doi: 10.1161/hs1201.099604
(Stroke. 2001;32:2905.)
© 2001 American Heart Association, Inc.
Original Contributions |
Targeted hsp70.1 Disruption Increases Infarction Volume After Focal Cerebral Ischemia in Mice
From the Department of Neurology, Seoul National University (S.-H.L., M.K., B.-W.Y., Y.-J.K., S.-J.M., J.-K.R.); Department of Biochemistry, Ilchun Molecular Medicine Institute MRC, Seoul National University (J.-S.L., J.-S.S.); and the Neuroscience Research Institute and Clinical Research Institute (B.-W.Y., M.K., J.-K.R.), Seoul, Korea. Drs Lee and Kim contributed equally to this work.
Correspondence to Byung-Woo Yoon, MD, PhD, Department of Neurology, Seoul National University Hospital, 28 Yongondong, Chongnogu, Seoul, 110-744, Korea. E-mail bwyoon{at}snu.ac.kr
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Abstract |
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Background and Purpose— Heat-shock proteins (HSPs) are highly conserved proteins that are induced by a variety of stresses. HSP70 is a 70-kDa HSP family known to have cytoprotective effects against various insults. The role of HSP70 in cerebral ischemia remains to be elucidated in vivo.
Methods— To investigate the effect of reduced HSP70 levels on cerebral ischemia, focal cerebral ischemia by intraluminal occlusion of the middle cerebral artery was induced in hsp70.1 knockout mice. The expressions of hsp70.1 and hsp70.3 mRNAs and HSP70 protein were determined, and infarction volumes were measured and compared.
Results— Northern blots confirmed the absence of hsp70.1 mRNA expression in the knockout mice. The mean infarction volume was significantly larger in hsp70.1 knockout mice (92.5±8.3 mm3) than in the wild-type mice (59.3±8.9 mm,3 P<0.001). Western blots showed increased HSP70 expression in the ischemic hemisphere in both knockout and wild-type mice, but HSP70 expression levels in knockout mice were significantly lower than those in their wild-type littermates. Immunohistochemistry did not show any significant differences between the knockout and wild-type animals and showed increased HSP70 immunoreactivity in the ischemic hemisphere, with predominance in the cerebral cortex, especially in the penumbra.
Conclusions— Our results suggest that hsp70.1 plays an important role in the early protection of the brain, at least after acute focal cerebral ischemia in mice.
Key Words: cytoprotection • heat-shock proteins • ischemia • mice
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Introduction |
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Heat-shock proteins (HSPs) are a group of highly conserved proteins. They are present in virtually all species from bacteria to humans and are induced under a variety of environmental stresses. The 70-kDa family of HSPs is one of the most extensively studied subgroups of HSP proteins. Functionally, the 70-kDa HSP (HSP70) family is a group of chaperones that assist in the folding, transport, and assembly of proteins in the cytoplasm, mitochondria, and endoplasmic reticulum.1 In mice, the HSP70 family contains at least 7 different proteins, including the 70-kDa heat-shock cognate (HSC70), the 75-kDa and 78-kDa glucose-regulated proteins (GRP75 and GRP78, respectively), testis-specific HSC70, and spermatocyte-specific HSP70.2.2 Environmental stresses induce the expression of 2 additional HSP70s (HSP70.1 and HSP70.3), and their genes (hsp70.1 and hsp70.3) show very high homology with human hsp70.2 and hsp70.1.3 It has been demonstrated that hsp70.2 is located in chromosome 12; moreover, hsp70.1, hsp70.3, and hsc70t are all located within a 50-kbp length of H-2 in chromosome 12.2,4 The organization of these genes, which were originally discovered in mice, resembles that in humans and rats. The relationships between the hsp70 genes in rats, humans, and mice can be summarized as follows: rhsp70-1=hhsp70-2=mhsp70.1, rhsp70-2=hhsp70-1=mhsp70.3, and rhsp70-3=hhsp70-Hom=mhsc70t (where r indicates rat; h, human; and m, mouse).
