Expression of brain-derived neurotrophic factor immunoreactivity and mRNA in the hippocampal CA1 and cortical areas after chronic ischemia in rats

We studied the expression of brain-derived neurotrophic factor (BDNF) immunoreactivity and mRNA in the ischemia-vulnerable cerebral hippocampal CA1 and cortical areas after permanent occlusion of bilateral internal carotid arteries. Four groups of rats were studied, including 1) young normotensive Wistar-Kyoto (WKY) rats, 2) aged normotensive WKY rats, 3) young spontaneous hypertensive rats (SHR), and 4) aged SHR. Each group contained rats from sham operation and 1 week, 4 weeks, and 8 weeks after cerebral ischemia (n = 3-5 at each time point). Hematoxylin and eosin staining and in situ apoptosis detection showed no neuronal damage from 1 week to 8 weeks in all the ischemic rats. Immunohistochemistry and Western blot showed that BDNF immunoreactivity increased only at 1 week in the CA1 area of young WKY rats (P < .001) and SHR (P = .002) and decreased only at 8 weeks in the cortical area of aged WKY rats (P = .02). In situ hybridization and TaqMan real-time RT-PCR showed that BDNF mRNA decreased consistently from 1 week to 8 weeks in both CA1 and cortical areas in young SHR (P < .05 and P < .01, respectively) and in aged WKY rats (P < .01 and P < .05, respectively) but was not changed in young WKY rats or aged SHR (P > .05) compared with the sham-operated rats. Our study demonstrates an expression disparity of BDNF immunoreactivity and mRNA in the hippocampal CA1 and cortical areas, especially in the young SHR and aged WKY rats after mild cerebral ischemia. Our study suggests that, under permanent occlusion of bilateral internal carotid arteries, aging and the level of blood pressure may have influence on the expression of BDNF.     

The expression of transforming growth factor-beta1 (TGF-beta1) in hippocampal neurons: a temporary upregulated protein level after transient forebrain ischemia in the rat

Exogenous TGF-beta1 has been shown to protect neurons from damage induced in vitro and in vivo. In this study we attempted to examine the expression of endogenous TGF-beta1 mRNA and protein in the hippocampus of non-ischemic and ischemic rats, and to localize TGF-beta1 protein and DNA fragmentation by double-staining. Transient ischemia was induced for 10 min in Wistar rats by clamping both common carotid arteries and lowering blood pressure to 40 mmHg. Bioactive TGF-beta1 was selectively determined in CA1 pyramidal neurons of non-ischemic rats. It was upregulated after 3 h and 6 h of reperfusion corresponding to the increase in TGF-beta1 mRNA level detected by RT-PCR. Lectin and GFAP staining showed no detectable activated microglial cells and astrocytes in the hippocampus 3 h and 6 h after ischemia. When neuronal damage proceeded through day 2 to day 4 after ischemia as demonstrated by TUNEL-staining, TGF-beta1 immunoreactivity (ir) disappeared in damaged neurons but persisted in viable neurons although TGF-beta1 mRNA levels continuously increased. Double-staining revealed that TUNEL-positive neurons did not express TGF-beta1, while TUNEL-negative neurons in the CA1 subfield exhibited a distinct TGF-beta1 ir. These data indicate that hippocampal CA1 neurons can express TGF-beta1 under physiological conditions and upregulate its expression during the first hours after ischemia, that is independent of the activation of glial cells. The endogenous TGF-beta1 expressed in neurons may play a role in the pathological process of DNA degradation and delayed neuronal death after transient forebrain ischemia.     

