Temporal and spatial distribution of heat shock mRNA and protein (hsp70) in the rabbit cerebellum in response to hyperthermia

We have previously investigated the expression of hsp70 genes in the hyperthermic rabbit brain at the mRNA level by Northern blot and in situ hybridization procedures. Our studies have now been extended to the protein level utilizing Western blot and immunocytochemistry. Using an antibody which is specific to inducible hsp70, a prominent induction of hsp70 protein in glial cells of hyperthermic animals was noted. In particular, Bergmann glial cells in the cerebellum are strongly immunoreactive while adjacent Purkinje neurons are immunonegative. Extension of our in situ hybridization studies to a time course analysis revealed that the initial glial induction events were followed by a delayed accumulation of inducible hsp70 mRNA in Purkinje neurons at 10 hr post-heat shock. In control animals, high levels of constitutively expressed hsc70 mRNA and protein were observed in Purkinje neurons. Similar hsc70 and hsp70 mRNA observations were also made in neurons of the deep cerebellar nuclei and in motor neurons of the spinal cord. Our results suggest that these neuronal cell types accmulate hsp70 mRNA in response to hyperthermic treatment; however, the response is delayed when compared to the rapid response seen in glial cells. The high constitutive levels of hsc70 in certain neuronal cell types may play a role in the initial dampening of the hsp70 induction response in these cells. © 1993 Wiley-Liss, Inc.     

Expression of heat shock genes (hsp70) in the rabbit spinal cord: localization of constitutive and hyperthermia-inducible mRNA species.

We have previously reported that hyperthermia induces the expression of a heat shock gene in the rabbit brain (Sprang and Brown, Mol Brain Res 3:89-93, 1987). Striking regional and cell type differences in the pattern of induction of the hsp70 mRNA were noted. Tissue injury also induces the rapid induction of hsp70 mRNA in the mammalian brain (Brown et al., Neuron 2:1559-1564, 1989). In the present study, in situ hybridization with 35S-labelled riboprobes specific for constitutive and inducible hsp70 mRNA species was employed to investigate the effect of fever-like temperatures on hsp70 gene expression in the rabbit spinal cord. Expression of constitutive hsp70 mRNA was detected in large motor neurons of both control and hyperthermic animals. Within 1 hr after hyperthermia, a massive induction of inducible hsp70 mRNA was noted in fibre tracts of the spinal cord, a pattern consistent with a strong glial response to heat shock. Induction was not observed in the large motor neurons.     

Induction of heat shock (stress) genes in the mammalian brain by hyperthermia and other traumatic events: a current perspective

Is the heat shock response physiologically relevant? For example, following hyperthermia or ischemia, what neural cell types show induction of heat shock genes and what is the time course of the effect? Initial experiments in this area demonstrated the prominent Induction of a 70 kDa heat shock protein (hsp70) when labeled brain proteins isolated from hyperthermic animals were analyzed. Recently, in situ hybridization and immunocytochemistry have been utilized to map out the pattern of expression of both constitutively expressed and stress-inducible members of the hsp70 multigene family. Different types of neural trauma have been found to induce characteristic cellular responses in the mammalian brain with regard to the type of brain cell that responds by inducing hsp70 and the timing of the induction response. Fever-like temperature causes a dramatic induction of hsp70 mRNA within 1 hr in fiber tracts of the fore-brain and cerebellum, a pattern consistent with a strong glial response to heat shock. Tissue injury, namely, a small surgical cut in the cerebral cortex, induces a rapid and highly localized induction of hsp70 mRNA in cells proximal to the injury site. Using an immunocytochemical approach, a neuronal pattern of induction of hsp70 has been demonstrated following ischemia or kainic acid–induced seizures. It is apparent that the pattern of induction of hsp70 may be a useful early marker of cellular injury and may identify previously unrecognized areas of vulnerability in the nervous system.     