The hsp70.1 and hsp70.3 genes are separated by only 7 kb on chromosome 17 and show 99% homology.5,6 The 2 genes are located on a single exon, which does not contain an intron, and the sequences controlling transcription are densely located in the promoter area. The hsp70 gene is scarcely expressed in the normal cells of the mouse but is abundant after cellular stress. Despite the very similar DNA sequences of the 2 genes, hsp70.1 generates a single mRNA, whereas hsp70.3 produces 2 mRNA products; this occurrence has been attributed to differences in the 3' untranslated region (UTR).7
Cytoprotective effects of HSP70 have been suggested by a number of in vitro studies.8–13 It has been reported that both heat shock and HSP70 overexpression protect neurons against glutamate-mediated toxicity,14 sublethal heat shock,15 glucose or oxygen deprivation,11,12,16 and simulated ischemia.10,16,17 Moreover, in vivo studies using transgenic mice have shown that the heart is protected by HSP70 from myocardial ischemic injury.18–20 In the nervous system, it has been reported that gene therapy with HSP72 improves neuron survival after focal cerebral ischemia21; however, recent studies using transgenic mice are controversial.22–24 Overexpression of HSP70 resulted in a 24-hour improvement in hippocampal neuron survival, but the overall infarct size was not affected.22 Another study using HSP70-transgenic mice reported that there is no relationship between infarct size and hippocampal neuron survival.23 Recently, Rajdev et al24 showed that cerebral infarction after 6 hours of ischemia is significantly lower in transgenic mice overexpressing rat HSP70 than in wild-type mice. This inconsistency may be related to differences in the level of HSP70 overexpression or the transgenic strains used. Thus, although several studies have suggested that HSP70 may protect the brain from various insults, the role of HSP70 in cerebral ischemia in vivo requires further clarification. In addition, because hsp70.1 and hsp70.3 are at different loci but encode an identical protein, it has been suggested that the 2 genes may be separately expressed in different situations or with different patterns. However, studies on this topic are limited, especially in the brain.
In the present study, we used hsp70.1 knockout mice to further investigate the neuroprotective function of HSP70 in cerebral ischemia. The present study used a middle cerebral artery (MCA) occlusion/reperfusion model in hsp70.1(-/-) mice and their wild-type littermates to investigate the role of HSP70 in cerebral ischemic injury.
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Materials and Methods |
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Animals
We produced mice bearing a null allele of hsp70.1 by using a gene-targeting technique described previously.25 In brief, a murine genomic clone of the hsp70.1 locus was cloned from a
FixII phage library prepared from 129/Sv embryonic stem (ES) cells with the use of a human hsp70 cDNA probe and then characterized by Southern blot analysis and DNA sequencing. The targeting vector contained a 7.5-kb NotI-XhoI fragment from the 5' promoter and a regulatory region of the hsp70.1 gene as the long arm, a 1.9-kb neomycin-resistance gene, an 0.8-kb NotI-SmaI fragment derived from the hsp70.1 exon as the short arm, and a 3.4-kb fragment containing 2 copies of the herpes simplex virus thymidine kinase gene (Figure 1A). The overall strategy for hsp70.1 gene targeting was to replace the promoter and some of the coding sequences with a phosphoglycerate kinase promoter with neomycin resistance gene (PGK-neo) expression cassette. Ten micrograms of NotI-linearized targeting vector was electroporated into E14/BK4 ES cells, and correctly targeted clones were selected with G418 (0.2 mg/mL, Life Technologies) and FIAU (200 µmol/L, Syntex) in DMEM medium. Southern blot analysis using a 520-bp 3' UTR as a probe recognized a 6-kb BamHI homologous recombinant and a 10-kb wild-type BamHI recombinant. Three independent homologous recombinant hsp70.1 ES cell clones (2D, 11E, and 28C) were injected into C57BL/6 blastocysts. The heterozygous mutant mice were generated from 1 line. The mutant mice used in all experiments had been backcrossed to the C57BL/6 strain for 9 generations. Genotypes were determined by using a 4-primer polymerase chain reaction (PCR) approach and confirmed by Southern blotting of genomic DNA isolated from tail biopsies. Mice were genotyped by isolating tail DNA and digesting it with BamHI for Southern blot analysis (Figure 1B). For PCR genotyping, the following 2 primer sets were used: (1) forward primer a (5'-AGGAGCTGACCCTTAA CAGC-3') and reverse primer a' (5'-GTCGTTGGCGATGATCTC-3'), annealing to the deleted part of genomic sequences, and (2) forward primer b (5'-CGAGATCAGCAGCCTCTGTTCC-3'), located within the PGK promoter in the neomycin-resistance cassette, and reverse primer c (5'-CCA AGCAGCTATCAAGTGTTCC-3'), annealing to the genomic sequences in the 3' arm homologous region. A 500-bp PCR fragment was generated from the wild-type allele with primers a and a', and a 1250-bp fragment was generated from the targeted allele with primers b and c. When the hsp70.1 knockout mice were produced, the targeted disruption of hsp70.1 was found not to be lethal, because the ratio of hsp70.1(-/-) to hsp70.1(+/-) to hsp70.1(+/+) was 1:2:1, in accord with Mendel’s law. Male mice weighing 25 to 30 g were used throughout the present study. Fifteen hsp70.1 knockout and 15 wild-type mice were used to determine the infarct volume, 12 (6 knockout and 6 wild-type) mice were used for immunohistochemistry, 12 (6 knockout and 6 wild-type) mice were used for Western blot analysis, and 4 (2 knockout and 2 wild-type) mice were used for Northern blot analysis. The protocols for the care and use of animals throughout these procedures were approved by the animal care committee at Seoul National University.
