Neurological Research A Journal of Progress in Neurosurgery, Neurology and Neurosciences
ISSN: 0161-6412 (Print) 1743-1328 (Online) Journal homepage: http://www.tandfonline.com/loi/yner20
Differential expression of heat shock protein 70 gene between the cortex and caudate after transient focal cerebral ischaemia in rats K. Abe, J. Kawagoe, T. Araki, M. Aoki & K. Kogure To cite this article: K. Abe, J. Kawagoe, T. Araki, M. Aoki & K. Kogure (1992) Differential expression of heat shock protein 70 gene between the cortex and caudate after transient focal cerebral ischaemia in rats, Neurological Research, 14:5, 381-385, DOI: 10.1080/01616412.1992.11740089 To link to this article: https://doi.org/10.1080/01616412.1992.11740089
Published online: 20 Jul 2016.
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Citing articles: 39 View citing articles
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Differential expression of heat shock protein 70 gene between the cortex and caudate after transient focal cerebral ischaemia in rats K. Abe, j. Kawagoe, T. Araki, M. Aoki and K. Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai, Japan
In relation to changes of total protein synthesis, induction of 70-kOa heat shock protein ( HSP70) mRNA was examined by Northern blot and in situ hybridization after 30 min of transient middle cerebral artery ( MCA ) occlusion of rats. HSP70 mRNA was not present in the control condition of brain. With reperfusion, the mRNA was greatly induced along with the recovery of total protein synthesis in the cerebral cortex and lateral caudate of the ipsilateral hemisphere. However, the level of the mRNA reached a maximum earlier in the lateral caudate (at 3 h) than in the cortex (at 8 h ), and the maximum amount of the mRNA was much smaller in the caudate than in the cortex. Total protein synthesis in the lateral caudate did not completely recover until 7 days. Histological examination showed a severe damage in cells of lateral caudate, while cells in the cortex were almost normal at 7 days. No difference in the brain temperature was observed between the two regions. These results show that the induction of HSP70 mRNA correlates · with the recovery of protein synthesis in brain cells after a transient ischaemia, and that the HSP70 gene expression is different at the transcriptional/eve/ between the cortical and caudate cells after the transient ischaemia. Keywords: Heat shock protein; ischaemia; protein synthesis
INTRODUCTION A role of molecular chaperone has been suggested for heat shock proteins (HSPs). HSP is induced in stressful conditions of cells such as heat shock or cerebral ischaemia2•18•22. Among HSPs, 70-kDa heat shock protein (HSP70) is known to be essential to restore normal ribosome assembly, promotes the synthesis of new ribosome, and accelerates the recovery of cell after heat shock17 . A role for protein folding has been recognized as the most important role of HSP7 . HSP recognizes and stabilizes partially folded intermediates during polypeptide folding, assembly and disassembly. HSP also disentangles malfolded or aggregated protein. A transient ischaemia causes profound reduction of total protein synthesis 4 and the disaggregation of polysome is among the earliest pathological findings in ischaemic neuronal damage9 . A role of HSP70 in t he recovery of protein synthesis has been indicated 17•18, and a recent report suggested a role of molecular chaperone of HSP70 for newly synthesized or degraded protein 3 with ATP-dependent mechanism 11 . Regional difference of the recovery from a transient ischaemia between cerebral cortex and lateral ·caudate has been reported 1 •15 . However, no report has shown a regional difference of an induction of molecular chaperone in relation to a recovery of protein synthesis between in the cortex and caudate after cerebral ischaemia. Correspondence to : Koji Abe, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-mac\li, Aobaku, Sendai 980, Japan. Accepted for publication February 1992.
© 1992 Forefront Publishing Group 0161-6412/92/ 050381- 05
Therefore, we examined a possible different induction of HSP70 mRNA between cortex and lateral caudate in relation to a change of total protein synthesis using a transient focal brain ischaemia model of rats. MATERIALS AND METHODS The model used for this study consisted of middle cerebral artery (MCA) occlusion using a microembolus of nylon thread 14. During the surgical preparation for MCA occlusion, male rats of Wistar strain, weighing 250-280 g, were lightly anaesthetized by inhalation of a nitrous oxide/ oxygen / halothane (69% :30% :1 %) mixture. When the animals began to awake after the stop of the anaesthesia, the origin of the right MCA was occluded for 30 min by insertion of a nylon thread via the internal carotid artery. After 30 min of ischaemia, the nylon thread was removed and the animals recovered for 1, 3, 8 h, 1, 2, and 7 days until decapitation. Sham animals were treated with the cervical preparation, but without the following insertion of nylon thread. During the surgical preparation and occlusion of MCA, rats were on a heat pad warmed at 37 Rectal temperature was monitored in all animals. The animals recovered in the room at 22- 25 °C. At the end of the reperfusio n, rats were decapitated, and the cerebral cortex and the lateral caudate of the occluded M CA territory were dissected on ice for the following Northern analysis. After the dissection, the brains were quickly frozen in liquid nitrogen, and st ored at -80 °C. Samples for 3 animals were combined in each time point t o yield about 500 mg for the cortical samples and about 200 mg for the caudat e samples
oc.
