was different (Fig. 2). It is known that HSP70 and HSC70 proteins bind de natured or unfolded proteins and degrade or refold abnormal proteins under stressful conditions (Pel ham, 1990; Hightower, 1991). HSP70 is essential to restore normal ribosome assembly, to promote the synthesis of new ribosomes and proteins, and to accelerate the recovery of cells (Pelham, 1984) by
ATP-dependent mechanisms (Lewis and Pelham, 1985). It has been suggested that HSC70 plays a role in disassembling the clathrin cage and in participat ing in posttranslational transmembrane targeting of proteins to cellular organelles (Flaherty et a!. , 1990). Thus, the co-induction of HSP70 and HSC70 mRNAs indicates a cooperative role of these pro teins under such stressful conditions as transient ischemia. The co-induction also suggests an active
folding of damaged and/or newly synthesized pro teins in several cell populations of the postischemic brain. At 8 h after reperfusion, while CA1 cells pro duced a greater amount of HSP70 mRNA than CA3 cells, the amount of HSC70 mRNA induction in CA1 cells was still smaller than that in CA3 cells (Fig. 2), suggesting possibly different transcrip tional mechanisms in the induction of HSP70 and HSC70 mRNAs in hippocampal CAl cells after transient ischemia. We suspect that HSP70 aids HSC70 in salvaging denatured proteins by solubi lizing them and facilitating refolding, or chaperon ing them to a degradative system (Hightower, 1991). In addition to the cooperative roles of HSP70 as a molecular chaperone in stressful conditions (Hightower, 1991; Gething and Sambrook, 1992), HSC70 has a role in disassembling the clathrin cage (Flaherty et a!. , 1990; Gething and Sambrook, 1992). Recently, Yoshimi et a!. (1991) demonstrated that the immunoreactivity of clathrin increased in CAl cells from 3 h to 2 days after ischemia. They also suggested that the abnormality in clathrin turn over after ischemia might be related to the patho genesis of delayed neuronal death in CAl cells. Be cause HSC70 is involved in clathrin metabolism, the lesser induction of HSC70 mRNA (right column J Cereb Blood Flow Metab, Vol.
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in Fig. 2) combined with the increase of clathrin (Yoshimi et aI. , 1991) strongly suggests a dysfunc tion of a membrane-associated vesicle transport system in CAl cells after transient ischemia. A weak induction of HSP70 and HSC70 mRNAs in the mediodorsal thalamic nuclei is consistent with a lesser inhibition of total protein synthesis by transient ischemia in this region (Thilmann et aI., 1986). These results indicate that this area of gerbil forebrain receives much less stress after transient ischemia plus reperfusion. The blood supply from the vertebral arteries to this region (Suzuki et aI., 1983; Araki et aI. , 1989) may explain this phenom enon. Many putative mechanisms have been proposed to explain delayed neuronal damage (Bodsh et aI. , 1985; lmdahl and Hossmann, 1986; Rothman and Olney, 1986; Westerberg et al., 1987); however, its precise mechanism is not fully understood. Our re sults showed that the disappearance of HSP70 and HSC70 mRNAs preceded the death of CAl cells. Further investigation of the distributions of other proteins and genes in brains under normal and post ischemic conditions will be acquired to clarify the mechanism of ischemia-induced selective vulnera bility. Acknowledgment: This work was partly supported by Monbusho grants 01044018 and 03404028, a grant of the Ministry of Welfare and Health of Japan, and the Kanae foundation. The authors would like to express their ap preciation to Drs. M. Aoki and H. Shozuhara and Mrs. M. Matsumoto for their excellent technical assistance.
