Journal of Cerebral Blood Flow and Metabolism 11:621--627 © 1991 The International Society of Cerebral Blood Flow and Metabolism Published by Raven Press, Ltd" New York
Heat Shock Protein hsp72 Induction in Cortical and Striatal Astrocytes and Neurons Following Infarction
*tFrank R. Sharp, t§Daniel Lowenstein, t§Roger Simon, and *tKinya Hisanaga *Department of Neurology, VA Medical Center, and Department of tNeurology, University of California at San Francisco, and §Department of Neurology, San Francisco General Hospital, San Francisco, California, U.S.A.
Summary: Transient global and transient focal ischemia induced the 72 kDa heat shock protein (hsp72) in neurons in cortex, striatum, and other regions known to be injured during transient ischemia. A novel finding was the induc tion of hsp72 in islands (cylinders in three dimensions) of cells composed of astrocytes around the perimeter and neurons in the interior. Since histology showed pale stain ing in these regions, it is proposed that these islands rep-
resent areas of focal infarction in the distribution of small cortical and lenticulostriate arteries. Although the factors responsible for hsp72 induction during ischemia and in farction are unknown, these results suggest differences in mechanisms of hsp72 induction in astrocytes compared to neurons. Key Words: Heat shock proteins-Cerebral ischemia-Astrocytes-Neurons-Stress proteins-Pro tein synthesis in ischemia.
Protein synthesis decreases during cerebral isch emia (Kleihues and Hossmann, 1971; Kleihues et aI., 1975; Dienel et aI., 1980, 1985; Takahashi et aI., 1984; Nowak et aI., 1985; Kiessling et aI., 1986; Dwyer et al., 1987). Whereas the decrease appears to be temporary in reversible ischemia, prolonged failure of protein synthesis occurs in regions and cells destined to die (Bodsch et aI., 1985; Thilmann et al., 1986). However, the synthesis of one general class of proteins, called the heat shock proteins, appears to increase during ischemia (Nowak, 1985; Dienel et aI., 1986; Jacewicz et aI., 1986; Nowak et aI., 1987, 1990; Vass et aI., 1988; Dwyer et aI., 1989; Gonzalez et aI., 1989. 1991; Ferriero et aI.. 1990). Heat shock proteins (hsp's) are a family of pro teins that perform many functions. They are in duced by a variety of stresses and injuries that denature proteins (Schlesinger et al., 1982; Craig,
1985; Anathan et aI.. 1986; P elham, 1986; Lindquist, 1986; Brown et aI., 1989). They chaper one protein movements across membranes and per form important protein-protein interactions in nor mal and stressed cells (Chappell et aI., 1986; Ellis, 1987; Chirico et aI., 1988; Cheng et aI., 1989; Chiang et aI., 1989; Deshaies et aI., 1989; Beckmann et aI., 1990). Once hsp's are induced, they may prevent proteins from assuming abnormal tertiary struc tures or being denatured (Pelham, 1986), and pro tect cells from subsequent injury (Schlesinger et aI., 1982; Johnston and Kucey, 1988; Riabowol et aI., 1988). For example, induction of hsp's by heating animals (a) correlates with protection of the retina from light-induced injury (Barbe et aI., 1988) and (b) correlates with protection of neurons from damage produced by global ischemia (Chopp et aI., 1989). In the brain, ischemia induces several hsp's (Di enel et aI., 1985, 1986; Nowak, 1985; Nowak et aI., 1985). The inducible hsp72 protein is not present in normal rat brain, (Lowenstein et aI., 1990) and is expressed primarily in neurons of the hippocampus, cortex, striatum, and other regions in the gerbil transient global ischemia model (Vass et aI., 1988) and rat transient global ischemia model (Gonzalez et aI., 1991). Transient focal ischemia (Gonzalez et
Received October 12, 1990; revised December 3, 1990; ac cepted December 7, 1990. Address correspondence and reprint requests to Dr. F. R. Sharp, Neurology (VI27), Veterans Administration Medical Center, 4150 Clement Street, San Francisco, CA 94121, U.S.A. Abbreviations used: HS, horse serum; hsp, heat shock pro tein; LI, -like immunoreactivity; MCA, middle cerebral artery; PB, phosphate buffer.
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aI., 1989; Ferriero et aI., 1990) and kainic acid (Uney et aI., 1988; Gonzalez et aI., 1989; Vass et aI., 1989) also induce hsp72 primarily in neurons. Here we report that hsp72 can also be induced in astrocytes in rat global and focal ischemia models at the margins of infarctions. MATERIALS AND METHODS Global forebrain ischemia was produced in ten 300400 g rats using a method similar to that of Smith et al. (1984). Rats were anesthetized with halothane and nitrous oxide. After clamping one or both carotid arteries, sys temic blood pressure was lowered to 50 mm Hg by with drawing blood from a femoral arterial catheter and infus ing Arfonad (Sigma, St. Louis, MO, U.S.A.). EEG iso electricity was maintained for 5 min. Global ischemia was reversed by removing the carotid clamps, stopping Ar fonad, and reinfusing the subject's blood. Four control rats were subjected to the same surgery but not rendered ischemic. Focal ischemia was produced using the method of Zea Longa et al. (1989) in six adult male rats. Subjects were anesthetized with metafane and xylazine (20 mg/kg). The right common carotid was exposed and the external ca rotid and its branches and pterygopalatine arteries were coagulated and ligated. A 4-0 monofilament suture, blunted at the tip, was threaded into the external carotid stump and up into the internal carotid for 17-18 mm until resistance was met. This partial occlusion of the proximal middle cerebral artery (MCA) was maintained for I h, after which the suture was removed and the external ca rotid stump was coagulated and the wound closed. Four control subjects underwent surgery but did not have the suture inserted. Following a 24-h survival in their home cages with food and water available ad libitum, all subjects were reanes thetized with ketamine (80 mg/kg) and xylazine (20 mg/kg) and perfused with 4% paraformaldehyde. Their brains were removed, postfixed for 4 h, and sectioned (50 ,..,m) on a vibratome. The sections were reacted for the induc ible hsp72 using a monoclonal antibody (sold by Amer,sham, Arlington Heights, IL, U.S.A.) developed and characterized by Welch and Suhan (1986) that reacts with one major protein (presumptive hsp72) and either a break down product or another minor protein on Western blots (Vass et aI., 1988). Immunocytochemistry was performed using the avi din-biotin/horseradish peroxidase technique (Elite Vectastain, Burlingame, CA, U.S.A.). Briefly, sections were placed in phosphate buffer (PB, 0.1 M. pH 7.4) con taining 2% horse serum (HS), 0.2% Triton X100, and 0.1% bovine serum albumin (BSA) (PB-HS) for 2 h at room temperature. This was followed by a 48 h incuba tion at SOC in the hsp72 monoclonal antibody (Amersham) diluted 1:2,000 in PB-HS. They were washed in PB sev eral times and incubated in biotinylated horse anti-mouse second antibody 1:50-1 :5,000 for 2-3 h. Sections were then placed in an avidin-horseradish peroxidase solution for 3 h, and reacted in 15 mg of diaminobenzidine (DAB) (Sigma) dissolved in 100 ml of PB to which 0.001% hy drogen peroxide was added a drop at a time. Sections were then washed and counterstained with either cresyl Echt violet or hematoxylin and eosin. Control sections
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were prepared by performing immunocytochemistry ex cept that primary antibody was deleted.
