Acta Neuropathol (1992) 84:94 - 99
pathoiogica (~) Springer-Verlag1992
Distribution of 72-kDa heat-shock protein in rat brain after hypertherrnia* Y. Li 1, M. Choppl,3, Y. Yoshida 2, and S. R. Levine 1 Departments of INeurology and 2Pathology, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202, USA 3Department of Physics, Oakland University, Rochester, MI 40309, USA Received October 18, 1991/Revised, accepted December 31, 1991
Summary. The distribution of the 72-kDa heat-shock protein (hsp72) in rat brain, 24 h following in vivo transient hyperthermia (41.5 ~ 15 min), was studied using immunohistochemistry (n=22). Tissue sections were also stained with hematoxylin and eosin, and with an anti-glial fibrillary acidic protein to evaluate neuronal and astrocytic response to transient hyperthermia, respectively, hsp72 was observed in glia and endothelial cells throughout brain, hsp72 was also found in neurons located in the: dentate gyrus, habenula, and hypothalamus, granular layer of the cerebellum and the olfactory area. Our data indicate, that hyperthermia causes neuronal expression of hsp72, particularly in cerebral neuronal populations which control the neuroendocrine stress response.
Immunohistological distribution of hsp72 in neurons by a hyperthermic stimulus has not been demonstrated in rat cerebrum. A recent study has shown that in vivo hyperthermia induces expression of neuronal hsp72 m R N A in brain regions controlling the neuroendocrine response to stress [2]; suggesting that hsp72 may be induce in neurons by hyperthermia, in concert with glial and endothelial cell expression of hsp72. Although the hsp72 m R N A has been detected in cerebral neurons after hyperthermia, there are no data demonstrating the expression of the 72-kDa stress protein in these cells. In this study, we employ immunohistochemical techniques, using a monoclonal antibody to the inducible 72-kDa stress protein, to demonstrate the expression of this protein in neurons of the cerebrum.
Key words: 72-kDa heat-shock protein - H y p e r t h e r m i a - Immunohistochemical localization - Rat brain
Materials and methods
T h e increased synthesis of highly conserved heat-shock proteins (hsp) has been recognized as a response of the central nervous system (CNS) to a variety of stressful stimuli. The 72-kDa hsp (hsp72), the most studied hsp, is induced in brain by direct heating or drug-induced hyperthermia [3, 4, 7, 8, 15, 22, 23, 25, 35], cerebral ischemia [5, 6, 9-13, 16-18, 24, 26, 29, 31, 32], traumatic injury [14], excitotoxins and seizures [12, 21, 33]. O f particular interest is the question, which cells in the CNS express hsp72? Available evidence suggests that hsp72 is induced primarily in neurons vulnerable to damage from ischemia or status epilepticus [5, 11-13, 17, 18, 21, 31-33], although hsp72 is also induced in microvascular elements and glia throughout the brain, and in cerebellar granule layer cells and the choroid plexus in rats subjected to a hyperthermic stress [22]. * Supported in part from NINDS grant PO1 NS23393 and an American Heart Association grant-in-aid (Michigan Affiliated) Correspondence to: Y. Li (address see above)
Twenty-two male Wistar rats weighing 260 +_ 20 g were used. Anesthesia was maintained with 1.0% halothane in N~O:O2 (69 % : 30 %) using a face mask. Nine animals were subjected to transient hyperthermia when placed on a floating platform in a high-humidity closed container. Rectal temperature was elevated over a period of approximately 40 rain to 41.5 _+0.1 ~ and was controlled by feedback regulation of water temperature in the container. The animals were maintained at this temperature for 15 min.They were then removed from the container and allowed to cool spontaneously at room temperature (19 ~ ~ To explore the effect of halothane anesthesia on the expression of hsp72 and temperature, two control groups were used: (1) rats (n = 2) were maintained at 37.5 + 0.1 ~ by a feedback-regulated temperature controller (YSI model 73-A,Yellow Springs Instrument Company, Yellow Springs, Ohio) under anesthesia with halothane for 55 min (40 min + 15 min); (2) rats (n = 2) were maintained under anesthesia with halothane (55 vain) at room temperature. To exlude the possibility that hypothermia occurring after transient hyperthermia itself evokes the expression of hsp72 (see results), we performed experiments on rats (n = 9) subjected to whole body hypothermia (30~ for 3 h. The animals were cooled using combination of alcohol evaporation and air cooling. Rectal temperature was monitored and regulated as in the animals subjected to hyperthermia. The immunohistochemical technique, described in detail by Vass et al. [32], was used to detect hsp72. Twenty-four hours
95 following treatment, all rats wre fixed by transcardial perfusion with sodium phosphate buffer (pH 7.4), followed by 4 % paraformaldehyde in buffer. Brains were removed and placed in the same fixative at 4 ~ overnight. The brains were then transferred to phosphate-buffered saline (PBS). Coronal sections (50 ~m) were cut on a vibratome. Blocking of nonspecific background staining was accomplished with normal sheep whole serum. A mouse monoclonal antibody to hsp72 (C92, Amersham, PRN 1197, Cleveland, Ohio) was used. This monoclonal antibody reacts only with the induced hsp72 of similar reactivity to the antibody reported by Welch and Suhan [34]. Endogenous peroxidase was blocked with H202 and methanol in PBS saline. Biotinylated sheep anti-mouse IgG was incubated for 8 h. Sections were incubated with streptavidin "bridge", followed by biotinylated horseradish peroxidase for 1 h each. Peroxidase was detected with diaminobenzidine. Sections were gelatin-mounted on slides for light microscopic evaluation. Control sections were prepared by performing immunohistochemistry, except that primary antibody was deleted. Vibratome sections (50 ~m) were cut and processed for anti-glial fibrillary acidic protein (GFAP) immunohistochemistry, using the same avidin-biotirdhorseradish peroxidase technique. The remaining coronal slices from these rat brains were embedded in paraffin, and the sections [6 ~xm)adjacent to those cut on the vibratome were stained with hematoxylin and eosin (H&E) for histopathological evaluation.
