Transient Global Brain Ischemia Research Articles

Expression of fatty acid‐binding proteins in adult hippocampal neurogenic niche of postischemic monkeys

Intracellular fatty acid (FA) chaperones known as FA-binding proteins (FABPs) are a group of molecules known to participate in cellular metabolic processes such as lipid storage, membrane synthesis, and β-oxidation or to coordinate transcriptional programs. However, their role in adult neurogenesis still remains obscure. The FABPs expressed in the central nervous system (CNS) are heart-type (FABP3), epidermal-type (FABP5), and brain-type (FABP7). These three FABPs possess a differential affinity for polyunsaturated fatty acids (PUFAs). Recently, we reported that GPR40, a receptor for free FAs and particularly for PUFAs, is expressed in the CNS of adult monkeys and upregulated after transient global brain ischemia in the hippocampal subgranular zone (SGZ), a neurogenic niche in adulthood. The SGZ showed a peak proliferation of progenitor cells and maximal expression of GPR40 during the second week after ischemia. As both FABPs and GPR40 might be closely related to the adult neurogenesis, here, we studied the expression of FABP 3, 5, and 7 in the SGZ, comparing normal and postischemic adult monkeys. Immunoblotting revealed that FABP5 and FABP7, but not FABP3, were significantly increased on day 15 after ischemia when compared with the nonischemic control. Immunohistochemistry showed that FABP5 was almost undetectable in the control SGZ but was abundant on day 15 after ischemia. FABP 3, 5, and 7 were expressed in S-100β-positive astrocytes and nestin-positive neural progenitors. However, only FABP 5 and 7 were found in bromodeoxyuridine (BrdU)-positive newly generated cells. FABPs were most frequently coexpressed with the S-100β-positive astrocytes, whereas βIII-tubulin-or polysialylated neural cell-adhesion molecule (PSA-NCAM)-positive newborn neurons in the vicinity of the astrocytes expressed none of the three FABPs. These results support a role of astrocyte- and/or neural progenitor-derived FABPs as components of the molecular machine regulating the progenitor cell niche in the adult primate brain.

EMF-1: Combination Therapy for Ischemic Brain Injury After Cardiac Arrest

This project focuses on evaluating combination therapy for global brain ischemia after resuscitation from cardiac arrest. The complexity of brain ischemia indicates that combination therapy will be required to intervene in the cascade of multiple, independently-lethal processes that ultimately result in neuronal death. We hypothesize that combination therapy utilizing hypothermia and ZGF-1, multiply-targeted to excitotoxicity, oxidative stress, and apoptosis, will improve neuronal survival and neurobehavioral outcome after transient global brain ischemia, to a degree greater than either treatment alone.

PKR-like endoplasmic reticulum kinase (PERK) activation following brain ischemia is independent of unfolded nascent proteins

Transient global brain ischemia results in an immediate inhibition of protein translation upon reperfusion. During early brain reperfusion protein synthesis is inhibited by alpha subunit of eukaryotic initiation factor 2 (eIF2α) phosphorylation by the PKR-like endoplasmic reticulum kinase (PERK). Normally, PERK is held in an inactive, monomeric state by the binding of the endoplasmic reticulum (ER) chaperone GRP78 to the lumenal end of PERK. The prevailing view is that ER stress leads to the accumulation of unfolded proteins in the ER lumen. GRP78 dissociates from PERK to bind these accumulated unfolded proteins, leading to PERK activation, phosphorylation of eIF2α, and inhibition of translation. To determine if an increase in unfolded nascent proteins following transient brain ischemia contributes to PERK activation, protein synthesis was blocked by intracerebral injection of anisomycin prior to induction of ischemia. Anisomycin inhibited protein synthesis by over 99% and reduced newly synthesized proteins in the ER to ∼20% of controls. With an ER nearly devoid of newly synthesized proteins, PERK was still activated and was able to phosphorylate eIF2α in CA1 neurons during reperfusion. These data strongly argue that PERK activation is independent of the large increase in unfolded nascent proteins within the ER following transient global brain ischemia.

Morgana/CHP-1 is a novel chaperone able to protect cells from stress

Morgana/CHP-1 (CHORD containing protein-1) has been recently shown to be necessary for proper cell divisions. However, the presence of the protein in postmitotic tissues such as brain and striated muscle suggests that morgana/CHP-1 has additional cellular functions. Here we show that morgana/CHP-1 behaves like an HSP90 co-chaperone and possesses an independent molecular chaperone activity towards denatured proteins. The expression time profile of morgana/Chp-1 in NIH3T3 cells in response to heat stress is similar to that of Hsp70, a classical effector of Heat Shock Factor-1 mediated stress response. Moreover, overexpression of morgana/CHP-1 in NIH3T3 cells leads to the increased stress resistance of the cells. Interestingly, morgana/Chp-1 upregulation in response to transient global brain ischemia lasts longer in ischemia-resistant regions of the gerbil hippocampus than in vulnerable ones, suggesting the involvement of morgana/CHP-1 in natural protective mechanisms in vivo.

