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 mRNA species encoding heat shock protein 90 (hsp90) in control and hyperthermic rabbit brain

Northern blot and in situ hybridization were employed to investigate regional and cell type differences in the expression of hsp90 mRNA species in control and hyperthermic rabbit brain. Riboprobes specific to hsp90 α and β mRNA species were utilized in time-course Northern blot studies on cerebral hemispheres and the cerebellum. Following hyperthermia, levels of hsp90 α and β mRNA were elevated in both brain regions; however, the magnitude of induction was more robust in the cerebellum than in cerebral hemispheres. The pattern of expression of hsp90 genes in rabbit brain was analyzed by in situ hybridization. These studies revealed that hsp90 genes are preferentially expressed in neuronal cell populations in the unstressed mammalian brain. The distribution of hsp90 α and β mRNA was similar, though the signal for the latter was stronger. Following hyperthermia, changes were not detected in the pattern of hsp90 β mRNA expression in the hippocampus. In the cerebellum, a rapid induction of hsp90 β mRNA was apparent in the neuron-enriched granule cell layer, followed by a delayed accumulation in Purkinje neurons. Unlike hsp70, induction of hsp90 was not detected in glial cells of hyperthermic rabbit brain. The localization of hsp90 to neurons suggests that this heat shock protein plays an important role in neuronal function. © 1996 Wiley-Liss, Inc.     

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.     

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.     

Effect of electro-acupuncture on the expression of heat shock protein-70 gene in rat spinal cords following spinal cord injury

BACKGROUND: It is generally believed that the mechanism by which heat shock protein-70 (HSP70) protects cells is related to its effectiveness in maintaining the normal stereochemical structure of intracellular proteins, and in participating in the process of cell apoptosis. Whether electro-acupuncture participates in HSP70 expression and produces neuroprotective effects remain unclear. OBJECTIVE: This study aimed at detecting HSP70 expression after electro-acupuncture in rats with transected spinal cord, in order to further validate the mechanism of electro-acupuncture-induced effects in the treatment of spinal cord injury. DESIGN: A controlled observational experiment. SETTING: Shanghai University of Traditional Chinese Medicine and Toho University, School of Medicine. MATERIALS: Seventy adult male Sprague-Dawley rats of SPF grade, weighing 200±20 g, were provided by the Laboratory Animal Center of Shanghai University of Traditional Chinese Medicine, with permission No. SYXK (hu) 2004–2005. The animals were handled in accordance with the requests from Animal Ethics Committees for guidance. A G6805-2 multiple purpose treatment machine was used (Shanghai Medical Instruments High-Tech Co.,Ltd., Shanghai, China). METHODS: This study was carried out in the state level laboratories of Shanghai University of Traditional Chinese Medicine and Toho University, School of Medicine between January 2005 and July 2007. The rats were randomly divided into the electro-acupuncture treated group, which received electro-acupuncture treatment in addition to spinal cord surgery and the control group, which received only spinal cord surgery, with 35 rats in each group. All the rats underwent the same surgery consisting of spinal cord transection at the T10 level. If the spinal cord was completely transected and the two posterior limbs were completely paralyzed, then the surgery was considered successful and the animal was kept for further analysis and testing. After surgery, rats in the experimental group were electro-acupunctured with a G6805-2 multiple purpose treatment machine. Two needle electrodes were inserted under the T7 and T10 spinal processes, The treatment was administered once a day for 20 minutes. Rats in the control group were not given any treatment after surgery. Five rats were sacrificed separately in each group on days 1, 2, 3, 7, 14, 21 and 28 after surgery. HSP70 gene expression at the site of lesion was located and quantitatively analyzed by immunohistochemistry and real-time PCR methods. Simultaneously, the spinal cord injury region and neurons were observed by HE and Klüver-Barrera stainings. MAIN OUTCOME MEASURES: ①HSP70 gene expression in the spinal cord injury region. ② The number of neurons in the spinal cord injury region. RESULTS: Seventy rats were involved in the final analysis. ①At the end of each pre-determined block of time, HSP70 mRNA level in the spinal cord injury region of rats in the electro-acupuncture treated group was significantly higher than that in the control group (P < 0.05). HSP70 gene expression in the two groups reached peak levels on day 2 after surgery. ② On days 7, 14, 21 and 28 after surgery, the number of neurons in the spinal cord injury region in the electro-acupuncture treated group was significantly higher than that in the control group (P < 0.05). CONCLUSION: Electro-acupuncture can effectively enhance HSP70 expression in the spinal cord injury region. HSP70 may participate in this apparent neuroprotective effect.     

