Protective Effect of Oren-gedoku-to Against Induction of Neuronal Death by Transient Cerebral Ischemia in the C57BL/6 Mouse

We examined the neuroprotective effects of oren-gedoku-to (TJ15), a herbal medicine, after transient forebrain ischemia. Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 15 min in C57BL/6 mice treated with TJ15. In the control ischemic group without TJ15 treatment, histologic examination of brain tissue collected seven days after reperfusion showed death of pyramidal cells in CA2-3 area of the hippocampus, unilaterally or bilaterally. In mice treated with oral TJ15 (845 mg/kg/day) for five weeks, the frequency of ischemic neuronal death was significantly lower. Immunohistochemistry for Cu/Zn-superoxide dismutase (Cu/Zn-SOD) showed strongly reactive astrocytes in the hippocampus of ischemic mice treated with TJ15. Damage to nerve cells by free radicals plays an important role in the induction of neuronal death by ischemia-reperfusion injury. Our results suggest that TJ15 protects against ischemic neuronal death by increasing the expression of Cu/Zn-SOD and suggest that oren-gedoku-to reduces the exposure of hippocampal neurons to oxidative stress.     

Effects of hyperglycemic and normoglycemic cerebral ischemia on phosphorylation of c-jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK).

Transient global cerebral ischemia leads to delayed neuronal cell death in the hippocampal CA1, caudate putamen and neocortex. If preischemic hyperglycemia exists, the same duration of ischemia recruits additional brain structures, such as dentate gyrus to become damaged. The objective of the present study is to determine whether activation of mitogen-activated protein kinases (MAPKs) plays a role in hyperglycemia-mediated ischemic neuronal damage. Using phopho-specific antibodies against c-jun NH2-terminal kinase (JNK) and p38 MAPK, we studied activation of these two MAPKs in ischemia-vulnerable neocortex and ischemia-resistant dentate gyrus in rats subjected to 15 min of forebrain ischemia and followed by 0.5, 1 and 3 hr of recirculation under normo- and hyperglycemic conditions. The results showed that levels of phosphorylated JNK increased in both normo- and hyperglycemic brains following blood reperfusion for 0.5 hr and persisted up to 3 hr in the neocortex but not in the dentate gyrus, implying JNK may play a role in mediating neuronal cell death after ischemia. However, since hyperglycemia did not further increase phospho-JNK, JNK may not contribute to the detrimental effect of hyperglycemia on neuronal cell death. The amount of phospho-p38 was not altered by ischemia under both normo- and hyperglycemic conditions, suggesting that p38 MAPK may not play a major role in mediating neuronal damage in these two structures.     

Astrocytes protect neurons from nitric oxide toxicity by a glutathione-dependent mechanism

Nitric oxide (NO) contributes to neuronal death in cerebral ischemia and other conditions. Astrocytes are anatomically well positioned to shield neurons from NO because astrocyte processes surround most neurons. In this study, the capacity of astrocytes to limit NO neurotoxicity was examined using a cortical co-culture system. Astrocyte-coated dialysis membranes were placed directly on top of neuronal cultures to provide a removable astrocyte layer between the neurons and the culture medium. The utility of this system was tested by comparing neuronal death produced by glutamate, which is rapidly cleared by astrocytes, and N-methyl-D-aspartate (NMDA), which is not. The presence of an astrocyte layer increased the LD(50) for glutamate by approximately four-fold, but had no effect on NMDA toxicity. Astrocyte effects on neuronal death produced by the NO donors S-nitroso-N-acetyl penicillamine and spermine NONOate were examined by placing these compounds into the medium of co-cultures containing either a control astrocyte layer or an astrocyte layer depleted of glutathione by prior exposure to buthionine sulfoximine. Neurons in culture with the glutathione-depleted astrocytes exhibited a two-fold increase in cell death over a range of NO donor concentrations. These findings suggest that astrocytes protect neurons from NO toxicity by a glutathione-dependent mechanism.     