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Gething MJ, Sambrook J (1992) Protein folding in the cell. Na ture 355:33-45 Gonzalez MF, Lowenstein D, Fernyak S, Hisanaga K, Simon R, Sharp FR (1991) Induction of heat shock protein 72-like im munoreactivity in the hippocampal formation following tran sient global ischemia. Brain Res Bull 26:241-250 Hatakeyama T, Matsumoto M, Brengman JM, Yanagihara T (1988) Immunohistochemical investigation of ischemic and postischemic damage after bilateral carotid occlusion in ger bils. Stroke 19: 1526-1534 Hightower LE (1991) Heat shock, stress proteins, chaperones, and proteotoxicity. Cell 66:191-197 Imdahl A, Hossmann K-A (1986) Morphometric evaluation of post-ischemic capillary perfusion in selectively vulnerable areas of gerbil brain. Acta Neuropathol (Ber/) 69: 267-271 Kirino T (1982) Delayed neuronal death in the gerbil hippocam pus following ischemia. Brain Res 239:57-69 Kirino T, Tsujita Y, Tamura A (1991) Induced tolerance to isch emia in gerbil hippocampal neurons. J Cereb Blood Flow Metab 1l:299-307 Lewis MT, Pelham HRB (1985) Involvement of ATP in the nu clear and nucleolar functions of the 70 kd heat shock protein. EMBO J 4: 3137-3143 Lindquist S (1986) The heat-shock response. Annu Rev Biochem 55: 1151-1191 Magnusson K, Wieloch T (1989) Impairment of protein ubiquit ination may cause delayed neuronal death. Neurosci Lett 96: 264-270 Nowak TS Jr, Bond U, Schlesinger MJ (1990) Heat shock RNA levels in brain and other tissues after hyperthermia and tran sient ischemia. J Neurochem 54:451-458 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 Pelham HRB (1984) HSP70 accelerates the recovery of nucleolar morphology after heat shock. EMBO J 3: 3095-3100 Pelham HRB (1990) Functions of the hsp70 protein family: an overview. In: Stress Proteins in Biology and Medicine (Morimoto RI, Tissieres A, Georgopoulos C, eds) New York, Cold Spring Harbor Laboratory Press, pp 287-299 Petito CK, Feldmann E, Pulsinelli WA, Plum F (1987) Delayed hippocampal damage in humans following cardiorespiratory arrest. Neurology 37: 1281-1286
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Pulsinelli WA, Brierley JB, Plum F (1982) Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol 11:491-498 Rothman SM, Olney JW (1986) Glutamate and the pathophysi ology of hypoxic-ischemic brain damage. Ann Neurol 19: 105-111 Sato S, Abe K, Kawagoe J, Saito A, Kogure K (1991) Molecular cloning of heat shock protein (HSP) 70 gene in postischemic gerbil brain. J Cereb Blood Flow Metab 11(suppl 2):S345 Smith M-L, Auer RN, Siesjo BK (1984) The density and distri bution of ischemic brain injury in the rat following 2-10 min of forebrain ischemia. Acta Neuropathol (Ber/) 64:319-332 Suzuki R, Yamaguchi T, Kirino T, Orzi F, Klatzo I (1983) The effects of 5-minute ischemia in Mongolian gerbils. 1. Blood brain barrier, cerebral blood flow, and local cerebral glucose utilization changes. Acta Neuropathol (Ber/) 60:207-216 Thilmann R, Xie Y, Kleihues P, Kiessling M (1986) Persistent inhibition of protein synthesis precedes delayed neuronal death in postischemic gerbil hippocampus. Acta Neuro pathol (Ber/) 71: 88-93 Vass K, Welch WJ, Nowak TS Jr (1988) Localization of 70-kDa stress protein induction in gerbil brain after ischemia. Acta Neuropathol (Ber/) 77: 128-135 Welch WJ, Suhan JP (1986) Cellular and biochemical events in mammalian cells during and after recovery from physiolog ical stress. J Cell Bioi 103:2035-2052 Westerberg E, Deshpande JK, Wieloch T (1987) Regional differ ences in arachidonic acid release in rat hippocampal CAl and CA3 regions during cerebral ischemia. J Cereb Blood Flow Metab 7: 189-192 Yoshidomi M, Hayashi T, Abe K, Kogure K (1989) Effects of a new calcium channel blocker, KB-2796, on protein synthesis of the CA l pyramidal cell and delayed neuronal death fol lowing transient forebrain ischemia. J Neurochem 53: 1589-1594 Yoshimi K, Takeda M, Nishimura T, Kudo T, Nakamura Y, Tada K, Iwata N (1991) An immunohistochemical study of MAP2 and clathrin in gerbil hippocampus after cerebral isch emia. Brain Res 560: 149-158 Yoshioka M, Nagano I, Nakamura S, Imaizumi M, Kimura N (1990) Detection of vasoactive intestinal polypeptide mes senger RNA in ganglioneuroblastoma by in situ hybridiza tion. Endocr Pathol 1:51-57