甲基强地松龙对大鼠脊髓损伤后热休克蛋白70表达的影响

目的 探讨大剂量甲基强地松龙㈣对大鼠脊髓损伤后脊髓内热休克蛋白70(HSP70)表达的影响.方法 125只SD大鼠按随机数字表法分为五组:单纯手术组30只、MP对照组30只、脊髓损伤组30只、MP治疗组30只、空白对照组5只,在实验开始后6个时间点:2h、6 h、12h、24h、48 h、72h(空白对照组任选一个时间点)取出脊髓标本进行脊髓病理形态学观察.并通过免疫组化染色法检测脊髓组织中HSP70的表达.结果 使用MP治疗后,大体标本及HE染色观察显示脊髓的继发性损伤减轻.免疫组化染色显示:空白对照组和MP对照组的脊髓组织中基本没有HSP70的表达:单纯手术组各时间点见少量HSP70表达;脊髓损伤组损伤后2h脊髓组织内出现HSP70的表达,损伤后24 h脊髓组织中HSP70表达达到高峰,并维持至损伤后72 h,HSP70表达主要出现在脊髓灰质的神经细胞和胶质细胞中;MP治疗组脊髓组织中HSP70的表达在所有的时间点较脊髓损伤组明显增强,差异有统计学意义(P<0.05),表达高峰提前至12h.同时脊髓白质神经纤维网内也可见HSP70的表达.结论 大剂量MP可以显著增强损伤的脊髓组织中HSP70的表达,提前其表达高峰时间,扩大其表达范围.通过诱导损伤后脊髓组织大量表达HSP70可能是MP保护脊髓的作用机理之一.     

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.     

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.     

Differential expression of class 3 and 4 semaphorins and netrin in the lamprey spinal cord during regeneration

To explore the role of axon guidance molecules during regeneration in the lamprey spinal cord, we examined the expression of mRNAs for semaphorin 3 (Sema3), semaphorin 4 (Sema4), and netrin during regeneration by in situ hybridization. Control lampreys contained netrin-expressing neurons along the length of the spinal cord. After spinal transection, netrin expression was downregulated in neurons close (500 mum to 10 mm) to the transection at 2 and 4 weeks. A high level of Sema4 expression was found in the neurons of the gray matter and occasionally in the dorsal and the edge cells. Fourteen days after spinal cord transection Sema4 mRNA expression was absent from dorsal and edge cells but was still present in neurons of the gray matter. At 30 days the expression had declined to some extent in neurons and was absent in dorsal and edge cells. In control animals, Sema3 was expressed in neurons of the gray matter and in dorsal and edge cells. Two weeks after transection, Sema3 expression was upregulated near the lesion, but absent in dorsal cells. By 4 weeks a few neurons expressed Sema3 at 20 mm caudal to the transection but no expression was detected 1 mm from the transection. Isolectin I-B(4) labeling for microglia/macrophages showed that the number of Sema3-expressing microglia/macrophages increased dramatically at the injury site over time. The downregulation of netrin and upregulation of Sema3 near the transection suggests a possible role of netrin and semaphorins in restricting axonal regeneration in the injured spinal cord.     