Results The time course of rectal temperature in rats exposed for 15 rain to 41.5 ~ hyperthermia followed by recovery at room temperature, and the two control groups, are presented in Fig. 1. The sham-operated rats (37.5 + 0.1~ in control group 1 awoke within 5 min following termination of anesthesia. Rectal temperature decreased I ~ after termination of anesthesia, and gradually returned to the normal by 3 h. Contraction of the pilomotor muscles, which reduces heat loss from skin, was noted. T h e rats in group 2 (at room tempera-
43
42 41 40 i.u nt< wcu LU F-IO IJA er
39 38 ~JJ
37
~
Z
ture) also awoke within 5 min following termination of anesthesia. Their core temperature decreased 2 ~ during anesthesia and returned to the normal range after 0.5 h. Contraction of the pilomotor muscles, assumption of the huddle position with the extremities held close to the body, and shivering and chattering of the teeth, were present after termination of anesthesia. The hyperthermic animals recovered consciousness within 20-30 rain. Transient hyperthermia was immediately followed by more than a 2 h period of hypothermia (32.9 + 1.1~ with a gradual return to normal temperature after 7 - 9 h. During the recovery, the animals exhibited contraction of the pilomotor muscles, assumption of the huddle position, shivering and chattering. No evidence of neuronal necrosis or hsp72 staining was detected in hypothermia or sham-operated animals. In the experimental animals subjected to hyperthermia, hsp72-positive staining was detected in neurons, glia, and endothelial cells.
Neurons hsp72 was detected in cell bodies of neurons in selected brain regions: dentate gyrus (Fig. 2a,b), medial habenula (Fig. 3a,b), and hypothalamus (Fig. 4a,b). No hsp72positive neurons were detected in the cerebral cortex and basal ganglia. In the cerebellum, granule cells exhibited intense hsp72 induction (Fig. 5a,b). hsp72 was not detected in purkinje cells and neurons in the dentate nuclei, nor in the brain stem. hsp72 was present in neurons in the olfactory area (Fig. 6).
Glia hsp72-positive glia, exhibiting many fine processes, were widespread in gray and white m a t t e r throughout the brain. No spinous or varicoid processes were observed. Some of the glial processes extended to the capillaries (Figs. 2b, 3b, 4b, 5b), indicating that these cells are astrocytes. The distribution of hsp72-positive glia paralleled that of GFAP-positive cells in most brain regions (Fig. 2c,d). Larger and more distinct nuclei of GFAPpositive cells were observed in hyperthermic animals than in control animals.
36 35
Endothelial cells
34 33 32 31
F i
30 O
1
2
3
4
5
6
_l
7
B
9
hsp72 distribution was widespread in endothelial cells of arterioles, veins and capillaries in the gray and white matter of cerebrum and cerebellum following hyperthermia.
TIME (HOUR)
Fig. 1. Time course of rectal temperature in rats exposed for 15 rain to 41.5~ hyperthermia, followed by recovery at room temperature. Sham-operated rats: the temperature profiles in group 1 (n=2) and group 2 (n=2) see procedure described in the text. Symbols indicate mean + SD on nine rats. O, hyperthermia; sham-operated: vq, group 1; O, group 2
Others hsp72 was induced in the choroid plexus of all animals. hsp72 was not detected in the ependymal cells, or in the leptomeninges.
