Regional expression of heat shock protein-70 mRNA and c-fos mRNA following focal ischemia in rat brain.

In situ hybridization was used to estimate regional levels of heat shock protein-70 (HSP-70) mRNA and c-fos mRNA in two related models of focal cerebral ischemia. In the first model, permanent occlusion of the distal middle cerebral artery (MCA) alone caused a patchy increase in HSP-70 mRNA by 1 h in the central zone of the MCA territory of the ipsilateral neocortex. Tissue levels of HSP-70 mRNA continued to increase for several hours and remained elevated at 24 h. In contrast to the focal expression of HSP-70, c-fos mRNA was increased throughout the ipsilateral cerebral cortex by 15 min and remained elevated for least 3 h. The wide distribution of c-fos expression suggests it may have been caused by spreading depression. In the second model, severe focal ischemia was produced with a combination of transient (1-h) bilateral carotid artery occlusion and permanent MCA occlusion. Combined occlusion for 1 h without reperfusion caused expression of HSP-70 mRNA only in regions adjacent to the central zone of the MCA territory of the neocortex. However, reperfusion of the carotids for 2 h generated intense expression of HSP-70 mRNA throughout most of the ipsilateral cerebral cortex, white matter, striatum, and hippocampus. The wide-spread increase in HSP-70 mRNA suggests that reperfusion triggered expression in all previously ischemic regions. However, at 24 h of reperfusion, increased levels of HSP-70 mRNA were restricted primarily to the ischemic core of the neocortex. These results suggest that expression of HSP-70 mRNA is prolonged in regions undergoing injury, but is transient in surrounding regions that recover.     

Distributions of heat shock protein-70 mRNAs and heat shock cognate protein-70 mRNAs after transient global ischemia in gerbil brain.

Distributions of heat shock protein (HSP)-70 mRNAs and heat shock cognate protein (HSC)-70 mRNAs after 10 min of transient global ischemia were investigated in gerbil forebrain by in situ hybridization using cloned cDNA probes selective for the mRNAs. Expression of HSP70 immunoreactivity was also examined in the same brains. In hippocampal CA1 neuronal cells, in which only a minimal induction of immunoreactive HSP70 protein was found, the strong hybridization for HSP70 mRNA disappeared at around 2 days before the death of CA1 cells became evident. Furthermore, in hippocampal CA3 cells, a striking induction of HSP70 mRNA was sustained even at 2 days along with a prominent accumulation of HSP70 immunoreactivity. In contrast to the case of HSP70 mRNA, HSC70 mRNA was present in most neuronal cells, especially dense in CA3 cells, of the sham brain. A co-induction of HSP70 and HSC70 mRNAs was observed in several cell populations after the reperfusion with a peak at 8 h, although the magnitude of HSC70 mRNA induction was lower than that of HSP70 mRNA, particularly in CA1 cells. The expression of HSC70 mRNA in CA1 cells also disappeared at around 2 days. All the induced signals of HSP70 and HSC70 mRNAs in other cell populations were diminished and returned to the sham level, respectively, by 7 days. These results are the first to show the time courses of distribution of HSP70 and HSC70 mRNAs and the immunoreactive HSP70 protein in the same gerbil brain after ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glial expression of small heat shock proteins following an excitotoxic lesion in the immature rat brain

Heat shock proteins (HSPs) are chaperones induced under pathological conditions and involved in protein stabilization and cellular protection. In this study, we have evaluated the expression pattern of the glial cell-related HSP27, HSP32, and HSP47 following an excitotoxic lesion in the immature rat brain. Postnatal day 9 rats received an intracortical injection of N-methyl-D-aspartate and tissue was processed immunohistochemically for HSPs and double labeling using astroglial and microglial markers. HSP expression was quantified by image analysis. Excitotoxic damage caused primary cortical degeneration and secondary damage in the corresponding thalamus. In the injured cortex, reactive microglia/macrophages expressed HSP32 from 10 h until 14 days postlesion (PL), showing maximal levels at days 3-5. In parallel, most cortical reactive astrocytes showed expression of HSP47 from 10 h until 14 days PL and a population of them also displayed HSP27 labeling from 1 day PL. In addition, some cortical reactive astrocytes showed a temporary expression of HSP32 at day 1. In general, astroglial HSP expression in the cortex achieved maximal levels at days 3-5 PL. In the damaged thalamus, HSP32 was not significantly induced, but reactive astrocytes expressed HSP47 and some of them also HSP27. Thalamic astroglial HSP induction was transient, peaked at 5 days PL and reached basal levels by day 14. The injury-induced expression of HSP32, HSP27, and HSP47 in glial cells may contribute to glial cell protection and adaptation to damage, therefore playing an important role in the evolution of the glial response and the excitotoxic lesion outcome. HSP32 may provide antioxidant protective mechanisms to microglia/macrophages, whereas HSP47 could contribute to extracellular matrix remodeling and HSP27 may stabilize the astroglial cytoskeleton and participate in astroglial antioxidant mechanisms.     