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Distributions of Heat Shock Protein-70 mRNAs and Heat Shock Cognate Protein-70 mRNAs After Transient Global Ischemia in Gerbil Brain
J. Kawagoe, K. Abe, S. Sato, I. Nagano, S. Nakamura, and K. Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai, Japan
Summary: Distributions of heat shock protein (HSP)-70 mRN As and heat shock cognate protein (HSC)-70 mRNAs after 10 min of transient global ischemia were investigated in gerbil forebrain by in situ hybridization using cloned eDNA probes selective for the mRNAs. Ex pression of HSP70 immunoreactivity was also examined in the same brains. In hippocampal CAl neuronal cells, in which only a minimal induction of immunoreactive HSP70 protein was found, the strong hybridization for HSP70 mRNA disappeared at around 2 days before the death of CAl cells became evident. Furthermore, in hip pocampal CA3 cells, a striking induction of HSP70 mRNA was sustained even at 2 days along with a prom inent accumulation of HSP70 immunoreactivity. In con trast to the case of HSP70 mRNA, HSC70 mRNA was present in most neuronal cells, especially dense in CA3 cells, of the sham brain. A co-induction of HSP70 and HSC70 mRNAs was observed in several cell populations after the reperfusion with a peak at 8 h, although the
magnitude of HSC70 mRNA induction was lower than that of HSP70 mRNA, particularly in CAl cells. The ex pression of HSC70 mRNA in CAl cells also disappeared at around 2 days. All the induced signals of HSP70 and HSC70 mRNAs in other cell populations were diminished and returned to the sham level, respectively, by 7 days. These results are the first to show the time courses of distribution of HSP70 and HSC70 mRNAs and the immu noreactive HSP70 protein in the same gerbil brain after ischemia. The results suggest that the weak induction of HSP70 protein in CAl cells, which may relate to the vul nerability of this cell population, is due to both transla tional and transcriptional deficits. The different roles un der normal condition and the cooperative role in the re covery process from ischemic injury between HSP70 and HSC70, and the involvement of HSC70 in CAl cell death, are suggested. Key Words: Heat shock protein-Heat shock cognate protein-Cerebral ischemia-In situ hy bridization-Gerbil.
Selective neuronal vulnerability after global isch emia has been demonstrated in human (Petito et al., 1987) and rodent brains (Kirino, 1982; Pulsinelli et al., 1982; Smith et al., 1984; Hatakeyama et al. , 1988; Araki et al. , 1989; Araki et al. , 1991). In gerbil brain, hippocampal CAl pyramidal neurons are killed after transient ischemia while other neurons such as dentate gyrus granule cells and hippocam pal CA3 cells are much less vulnerable (Kirino, 1982; Hatakeyama et al., 1988; Araki et al., 1989;
Araki et al. , 1991). It is also demonstrated that tran sient global ischemia induces 70 kDa-heat shock protein (HSP70), which has been suggested to play a protective role under stressful conditions (Pel ham, 1984; Lindquist, 1986; Barbe et al. , 1988; Kirino et al. , 1991; Abe et al. , 1991). Recent immunohistochemical studies have re vealed a regional difference in the induction of im munoreactive HSP70 protein between the most vul nerable CAl cells and the more resistant CA3 and dentate granule cells after ischemia. Vass et al. (1988) reported a minimal induction of the HSP70 immunoreactivity in CAl cells of gerbil brain after ischemia, while CA3 and dentate granule cells showed pronounced immunoreactivity of HSP70. Magnusson and Wieloch (1989) demonstrated that the decreased immunostaining of ubiquitin, a stress inducible protein, in the CAl cells after ischemia
Received December II, 1991; final revision received March 5, 1992; accepted March 25, 1992. Address correspondence and reprint requests to Dr. 1. Kawagoe, Department of Neurology, Institute of Brain Dis eases, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan. Abbreviations used: ABC, avidin-biotin-peroxidase; HSP, heat shock protein; HSC, heat shock cognate protein; SSC, so dium chloride/sodium citrate; SDS, sodium dodecyl sulfate.