96
Fig. 2a-d. Dentate gyrus after 15 min hyperthermia at 41.5 ~ and 24 h recovery, exhibiting 72-kDa heat-shock protein (hsp72) and GFAP immunoreactivity in a vibratome section (50 ~m). a hsp72positive neurons (asterisks) are present in the granule cell layer along with widespread hsp 72-positive glia and endothelial cells, b The enlarged area from the box outlined in a shows hsp72-positive neurons (straight arrows), glia (curve arrows) and endothelial cells (arrowheads). hsp72-positive glia exhibited many fine processes. Some of these processes extended to the capillaries, indicating that these cells are astrocytes, c An adjacent GFAP-positive section from the dentate gyrus in the same rat demonstrates abundant astrocyte cells parallel to hsp72-positive glia. d An enlarged area from the box outline in c. a,c x 33; b,d • 83
Fig. 3. a Neurons in the medial habenula (white asterisks) expressed hsp72, while hsp72 was not detected in neurons of the lateral habenula (black asterisks), hsp72-positive glia and endothelial cells were widespread, h A n enlarged area from the box outlined in a. hsp72 in neurons (straight arrows) in the medial habenula with hsp72-positive glia (curve arrows) and endothelial cells (arrowheads). a • 33; b • 165
97
Fig. 4. a Hypothalamus, exhibiting hsp72 in neurons, glia and endothelial cells, b A n enlarged area from the box outline in a. hsp72 is found in scattered neurons (straight arrows), glia (curve arrows) and endothelial cells (arrowheads). a x 33; b • 165 Fig. 5. a Intense hsp72 staining was present in neurons in the granule cell layer (arrows). hsp72-positive glia and endothelial cells were present through out the cerebellum, b An enlarged area from the box outlined in a. hsp72 is present in neurons (asterisks), glia (curve arrows), and endothelial cells (arrowheads). a • 9; b x 80 Fig. 6. Olfactory area, exhibiting hsp72 in neurons of the plexiform layer endothelial cells. • 18
(straight arrows), with
widespread hsp72-positive glia and
98
Discussion Our data demonstrate that transient hyperthermia induces hsp72 in cell bodies of neurons, glia - likely astrocytes, and endothelial cells in the brain. These observations are the first to demonstrate hyperthermiainduced hsp72 in neurons of cerebrum. Our data, therefore, complement those of Blake et al. [2], who, using in situ hybridization, found that hyperthermia in Wistar rats induced hsp72 m R N A in neurons of the hip'pocampal dentate gyms, hypothalamus and the medial habenula. Thus, the 72-kDa protein as well as the m R N A are induced in neurons of these regions. Previous measurements of hsp72 immunoreactivity in the Sprague Dawley rat after hyperthermic stress have shown that hsp72 is induced in glia and endothelial cells throughout the brain, in granule layer cells in the cerebellum, and not in neurons of cerebrum [22]. We speculate, that the absence of hsp72 in neurons of cerebrum, which contrasts to our findings, may be attributed to a different hyperthermic protocol in a different species of rat. Rectal temperatures of 42.5 ~ were induced for approximately 1 h of hyperthermic exposure in the Sprague Dawley rats. This constitutes a level of hyperthermia that exceeds the apparent threshold for hsp72 induction, as determined in mice [26], and the combination of excessive and a long duration of hyperthermia may cause neuronal necrosis, or inhibit hsp72 induction. No neuronal necrosis was detected in any of the animals in the present study. Since hsp72 expression is associated with tissue insult and stress [19, 20], our data suggest that non-lethal stress such as transient hyperthermia induces hsp72 in neurons, glia and endothelial cells in the brain. Our data indicate that hyperthermia causes impairment of post anesthesia regulation of body temperature. The hsp72 expression in brain regions coordinating the neuroendocrine response to stress may reflect dysfunction of temperature regulation. Body temperature is regulated by the thermoregulatory center in the hypothalamus [27], in which selective neuronal induction of hsp72 occurs. The hypothalamic-pituitary-adrenal axis regulates levels of adrenocortical hormones (mainly cortisol).The production of cortisol by the adrenal gland is controlled by the adrenocorticotropic h o r m o n e ( A C T H ) from the anterior pituitary. A C T H , in turn, is controlled by the corticotropin-releasing factor from the hypothalamus. Cortisol also regulates vascular responsiveness [1]. The hippocampus and habenula are assod a t e d with neuronal networks involved with the neuroendocrine stress response [2]. Electrophysiological studies have demonstrated both direct and trans-synaptic connections from the hippocampus to the hypothalamus [28, 30]. Selective neuronal induction of hsp72 is present in the hypothalamus, hippocampus, and habenula. Impaired thermoregulation may, therefore, reflect a subthreshold of neuronal damage to the brain regions controlling neuroendocrine response to stress, hsp72positive cells are, thus, suggestive of stress, and possibly damage, to the anatomical sites associated with temperature control.
Acknowledgements. The authors gratefully acknowledge Lisa Pietrantoni, HTL (ASCP) for assistance with histological preparations, and Zhuangxian Qing and Patricia Ruffin for manuscript preparation.