RESULTS
hsp72-like immunoreactivity (hsp72LI) was not detected in any neurons or astrocytes in the brains of the eight control subjects. Moreover, no hsp72LI staining was observed in subjects in which the horse anti-mouse second antibody was preabsorbed with rat serum. Some staining, confined mostly to neu ronal cell bodies in the centers of infarctions, was observed when second antibody was not preab sorbed with rat serum. As noted previously by Vass et al. (1989), some injured neurons appear to take up immunoglobulins (and other proteins), and prob ably because of cross-reactivity of rodent immuno globulins, incubation with anti-mouse second antisera (which have not been preabsorbed) can produce nonspecific cellular staining of immuno globulins in injured neurons and other cells. This nonspecific effect generally produced staining of cell bodies. An additional artefact was a light, dif fuse brown staining in infarcted regions that was present when the first antibody was deleted from the protocol. The brown staining was decreased by pretreating sections with 0.00 I % hydrogen perox ide, suggesting that endogenous peroxidase activity had increased following infarction (Gonzalez et aI., 1989). Specific hsp72LI was induced in the brains of all ten globally ischemic subjects and all six focally ischemic subjects. During systemic hypotension with both carotids occluded, hsp72LI was induced only in neurons in many regions: in cortical neurons in the MCA distribution primarily in layers 2, 3, 5, and 6 of the neocortex; diffusely in medium-sized striatal neurons; in pyramidal neurons in CA3 and to some extent in pyramidal neurons in CAl of the hippocampus; in dentate granule cell neurons; in thalamic reticular nucleus neurons; and in large dor sal septal neurons. Cresyl violet and hematoxylin and eosin stains showed no evidence of regional infarction in regions where hsp72 was only induced in neurons, but instead showed shrunken and pyk notic neurons typical of neuronal cell death, A sim ilar pattern of hsp72 induction has been described in the gerbil global ischemia model (Vass et aI., 1988). However, on many occasions (8/10 rats) follow ing systemic hypotension combined with carotid oc clusion, hsp72LI was induced in islands of cells in both neocortex (Figs. IF-H) and the striatum (Figs. 2A,C, E-G). Similar islands were induced during fo cal ischemia (6/6 rats) in the neocortex (Figs. l A-E) and striatum (Figs. 2B,D). The islands varied in size from a few hundred microns (Figs. 1G and 2C) to
HSP72 INDUCTION IN GLIA AND NEURONS
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FIG. 1. hsp72-like immunoreactivity is pictured in coronal sections of neocortex from subjects (a) 18 h after being subjected to temporary (1 h) occlusion of the left middle cerebral artery (panels A-E) and (b) 18 h after being subjected to 5 min of carotid occlusion combined with systemic hypotension (panels F-H). Note the patchy involvement of neocortex (panels A-G). The patches consisted of islands of cells in which astrocytes (solid arrows) formed a perimeter (8-G) around regions of variable numbers of immunoreactive neurons (open arrows). The cell body and processes of neurons visualized. Calibration bars: A 2 mm; B 500 ILm; C-G 200 ILm; H 50 ILm. =
=
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many millimeters (Figs. IB and 2A,B) across. These islands occurred in deep (Fig. 2A,B) as well as superficial (Fig. 2C,D) portions of the cortex and could involve any portion of striatum (Figs. 2A,B).
(G)
and astrocytes
(H)
could be
=
Nissl and hematoxylin and eosin histology showed pale staining in the hsp72 islands of cells that ap peared to be characteristic of focal infarctions. The perimeters of all of the islands were com-
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FIG. 2.
