Regionally selective effects of NMDA receptor antagonists against ischemic brain damage in the gerbil

This study compared the ability of three N-methyl-D-aspartate (NMDA) receptor antagonists to prevent neuronal degeneration in an animal model of global cerebral ischemia. The model employed is characterized by damage to the striatum, hippocampus, and neocortex. Antagonists were administered to gerbils either before or after a 5-min bilateral carotid occlusion. The intraischemic rectal temperature was either maintained at 36-37 degrees C or allowed to fall passively to 28-32 degrees C. Antagonists and doses tested were 1 and 10 mg/kg of MK-801 (pre- or postischemia), 30 mg/kg of CGS 19755 preischemia, four 25 mg/kg doses of CGS 19755 administered between 0.5 and 6.5 h postischemia, and 40 mg/kg of MDL 27,266 (pre- or postischemia). All three NMDA receptor antagonists exhibited some degree of neuroprotective activity when the carotid occlusion was performed under normothermic conditions. Most of the treatments with antagonist markedly reduced striatal damage. CA1 hippocampal and neocortical pyramidal cells were spared by only three of the treatments, however, and the extent of neuroprotection varied widely from case to case. Toxic doses of antagonist were required to protect CA1 pyramidal cells from ischemic damage. Ischemic damage to hippocampal areas CA2-CA3a and CA4 appeared to be resistant to all of these treatments. Most CA1 pyramidal cells that were protected from degeneration by an NMDA receptor antagonist were histologically abnormal. The neuroprotective effects of MK-801 and intraischemic hypothermia appeared to be additive. MK-801 (10 mg/kg) consistently reduced the postischemic brain temperature, but only the magnitude of hypothermia produced soon after reperfusion correlated with its neuroprotective action. These results suggest that NMDA receptor antagonists are relatively poor neuroprotective agents against a moderately severe ischemic insult.     

Therapeutic hypothermia influences cell genesis and survival in the rat hippocampus following global ischemia

Delayed hypothermia salvages CA1 neurons from global ischemic injury. However, the effects of this potent neuroprotectant on endogenous repair mechanisms, such as neurogenesis, have not been clearly examined. In this study, we quantified and phenotyped newly generated cells within the hippocampus following untreated and hypothermia-treated ischemia. We first show that CA1 pyramidal neurons did not spontaneously regenerate after ischemia. We then compared the level of neuroprotection when hypothermia was initiated either during or after ischemia. Treatment efficacy decreased with longer delays, but hypothermia delayed for up to 12 hours was neuroprotective. Although bromodeoxyuridine (BrdU) incorporation was elevated in ischemic groups, CA1 neurogenesis did not occur as the BrdU label did not colocalize with neuronal nuclei (NeuN) in any of the groups. Instead, the majority of BrdU-labeled cells were Iba-positive microglia, and neuroprotective hypothermia decreased the delayed generation of microglia during the third postischemic week. Conversely, hypothermia delayed for 12 hours significantly increased the survival of newly generated dentate granule cells at 4 weeks after ischemia. Thus, our findings show that CA1 neurogenesis does not contribute to hypothermic neuroprotection. Importantly, we also show that prolonged hypothermia positively interacts with postischemic repair processes, such as neurogenesis, resulting in improved functional outcome.     

Postischemic Hypothermia and IL-10 Treatment Provide Long-Lasting Neuroprotection of CA1 Hippocampus Following Transient Global Ischemia in Rats

Experimental studies have demonstrated that postischemic therapeutic interventions may delay rather than provide long-lasting neuroprotection. The purpose of this study was to determine whether mild hypothermia (33-34 degrees C) combined with the anti-inflammatory cytokine interleukin-10 (IL-10) would protect the CA1 hippocampus 2 months after ischemia. Rats were subjected to 12.5 min of normothermic (37 degrees C) forebrain ischemia by two-vessel occlusion followed immediately by: (a) 4 h of normothermic (37 degrees C) reperfusion (n = 5); (b) 4 h of postischemic hypothermia (33-34 degrees C) (n = 5); (c) 4 h of normothermia plus IL-10 (5 micrograms) treatment 30 min after ischemia and at 3 days (n = 5); or (d) 4 h of hypothermia plus IL-10 treatment (n = 5). Rats survived for 2 months and were perfusion fixed for quantitative histopathological assessment of CA1 hippocampus. Postischemic normothermia and hypothermia, as well as normothermia plus IL-10 treatment led to severe damage of the CA1 hippocampus. In contrast, the combined treatment of hypothermia with IL-10 treatment improved overall neuronal survival by 49% compared to normothermic ischemia (P < 0.01). These data emphasize the detrimental consequences of secondary inflammatory responses on ischemic neuronal damage after transient global ischemia. In postinjury settings where restricted durations of mild hypothermia can be induced, anti-inflammatory treatments, including IL-10, may promote chronic neuroprotection.     