References 1. Berne RM, Levy MN (1988) Physiology, 2nd edn. CV Mosby, Washington, D.C., pp 905-909 2. Blake MJ, Nowak TS Jr, Holbrook NJ (1990) In vivo hyperthermia induces expression of HSP70 mRNA in brain regions controlling the neuroendocrine response to stress. Mol Brain Res 8:89-92 3. Brown IR (1983) Hyperthermia induces synthesis of a heat shock protein by polysomes isolated from the fetal and neonatal mammalian brain. J Neurochem 40:1490-1493 4. Brown IR (1990) Induction of heat shock (stress) genes in the mammalian brain by hyperthermia and other traumatic events: a current perspective. J Neurosci Res 27:247-255 5. Chopp M, Tidwell CD, Lee YJ, Knight R, Helpern JA,Welch KMA (1989) Reduction of hyperthermic ischemic acidosis by a conditioning event in cats. Stroke 20:1357-1360 6. Chopp M, Li Y, Dereski MO, Levine SR,YoshidaY, Garcia JH (1991) Neuronal injury and expression of 72-kDa heat shock protein after forebrain ischemia in the rat. Acta Neuropathol 83:66-71 7. Cosgrove JW, Brown IR (1983) Heat shock protein in mammalian brain and other organs after a physiologically relevant increase in body temperature induced by D-lysergic acid diethylamide. Proc Natl Acad Sci USA 80:569-573 8. Currie RW,White FP (1983) Characterization ofthe synthesis and accumulation of a 71-kilodalton protein induced in rat tissues after hyperthermia. Can J Biochem Cell Biol 61:438-446 9. Dienel GA, Kiessling M, Jacewicz M, Pulsinelli WA (1986) Synthesis of heat shock protein in rat brain cortex after transient ischemia. J Cereb Blood Flow Metab 6:505-510 10. 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 11. Ferriero DM, Soberano HQ, Simon RR Sharp FR (1990) Hypoxia-ischemia induces heat shock protein-like (HSP72) immunoreactivity in neonatal rat brain. Dev Brain Res 53:145-150 12. Gonzalez MF, Shiraishi K, Hisanaga K, Sagar SM, Mandabach M, Sharp FR (1989) Heat shock proteins as markers of neural injury. Mol Brain Res 6:93-100 13. Gonzalez MF, Lowenstein D, Fernyak S, Hisanaga K, Simon R, 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 14. Gower D J, Hollman C, Lee KS, Tytell M (1989) Spinal cord injury and the stress protein response. J Neurosurg 70: 605-611 15. Inasi BS, Brown IR (1982) Synthesis of a heat shock protein in the microvascular system of the rabbit brain following elevation of body temperature. Biochem Biophy Res Commun 106:881-887 16. Kiessling M, Dienel GA, Jacewicz M, Pulsinelli WA (1986) Protein synthesis in postischemic rat brain: a two-dimensional electrophoretic analysis. J Cereb Blood Flow Metab 6: 642-649 17. Kirino T, Tsujita Y, Tamura A (1991) Induced tolerance to ischemia in gerbil hippocampal neurons. J Cereb Blood Flow Metab 11:299-307 18. Kitagawa K, Matsumoto M, Tagaya M, Hata R, Ueda H, Niinobe M, Handa N, Fukunaga R, Kimura K, Mikoshiba K, Kamada T (1990) 'Ischemic tolerance' phenomenon found in the brain. Brain Res 528:21-24
99 19. Lindquist S (1986) The heat-shock response. Annu Rev Biochem 55:1151-1191 20. Lindquist S, Craig E A (1988) The heat-shock proteins. Annu Rev Genet 22:631-677 21. 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 epilepticus. Brain Res 531:173-182 22. Marini AM, Kozuka M, Lipsky RH, Nowak TS Jr (1990) 70-Kilodalton heat shock protein induction in cerebellar astrocytes and cerebellar granule cells in vitro: comparison with immunocytochemical localization after hyperthermia in vivo. J Neurochem 54:1509-1516 23. Miller EK, Raese JD, Morrison-Bogorad M (1991) Expression of heat-shock protein 70 and heat shock cognate 70 messenger RNAs in rat cortex and cerebellum after heat shock or amphetamine treatment. J Neurochem 56:2060-2071 24. Nowak TS Jr (1985) Synthesis of a stress protein following transient ischemia in the gerbil. J Neurochem 45:1635-1641 25. Nowak TS Jr (1988) Effects of amphetamine of protein synthesis and energy metabolism in mouse brain: role of drug-induced hyperthermia. J Neurochem 50:285-294 26. Nowak TS Jr, Bond U, Schlesinger MJ (1990) Heat shock RNA levels in brain and other tissues after hyperthermia and transient ischemia. J Neurochem 54:451-456 27. Porth CM (1990) Pathophysiology Concepts of altered health states, 3rd edn. JB Lippincott, Phihladelphia, pp 95-106
28. Saphier D, Feldman S (1987) Effects of septal and hippocampal stimuli on paraventricular nucleus neurons. Neurosci 20: 749-755 29. Sharp FR, Lowenstein D, Simon R, Hisanaga K (1991) Heat shock protein hsp72 induction in cortical and striatal astrocytes and neurons following infarction. J Cereb Blood Flow Metab 11: 621-627 30. Silverman AJ, Oldfield BJ (1984) Synaptic input to vasopressin neurons of the paraventricular nucleus (PVN). Peptides 5 [Suppl l]: 139-150 31. Simon RP, Cho H, Gwinn R, Lowenstein DH (1991) The temporal profile of 72-kDa heat-shock protein expression following global ischemia. J Neurosci 11:881-889 32. Vass K,Welch WJ, Nowak TS Jr (1988) Localization of 70-kDa stress protein induction in gerbil brain after ischemia. Acta Neuropathol 77:128-135 33. 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. Neurosci Lett 100:259-264 34. Welch WJ, Suhan JP (1986) Cellular and biochemical events in mammalian cells during and after recovery from physiological stress. Cell Biochem 103:2035-2052 35. White FP (1981) The induction of "stress" proteins in organ slices from brain, heart, and lung as a function of postnatal development. J Neurosci 1:1312-1319
pathoiogica (~) Springer-Verlag1992
Distribution of 72-kDa heat-shock protein in rat brain after hypertherrnia* Y. Li 1, M. Choppl,3, Y. Yoshida 2, and S. R. Levine 1 Departments of INeurology and 2Pathology, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202, USA 3Department of Physics, Oakland University, Rochester, MI 40309, USA Received October 18, 1991/Revised, accepted December 31, 1991
Summary. The distribution of the 72-kDa heat-shock protein (hsp72) in rat brain, 24 h following in vivo transient hyperthermia (41.5 ~ 15 min), was studied using immunohistochemistry (n=22). Tissue sections were also stained with hematoxylin and eosin, and with an anti-glial fibrillary acidic protein to evaluate neuronal and astrocytic response to transient hyperthermia, respectively, hsp72 was observed in glia and endothelial cells throughout brain, hsp72 was also found in neurons located in the: dentate gyrus, habenula, and hypothalamus, granular layer of the cerebellum and the olfactory area. Our data indicate, that hyperthermia causes neuronal expression of hsp72, particularly in cerebral neuronal populations which control the neuroendocrine stress response.