Selective induction of a heat shock gene in fibre tracts and cerebellar neurons of the rabbit brain detected by in situ hybridization

In situ hybridization reveals striking regional differences in the expression of constitutive and inducible heat shock genes in the rabbit brain. Fibre tracts throughout the brain show a dramatic induction of heat shock mRNA after hyperthermia, a pattern consistent with a strong glial response to heat shock. While neurons in the cerebellum exhibit a marked induction of heat shock mRNA, neurons of the hippocampus do not. Constitutive expression of a heat shock gene is observed in neuronal laminae of the hippocampus and cerebellum.     

Intracellular localization of heat shock mRNAs (hsc70 and hsp70) to neural cell bodies and processes in the control and hyperthermic rabbit brain

Heat shock proteins are essential cellular proteins that may play important roles in cellular repair and/or protection. This report focuses on the expression of two members of the hsp70 multigene family, namely, constitutive hsc70 mRNA and stress-inducible hsp70 mRNA in the control and hyperthermic rabbit brain. The intracellular localization of these heat shock mRNAs was examined using high-resolution nonradioactive in situ hybridization. The distribution of hsc70 mRNA and hsp70 mRNA was examined in (1) neuronal cell bodies and their dendritic processes and (2) oligodendrocytes and their cellular processes. In control animals, hsc70 mRNA was detected in the apical dendritic processes and cell bodies of cortical layer II and V neurons, CA3 and CA4 neurons, deep cerebellar neurons, and brainstem neurons. A time course analysis of hsc70 mRNA, after a physiologically relevant increase in body temperature of 2.6°C, revealed more distal transport of this constitutive message into dendrites of these neuronal populations. In the same neuronal populations, basal levels of hsp70 mRNA were observed in the cell body; however, this mRNA was not detected in dendritic processes in control or hyperthermic animals. After hyperthermia, hsp70 mRNA was strongly induced in oligodendrocytes and transported to the processes of these glial cells. The localization of heat shock messages in the processes of these neural cell types could provide a mechanism for local control of synthesis of heat shock proteins in cellular compartments that are remote from the cell body. © 1996 Wiley-Liss, Inc.     

Localization of constitutive and hyperthermia-inducible heat shock mRNAS (hsc70 and hsp70) in the rabbit cerebellum and brainstem by non-radioactive in situ hybridization

Neural expression of constitutive hsc70 mRNA and hyperthermia-inducible hsp70 mRNA is examined using radioactive and non-radioactive in situ hybridization procedures. A strong induction of hsp70 mRNA was noted in cell populations in cerebellar layers and in the brainstem which demonstrated expression of mRNA encoding proteolipid protein, an oligodendrocyte marker. The non-radioactive in situ hybridization procedure using digoxigenin (DIG)-UTP-labeled riboprobes permitted improved signal localization, and stress-inducible hsp70 mRNA was detected at the cytoplasmic cap areas of individual oligodendrocytes. Cell types which express constitutive members of the hsc/hsp70 multigene family were also identified. Neurons in the brainstem and in the deep white matter and molecular layer of the cerebellum showed expression of hsc70 mRNA while signal was not detected in adjacent glial cells. A neuron-specific enolase riboprobe aided in the identification of neuronal cell types. The non-radioactive DIG riboprobe revealed that hsc70 mRNA was highly localized to the cyto-plasm of individual neurons. High constitutive levels of hsc70 in certain neurons may dampen hsp70 induction after hyperthermia in these cell populations. © 1995 Wiley-Liss, Inc.     

10 kD mitochondrial matrix heat shock protein mRNA is induced following global brain ischemia in the rat

Heat shock proteins (HSP's) are a family of highly conserved proteins whose expression is increased by stress. The expression of many HSP's is induced in neurons by ischemia; however, the response of the 10 kDa mitochondrial matrix HSP (HSP10) is less well characterized. To address this issue, asphyxial cardiac arrest was induced in 28 male Sprague-Dawley rats. Northern blot analysis revealed that hsp10 mRNA was increased 2.7-fold in asphyxiated rats compared to sham-operated controls. In situ hybridization demonstrated increased mRNA in the cortex, septal nuclei, hippocampus, thalamic nuclei, purkinje cell layer of the cerebellum, and isolated brainstem nuclei of asphyxiated rats. The increase of mRNA was most robust 8 h after the injury but remained increased for 72 h. These results show that hsp10 mRNA is increased following asphyxial cardiac arrest in rats and suggest that hsp10 could be another determinate of neuronal survival after ischemia.     