Reduction in brain-derived neurotrophic factor protein level in the hippocampal CA1 dendritic field precedes the delayed neuronal damage in the rat brain

Utilizing a specific polyclonal antibody against a peptide unique for brain-derived neurotrophic factor (BDNF), we investigated the regional and temporal profiles of immunoreactivity of the BDNF protein in the rat hippocampus after transient forebrain ischemia. The pattern of immunoreactivity for the BDNF receptor (TrkB) was also examined and compared with that for BDNF. In the early phase after ischemia, we observed a distinct regional difference in immunoreactivity between the pyramidal cell layer and the stratum radiatum of the CA1 subfield. In the pyramidal cell layer, there was a rapid and transient increase in the positive immunostaining for both BDNF and TrkB. By contrast, in the stratum radiatum there was a marked decrease in BDNF immunoreactivity, but not one in that of TrkB. One week after ischemia, high immunoreactivity for both BDNF and TrkB was observed in the reactive astrocytes in the dendritic field of the CA1 subfield. These findings suggest that a transport of BDNF from the neuronal soma to the dendrites of the stratum radiatum might be ceased after the ischemic insult. Thus, a dysfunctional autocrine mechanism of BDNF within the CA1 neuron may be involved in the pathogenesis of selective neuronal damage after ischemia. J. Neurosci. Res. 53:318–329, 1998. © 1998 Wiley-Liss, Inc.     

The Distribution of Brain-derived Neurotrophic Factor and its Receptor trkB in Parvlbumin-containing Neurons of the Rat Visual Cortex

We analysed the distribution of brain-derived neurotrophic factor (BDNF) and its receptor trkB in the adult rat visual cortex, paying particular attention to a GABAergic neuronal subpopulation—the parvalburnin-positive cells. We found expression of trkB in the cell body and apical dendrite of pyramidal neurons and in the cell body of non-pyramidal neurons. Double labelling experiments revealed extensive colocalization of parvalbumin and trkB immunoreactivity in non-pyramidal neurons. Interestingly, the trkB-positive pyramidal neurons appeared surrounded by parvalbumin-labelled boutons. The use of double immunohistochemistry and in situ hybridization histochemistry showed that parvalbumin-positive neurons express trkB mRNA. BDNF rnRNA was found in several cells. Coexpression of BDNF mRNA and parvalbumin immunoreactivity was extremely rare. These data strongly suggest that BDNF synthesized by cortical neurons acts as a postsynaptically derived factor for parvalbumin-positive neurons in the adult rat visual cortex.     

Changes in spinal GDNF, BDNF, and NT-3 expression after transient spinal cord ischemia in the rat

Previous studies have demonstrated that the expression of several growth factors including glial cell-derived neurotrophic factor (GDNF), brain-derived growth factor (BDNF), and neurotrophin-3 (NT-3) play an important role in defining neuronal survival after brain ischemia. In the present study, using a well-defined model of transient spinal ischemia in rat, we characterized the changes in spinal GDNF, BDNF, and NT-3 expression as defined by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence coupled with deconvolution microscopy. In control animals, baseline levels of GDNF, BDNF, and NT-3 (74 +/- 22, 3,600 +/- 270, 593 +/- 176 pg/g tissue, respectively) were measured. In the ischemic group, 6 min of spinal ischemia resulted in a biphasic response with increases in tissue GDNF and BDNF concentrations at the 2-hr and 72-hr points after ischemia. No significant differences in NT-3 concentration were detected. Deconvolution analysis revealed that the initial increase in tissue GDNF concentration corresponded to a neuronal upregulation whereas the late peak seen at 72 hr corresponded with increased astrocyte-derived GDNF synthesis. Increased expression of BDNF was seen in neurons, astrocytes, and oligodendrocytes. These data suggest that the early increase in neuronal GDNF/BDNF expression likely modulates neuronal resistance/recovery during the initial period of postischemic reflow. Increased astrocyte-derived BDNF/GDNF expression corresponds with transient activation of astrocytes and may play an active role in neuronal plasticity after non-injurious intervals of spinal ischemia.     

Differential cellular expression of tumor necrosis factor-alpha and Type I tumor necrosis factor receptor after transient global forebrain ischemia.