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of each group. Total RNA was extracted by the method of Chmoczynski and Sacchi 5 using guanidine isothiocyanate. Northern blot analyses for hybridization were performed by the method of our previous report2 . Two probes for HSP70 gene (pH 2.3: Reference 24) and a-tubulin gene10 were used against the Northern blot. After hybridization, the filtre was finally washed with 0.1 • SSC (1 • SSC = 150 mM NaCI + 15 mM sodium citrate)+ 0.1% SDS (sodium dodecylsulphate) at 65 °C. Filtres were exposed to Kodak X-ray film for 20 h at -80 °C. The filtre was then stripped of the radiolabelled pH 2.3 probe, then hybridized with the probe for tubulin. The two Northern blot membranes (for the cortical tissue and the caudate tissue) were always hybridized together in a same hybridization bag. The level of tubulin mRNA was measured as an internal standard. The forebrain obtained by decapitation were frozen in powdered dry ice. Sections (10 JJ.m) at caudate nucleus levels were cut on a cryostat at -18 °C, and collected on Histostick (Accurate Chemical and Scientific Corp., Westbury, NY, USA)- coated slides. In situ hybridization was performed by the method of a previous report from our laboratol)'26 . The insert of pH 2.3 were radiolabelled with a- 35 S dCTP (1000 Ci / mmol, Amersham, UK) by random primer labelling kit (Boehringer Mannheim, Germany), resulting in a specific activity of about 5 x 108 dpm / JJ.g. The 1.1 Kb fragment of pHSG396 plasmid (Takara, Tokyo, Japan) digested by Hinf I was similarly labelled as a control probe. Hybridization signals were visualized by exposure against an X-ray film, then the slides were dipped in a liquid emulsion (NR-M2, Konica Corp., Tokyo, Japan) diluted (1:1) with 0.6M ammonium for 3 weeks, acetate. Then they were exposed at 4 developed, and counter stained with haematoxylin. Several sections at 8 h of reperfusion were treated with 100 JJ.g/ ml RNase A and 10 units / ml RNase T1 (Sigma, St. Louis, MO, USA) at 37 °C for 30 min prior to prehybridization and were hybridized with the probe. Three animals in each time point were examined. Autoradiographic analysis for the total protein synthesis was performed as described before1 . In brief, three animals in each time point were treated . with ischaemia / reperfusion as described above. Forty five minutes before the time of decapitation, 14 Cmethionine (51.8 mCi / mmol) was intravenously injected, then 20 JJ.m coronal brain sections were obtained. The sections were treated with 0.5 % TCA (trichloroacetate) solution and were exposed to an X-ray film for 4 weeks. Rat brains at 7 days after the reperfusion were fixed by perfusion fixation using 40% formaldehyde / glacial acetic acid / methanol (1 :1 :8) according to the method of a previous report1 "'. Brain sections (10 JJ.m) from three animals in each time point were stained with cresyl violet_?nd examined by light microscopy. With different series of animals, brain temperatures were measured during and after MCA occlusion. Two days before the experiment, incision of midline parietal scalp revealed parietal bone under a pentobarbital anaesthesia (50 mg/ kg, i.p.). A small hole of the bone was made by a dental drill at just caudal to the coronal suture which was 4 mm right side from the sagittal suture. The animals recovered at the room temperature. On the day of experiment, cervical preparations were performed under the nitrous oxide / oxygen / halothane
oc
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anaesthesia for the following MCA occlusion. After stop of the anaesthesia, temperatures of cerebral cortex and lateral caudate of the side of MCA occlusion were monitored by insertion of a needle microprobe (t ype MT26 / 2, diameter0.46 mm, Physitemp Instruments Inc., NJ, USA) during and after the MCA occlusion. In order to compare with sham control brain, brain temperatures of sham treated animals were monitored in the same way. Rectal temperatures were also monitored with a rectal probe. Each series consisted of 6 animals. Statistical analyses were performed with a non-parametrical test such as the Wilcoxon rank-sum (Wilcoxon' s U) test.
RESULTS As shown in Figure 1, the HSP70 mRNA is not present in a detectable amount both in the cerebral cortex and the lateral caudate of sham rats. It is induced in the cerebral cortex at 3 h to 1 day after the transient ischaemia with a maximum at 8 h. The mRNA was also induced in the lateral caudate at 3 to 8 h of the reperfusion. However, the maximum induction in the caudate was at 3 h, and the maximum amount of the induction was significantly smaller than that in the cerebral cortex at 8 h. The amount of HSP70 mRNA returned to the undetectable level by 2 days in the cortex, and by 1 day in the caudate. The level of tubulin mRNA showed a very small decrease at 3 h to 24 h in the cortex, and a small decrease at 1 h to 8 h in the caudate. Two sizes of mRNA for HSP70 were detected after ischaemia (2.8 and 3.0 Kb ). A synthetic oligonucleotide which selectively detected inducible species of HSP70 family mRNA also detected two closely spaced doublet of inducible mRNA in rats12 . The two sizes of mRNA for HSP70 changed almost in a same manner after ischaemia both in the cortex and caudate (Figure 1). In situ hybridization study showed spacial distribution
of the mRNA. The time course and the amount of mRNA is compatible to the result of Northern blot analysis. Typical cases are shown in Figure 2. Observation of sections dipped in the liquid emulsion revealed the localization of high density autoradiographic grains for HSP70 mRNA in the cell body (data not shown ). No detective signal was found after the treatment of RNases and in the case with plasmid DNA (pHSG 396) as the probe (data not shown ). These data support the specificity of the hybridization. Autoradiographic analysis showed that total protein synthesis markedly decreased at 1 h in the cerebral
CORTEX
CAUDATE
C 1h 3h Sh 1d 2d 7d
C lh 3h Sh 1d 2d 7d
HSP 70
,_,. ,;,.;~
TUBULIN ...