Induction of heat shock (stress) protein 70 and its mRNA in the normal and light-damaged rat retina after whole body hyperthermia

In situ hybridization and immunocytochemistry were used to investigate the distribution of the 70 kDa heat shock or stress protein (hsp70) and its mRNA in specific layers of the retina of adult rats at 0, 4, 18, and 48 or 50 hr after a brief whole body hyperthermic treatment. Induction of hsp70 mRNA was noted in the photoreceptor layer of the retina within 4 hr after hyperthermia. Pronounced accumulation of inducible hsp70 immunoreactivity was observed in cytoplasmic extensions of the photoreceptor cells, especially the inner segment zone which attained peak levels at the 18 hr time point. Selective destruction of photoreceptors by light damage prior to hyperthermia inhibited the post-hyperthermic rise in newly synthesized retinal hsp70. Our results suggest that the photoreceptor cell layer is the primary site of synthesis of hsp70 in the rat retina and that the greatest increase in hsp70 immunoreactivity following such a hyperthermic stress occurs in that layer. This stress response of the photoreceptors is discussed in relation to their location and function in the retina. © 1994 Wiley-Liss, Inc.     

Comparative distribution of estrogen receptor-α and -β mRNA in the rat central nervous system

Estrogen plays a profound role in regulating the structure and function of many neuronal systems in the adult rat brain. The actions of estrogen were thought to be mediated by a single nuclear estrogen receptor (ER) until the recent cloning of a novel ER (ER-β). To ascertain which ER is involved in the regulation of different brain regions, the present study compared the distribution of the classical (ER-α) and novel (ER-β) forms of ER mRNA-expressing neurons in the central nervous system (CNS) of the rat with in situ hybridization histochemistry. Female rat brain, spinal cord, and eyes were frozen, and cryostat sections were collected on slides, hybridized with [³⁵S]-labeled antisense riboprobes complimentary to ER-α or ER-β mRNA, stringently washed, and opposed to emulsion. The results of these studies revealed the presence of ER-α and ER-β mRNA throughout the rostral-caudal extent of the brain and spinal cord. Neurons of the olfactory bulb, supraoptic, paraventricular, suprachiasmatic, and tuberal hypothalamic nuclei, zona incerta, ventral tegmental area, cerebellum (Purkinje cells), laminae III–V, VIII, and IX of the spinal cord, and pineal gland contained exclusively ER-β mRNA. In contrast, only ER-α hybridization signal was seen in the ventromedial hypothalamic nucleus and subfornical organ. Perikarya in other brain regions, including the bed nucleus of the stria terminalis, medial and cortical amygdaloid nuclei, preoptic area, lateral habenula, periaqueductal gray, parabrachial nucleus, locus ceruleus, nucleus of the solitary tract, spinal trigeminal nucleus and superficial laminae of the spinal cord, contained both forms of ER mRNA. Although the cerebral cortex and hippocampus contained both ER mRNAs, the hybridization signal for ER-α mRNA was very weak compared with ER-β mRNA. The results of these in situ hybridization studies provide detailed information about the distribution of ER-α and ER-β mRNAs in the rat CNS. In addition, this comparative study provides evidence that the region-specific expression of ER-α, ER-β, or both may be important in determining the physiological responses of neuronal populations to estrogen action. J. Comp. Neurol. 388:507–525, 1997. © 1997 Wiley-Liss, Inc.     