We examined the expression of tumor necrosis factor-alpha (TNF-alpha) and the Type I tumor necrosis factor receptor, (TNFR1), in relation to c-fos, a known regulator gene of immediate cellular responses, after an extended period of global ischemia. The number of TNF-alpha mRNA expressing cells peaked in most brain areas after 8 h of reperfusion. Significant increases in TNFR1 mRNA expression were evident in the cortex at 2 and 8 h of reperfusion and after 8 h of reperfusion in the CA3/CA4 region of the hippocampus. Transient neuronal c-fos mRNA expression preceded these responses. TNF-alpha immunoreactivity was seen in neurons>oligodendrocytes=perivascular cells=ependymal cells=vessel wall structures. After ischemia/reperfusion, increased TNF-alpha immunoreactivity was evident only in oligodendrocytes. TNFR1 immunoreactivity in sham brains manifested in bundles of cellular fibers of variable length and thickness. In post-ischemic brains, immunoreactivity in these cellular processes representing mainly astroglial extensions was suppressed at 2 h but recovered partially by 8 and 24 h of reperfusion. In contradiction, transient ischemia-induced TNFR1 immunoreactivity was observed in somas of large cortical neurons, in activated microglia/macrophages, perivascular and endothelial cells.Taken together, the increase in neuronal TNF-alpha mRNA appeared not to be followed by substantial translation to protein in the cerebral tissue after an extended period of global ischemia. However, there was increased neuronal TNFR1 immunostaining in conjunction with increased immunostaining for TNF-alpha in oligoglial elements, which suggests signaling to neurons by enhanced oligoglial TNF-alpha.     

Expression and changes of galanin in neurons and microglia in the hippocampus after transient forebrain ischemia in gerbils

In the present study, we investigated chronological changes of galanin (GAL), well known as the potassium channel opener, immunoreactivity and GAL protein level in the hippocampus of the gerbil at the various times after 5 min transient forebrain ischemia. In the sham-operated group, weak GAL immunoreactivity was found in non-pyramidal cells. At 12 h after ischemia-reperfusion, the number of GAL-immunoreactive neurons and GAL immunoreactivity were significantly increased in the hippocampus compared to 3 h after ischemic insult, especially in the hippocampal CA1 region. Thereafter the number of GAL-immunoreactive neurons and GAL immunoreactivity decrease time-dependently in the hippocampus. Four days after transient ischemia, GAL immunoreactivity was low as compared with the sham-operated group. At this time point after ischemic insult, GAL immunoreactivity was shown in microglia in the CA1 region because delayed neuronal death happened in the CA1 pyramidal cells. The result of Western blot showed the pattern of GAL expression similar to that of immunohistochemical data. These results suggest that the early increase of GAL in the CA1 pyramidal cells may be associated with the reduction of the excitotoxic damage, that long-lasting enhanced expression of endogenous GAL at 12 h-2 days after ischemia may be associated with efflux of potassium ion into the extracellular space, and that GAL expression in microglia 4 days after ischemia may be associated with reduction of ischemic damage.     

Differential expression of HSC73 and HSP72 mRNA and proteins between young and adult gerbils after transient cerebral ischemia: relation to neuronal vulnerability.

This study presents a quantitative comparison of the time courses and regional distribution of both constitutive HSC73 and inducible HSP72 mRNA expression and their respective encoded proteins between young (3-week-old) and adult (3-month-old) gerbil hippocampus after transient global ischemia. The constitutive expression of HSC73 mRNA and protein in the hippocampus of the young sham-operated gerbils was significantly higher than in the adults. The HSC73 mRNA expression after ischemia in the CA1 layer of young gerbils was greater than in adult gerbils. HSC73 immunoreactivity was not significantly changed after ischemia-reperfusion in adult hippocampus, whereas it decreased in young gerbils. Ischemia-reperfusion led to induction of HSP72 mRNA expression throughout the hippocampus of both young and adult gerbils. HSP72 mRNA induction was more intense and sustained in the CA1 subfield of young gerbils; this was associated with a marked induction of HSP72 proteins and neuronal survival. The transient expression of HSP72 mRNA in the CA1 layer of adult gerbils was not associated with a subsequent synthesis of HSP72 protein but was linked to neuronal loss. Expression of HSP72 mRNA was shifted to an earlier period of reflow in CA3 and dentate gyrus (DG) subfields of young animals. These findings suggest that the induction of both HSP72 mRNA and proteins in the CA1 pyramidal neurons of young gerbils, as well as the higher constitutive expression of HSC73, may partially contribute to higher neuronal resistance of young animals to transient cerebral ischemia.     