Immunohistological distribution of hsp72 in neurons by a hyperthermic stimulus has not been demonstrated in rat cerebrum. A recent study has shown that in vivo hyperthermia induces expression of neuronal hsp72 m R N A in brain regions controlling the neuroendocrine response to stress [2]; suggesting that hsp72 may be induce in neurons by hyperthermia, in concert with glial and endothelial cell expression of hsp72. Although the hsp72 m R N A has been detected in cerebral neurons after hyperthermia, there are no data demonstrating the expression of the 72-kDa stress protein in these cells. In this study, we employ immunohistochemical techniques, using a monoclonal antibody to the inducible 72-kDa stress protein, to demonstrate the expression of this protein in neurons of the cerebrum.
Key words: 72-kDa heat-shock protein - H y p e r t h e r m i a - Immunohistochemical localization - Rat brain
Materials and methods
T h e increased synthesis of highly conserved heat-shock proteins (hsp) has been recognized as a response of the central nervous system (CNS) to a variety of stressful stimuli. The 72-kDa hsp (hsp72), the most studied hsp, is induced in brain by direct heating or drug-induced hyperthermia [3, 4, 7, 8, 15, 22, 23, 25, 35], cerebral ischemia [5, 6, 9-13, 16-18, 24, 26, 29, 31, 32], traumatic injury [14], excitotoxins and seizures [12, 21, 33]. O f particular interest is the question, which cells in the CNS express hsp72? Available evidence suggests that hsp72 is induced primarily in neurons vulnerable to damage from ischemia or status epilepticus [5, 11-13, 17, 18, 21, 31-33], although hsp72 is also induced in microvascular elements and glia throughout the brain, and in cerebellar granule layer cells and the choroid plexus in rats subjected to a hyperthermic stress [22]. * Supported in part from NINDS grant PO1 NS23393 and an American Heart Association grant-in-aid (Michigan Affiliated) Correspondence to: Y. Li (address see above)
Twenty-two male Wistar rats weighing 260 +_ 20 g were used. Anesthesia was maintained with 1.0% halothane in N~O:O2 (69 % : 30 %) using a face mask. Nine animals were subjected to transient hyperthermia when placed on a floating platform in a high-humidity closed container. Rectal temperature was elevated over a period of approximately 40 rain to 41.5 _+0.1 ~ and was controlled by feedback regulation of water temperature in the container. The animals were maintained at this temperature for 15 min.They were then removed from the container and allowed to cool spontaneously at room temperature (19 ~ ~ To explore the effect of halothane anesthesia on the expression of hsp72 and temperature, two control groups were used: (1) rats (n = 2) were maintained at 37.5 + 0.1 ~ by a feedback-regulated temperature controller (YSI model 73-A,Yellow Springs Instrument Company, Yellow Springs, Ohio) under anesthesia with halothane for 55 min (40 min + 15 min); (2) rats (n = 2) were maintained under anesthesia with halothane (55 vain) at room temperature. To exlude the possibility that hypothermia occurring after transient hyperthermia itself evokes the expression of hsp72 (see results), we performed experiments on rats (n = 9) subjected to whole body hypothermia (30~ for 3 h. The animals were cooled using combination of alcohol evaporation and air cooling. Rectal temperature was monitored and regulated as in the animals subjected to hyperthermia. The immunohistochemical technique, described in detail by Vass et al. [32], was used to detect hsp72. Twenty-four hours
95 following treatment, all rats wre fixed by transcardial perfusion with sodium phosphate buffer (pH 7.4), followed by 4 % paraformaldehyde in buffer. Brains were removed and placed in the same fixative at 4 ~ overnight. The brains were then transferred to phosphate-buffered saline (PBS). Coronal sections (50 ~m) were cut on a vibratome. Blocking of nonspecific background staining was accomplished with normal sheep whole serum. A mouse monoclonal antibody to hsp72 (C92, Amersham, PRN 1197, Cleveland, Ohio) was used. This monoclonal antibody reacts only with the induced hsp72 of similar reactivity to the antibody reported by Welch and Suhan [34]. Endogenous peroxidase was blocked with H202 and methanol in PBS saline. Biotinylated sheep anti-mouse IgG was incubated for 8 h. Sections were incubated with streptavidin "bridge", followed by biotinylated horseradish peroxidase for 1 h each. Peroxidase was detected with diaminobenzidine. Sections were gelatin-mounted on slides for light microscopic evaluation. Control sections were prepared by performing immunohistochemistry, except that primary antibody was deleted. Vibratome sections (50 ~m) were cut and processed for anti-glial fibrillary acidic protein (GFAP) immunohistochemistry, using the same avidin-biotirdhorseradish peroxidase technique. The remaining coronal slices from these rat brains were embedded in paraffin, and the sections [6 ~xm)adjacent to those cut on the vibratome were stained with hematoxylin and eosin (H&E) for histopathological evaluation.