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failed to recover although that in the other resistant cells of rat hippocampus recovered. Furthermore, a lower level of HSP70 induction in hippocampal CA1 cells was detected at the transcriptional level by Northern blot analysis in a to-min ischemia study in gerbils (Abe et aI. , 1991). In contrast to this result, the in situ hybridization technique revealed a strong and prolonged hybridization of HSP70 mRNA in CAl cells of gerbil brain after 5-min ischemia (Nowak, 1991). These results indicate the impor tance of investigating the regional distributions of immunoreactivity and mRNA of the HSP70 family in the same ischemic brain in order to consider mechanisms of ischemia-induced selective vulnera bility. Heat shock cognate protein (HSC)-70 is a mem ber of the HSP70 family, and is constitutively ex pressed in normal development and cell differenti ation (Flaherty et aI. , 1990). Northern blot analyses revealed an induction of HSC70 mRNA in the post ischemic gerbil brain (Nowak et aI. , 1990; Abe et aI., 1991). Abe et ai. (1991) also observed a partial regional difference in the amount of HSC70 mRNA under normal and postischemic conditions, and pro posed a possible protective role of HSC70 protein in CAl cell damage after ischemia. However, the spa tial distributions of HSC70 mRNA in normal and postischemic gerbil brains have not yet been deter mined. Therefore, it is of interest to investigate the distribution of HSC70 mRNA in subregions of nor mal and ischemic brains, especially in the selec tively vulnerable hippocampal formation. We recently isolated cDNA clones selective for HSP70 and HSC70 mRNAs from gerbil brain (Sato et aI., 1991). Using these cDNA clones as probes, we investigated the spatial distributions of HSP70 and HSC70 mRNAs by in situ hybridization in the same gerbil brain after to-min ischemia. The distri bution of HSP70 immunoreactivity was also deter mined. MATERIALS AND METHODS Animal model Male Mongolian gerbils (Meriones unguiculatus), aged 10--11 weeks and weighing 70--75 g, were lightly anesthe tized by inhalation of a nitrous oxide/oxygen/halothane (69%:30%:1%) mixture. Both common carotid arteries were exposed and anesthesia was then discontinued. When the animal began to regain consciousness, the ar teries were occluded for 10 min with surgical clips (Abe et aI., 1989). Body temperature was monitored in all animals with a rectal probe and was maintained at 37°C using a heating pad during surgical preparation and occlusion of the carotid arteries. After the restoration of blood flow, no attempt was made to maintain constant body temper ature. The animals recovered for 1,3,8 h,1,2,and 7 days
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3 for each time point) at ambient temperature (2123°C) and were then decapitated. Sham animals were killed 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 J.Lm) at the 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). Histological observation with cresyl violet staining, in situ hybridization for HSP70 and HSC70 mRNAs, and immunostaining against HSP70 protein were performed on the sections.
(n
=
Hybridization experiments The inserts of cloned cDNAs used as probes in the present study were derived originally from cerebral cor tex of gerbils. The sizes of the inserts selective for HSP70 and HSC70 mRNAs were 1.0 (pGA3) and 1.4 kb (pGD 3), respectively (Sato et aI., 1991). The specificities of these probes for HSP70 and HSC70 mRNAs in gerbil brain were examined by Northern blot analysis. Total RNAs were extracted from cerebral cortices of the sham and the postischemic gerbil brains, electrophoresed, and trans ferred to a nylon membrane as in our previous report (Abe et aI., 1991). The probes were radiolabeled with et-[32p]_dATP (6000 Ci/mmol, Amersham, Tokyo, Japan) by random primer labeling and hybridized against North ern blots (15 J.Lg of total RNA/lane) at 42°C for 20 h in a hybridization solution containing formamide. After the hybridization, the filters were washed with 2 x SCC (1 x SSC 150 mM NaCI/15 mM sodium citrate), 1 x SSC, and 0.5 x SSC with 0.2% sodium dodecyl sulfate (SDS) at room temperature or 65°C. The filters were exposed to x-ray film for 20 h at -80°C. In situ hybridization was performed by a method of Yoshioka et al. (1990) with a slight modification. Briefly, the sections were fixed for 10 min in an ethanol/acetic acid (3:1) mixture, transferred to 0.2 M HCI for 20 min, and immersed for 20 min at 50°C in 2 x SSC (pH 7.0). They were then digested for 15 min at 37°C with 100 J.Lg/ml of proteinase K (Merck and Co. Rahway NJ) in 20 mM Tris-HCI buffer (pH 7.4) with 2 mM CaC!2 and dehy drated through graded ethanol. Slides were prehybridized for 2 h at room temperature in a solution containing 50% formamide, 600 mM NaC!, 10 mM Tris-HCI (pH 7.5), 1 mM EDTA, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% bovine serum albumin, 100 J.Lg/ml sonicated, dena tured calf thymus DNA, and 0.5 mg/ml calf liver RNA. Hybridization was carried out for 20 h at 42°C using 35S_ labeled cDNA probe at a concentration of 0.1 ng/J.LI in prehybridization solution supplemented with 10% dex tran sulfate and 10 mM dithiothreitol. The cDNA inserts were radiolabeled with et-e5S]deoxycytidine triphosphate (1000 Ci/mmol, Amersham) by random primer labeling (Feinberg and Vogelstein, 1983) using a kit (Boehringer Mannheim Yamanouchi Co., Tokyo, Japan), resulting in specific activities of 5-6 x 108 dpm/J.Lg. The control probe was a similarly labeled plasmid vector (pHSG396,Takara Shuzo Co., Kyoto, Japan) fragment (Ll kb) digested by Hinfl (Takara). The sections were washed at 42°C for 2 h in 2 x SSC and for 1 h in 1 x SSC, and then dehydrated through a graded ethanol series containing 0.3 M ammo nium acetate. After visualization of the hybridization by exposure to x-ray film for 24 h at room temperature, the sections were dipped in a liquid emulsion (NR-M2, Kon ica Co., Tokyo, Japan) diluted (1:1) with 0.6 M ammo=
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nium acetate. They were then exposed at 4°C for approx imately 3 weeks, developed, and counterstained with he matoxylin. Several slides were treated with lOO j-Lg/ml RNase A and lO units/ml RNase Tl (Sigma, St. Louis, MO U.S.A.) in 2 x SSC at 37°C for 2 h prior to prehy bridization and were then hybridized with the probe for HSP70 or HSC70 mRNA.