Delayed hippocampal neuronal death in young gerbil following transient global cerebral ischemia is related to higher and longer-term expression of p63 in the ischemic hippocampus

The tumor suppressor p63 is one of p53 family members and plays a vital role as a regulator of neuronal apoptosis in the development of the nervous system. However, the role of p63 in mature neuronal death has not been addressed yet. In this study, we first compared ischemia-induced effects on p63 expression in the hippocampal regions (CA1–3) between the young and adult gerbils subjected to 5 minutes of transient global cerebral ischemia. Neuronal death in the hippocampal CA1 region of young gerbils was significantly slow compared with that in the adult gerbils after transient global cerebral ischemia. p63 immunoreactivity in the hippocampal CA1 pyramidal neurons in the sham-operated young group was significantly low compared with that in the sham-operated adult group. p63 immunoreactivity was apparently changed in ischemic hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. In the ischemia-operated adult groups, p63 immunoreactivity in the hippocampal CA1 pyramidal neurons was significantly decreased at 4 days post-ischemia; however, p63 immunoreactivity in the ischemia-operated young group was significantly higher than that in the ischemia-operated adult group. At 7 days post-ischemia, p63 immunoreactivity was decreased in the hippocampal CA1 pyramidal neurons in both ischemia-operated young and adult groups. Change patterns of p63 level in the hippocampal CA1 region of adult and young gerbils after ischemic damage were similar to those observed in the immunohistochemical results. These findings indicate that higher and longer-term expression of p63 in the hippocampal CA1 region of the young gerbils after ischemia/reperfusion may be related to more delayed neuronal death compared to that in the adults.     

Effects of fluoxetine on ischemic cells and expressions in BDNF and some antioxidants in the gerbil hippocampal CA1 region induced by transient ischemia

Fluoxetine, a selective serotonin reuptake inhibitor, alters several physiological processes, for example, elevating intracellular cAMP level, in the hippocampus. We examined the effect of fluoxetine on ischemia-induced neuronal death, the expression of brain-derived neurotrophic factor (BDNF) and changes in some antioxidative enzymes in the hippocampal CA1 region induced by transient ischemia. In addition, we also studied the effect of fluoxetine on locomotor activity in gerbils after ischemia/reperfusion. Animals were administered with various doses of fluoxetine (10, 20, and 40 mg/kg, i.p.) once daily for 3 days before the ischemic surgery. The treatment of 10 mg/kg and 20 mg/kg fluoxetine did not show significant neuroprotective effects on CA1 pyramidal cells 4 days after ischemia/reperfusion, while the treatment with 40 mg/kg fluoxetine in ischemic animals showed about 77% neuronal survival rate compared to the control group. The treatment of 40 mg/kg fluoxetine in ischemic animals enhanced significantly BDNF, catalase (CAT), glutathione peroxidase (GPX), and superoxide dismutase-1 (SOD1) immunoreactivity in the CA1 region compared to those in the saline-treated group 4 days after ischemia/reperfusion. In addition, the treatment of fluoxetine (10, 20, 40 mg/kg) significantly inhibited post-ischemic hyperactivity. In brief, treatment with fluoxetine protects neuronal damage after transient ischemia, and the neuroprotective effect of fluoxetine in an ischemic animal model may be related with the up-regulation of BDNF, CAT, GPX, and SOD1 expression.     

Transient ischemia-induced changes of excitatory amino acid carrier 1 in the ventral horn of the lumbar spinal cord in rabbits

OBJECTIVES: To examine temporal changes of EAAC1 immunoreactivity and its protein level in the spinal ventral horn after transient ischemia in the rabbit to investigate the correlation between neuronal cell death and EAAC1 in the ventral horn of spinal cord. METHODS: White rabbits weighing 2.5-3.0 kg were anesthetized with a mixture of 2.5% isoflurane in 30% oxygen and 70% nitrous oxide, and the abdominal aortic artery below the left renal artery was occluded for 15 minutes. At designated times after reperfusion, the immunohistochemical and Western blot analysis for EAAC1 was conducted using tissues of the seventh lumbar spinal segment. RESULTS: EAAC1 immunoreactivity was detected in the neurons of the normal spinal cord. EAAC1 immunoreactivity and protein level reduced significantly 30 minutes after ischemia/reperfusion, but EAAC1 immunoreactivity and protein level again increased by 80% versus sham 3 hours after ischemia. At this time point, neurological defect in hindlimb was also detected. Thereafter, EAAC1 immunoreactivity and protein levels remained to be attenuated in the ventral horn of spinal cord until 48 hours after ischemia. CONCLUSION: The significant change in EAAC1 expression and motor defects at early time after transient spinal cord ischemia relates to the acute events following ischemia/reperfusion. These results indicate that EAAC1 has an important role in the modulation of glutamate homeostasis in ischemic neurons in the spinal ventral horn.     