Resveratrol prevents CA1 neurons against ischemic injury by parallel modulation of both GSK‐3β and CREB through PI3‐K/Akt pathways

Accumulating evidence indicates that resveratrol potently protects against cerebral ischemia damage due to its oxygen free radicals scavenging and antioxidant properties. However, cellular mechanisms that may underlie the neuroprotective effects of resveratrol in brain ischemia are not fully understood yet. This study aimed to investigate the potential association between the neuroprotective effect of resveratrol and the apoptosis/survival signaling pathways, in particular the glycogen synthase kinase 3 (GSK‐3β) and cAMP response element‐binding protein (CREB) through phosphatidylinositol 3‐kinase (PI3‐K)‐dependent pathway. An experimental model of global cerebral ischemia was induced in rats by the four‐vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl staining indicated extensive neuronal death at 7 days after ischemia/reperfusion. Administration of resveratrol by i.p. injections (30 mg/kg) for 7 days before ischemia significantly attenuated neuronal death. Both GSK‐3β and CREB appear to play a critical role in resveratrol neuroprotection through the PI3‐K/Akt pathway, as resveratrol pretreatment increased the phosphorylation of Akt, GSK‐3β and CREB in 1 h in the CA1 hippocampus after ischemia/reperfusion. Furthermore, administration of LY294002, an inhibitor of PI3‐K, compromised the neuroprotective effect of resveratrol and decreased the level of p‐Akt, p‐GSK‐3β and p‐CREB after ischemic injury. Taken together, the results suggest that resveratrol protects against delayed neuronal death in the hippocampal CA1 by maintaining the pro‐survival states of Akt, GSK‐3β and CREB pathways. These data suggest that the neuroprotective effect of resveratrol may be mediated through activation of the PI3‐K/Akt signaling pathway, subsequently downregulating expression of GSK‐3β and CREB, thereby leading to prevention of neuronal death after brain ischemia in rats.     

Cellular localization of epidermal‐type and brain‐type fatty acid‐binding proteins in adult hippocampus and their response to cerebral ischemia

This study aimed at an analysis of expression of epidermal‐type and brain‐type fatty acid‐binding proteins (E‐FABP and B‐FABP, also called FABP5 and FABP7, respectively) in adult hippocampus and their potential value as neuroprotective factors after ischemic brain damage in monkey model. The immunostaining and Western blotting results show that FABP5 was mainly expressed in neurons, whereas FABP7 was primarily expressed in astrocytes and progenitors of the subgranular zone (SGZ). Interestingly, FABP5 expression in neurons increased in cornu Ammonis 1 (CA1) and remains stable within dentate gyrus (DG) after ischemia; FABP7 expression increased within both CA1 and SGZ. This indicates a potential role for FABP5 and FABP7 in intracellular fatty acid transport within different neural cells. The change in FABP5–7 expression within CA1 and DG of the adult postischemic hippocampus was compatible with previous findings of downregulation in CA1 neurons and upregulation in SGZ progenitor cells after ischemia. Altogether, the present data suggest that polyunsaturated fatty acids, such as docosahexaenoic acid, may act via FABP5 or 7 to regulate adult postischemic hippocampal neuronal antiapoptosis or neurogenesis in primates. © 2009 Wiley‐Liss, Inc.     

