Hippocampal CA1 Neuronal Death Research Articles

Activation of BDNF- and VEGF-mediated Neuroprotection by Treadmill Exercise Training in Experimental Stroke.

Early treatment of ischemic stroke is one of the most effective ways to reduce brains' cell death and promote functional recovery. This study was designed to examine the effect of aerobic exercise on post ischemia/reperfusion injury on concentration and expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) after inducing a neuronal loss in CA1 region of hippocampus in Male Wistar rats. Three experimental groups including sham(S), ischemia/reperfusion-control (IRC) and ischemia/reperfusion exercise (IRE) were used for this purpose. The rats in the IRE group received a bilateral carotid artery occlusion treatment. They ran for 45 minutes on a treadmill five days per week for eight consecutive weeks. Cresyl violet (Nissl), Hematoxylin (H & E) and Eosin staining procedure were used to determine the extent of damage. A ladder rung walking task was used to assess the functional impairments and recovery after the ischemic lesion. ELISA and immunohistochemistry method were employed to measure BDNF and VEGF protein expressions. The result showed that the brain ischemia/reperfusion condition increased the cell death in hippocampal CA1 neurons and impaired motor performance on the ladder rung task whereas the aerobic exercise program significantly decreased the brain cell's death and improved motor skill performance. It was concluded that ischemic brain lesion decreased the BDNF and VEGF expression. It seems that the aerobic exercise following the ischemia/reperfusion potentially promotes neuroprotective mechanisms and neuronal repair and survival mediated partly by BDNF and other pathways.

Disassembly of Death-associated Protein Kinase and DANGER Interaction Mediates Hippocampal CA1 Neuron Death in Rat Cerebral Ischemic Reperfusion

Death-associated protein kinase (DAPK) is a Ca2+/CaM-regulated protein kinase that is involved in cell death processes by multiple pathways. It has been reported that DAPK may play a role in brain ischemia-induced neuronal death, but this mechanism is not well understood. DANGER, a membrane-associated protein that binds to DAPK physiologically, inhibits DAPK activation. In the present study, we used a transient global brain ischemia and reperfusion (I/R) rat model to investigate whether the interaction between DAPK and DANGER is involved in neuronal cell death following brain ischemia, and to reveal the mechanism of action. Our results indicate that the DAPK/DANGER interaction in the hippocampal CA1 region was significantly reduced after I/R with a peak reduction at 6 h. We further demonstrate that the NMDA inhibitor MK-801, DAPK inhibitor, or calcineurin inhibitor FK-506 prevented the dissociation of DANGER from DAPK 6 h after I/R. This was accompanied by a significantly decreased I/R-induced dephosphorylation of DAPK(ser-308), inhibiting DAPK catalytic activity. Moreover, the expression of DANGER and the interaction between DANGER and IP3R on the endoplasmic reticulum was significantly increased at I/R 6 h, which may be related to a reduction of DAPK/DANGER binding under I/R condition. Furthermore, MK-801, DAPK inhibitor and FK-506 had neuroprotective effects against hippocampal CA1 neuronal death 5 days after I/R. In conclusion, our data suggest that the dissociation of DANGER from DAPK may mediate DAPK activation, which is involved in DAPK-related neuronal death following I/R injury.

Embryonic lethal abnormal vision proteins and adenine and uridine-rich element mRNAs after global cerebral ischemia and reperfusion in the rat

Prolonged translation arrest correlates with delayed neuronal death of hippocampal CA1 neurons following global cerebral ischemia and reperfusion. Many previous studies investigated ribosome molecular biology, but mRNA regulatory mechanisms after brain ischemia have been less studied. Here we investigated the embryonic lethal abnormal vision/Hu isoforms HuR, HuB, HuC, and HuD, as well as expression of mRNAs containing adenine and rich uridine elements following global ischemia in rat brain. Proteomics of embryonic lethal abnormal vision immunoprecipitations or polysomes isolated from rat hippocampal CA1 and CA3 from controls or following 10 min ischemia plus 8 h of reperfusion showed distinct sets of mRNA-binding proteins, suggesting differential mRNA regulation in each condition. Notably, HuB, HuC, and HuD were undetectable in NIC CA1. At 8 h reperfusion, polysome-associated mRNAs contained 46.1% of ischemia-upregulated mRNAs containing adenine and rich uridine elements in CA3, but only 18.7% in CA1. Since mRNAs containing adenine and rich uridine elements regulation are important to several cellular stress responses, our results suggest CA1 is disadvantaged compared to CA3 in coping with ischemic stress, and this is expected to be an important contributing factor to CA1 selective vulnerability. (Data are available via ProteomeXchange identifier PXD004078 and GEO Series accession number GSE82146).