Infarct tolerance induced by intra-cerebral infusion of recombinant brain-derived neurotrophic factor

Neuronal expression of brain-derived neurotrophic factor (BDNF) has been implicated in the mechanism of infarct tolerance (resistance to stroke) (H. Yanamoto et al., Infarct tolerance accompanied enhanced BDNF-like immunoreactivity in neuronal nuclei, submitted to Brain Res.), a process that takes more than 7 days following a preconditioning of repetitive cortical spreading depression (CSD). To investigate whether an elevated level of BDNF protein in the brain solely protects neurons against temporary focal ischemia, recombinant (r)BDNF was infused into the rat neocortex. Recombinant BDNF (or vehicle: saline) was administered into the left neocortex via an implanted osmotic minipump for 2.5, 7, 10 or 14 days pre-ischemia, during ischemia and for 2 days post-ischemia (8 microgram in total) in male Sprague-Dawley rats (n=6 each). Temporary focal ischemia was induced in the left middle cerebral artery (MCA) territory by three-vessel occlusion of bilateral common carotid arteries (CCAs) and MCA for 2 h, and the cerebral infarct volume was analyzed 2 days after ischemia using TTC staining. Regional cerebral blood flow (rCBF) of the left neocortex was monitored after 14 days of intracerebral administration of BDNF or vehicle (n=10 each). The distribution of BDNF following different periods of rBDNF or vehicle-infusion was analyzed using immunohistochemical techniques (n=5 each). In the groups treated with 8 microgram of rhBDNF for 7, 10, or 14 days pre-ischemia, there were significant reductions of neocortical infarct volume compared to in the control or vehicle-treated groups (p<0.05). In the rCBF study, there was no significant change after the infusion of 8 microgram rhBDNF for 14 days. In the histological study, a wide distribution of BDNF-like immunoreactivity in the neuronal nuclei in the ipsilateral neocortex was demonstrated after the infusion of 8 microgram rhBDNF for 14 days. The BDNF-like immunoreactivity in the neuronal nuclei was enhanced at the time that the resistance to stroke was achieved by direct intra-cerebral infusion of exogenous rBDNF. Elucidating the function of the BDNF-like protein located in the neuronal nuclei should reveal a new strategy for neuroprotection against ischemic brain attack in humans.     

Expression of glial cell line-derived neurotrophic factor induced by transient forebrain ischemia in rats.

This study examined the expression of glial cell line-derived neurotrophic factor (GDNF) mRNA and the cellular localization of GDNF production in rats subjected to transient forebrain ischemia induced by four-vessel occlusion. Transient forebrain ischemia induced GDNF mRNA expression in the hippocampus from 3 h to 3 days after the ischemic episode, with peak expression at 6 h. The GDNF mRNA increase in the cerebral cortex was similar to that in the hippocampus, whereas no increase in GDNF mRNA was observed in the striatum and brainstem. Western blot analysis showed that GDNF in the hippocampal CA1 region was increased slightly from 3 to 24 h after the ischemia, and then subsequently declined to below the baseline level. In the hippocampus, GDNF was evenly produced in pyramidal neurons of both sham-operated rats and normal rats, as determined by immunohistochemistry. Interestingly, we found that ischemia-induced reactive astrocytes, as well as surviving neurons, produced GDNF in 3-7 days after the ischemia. On the other hand, in other regions, such as the cerebral cortex, striatum, and brainstem, there was no change in GDNF-positive cells secondary to ischemia. These findings suggest that expression of GDNF mRNA is regulated in part via ischemia-induced neuronal degeneration. They also suggest that ischemia-induced reactive astrocytes may produce GDNF to protect against neuronal death. Therefore, GDNF may play an important role in ischemia-induced neuronal death in the brain.     