Heat shock protein (hsx70) mRNA expression in human brain: effects of neurodegenerative disease and agonal state

Heat shock proteins (hsps) are involved in the response by cells to stress including hyperthermia, hypoxia and injury. Previous work has demonstrated expression of messenger RNA (mRNA) encoding 70 kDa hsps (hsp70) in animal brain in response to stimuli such as these. We have used in situ hybridization histochemistry to assess the distribution and quantity of a specific hsp70 (called hsx70) mRNA in frontal cortex and cerebellum from normal and demented patients whose pre-mortem course had been documented. In cortex, hsx70 mRNA was concentrated over pia mater and glia but was also present over neurons; in cerebellum, hsx70 mRNA was prominent over granule cells but absent from Purkinje cells. Detection of hsx70 mRNA did not correlate with pre-mortem factors such as pyrexia or coma. Increased hsx70 mRNA was found in frontal cortex white matter in Alzheimer's disease and in a mixed group of other neurodegenerative disorders. No changes occurred in cerebellum. The data provide further evidence for altered hsp gene expression in dementia, and support the existence of a stress response occurring in brains affected by such diseases.     

急性脑创伤后神经元Caspase 3表达、激活及作用的实验研究

目的 观察急性脑创伤后Caspase3在神经元凋亡中的作用。方法 采用大鼠脑创伤模型，以TUNEL法观察皮层，海马区神经元凋亡；以Northern杂交，原位杂交，免疫组化，Western杂交等对Caspase3在mRNA与蛋白质水平表达和酶活性变化进行观察；通过脑室注射给予Z-DEVD，fmk,观察其对神经元凋亡的治疗作用。结果 大鼠打击后2小时可见TUNEL染色阳性神经元，1天最明显，7天时仍高，Northern杂交和原位杂交显示，皮层，海马区神经元致伤后2小时Caspase3mRNA表达增高，1天最明显，7天仍高于正常，免疫组化法见Caspase3P20亚单位在打击后2小时表达增高，1-3天最明显，可持续至7天，Western杂交显示Caspase3下游底物PARP被降解，脑室内注射Z-DE-VD，fmk可使神经元凋亡减少。结论 急性脑创伤后神经元中Caspase3表达增高，酶活性增强，可导致神经元凋亡，Caspase3特异性酶活性抑制剂能减少神经元凋亡。     

Polysaccharide mannan components of Candida albicans and Saccharomyces cerevisiae cell wall produce fever by intracerebroventricular injection in rats

The cell wall mannan components of Candida albicans and Saccharomyces cerevisiae produced hyperthermic responses when injected intracerebroventricularly at doses of 10 microg in rats. Indomethacin treatment (5 mg/kg subcutaneously) completely abolished these responses. Serum interferon-gamma, tumor necrosis factor-alpha and interleukin-1beta levels showed an upward trend during the initial phase of the hyperthermic response induced by S. cerevisiae mannan. Meanwhile, serum levels of these proinflammatory cytokines did not increase at all at the initial phase of C. albicans mannan-induced hyperthermia. Histopathological examination of the brain tissue samples revealed no specific change throughout the parenchyma of rats given either mannan. These results indicate that the polysaccharide mannan components of yeasts, regardless of the pathogenicity, produce a pyrogenic response by a direct injection into the brain in rats. This response is not accompanied by proinflammatory cytokine induction in the periphery.     

Expression of tyrosine hydroxylase in cerebellar Purkinje neurons of the mutant tottering and leaner mouse.