Nicotinamide mononucleotide inhibits post-ischemic NAD+ degradation and dramatically ameliorates brain damage following global cerebral ischemia

Nicotinamide adenine dinucleotide (NAD+) is an essential cofactor for multiple cellular metabolic reactions and has a central role in energy production. Brain ischemia depletes NAD+ pools leading to bioenergetics failure and cell death. Nicotinamide mononucleotide (NMN) is utilized by the NAD+ salvage pathway enzyme, nicotinamide adenylyltransferase (Nmnat) to generate NAD+. Therefore, we examined whether NMN could protect against ischemic brain damage. Mice were subjected to transient forebrain ischemia and treated with NMN or vehicle at the start of reperfusion or 30min after the ischemic insult. At 2, 4, and 24h of recovery, the proteins poly-ADP-ribosylation (PAR), hippocampal NAD+ levels, and expression levels of NAD+ salvage pathway enzymes were determined. Furthermore, animal's neurologic outcome and hippocampal CA1 neuronal death was assessed after six days of reperfusion. NMN (62.5mg/kg) dramatically ameliorated the hippocampal CA1 injury and significantly improved the neurological outcome. Additionally, the post-ischemic NMN treatment prevented the increase in PAR formation and NAD+ catabolism. Since the NMN administration did not affect animal's temperature, blood gases or regional cerebral blood flow during recovery, the protective effect was not a result of altered reperfusion conditions. These data suggest that administration of NMN at a proper dosage has a strong protective effect against ischemic brain injury.

Resveratrol protects CA1 neurons against focal cerebral ischemic reperfusion-induced damage via the ERK-CREB signaling pathway in rats

Ischemic stroke is a primary cause of mortality and disability in the aged population. Resveratrol (Res), a natural polyphenol enriched in plants, presents diverse biological activities, e.g., antiinflammatory and anti-oxidation effects. Here, we evaluated whether Res pretreatment influenced focal cerebral ischemia-induced cognitive impairment, and we explored the underlying mechanisms in rats. The results showed that a single administration of Res (30mg/kg, i.p.) at 1 or 4h, but not at 24h before focal cerebral ischemia exerted significant neuroprotective effects, including a reduction in hippocampal CA1 neuronal death and spatial cognition deficits caused by ischemia. The neuroprotective effects of Res were suppressed by pretreatment with MK801, an NMDA receptor blocker, or U0126, an extracellular signal regulated kinase 1/2 (ERK1/2) kinase inhibitor. A western blot analysis revealed that Res treatment at 1h before ischemia significantly increased ERK1/2 phosphorylation and cyclic-AMP response element binding protein (CREB) phosphorylation in the CA1 region of the hippocampus, which can be prevented with U0126 pretreatment. The results showed that the NMDA receptor-mediated ERK-CREB signaling pathway might participates in Res-induced neuroprotection in rats with focal cerebral ischemia.

Abstract 17177: Crosstalk and Synergism Between Local Brain-derived and Exogenous 17β-Estradiol in Regulating Rapid, Pro-survival Signaling in the Hippocampus Following Global Cerebral Ischemia