_
....... ...
::_:.;_:~_. ..J;;~
,j_...-:_ ..
--...------
...
Northern blot analyses of HSP70 and tubulin mRNAs in the cerebral cortex and the lateral caudate at 1, 3, 8 h, 1, 2, and 7 days after 30 min of transie nt MCA occlusion. C re presents the sham control. Each lane contains 15 f./,g of total RNA. Arrowheads re present the riboso mal RNAs Fiture 1:
Heat shock protein mRNA: K. Abe et al.
cortex and caudate of the occluded MCA territory, then gradually recovered (Figure 3). The synthesis recovered to the control level in the cortex by 2 days, while it did not completely recover in the lateral caudate until 7 days. All three cases showed almost the same results in each time point. Typical cases are shown in Figure 3. Histological examination showed almost normal finding in the cortex (Figure 4C) as compared to more severe damage in the lateral caudate (Figure 40) at 7 days after the reperfusion. Caudate cells (small to medium and large neurons) are almost lost, and reactive gliosis is detected (Figure 40). Table 1 shows changes of temperatures of brain and rectum during and after MCA occlusion. Temperatures of the cerebral cortex and the lateral caudate of the occluded MCA territory, and rectum significantly decreased during ischaemia. The temperatures i"ncreased after the reperfusion with a peak at 2 h, then gradually recovered to the pre-ischaemic level by 10 h. No significant difference of the temperature between the
Figure 3:
Autoradiographic demonstrations of regional total protein synthesis in rat brain after the transient MCA occlusion. C represents the case of the sham control. 1, 3, 8, 1d, 2d, and 7d represent the typical cases of 1 h, 3 h, 8 h, 1 day, 2 days, and 7 days of reperfusion after the transient ischaemia, respectively
c 3H
BH
70· Figure 2:
In situ hybridization with HSP70 probe show inductions of the mRNA in the cerebral cortex and lateral caudate. C, 3H, 8H, and 7D represent the typical cases of the sham control, 3 h, 8 h, and 7 days after the reperfusion, respectively. Note that the induction in the caudate is smaller and shorte~ than in the cerebral cortex
Table 1:
Histology of cerebral cortex (A and C) and lateral caudate (B and D). A and B are from normal control brain. C and D are from the ipsilateral hemisphere at -7 days after the 30 min MCA occlusion. Bar represents 130 Jl.m Figure 4:
Changes of temperature of brain and rectum during and after MCA occlusion in rats Period of ischaemia (min)
COR CAD REC
Period after recirculation (h)
pre
5
15
30
0.5
1
1.5
2
2.5
3
10
37.6 ±0.3 37.6 ± 0.3 37.1 ± 0.3
35.oa ±0.6 35.1a ± 0.6 35.0a ± 0.7
34.9a ±0.6 35.1a ± 0.7 34.8a ± 0.8
35.3a ± 0.4 35.4a ± 0.3 35.3a ± 0.4
37.1 ± 0.4 37.2 ± 0.4 37.2 ± 0.3
37.8 ± 0.6 37.8 ±0.4 37.5 ± 0.5
38.3b ± 0.5 38.4b ± 0.4 38.2b ± 0.7
38.6b ± 0.8 38.7b ± 0.7 38.8b ± 0.9
38.5b ± 0.8 38.6b ± .0.8 38.6b ± 0.9
38.2 ± 0.9 38.3 ± 0.8 38.2 ±0.5
37.5 ±0.1 37.6 ± 0.1 37.2 ± 0.7
Data represent mean ± SD of temperatures (n = 6). COR, CAD, and REC represent the temperatures of the cortex and lateral caudate of the occluded MCA territory, and rectum, respectively. • p < 0.01, b p < O.OS against the values of pre-MCA occlusion.
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Heat shock protein mRNA: K. Abe et al.
cerebral cortex and lateral caudate was observed. Sham operation did not change temperatures significantly up to 3.5 h after the operation (data not shown).