Regional expression of c-fos mRNA in rat brain during the evolution of amygdala kindled seizures.

The biochemical alterations eliciting the growth and spread of afterdischarge and accompanying the evolution of behavioral seizure stages in electrical kindling are not known. In situ hybridization for c-fos mRNA was used to map potential brain structures recruited during the evolution of major seizures from electrical kindling of the amygdala in rats. Two different patterns of c-fos induction were observed in the earliest stages of kindling (stages 1 and 2). A unilateral cortical distribution included the insular, temporal, perirhinal and parietal cortices and the amygdala. No changes in the hippocampus were noted in this group. The second distribution pattern was limited to the hippocampus (either unilateral or bilateral) and amygdala (unilateral) with no changes in the cortical areas. The afterdischarge durations were significantly (2 fold) longer in the 'hippocampal' group as compared to the 'cortical' group. In the later stages of kindling (stages 4 and 5) the distribution of c-fos mRNA was uniformly bilateral and involved a combination of the hippocampal and cortical distributions observed in the earlier stages and including the amygdala bilaterally as well. The induction of c-fos mRNA appears to provide a map of two different routes in the sequential pathways involved in the evolution of kindled seizures; it may also ultimately prove to be an important component of the kindling process itself. Additionally, c-fos mRNA was elevated bilaterally in the inferior colliculus of animals exhibiting running fits with their seizures. The inferior colliculus was previously shown by others to be involved in running fits accompanying convulsions.     

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.     

Different thresholds of HSP70 and HSC70 heat shock mRNA induction in post-ischemic gerbil brain.

Thresholds of induction of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs after transient global ischemia in gerbil brain were investigated by in situ hybridization using cloned cDNA probes selective for each mRNA species. In sham control brain, HSP70 mRNA was little present, while HSC70 mRNA was present in most cell populations. A 0.5-min occlusion of bilateral common carotid arteries did not affect the amount of HSP70 and HSC70 mRNAs. The selective induction of HSC70 mRNA was observed in dentate granule cells at 1 h, and in most cells of hippocampus especially dentate gyrus at 3 h after 1 min of ischemia when induction of HSP70 mRNA was not evident in the identical brain. The selective induction diminished by 2 days. However, after 2 min of ischemia, HSP70 and HSC70 mRNAs were induced together in hippocampal cells from 1 h of the reperfusion, and the co-induction prolonged in CA1 cells until 2 days. Body temperatures monitored at rectum increased after the reperfusion with a peak at 30 min. The degree of increase of the body temperature was significantly higher in the case after 2-min ischemia than in the cases after 0.5- and 1-min ischemia. Although HSP70 and HSC70 mRNAs are generally co-induced in stressful conditions, our results suggest the different thresholds of the induction between HSP70 and HSC70 mRNAs after transient brain ischemia. The selective induction of HSC70 mRNA which is not accompanied by the induction of HSP70 mRNA may relate to the differences of the duration of ischemia and the degree of the increase of body temperature after ischemia.     