Cardiac arrest-induced global cerebral ischemia (GCI) can lead to death of hippocampal CA1 neurons and cognitive impairment. Previous work has shown that the steroid hormone, 17β-estradiol (E2) is strongly neuroprotective against GCI, an effect that involves, in part, rapid signaling via the membrane estrogen receptor, GPR30. To gain greater insight into E2 actions, the current study examined: 1) whether GPR30 expression in the hippocampus changes after CGI, and 2) the role and potential crosstalk/synergism between local (brain-derived) and exogenous E2 in regulation of GPR30 and rapid prosurvival signaling after GCI. GCI was induced in adult ovariectomized female rats by a 4-vessel occlusion model (12-min occlusion). Vehicle or low diestrous levels of E2 were administered chronically by minipump beginning at the time of ovariectomy. The results showed that GPR30 expression in the hippocampal CA1 was unchanged at 3h, but significantly decreased at 1d and 3d after GCI. In contrast, aromatase expression showed an initial decrease at 3h, a rebound at 1day, and a robust elevation at 3 days after CGI. Administration of exogenous E2 increased both GPR30 and aromatase expression at 3h, 1d and 3d after GCI. Interestingly, central administration of the aromatase inhibitor, letrozole or aromatase antisense (AS) oligonucleotides abolished exogenous E2 enhancement of GPR30 and aromatase at 3d after GCI. Furthermore, letrozole and aromatase-AS treatment attenuated exogenous E2-induced phosphorylation/activation of prosurvival ERK1/2 and CREB following GCI, as well as exogenous E2-induced enhanced expression of BDNF, PSD95 and synaptophsin. As a whole, the results suggest that local-derived E2 may help mediate beneficial protective effects of exogenous E2 after GCI to enhance GPR30 expression, increase activation of ERK1/2-CREB-BDNF pro-survival signaling, and improve synaptic function.

Protective mechanisms of CA074-me (other than cathepsin-B inhibition) against programmed necrosis induced by global cerebral ischemia/reperfusion injury in rats.

Many studies have demonstrated the key role of lysosomes in ischemic cell death in the brain and have led to the "lysosomocentric" hypothesis. In this hypothesis, the release of cathepsin-B due to a change of lysosomal membrane permeabilization (LMP) or rupture is critical, and this can be prevented by its inhibitors CA074 and CA074-me. However, the role of CA074-me in neuronal death and its effect on the change of lysosomal membrane integrity after global cerebral ischemia/reperfusion (I/R) injury is not clear, so we investigated this here. Rat hippocampal CA1 neuronal death was evaluated after 20-min global cerebral I/R injury. CA074-me (1 μg, 10 μg) were given intracerebroventricularly 1h before ischemia or 1h post reperfusion. The changes of heat shock protein 70 (Hsp70), cathepsin-B, lysosomal-associated membrane protein 1 (LAMP-1), receptor-interacting protein 3 (RIP3), and the change of lysosomal pH were evaluated respectively. Hippocampal CA1 neuronal programmed necrosis induced by global cerebral I/R injury was prevented by CA074-me both pre-treatment and post-treatment. Diffuse cytoplasmic cathepsin-B and LAMP-1 immunostaining synchronized with the pyknotic nuclear changes 2 days post reperfusion, and a rise of lysosomal pH with the leakage of DND-153, a dye of lysosomes, after oxygen-glucose deprivation (OGD) was detected. Both of these changes demonstrated the rupture of lysosomal membrane and the leakage of cathepsin-B, and this was strongly inhibited by CA074-me pre-treatment. The overexpression and nuclear translocation of RIP3 and the reduction of NAD(+) level after I/R injury were also inhibited, while the upregulation of Hsp70 was strengthened by CA074-me pre-treatment. Delayed fulminant leakage of cathepsin-B due to lysosomal rupture is a critical harmful factor in neuronal programmed necrosis induced by 20-min global I/R injury. In addition to being an inhibitor of cathepsin-B, CA074-me may have an indirect neuroprotective effect by maintaining lysosomal membrane integrity and protecting against lysosomal rupture.

Ischemic Preconditioning Mediates Neuroprotection against Ischemia in Mouse Hippocampal CA1 Neurons by Inducing Autophagy.