Distribution of hsp72-like immunoreactivity in caudate-putamen (cp) from subjects (a) 18 h after being subjected to temporary (1 h) occlusion of the left middle cerebral artery (panels B and D) and (b) 18 h after being subjected to 5 min of carotid occlusion combined with systemic hypotension (panels A, C, and E-G). Note the patchy induction of hsp72 in caudate-putamen
(panels A-D). The patches consisted of islands of positive cells (A-D) in which astrocytes (solid arrows, A-E) formed a perimeter or border (A-E) around regions of immunoreactive neurons (F, right side of E, and center of D). The perikarya and dendrites of
neurons were stained (E and F) and the cell body and processes of astrocytes were immunostained (solid arrow in E and G). There was no staining of white matter tracts (0, E, and F). Calibration bars: A,B 1 mm; C,D 200 ILm; E,F 50 ILm; G 20 ILm. =
posed of hsp72-stained astrocytes often one cell thick (Figs. lB, E and 2D, E), but sometimes four to five astrocytes thick (Figs. IG and 2e). The hsp72 induction in cortical (Figs. IC-H) and striatal (Figs. 2C-G) astrocytes produced Golgi-like images (Figs. IH and 2G). hsp72 was induced in dendrites,
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perikarya, and axons of neurons within the islands in the cortex (Figs. I D-G) and striatum (Figs. 2D F). The "islands" continued through many succes sive sections so that they formed "cylinders" in three dimensions. Cresyl violet and hematoxylin and eosin stains showed that the cylinders were in-
HSP72 INDUCTION IN GLIA AND NEURONS
farctions, manifested by pale staining anri pyknotic neurons, and that the induction of hsp72LI in astro cytes occurred at the borders of the infarctions. Although the histological identification of the glia-like cells in which hsp72 was induced by infarc tion was not proven in these studies, the glia are almost certainly astrocytes. The cells were associ ated with blood vessels (Fig. 1 H) and not with white matter bundles, and had processes characteristic of astrocytes seen with glial fibrillary acidic protein staining (Figs. IH and 2G). DISCUSSION Vass et al. (1988) found that temporary bilateral carotid occlusion in adult gerbils induced hsp72 bi laterally in neurons in the neocortex, entorhinal cortex, striatum, hippocampus, dentate gyrus, and other regions. Our work confirmed induction in hip pocampal (Gonzalez et aI., 1991) and cortical (Gonzalez et aI., 1989) neurons using transient rat global and focal ischemia models, respectively. Fer riero et al. (1990) also found that hsp72 was induced primarily in neurons in a transient neonatal rat isch emia/hypoxia model. The degrees of ischemia used in these models would be expected to produce neu ronal death without evidence of cerebral infarction. The present results demonstrate glial as well neu ronal induction of hsp72 in islands of cortical and striatal cells following global and focal ischemia that results in infarction. In their model of global isch emia, Smith et al. (1984) noted circumscribed areas of dense neuronal necrosis in cortex and striatum that they attributed to damage in the distribution of arteries and possibly veins. Our data confirm small focal infarctions using the Smith et al. (1984) global ischemia model and the Zea Longa et al. (1989) fo cal ischemia model. Moreover, our data show that the islands (cylinders in three dimensions) of hsp72 cells in the neocortex and striatum observed in this study occur in infarctions-presumably in the dis tribution of one or several small blood vessels. These infarctions likely occurred because of poor reflow through these vessels following restoration of blood pressure in the global ischemia model and following restoration of flow following removal of the suture in the focal ischemia model. Induction of hsp's in glia has been previously de scribed in white matter of heated rats (Sprang and Brown, 1987; Manzerra and Brown, 1990; Nowak et aI., 1990). Nishimura et al. (1988, 1989) also showed that hsp's were induced in cultured astro cytes following heating and exposure to low pH. Marini et al. (1990) showed that heat induced hsp72 in cerebellar astrocytes but not cerebellar granule cell neurons in vitro, whereas heat induced hsp72 in
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both cerebellar cell types in vivo. The present study shows that infarction induces hsp72 in cortical as trocytes and neurons in vivo. Although the precise mechanism of hsp induction is still unclear, it is believed that denatured proteins somehow "acti vate" a heat shock factor (a pwtein) that binds to heat shock elements on the DN A that then induce heat shock genes (Anathan et aI., 1986; Wu et aI., 1987; Zimarino et aI., 1990). Any process that de natures proteins or leads to production of proteins with abnormal tertiary structures might induce hsp's (Anathan et aI., 1986; Pelham, 1986). It is im portant to point out that hsp72 is induced in only a fraction of the neurons even in the core of an in farction, making the mechanism of induction in some but not all neurons uncertain. The mechanism by which hsp72LI is induced in a rim of astrocytes surrounding an ischemic vessel is also difficult to explain. Presumably, intracellular pH would be low and intracellular calcium would be high in all glia and neurons in the center and in the periphery of acute infarctions. Increased calcium and decreased pH could both denature intracellular proteins and could explain the hsp72 induction in an island of neurons surrounding a vessel. What then explains hsp72 induction in glia at the periphery of infarctions? It is possible that certain concentra tions or durations of the hsp-inducing factors are required for glial hsp72 induction, and that this only occurs at the outer borders (penumbra) of ischemic blood vessels. It is also possible that different fac tors denature proteins and induce hsp72 in glia and neurons, and that the glial-inducing factor(s) are only present in the penumbra of infarcted vessels. It is notable that increases in 2-deoxyglucose uptake occur at the margins of chronic MCA infarctions that may be due to release of excitatory amino ac ids, increased lactate production, or some other metabolic abnormality present in the penumbra but not in the core of the infarction (Shiraishi et aI., 1989). Whatever the mechanism of hsp72 induction in astrocytes at the margins of infarctions, it is tempt ing to speculate that the glia at the periphery of these islands could have a functional role in isolat ing normal brain from the abnormal ionic, pH, and excitatory amino acid gradients in infarcted brain. Moreover, since transfer of hsp's from glia to neu ronal axons has been noted (Tytell et aI., 1986), it is possible that there is some interaction between in farcted neurons and glia in the penumbra. Lastly, hsp72 induction in astrocytes at the margins of in farctions might provide a biochemical marker for defining the edges of infarctions that could be used to quantitate infarction volumes rather than using