ASP 70
HSC 70
S
S
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8h
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Histochemical study Immunostaining against HSP70 in gerbil brain sections was performed by avidin-biotin-peroxidase (ABC) method using a kit (PK-6lO2, Vector Laboratories, Bur lingame CA). The sections were fixed for 10 min in ice cold acetone, air-dried, and rinsed in 0.01 M phosphate buffer containing 0.5 M NaCI (pH 7.4). After blocking with 10% normal horse serum for 2 h, the slides were washed and incubated for 12-15 h at 4°C with a mouse monoclonal antibody against the stress-inducible species of the HSP70 family (RPN1197, which was originally de signed as clone C92, Amersham) diluted in the buffer (11 200) containing 10% normal horse serum and 0.3% Triton X-I00. Specificity of the antibody has been described elsewhere (Welch and Suhan, 1986; Vass et aI., 1988; Kirino et ai., 1991). Endogenous peroxidase was blocked for 20 min with 0.3% H 202 and lO% methanol. Then the sections were washed and incubated for 3 h with biotiny lated anti-mouse IgG (11200) in the buffer, followed by incubation for 30 min with avidin-biotin-horseradish per oxidase complex. Staining was developed with 3,3' diaminobenzidine tetrahydrochloride (0.5 mg/ml in 100 mM Tris-HCI buffer, pH 7.2) in the presence of 0.02% H202•
Ischemia-induced cell damage was estimated by light microscopic observation of cresyl violet-stained brain sections (Pulsinelli et aI., 1982; Araki et aI., 1989; Araki et aI., 1991).
RESULTS The results obtained in the histochemical study and in situ hybridization were reproducible in all animals used at each time point. Hybridization experiments
Figure 1 shows the results of Northern blot anal ysis using cloned HSP70 and HSC70 cDNA probes against total RNA isolated from gerbil cerebral cor tex in the present ischemia model. Figure 1 shows the autoradiogram of the membrane filter finally washed with 0. 5 x SSC at 65°C. Final wash with 2 or 1 x SSC at room temperature yielded the same results (data not shown). In the sham control brain, there was no hybridization with the HSP70 mRNA while HSC70 mRNA was identified. At 8 h after the reperfusion, HSP70 mRNA was greatly induced and a slight increase of HSC70 mRNA level was also observed. Thus, HSP70 and HSC70 probes used in the present study selectively detected the HSP70 (2.8 kb) and the HSC70 (2.4 kb) mRNA spe cies, respectively, in the gerbil brain as reported by Sato et a1. (1991). The amount of HSP70 mRNA decreased, and that of HSC70 mRNA returned to
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FIG. 1. Northern blot analysis of HSP70 and HSC70 mRNAs in gerbil cerebral cortices of sham control (S) and postisch emie brains at B h (Bh) and 2 days (2d) after reperfusion. Note that HSP70 and HSC70 probes detected different mRNA spe cies in the gerbil brain and did not cross-react. Small and large arrowheads show positions of the detected HSP70 (2.B kb) and HSC70 (2.4 kb) mRNAs, respectively.