The hippocampal CA1 region is sensitive to hypoxic and ischemic injury but can be protected by ischemic preconditioning (IPC). However, the mechanism through which IPC protects hippocampal CA1 neurons is still under investigation. Additionally, the role of autophagy in determining the fate of hippocampal neurons is unclear. Here, we examined whether IPC induced autophagy to alleviate hippocampal CA1 neuronal death in vitro and in vivo with oxygen glucose deprivation (OGD) and bilateral carotid artery occlusion (BCCAO) models. Survival of hippocampal neurons increased from 51.5% ± 6.3% in the non-IPC group (55 min of OGD) to 77.3% ± 7.9% in the IPC group (15 min of OGD, followed by 55 min of OGD 24 h later). The number of hippocampal CA1 layer neurons increased from 182 ± 26 cells/mm2 in the non-IPC group (20 min of BCCAO) to 278 ± 55 cells/mm2 in the IPC group (1 min × 3 BCCAO, followed by 20 min of BCCAO 24 h later). Akt phosphorylation and microtubule-associated protein light chain 3 (LC3)-II/LC3-I expression were increased in the preconditioning group. Moreover, the protective effects of IPC were abolished only by inhibiting the activity of autophagy, but not by blocking the activation of Akt in vitro. Using in vivo experiments, we found that LC3 expression was upregulated, accompanied by an increase in neuronal survival in hippocampal CA1 neurons in the preconditioning group. The neuroprotective effects of IPC on hippocampal CA1 neurons were completely inhibited by treatment with 3-MA. In contrast, hippocampal CA3 neurons did not show changes in autophagic activity or beneficial effects of IPC. These data suggested that IPC may attenuate ischemic injury in hippocampal CA1 neurons through induction of Akt-independent autophagy.

17-AAG post-treatment ameliorates memory impairment and hippocampal CA1 neuronal autophagic death induced by transient global cerebral ischemia

Neuro-inflammation plays an important role in global cerebral ischemia (GCI). The 72-kDa heat shock protein (Hsp70) has been reported to be involved in the inflammatory response of many central nervous system diseases. Preclinical findings implicate that 17-allylamino-demethoxygeldanamycin (17-AAG), an anticancer drug in clinical, provide neuroprotection actions in a rat model of traumatic brain injury, and the beneficial effects of 17-AAG were specifically due to up-regulation of Hsp70. However, no experiments have tested whether 17-AAG has beneficial or harmful effects in the setting of GCI. The present study was designed to determine the hypothesis that administration of 17-AAG could attenuate cerebral infarction and improve neuronal survival, thereby ameliorating memory impairment in a rat model of GCI. Furthermore, to test whether any neuroprotective effect of 17-AAG was associated with inflammatory response and neuronal autophagy, we examined the expression of multiplex inflammatory cytokine levels as well as autophagy-associate protein in hippocampal CA1 of rat brain. Our results showed that post-GCI administration of 17-AAG significantly protected rats against GCI induced brain injury, and 17-AAG is also an effective antagonist of the inflammatory response and thereby ameliorates hippocampal CA1 neuronal autophagic death. We therefore believe that the present study provides novel clues in understanding the mechanisms by which 17-AAG exerts its neuroprotective activity in GCI. All data reveal that 17-AAG might be a potential neuroprotective agent for ischemic stroke.

(+)-Naltrexone is neuroprotective and promotes alternative activation in the mouse hippocampus after cardiac arrest/cardiopulmonary resuscitation.

Despite dramatic improvement in cardiopulmonary resuscitation (CPR) and other techniques for cardiac arrest (CA), the majority of survivors continue to show signs of decreased memory or executive cognitive function. Such memory impairment may be due to hippocampal CA1 neuronal death, which is delayed by several days after CA/CPR. Classical microgliosis in the CA1 region may contribute to neuronal death, yet the role of a key activation receptor Toll Like Receptor 4 (TLR4) has not been previously investigated for such neuronal death after CA/CPR. We show that (+)-naltrexone was neuroprotective after CA/CPR. TLR4 blockade was associated with decreased expression of markers for microglial/macrophage activation and T cell and B cell infiltration, as well as decreased pro-inflammatory cytokine levels. Notably, IL-10 expression was elevated in response to CA/CPR, but was not attenuated by (+)-naltrexone, suggesting that the local monocyte/microglial phenotype had shifted towards alternative activation. This was confirmed by elevated expression of Arginase-1, and decreased expression of NFκB p65 subunit. Thus, (+)-naltrexone and other TLR4 antagonists may represent a novel therapeutic strategy to alleviate the substantial burden of memory or executive cognitive function impairment after CA/CPR.