Postischemic inhibition of GABA reuptake by tiagabine slows neuronal death in the gerbil hippocampus

The neuroprotective effects of enhancing neuronal inhibition with a γ-aminobutyric acid (GABA) uptake inhibitor were studied in gerbil hippocampus following transient ischemia. We used in vivo microdialysis to determine a suitable dosing regimen for tiagabine (NNC 328) to elevate extracellular levels of GABA within the hippocampus. In anesthetized (normothermic) gerbils, tiagabine (45 mg/kg, i. p.) selectively elevated extracellular GABA levels 450% in area CA1 of the hippocampus. In gerbils subjected to cerebral ischemia via 5-min bilateral carotid occlusion, extracellular GABA levels increased 13-fold in area CA1, returning to baseline within 30–45 min. When tiagabine was injected 10 min following onset of reperfusion, GABA levels remained elevated (200–470%) for 90 min. In addition, tiagabine significantly reduced the ischemic-induced elevation of glutamate levels in area CA1 during the postischemic period when GABA levels were elevated. There was no effect of postischemic tiagabine on aspartate or six other amino acids. Using the same dosing regimen, we evaluated the degree of neuroprotection in the hippocampus of gerbils 4 and 21 days after ischemia. Tiagabine decreased body temperature a maximum of 2.7°C beginning 30 min into reperfusion and lasting 90 min. In untreated gerbils sacrificed 4 and 21 days after ischemia, there was severe necrosis (99%) of the pyramidal cell layer in area CA1. Whereas tiagabine significantly protected the CA1 pyramidal cell layer in ischemic gerbils at 4 days (overt necrosis confined to about 17% of area CA1), the protection diminished significantly 21 days postischemia. When normothermia was maintained both during and after ischemia in a separate group of tiagabine-treated animals, approximately 77% of the CA1 pyramidal cell layer was necrotic at 4 days. Based on these findings, we suggest that (1) tiagabine slows the development of hippocampal degeneration following ischemia, and (2) that mild, postischemic hypothermia is responsible, in large part, for the neuroprotective actions of this drug. We conclude that the histological outcome after administration of cerebral neuroprotectants should be assessed following long-term survival. © 1995 Wiley-Liss, Inc.     

亚低温对大鼠短暂全脑缺血后神经元凋亡的影响

目的 探讨亚低温对大鼠脑缺血后神经元凋亡的影响，揭示亚低温的部分神经保护机制。方法 采用“双侧颈总动脉阻断＋全身低血压”方法来建立大鼠短暂性全脑缺血模型。用神经元尼氏体亚甲兰特殊染色法观察大鼠脑缺血后海马CA1区神经元损害情况；原位细胞凋亡检测法（TUNEL染色）及电镜观察脑缺血后CA1区神经元凋亡情况。结果 与假手术组、低温缺血组相比，常温缺血组海马CA1区神经元缺失明显（P＜0．01）。常温及低温缺血组海马CA1区均存在神经元凋亡，但低温缺血组海马CA1区凋亡神经元数明显少于缺血组（P＜0．01）。结论 经“双侧颈总动脉阻断＋全身低血压”方法建立的大鼠短暂全脑缺血模型证实了亚低温的脑保护作用。全脑缺血后的迟发性神经元死亡很可能经由凋亡途径，而亚低温可通过抑制缺血性神经元凋亡而发挥一定的神经保护作用。     

Overexpression of UCP2 protects thalamic neurons following global ischemia in the mouse.

Uncoupling protein 2 (UCP2) is upregulated in the brain after sublethal ischemia, and overexpression of UCP2 is neuroprotective in several models of neurodegenerative disease. We investigated if increased levels of UCP2 diminished neuronal damage after global brain ischemia by subjecting mice overexpressing UCP2 (UCP2/3tg) and wild-type littermates (wt) to a 12-min global ischemia. The histopathological outcome in the cortex, hippocampus, striatum, and thalamus was evaluated at 4 days of recovery, allowing maturation of the selective neuronal death. Global ischemia led to extensive cell death in the striatum, thalamus, and in the CA1 and CA2, and less-pronounced cell death in the CA3 and dentate gyrus (DG) hippocampal subfields. Histologic damage was significantly lower in the ventral posterolateral VPL and medial VPM thalamic nuclei in UCP2/3tg animals compared with wt. These thalamic regions showed a larger increase in UCP2 expression in UCP2/3tg compared with wt animals relative to the nonprotected DG. In the other regions studied, the histologic damage was lower or equal in UCP2/3tg animals compared with wt. Consequently, neuroprotection in the thalamus correlated with a high expression of UCP2, which is neuroprotective in a number of models of neurodegenerative diseases.     