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differences in histological staining intensity (Swan son et ai., 1990). Acknowledgment: We wish to thank Dr. Stephen M. Sagar for his helpful comments on this manuscript, and we particularly appreciate the excellent technical assis tance of Pat Jasper, Theresa Marsh, and Matt Morton. This work was supported by the VA Medical Research Service and NIH grants NS24666 and NS281 67 to F.R.S., NS01424 to D. L., and NS24728 to R. S. REFERENCES Anathan J. Goldberg AL, Voellmy R (1986) Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 232:522-524 Barbe MF, Tytell M, Gower DJ, Welch WJ (1988) Hyperthermia protects against light damage in the rat retina. Science 241:1817-1820 Beckmann RP, Mizzen LA, Welch WJ (1990) Interaction of HSP70 with newly synthesized proteins: Implications for protein folding and assembly. Science 248:850--854 Bodsch W, Takahashi K, Barbier A, Grosse Ophoff B, Hoss mann K-A (1985) Cerebral protein synthesis and ischemia. Prog Brain Res 63:197-210 Brown IR, Rush S, Ivy GO (1989) Induction of a heat shock gene at the site of tissue injury in the rat brain. Neuron 2:15591564 Chappell T, Welch W, Schlossmann D, Palter K, Schlesinger M. Rothman J (1986) Uncoating ATPase is a member of the 70kD family of stress proteins. Cell 45:3-13 Cheng MY, Hartl F-U, Martin J, Pollock RA, Kalousek F, Neupert W, Hallberg EM, Hallberg RL, Horwich AL (1989) Mitochondrial heat-shock protein hsp60 is essential for as sembly of proteins imported into yeast mitochondria. Na ture (Lond) 337:620--625 Chiang H-L, Terlecky SR, Plant CP, Dice JF (1989) A role for a 70-kiloDalton heat shock protein in lysosomal degradation of intracellular proteins. Science 246:382-385 Chirico WJ, Waters MG, Blobel G (1988) 70kD heat shock re lated proteins stimulate protein translocation into mi crosomes. Nature (Lond) 332:805-810 Chopp M, Chen H, Ho K-L, Dereski MO, Brown E, Hetzel FW, Welch KMA (1989) Transient hyperthermia protects against subsequent forebrain ischemic cell damage in the rat. Neu rology 39:1396--1398 Craig EA (1985) The heat shock response. CRC Crit Rev Bio chem 18:239-280 Deshaies RJ, Koch BD, Schenkman R (1989) The role of stress proteins in membrane biogenesis. Trends Bioi Sci 13:384388 Dienel GA, Pulsinelli WA, Duffy TE (1980) Regional protein synthesis in rat brain following acute hemispheric ischemia. J Neurochem 35:1216--1226 Dienel GA, Cruz NF, Rosenfeld SJ (1985) Temporal profiles of proteins responsive to ischemia. J Neurochem 44:600--610 Dienel GA, Kiessling M, Jacewicz M, Pulsinelli WA (1986) Syn thesis of heat shock proteins in rat brain cortex after tran sient ischemia. J Cereb Blood Flow Metab 6:505-510 Dwyer BE, Nishimura RN, Powell CL, Mailheau SL (1987) Fo cal protein synthesis inhibition in a model of neonatal hyp oxic-ischemic injury. Exp NeuroI95:277-289 Dwyer BE, Nishimura RN, Brown IR (1989) Synthesis of the major inducible heat shock protein in rat hippocampus after neonatal hypoxia-ischemia. Exp Neurol 104:28-31 Ellis RJ (1987) Proteins as molecular chaperones. Nature (Lond) 328:378-379 Ferriero DM, Soberano HQ, Simon RP, Sharp FR (1990) Hyp oxia-ischemia induces heat shock protein-like (HSP72) im munoreactivity in neonatal rat brain. Dev Brain Res 53:145150
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Gonzalez MF, Shiraishi K, Hisanaga K, Sagar SM, Mandabach M, Sharp FR (1989) Heat shock proteins as markers of neu ral injury. Mol Brain Res 6:93-100 Gonzalez MF, Lowenstein DH, Hisanga K, Simon RP, Sagar SM, Sharp FR (1991) Induction of heat shock protein 72-like immunoreactivity in the hippocampal formation following transient global ischemia. Brain Res Bull 26:241-250 Jacewicz M, Kiessling M, Pulsinelli WA (1986) Selective gene expression in focal cerebral ischemia. J Cereb Blood Flow Metab 6:263-272 Johnston RN, Kucey BF (1988) Competitive inhibition of hsp70 gene expression causes thermo sensitivity . Science 242: 1551-1553 Kiessling M, Dienel GA, Jacewicz M, Pulsinelli WA (1986) Pro tein synthesis in postischemic rat brain: A two-dimensional electrophoretic analysis. J Cereb Blood Flow Metab 6:642649 Kleihues P, Hossmann K-A (1971) Protein synthesis in the cat brain after prolonged cerebral ischemia. Brain Res 35:409418 Kleihues P, Hossmann K-A, Pegg AE, Kobayashi K, Zimmer mann V (1975) Resuscitation of the monkey brain after one hour complete ischemia, III. Indications of metabolic recov ery, Brain Res 95:61-73 Lindquist S (1986) The heat shock response. Annu Rev Biochem 55:1151-1191 Lowenstein DH, Simon RP, Sharp FR (1990) The pattern of 72-kDa heat shock protein-like immunoreactivity in the rat brain following flurothyl-induced status epilepticies. Brain Res 531:173-182. Manzerra P, Brown IR (I990) Time course of induction of a heat shock gene (hsp70) in the rabbit cerebellum after LSD in vivo: Involvement of drug-induced hyperthermia. Neuro chem Res 15:53-59 Marini AM, Kozuka M, Lipsky RH, Nowak TS Jr (1990) 70Kilodalton heat shock protein induction in cerebellar astro cytes and cerebellar granule cells in vitro: Comparison with immunocytochemical localization after hyperthermia in vivo. J Neurochem 54:1509-1516 Nishimura RN, Dwyer BE, Welch W, Cole R, de Vellis J, Liotta K (1988) The induction of the major heat-stress protein in purified rat glial cells. J Neurosci Res 20:12-18 Nishimura RN, Dwyer BE, Cole R, de Vellis J, Picard K (1989) Induction of the major inducible 68-kDa heat-shock protein after rapid changes in extracellular pH in cultured rat astro cytes. Exp Cell Res 180:276--280 Nowak TS Jr (1985) Synthesis of a stress protein following tran sient ischemia in the gerbil. J Neurochem 45:1635-1641 Nowak TS Jr, Fried RL, Lust WD, Passonneau JV (1985) Changes in brain energy metabolism and protein synthesis following transient bilateral ischemia in the gerbil. J Neuro chem 44:487-494 Nowak TS Jr, Vass K, Welch WJ (1987) Localization of 70kd heat shock protein induction in gerbil brain 'lfter transient ischemia. Soc Neurosci Abstr 13:1688 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 Pelham HRB (1986) Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell 46:959-961 Riabowol KT, Mizzen LA, Welch WJ (1988) Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70. Science 242:433-436 Schlesinger MJ, Ashburner M, Tissieres A (eds) (1982) Heat Shock: from Bacteria to Man, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Shiraishi K, Sharp FR, Simon RP (1989) Sequential metabolic changes in rat brain following middle cerebral artery occlu sion: A 2-deoxyglucose study. J Cereb Blood Flow Metab 9:765-773 Smith M-L, Benedek G, Dahlgren N, Rosen I, Wieloch T, Siesjo BK (1984) Models for studying long-term recovery following