Expression and dendritic mRNA localization of GABAC receptor rho1 and rho2 subunits in developing rat brain and spinal cord

The cellular distribution of GABAC receptor rho1 and rho2 subunits in the rat central nervous system remains controversial. We investigated how these subunits were distributed in cerebellum, hippocampus and spinal cord at postnatal day 1, 7 or in adult life. We found that in the adult cerebellum rho1 and rho2 mRNAs were expressed in Purkinje cells and basket-like cells only. In the hippocampus both subunits were expressed throughout the CA1 pyramidal layer, dentate gyrus and scattered interneurons with maximum staining intensity at P7. In the adult hippocampus in situ staining was predominantly found on interneurons. GABAC antibody labelling in P7 and adult hippocampus was largely overlapping with the in situ staining. Western blot analysis showed GABAC receptor in retina, ovary and testis. In the spinal cord the rho2 signal was consistently stronger than rho1 with overlapping expression patterns. At P1, the most intensely labelled cells were the motoneurons while on P7 and adult sections, interneurons and motoneurons were likewise labelled. On spinal neurons both rho1 and rho2 mRNAs showed somatodendritic localization, extending out for >100 microm with punctate appearance especially in adult cells. A similar spinal distribution pattern was provided with polyclonal antibody labelling, suggesting close correspondence between mRNA and protein compartmentalization. Electrophysiological experiments indicated that P1 spinal motoneurons did possess functional GABAC receptors even though GABAC receptors played little role in evoked synaptic transmission. Our results suggest a pattern of rho1 and rho2 subunit distribution more widespread than hitherto suspected with strong developmental regulation of subunit occurrence.     

Colocalization of Oestrogen Receptor lmmunoreactivity and Preproenkephalin mRNA Expression to Neurons in the Superficial Laminae of the Spinal and Medullary Dorsal Horn of Rats

A double-labelling procedure combining immunohistochemical staining with in situ hybridization using a radiolabelled cRNA probe was employed to demonstrate oestrogen receptor-like immunoreactivity and preproenkephalin-A mRNA in the medullary and spinal dorsal horn of female rats. Both markers labelled large numbers of neurons in the substantia gelatinosa and its trigeminal homologue. Many of these neurons were double-labelled, displaying both oestrogen receptor-like- immunoreactivity and preproenkephalin-A mRNA; cell counts showed that 40–60% of the of the oestrogen receptor-like-immunoreactive cells in the superficial laminae also were labelled for preproenkephalin-A mRNA, and that 60–70% of the preproenkephalin-A mRNA-labelled neurons in the same laminae displayed oestrogen receptor-like immunoreactivity. Previous studies have shown that oestrogen receptors can bind to the promoter region of the preproenkephalin-A gene, and studies on the hypothalamus have demonstrated that oestrogen regulates enkephalin expression in select neuronal populations. The present results demonstrate that enkephalinergic neurons in the superficial dorsal horn contain oestrogen receptors and suggest that oestrogen may play an important role in the modulation of sensory and nociceptive processing in the lower medulla and spinal cord.     

Intracellular and extracellular expression of the major inducible 70kDa heat shock protein in experimental ischemia-reperfusion injury of the spinal cord

Inflammatory responses exacerbate ischemia-reperfusion (IR) injury of spinal cord, although understanding of mediators is incomplete. The major inducible 70kDa heat shock protein (hsp70) is induced by ischemia and extracellular hsp70 (e-hsp70) can modulate inflammatory responses, but there is no published information regarding e-hsp70 levels in the cerebrospinal fluid (CSF) or serum as part of any neurological disease state save trauma. The present work addresses this deficiency by examining e-hsp70 in serum and CSF of dogs in an experimental model of spinal cord IR injury. IR injury of spinal cord caused hind limb paraplegia within 2–3 h that was correlated to lumbosacral poliomalacia with T cell infiltrates at 3 d post-ischemia. In this context, we showed a 5.2-fold elevation of e-hsp70 in CSF that was induced by ischemia and was sustained for the following 3 d observation interval. Plasma e-hsp70 levels were unaffected by IR injury, indicating e-hsp70 release from within the central nervous system. A putative source of this e-hsp70 was ependymal cells in the ischemic penumbra, based upon elevated i-hsp70 levels detected within these cells. Results warrant further investigation of e-hsp70's potential to modulate spinal cord IR injury.     

Immunocytochemical localization of corticotropin releasing factor (CRF) in the rat spinal cord

The presence of corticotropin releasing factor (CRF)-immunoreactive nerve fibers and cell bodies in the spinal cord is demonstrated. Immunopositive fibers were found in the lateral column of the white matter, in laminae I, V–IV, X, and in the intermediolateral column of the spinal cord. Complete transection of the spinal cord showed that the majority of the fibers in the lateral funiculus formed an ascending pathway; however, a few descending fibers were also detected. Hypophysectomy resulted in enhanced immunoreactivity to the fibers and staining of CRF-immunoreactive cell bodies in laminae V–VII, X, and in the intermediolateral sympathetic column. The results suggest that CRF is not merely an ACTH releasing factor, but also a regulatory peptide which may be involved in several stress-related neural responses.     