A synthetic NCAM-derived peptide, FGL, protects hippocampal neurons from ischemic insult both in vitro and in vivo

There is a major unmet need for development of innovative strategies for neuroprotection against ischemic brain injury. Here we show that FGL, a neural cell adhesion molecule (NCAM)-derived peptide binding to and inducing phosphorylation of the fibroblast growth factor receptor (FGFR), acts neuroprotectively after an ischemic insult both in vitro and in vivo. The neuroprotective activity of FGL was tested in vitro on dissociated rat hippocampal neurons and hippocampal slice cultures, using a protocol of oxygen-glucose deprivation (OGD). FGL protected hippocampal neurons from damage and maintained or restored their metabolic and presynaptic activity, both if employed as a pretreatment alone to OGD, and if only applied after the insult. In vivo 24 h pretreatment with a single suboccipital injection of FGL significantly protected hippocampal CA1 neurons from death in a transient global ischemia model in the gerbil. We conclude that FGL promotes neuronal survival after ischemic brain injury.     

Brain damage in a mouse model of global cerebral ischemia. Effect of NMDA receptor blockade

The importance of particular genes in neuronal death following global cerebral ischemia can readily be studied in genetically modified mice provided a reliable model of ischemia is available. For that purpose, we developed a mouse model of global cerebral ischemia that induces consistent damage to different regions of the brain and with a low mortality rate. Twelve minutes of ischemia was induced in C57BL/6 mice by bilateral common carotid artery occlusion under halothane anesthesia and artificial ventilation. Body and brain temperature were monitored and cortical cerebral blood flow in each hemisphere was measured by laser Doppler flowmeter before, during, and for 5 min after ischemia. Extensive damage was found in the striatum and marked cell damage was observed in the CA1 and CA2 regions of hippocampus and in thalamus. Mild damage was seen in the CA3 region, dentate gyrus and cortex. Hippocampal damage in the CA1 region is delayed and developed over 48 h. Intraischemic hypothermia of 33 degrees C provided a robust neuroprotection. The non-competitive N-methyl-D-aspartate receptor blocker, MK-801, did not provide protection in the hippocampus, cortex, striatum or thalamus when administered 30 min prior to ischemia or 2 h after the end of ischemia, but selectively mitigated damage in the hippocampus, when administered immediately following ischemia. This model of global cerebral ischemia may be useful in pharmacological and genomic studies of ischemic brain damage.     

Mild hypothermia ameliorates ubiquitin synthesis and prevents delayed neuronal death in the gerbil hippocampus.

BACKGROUND AND PURPOSE: The purpose of the present study is to determine the effect of mild hypothermia on the synthesis of ubiquitin, an important protein for maintenance of cell viability, in the hippocampal neurons following transient cerebral ischemia. METHODS: Transient ischemia was induced by occluding both common carotid arteries for 5 minutes. In experiment 1, the animals were divided into four groups according to the rectal and scalp temperatures during ischemia: the normothermia group and the graded hypothermia A, B, and C groups (n = 9 per group). CA1 neuronal density was assessed at 7 days after ischemia. In experiment 2, the animals were divided into two groups designated the normothermia and the hypothermia groups (n = 6 per group). The presence of ubiquitin was examined by immunohistochemistry at 6, 24, and 48 hours after transient ischemia in various regions of the hippocampus. RESULTS: In experiment 1, the mean +/- SEM neuronal density per millimeter was 12 +/- 1 in the normothermia group and 126 +/- 25, 225 +/- 10, and 214 +/- 9 in hypothermia groups A, B, and C, respectively. Mild hypothermia in groups B and C, in which the brain temperature was below 33 degrees C, ameliorated markedly the extent of ischemic neuronal damage in the CA1 sector (p less than 0.01). In experiment 2, ubiquitin immunoreactivity had disappeared in all regions of the hippocampus at 6 hours after ischemia and showed no subsequent recovery in the CA1 pyramidal neurons under normothermic conditions. Under hypothermic conditions, however, it had recovered significantly in the CA1 pyramidal neurons at 24 and 48 hours after ischemia (p less than 0.01). CONCLUSIONS: We conclude that mild hypothermia, in which the brain temperature is below 33 degrees C, markedly improves the ischemic delayed neuronal damage in the CA1 sector, and that increased ubiquitin synthesis and protein ubiquitination could be one essential part of the protective mechanism afforded by mild hypothermia against delayed neuronal death.     