Autophagy was activated in injured astrocytes and mildly decreased cell survival following glucose and oxygen deprivation and focal cerebral ischemia

The present study evaluated autophagy activation in astrocytes and its contribution to astrocyte injury induced by cerebral ischemia and hypoxia. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO) in rats. In vitro hypoxia in cultured primary astrocytes was induced by the oxygen-glucose deprivation (OGD). Alterations of astrocytes were evaluated with astroglia markers glial fibrillary acidic protein (GFAP). The formation of autophagosomes in astrocytes was examined with transmission electron microscopy (TEM). The expression of autophagy-related proteins were examined with immunoblotting. The role of autophagy in OGD or focal cerebral ischemia-induced death of astrocytes was assessed by pharmacological inhibition of autophagy with 3-methyladenine (3-MA) or bafilomycin A1 (Baf). The results showed that GFAP staining was reduced in the infarct brain areas 3-12 h following pMCAO. Cerebral ischemia or OGD induced activation of autophagy in astrocytes as evidenced by the increased formation of autophagosomes and autolysosomes and monodansylcadaverine (MDC)-labeled vesicles; the increased production of microtubule-associated protein 1 light chain 3 (LC3-II); the upregulation of Beclin 1, lysosome-associated membrane protein 2 (LAMP2) and lysosomal cathepsin B expression; and the decreased levels of cytoprotective Bcl-2 protein in primary astrocytes. 3-MA inhibited OGD-induced the increase in LC3-II and the decline in Bcl-2. Furthermore, 3-MA and Baf slightly but significantly attenuated OGD-induced death of astrocytes. 3-MA also significantly increased the number of GFAP-positive cells and the protein levels of GFAP in the ischemic cortex core 12 h following pMCAO. These results suggest that ischemia or hypoxia-induced autophagic/lysosomal pathway activation may at least partly contribute to ischemic injury of astrocytes.     

Bradykinin Postconditioning Protects Pyramidal CA1 Neurons Against Delayed Neuronal Death in Rat Hippocampus

Aims The present study was undertaken to evaluate possible neuroprotective effect of bradykinin against delayed neuronal death in hippocampal CA1 neurons if applied two days after transient forebrain ischemia in the rat. Methods Transient forebrain ischemia was induced in male Wistar rats by four-vessel occlusion for 8 min. To assess efficacy of bradykinin as a new stressor for delayed postconditioning we used two experimental groups of animals: ischemia 8 min and 3 days of survival, and ischemia 8 min and 3 days of survival with i.p. injection of bradykinin (150 μg/kg) applied 48 h after ischemia. Results We found extensive neuronal degeneration in the CA1 region at day 3 after ischemia/reperfusion. The postischemic neurodegeneration was preceded by increased activity of mitochondrial enzyme MnSOD in cytoplasm, indicating release of MnSOD from mitochondria in the process of delayed neuronal death. Increased cytosolic cytochrome c and subsequently caspase-3 activation are additional signs of neuronal death via the mitochondrial pathway. Bradykinin administration significantly attenuated ischemia-induced neuronal death, and also suppressed the release of MnSOD, and cytochrome c, and prevented caspase-3 activation. Conclusions Bradykinin can be used as an effective stressor able to prevent mitochondrial failure leading to apoptosis-like delayed neuronal death in postischemic rat hippocampus.     

Phosphorylation of JNK Increases in the Cortex of Rat Subjected to Diabetic Cerebral Ischemia

Previous studies have demonstrated that the c-Jun N-terminal kinase (JNK) pathway plays an important role in inducing neuronal apoptosis following cerebral ischemic injury. JNK signaling pathway in activated during cerebral ischemic injury. It participates in ischemia-induced neuronal apoptosis. However, whether JNK signaling is involved in the process of neuronal apoptosis of diabetes-induced cerebral ischemia is largely unknown. This study was undertaken to evaluate the influence of cerebral ischemia–reperfusion injury on phosphorylation of JNK in diabetic rats. Twenty-four adult streptozotocin induced diabetic and 24 adult non-diabetic rats were randomly subjected to 15 min of forebrain ischemia followed by reperfusion for 0, 1, 3, and 6 h. Sixteen sham-operated diabetic and non-diabetic rats were used as controls. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). Protein expression of phospho-JNK was examined by immunohistochemistry and Western blot. The numbers of TUNEL-positive cells and phospho-JNK protein expression in the cerebral cortices after 1, 3 and 6 h reperfusion was significantly higher in diabetic rats compared to non-diabetic animals subjected to ischemia and reperfusion (p < 0.05). Western blot analysis showed significantly higher phospho-JNK protein expression in the cerebral cortices of the diabetic rats after 1 and 3 h reperfusion than that was presented in non-diabetic animals subjected to ischemia and reperfusion (p < 0.05). These findings suggest that increased phosphorylation of JNK may be associated with diabetes-enhanced ischemic brain damage.     