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Carbon Black Perfusion Study
Carbon black perfusion testing was used to compare cerebrovascular anatomy. Animals were anesthetized and perfused with 2 mL of a concentrated solution of carbon black ink via the left cardiac ventricle, until the tissues (eg, tongue) turned black. After decapitation, the brains were carefully removed and placed in 10% buffered formalin for 24 hours before examination. The vessels of the circle of Willis and their branches were examined under a microscope.
Model of Focal Ischemia
Focal cerebral ischemia was induced by using a minor modification of the endovascular internal carotid artery suture method developed by Longa et al.26 In brief, after an intraperitoneal injection of 1% ketamine (30 mg/kg) and xylazine hydrochloride (4 mg/kg), the left common carotid artery was exposed at its bifurcation by a midline cervical incision. The branches from the external carotid artery (ECA) were coagulated, and the pterygopalatine artery was ligated with a 5-0 silk suture. The ECA was then transected, and a 5-0 nylon monofilament suture (with its tip rounded by heating) was inserted into the ECA stump. To occlude the origins of the MCA and proximal anterior cerebral artery, the suture was advanced into the internal carotid artery for a distance of 6 mm beyond the internal carotid artery (the pterygopalatine artery bifurcation). The suture was secured in place with a ligature, and the wound was closed. The monofilament was removed 120 minutes after occlusion. The animals were allowed food and water ad libitum. Seizures were not observed during the experiments at any time after the MCA occlusion. Rectal temperature was maintained at 37±0.5°C by using a thermistor-controlled heating blanket. Because the knockout mice were very weak under stressful conditions, physiological measurements were carried out in a separate group of mice. Mean arterial blood pressure, arterial blood gases, pH, blood glucose, and rectal temperature were measured. Mean arterial blood pressures were determined during occlusion and the first 30 minutes of reperfusion. Arterial blood samples obtained with use of a femoral catheter were analyzed at baseline and at the end of ischemia. The unpaired Student t test was used to compare the physiological variables of the 2 groups.
Measurement of Infarct Volume
The brain was removed and cut from the frontal tip into 1-mm-thick slices and immersed in a 2% solution of 2,3,7-triphenyltetrazolium chloride (TTC). The stained slices were then fixed in phosphate-buffered 4% paraformaldehyde, and the infarcted and total hemispheric areas of each section were traced and measured by using an image analysis system (Image-Pro Plus, Media Cybernetics). The infarction volumes of the hsp70.1 knockout and wild-type mice were compared by using the Mann-Whitney U test (mean±SD).
Immunohistochemistry
After the heart was perfused with 4% paraformaldehyde in PBS, the brain tissues were embedded in paraffin blocks and sectioned at 4-µm thickness. Sections, mounted on slides, were deparaffinized and exposed to 3% hydrogen peroxide in methanol (methanol to 3% H2O2 ratio 3:1). For blocking, sections were incubated in 3% BSA, followed by 10% normal goat serum in PBS for 30 minutes. Anti-HSP70 antibody (1:500, HSP70 W27 catalogue No. sc-24 mouse monoclonal IgG2a, Santa Cruz Biotechnology Inc) was used in a humidified chamber at 37°C for 30 minutes. Slides were then washed 3 times with PBS, secondary antibody (DAKO Corp) was applied at 37°C for 30 minutes, and the slides were washed with 50 mmol/L Tris-HCl (pH 7.6) and treated with diaminobenzidine chromogen.