Isolation of complementary DNAs for heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 genes and the expressions in post-ischaemic gerbil brain

Abstract

A complementary DNA (cDNA) library was constructed with a plasmid vector from cerebral cortices of gerbils at 8 h of reperfusion after 10 min of bilateral common carotid ligation. After the 3rd screening of this cDNA library with a human genomic DNA probe for HSP70 (pH2.3), 4 cDNA clones were isolated (named pGAy pGBy pGD3 and pGE^ respectively). Southern and Northern blot analysisand partial nucleotide sequence analysis indicated that pGA3 and pGE4 were the HSP70 cDNA clones, and that pGB1 and pGD3 were the HSC70 cDNA clones; which selectively recognized HSP70 or HSC70 mRNA, respectively. HSP70 mRNA is present in a very small amount in normal controls, and is greatly induced after the transient ischaemia. HSC70 mRNA is constitutively expressed in a normal gerbil brain, but is still inducible. In situ hybridization study demonstrated that the HSP70 mRNA was present in a very small amount in the hippocampal pyramidal and dentate granule cells in the sham controland that the mRNA was greatly induced in the cells of hippocampus, dentate gyrusmedial habenula, ventral thalamic nuclei, caudate putamen, ventromedial and arcuate hypothalamic nuclei, amygdaloid nuclei and cerebral cortex after 8 h of reperfusion. HSC70 mRNA was present in almost the same areas of sham control and was slightly induced after 8 h of reperfusion. Our results indicate that HSP70 and HSC70 cDNA clones were first isolated from post-ischaemic gerbil brain, and selectively recognize HSP70 or HSC70 mRNA, respectively. A regional difference in the induction of the HSP70 and HSC70 mRNA in post-ischaemic gerbil brain was observed by in situ hybridization. [Neurol Res 1992; 14: 000-000]     

RhoA, RhoB, RhoC, Rac1, Cdc42, and Tc10 mRNA levels in spinal cord, sensory ganglia, and corticospinal tract neurons and long-lasting specific changes following spinal cord injury

Inhibition of RhoA has been shown to enhance axonal regeneration following spinal cord injury. Here we mapped mRNA expression patterns of RhoA, B, and C, Rac1, Cdc42, and Tc10 in spinal cord, sensory ganglia, and sensorimotor cortex in uninjured rats, and following spinal cord injury or sham laminectomy. In the intact spinal cord, neurons displayed high levels of Rac1, Cdc42, and Tc10 mRNA hybridization signal. GFAP-immunoreactive astrocytes expressed primarily RhoB and Rac1, while oligodendrocyte-like cells expressed RhoA, Rac1, and Cdc42. Injury caused profound, long-lasting upregulation of RhoA, Rac1, Cdc42, and Tc10 mRNA in the spinal cord, while RhoB was modestly increased and RhoC did not change. GFAP-immunoreactive reactive astrocytes exhibited a dramatic increase of RhoA mRNA expression along with increases of Rac1 and Cdc42. Injury also led to elevation of RhoA, Cdc42, and Tc10 in neurons and modest increases of RhoA, Rac1, and Tc10 in oligodendrocyte-like cells. Laminectomy caused similar, but less pronounced alterations of investigated mRNA species. In dorsal root ganglia neuronal RhoA, Rac1, Cdc42, and Tc10 mRNA levels were increased similarly by spinal cord injury and sham surgery. The CST pyramidal cells expressed Tc10 mRNA and the CST itself was Tc10-immunoreactive. Tc10-immunoreactivity disappeared distal to injury. We conclude that there are gene-specific patterns of expression of the six different Rho-GTPases in normal spinal cord and dorsal root ganglia, and that specific changes of temporal and spatial expression patterns occur in response to spinal cord injury, suggesting different roles of these GTPases in the cellular sequelae of CNS injury.