Two caspase-2 transcripts are expressed in rat hippocampus after global cerebral ischemia

Caspase family genes play a critical role in the initiation and execution of programmed cell death. Programmed cell death is an important contributor to neuronal loss following cerebral ischemia. We have performed a series of experiments to investigate the role of a specific caspase, caspase-2, in the development of delayed neuronal death following transient global ischemia in the rat. A rat ischemic brain cDNA library was screened, and two splice-variants of caspase-2 mRNA were identified, caspase-2S and caspase-2L, which were highly homologous with the sequences of human and mouse caspase-2S and caspase-2L genes, respectively. RT-PCR demonstrated an increase in expression of both caspase-2S and caspase-2L mRNA at 8, 24 and 72 h of reperfusion after global ischemia. The ratio of the two PCR fragments did not change significantly throughout the time course of reperfusion. Western blot with monoclonal antibody specific to the pro-apoptotic caspase-2L splice variant revealed an increase in procaspase-2 (51 kDa) protein from 4 to 72 h following ischemia compared with sham-operated controls. Furthermore, an approximately 30-kDa cleavage product appeared at 8 h and increased with increasing duration of reperfusion. Thus, caspase-2L is both translated and activated following transient global ischemia. Finally, intraventricular administration of the caspase-2-like inhibitor (VDVAD-FMK) 30 min before induction of ischemia decreased the number of CA1 neurons staining positively for DNA damage (Klenow-labeling assay) and increased the number of healthy-appearing CA1 neurons (cresyl violet) compared with vehicle-treated controls. Taken together, the data suggest that caspase-2 induction and activation are important mediators of delayed neuronal death following transient global ischemia.     

Neuronal Damage Using Fluoro-Jade B Histofluorescence and Gliosis in the Gerbil Septum Submitted to Various Durations of Cerebral Ischemia

The extent of neuronal damage/death in some brain regions is highly correlated to duration time of transient ischemia. In the present study, we carried out neuronal degeneration/death and glial changes in the septum 4 days after 5, 10, 15, and 20 min of transient cerebral ischemia using gerbils. To examine neuronal damage, Fluoro-Jade B (F-J B, a marker for neuronal degeneration) histofluorescence staining was used. F-J B positive (⁺) cells were detected in the septo-hippocampal nucleus (SHN) of the septum only in the 20 min ischemia-group; the mean number of F-J B⁺ neurons was 14.9 ± 2.5/400 μm² in a section. Gliosis of astrocytes and microglia was examined using anti-glial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1), respectively. In all the ischemia-groups, GFAP- and Iba-1-immunoreactive astrocytes and microglia, respectively, were increased in number, and apparently tended to be increased in their immunoreactivity. Especially, in the 20 min ischemia-group, the number and immunoreactivity of Iba-immunoreactive microglia was highest and strongest in the ischemic SHN 4 days after ischemia–reperfusion. In brief, our findings showed that neuronal damage/death in the SHN occurred and gliosis was apparently increased in the 20 min ischemia-group at 4 days after ischemia–reperfusion.     

Temporal Profile of Astrocytes and Changes of Oligodendrocyte-Based Myelin Following Middle Cerebral Artery Occlusion in Diabetic and Non-diabetic Rats

The long-term impacts of cerebral ischemia and diabetic ischemia on astrocytes and oligodendrocytes have not been defined. The objective of this study is to define profile of astrocyte and changes of myelin in diabetic and non-diabetic rats subjected to focal ischemia.Focal cerebral ischemia of 30-min duration was induced in streptozotocin-induced diabetic and vehicle-injected normoglycemic rats. The brains were harvested for immunohistochemistry of glial fibrillary acidic protein (GFAP) and 2'', 3''-cyclic nucleotide 3''-phosphodiesterase (CNPase) at various reperfusion endpoints ranging from 30 min up to 28 days. The results showed that activate astrocytes were observed after 30 min and peaked at 3 h to 1 day after reperfusion in ischemic penumbra, and peaked at 7 days of reperfusion in ischemic core. Diabetes inhibited the activation of astrocytes in ischemic hemisphere. Demyelination occurred after 30 min of reperfusion in ischemic core and peaked at 1 day. Diabetes caused more severe demyelination compared with non-diabetic rats. Remyelination started at 7 days and completed at 14 and 28 days in ischemic region. Diabetes inhibited the remyelination processes. It is concluded that ischemia activates astrocytes and induces demyelination. Diabetes inhibits the activation of astrocytes, exacerbates the demyelination and delays the remyelination processes. These may contribute to the detrimental effects of hyperglycemia on ischemic brain damage.     