Morphological investigation of the neuroprotective effects of graded hypothermia after diverse periods of global cerebral ischemia in gerbils

Hypothermia is known to be the most effective method to protect the neuronal damage induced by ischemia. In the present study, we investigated the histopathological consequences of hippocampal CA1 pyramidal neurons as well as the glial reactions in the hippocampus, after diverse periods of ischemic insult at graded intra-ischemic hypothermia ranging from 32 to 20°C. Gerbils were exposed to forebrain ischemia by clamping the bilateral common carotid arteries for 5–120 min depending upon the temperatures. The morphological study was performed 7 days after ischemia or sham-operation. Histopathological evaluation of delayed neuronal death (DND) was performed by Cresyl violet (CV) staining and MAP2 immunoreactivity. Glial reactions were examined by GFAP immunostaining and isolectin B4 histochemistry, corresponding to astrocytes and microglia, respectively. The forebrain ischemia at 32°C for 10 min and at 28°C for 20 min did not induce DND in the CA1 region. However, the ischemia at 32°C for 20 min and at 28°C for 30 min caused extensive degeneration of CA1 pyramidal neurons as observed in normothermic ischemic animals. Under the condition of deep hypothermia, the ischemia for 60 min at 24°C and for 120 min at 20°C which were the longest durations of each temperature within the limitation of the animal survival following 7 days, induced no DND in CA1 pyramidal neurons. The reactive changes of astrocytes were observed not only in ischemic animals with DND, but also in ischemic animals without DND. Computer image analysis showed that the area fraction of GFAP-positive structures in the CA1 region was significantly increased in both ischemic cases with and without DND compared with each sham group. In contrast, the distribution of activated microglia was much more restricted to the CA1 region and they were always accompanied by DND at 7 days postischemia. The present results demonstrate the remarkable neuroprotective effect of deep hypothermia that has been widely used in cardiovascular surgeries as the cerebroprotective strategy during total circulatory cessation. The findings also suggest that even under the condition of hypothermia, glial reactions may play an important role in neuronal survival and death after ischemia.     

Protective effects of peroxisome proliferator‐activated receptors γ coactivator‐1α against neuronal cell death in the hippocampal CA1 subfield after transient global ischemia

Peroxisome proliferator‐activated receptors γ coactivator‐1α (PGC‐1α) may regulate the mitochondrial antioxidant defense system under many neuropathological settings. However, the exact role of PGC‐1α in ischemic brain damage is still under debate. Based on an experimental model of transient global ischemia (TGI), this study evaluated the hypothesis that the activation of PGC‐1α signaling pathway protects hippocampal CA1 neurons against delayed neuronal death after TGI. In Sprague‐Dawley rats, significantly increased content of oxidized proteins in the hippocampal CA1 tissue was observed as early as 30 min after TGI, followed by augmentation of PGC‐1α expression at 1 hr. Expression of uncoupling protein 2 (UCP2) and superoxide dismutases 2 (SOD2) in the hippocampal CA1 neurons was upregulated 4–48 hr after TGI. In addition, knock‐down of PGC‐1α expression by pretreatment with a specific antisense oligodeoxynucleotide in the hippocampal CA1 subfield downregulated the expression of UCP2 and SOD2 with resultant exacerbation of oxidative stress and augmentation of delayed neuronal cell death in the hippocampus after TGI. Overall, our results indicate that PGC‐1α is induced by cerebral ischemia leading to upregulation of UCP2 and SOD2, thereby providing a neuroprotective effect against ischemic brain injury in the hippocampus by ameliorating oxidative stress. © 2009 Wiley‐Liss, Inc.     

Decreased epileptic susceptibility correlates with neuropeptide Y overexpression in a model of tolerance to excitotoxicity

Prior epileptic episodes have been shown to decrease markedly the neuronal damage induced by a second epileptic episode, similar to the tolerance following an episode of mild ischemia. Endogenous neuroprotective effects mediated by various mechanisms have been put forward. This study investigated whether neuroprotection against the excitotoxic damage induced by re-exposure to an epileptic challenge can reflect a change in epileptic susceptibility. Tolerance was elicited in rats by a preconditioning session using intrahippocampal kainic acid (KA) administration followed at 1, 7 and 15-day intervals by a subsequent intraventricular KA injection. The degree of pyramidal cell loss in the vulnerable CA3 subfield contralateral to the KA-injected hippocampus was extensively reduced in animals experiencing KA ventricular administration. This neuroprotection was highly significant 1 and 7 days after injection, but not 15 days after injection. In preconditioned animals, the after-discharge threshold was assessed as an index of epileptic susceptibility. It increased significantly from 1 to 15 days after intrahippocampal KA administration. Finally, an enhancement of neuropeptide Y expression in both non-principal cells and mossy fibers was detected, occurring at the same time as the decrease in epileptic susceptibility. These results provide further evidence of an 'epileptic tolerance' as shown by the substantial neuroprotective effect of a prior episode of epileptic activity upon subsequent epileptic insult and suggest that the prevention of excitotoxic damage after preconditioning results from an endogenous neuroprotective mechanism against hyperexcitability and seizures.     