Enhancement of Hippocampal Ca1 Neuronal Death On the Line of Ageing in Rats Brain

Aging related with specific numbers of disorders, including from cognitive decline to Parkinson's disease, Alzheimer's disease, schizophrenia, and even depression, which mostly are similar to those caused by hippocampal cell damages. There is an increasing concern about age related disorders. There may be several complex circuits, which involve hippocampus in the mechanisms of aforementioned disorders. Moreover, hippocampus appears to be an early target of stress and hormones, excitatory amino acids and a bunch of neurotransmitters as well as age-related structural changes. Hence, the study of hippocampal anatomical changes during aging results in better understanding of age-related brain disorders. Previous study showed ionic, channel as well as receptors disturbance in CA1 hippocampal region during aging process. In the present experiment the structural changes of hippocampal CA1 region of aged rat were studied. In this study 21 male Wistar rats with the age of 3, 6, and 12 months were deeply anesthetized and perfused transcardially, the brain of each rat was fixed. Histological assessments were done on hippocampal region and mean number of cell death were analyzed. Mean numbers of neural cell death in CA1 hippocampal region was significantly increased in the third and twelfth months compared to the month 6. However, there was no statistical significant difference between the third and twelfth month rats in their hippocampal cell death. Concluded those middle age rats are less vulnerability to cell death. Data showed that CA1 related disorder may be less abundant in middle age rats compare to younger and older age animals.

Consistent delayed unilateral neuronal death after modified transient focal cerebral ischemia in mice that mimics neuronal injury after transient global cerebral ischemia.

Numerous studies have attempted to reveal the pathophysiology of ischemic neuronal injury using a representative transient global cerebral ischemia (tGCI) model in rodents; however, most of them have used gerbil or rat models. Recent advances in transgene and gene-knockout technology have enabled the precise molecular mechanisms of ischemic brain injury to be investigated. Because the predominant species for the study of genetic mutations is the mouse, a representative mouse model of tGCI is of particular importance. However, simple mouse models of tGCI are less reproducible; therefore, a more complex process or longer duration of ischemia, which causes a high mortality rate, has been used in previous tGCI models in mice. In this study, the authors aimed to overcome these problems and attempted to produce consistent unilateral delayed hippocampal CA1 neuronal death in mice. C57BL/6 mice were subjected to short-term unilateral cerebral ischemia using a 4-mm silicone-coated intraluminal suture to obstruct the origin of the posterior cerebral artery (PCA), and regional cerebral blood flow (rCBF) of the PCA territory was measured using laser speckle flowmetry. The mice were randomly assigned to groups of different ischemic durations and histologically evaluated at different time points after ischemia. The survival rate and neurological score of the group that experienced 15 minutes of ischemia were also evaluated. Consistent neuronal death was observed in the medial CA1 subregion 4 days after 15 minutes of ischemia in the group of mice with a reduction in rCBF of < 65% in the PCA territory during ischemia. Morphologically degenerated cells were mostly positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and cleaved caspase 3 staining 4 days after ischemia. The survival rates of the mice 24 hours (n = 24), 4 days (n = 15), and 7 days (n = 7) after being subjected to 15 minutes of ischemia were 95.8%, 100%, and 100%, respectively, and the mice had slight motor deficits. The authors established a model of delayed unilateral hippocampal neuronal death in C57BL/6 mice by inducing ischemia in the PCA territory using an intraluminal suture method and established inclusion criteria for PCAterritory rCBF monitored by laser speckle flowmetry. This model may be useful for investigating the precise molecular mechanisms of ischemic brain injury.

Abstract T P84: Neuroprotective Effect of Probenecid in a Rat Model of Transient Global Cerebral Ischemia-Reperfusion