DISCUSSION This is the first observation on the different induction of HSP70 mRNA between the cerebral cortex and the lateral caudate in a transient MCA occlusion model of rats. Our results indicate that HSP70 mRNA was greatly induced along with the recovery of total protein synthesis in the cortex, but that the mRNA was induced earlier and more-weakly in the lateral caudate where protein synthesis did not completely recover. The profound correlation between the induction of HSP70 mRNA and the recovery of the total protein synthesis strongly suggests the role of HSP70 as a molecular chaperone in the recovery of brain cells after a transient ischaemic stress. A histopathological study showed that the 30 min transient MCA occlusion causes severe damage in neuronal cells of lateral caudate, while the same ischaemic treatment resulted in only a minimal damage to cortical neurons (Figure 4, Reference 15). It should be of interest that cortical cells, which are relatively resistant to the transient ischaemia, produced a greater amount of HSP70 mRNA, but that the caudate cells, which are more vulnerable to the transient ischaemic stress, produced a smaller amount of the mRNA, and the induction quickly diminished as early as at 8 h (Figures 1 and 2). Hyperthermia aggravates brain cell damage after transient ischaemia13, and hypothermia protects or ameliorates the damage25 . Even though brain temperature of the MCA territory significantly changed during and after the MCA occlusion, no regional difference was found between the cerebral cortex and lateral caudate (Table 1). Regional cerebral blood flow (rCBF) during ischaemia and reperfusion is one of the critical factors for the prognosis of the damage to neuronal cells caused by transient ischaemia. A previous study revealed that rCBF in the cortex and the lateral caudate are not significantly different during the ischaemia and reperfusion in this MCA occlusion model 1 ' 14 . Therefore, the different vulnerability between regions should not be due to a difference of brain temperature nor rCBF, but rather be due to other mechanisms such as regionally different transneuronal mechanism, or gene expressiol} of putative cell protective proteins. The laterar caudate is rich in receptors of excitatory neurotransmitters, such as glutamate and aspartate6 . Receptors of inhibitory neurotransmitters, such as y-aminobutyric acid (GABA) and benzodiazepine exist in smaller amounts in the caudate than in the . cortex16. The striatal cells are also richly innervated by nigrostriatal dopaminergic projections8 . The nigrostriatal dopaminergic activity has been reported to play an important role in the striatal neuronal damage as well as glutamatergic activity from the cortex15' 23 . Thus, imbalance of excitatory /inhibitory neuronal innervation might aggravate the damage of caudate neurons. There is a relationship between excitatory neurotransmission and HSP70 gene expression. A stereotaxic injection of kainic acid into the striatum of rats induces synthesis of mRNA for HSP7021 . Because of the potential imbalance of excitatory /inhibitory innervation, cells in lateral caudate are under more stressful conditions than
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cortical cells during and after the transient ischaemia. Therefore, the induction of the HSP70 mRNA should be greater in the caudate than in the cortex by the transcriptional mechanism20. However, our results showed that the maximum induction in the caudate was smaller and shorter than in the cortex, indicating that there may be an alternative mechanism to account for the less induction in the caudate cells. It is possible that the excitotoxicity is too strong to induce full scale transcription of the HSP70 gene2 . An elucidation of the exact mechanism of the vulnerability of caudate neurons in this model will be further required.
ACKNOWLEDGEMENT This work was partly supported by Monbusho grant 01044018, 03404028, and the Kanae-Sinyaku foundation. REFERENCES
2
3
4
5
6
7 8
9 10
11
12
13
14
15
16
Abe K, Araki K, Kogure K. Recovery from edema and of protein synthesis differs between the cortex and caudate following transient focal cerebral ischemia in rats, 1. Neurochem 1988; 51: 1470-1476 Abe K, Tanzi RE, Kogure K. Induction of HSP70 mRNA after transient ischemia in gerbil brain, Neurosci Lett 1991; 125: 166-168 Beckmann RP, Mizzen LA, Welch W]. Interaction of HSP70 with newly synthesized proteins: implications for protein folding and assembly, Science 1990; 248 : 850- 853 Bodsh W , Takahashi K, Barbier B, Ophoff G, Hossmann KA. Cerebral proteins and ischemia, Progr Brain Res 1985; 63: 197-210 Chmoczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidine thiocyanate-phenol-chloroform extraction, Anal Biochem 1987; 162: 156-159 Fagg GE, Foster AC. Amino acid neurotransmitters and their pathways in the mammalian central nervous system, Neuroscience 1983; 9: 701-719 Gething M], Sambrook ]. Protein folding in the cell, Nature 1992; 355: 33-45 Globus MYT, Ginsberg MD, Dietrich WD, Busto R, Scheinberg P. Substantia nigra lesion protects against ischemic damage in the striatum, Neurosci Lett 1987; 80 : 251-256 Kirino T. Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res 1982; 239: 57- 69 Lemishka IR, Farmer S, Racaniello VR, Sharp PA, Nucleotide sequence and evolution of mammalian a-tubulin messenger RNA, I Mol Bioi 1981; 151 : 101-120 Lewis MT, Pelham HB. Involvement of ATP in the nuclear and nucleolar funct ions of the 70kd heat shock protein, EMBO I 1985; 4: 3137-3143 Marini AM, Kozuka M, Lipsky RH, Nowak TS Jr. 70-kilodalton heat shock protein induction in cerebellar astrocytes and cerebellar granule cells in vitro : comparison w ith immunocytochemical location after hyperthermia in vivo, I Neurochem 1990; 54: 1509-1516 Minamisawa H, Smith ML, Siesjo, BK. The effect of moderate hyperthermia (39 °C) and hypothermia (35 ° C) on brain damage following 10 and 15 minutes of forebrain ischemia, 1 Cereb Blood Flow Metabol 1989; 9 (suppl.) : 265 Nagasawa H, Kogure K. Correlation between cerebral blood flow and histologic changes in a new model of middle cerebral artery occlusion, Stroke 1989; 20: 1037-1043 Nakano S, Kogure K, Fujikura H. Ischemia-induced slowly progressive neuronal damage in the rat brain, Neuroscience 1990; 38: 115-124 Onodera H, Sato G, Kogure K. GABA and benzodiazepine receptors in the gerbil brain after transient ischemia: demonstration by quantitative receptor autoradiography, 1 Cereb Blood Flow M etabol 1987; 7 : 82-88