Behavioral changes and expression of heat shock protein hsp-70 mRNA, brain-derived neurotrophic factor mRNA, and cyclooxygenase-2 mRNA in rat brain following seizures induced by systemic administration of kainic acid

Kainic acid-induced seizures in rats represent an established animal model for human temporal lobe epilepsy. However, it is well-known that behavioral responses to the systemic administration of kainic acid are inconsistent between animals. In this study, we examined the relationship between expression of genes, neuropathological damage, and behavioral changes (seizure intensity and body temperature) in rats after systemic administration of kainic acid. The considerable differences in the response to kainic acid-induced seizures were observed in rats after a single administration of kainic acid (12 mg/kg i.p.). There was no detection of the expression of heat shock protein hsp-70 mRNA and HSP-70 protein in brain of vehicle-treated controls and in animals exhibiting weak behavioral changes (stage 1–2). A moderate expression of hsp-70 mRNA was detected throughout all regions (the pyramidal cell layers of CA1–3 and dentate gyrus) of the hippocampus, the basolateral, lateral, central and medial amygdala, the piriform cortex, and the central medial thalamic nucleus of rats that developed moderate seizures (stage 3–4). Marked expression of hsp-70 mRNA was detected in the all regions (cingulate, parietal, somatosensory, insular, entorhinal, piriform cortices) of cerebral cortex and all regions of hippocampus, and the central medial thalamic nucleus of the rats that developed severe seizures (stage 4–5). In addition, marked HSP-70 immunoreactivity was detected in the pyramidal cell layers of CA1 and CA3 regions of hippocampus, all regions (cingulate, parietal, somatosensory, insular, piriform cortices) of cerebral cortex, and the striatum of rats that developed severe seizures (stage 4–5). Furthermore, a marked expression of cyclooxygenase-2 (COX-2) mRNA and brain-derived neurotrophic factor (BDNF) mRNA levels by kainic acid-induced behavioral seizures (stage 3–4 or stage 4–5) was detected in all hippocampal pyramidal cell layers, granule layers of dentate gyrus, piriform cortex, neocortex, and amygdala. The present study suggest that the behavioral changes (seizure intensity and body temperature) and neuropathological damage after systemic administration of kainic acid are inconsistent between animals, and that these behavioral changes (severity of kainic acid-induced limbic seizures) might be correlated with gene expression of hsp-70 mRNA, COX-2 mRNA, and BDNF mRNA in rat brain.     

Methylphenidate regulates c-fos and fosB expression in multiple regions of the immature rat brain

Methylphenidate (Ritalin, MPH) is a common psychostimulant used to treat childhood attention-deficit hyperactivity disorder (ADHD). Little is known about the long-term developmental effects on gene expression and behavior, which may occur with extended MPH use. We reported previously that the striatum is a major target of MPH, consistent with human MRI studies. In the present study, we tested the hypothesis that MPH is likely to have widespread effects in extra-striatal regions of the brain. We used the expression of two immediate early genes, c-fos and fosB, as probes to map the response of the immature rat brain to single (1 day) versus repeated (14 days) MPH treatment (2 or 10 mg/kg; s.c.) from postnatal day 25 to 38. Consistent with previous reports, the striatum is a major target of acute MPH action, as indicated by elevated levels of cFOS-immunoreactivity (-ir). Increases in c-fos expression were also seen in the nucleus accumbens, cingulate/frontal cortex and piriform cortex, and Islands of Calleja. FosB expression was elevated only in the striatum following a single stimulation. Chronic MPH treatment (10 mg/kg/day for 14 days) resulted in an attenuation of c-fos expression in the striatum and Islands of Calleja. However, levels of cFOS-ir remained elevated in the nucleus accumbens and frontal cortex. In contrast to the inhibitory effect of repeated MPH exposure on c-fos expression, FOSB-ir was further elevated in the striatum, and an increase was observed in the cingulate/frontal and piriform cortices. Thus, chronic MPH differentially regulated expression of c-fos and fosB in several brain regions. Our data suggest that MPH may exert its stimulant effects at multiple sites in the immature brain, which has implications for long-term treatment in children.     