Preparation of Brain Protein Extracts and Western Blot Analysis
Mice were euthanized by cervical dislocation, and the brains were quickly removed and dissected into right and left hemispheres. These were then placed on ice in 10 vol cold homogenization buffer (50 mmol/L Tris and 120 mmol/L NaCl, pH 7.4) to which protease inhibitors (complete Mini, GIBCO) had been freshly added. The tissue was then homogenized and stored at -70°C. Protein concentrations were determined by using the Bradford method (Bio-Rad). Protein extracts from brain tissue (20 µg) were separated by SDS-PAGE. Protein separation was performed in 10% polyacrylamide with 0.05% bis-acrylamide. The proteins were then transferred to cellulose membrane, and the blots were probed with anti-HSP70 antibody. Signals were detected by enhanced chemiluminescence (Supersignal, Pierce). Film autoradiograms were exposed from 1 second to 30 minutes. The films were scanned with a GS-700 imaging densitometer (Bio-Rad), and the results were quantified by using the Multi-Analyst software program (Bio-Rad). Each blot was probed for 
-tubulin as an internal control to ensure equivalent protein loading and protein integrity. Relative optical densities were obtained from each mouse, and the results were compared by using the unpaired Student t test. Statistical significance was accepted at a value of P<0.05.
Preparation of Total RNA and Northern Blot Analysis
The right and left hemispheres were frozen immediately in liquid nitrogen after dissection and stored at -70°C. Total RNA was prepared by homogenizing the brain tissues in an acid guanidinium thiocyanate solution and extracted with phenol and chloroform, as previously described.27 The final RNA pellet was dissolved in diethyl pyrocarbonate–treated H2O. Ten micrograms of RNA was separated by electrophoresis on denaturing agarose gels and subsequently transferred to a nylon membrane (Amersham Pharmacia Biotech). Membranes were hybridized by using hsp70.1- or hsp70.3-specific probes labeled with [-32P]dCTP (Amersham Pharmacia Biotech) with a random-priming DNA labeling kit (Amersham Pharmacia Biotech). The following 2 primer sets (accession No. M35021) were used to produce the following hsp70.1 mRNA-specific probes: forward primer (5'-TGCACTTGATAGCTGCTTGG; start point, 2779) and reverse primer (5'-GCAGTGTAGACATGTATGCA; start point, 3290). For the hsp70.3 mRNA-specific probe (accession No. M76613), the following primer sets were used: forward primer (5'-CTGGCTAGGAGACAGATATG; start point, 2990) and reverse primer (5'-GGGCAGTGCTGAATTGAAGA; start point, 3217). Hybridization was performed at 63°C for 20 hours in a hybridization solution containing 0.2 mmol/L Na2HPO4 (pH 7.2), 7% SDS, 1% BSA, and 1 mmol/L EDTA. Autoradiography was performed with a Bio-Imaging Analyzer BAS 1000 (FUJI Photo Film).
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Results |
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Targeted deletion of the hsp70.1 gene did not produce any remarkable effect on the natural development of the mice. In terms of phenotype, no remarkable differences were observed between the knockout and wild-type mice: they were all black, and their sizes were similar. Gross morphology of the brains from the knockout mice was indistinguishable from that of their wild-type littermates, and the carbon black perfusion study did not reveal any notable differences between the cerebrovascular structures of the 2 groups.
Physiological parameters were measured in 4 knockout and 4 wild-type mice, as shown in the Table. None of the physiological parameters of the knockout and the wild-type mice were found to be significantly different before, during, or after the operation.
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Infarction Volume
In the present study, most of the hsp70.1 knockout mice were unable to survive 24 hours after a 2-hour MCA occlusion. First, 6 hsp70.1 knockout mice that underwent 2-hour occlusion and 24-hour reperfusion died from extensive brain infarction and swelling. Even 4 hours after reperfusion, the brain sections of the hsp70.1 knockout mice, compared with those of their wild-type littermates, showed extended ischemic regions, as evidenced around the central core of the ischemia. The infarction covered a wide area, from the basal ganglia to the cerebral cortex, in knockout mice but was mainly confined to the basal ganglia in wild-type mice (Figure 2A).