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17
Pelham HRB. HSP70 accelerates the recovery of nucleolar morphology after heat shock, EMBO I 1984; 3: 3095-3100 18 Pelham HRB. Heat shock and the sorting of luminal ER proteins, EMBO I 1989; 8: 3171-3176 19 Pulsinelli WA, Brierly JB, Plum F. Temporal profile of neuronal damage in a model of transient forebrain ischemia, Ann Neural 1982; 11 : 491-498 20 Theodorakis NG, Morimoto Rl. Posttranscriptional regulation of HSP70 expression in human cells, Mol Cell Bioi 1987; 7: 4357-4368 21 Uney JB, Leigh PN, Marsden CD, Lee A, Anderson BH. Stereotaxic injection of kinic acid into the striatum of rats induces synthesis of mRNA for heat shock protein 70, FEBS Lett 1988; 235: 215-218 22 Vass K, Welch WJ, Nowak TS Jr. Localization of 70-kDa stress
protein
induction
in gerbil
brain
after ischemia, Acta
Neuropathol. 1988; 77: 128-135
23 24
25
26
Wieloch T. Neurochemical correlates to selective neuronal vulnerability, Progr Brain Res 1985; 63: 69-85 Wu B, Hunt C, Morimoto R. Structure and expression of the human gene encoding major heat shock protein HSP70, Mol Cell Bio/1985; 5: 330-341 Yamashita K, Eguchi Y, Kajiwara K, Ito H. Mild hypothermia ameliorates ubiquitin synthesis and prevents delayed neuronal death in the gerbil hippocampus, Stroke 1991 ; 22:1574-1581 Yoshioka M, Nagano I, Nakamura S, lmaizumi M, Kimura N. Detection of vasoactive intestinal polypeptide mTNA in ganglia neuroblastoma by in situ hybridization, Endocr Pathol 1990; 1: 51-57
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ISSN: 0161-6412 (Print) 1743-1328 (Online) Journal homepage: http://www.tandfonline.com/loi/yner20
Differential expression of heat shock protein 70 gene between the cortex and caudate after transient focal cerebral ischaemia in rats K. Abe, J. Kawagoe, T. Araki, M. Aoki & K. Kogure To cite this article: K. Abe, J. Kawagoe, T. Araki, M. Aoki & K. Kogure (1992) Differential expression of heat shock protein 70 gene between the cortex and caudate after transient focal cerebral ischaemia in rats, Neurological Research, 14:5, 381-385, DOI: 10.1080/01616412.1992.11740089 To link to this article: https://doi.org/10.1080/01616412.1992.11740089
Published online: 20 Jul 2016.
Submit your article to this journal
Citing articles: 39 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=yner20
Differential expression of heat shock protein 70 gene between the cortex and caudate after transient focal cerebral ischaemia in rats K. Abe, j. Kawagoe, T. Araki, M. Aoki and K. Kogure Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai, Japan
In relation to changes of total protein synthesis, induction of 70-kOa heat shock protein ( HSP70) mRNA was examined by Northern blot and in situ hybridization after 30 min of transient middle cerebral artery ( MCA ) occlusion of rats. HSP70 mRNA was not present in the control condition of brain. With reperfusion, the mRNA was greatly induced along with the recovery of total protein synthesis in the cerebral cortex and lateral caudate of the ipsilateral hemisphere. However, the level of the mRNA reached a maximum earlier in the lateral caudate (at 3 h) than in the cortex (at 8 h ), and the maximum amount of the mRNA was much smaller in the caudate than in the cortex. Total protein synthesis in the lateral caudate did not completely recover until 7 days. Histological examination showed a severe damage in cells of lateral caudate, while cells in the cortex were almost normal at 7 days. No difference in the brain temperature was observed between the two regions. These results show that the induction of HSP70 mRNA correlates · with the recovery of protein synthesis in brain cells after a transient ischaemia, and that the HSP70 gene expression is different at the transcriptional/eve/ between the cortical and caudate cells after the transient ischaemia. Keywords: Heat shock protein; ischaemia; protein synthesis
INTRODUCTION A role of molecular chaperone has been suggested for heat shock proteins (HSPs). HSP is induced in stressful conditions of cells such as heat shock or cerebral ischaemia2•18•22. Among HSPs, 70-kDa heat shock protein (HSP70) is known to be essential to restore normal ribosome assembly, promotes the synthesis of new ribosome, and accelerates the recovery of cell after heat shock17 . A role for protein folding has been recognized as the most important role of HSP7 . HSP recognizes and stabilizes partially folded intermediates during polypeptide folding, assembly and disassembly. HSP also disentangles malfolded or aggregated protein. A transient ischaemia causes profound reduction of total protein synthesis 4 and the disaggregation of polysome is among the earliest pathological findings in ischaemic neuronal damage9 . A role of HSP70 in t he recovery of protein synthesis has been indicated 17•18, and a recent report suggested a role of molecular chaperone of HSP70 for newly synthesized or degraded protein 3 with ATP-dependent mechanism 11 . Regional difference of the recovery from a transient ischaemia between cerebral cortex and lateral ·caudate has been reported 1 •15 . However, no report has shown a regional difference of an induction of molecular chaperone in relation to a recovery of protein synthesis between in the cortex and caudate after cerebral ischaemia. Correspondence to : Koji Abe, Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, 1-1 Seiryo-mac\li, Aobaku, Sendai 980, Japan. Accepted for publication February 1992.