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gene in fiber tracts and cerebellar neurons of the rabbit brain detected by in situ hybridization. Mol Brain Res 3:89-93 Swanson RA, Morton MT, Wu GT, Savalos R, Davidson C, Sharp FR (1990) A semi-automated method for measuring brain infarct volume. J Cereb Blood Flow Metab 10:290--293 Takahashi K, Bodsch W, Hossmann K-A (1984) Susceptibility of hippocampal protein synthesis to transient forebrain isch emia of adult and infant gerbil brain. Drugs Dis 1:72-78 Thilmann R, Xie Y, Kleihues P, Kiessling M (1986) Persistent inhibition of protein synthesis precedes delayed neuronal death in postischemic gerbil hippocampus. Acta Neuro pathoI71:88-91
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Vass K, Welch WJ, Nowak TS Jr (1988) Localization of 70kD stress protein induction in gerbil brain after ischemia. Acta Neuropathol 77:128-\35 Vass K, Berger ML, Nowak TS Jr, Welch WJ, Lassmann H (1989) Induction of stress protein hsp70 in nerve cells after status epilepticus in the rat. Neurasci Lett 100:259-264 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 Wu C, Wilson S, Walker B, Dawid I, Paisley T, Zimarino V, Ueda H (1987) Purification and properties of Drosophila heat shock activator protein. Science 238:1247-1253 Zea Longa E, Weinstein PR, Carlson S, Cummins R (1989) Re versible middle cerebral artery occlusion without craniec tomy in rats. Stroke 20:84-91 Zimarino V, Tsai C, Wu C (1990) Complex modes of heat shock factor activation. Mol Cell Bioi 10:752-759
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Heat Shock Protein hsp72 Induction in Cortical and Striatal Astrocytes and Neurons Following Infarction
*tFrank R. Sharp, t§Daniel Lowenstein, t§Roger Simon, and *tKinya Hisanaga *Department of Neurology, VA Medical Center, and Department of tNeurology, University of California at San Francisco, and §Department of Neurology, San Francisco General Hospital, San Francisco, California, U.S.A.
Summary: Transient global and transient focal ischemia induced the 72 kDa heat shock protein (hsp72) in neurons in cortex, striatum, and other regions known to be injured during transient ischemia. A novel finding was the induc tion of hsp72 in islands (cylinders in three dimensions) of cells composed of astrocytes around the perimeter and neurons in the interior. Since histology showed pale stain ing in these regions, it is proposed that these islands rep-
resent areas of focal infarction in the distribution of small cortical and lenticulostriate arteries. Although the factors responsible for hsp72 induction during ischemia and in farction are unknown, these results suggest differences in mechanisms of hsp72 induction in astrocytes compared to neurons. Key Words: Heat shock proteins-Cerebral ischemia-Astrocytes-Neurons-Stress proteins-Pro tein synthesis in ischemia.
Protein synthesis decreases during cerebral isch emia (Kleihues and Hossmann, 1971; Kleihues et aI., 1975; Dienel et aI., 1980, 1985; Takahashi et aI., 1984; Nowak et aI., 1985; Kiessling et aI., 1986; Dwyer et al., 1987). Whereas the decrease appears to be temporary in reversible ischemia, prolonged failure of protein synthesis occurs in regions and cells destined to die (Bodsch et aI., 1985; Thilmann et al., 1986). However, the synthesis of one general class of proteins, called the heat shock proteins, appears to increase during ischemia (Nowak, 1985; Dienel et aI., 1986; Jacewicz et aI., 1986; Nowak et aI., 1987, 1990; Vass et aI., 1988; Dwyer et aI., 1989; Gonzalez et aI., 1989. 1991; Ferriero et aI.. 1990). Heat shock proteins (hsp's) are a family of pro teins that perform many functions. They are in duced by a variety of stresses and injuries that denature proteins (Schlesinger et al., 1982; Craig,
1985; Anathan et aI.. 1986; P elham, 1986; Lindquist, 1986; Brown et aI., 1989). They chaper one protein movements across membranes and per form important protein-protein interactions in nor mal and stressed cells (Chappell et aI., 1986; Ellis, 1987; Chirico et aI., 1988; Cheng et aI., 1989; Chiang et aI., 1989; Deshaies et aI., 1989; Beckmann et aI., 1990). Once hsp's are induced, they may prevent proteins from assuming abnormal tertiary struc tures or being denatured (Pelham, 1986), and pro tect cells from subsequent injury (Schlesinger et aI., 1982; Johnston and Kucey, 1988; Riabowol et aI., 1988). For example, induction of hsp's by heating animals (a) correlates with protection of the retina from light-induced injury (Barbe et aI., 1988) and (b) correlates with protection of neurons from damage produced by global ischemia (Chopp et aI., 1989). In the brain, ischemia induces several hsp's (Di enel et aI., 1985, 1986; Nowak, 1985; Nowak et aI., 1985). The inducible hsp72 protein is not present in normal rat brain, (Lowenstein et aI., 1990) and is expressed primarily in neurons of the hippocampus, cortex, striatum, and other regions in the gerbil transient global ischemia model (Vass et aI., 1988) and rat transient global ischemia model (Gonzalez et aI., 1991). Transient focal ischemia (Gonzalez et
Received October 12, 1990; revised December 3, 1990; ac cepted December 7, 1990. Address correspondence and reprint requests to Dr. F. R. Sharp, Neurology (VI27), Veterans Administration Medical Center, 4150 Clement Street, San Francisco, CA 94121, U.S.A. Abbreviations used: HS, horse serum; hsp, heat shock pro tein; LI, -like immunoreactivity; MCA, middle cerebral artery; PB, phosphate buffer.