Results The time course of rectal temperature in rats exposed for 15 rain to 41.5 ~ hyperthermia followed by recovery at room temperature, and the two control groups, are presented in Fig. 1. The sham-operated rats (37.5 + 0.1~ in control group 1 awoke within 5 min following termination of anesthesia. Rectal temperature decreased I ~ after termination of anesthesia, and gradually returned to the normal by 3 h. Contraction of the pilomotor muscles, which reduces heat loss from skin, was noted. T h e rats in group 2 (at room tempera-
43
42 41 40 i.u nt< wcu LU F-IO IJA er
39 38 ~JJ
37
~
Z
ture) also awoke within 5 min following termination of anesthesia. Their core temperature decreased 2 ~ during anesthesia and returned to the normal range after 0.5 h. Contraction of the pilomotor muscles, assumption of the huddle position with the extremities held close to the body, and shivering and chattering of the teeth, were present after termination of anesthesia. The hyperthermic animals recovered consciousness within 20-30 rain. Transient hyperthermia was immediately followed by more than a 2 h period of hypothermia (32.9 + 1.1~ with a gradual return to normal temperature after 7 - 9 h. During the recovery, the animals exhibited contraction of the pilomotor muscles, assumption of the huddle position, shivering and chattering. No evidence of neuronal necrosis or hsp72 staining was detected in hypothermia or sham-operated animals. In the experimental animals subjected to hyperthermia, hsp72-positive staining was detected in neurons, glia, and endothelial cells.
Neurons hsp72 was detected in cell bodies of neurons in selected brain regions: dentate gyrus (Fig. 2a,b), medial habenula (Fig. 3a,b), and hypothalamus (Fig. 4a,b). No hsp72positive neurons were detected in the cerebral cortex and basal ganglia. In the cerebellum, granule cells exhibited intense hsp72 induction (Fig. 5a,b). hsp72 was not detected in purkinje cells and neurons in the dentate nuclei, nor in the brain stem. hsp72 was present in neurons in the olfactory area (Fig. 6).
Glia hsp72-positive glia, exhibiting many fine processes, were widespread in gray and white m a t t e r throughout the brain. No spinous or varicoid processes were observed. Some of the glial processes extended to the capillaries (Figs. 2b, 3b, 4b, 5b), indicating that these cells are astrocytes. The distribution of hsp72-positive glia paralleled that of GFAP-positive cells in most brain regions (Fig. 2c,d). Larger and more distinct nuclei of GFAPpositive cells were observed in hyperthermic animals than in control animals.
36 35
Endothelial cells
34 33 32 31
F i
30 O
1
2
3
4
5
6
_l
7
B
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hsp72 distribution was widespread in endothelial cells of arterioles, veins and capillaries in the gray and white matter of cerebrum and cerebellum following hyperthermia.
TIME (HOUR)
Fig. 1. Time course of rectal temperature in rats exposed for 15 rain to 41.5~ hyperthermia, followed by recovery at room temperature. Sham-operated rats: the temperature profiles in group 1 (n=2) and group 2 (n=2) see procedure described in the text. Symbols indicate mean + SD on nine rats. O, hyperthermia; sham-operated: vq, group 1; O, group 2
Others hsp72 was induced in the choroid plexus of all animals. hsp72 was not detected in the ependymal cells, or in the leptomeninges.