almost the sham control level by 2 days, respec tively. Neither the HSP70 probe nor the HSC70 probe cross-hybridized with any other mRNA spe cies. Distribution of HSP70 mRNA in postischemic gerbil brain is shown in the left column of Fig. 2. HSP70 mRNA was not abundantly present in sham brain; however, the mRNA was greatly induced in several cell popUlations even at 1 h after reperfu sion, with a significant induction in dentate gyrus granule cells, which continued until 1 day. In hip pocampal CAl neuronal cells, HSP70 mRNA in duction was gradually intensified with a peak at 8 h to 1 day, and was diminished at around 2 days. Maximum induction of HSP70 mRNA in the cere bral cortex, lateral geniculate body, medial habe nula, and ventral thalamic nuclei (especially the dorsomedial part) was also observed at 8 h. Lami nar pattern of hybridization in the cortex and less expression in medial ventral thalamic nuclei were noted at this time. In hippocampal CA3 pyramidal cells, the induction of HSP70 mRNA began at 1 h, reached maximum at 1 day, and was sustained even at 2 days. All of the induced signal of HSP70 mRNA disappeared by 7 days after the reperfusion. Localizations of HSC70 mRNA in sham and post-
HSP70 AND HSC70 mRNAs IN POSTISCHEMIC GERBIL BRAIN
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HSC70
s
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3h FIG. 2. Distributions of HSP70
umn)
(left col
and HSC70 (right column) mRNAs in the sham control (S) and the postischemic gerbil brains. Note a strong hybridization of HSP70 mRNA in CA1 cells up to 1 day and the co-induction of HSP70 and HSC70 mRNAs in most cell populations of the brain after ischemia. Bar 2 mm.
8h
=
1d
2d
,-
7d
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ischemic gerbil brains are documented in the right column of Fig. 2. In contrast to the case of HSP70 mRNA, HSC70 mRNA was present in most cell populations of the sham brain. A relatively high level of HSC70 mRNA density was observed in CA3 cells, cells of medial habenula, medial amyg daloid, arcuatal nuclei, and ventromedial hypothal amus. Transient ischemia increased the amount of
HSC70 mRNA in several neuronal cell populations, including CAl cells, from 1 h with a peak at 8 h, although the magnitude of HSC70 mRNA induction was lower than that of HSP70 mRNA, particularly in CAl cells. At around 2 days, HSC70 mRNA in CAl cells was lost. The induction of HSC70 mRNA returned to the sham level by 7 days except for the CAl region.
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Observation of sections dipped in liquid emulsion revealed that grains for HSP70 and HSC70 probes were predominantly located in the neuronal cell body (data not shown). Neither the sections hybrid ized with the probe from the plasmid vector nor the sections treated with RNases before incubation with the probe for HSP70 or HSC70 mRNA exhib ited any specific hybridization (data not shown). Histochemical study
Results obtained in the immunostaining against HSP70 protein were almost consistent with those of Vass et al. ( 1988). Sham brains did not express HSP70 immunoreactivity in the hippocampal for mation (Fig. 3A). After transient ischemia, CA t cells showed a weak immunoreactivity of HSP70 from t to 2 days (Fig. 3B), while a prominent im munoreactivity of HSP70 in the cell body and den drites of CA3 neurons was found (Fig. 3B and C).
FIG. 4. High-magnification photographs of cresyl violet stained hippocampal CA1 cells of sham (A) and postischemic brains at 2 days (8) and 7 days (C) after reperfusion. Note that the majority of CA1 cells is still present at 2 days (8) although the staining begins to decrease (B) as compared with sham control (A). Delayed neuronal cell death in CA1 is apparent at 7 days (C). Bar 1 0 f.Lm. =
FIG. 3. Representative photographs of immunostaining against HSP70 protein in the hippocampal formation of sham (A) or 1 0-min ischemia plus 2-day reflow (8 and C) gerbil brain. Note the minimal immunostaining of CA1 cells as com pared with pronounced staining in CA3 and dentate gyrus granule cells at 2 days. Bar 500 f.Lm in A and B, and 25 f.Lm inC. =
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Staining of dentate gyrus granule cells was also ob served (Fig. 3B). Cresyl violet staining of sections from identical ischemic brain showed that the majority of hippo campal CAl cells was still preserved at 2 days (Fig. 4B), when HSP70 and HSC70 mRNAs were almost lost (in 2d of Fig. 2). At this time, however, the staining of CAl cells began to decrease (Fig. 4B) as compared with sham brains (Fig. 4A). Hippocampal CA t neuronal cells completely disappeared at 7 days after reperfusion (Fig. 4C).