Delayed cerebral atrophy following moderate hypoxia-ischemia in the immature rat

Hypoxia-ischemia (H/I) damages cells in the immature brain and interferes with subsequent brain development; the extent of the damage has been related to the severity, or duration, of the initial insult. This study examined the effects of both severe and moderate duration of H/I on the evolution of damage through 8 weeks of recovery. Seven-day-old rat pups were subjected to either 75 min or 2 h of 8% oxygen following a unilateral carotid artery ligation. Evaluation of brain damage included morphometric analysis of hemispheric diameter at 2, 4, and 8 weeks of recovery, and hematoxylin and eosin for evaluation of pathology at 8 weeks. Two hours of H/I produced severe infarction in the ipsilateral hemisphere in the majority of the survivors, apparent by 2 weeks of recovery with no change at 4 or 8 weeks. In marked contrast, 75 min of H/I produced no significant damage during the initial 2 weeks of recovery but resulted in progressive cerebral atrophy with delayed infarction such that the extent of damage at 8 weeks was not different from the 2-hour group. Thus, even a mild-moderate ischemic insult to the perinatal brain establishes a vulnerable region which ultimately dies without intervention.     

Regulation of glucocorticoid receptor mRNA and heat shock protein 70 mRNA in the developing sheep brain

Fetal hypothalamo-pituitary-adrenal (HPA) activity increases dramatically at term in sheep, however, little is known about the regulation of glucocorticoid feedback in the developing brain. Heat shock protein 70 (hsp70) is closely associated with glucocorticoid actions within the cell. We hypothesized that there is a decrease in glucocorticoid negative feedback in the brain, near term, resulting from changes in the expression of glucocorticoid receptors (GR) and hsp70. Brains were removed at various stages of development. GR mRNA levels in the paraventricular nucleus (PVN) and cortex, and hsp70 mRNA in the PVN were determined by in situ hybridization. In the hippocampus, GR mRNA levels were measured by Northern analysis. In the PVN, GR mRNA was present by d60. GR mRNA levels reached a peak at d100-110, but then decreased significantly with progression of gestation, and were lowest at term. Hippocampal GR mRNA levels were highest on day 130 of gestation, decreasing to low levels at term. In the cerebral cortex, GR mRNA levels were expressed at high levels in all layers of the cortex by day 110 of gestation with levels decreasing to term. Hsp70 mRNA was present in both parvocellular and magnocellular regions of the PVN, and there was no significant change in late gestation. In conclusion, (1) The high levels of GR mRNA present in the PVN, hippocampus and cerebral cortex during fetal life are likely important in development of these structures at a time when circulating glucocorticoids are low. (2) Changes in GR mRNA levels in the PVN are not associated with alterations in the expression of hsp70. (3) The decrease in GR mRNA in the hippocampus and PVN in late gestation, at a time when fetal plasma cortisol is increasing, likely facilitates maintained hypothalamic drive to the pituitary corticotroph.     

Flunarizine limits hypoxia-ischemia induced morphologic injury in immature rat brain

We examined the impact of pre-treatment with the calcium antagonist flunarizine on the development of hypoxic-ischemic brain injury in the immature rat. Unilateral carotid artery ligation and subsequent exposure to 2 hours of 8% oxygen in 7-day-old rats was used as a model for perinatal hypoxic-ischemic encephalopathy. This procedure leads to atrophy in the cerebral hemisphere ipsilateral to carotid occlusion, with prominent foci of neuronal infarction in the caudate-putamen (striatum). The morphologic injury develops after 1 1/2 hours of hypoxia; and there is an equivalent time threshold for duration of hypoxic exposure needed to acutely stimulate dopamine release in the ipsilateral striatum. Parenteral administration of 30 mg/kg of flunarizine before hypoxic exposure limited both the release of dopamine acutely and the extent of morphologic damage observed two weeks after the insult. Oral administration of 30 mg/kg of flunarizine in a different vehicle prevented morphologic damage but had no effect on stimulated dopamine release. The drug vehicle for the parenteral preparation also prevented tissue injury, but to a lesser degree than flunarizine. However the parenteral vehicle was equipotent with parenteral flunarizine in limiting acute stimulation of dopamine release. The results demonstrate that flunarizine has potent neuroprotective properties against morphologic brain injury from hypoxia-ischemia, acting by a mechanism which is independent of effects on dopamine release.     