Bid-mediated mitochondrial pathway is critical to ischemic neuronal apoptosis and focal cerebral ischemia

We have investigated the role of the BH3-only pro-death Bcl-2 family protein, Bid, in ischemic neuronal death in a murine focal cerebral ischemia model. Wild-type and bid-deficient mice of inbred C57BL/6 background were subjected to 90-min ischemia induced by left middle cerebral artery occlusion followed by 72-h reperfusion. The volume of ischemic infarct was significantly smaller in the bid-deficient brains than in the wild-type brains, suggesting that Bid participated in the ischemic neuronal death. Indeed, following the ischemic treatment there was a significant reduction of apoptosis in the ischemic areas, particularly in the inner infarct border zone (the penumbra), of the bid-deficient brains. In addition, activation of Bid in the wild-type brains could be readily detected at approximately 3 h after ischemia, as evidenced by its proteolytic cleavage and translocation to the mitochondria as determined using Western blot analysis and immunofluorescence staining. Correspondingly, mitochondrial release of cytochrome c could be detected around the same time Bid was cleaved in the wild-type brains. However, no significant cytochrome c release was detected in the bid-deficient brains until 24 h later. This suggests that, although the mitochondrial apoptosis pathway might be activated by multiple mechanisms during focal cerebral ischemia, Bid is critical to its early activation. This notion was further supported by the finding that caspase-3 activation was severely impaired in the bid-deficient brains, whereas activation of caspase-8 was much less affected. Taken together, these data suggest that Bid is activated early in neuronal ischemia in a caspase-8-dependent fashion and that Bid is perhaps one of the earliest and most potent activators of the mitochondrial apoptosis pathway. Thus, the role of Bid in the induction of ischemic neuronal death may render this molecule an attractive target for future therapeutic intervention.     

Cdh1 inhibits reactive astrocyte proliferation after oxygen–glucose deprivation and reperfusion

Anaphase-promoting complex (APC) and its co-activator Cdh1 are required for cell cycle regulation in proliferating cells. Recent studies have defined diverse functions of APC–Cdh1 in nervous system development and injury. Our previous studies have demonstrated the activity of APC–Cdh1 is down-regulated in hippocampus after global cerebral ischemia. But the detailed mechanisms of APC–Cdh1 in ischemic nervous injury are unclear. It is known that astrocyte proliferation is an important pathophysiological process following cerebral ischemia. However, the role of APC–Cdh1 in reactive astrocyte proliferation is not determined yet. In the present study, we cultured primary cerebral astrocytes and set up in vitro oxygen–glucose deprivation and reperfusion model. Our results showed that the expression of Cdh1 was decreased while Skp2 (the downstream substrate of APC–Cdh1) was increased in astrocytes after 1 h oxygen–glucose deprivation and reperfusion. The down-regulation of APC–Cdh1 was coupled with reactive astrocyte proliferation. By constructing Cdh1 expressing lentivirus system, we also found exogenous Cdh1 can down-regulate Skp2 and inhibit reactive astrocyte proliferation induced by oxygen–glucose deprivation and reperfusion. Moreover, Western blot showed that other downstream proteins of APC–Cdh1, PFK-1 and SnoN, were decreased in the inhibition of reactive astrocyte proliferation with Cdh1 expressing lentivirus treatment. These results suggest that Cdh1 plays an important role in the regulation of reactive astrocyte proliferation induced by oxygen–glucose deprivation and reperfusion.     