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The infarction volume was measured and compared by using a transient focal ischemia model involving 2 hours of MCA occlusion and 4 hours of reperfusion. Quantitatively, the infarction volume in the hsp70.1 knockout mice was 30% greater than that of their wild-type littermates (value for knockout mice was 92.5±8.3 mm3; P<0.001 versus wild-type [59.3±8.9 mm3]) (Figure 2B).
HSP70 Protein Expression
Immunohistochemistry showed increased HSP70 immunoreactivity in the infarcted area in both groups (Figure 3). HSP70 was preferentially localized to the basal ganglionic and cortical neurons in the left hemisphere, and cortical immunoreactivity was more relatively strong. In particular, compared with the results of TTC staining, the cortical immunoreactivity was denser in the penumbral areas. In the contralateral hemisphere, both groups showed scant HSP70 immunostaining.
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Western blots showed increased HSP70 expression in the ischemic hemisphere in both wild-type and hsp70.1 knockout mice (Figure 4A and 4B). However, HSP70 expression levels in hsp70.1 knockout mice were significantly lower than those in their wild-type littermates (P<0.05). No significant differences in -tubulin immunoreactivity were found among the different groups, underscoring the validity of using -tubulin as an internal control. All experiments were repeated at least 6 times.
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HSP70 mRNA Expression
In the ischemic hemispheres of wild-type mice, both hsp70.3 and hsp70.1 mRNAs were prominently expressed, as shown by Northern blot analysis (Figure 4C). However, in the hsp70.1 knockout mice, hsp70.1 mRNA expression was absent from both hemispheres, whereas hsp70.3 mRNA was significantly elevated in the left hemisphere. Interestingly, a moderate signal for hsp70.3 mRNA was detected in the contralateral nonischemic hemisphere in the wild-type group.
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Discussion |
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The present study was undertaken to investigate the effect of HSP70 reduction on cerebral ischemia. The results show that reduced HSP70 protein expression increases cellular damage during acute focal cerebral ischemia in hsp70.1 knockout mice. Moreover, this is the first study that demonstrates the neuroprotective effect of HSP70 after cerebral ischemia in the hsp70.1 knockout mouse model. Our findings are consistent with previous observations that the overexpression of HSP70 confers neuroprotection against cerebral ischemia.22,24 The findings also strongly support the hypothesis that HSP70 is a cytoprotective protein within the brain and that it is at least partially responsible for cerebral protection after transient MCA occlusion.
In the present study, we used a transient focal ischemia model consisting of 2-hour MCA occlusion and 4-hour reperfusion. We used this model because the majority of hsp70.1 knockout mice were unable to survive even 24 hours after 2-hour MCA occlusion, although the hsp70.1 gene knockout in itself was not lethal. Therefore, there is a possibility that the differences observed were not due to differences in infarct volume but to differences caused by infarct evolution. However, the mean infarct volume in the wild-type littermates even at the 24-hour stage (data not shown) was significantly smaller than that in the knockout mice after the 2-hour occlusion/4-hour reperfusion, and this finding cannot be explained by differences in lesion evolution. It has been reported that after 60 minutes of focal ischemia, HSP70 synthesis increased within 4 hours, peaked at 24 hours, and persisted for up to 7 days,28 which suggests that HSP70 might not be fully expressed in the brains of either the hsp70.1(-/-) mice or the wild-type littermates during our study period. Thus, our investigation is limited in not examining the long-term effects of HSP70. In addition, the anti-HSP70 antibody used in the present study recognizes both HSP70 and the HSC70. This cross-reaction might influence the baseline level of HSP70 protein in sham-operated animals. However, it is well known that HSP70 protein increases greatly after stress, and the considerable increase of protein in the ischemic hemisphere was most likely due to the expression of HSP70. Because we did not directly compare the level of HSP70 with that of HSC70 in the present study, we could not identify the fraction of HSC70 in the expressed protein. Prior studies29,30 indicate that the HSC70 abundant in normal cells exhibits a slight increase after stress compared with a robust increase in the expression of HSP70 protein. In addition, the HSC70 gene was not under the influence of targeted deletion in the present study; thus, we believe that the comparison between the 2 groups under the same conditions was valid.