Rubrospinal neurons fail to respond to brain-derived neurotrophic factor applied to the spinal cord injury site 2 months after cervical axotomy

Numerous experimental therapies to promote axonal regeneration have shown promise in animal models of acute spinal cord injury, but their effectiveness is often found to diminish with a delay in administration. We evaluated whether brain-derived neurotrophic factor (BDNF) application to the spinal cord injury site 2 months after cervical axotomy could promote a regenerative response in chronically axotomized rubrospinal neurons. BDNF was applied to the spinal cord in three different concentrations 2 months after cervical axotomy of the rubrospinal tract. The red nucleus was examined for reversal of neuronal atrophy, GAP43 and Talpha1 tubulin mRNA expression, and trkB receptor immunoreactivity. A peripheral nerve transplant paradigm was used to measure axonal regeneration into peripheral nerve transplants. Rubrospinal axons were anterogradely traced and trkB receptor immunohistochemistry performed on the injured spinal cord. We found that BDNF treatment did not reverse rubrospinal neuronal atrophy, nor promote GAP-43 and Talpha1 tubulin mRNA expression, nor promote axonal regeneration into peripheral nerve transplants. TrkB receptor immunohistochemistry demonstrated immunoreactivity on the neuronal cell bodies, but not on anterogradely labeled rubrospinal axons at the injury site. These findings suggest that the poor response of rubrospinal neurons to BDNF applied to the spinal cord injury site 2 months after cervical axotomy is not related to the dose of BDNF administered, but rather to the loss of trkB receptors on the injured axons over time. Such obstacles to axonal regeneration will be important to identify in the development of therapeutic strategies for chronically injured individuals.     

Expression of the glial cell line-derived neurotrophic factor gene in rat brain after transient MCA occlusion

Change of the glial cell line-derived neurotrophic factor (GDNF) gene expression in rat brain was examined after transient middle cerebral artery (MCA) occlusion of adult rats. Northern blot analysis showed that the mRNA began to be induced in the occluded MCA from 1 h of reperfusion with a peak at 3 h, and almost diminished by 1 day of reperfusion. Immunohistochemical analysis with brain sections showed an expression of GDNF-like immunoreactivity in neurons of the cerebral cortex and caudate after 90 min of ischemia in a similar way to the mRNA, but the staining was more disseminated and stronger in the cerebral cortex than the caudate. No glial cell was stained in the brain sections. The present results indicate that the GDNF gene was expressed in an early stage of reperfusion in neuronal cells of the MCA territory, but that the staining property was different between in the cerebral cortex and caudate.     

Changes in immunoreactivity of HSP60 and its neuroprotective effects in the gerbil hippocampal CA1 region induced by transient ischemia

Heat shock proteins act as molecular chaperones and are involved in protein folding, refolding, transport, and translocation. In the present study, we observed changes in heat shock protein 60 (HSP60) immunoreactivity and protein level in the gerbil hippocampal CA1 region after 5 min of transient forebrain ischemia and its neuroprotective effect against ischemic damage. HSP60 immunoreactivity in the CA1 region began to increase in the stratum pyramidale at 30 min after ischemia/reperfusion, and peaked 24 h after ischemia/reperfusion. Thereafter, HSP60 immunoreactivity was decreased in the CA1 region with time. Seven days after ischemia/reperfusion, HSP60 immunoreactivity was increased again in the CA1 region: at this time point after ischemia/reperfusion, HSP60 immunoreactivity was expressed in glial cells in the ischemic CA1 region. HSP60 immunoreactive glial cells were astrocytes containing glial fibrillar acidic protein. In contrast, change in HSP60 immunoreactivity in the ischemic CA2/3 region was not significant compared with that in the ischemic CA1 region. In Western blot study, HSP60 protein level in the CA1 region was increased after ischemia/reperfusion and highest 24 h after ischemia/reperfusion. Animals treated with recombinant adenoviruses expressing Hsp60 (Ad-Hsp60) showed the neuroprotection of CA1 pyramidal neurons from ischemic damage. These results suggest that HSP60 may be associated with delayed neuronal death of CA1 pyramidal neurons after transient ischemia, and the induction of HSP60 protects the neurons from ischemic damage.     