N-Myc Downstream-Regulated Gene 2 (Ndrg2) Is Involved in Ischemia–Hypoxia-Induced Astrocyte Apoptosis: a Novel Target for Stroke Therapy

Nearly all clinical trials that have attempted to develop effective strategies against ischemic stroke have failed, excluding those for thrombolysis, and most of these trials focused only on preventing neuronal loss. However, astrocytes have gradually become a target for neuroprotection in stroke. In previous studies, we showed that the newly identified molecular N-myc downstream-regulated gene 2 (Ndrg2) is specifically expressed in astrocytes in the brain and involved in some neurodegenerative diseases. However, the role of NDRG2 in ischemic stroke remained unclear. In this study, we investigated the role of NDRG2 in middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia and in oxygen–glucose deprivation (OGD)-induced cellular apoptosis in the M1800 astrocyte cell line. NDRG2 mRNA and protein expression began to increase at 6 and 2 h after reperfusion and peaked at 24 h in the ischemic penumbra and in M1800 cells, as detected by RT-PCR and Western blotting. Double immunofluorescence staining showed that the number of apoptotic cells increased as the NDRG2-positive signal increased and that the NDRG2 signal was sometimes co-localized with TUNEL-positive cells and translocated from the cytoplasm to the nucleus in both the ischemic penumbra and the M1800 cells. Using a lentivirus, we successfully constructed two stable astrocytic cell lines in which NDRG2 expression was significantly up- or down-regulated. NDRG2 silencing had a proliferative effect and reduced the percentage of apoptotic cells, reactive oxygen species (ROS) production, and cleaved Caspase-3 protein expression following OGD, whereas NDRG2 over-expression had the opposite effects. In conclusion, NDRG2 is involved in astrocyte apoptosis following ischemic–hypoxic injury, and inhibiting NDRG2 expression significantly reduces ROS production and astrocyte apoptosis. These findings provide insight into the role of NDRG2 in ischemic–hypoxic injury and provide potential targets for future clinical therapies for stroke.     

Extended therapeutic window for caspase inhibition and synergy with MK-801 in the treatment of cerebral histotoxic hypoxia

In rats, striatal histotoxic hypoxic lesions produced by the mitochondrial toxin malonate resemble those of focal cerebral ischemia. Intrastriatal injections of malonate induced cleavage of caspase-2 beginning at 6 h, and caspase-3-like activity as identified by DEVD biotin affinity-labeling within 12 h. DEVD affinity-labeling was prevented and lesion volume reduced in transgenic mice overexpressing BCL-2 in neuronal cells. Intrastriatal injection of the tripeptide, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a caspase inhibitor, at 3 h, 6 h, or 9 h after malonate injections reduced the lesion volume produced by malonate. A combination of pretreatment with the NMDA antagonist, dizocilpine (MK-801), and delayed treatment with zVAD-fmk provided synergistic protection compared with either treatment alone and extended the therapeutic window for caspase inhibition to 12 h. Treatment with cycloheximide and zVAD-fmk, but not with MK-801, blocked the malonate-induced cleavage of caspase-2. NMDA injections alone resulted in a weak caspase-2 cleavage. These results suggest that malonate toxicity induces neuronal death by more than one pathway. They strongly implicate early excitotoxicity and delayed caspase activation in neuronal loss after focal ischemic lesions and offer a new strategy for the treatment of stroke.     

Astrocyte Mitochondria in In Vitro Models of Ischemia

There is growing evidence that preservation of mitochondrial respiratory function during cerebral ischemia-reperfusion predicts the ultimate extent of tissue injury. Because neurons are selectively vulnerable to ischemic injury, many studies have focused on neuronal mitochondrial dysfunction in ischemia. However, positron emission tomography (PET) studies in animals and humans suggest that non-neuronal cells such as astrocytes may also experience mitochondrial metabolic compromise that contributes to ischemic necrosis. Astrocytes carry out a number of functions that are critical to normal nervous system function, including uptake of neurotransmitters, regulation of pH and ion concentrations, and metabolic support of neurons. Mitochondria are important for many of these actions. We have used a cell culture model of stroke, oxygen-glucose deprivation (OGD), to study the response of astrocyte mitochondria to ischemia, and to evaluate how changes in astrocyte mitochondrial function might affect neuronal survival and recovery after ischemia.     