Background and Purpose: Probenecid (PROB) has been used for decades to treat gouty arthritis with few side effects and recent studies revealed that it is also a specific inhibitor of pannexin-1 channel. Panx1 channel was activated by ischemic injury and inhibition of the panx1 channel maybe efficacious in stroke treatment. However, the role of PROB in cerebral ischemia /reperfusion (I/R) injury remains unclear. The aim of this study was to investigate the role of PROB in the transient global cerebral I/R injury in rats and its protective mechanisms. Methods: Twenty minutes of transient global cerebral I/R was induced using the four-vessel occlusion (4-VO) method in rats. PROB was given in the different dose, time and administration routes to verify its neuroprotective effects. Neuronal death in the hippocampal CA1 region was observed using H &amp; E staining 7 days after ischemia. Molecular mechanisms of activation of calpain-cathepsin pathway and inflammatory cells by I/R injury were also investigated. Results: Treatment with PROB (0.1, 1 and 10 mg/kg ) 10 min before ischemia protected against I/R-induced hippocampal CA1 neuronal death significantly, and 1 mg/kg has best protective effect. Post-insult treatment 2h after reperfusion also protected against neuronal death and prolonged use for continuous 7 days could improve its protective effects compared to the single use 6h after reperfusion.Furthermore,oral administration also had protective effect. Cathepsin B expression was inceased significantly in CA-1 region after ischemia and PROB treatment could inhibit its expression. Expression of both calpain-1 and hsp70 at 1d ,2d and 3d after reperfusion were upregulated, whereas the expression of calpain-1 was inhibited and hsp70 was strengthened by pre-treatment with PROB. Prolonged PROB treatment suppressed the activation of microglia and astrocytes, reduced the number of microglia in CA1 region. Conclusions: Our study indicates that PROB protects against transient global cerebral I/R injury administrated before ischemia and even 6h after reperfusion by reducing calpain-1 expression , inhibiting lysosomal rupture and the activation of the glia, which suggests RPOB may be a promising therapeutic drug for clinical treatment of ischemic cerebral injury.

EGB1212 Post-treatment Ameliorates Hippocampal CA1 Neuronal Death and Memory Impairment Induced by Transient Global Cerebral Ischemia/Reperfusion

Extracts of Ginkgo biloba have been used in traditional medicines for centuries, and have potential for clinical applications in cerebral ischemia/reperfusion injury. However, standardized extracts have proven protective only as pre-treatments, and the major mechanisms of action remain unclear. We explored the potential of the novel extract EGB1212, which meets the United States Pharmacopeia (USP) 31 standardization criteria for pharmaceutical use, as a post-treatment after global cerebral ischemia/reperfusion (GCI/R) injury in a rat model. The pre-treated group was administered EGB1212 for 7 d prior to common carotid artery occlusion (i.e., ischemia, for 20 min). Post-treated rats received the same but starting 2 h after ischemia and continuing for 7 d. Seven days after GCI/R, brains of each group were processed for H&E staining of hippocampal CA1 neurons. Remaining rats underwent the Morris water maze and Y-maze tests of spatial learning and memory, beginning eight days after reperfusion. To assess hippocampal autophagy, light chain (LC)-3-I/LC3-II and Akt/pAkt were determined via a Western blot of rat hippocampi harvested 12, 24, or 72 h after reperfusion. EGB1212 pre- and post-treatments both improved neuronal survival and spatial learning and memory functions. Pre-treatment effectively reduced LC3-II levels and post-treatment resulted in significantly elevated pAkt levels. We conclude that EGB1212 exerted significant neuroprotection in GCI/R in both preventative and post-treatment settings. This extract shows great potential for clinical applications.