© 1992 Forefront Publishing Group 0161-6412/92/ 050381- 05
Therefore, we examined a possible different induction of HSP70 mRNA between cortex and lateral caudate in relation to a change of total protein synthesis using a transient focal brain ischaemia model of rats. MATERIALS AND METHODS The model used for this study consisted of middle cerebral artery (MCA) occlusion using a microembolus of nylon thread 14. During the surgical preparation for MCA occlusion, male rats of Wistar strain, weighing 250-280 g, were lightly anaesthetized by inhalation of a nitrous oxide/ oxygen / halothane (69% :30% :1 %) mixture. When the animals began to awake after the stop of the anaesthesia, the origin of the right MCA was occluded for 30 min by insertion of a nylon thread via the internal carotid artery. After 30 min of ischaemia, the nylon thread was removed and the animals recovered for 1, 3, 8 h, 1, 2, and 7 days until decapitation. Sham animals were treated with the cervical preparation, but without the following insertion of nylon thread. During the surgical preparation and occlusion of MCA, rats were on a heat pad warmed at 37 Rectal temperature was monitored in all animals. The animals recovered in the room at 22- 25 °C. At the end of the reperfusio n, rats were decapitated, and the cerebral cortex and the lateral caudate of the occluded M CA territory were dissected on ice for the following Northern analysis. After the dissection, the brains were quickly frozen in liquid nitrogen, and st ored at -80 °C. Samples for 3 animals were combined in each time point t o yield about 500 mg for the cortical samples and about 200 mg for the caudat e samples
oc.
Neurological Research, 1992, Volume 14, December
381
Heat shock protein mRNA: K. Abe et al.
of each group. Total RNA was extracted by the method of Chmoczynski and Sacchi 5 using guanidine isothiocyanate. Northern blot analyses for hybridization were performed by the method of our previous report2 . Two probes for HSP70 gene (pH 2.3: Reference 24) and a-tubulin gene10 were used against the Northern blot. After hybridization, the filtre was finally washed with 0.1 • SSC (1 • SSC = 150 mM NaCI + 15 mM sodium citrate)+ 0.1% SDS (sodium dodecylsulphate) at 65 °C. Filtres were exposed to Kodak X-ray film for 20 h at -80 °C. The filtre was then stripped of the radiolabelled pH 2.3 probe, then hybridized with the probe for tubulin. The two Northern blot membranes (for the cortical tissue and the caudate tissue) were always hybridized together in a same hybridization bag. The level of tubulin mRNA was measured as an internal standard. The forebrain obtained by decapitation were frozen in powdered dry ice. Sections (10 JJ.m) at caudate nucleus levels were cut on a cryostat at -18 °C, and collected on Histostick (Accurate Chemical and Scientific Corp., Westbury, NY, USA)- coated slides. In situ hybridization was performed by the method of a previous report from our laboratol)'26 . The insert of pH 2.3 were radiolabelled with a- 35 S dCTP (1000 Ci / mmol, Amersham, UK) by random primer labelling kit (Boehringer Mannheim, Germany), resulting in a specific activity of about 5 x 108 dpm / JJ.g. The 1.1 Kb fragment of pHSG396 plasmid (Takara, Tokyo, Japan) digested by Hinf I was similarly labelled as a control probe. Hybridization signals were visualized by exposure against an X-ray film, then the slides were dipped in a liquid emulsion (NR-M2, Konica Corp., Tokyo, Japan) diluted (1:1) with 0.6M ammonium for 3 weeks, acetate. Then they were exposed at 4 developed, and counter stained with haematoxylin. Several sections at 8 h of reperfusion were treated with 100 JJ.g/ ml RNase A and 10 units / ml RNase T1 (Sigma, St. Louis, MO, USA) at 37 °C for 30 min prior to prehybridization and were hybridized with the probe. Three animals in each time point were examined. Autoradiographic analysis for the total protein synthesis was performed as described before1 . In brief, three animals in each time point were treated . with ischaemia / reperfusion as described above. Forty five minutes before the time of decapitation, 14 Cmethionine (51.8 mCi / mmol) was intravenously injected, then 20 JJ.m coronal brain sections were obtained. The sections were treated with 0.5 % TCA (trichloroacetate) solution and were exposed to an X-ray film for 4 weeks. Rat brains at 7 days after the reperfusion were fixed by perfusion fixation using 40% formaldehyde / glacial acetic acid / methanol (1 :1 :8) according to the method of a previous report1 "'. Brain sections (10 JJ.m) from three animals in each time point were stained with cresyl violet_?nd examined by light microscopy. With different series of animals, brain temperatures were measured during and after MCA occlusion. Two days before the experiment, incision of midline parietal scalp revealed parietal bone under a pentobarbital anaesthesia (50 mg/ kg, i.p.). A small hole of the bone was made by a dental drill at just caudal to the coronal suture which was 4 mm right side from the sagittal suture. The animals recovered at the room temperature. On the day of experiment, cervical preparations were performed under the nitrous oxide / oxygen / halothane
oc
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anaesthesia for the following MCA occlusion. After stop of the anaesthesia, temperatures of cerebral cortex and lateral caudate of the side of MCA occlusion were monitored by insertion of a needle microprobe (t ype MT26 / 2, diameter0.46 mm, Physitemp Instruments Inc., NJ, USA) during and after the MCA occlusion. In order to compare with sham control brain, brain temperatures of sham treated animals were monitored in the same way. Rectal temperatures were also monitored with a rectal probe. Each series consisted of 6 animals. Statistical analyses were performed with a non-parametrical test such as the Wilcoxon rank-sum (Wilcoxon' s U) test.