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.     

Autoradiographic localization of N-type VGCCs in gerbil hippocampus and failure of omega-conotoxin MVIIA to attenuate neuronal injury after transient cerebral ischemia.

In the mammalian central nervous system, transient global ischemia of specific duration causes selective degeneration of CA1 pyramidal neurons in hippocampus. Many of the ischemia-induced pathophysiologic cascades that destroy the neurons are triggered by pre- and postsynaptic calcium entry. Consistent with this, many calcium channel blockers have been shown to be neuroprotective in global models of ischemia. omega-Conotoxin MVIIA, a selective N-type VGCC blocker isolated from the venom of Conus magus, protects CA1 neurons in the rat model of global ischemia, albeit transiently. The mechanism by which this peptide renders neuroprotection is unknown. We performed high-resolution receptor autoradiography with the radiolabeled peptide and observed highest binding in stratum lucidum of CA3 subfield, known to contain inhibitory neurons potentially important in the pathogenesis of delayed neuronal death. This finding suggested that the survival of stratum lucidum inhibitory neurons might be the primary event, leading to CA1 neuroprotection after ischemia. Testing of this hypothesis required the reproduction of its neuroprotective effects in the gerbil model of global ischemia. Surprisingly, we found that omega-MVIIA did not attenuate CA1 hippocampal injury after 5 min of cerebral ischemia in gerbil. Possible reasons are discussed. Lastly, we show that the peptide can be used as a synaptic marker in assessing short and long-term changes that occur in hippocampus after ischemic injury.     

Postconditioning mitigates cell death following oxygen and glucose deprivation in PC12 cells and forebrain reperfusion injury in rats

Postconditioning mitigates ischemia‐induced cellular damage via a modified reperfusion procedure. Mitochondrial permeability transition (MPT) is an important pathophysiological change in reperfusion injury. This study explores the role of MPT modulation underlying hypoxic postconditioning (HPoC) in PC12 cells and studies the neuroprotective effects of ischemic postconditioning (IPoC) on rats. Oxygen‐glucose deprivation (OGD) was performed for 10 hr on PC12 cells. HPoC was induced by three cycles of 10‐min reoxygenation/10‐min rehypoxia after OGD. The MPT inhibitor N‐methyl‐4‐isoleucine cyclosporine (NIM811) and the MPT inducer carboxyatractyloside (CATR) were administered to selective groups before OGD. Cellular death was evaluated by flow cytometry and Western blot analysis. JC‐1 fluorescence signal was used to estimate the mitochondrial membrane potential (△Ψ_m). Transient global cerebral ischemia (tGCI) was induced via the two‐vessel occlusion and hypotension method in male Sprague Dawley rats. IPoC was induced by three cycles of 10‐sec reperfusion/10‐sec reocclusion after index ischemia. HPoC and NIM811 administration attenuated cell death, cytochrome c release, and caspase‐3 activity and maintained △Ψ_m of PC12 cells after OGD. The addition of CATR negated the protection conferred by HPoC. IPoC reduced neuronal degeneration and cytochrome c release and cleaved caspase‐9 expression of hippocampal CA1 neurons in rats after tGCI. HPoC protected PC12 cells against OGD by modulating the MPT. IPoC attenuated degeneration of hippocampal neurons after cerebral ischemia. © 2014 Wiley Periodicals, Inc.     