HSP70 AND HSC70 mRNAs IN POSTISCHEMIC GERBIL BRAIN
DISCUSSION
Northern blot analysis demonstrated 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 (Fig. 1). Furthermore, the temporal and quantitative changes of HSP70 and HSC70 mRNA levels in Northern blot analysis (Fig. 1) were consistent with those of the in situ hybridiza tion analysis (Fig. 2). In the in situ hybridization analysis, grains for the HSP70 and HSC70 probes were located predominantly in the cell body. The specificities of these probes were further supported by the negative signal after hybridization with the irrelevant plasmid vector probe and by the elimina tion of the signal after pretreatment with RNases prior to incubation with the probe for HSP70 or HSC70 mRNA. These results prove that the HSP70 and HSC70 cDNA probes used here selectively rec ognize HSP70 and HSC70 mRNAs of gerbil brain in situ, respectively. HSP70 mRNA was induced with regional differ ences among the subregions of ischemic gerbil brain (left column in Fig. 2). However, the temporal pro file of the induction of HSP70 mRNA detected with cDNA probe in the present study using a JO-min ischemic gerbil brain differed somewhat from the results reported by Nowak (1991) using a 5-min ischemic gerbil brain and a synthetic oligonucle otide probe selective for HSP70 mRNA. The present study revealed that significant HSP70 mRNA induction occurred in hippocampal CAl cells and disappeared at around 2 days, and that the more resistant hippocampal CA3 and dentate gran ule cells showed a sustained, striking induction of HSP70 mRNA to at least 2 days and 1 day, respec tively (left column in Fig. 2). On the other hand, in the 5-min ischemic gerbil forebrain, a more sus tained induction of HSP70 mRNA in CAl cells up to 2 days and a great reduction of hybridization in cells of the dentate granule and hippocampal CA3 by 1 day were detected in the study of Nowak (1991). Although ischemia was induced during anes thesia (Nowak, 1991) or after the recovery from an esthesia (present study), the difference in HSP70 mRNA induction may mainly be due to the differ ence in the duration of ischemia. In cells of the dentate granule and hippocampal CA3, it is reason able to infer that 10-min ischemia induced stronger and longer-lasting hybridization of HSP70 mRNA than 5-min ischemia, since the greater stress condi tion may produce greater stress response in general. However, in CAl cells, HSP70 mRNA induction disappeared at around 2 days after 10-min ischemia
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(left column in Fig. 2), in contrast to the "superin duction" of HSP70 mRNA after 5-min ischemia (Nowak, 1991). The majority of CAl cells was still spared at 2 days in the present experiment (Fig. 4B), and calcium accumulation was not yet ob served at this time point in the same 10-min gerbil ischemia model (Araki et aI. , 1989). Therefore, these results suggest that a transcriptional inhibition of HSP70 production may begin at around 2 days in CAl cells after JO-min ischemia before the death of cells becomes evident. Abe et al. (1991) showed less expression of HSP70 mRNA in CAl cells than in the cerebral cortex at 8 h after 10-min transient isch emia by Northern blot analysis using a human ge nomic DNA probe which detected both HSP70 and HSC70 mRNAs. However, it is difficult to compare directly these results because of the differences in the technique of detecting mRNA and the probes used. HSP70 has been demonstrated to be induced by transient ischemia in rodent brain (Vass et aI., 1988; Gonzalez et aI. , 1991; Kirino et aI. , 1991) and to play a protective role under various stressful con ditions (Pelham, 1984; Lindquist, 1986; Barbe et aI., 1988; Abe et aI. , 1991; Kirino et aI., 1991). It is interesting that CAl cells produce only a minimal HSP70 immunoreactivity from 1 to 2 days post ischemia (Fig. 3B and Vass et aI., 1988) in spite of the prolonged, striking induction of HSP70 mRNA at least by 1 day (left column in Fig. 2). On the other hand, more resistant cells, such as dentate granule and hippocampal CA3, successfully produced both HSP70 mRNA and protein even at 1 day (left col umn in Fig. 2 and Vass et aI. , 1988). These results suggest that a translational deficit in HSP70 produc tion may begin earlier than the beginning of tran scriptional failure. Our results are the first to demonstrate the spatial distribution of HSC70 mRNA in normal and post ischemic gerbil brains (right column in Fig. 2). In contrast to the case of HSP70 mRNA, HSC70 mRNA was present in most cell populations of the gerbil brain under sham control conditions. These results suggest different roles for HSP70 and HSC70 under normal conditions. HSC70 has been suggested to be associated with nascent polypeptide chains (Beckmann et aI. , 1990; Hightower, 1991). It is interesting that the distribution of total protein synthesis in the sham gerbil brain (Thilmann et aI. , 1986; Yoshidomi et al. , 1989) is similar to that of HSC70 mRNA. A possibility might be that the rel ative densities of cells were higher in those regions. However, in cresyl violet-stained sham control brains, the neuronal densities among the cell popu lations such as the CA3 area and the dorsomedial