96
Fig. 2a-d. Dentate gyrus after 15 min hyperthermia at 41.5 ~ and 24 h recovery, exhibiting 72-kDa heat-shock protein (hsp72) and GFAP immunoreactivity in a vibratome section (50 ~m). a hsp72positive neurons (asterisks) are present in the granule cell layer along with widespread hsp 72-positive glia and endothelial cells, b The enlarged area from the box outlined in a shows hsp72-positive neurons (straight arrows), glia (curve arrows) and endothelial cells (arrowheads). hsp72-positive glia exhibited many fine processes. Some of these processes extended to the capillaries, indicating that these cells are astrocytes, c An adjacent GFAP-positive section from the dentate gyrus in the same rat demonstrates abundant astrocyte cells parallel to hsp72-positive glia. d An enlarged area from the box outline in c. a,c x 33; b,d • 83
Fig. 3. a Neurons in the medial habenula (white asterisks) expressed hsp72, while hsp72 was not detected in neurons of the lateral habenula (black asterisks), hsp72-positive glia and endothelial cells were widespread, h A n enlarged area from the box outlined in a. hsp72 in neurons (straight arrows) in the medial habenula with hsp72-positive glia (curve arrows) and endothelial cells (arrowheads). a • 33; b • 165
97
Fig. 4. a Hypothalamus, exhibiting hsp72 in neurons, glia and endothelial cells, b A n enlarged area from the box outline in a. hsp72 is found in scattered neurons (straight arrows), glia (curve arrows) and endothelial cells (arrowheads). a x 33; b • 165 Fig. 5. a Intense hsp72 staining was present in neurons in the granule cell layer (arrows). hsp72-positive glia and endothelial cells were present through out the cerebellum, b An enlarged area from the box outlined in a. hsp72 is present in neurons (asterisks), glia (curve arrows), and endothelial cells (arrowheads). a • 9; b x 80 Fig. 6. Olfactory area, exhibiting hsp72 in neurons of the plexiform layer endothelial cells. • 18
(straight arrows), with
widespread hsp72-positive glia and
98
Discussion Our data demonstrate that transient hyperthermia induces hsp72 in cell bodies of neurons, glia - likely astrocytes, and endothelial cells in the brain. These observations are the first to demonstrate hyperthermiainduced hsp72 in neurons of cerebrum. Our data, therefore, complement those of Blake et al. [2], who, using in situ hybridization, found that hyperthermia in Wistar rats induced hsp72 m R N A in neurons of the hip'pocampal dentate gyms, hypothalamus and the medial habenula. Thus, the 72-kDa protein as well as the m R N A are induced in neurons of these regions. Previous measurements of hsp72 immunoreactivity in the Sprague Dawley rat after hyperthermic stress have shown that hsp72 is induced in glia and endothelial cells throughout the brain, in granule layer cells in the cerebellum, and not in neurons of cerebrum [22]. We speculate, that the absence of hsp72 in neurons of cerebrum, which contrasts to our findings, may be attributed to a different hyperthermic protocol in a different species of rat. Rectal temperatures of 42.5 ~ were induced for approximately 1 h of hyperthermic exposure in the Sprague Dawley rats. This constitutes a level of hyperthermia that exceeds the apparent threshold for hsp72 induction, as determined in mice [26], and the combination of excessive and a long duration of hyperthermia may cause neuronal necrosis, or inhibit hsp72 induction. No neuronal necrosis was detected in any of the animals in the present study. Since hsp72 expression is associated with tissue insult and stress [19, 20], our data suggest that non-lethal stress such as transient hyperthermia induces hsp72 in neurons, glia and endothelial cells in the brain. Our data indicate that hyperthermia causes impairment of post anesthesia regulation of body temperature. The hsp72 expression in brain regions coordinating the neuroendocrine response to stress may reflect dysfunction of temperature regulation. Body temperature is regulated by the thermoregulatory center in the hypothalamus [27], in which selective neuronal induction of hsp72 occurs. The hypothalamic-pituitary-adrenal axis regulates levels of adrenocortical hormones (mainly cortisol).The production of cortisol by the adrenal gland is controlled by the adrenocorticotropic h o r m o n e ( A C T H ) from the anterior pituitary. A C T H , in turn, is controlled by the corticotropin-releasing factor from the hypothalamus. Cortisol also regulates vascular responsiveness [1]. The hippocampus and habenula are assod a t e d with neuronal networks involved with the neuroendocrine stress response [2]. Electrophysiological studies have demonstrated both direct and trans-synaptic connections from the hippocampus to the hypothalamus [28, 30]. Selective neuronal induction of hsp72 is present in the hypothalamus, hippocampus, and habenula. Impaired thermoregulation may, therefore, reflect a subthreshold of neuronal damage to the brain regions controlling neuroendocrine response to stress, hsp72positive cells are, thus, suggestive of stress, and possibly damage, to the anatomical sites associated with temperature control.
Acknowledgements. The authors gratefully acknowledge Lisa Pietrantoni, HTL (ASCP) for assistance with histological preparations, and Zhuangxian Qing and Patricia Ruffin for manuscript preparation.