Neuroprotective Mechanism of Taurine due to Up-regulating Calpastatin and Down-regulating Calpain and Caspase-3 during Focal Cerebral Ischemia

Aims Taurine as an endogenous substance possesses a number of cytoprotective properties. In the study, we have evaluated the neuroprotective effect of taurine and investigated whether taurine exerted neuroprotection through affecting calpain/calpastatin or caspase-3 actions during focal cerebral ischemia, since calpain and caspase-3 play central roles in ischemic neuronal death. Methods Male Sprague–Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAo), and 22 h of reperfusion. Taurine was administrated intravenously 1 h after MCAo. The dose–responses of taurine to MCAo were determined. Next, the effects of taurine on the activities of calpain, calpastatin and caspase-3, the levels of calpastatin, microtubule-associated protein-2 (MAP-2) and αII-spectrin, and the apoptotic cell death in penumbra were evaluated. Results Taurine reduced neurological deficits and decreased the infarct volume 24 h after MCAo in a dose-dependent manner. Treatment with 50 mg/kg of taurine significantly increased the calpastatin protein levels and activities, and markedly reduced the m-calpain and caspase-3 activities in penumbra 24 h after MCAo, however, it had no significant effect on μ-calpain activity. Moreover, taurine significantly increased the MAP-2 and αII-spectrin protein levels, and markedly reduced the ischemia-induced TUNEL staining positive score within penumbra 24 h after MCAo. Conclusions Our data demonstrate the dose-dependent neuroprotection of taurine against transient focal cerebral ischemia, and suggest that one of protective mechanisms of taurine against ischemia may be blocking the m-calpain and caspase-3-mediated apoptotic cell death pathways.     

Post-ischemic activation of caspase-3 in the rat hippocampus: evidence of an axonal and dendritic localisation

The relationship between caspase-3 activation and delayed neuronal death after ischemia was examined. Expression of caspase-3 was evaluated by colorimetric assay, immunoblotting and by immunohistochemistry. Apoptosis was characterised by terminal desoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labelling. Immunohistochemistry showed caspase-3 activation in the whole hippocampus as early as 30 min after ischemia with exclusive localisation in fiber systems, especially in the perforant path and mossy fibers, Schaffer-collaterals, as well as apical and basal dendrites of pyramidal cells. One day post-ischemia, the 18 kDa cleavage product of caspase-3 (p18) was seen in all cell compartments (nucleus, cytosol and dendrites) throughout the entire subfields and the dentate gyrus with high distribution in mossy fibers. Two days post-ischemia, p18 kDa was only seen in the nuclei and cytosol of hippocampal cells without specific regional differences among hippocampal subfields. A significant number of apoptotic cells appeared only in the CA1 pyramidal cells at 2-3 days post-ischemia. Our data provides the first evidence that caspase-3 activation was detectable in the trisynaptic pathway fiber bundles which probably correspond to perforant path, alvear path and collaterals of Schaffer, and that activation of caspase-3 led to execution of apoptotic cell death program in selectively vulnerable areas, but not in the resistant area of the hippocampus.     

Time-dependent contribution of non neuronal cells to BDNF production after ischemic stroke in rats

Although brain-derived neurotrophic factor (BDNF) plays a central role in recovery after cerebral ischemia, little is known about cells involved in BDNF production after stroke. The present study testes the hypothesis that neurons are not the unique source of neosynthesized BDNF after stroke and that non neuronal-BDNF producing cells differ according to the delay after stroke induction. For this purpose, cellular localization of BDNF and BDNF content of each hemisphere were analysed in parallel before and after (4h, 24h and 8d) ischemic stroke in rats. Stroke of different severities was induced by embolization of the brain with variable number of calibrated microspheres allowing us to explore the association between BDNF production and neuronal death severity. The main results are that (a) unilateral stroke increased BDNF production in both hemispheres with a more intense and long-lasting effect in the lesioned hemisphere, (b) BDNF levels either of the lesioned or unlesioned hemispheres were not inversely correlated to neuronal death severity whatever the delay after stroke onset, (c) in the unlesioned hemisphere, stroke resulted in increased BDNF staining in neurons and ependymal cells (at 4h and 24h), (d) in the lesioned hemisphere, beside neurons and ependymal cells, microglial cells (at 24h), endothelial cells of cerebral arterioles (at 4h and 24h) and astrocytes (at 8d) exhibited a robust BDNF staining as well. Taken together, overall data suggest that non neuronal cells are able to produce substantial amount of BDNF after ischemic stroke and that more attention should be given to these cells in the design of strategies aimed at improving stroke recovery through BDNF-related mechanisms.     