RESULTS As shown in Figure 1, the HSP70 mRNA is not present in a detectable amount both in the cerebral cortex and the lateral caudate of sham rats. It is induced in the cerebral cortex at 3 h to 1 day after the transient ischaemia with a maximum at 8 h. The mRNA was also induced in the lateral caudate at 3 to 8 h of the reperfusion. However, the maximum induction in the caudate was at 3 h, and the maximum amount of the induction was significantly smaller than that in the cerebral cortex at 8 h. The amount of HSP70 mRNA returned to the undetectable level by 2 days in the cortex, and by 1 day in the caudate. The level of tubulin mRNA showed a very small decrease at 3 h to 24 h in the cortex, and a small decrease at 1 h to 8 h in the caudate. Two sizes of mRNA for HSP70 were detected after ischaemia (2.8 and 3.0 Kb ). A synthetic oligonucleotide which selectively detected inducible species of HSP70 family mRNA also detected two closely spaced doublet of inducible mRNA in rats12 . The two sizes of mRNA for HSP70 changed almost in a same manner after ischaemia both in the cortex and caudate (Figure 1). In situ hybridization study showed spacial distribution
of the mRNA. The time course and the amount of mRNA is compatible to the result of Northern blot analysis. Typical cases are shown in Figure 2. Observation of sections dipped in the liquid emulsion revealed the localization of high density autoradiographic grains for HSP70 mRNA in the cell body (data not shown ). No detective signal was found after the treatment of RNases and in the case with plasmid DNA (pHSG 396) as the probe (data not shown ). These data support the specificity of the hybridization. Autoradiographic analysis showed that total protein synthesis markedly decreased at 1 h in the cerebral
CORTEX
CAUDATE
C 1h 3h Sh 1d 2d 7d
C lh 3h Sh 1d 2d 7d
HSP 70
,_,. ,;,.;~
TUBULIN ...
_
....... ...
::_:.;_:~_. ..J;;~
,j_...-:_ ..
--...------
...
Northern blot analyses of HSP70 and tubulin mRNAs in the cerebral cortex and the lateral caudate at 1, 3, 8 h, 1, 2, and 7 days after 30 min of transie nt MCA occlusion. C re presents the sham control. Each lane contains 15 f./,g of total RNA. Arrowheads re present the riboso mal RNAs Fiture 1:
Heat shock protein mRNA: K. Abe et al.
cortex and caudate of the occluded MCA territory, then gradually recovered (Figure 3). The synthesis recovered to the control level in the cortex by 2 days, while it did not completely recover in the lateral caudate until 7 days. All three cases showed almost the same results in each time point. Typical cases are shown in Figure 3. Histological examination showed almost normal finding in the cortex (Figure 4C) as compared to more severe damage in the lateral caudate (Figure 40) at 7 days after the reperfusion. Caudate cells (small to medium and large neurons) are almost lost, and reactive gliosis is detected (Figure 40). Table 1 shows changes of temperatures of brain and rectum during and after MCA occlusion. Temperatures of the cerebral cortex and the lateral caudate of the occluded MCA territory, and rectum significantly decreased during ischaemia. The temperatures i"ncreased after the reperfusion with a peak at 2 h, then gradually recovered to the pre-ischaemic level by 10 h. No significant difference of the temperature between the
Figure 3:
Autoradiographic demonstrations of regional total protein synthesis in rat brain after the transient MCA occlusion. C represents the case of the sham control. 1, 3, 8, 1d, 2d, and 7d represent the typical cases of 1 h, 3 h, 8 h, 1 day, 2 days, and 7 days of reperfusion after the transient ischaemia, respectively
c 3H
BH
70· Figure 2:
In situ hybridization with HSP70 probe show inductions of the mRNA in the cerebral cortex and lateral caudate. C, 3H, 8H, and 7D represent the typical cases of the sham control, 3 h, 8 h, and 7 days after the reperfusion, respectively. Note that the induction in the caudate is smaller and shorte~ than in the cerebral cortex
Table 1:
Histology of cerebral cortex (A and C) and lateral caudate (B and D). A and B are from normal control brain. C and D are from the ipsilateral hemisphere at -7 days after the 30 min MCA occlusion. Bar represents 130 Jl.m Figure 4:
Changes of temperature of brain and rectum during and after MCA occlusion in rats Period of ischaemia (min)
COR CAD REC
Period after recirculation (h)
pre
5
15
30
0.5
1
1.5
2
2.5
3
10
37.6 ±0.3 37.6 ± 0.3 37.1 ± 0.3
35.oa ±0.6 35.1a ± 0.6 35.0a ± 0.7
34.9a ±0.6 35.1a ± 0.7 34.8a ± 0.8
35.3a ± 0.4 35.4a ± 0.3 35.3a ± 0.4
37.1 ± 0.4 37.2 ± 0.4 37.2 ± 0.3
37.8 ± 0.6 37.8 ±0.4 37.5 ± 0.5
38.3b ± 0.5 38.4b ± 0.4 38.2b ± 0.7
38.6b ± 0.8 38.7b ± 0.7 38.8b ± 0.9
38.5b ± 0.8 38.6b ± .0.8 38.6b ± 0.9
38.2 ± 0.9 38.3 ± 0.8 38.2 ±0.5
37.5 ±0.1 37.6 ± 0.1 37.2 ± 0.7
Data represent mean ± SD of temperatures (n = 6). COR, CAD, and REC represent the temperatures of the cortex and lateral caudate of the occluded MCA territory, and rectum, respectively. • p < 0.01, b p < O.OS against the values of pre-MCA occlusion.