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Introduction: Cardiac arrest (CA) is an important cause of morbidity and mortality in the adult and pediatric populations. Women and adult female animals demonstrate better outcomes after CA compared to male counterparts, due in large part to their higher levels of circulation estrogen (E2). Consistent with this, E2 treatment is neuroprotective in adult male and female animals following CA via estrogen receptor? (ER?) binding. We investigated the neuroprotective effects of treatment with E2 and ER? agonist DPN in our novel mouse model of pediatric CA. Hypothesis: E2 treatment after CA/cardiopulmonary resuscitation will reduce CA1 hippocampal neuronal death via ER? signaling in male and female juvenile mice. Methods: Male and female juvenile (prepubertal; postnatal day 20-25) mice (C57Bl/6, n=18 and 20, respectively) underwent CA induced by intravenous potassium chloride injection. Following 6 min of ischemia, mice were resuscitated with mechanical ventilation, epinephrine, and chest compressions. 30 min after return of circulation, mice were treated with intravenous E2 (100 µg/kg), ER? agonist diarylpropiolnitrile (DPN, 240 µg/kg), or vehicle. Histopathology with hematoxylin and eosin staining for analysis of CA1 hippocampal neuronal death was performed 3 d after CA/CPR. Neuronal death was calculated as % dead/total neurons, data expressed as mean±SEM. Results: Male and female juvenile mice treated with vehicle exhibited a similar degree of hippocampal CA1 neuronal death after CA/CPR (74 ± 3 and 68 ± 6%, respectively). Treatment with E2 resulted in significantly less neuronal death in male but not female mice (40 ± 11 and 58 ± 6%, respectively). DPN treatment did not provide significant neuroprotection to either male or female mice (64 ± 4% and 52 ± 14%, respectively). Conclusions: Our findings suggest that E2-mediated protection in the juvenile brain after CA/CPR occurs via a different receptor than in the adult brain, and demonstrates gender specificity that is unique to this prepubertal population. We are now investigating the neuroprotective effects of other estrogen receptors, ER? and the non-classical ER GPR30 to determine the mechanism of E2 neuroprotection observed in juvenile males.

Inhibition of Nitric Oxide Production and Inducible Nitric Oxide Synthase Expression by a Polymethoxyflavone from Young Fruits ofCitrus unshiuin Rat Primary Astrocytes

Abnormal activation of astrocytes (e.g., the overproduction of cytokines and nitric oxide) is relevant to neurodegenerative disease. It is important, therefore, to search for inhibitors of the abnormal activation of astrocytes that can be derived from natural substances. This study focused on the effects of extracts from young fruits of Citrus unshiu on lipopolysaccharide (LPS)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in rat primary astrocytes. The methanol extract of young citrus inhibited NO production in a concentration-dependent manner. After reverse-phase extraction of the extract, we found that polymethoxyflavone, nobiletin, 3,5,6,7,8,3',4'-heptamethoxyflavone, and tangeletin inhibited NO production by primary astrocytes. These polymethoxyflavones also inhibited LPS-induced iNOS protein and mRNA expression by suppressing nuclear factor-κB (NF-κB) activation and p38-mitogen-activated protein kinase (MAPK) phosphorylation. To evaluate possible applications of these neuroprotective agents in vivo, we examined the effects of young citrus fruit on delayed neurodegeneration in hippocampal CA1 neurons of the Mongolian gerbil after global ischemia. Oral administration of young citrus fruit significantly suppressed delayed neuronal death in hippocampal CA1 neurons. This suggests a possible application of young citrus fruit as a neuroprotective agent.

Neuron-Specific Prolyl-4-Hydroxylase Domain 2 Knockout Reduces Brain Injury After Transient Cerebral Ischemia

Numerous factors involved in the adaptive response to hypoxia, including erythropoietin and vascular endothelial growth factor are transcriptionally regulated by hypoxia-inducible factors (HIFs). During normoxia, prolyl-4-hydroxylase domain (PHD) proteins hydroxylate HIF-α subunits, resulting in their degradation. We investigated the effect of neuronal deletion of PHD2, the most abundant isoform in brain, for stroke outcome. We generated neuron-specific Phd2 knockout mice and subjected animals to systemic hypoxia or transient middle cerebral artery occlusion. Infarct volume and cell death were determined by histology. HIF-1α, HIF-2α, and HIF target genes were analyzed by immunoblotting and real-time polymerase chain reaction, respectively. Neuron-specific ablation of Phd2 significantly increased protein stability of HIF-1α and HIF-2α in the forebrain and enhanced expression of the neuroprotective HIF target genes erythropoietin and vascular endothelial growth factor as well as glucose transporter and glycolysis-related enzymes under hypoxic and ischemic conditions. Mice with Phd2-deficient neurons subjected to transient cerebral ischemia exhibited a strong reduction in infarct size, and cell death of hippocampal CA1 neurons located in the peri-infarct region was dramatically reduced in these mice. Vessel density in forebrain subregions, except for caudate-putamen, was not altered in Phd2-deficient animals. Our findings denote that the endogenous adaptive response on hypoxic-ischemic insults in the brain is at least partly dependent on the activity of HIFs and identify PHD2 as the key regulator for the protective hypoxia response. The results suggest that specific inhibition of PHD2 may provide a useful therapeutic strategy to protect brain tissue from ischemic injury.