Ischemia-induced changes in glucagon-like peptide-1 receptor and neuroprotective effect of its agonist, exendin-4, in experimental transient cerebral ischemia

Glucagon-like peptide-1 receptor (GLP-1R) protects against neuronal damages in the brain. In the present study, ischemia-induced changes in GLP-1R immunoreactivity in the gerbil hippocampal CA1 region were evaluated after transient cerebral ischemia; in addition, the neuroprotective effect of the GLP-1R agonist exendin-4 (EX-4) against ischemic damage was studied. GLP-1R immunoreactivity and its protein levels in the ischemic CA1 region were highest at 1 day after ischemia/reperfusion (I/R). At 4 days after I/R, GLP-1R immunoreactivity was hardly detected in CA1 pyramidal neurons, and its protein level was lowest. GLP-1R protein level was increased again at 10 days after I/R, and GLP-1R immunoreactivity was found in astrocytes and GABAergic interneurons. In addition, EX-4 treatment attenuated ischemia-induced hyperactivity, neuronal damage, and microglial activation in the ischemic CA1 region in a dose-dependent manner. EX-4 treatment also induced the elevation of GLP-1R immunoreactivity and protein levels in the ischemic CA1 region. These results indicate that GLP-1R is altered in the ischemic region after an ischemic insult and that EX-4 protects against ischemia-induced neuronal death possibly by increasing GLP-1R expression and attenuating microglial activation against transient cerebral ischemic damage.     

Alterations of oxidative stress markers and apoptosis markers in the striatum after transient focal cerebral ischemia in rats

Cumulative evidence demonstrates that apoptosis caused by oxidative stress plays a key role in neuronal cell death after transient focal cerebral ischemia. In this study, we investigated exactly the immunohistochemical alterations of neuronal nuclei (NeuN), Cu/Zn-SOD (superoxide dismutase), Mn-SOD, 4-hydroxy-2-nonenal (HNE), and single strand DNA (ssDNA) in the striatum from 3 h up to 15 days after transient focal cerebral ischemia in rats under the same conditions. A conspicuous decrease of NeuN immunoreactive neurons was observed in the ipsilateral striatum from 3 h up to 15 days after focal ischemia. For Cu/Zn-SOD, Mn-SOD and HNE immunostainings, the alteration of Cu/Zn-SOD and HNE immunoreactivity was more pronounced than that of Mn-SOD immunoreactivity in the shrunken or atrophic neurons of ipsilateral striatum 3 h after focal ischemia. Thereafter, a significant increase of HNE immunoreactivity was observed in the shrunken or atrophic neurons of ipsilateral striatum up to 15 days after focal ischemia. In contrast, a significant decrease of Cu/Zn-SOD immunoreactivity was found in the ipsilateral striatum from 3 up to 15 days after focal ischemia. On the other hand, a significant increase of Mn-SOD immunereactivity was observed in the ipsilateral striatum from 1 up to 7 days after focal ischemia. In addition, our Western blot analysis also showed a significant increase of Cu/Zn-SOD and Mn-SOD in the ipsilateral striatum 1 day after focal ischemia, as compared to sham-operated group. In contrast, a significant increase in the number of ssDNA immunoreactive apoptotic neurons was observed in the ipsilateral striatum from 3 h to 3 days after focal cerebral ischemia. The present results also suggest that increased reactive oxygen species (ROS) production during reperfusion may contribute to the induction of the alteration of lipid peroxidation and could thereby lead to apoptosis in neurons of the ipsilateral striatum after transient focal ischemia, because of an insufficient expression of Cu/Zn-SOD and Mn-SOD. Furthermore, our findings demonstrate that the lipid peroxidation against mitochondrial membrane may contribute to apoptosis of striatal neurons after transient focal ischemia. Thus our findings demonstrate that the protection of lipid peroxidation against mitochondrial membrane may offer a novel therapeutic strategy for brain stroke in humans.