In situ hybridization histochemistry, Northern blot analysis and immunohistochemistry were used to examine tyrosine hydroxylase (TH) mRNA concentrations and immunoreactivity in the locus coeruleus and cerebellum of the tottering (tg/tg), leaner (tgla/tgla), compound heterozygous (tg/tgla) and wild type control (+/+) mice, bred on a C57BL/6J background. Cerebellar Purkinje neurons, long considered to be GABAergic, showed high levels of TH mRNA in the caudal vermis and the lateral hemispheres of the cerebellum of tg/tg, tg/tgla, and tgla/tgla mice. Analysis of grain density over individual Purkinje cells showed significantly greater concentrations of TH mRNA in tg/tg, tg/tgla, and tgla/tgla mice as compared to +/+ wild type control mice. Comparison of adult (greater than or equal to 2 months) and young, pre-seizure (less than or equal to 3 weeks) mutant mice showed Purkinje cells densely labelled for TH mRNA at both ages, suggesting that TH gene expression in Purkinje cells is independent of the onset of seizures. Northern blot analysis confirmed the findings from the in situ hybridization studies, demonstrating a single band identical to TH mRNA. Immunohistochemistry confirmed the presence of TH protein in Purkinje cells of the caudal vermis and the lateral hemispheres of the cerebellum in both control and mutant mice. Quantitation of mRNA for TH and the coexisting neuropeptide, galanin, in the locus coeruleus detected no significant differences between adult tg/tg, tg/tgla and +/+ control mice. The present findings demonstrate that the classically GABAergic Purkinje cells in the cerebellum express low levels of TH, and that the mutant tottering and leaner strains of mice express extremely high levels of mRNA and protein for TH.     

Expression of angiotensin converting enzyme mRNA in rat brain.

Using molecular genetic approaches, two forms of angiotensin-converting enzyme (ACE) have so far been identified; a pulmonary form and a testicular form. This study utilized both northern blot analysis and in-situ hybridization to examine the expression of ACE mRNA in the rat brain. Northern blot analysis using oligonucleotide probes specific to the testicular or pulmonary forms of ACE demonstrated expression of a 4-5kb mRNA doublet in the rat brain, identical in size to pulmonary ACE mRNA. No expression of the testicular form of ACE mRNA was detected in the brain. In situ hybridization demonstrated that ACE mRNA was expressed in the choroid plexus, caudate putamen and cerebellum. A weak signal was also observed in the hippocampus. This distribution correlated well with the expression of ACE mapped by radioligand binding and autoradiography.     

Induction of constitutive heat shock protein 73 mRNA in the dentate gyrus by seizures.

We examined the effects of generalized seizures on heat shock protein (hsp) mRNA induction in the rat brain using in situ hybridization. Seizures induced by electroconvulsive shock, electrical or cocaine kindling caused a selective induction of the constitutive hsp 73 gene in the dentate gyrus. In these seizure paradigms, not thought to induce widespread tissue damage, neither the heat-inducible hsp 72 gene nor a member of the hsp 90 family (hsp 84) were induced. Hsp 73 may play a role in the adaptation and/or in the maintenance of dentate granule cell integrity following seizures.     

Morphine differentially regulates hsp90beta expression in the nucleus accumbens of Lewis and Fischer 344 rats

We have comparatively studied hsp90beta gene and protein expression in the nucleus accumbens of Lewis and Fischer 344 (F344) rats, two inbred strains that exhibit prominent behavioural differences in drug-seeking behaviours. Phenotypical studies confirmed that Lewis rats developed a higher preference for morphine-paired environments after conditioning. RT-PCR assays did not reveal strain-related differences in hsp90beta gene expression in basal conditions; however, acute morphine treatment provoked an increase of hsp90beta mRNA 2h after injection only in the case of Lewis rats. We also found a significant upregulation of the Hsp90beta protein in both strains 8h after morphine injection, this increase being significantly higher in Lewis rats. Taking into account the suggested roles for Hsp90 in the brain, the data suggest that Lewis and F344 strain differences concerning opioid-seeking behaviours could be related to differential sensitivity to opioid-induced neuronal plasticity within the brain reward system, an effect that could be mediated (at least partially) by stress proteins.     

Distribution of 72-kDa heat-shock protein in rat brain after hyperthermia

Summary The distribution of the 72-kDa heat-shock protein (hsp72) in rat brain, 24 h following in vivo transient hyperthermia (41.5°C, 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. hsp 72 was observed in glia and endothelial cells throughout brain. hsp 72 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.Supported in part from NINDS grant PO1 NS23393 and an American Heart Association grant-in-aid (Michigan Affiliated)