Coumestrol has neuroprotective effects before and after global cerebral ischemia in female rats

Global ischemia arising during cardiac arrest or cardiac surgery causes highly selective, delayed death of hippocampal CA1 neurons. Phytoestrogens are naturally occurring plant-derived compounds that are present in the human diet and are considered selective estrogen receptor (ER) modulators. The phytoestrogen coumestrol is a potent isoflavonoid, with binding affinities for both ER-α and ER-β that are comparable to those of 17b-estradiol. The present study examined the hypothesis that coumestrol protects hippocampal neurons in ovariectomized rats in a model of cerebral global ischemia. Ovariectomized rats were subjected to global ischemia (10min) or sham surgery and received a single intracerebroventricular or peripheral infusion of 20μg of coumestrol, 20μg of estradiol or vehicle 1h before ischemia or 0h, 3h, 6h or 24h after reperfusion. Estradiol and coumestrol afforded significant neuroprotection in all times of administration, with the exception of estradiol given 24h after the ischemic insult. Animals received icv infusion of the broad-spectrum ER antagonist ICI 182,780 (50μg) or vehicle into the lateral ventricle just before the E2 or coumestrol administration. The ER antagonist abolished estradiol protection, consistent with a role of classical ERs. In contrast, ICI 182,780 effected only partial reversal of the neuroprotective actions of coumestrol, suggesting that other cellular mediators in addition to classical ERs may be important. Additional research is needed to determine the molecular targets mediating the neuroprotective action of coumestrol and the therapeutic potential of this phytoestrogen in the mature nervous system.

N-terminally cleaved Bcl-xL mediates ischemia-induced neuronal death

Transient global ischemia in rats induces delayed death of hippocampal CA1 neurons. Early events include caspase activation, cleavage of anti-death Bcl-2 family proteins and large mitochondrial channel activity. However, a causal role of these events in ischemia-induced neuronal death is unclear. Unexpectedly, we found that the Bcl-2/Bcl-xL inhibitor ABT-737, which enhances death of tumor cells, protects rats against neuronal death in a clinically relevant model of brain ischemia. Bcl-xL is prominently expressed in adult neurons and can be cleaved by caspases to generate a pro-death fragment ΔN-Bcl-xL. We found that ABT-737 administered before or after ischemia inhibited ΔN-Bcl-xL-induced mitochondrial channel activity and neuronal death. To establish a causal role for ΔN-Bcl-xL, we generated knockin mice expressing caspase-resistant Bcl-xL. The knockin mice exhibit strikingly reduced mitochondrial channel activity and reduced vulnerability to ischemia-induced neuronal death. These findings point to truncated Bcl-xL as a potentially important therapeutic target in ischemic brain injury.

Mouse Transient Global Ischemia Two-Vessel Occlusion Model.

Transient global ischemia in rodents induces delayed death of hippocampal CA1 neurons, as well as in some hilar neurons of the dentate gyrus, medium aspiny neurons of the striatum, pyramidal neurons in neocortical layers II, V and VI, and Purkinje neurons of the cerebellum. In contrast to focal ischemia that mimics regional stroke in humans, this model of global ischemia mimics the brain injury that occurs after human cardiac arrest. Early events include caspase activation, cleavage of anti-death Bcl-2 family proteins and large mitochondrial channel activity. Genetically engineered mice provide opportunities for study such as the knock-in mouse expressing a caspase-resistant form of Bcl-xL found to exhibit markedly reduced mitochondrial channel activity and reduced vulnerability to ischemia-induced neuronal death1. It is therefore relevant to adapt and develop a simple protocol for producing transient global ischemia in mouse2. The two-vessel occlusion model has been specifically developed to provide optimal outcomes in mouse and offers several advantages over the four-vessel occlusion model traditionally used in rat including the relative ease of the procedure as well as only a single day of surgery. However it should be noted that this procedure has a higher morbidity rate compared to other ischemia models as well as a higher degree of variability. These two disadvantages necessitate the use of a larger cohort of animals, which for many healthy breeding transgenic animals is a non-deterring factor.