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aI., 1989; Ferriero et aI., 1990) and kainic acid (Uney et aI., 1988; Gonzalez et aI., 1989; Vass et aI., 1989) also induce hsp72 primarily in neurons. Here we report that hsp72 can also be induced in astrocytes in rat global and focal ischemia models at the margins of infarctions. MATERIALS AND METHODS Global forebrain ischemia was produced in ten 300400 g rats using a method similar to that of Smith et al. (1984). Rats were anesthetized with halothane and nitrous oxide. After clamping one or both carotid arteries, sys temic blood pressure was lowered to 50 mm Hg by with drawing blood from a femoral arterial catheter and infus ing Arfonad (Sigma, St. Louis, MO, U.S.A.). EEG iso electricity was maintained for 5 min. Global ischemia was reversed by removing the carotid clamps, stopping Ar fonad, and reinfusing the subject's blood. Four control rats were subjected to the same surgery but not rendered ischemic. Focal ischemia was produced using the method of Zea Longa et al. (1989) in six adult male rats. Subjects were anesthetized with metafane and xylazine (20 mg/kg). The right common carotid was exposed and the external ca rotid and its branches and pterygopalatine arteries were coagulated and ligated. A 4-0 monofilament suture, blunted at the tip, was threaded into the external carotid stump and up into the internal carotid for 17-18 mm until resistance was met. This partial occlusion of the proximal middle cerebral artery (MCA) was maintained for I h, after which the suture was removed and the external ca rotid stump was coagulated and the wound closed. Four control subjects underwent surgery but did not have the suture inserted. Following a 24-h survival in their home cages with food and water available ad libitum, all subjects were reanes thetized with ketamine (80 mg/kg) and xylazine (20 mg/kg) and perfused with 4% paraformaldehyde. Their brains were removed, postfixed for 4 h, and sectioned (50 ,..,m) on a vibratome. The sections were reacted for the induc ible hsp72 using a monoclonal antibody (sold by Amer,sham, Arlington Heights, IL, U.S.A.) developed and characterized by Welch and Suhan (1986) that reacts with one major protein (presumptive hsp72) and either a break down product or another minor protein on Western blots (Vass et aI., 1988). Immunocytochemistry was performed using the avi din-biotin/horseradish peroxidase technique (Elite Vectastain, Burlingame, CA, U.S.A.). Briefly, sections were placed in phosphate buffer (PB, 0.1 M. pH 7.4) con taining 2% horse serum (HS), 0.2% Triton X100, and 0.1% bovine serum albumin (BSA) (PB-HS) for 2 h at room temperature. This was followed by a 48 h incuba tion at SOC in the hsp72 monoclonal antibody (Amersham) diluted 1:2,000 in PB-HS. They were washed in PB sev eral times and incubated in biotinylated horse anti-mouse second antibody 1:50-1 :5,000 for 2-3 h. Sections were then placed in an avidin-horseradish peroxidase solution for 3 h, and reacted in 15 mg of diaminobenzidine (DAB) (Sigma) dissolved in 100 ml of PB to which 0.001% hy drogen peroxide was added a drop at a time. Sections were then washed and counterstained with either cresyl Echt violet or hematoxylin and eosin. Control sections
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were prepared by performing immunocytochemistry ex cept that primary antibody was deleted.
RESULTS
hsp72-like immunoreactivity (hsp72LI) was not detected in any neurons or astrocytes in the brains of the eight control subjects. Moreover, no hsp72LI staining was observed in subjects in which the horse anti-mouse second antibody was preabsorbed with rat serum. Some staining, confined mostly to neu ronal cell bodies in the centers of infarctions, was observed when second antibody was not preab sorbed with rat serum. As noted previously by Vass et al. (1989), some injured neurons appear to take up immunoglobulins (and other proteins), and prob ably because of cross-reactivity of rodent immuno globulins, incubation with anti-mouse second antisera (which have not been preabsorbed) can produce nonspecific cellular staining of immuno globulins in injured neurons and other cells. This nonspecific effect generally produced staining of cell bodies. An additional artefact was a light, dif fuse brown staining in infarcted regions that was present when the first antibody was deleted from the protocol. The brown staining was decreased by pretreating sections with 0.00 I % hydrogen perox ide, suggesting that endogenous peroxidase activity had increased following infarction (Gonzalez et aI., 1989). Specific hsp72LI was induced in the brains of all ten globally ischemic subjects and all six focally ischemic subjects. During systemic hypotension with both carotids occluded, hsp72LI was induced only in neurons in many regions: in cortical neurons in the MCA distribution primarily in layers 2, 3, 5, and 6 of the neocortex; diffusely in medium-sized striatal neurons; in pyramidal neurons in CA3 and to some extent in pyramidal neurons in CAl of the hippocampus; in dentate granule cell neurons; in thalamic reticular nucleus neurons; and in large dor sal septal neurons. Cresyl violet and hematoxylin and eosin stains showed no evidence of regional infarction in regions where hsp72 was only induced in neurons, but instead showed shrunken and pyk notic neurons typical of neuronal cell death, A sim ilar pattern of hsp72 induction has been described in the gerbil global ischemia model (Vass et aI., 1988). However, on many occasions (8/10 rats) follow ing systemic hypotension combined with carotid oc clusion, hsp72LI was induced in islands of cells in both neocortex (Figs. IF-H) and the striatum (Figs. 2A,C, E-G). Similar islands were induced during fo cal ischemia (6/6 rats) in the neocortex (Figs. l A-E) and striatum (Figs. 2B,D). The islands varied in size from a few hundred microns (Figs. 1G and 2C) to
HSP72 INDUCTION IN GLIA AND NEURONS
623
r "
FIG. 1. hsp72-like immunoreactivity is pictured in coronal sections of neocortex from subjects (a) 18 h after being subjected to temporary (1 h) occlusion of the left middle cerebral artery (panels A-E) and (b) 18 h after being subjected to 5 min of carotid occlusion combined with systemic hypotension (panels F-H). Note the patchy involvement of neocortex (panels A-G). The patches consisted of islands of cells in which astrocytes (solid arrows) formed a perimeter (8-G) around regions of variable numbers of immunoreactive neurons (open arrows). The cell body and processes of neurons visualized. Calibration bars: A 2 mm; B 500 ILm; C-G 200 ILm; H 50 ILm. =
=
=
many millimeters (Figs. IB and 2A,B) across. These islands occurred in deep (Fig. 2A,B) as well as superficial (Fig. 2C,D) portions of the cortex and could involve any portion of striatum (Figs. 2A,B).