Erythropoietin protects CA1 neurons against global cerebral ischemia in rat: potential signaling mechanisms

Erythropoietin (EPO) is a hormone that is neuroprotective in models of neurodegenerative diseases. This study examined whether EPO can protect against neuronal death in the CA1 region of the rat hippocampus following global cerebral ischemia. Recombinant human EPO was infused into the intracerebral ventricle either before or after the induction of ischemia produced by using the four-vessel-occlusion model in rat. Hippocampal CA1 neuron damage was ameliorated by infusion of 50 U EPO. Administration of EPO was neuroprotective if given 20 hr before or 20 min after ischemia, but not 1 hr following ischemia. Coinjection of the phosphoinositide 3 kinase inhibitor LY294002 with EPO inhibited the protective effects of EPO. Treatment with EPO induced phosphorylation of both AKT and its substrate, glycogen synthase kinase-3beta, in the CA1 region. EPO also enhanced the CA1 level of brain-derived neurotrophic factor. Finally, we determined that ERK activation played minor roles in EPO-mediated neuroprotection. These studies demonstrate that a single injection of EPO ICV up to 20 min after global ischemia is an effective neuroprotective agent and suggest that EPO is a viable candidate for treating global ischemic brain injury.     

TrkB‐mediated activation of the phosphatidylinositol‐3‐kinase/Akt cascade reduces the damage inflicted by oxygen–glucose deprivation in area CA3 of the rat hippocampus

The selective vulnerability of hippocampal area CA1 to ischemia‐induced injury is a well‐known phenomenon. However, the cellular mechanisms that confer resistance to area CA3 against ischemic damage remain elusive. Here, we show that oxygen–glucose deprivation–reperfusion (OGD‐RP), an in vitro model that mimic the pathological conditions of the ischemic stroke, increases the phosphorylation level of tropomyosin receptor kinase B (TrkB) in area CA3. Slices preincubated with brain‐derived neurotrophic factor (BDNF) or 7,8‐dihydroxyflavone (7,8‐DHF) exhibited reduced depression of the electrical activity triggered by OGD‐RP. Consistently, blockade of TrkB suppressed the resistance of area CA3 to OGD‐RP. The protective effect of TrkB activation was limited to area CA3, as OGD‐RP caused permanent suppression of CA1 responses. At the cellular level, TrkB activation leads to phosphorylation of the accessory proteins SHC and Gab as well as the serine/threonine kinase Akt, members of the phosphoinositide 3‐kinase/Akt (PI‐3‐K/Akt) pathway, a cascade involved in cell survival. Hence, acute slices pretreated with the Akt antagonist MK2206 in combination with BDNF lost the capability to resist the damage inflicted with OGD‐RP. Consistently, with these results, CA3 pyramidal cells exhibited reduced propidium iodide uptake and caspase‐3 activity in slices pretreated with BDNF and exposed to OGD‐RP. We propose that PI‐3‐K/Akt downstream activation mediated by TrkB represents an endogenous mechanism responsible for the resistance of area CA3 to ischemic damage.     

Indole‐3‐propionic acid attenuates neuronal damage and oxidative stress in the ischemic hippocampus

Tryptophan‐derived indole compounds have been widely investigated as antioxidants and as free‐radical scavengers. Indole‐3‐propionic acid (IPA), one of these compounds, is a deamination product of tryptophan. In the present study, we used Mongolian gerbils to investigate IPA's neuroprotective effects against ischemic damage and its antioxidative effects in the hippocampal CA1 region (CA1) after 5 min of transient forebrain ischemia. The repeated oral administration of IPA (10 mg/kg) for 15 days before ischemic surgery protected neurons from ischemic damage. In this group, the percentage of cresyl violet–positive neurons in the CA1 was 56.8% compared with that in the sham group. In the vehicle‐treated group, glial fibrillary acidic protein (GFAP)‐, S‐100‐, and vimentin‐immunoreactive astrocytes and ionized calcium‐binding adapter molecule 1 (Iba‐1)– and isolectin B4 (IB4)–immunoreactive microglia were activated 4 days after ischemia/reperfusion, whereas in the IPA‐treated ischemic group, GFAP, S‐100, Iba‐1, and IB4, but not vimentin, immunoreactivity was distinctly lower than that in the vehicle‐treated ischemic groups. The administration of IPA significantly decreased the level of 4‐hydroxy‐2‐nonenal, a marker of lipid peroxidation, in ischemic hippocampal homogenates compared with that in the vehicle‐treated ischemic groups at various times after ischemia/reperfusion. In addition, immunostaining for 8‐hydroxy‐2′‐deoxyguanosine showed DNA damage in pyramidal neurons in the ischemic CA1 was significantly lower in the IPA‐treated ischemic groups than in the vehicle‐treated ischemic groups. These results suggest that IPA protects neurons from ischemia‐induced neuronal damage by reducing DNA damage and lipid peroxidation. © 2009 Wiley‐Liss, Inc.