References 1. Berne RM, Levy MN (1988) Physiology, 2nd edn. CV Mosby, Washington, D.C., pp 905-909 2. Blake MJ, Nowak TS Jr, Holbrook NJ (1990) In vivo hyperthermia induces expression of HSP70 mRNA in brain regions controlling the neuroendocrine response to stress. Mol Brain Res 8:89-92 3. Brown IR (1983) Hyperthermia induces synthesis of a heat shock protein by polysomes isolated from the fetal and neonatal mammalian brain. J Neurochem 40:1490-1493 4. Brown IR (1990) Induction of heat shock (stress) genes in the mammalian brain by hyperthermia and other traumatic events: a current perspective. J Neurosci Res 27:247-255 5. Chopp M, Tidwell CD, Lee YJ, Knight R, Helpern JA,Welch KMA (1989) Reduction of hyperthermic ischemic acidosis by a conditioning event in cats. Stroke 20:1357-1360 6. Chopp M, Li Y, Dereski MO, Levine SR,YoshidaY, Garcia JH (1991) Neuronal injury and expression of 72-kDa heat shock protein after forebrain ischemia in the rat. Acta Neuropathol 83:66-71 7. Cosgrove JW, Brown IR (1983) Heat shock protein in mammalian brain and other organs after a physiologically relevant increase in body temperature induced by D-lysergic acid diethylamide. Proc Natl Acad Sci USA 80:569-573 8. Currie RW,White FP (1983) Characterization ofthe synthesis and accumulation of a 71-kilodalton protein induced in rat tissues after hyperthermia. Can J Biochem Cell Biol 61:438-446 9. Dienel GA, Kiessling M, Jacewicz M, Pulsinelli WA (1986) Synthesis of heat shock protein in rat brain cortex after transient ischemia. J Cereb Blood Flow Metab 6:505-510 10. 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 11. Ferriero DM, Soberano HQ, Simon RR Sharp FR (1990) Hypoxia-ischemia induces heat shock protein-like (HSP72) immunoreactivity in neonatal rat brain. Dev Brain Res 53:145-150 12. Gonzalez MF, Shiraishi K, Hisanaga K, Sagar SM, Mandabach M, Sharp FR (1989) Heat shock proteins as markers of neural injury. Mol Brain Res 6:93-100 13. Gonzalez MF, Lowenstein D, Fernyak S, Hisanaga K, Simon R, 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 14. Gower D J, Hollman C, Lee KS, Tytell M (1989) Spinal cord injury and the stress protein response. J Neurosurg 70: 605-611 15. Inasi BS, Brown IR (1982) Synthesis of a heat shock protein in the microvascular system of the rabbit brain following elevation of body temperature. Biochem Biophy Res Commun 106:881-887 16. Kiessling M, Dienel GA, Jacewicz M, Pulsinelli WA (1986) Protein synthesis in postischemic rat brain: a two-dimensional electrophoretic analysis. J Cereb Blood Flow Metab 6: 642-649 17. Kirino T, Tsujita Y, Tamura A (1991) Induced tolerance to ischemia in gerbil hippocampal neurons. J Cereb Blood Flow Metab 11:299-307 18. Kitagawa K, Matsumoto M, Tagaya M, Hata R, Ueda H, Niinobe M, Handa N, Fukunaga R, Kimura K, Mikoshiba K, Kamada T (1990) 'Ischemic tolerance' phenomenon found in the brain. Brain Res 528:21-24
99 19. Lindquist S (1986) The heat-shock response. Annu Rev Biochem 55:1151-1191 20. Lindquist S, Craig E A (1988) The heat-shock proteins. Annu Rev Genet 22:631-677 21. 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 epilepticus. Brain Res 531:173-182 22. Marini AM, Kozuka M, Lipsky RH, Nowak TS Jr (1990) 70-Kilodalton heat shock protein induction in cerebellar astrocytes and cerebellar granule cells in vitro: comparison with immunocytochemical localization after hyperthermia in vivo. J Neurochem 54:1509-1516 23. Miller EK, Raese JD, Morrison-Bogorad M (1991) Expression of heat-shock protein 70 and heat shock cognate 70 messenger RNAs in rat cortex and cerebellum after heat shock or amphetamine treatment. J Neurochem 56:2060-2071 24. Nowak TS Jr (1985) Synthesis of a stress protein following transient ischemia in the gerbil. J Neurochem 45:1635-1641 25. Nowak TS Jr (1988) Effects of amphetamine of protein synthesis and energy metabolism in mouse brain: role of drug-induced hyperthermia. J Neurochem 50:285-294 26. Nowak TS Jr, Bond U, Schlesinger MJ (1990) Heat shock RNA levels in brain and other tissues after hyperthermia and transient ischemia. J Neurochem 54:451-456 27. Porth CM (1990) Pathophysiology Concepts of altered health states, 3rd edn. JB Lippincott, Phihladelphia, pp 95-106
28. Saphier D, Feldman S (1987) Effects of septal and hippocampal stimuli on paraventricular nucleus neurons. Neurosci 20: 749-755 29. Sharp FR, Lowenstein D, Simon R, Hisanaga K (1991) Heat shock protein hsp72 induction in cortical and striatal astrocytes and neurons following infarction. J Cereb Blood Flow Metab 11: 621-627 30. Silverman AJ, Oldfield BJ (1984) Synaptic input to vasopressin neurons of the paraventricular nucleus (PVN). Peptides 5 [Suppl l]: 139-150 31. Simon RP, Cho H, Gwinn R, Lowenstein DH (1991) The temporal profile of 72-kDa heat-shock protein expression following global ischemia. J Neurosci 11:881-889 32. Vass K,Welch WJ, Nowak TS Jr (1988) Localization of 70-kDa stress protein induction in gerbil brain after ischemia. Acta Neuropathol 77:128-135 33. 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. Neurosci Lett 100:259-264 34. Welch WJ, Suhan JP (1986) Cellular and biochemical events in mammalian cells during and after recovery from physiological stress. Cell Biochem 103:2035-2052 35. White FP (1981) The induction of "stress" proteins in organ slices from brain, heart, and lung as a function of postnatal development. J Neurosci 1:1312-1319