(G)
and astrocytes
(H)
could be
=
Nissl and hematoxylin and eosin histology showed pale staining in the hsp72 islands of cells that ap peared to be characteristic of focal infarctions. The perimeters of all of the islands were com-
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FIG. 2.
Distribution of hsp72-like immunoreactivity in caudate-putamen (cp) from subjects (a) 18 h after being subjected to temporary (1 h) occlusion of the left middle cerebral artery (panels B and D) and (b) 18 h after being subjected to 5 min of carotid occlusion combined with systemic hypotension (panels A, C, and E-G). Note the patchy induction of hsp72 in caudate-putamen
(panels A-D). The patches consisted of islands of positive cells (A-D) in which astrocytes (solid arrows, A-E) formed a perimeter or border (A-E) around regions of immunoreactive neurons (F, right side of E, and center of D). The perikarya and dendrites of
neurons were stained (E and F) and the cell body and processes of astrocytes were immunostained (solid arrow in E and G). There was no staining of white matter tracts (0, E, and F). Calibration bars: A,B 1 mm; C,D 200 ILm; E,F 50 ILm; G 20 ILm. =
posed of hsp72-stained astrocytes often one cell thick (Figs. lB, E and 2D, E), but sometimes four to five astrocytes thick (Figs. IG and 2e). The hsp72 induction in cortical (Figs. IC-H) and striatal (Figs. 2C-G) astrocytes produced Golgi-like images (Figs. IH and 2G). hsp72 was induced in dendrites,
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=
=
=
perikarya, and axons of neurons within the islands in the cortex (Figs. I D-G) and striatum (Figs. 2D F). The "islands" continued through many succes sive sections so that they formed "cylinders" in three dimensions. Cresyl violet and hematoxylin and eosin stains showed that the cylinders were in-
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farctions, manifested by pale staining anri pyknotic neurons, and that the induction of hsp72LI in astro cytes occurred at the borders of the infarctions. Although the histological identification of the glia-like cells in which hsp72 was induced by infarc tion was not proven in these studies, the glia are almost certainly astrocytes. The cells were associ ated with blood vessels (Fig. 1 H) and not with white matter bundles, and had processes characteristic of astrocytes seen with glial fibrillary acidic protein staining (Figs. IH and 2G). DISCUSSION Vass et al. (1988) found that temporary bilateral carotid occlusion in adult gerbils induced hsp72 bi laterally in neurons in the neocortex, entorhinal cortex, striatum, hippocampus, dentate gyrus, and other regions. Our work confirmed induction in hip pocampal (Gonzalez et aI., 1991) and cortical (Gonzalez et aI., 1989) neurons using transient rat global and focal ischemia models, respectively. Fer riero et al. (1990) also found that hsp72 was induced primarily in neurons in a transient neonatal rat isch emia/hypoxia model. The degrees of ischemia used in these models would be expected to produce neu ronal death without evidence of cerebral infarction. The present results demonstrate glial as well neu ronal induction of hsp72 in islands of cortical and striatal cells following global and focal ischemia that results in infarction. In their model of global isch emia, Smith et al. (1984) noted circumscribed areas of dense neuronal necrosis in cortex and striatum that they attributed to damage in the distribution of arteries and possibly veins. Our data confirm small focal infarctions using the Smith et al. (1984) global ischemia model and the Zea Longa et al. (1989) fo cal ischemia model. Moreover, our data show that the islands (cylinders in three dimensions) of hsp72 cells in the neocortex and striatum observed in this study occur in infarctions-presumably in the dis tribution of one or several small blood vessels. These infarctions likely occurred because of poor reflow through these vessels following restoration of blood pressure in the global ischemia model and following restoration of flow following removal of the suture in the focal ischemia model. Induction of hsp's in glia has been previously de scribed in white matter of heated rats (Sprang and Brown, 1987; Manzerra and Brown, 1990; Nowak et aI., 1990). Nishimura et al. (1988, 1989) also showed that hsp's were induced in cultured astro cytes following heating and exposure to low pH. Marini et al. (1990) showed that heat induced hsp72 in cerebellar astrocytes but not cerebellar granule cell neurons in vitro, whereas heat induced hsp72 in