Reduced basal and phencyclidine-induced expression of heat shock protein-70 in rat prefrontal cortex by the atypical antipsychotic abaperidone

The effect of antipsychotic treatment on basal and phencyclidine (PCP)-induced heat shock protein-70 (hsp70) mRNA expression was studied in the rat striatum and in the prefrontal cortex. Abaperidone, a novel drug with an atypical antipsychotic profile, was compared, at pharmacologically equivalent doses, with the atypical antipsychotics clozapine and risperidone and also with haloperidol, a classical antipsychotic. Abaperidone and clozapine reduced basal hsp70 mRNA expression in the rat striatum and in the prefrontal cortex. No change in either region was found after haloperidol, whereas risperidone reduced hsp70 mRNA in the striatum but not in the prefrontal cortex. The N-methyl-D-aspartate (NMDA) receptor antagonist PCP significantly elevated hsp70 mRNA levels in the prefrontal cortex, an elevation that was potentiated by haloperidol and prevented by all of the atypical antipsychotics tested. Since hsp70 has been associated to some schizophrenia symptoms, we suggest that reduced hsp70 in the prefrontal cortex, a cortical area that plays a critical role in the etiology of many schizophrenia symptoms, may be linked to an atypical profile of antipsychotics, such as clozapine, and possibly also abaperidone.     

Bcl-2, Bax and Bcl-x expression following hypoxia-ischemia in the infant rat brain

Severe hypoxic-ischemic cerebral damage was produced in 8-day-old rats following permanent bilateral carotid artery occlusion and 15 min of ischemia. Cellular damage consisted of early necrosis and appearance of cells with apoptotic-like morphology (karyorrhectic cells) and cells with granular chromatin degeneration in the cerebral cortex, hippocampus, thalamus, striatum and amygdala. Expression of Bcl-2, Bax and Bcl-x was examined in control and hypoxic-ischemic rats using immunohistochemistry and Western blotting. Members of the Bcl-2 family were expressed in the vast majority of, if not all, neurons in control pups. Bcl-2, Bax and Bcl-x immunoreactivity decreased in necrotic cells, but about 60% of cells with apoptotic-like morphology and cells with granular chromatin degeneration were stained with antibodies to Bcl-2, Bax or Bcl-x. Although the total number of stained cells decreased with time, recruitment of cells with apoptotic morphology and cells with granular chromatin degeneration was still found up to 48 h. Western blots showed a band at about p28 and p21, respectively for Bcl-2 and Bax in control and hypoxic-ischemic pups at 6, 12 and 24 h. Two bands at about p37 and p30, representing Bcl-xL and Bcl-xS, respectively, were found in samples stained with antibodies to Bcl-x. No gross changes in the intensity of these bands were observed in ischemic pups at 6, 12 and 24 h, except for a slight decrease in Bcl-2 at 24 h, and a slight and inconstant increase of the putative Bcl-xS at 12 h. These results suggest that Bcl-2, Bax, Bcl-xL and Bcl-xS do not play a leading role in the fate of damaged nerve cells following a severe hypoxic-ischemic insult of the developing brain. Received: 24 April 1997 / Accepted: 18 June 1997     

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.     

NF-kappaB and IkappaBalpha expression following traumatic brain injury to the immature rat brain

NF-kappaB is one of the most important modulators of stress and inflammatory gene expression in the nervous system. In the adult brain, NF-kappaB upregulation has been demonstrated in neurons and glial cells in response to experimental injury and neuropathological disorders, where it has been related to both neurodegenerative and neuroprotective activities. Accordingly, the aim of this study was to evaluate the cellular and temporal patterns of NF-kappaB activation and the expression of its endogenous inhibitor IkappaBalpha following traumatic brain injury (TBI) during the early postnatal weeks, when the brain presents elevated levels of plasticity and neuroprotection. Our results showed that cortical trauma to the 9-day-old rat brain induced a very fast upregulation of NF-kappaB, which was maximal within the first 24 hours after injury. NF-kappaB was mainly observed in neuronal cells of the degenerating cortex as well as in astrocytes located in the corpus callosum adjacent to the injury, where a pulse-like pattern of microglial NF-kappaB activation was also found. In addition, astrocytes of the corpus callosum, and microglial cells to a lower extent, also showed de novo expression of IkappaBalpha within the time of NF-kappaB activation. This study suggests an important role of NF-kappaB activation in the early mechanisms of neuronal death or survival, as well as in the development of the glial and inflammatory responses following traumatic injury to the immature rat brain.