Our results showed prominent expression of HSP70 in endothelial cells and minimal expression within the basal ganglia. This is partially consistent with previous observations indicating that HSP70 protein is localized in neurons outside the infarcted area and in endothelial cells within the infarction after permanent MCA occlusion, although hsp70 mRNA has been shown to be expressed in infarcted areas and noninfarcted areras.28,31 In the present study, it was evident that HSP70 protein expression was elevated more in the cerebral cortex and in the ischemic penumbra than in the basal ganglia. Several earlier reports have also concluded that the protective effect of HSP70 occurs mainly in the ischemic penumbra.30,32,33 In the present study, TTC staining showed that ischemic damage and density in hsp70.1 were greater in knockout mice than in wild-type mice. Overall, the infarction-reducing effect in wild-type mice, which was prominent in the cerebral cortex, may be due to HSP70 expression. In the ischemic core, the neuroprotective effects may be limited by a translational block of hsp70 mRNA caused by severe ATP loss,31,34,35 because HSP70 generation is an energy-consuming process, but this possibility was not confirmed in the present study.
We measured hsp70.3 expression with hsp70.1. However, the individual functions of the 2 genes in stressful conditions are unclear. It is known that their nucleotide sequences are strikingly similar,2,5,6 and although they are considerably different in the 3' UTR, both genes are known to initiate transcription after stress and to produce the same HSP70 proteins.30,33 Because there is no definitive evidence that the 2 genes show different responses to stress, it is believed that they might respond differently to various stimuli. Recently, it has been suggested that the 2 genes may respond separately to a single stress. Akçetin et al36 reported that rhsp70.2 mediates a short and sensitive transient response after ischemia/reperfusion injury in rat kidney, whereas rhsp70.1 mediates the major and long-lasting protective defense mechanism. However, there has been no corresponding report regarding the nervous system. Although we did not compare the neuroprotective effects of hsp70.1 and hsp70.3, it may at least be true that the expression of hsp70.1 is important in "early" neuroprotection after focal cerebral ischemia. To confirm the role of hsp70.3 in cerebral ischemia, an hsp70.3 knockout or combined hsp70.1–70.3 knockout study would be helpful.
Northern blot analyses produced another interesting result. In the contralateral hemisphere, hsp70.3 mRNA was clearly expressed in wild-type mice but not in knockout mice. There have been a few studies reporting that focal ischemia induced expression of the HSP70 protein in the contralateral hemisphere,37–40 but the mechanism has not been established. Some of the suggested mechanisms were elevated intracranial pressure due to edema37 and secondary events, such as deafferentation or transsynaptic activation.38 A recent study reported that the synthesis of HSP70 might be an index of ongoing repair or a compensatory mechanism related to neuronal remodeling, contributing to facilitate the recovery of postischemic neurological deficits.40 Whatever the mechanism, it was interesting that no hsp70.3 mRNA in knockout mice and no hsp70.1 mRNA in wild-type mice was found in the contralateral hemisphere. We do not consider these results to have been caused by experimental error, because repeated analyses showed the same result in other animals. We believe that this may be associated with possible differences in expression pattern or a functional interaction between hsp70.1 and hsp70.3, but this was not investigated in the present study. Further investigation of the different responses and individual functions of hsp70.1 and hsp70.3 is needed.
In summary, our results demonstrate that ischemic damage is increased without hsp70.1 and that this is probably due to the detrimental effect of hsp70.1 deficiency on HSP70 expression. However, the long-term effects of hsp70.1 expression, which were not examined in the present study, remain to be resolved. HSP70 protein is known to play a very important role in the protection of cells against various stresses, including cerebral ischemia, but the individual functions of hsp70.1 and hsp70.3 and the molecular mechanisms of HSP70 (apoptosis block or chaperone function) are not understood. Further investigation is required to successfully develop novel therapeutic interventions based on HSP70 protein for stroke.
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Acknowledgments |
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This study was supported by the Korea Science and Engineering Foundation (grant 98-0403-1901-5).
Received April 27, 2001; revision received August 22, 2001; accepted August 23, 2001.
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References |
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