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Heat shock protein mRNA: K. Abe et al.
cerebral cortex and lateral caudate was observed. Sham operation did not change temperatures significantly up to 3.5 h after the operation (data not shown).
DISCUSSION This is the first observation on the different induction of HSP70 mRNA between the cerebral cortex and the lateral caudate in a transient MCA occlusion model of rats. Our results indicate that HSP70 mRNA was greatly induced along with the recovery of total protein synthesis in the cortex, but that the mRNA was induced earlier and more-weakly in the lateral caudate where protein synthesis did not completely recover. The profound correlation between the induction of HSP70 mRNA and the recovery of the total protein synthesis strongly suggests the role of HSP70 as a molecular chaperone in the recovery of brain cells after a transient ischaemic stress. A histopathological study showed that the 30 min transient MCA occlusion causes severe damage in neuronal cells of lateral caudate, while the same ischaemic treatment resulted in only a minimal damage to cortical neurons (Figure 4, Reference 15). It should be of interest that cortical cells, which are relatively resistant to the transient ischaemia, produced a greater amount of HSP70 mRNA, but that the caudate cells, which are more vulnerable to the transient ischaemic stress, produced a smaller amount of the mRNA, and the induction quickly diminished as early as at 8 h (Figures 1 and 2). Hyperthermia aggravates brain cell damage after transient ischaemia13, and hypothermia protects or ameliorates the damage25 . Even though brain temperature of the MCA territory significantly changed during and after the MCA occlusion, no regional difference was found between the cerebral cortex and lateral caudate (Table 1). Regional cerebral blood flow (rCBF) during ischaemia and reperfusion is one of the critical factors for the prognosis of the damage to neuronal cells caused by transient ischaemia. A previous study revealed that rCBF in the cortex and the lateral caudate are not significantly different during the ischaemia and reperfusion in this MCA occlusion model 1 ' 14 . Therefore, the different vulnerability between regions should not be due to a difference of brain temperature nor rCBF, but rather be due to other mechanisms such as regionally different transneuronal mechanism, or gene expressiol} of putative cell protective proteins. The laterar caudate is rich in receptors of excitatory neurotransmitters, such as glutamate and aspartate6 . Receptors of inhibitory neurotransmitters, such as y-aminobutyric acid (GABA) and benzodiazepine exist in smaller amounts in the caudate than in the . cortex16. The striatal cells are also richly innervated by nigrostriatal dopaminergic projections8 . The nigrostriatal dopaminergic activity has been reported to play an important role in the striatal neuronal damage as well as glutamatergic activity from the cortex15' 23 . Thus, imbalance of excitatory /inhibitory neuronal innervation might aggravate the damage of caudate neurons. There is a relationship between excitatory neurotransmission and HSP70 gene expression. A stereotaxic injection of kainic acid into the striatum of rats induces synthesis of mRNA for HSP7021 . Because of the potential imbalance of excitatory /inhibitory innervation, cells in lateral caudate are under more stressful conditions than
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cortical cells during and after the transient ischaemia. Therefore, the induction of the HSP70 mRNA should be greater in the caudate than in the cortex by the transcriptional mechanism20. However, our results showed that the maximum induction in the caudate was smaller and shorter than in the cortex, indicating that there may be an alternative mechanism to account for the less induction in the caudate cells. It is possible that the excitotoxicity is too strong to induce full scale transcription of the HSP70 gene2 . An elucidation of the exact mechanism of the vulnerability of caudate neurons in this model will be further required.
ACKNOWLEDGEMENT This work was partly supported by Monbusho grant 01044018, 03404028, and the Kanae-Sinyaku foundation. REFERENCES
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3
4
5
6
7 8
9 10
11
12
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