Astrogliosis Is a Possible Player in Preventing Delayed Neuronal Death

Mitigating secondary delayed neuronal injury has been a therapeutic strategy for minimizing neurological symptoms after several types of brain injury. Interestingly, secondary neuronal loss appeared to be closely related to functional loss and/or death of astrocytes. In the brain damage induced by agonists of two glutamate receptors, N-ethyl-D-aspartic acid (NMDA) and kainic acid (KA), NMDA induced neuronal death within 3 h, but did not increase further thereafter. However, in the KA-injected brain, neuronal death was not obviously detectable even at injection sites at 3 h, but extensively increased to encompass the entire hemisphere at 7 days. Brain inflammation, a possible cause of secondary neuronal damage, showed little differences between the two models. Importantly, however, astrocyte behavior was completely different. In the NMDA-injected cortex, the loss of glial fibrillary acidic protein-expressing (GFAP⁺) astrocytes was confined to the injection site until 7 days after the injection, and astrocytes around the damage sites showed extensive gliosis and appeared to isolate the damage sites. In contrast, in the KA-injected brain, GFAP⁺ astrocytes, like neurons, slowly, but progressively, disappeared across the entire hemisphere. Other markers of astrocytes, including S100β, glutamate transporter EAAT2, the potassium channel Kir4.1 and glutamine synthase, showed patterns similar to that of GFAP in both NMDA- and KA-injected cortexes. More importantly, astrocyte disappearance and/or functional loss preceded neuronal death in the KA-injected brain. Taken together, these results suggest that loss of astrocyte support to neurons may be a critical cause of delayed neuronal death in the injured brain.     

UV-vulnerability of human papilloma virus type-16 E7-expressing astrocytes is associated with mitochondrial membrane depolarization and caspase-3 activation

The human papilloma virus-type 16 (HPV-16) E6 and E7 proteins interact with the p53 and pRb tumor suppressor proteins, respectively. The effect of E6 or E7 expression on UV irradiation (5 and 20 J/m2)-induced genotoxic injury of confluent primary murine astrocytes was determined. Retroviral vectors were used to overexpress E6 and E7. Astrocytes expressing E7 showed increased vulnerability to UV-induced apoptosis while E6 over expressing astrocytes were protected from the same insults. Cell death in the E7 overexpressing cells was apoptotic because it showed DNA ladders, activation of caspase-3, formation of apoptotic bodies and decreased DNA content to less than the G0 level. After UV-irradiation the level of E2F1 in E7-expressing astrocytes was higher than E6-, LXSN- or mock-infected cells, and caspase-3 was activated to a greater extent. E7-expressing astrocytes showed the highest levels of Bax under normal growth conditions. The mitochondrial membrane potential of E7-expressing astrocytes was depolarized by 90% after UV-irradiation while the depolarization in control cells was about 50%. E6 overexpression decreased while E7 overexpression increased UV-induced astrocyte apoptosis.     

Metabolic and Biosynthetic Alterations in Cultured Astrocytes Exposed to Hypoxia/Reoxygenation

To investigate the astrocyte response to hypoxia/reoxygenation, as a model relevant to the pathogenesis of ischemic injury, cultured rat astrocytes were exposed to hypoxia. On restoration of astrocytes to normoxia, there was a dramatic increase in protein synthesis within 3 h, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocyte lysates showed multiple induced bands on fluorograms. Levels of cellular ATP declined during the first 3 h of reoxygenation and the concentration of AMP increased to ± 3.6 nmol/mg of protein within 1 h of reoxygenation. Reoxygenated astrocytes generated oxygen free radicals early after replacement into ambient air, and addition of diphenyliodonium, an NADPH oxidase inhibitor, diminished the generation of free radicals as well as the induction of several bands on fluorogram. Although addition of cycloheximide on reoxygenation resulted in inhibition of both astrocyte protein synthesis and accumulation of cellular AMP, it caused cell death within 6 h, suggesting the importance of protein synthesis in adaptation of hypoxic astrocytes to reoxygenation. Potential physiologic significance of biosynthetic products of astrocytes in hypoxia/reoxygenation was suggested by the recovery of glutamate uptake. These results indicate that the astrocyte response to hypoxia/reoxygenation includes generation of oxygen free radicals and de novo synthesis of products that influence cell viability and function in ischemia.