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thalamic nuclei were not significantly different from those in the CAl area and the ventromedial tha lamic nuclei, respectively (data not shown). There fore, the relatively high level of expression of HSC70 mRNA in some cell populations including CA3 cells may relate to active protein synthesis in these regions rather than to the density of cell pop ulations. The obvious co-induction of HSP70 and HSC70 mRNAs was observed in several cell populations of the postischemic brain although the magnitude of induction of these mRNAs was different (Fig. 2). It is known that HSP70 and HSC70 proteins bind de natured or unfolded proteins and degrade or refold abnormal proteins under stressful conditions (Pel ham, 1990; Hightower, 1991). HSP70 is essential to restore normal ribosome assembly, to promote the synthesis of new ribosomes and proteins, and to accelerate the recovery of cells (Pelham, 1984) by
ATP-dependent mechanisms (Lewis and Pelham, 1985). It has been suggested that HSC70 plays a role in disassembling the clathrin cage and in participat ing in posttranslational transmembrane targeting of proteins to cellular organelles (Flaherty et a!. , 1990). Thus, the co-induction of HSP70 and HSC70 mRNAs indicates a cooperative role of these pro teins under such stressful conditions as transient ischemia. The co-induction also suggests an active
folding of damaged and/or newly synthesized pro teins in several cell populations of the postischemic brain. At 8 h after reperfusion, while CA1 cells pro duced a greater amount of HSP70 mRNA than CA3 cells, the amount of HSC70 mRNA induction in CA1 cells was still smaller than that in CA3 cells (Fig. 2), suggesting possibly different transcrip tional mechanisms in the induction of HSP70 and HSC70 mRNAs in hippocampal CAl cells after transient ischemia. We suspect that HSP70 aids HSC70 in salvaging denatured proteins by solubi lizing them and facilitating refolding, or chaperon ing them to a degradative system (Hightower, 1991). In addition to the cooperative roles of HSP70 as a molecular chaperone in stressful conditions (Hightower, 1991; Gething and Sambrook, 1992), HSC70 has a role in disassembling the clathrin cage (Flaherty et a!. , 1990; Gething and Sambrook, 1992). Recently, Yoshimi et a!. (1991) demonstrated that the immunoreactivity of clathrin increased in CAl cells from 3 h to 2 days after ischemia. They also suggested that the abnormality in clathrin turn over after ischemia might be related to the patho genesis of delayed neuronal death in CAl cells. Be cause HSC70 is involved in clathrin metabolism, the lesser induction of HSC70 mRNA (right column J Cereb Blood Flow Metab, Vol.
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in Fig. 2) combined with the increase of clathrin (Yoshimi et aI. , 1991) strongly suggests a dysfunc tion of a membrane-associated vesicle transport system in CAl cells after transient ischemia. A weak induction of HSP70 and HSC70 mRNAs in the mediodorsal thalamic nuclei is consistent with a lesser inhibition of total protein synthesis by transient ischemia in this region (Thilmann et aI., 1986). These results indicate that this area of gerbil forebrain receives much less stress after transient ischemia plus reperfusion. The blood supply from the vertebral arteries to this region (Suzuki et aI., 1983; Araki et aI. , 1989) may explain this phenom enon. Many putative mechanisms have been proposed to explain delayed neuronal damage (Bodsh et aI. , 1985; lmdahl and Hossmann, 1986; Rothman and Olney, 1986; Westerberg et al., 1987); however, its precise mechanism is not fully understood. Our re sults showed that the disappearance of HSP70 and HSC70 mRNAs preceded the death of CAl cells. Further investigation of the distributions of other proteins and genes in brains under normal and post ischemic conditions will be acquired to clarify the mechanism of ischemia-induced selective vulnera bility. Acknowledgment: This work was partly supported by Monbusho grants 01044018 and 03404028, a grant of the Ministry of Welfare and Health of Japan, and the Kanae foundation. The authors would like to express their ap preciation to Drs. M. Aoki and H. Shozuhara and Mrs. M. Matsumoto for their excellent technical assistance.
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J Cereb Blood Flow Metab. Vol. 12. No.5. 1992