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both cerebellar cell types in vivo. The present study shows that infarction induces hsp72 in cortical as trocytes and neurons in vivo. Although the precise mechanism of hsp induction is still unclear, it is believed that denatured proteins somehow "acti vate" a heat shock factor (a pwtein) that binds to heat shock elements on the DN A that then induce heat shock genes (Anathan et aI., 1986; Wu et aI., 1987; Zimarino et aI., 1990). Any process that de natures proteins or leads to production of proteins with abnormal tertiary structures might induce hsp's (Anathan et aI., 1986; Pelham, 1986). It is im portant to point out that hsp72 is induced in only a fraction of the neurons even in the core of an in farction, making the mechanism of induction in some but not all neurons uncertain. The mechanism by which hsp72LI is induced in a rim of astrocytes surrounding an ischemic vessel is also difficult to explain. Presumably, intracellular pH would be low and intracellular calcium would be high in all glia and neurons in the center and in the periphery of acute infarctions. Increased calcium and decreased pH could both denature intracellular proteins and could explain the hsp72 induction in an island of neurons surrounding a vessel. What then explains hsp72 induction in glia at the periphery of infarctions? It is possible that certain concentra tions or durations of the hsp-inducing factors are required for glial hsp72 induction, and that this only occurs at the outer borders (penumbra) of ischemic blood vessels. It is also possible that different fac tors denature proteins and induce hsp72 in glia and neurons, and that the glial-inducing factor(s) are only present in the penumbra of infarcted vessels. It is notable that increases in 2-deoxyglucose uptake occur at the margins of chronic MCA infarctions that may be due to release of excitatory amino ac ids, increased lactate production, or some other metabolic abnormality present in the penumbra but not in the core of the infarction (Shiraishi et aI., 1989). Whatever the mechanism of hsp72 induction in astrocytes at the margins of infarctions, it is tempt ing to speculate that the glia at the periphery of these islands could have a functional role in isolat ing normal brain from the abnormal ionic, pH, and excitatory amino acid gradients in infarcted brain. Moreover, since transfer of hsp's from glia to neu ronal axons has been noted (Tytell et aI., 1986), it is possible that there is some interaction between in farcted neurons and glia in the penumbra. Lastly, hsp72 induction in astrocytes at the margins of in farctions might provide a biochemical marker for defining the edges of infarctions that could be used to quantitate infarction volumes rather than using
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differences in histological staining intensity (Swan son et ai., 1990). Acknowledgment: We wish to thank Dr. Stephen M. Sagar for his helpful comments on this manuscript, and we particularly appreciate the excellent technical assis tance of Pat Jasper, Theresa Marsh, and Matt Morton. This work was supported by the VA Medical Research Service and NIH grants NS24666 and NS281 67 to F.R.S., NS01424 to D. L., and NS24728 to R. S. REFERENCES Anathan J. Goldberg AL, Voellmy R (1986) Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 232:522-524 Barbe MF, Tytell M, Gower DJ, Welch WJ (1988) Hyperthermia protects against light damage in the rat retina. Science 241:1817-1820 Beckmann RP, Mizzen LA, Welch WJ (1990) Interaction of HSP70 with newly synthesized proteins: Implications for protein folding and assembly. Science 248:850--854 Bodsch W, Takahashi K, Barbier A, Grosse Ophoff B, Hoss mann K-A (1985) Cerebral protein synthesis and ischemia. Prog Brain Res 63:197-210 Brown IR, Rush S, Ivy GO (1989) Induction of a heat shock gene at the site of tissue injury in the rat brain. Neuron 2:15591564 Chappell T, Welch W, Schlossmann D, Palter K, Schlesinger M. Rothman J (1986) Uncoating ATPase is a member of the 70kD family of stress proteins. Cell 45:3-13 Cheng MY, Hartl F-U, Martin J, Pollock RA, Kalousek F, Neupert W, Hallberg EM, Hallberg RL, Horwich AL (1989) Mitochondrial heat-shock protein hsp60 is essential for as sembly of proteins imported into yeast mitochondria. Na ture (Lond) 337:620--625 Chiang H-L, Terlecky SR, Plant CP, Dice JF (1989) A role for a 70-kiloDalton heat shock protein in lysosomal degradation of intracellular proteins. Science 246:382-385 Chirico WJ, Waters MG, Blobel G (1988) 70kD heat shock re lated proteins stimulate protein translocation into mi crosomes. Nature (Lond) 332:805-810 Chopp M, Chen H, Ho K-L, Dereski MO, Brown E, Hetzel FW, Welch KMA (1989) Transient hyperthermia protects against subsequent forebrain ischemic cell damage in the rat. Neu rology 39:1396--1398 Craig EA (1985) The heat shock response. CRC Crit Rev Bio chem 18:239-280 Deshaies RJ, Koch BD, Schenkman R (1989) The role of stress proteins in membrane biogenesis. Trends Bioi Sci 13:384388 Dienel GA, Pulsinelli WA, Duffy TE (1980) Regional protein synthesis in rat brain following acute hemispheric ischemia. J Neurochem 35:1216--1226 Dienel GA, Cruz NF, Rosenfeld SJ (1985) Temporal profiles of proteins responsive to ischemia. J Neurochem 44:600--610 Dienel GA, Kiessling M, Jacewicz M, Pulsinelli WA (1986) Syn thesis of heat shock proteins in rat brain cortex after tran sient ischemia. J Cereb Blood Flow Metab 6:505-510 Dwyer BE, Nishimura RN, Powell CL, Mailheau SL (1987) Fo cal protein synthesis inhibition in a model of neonatal hyp oxic-ischemic injury. Exp NeuroI95:277-289 Dwyer BE, Nishimura RN, Brown IR (1989) Synthesis of the major inducible heat shock protein in rat hippocampus after neonatal hypoxia-ischemia. Exp Neurol 104:28-31 Ellis RJ (1987) Proteins as molecular chaperones. Nature (Lond) 328:378-379 Ferriero DM, Soberano HQ, Simon RP, Sharp FR (1990) Hyp oxia-ischemia induces heat shock protein-like (HSP72) im munoreactivity in neonatal rat brain. Dev Brain Res 53:145150
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