Blocking TRAIL-DR5 signaling with soluble DR5 reduces delayed neuronal damage after transient global cerebral ischemia

Mechanisms underlying delayed selective neuronal death after global cerebral ischemia remain to be clarified. Here, we report a critical role for tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in the pathogenesis of cerebral ischemia. C57BL/6j mice were subjected to transient global brain ischemia. RT-PCR and immunohistochemistry showed that the expression of TRAIL and DR5 was upregulated following transient ischemia–reperfusion. Dual immunofluorescence analysis indicated that TRAIL expression was significantly more pronounced in astrocytes and activated microglia/macrophages, whereas DR5 expression was more pronounced in neurons, which had a good correlation with the distribution of apoptotic cells. Treatment with soluble DR5 reduced ischemic cell death after transient global ischemia through blocking the interaction of endogenous TRAIL with DR5. These results indicate that TRAIL plays a deleterious role in the pathogenesis of delayed neuronal damage after global cerebral ischemia and inhibition of TRAIL function in the brain may represent a novel neuroprotective strategy to treat ischemic stroke.

Insulin activates the PI3K-Akt survival pathway in vulnerable neurons following global brain ischemia

Insulin is neuroprotective following transient global brain ischemia; however, the mechanisms by which insulin exerts its salutary effects remain unclear.Objective: We assessed insulin's effect on the PI3K-Akt survival system and consequent modulation of the pro-apoptotic proteins Bim, Bad and FoxO3a.Methods: We utilized rats subjected to 10 minutes of global brain ischemia, with or without insulin administered at the onset of reperfusion.Results: In sham-operated animals, minimal pAkt immunofluorescence was detected in the CA1. Moreover, at 30 minute reperfusion, there was no change in pAkt in CA1 neurons. Single bolus high-dose insulin treatment resulted in an early increase in pAkt after 30 minutes, preservation of CA1 neurons to 14 days of reperfusion and preservation of spatial learning ability. Insulin treatment increased cytoplasmic and nuclear staining for pAkt in both CA1 and cortex. Insulin-induced Akt phosphorylation was suppressed by the PI3K inhibitor wortmannin. Neither reperfusion nor insulin induced any change in the phosphorylation or subcellular localization of FoxO3a, Bim or Bad. A single bolus of high-dose insulin reduced CA1 neuronal cell death and thus represents a potential therapeutic intervention for global brain ischemia.Discussion: These results reveal that proximal elements of a known cell-survival pathway are triggered by high-dose insulin during early reperfusion. Insulin induces robust PI3K-dependent phosphorylation of Akt by 30 minute reperfusion and results in improvement of hippocampal structure and function. However, the Akt substrates FoxO3a, Bim and Bad do not undergo corresponding changes in phosphorylation or subcellular localization in this model of global brain ischemia. The downstream components of insulin-induced Akt survival signaling after transient global brain ischemia remain to be identified.

EMF-5: Combination Therapy for Ischemic Brain Injury After Cardiac Arrest

This project focuses on evaluating combination therapy for global brain ischemia after resuscitation from cardiac arrest. This approach is likely to also have implications for the treatment of focal ischemic stroke. The complexity of these disorders indicates that combination therapy will be required to intervene in the cascade of multiple, independently lethal biochemical reactions that ultimately result in neuronal death. We hypothesize that combination therapy utilizing hypothermia and IGF-1, multiply targeted to excitotoxicity, oxidative stress, and apoptosis, will improve neuronal survival and neurobehavioral outcome after transient global brain ischemia, to a degree greater than either treatment alone.

Neuroprotective effects of a standardized flavonoid extract from Diospyros kaki leaves

Ethnopharmacological relevance Flavonoids, extracted from the leaves of Diospyros kaki, are the main therapeutic components of NaoXingQing (NXQ), a potent and patented Chinese herbal remedy widely used in China for the treatment of apoplexy syndrome. Aim of the study To investigate the neuroprotective effects of FLDK-P70, a standardized flavonoid extract, using in vivo rat models of both focal ischemia/reperfusion (I/R) injury induced by middle cerebral artery occlusion (MCAO) and on transient global brain ischemia induced by four-vessel occlusion (4-VO). We also aim to examine the effects of FLDK-P70 on glutamate-induced cell injury of hippocampal neurons as well as on hypoxia-induced injury of cortical neurons in primary cell culture. Materials and methods and results Administration of FLDK-P70 for 12 days (40, 80 mg/kg body weight, p.o., 5 days before and 7 days after 4-VO) increased the survival of hippocampal CA1 pyramidal neurons after transient global brain ischemia. Similarly, administration of FLDK-P70 for 7 days (40, 80 mg/kg body weight, p.o., 3 days before and 4 days after MCAO) significantly reduced the lesion of the insulted brain hemisphere and improved the neurological behavior of rats. In primary rat hippocampal neuronal cultures, pretreatment with FLDK-P70 (5, 10 μg/ml) protected neurons from glutamate-induced excitotoxic neuronal death in a dose-dependent manner. In primary rat cerebral cortical neuronal culture, pretreatment with FLDK-P70 (25, 100 μg/ml) also reduced hypoxia–reoxygen induced neuronal death and apoptosis in a dose-dependent manner. Conclusions These in vivo and in vitro results suggest that FLDK-P70 significantly protects rats from MCAO and 4-VO ischemic injury in vivo and protects hippocampal neurons from glutamate-induced excitotoxic injury as well as cortical neurons from hypoxia-induced injury in vitro. The mechanisms of these effects may be due to the antioxidative activity of the flavonoids. These results convincingly demonstrate that FLDK-P70 may be useful for the prevention and treatment of ischemia/reperfusion injury and other related neurodegenerative diseases.

Post-ischemic hypothermia attenuates loss of the vascular basement membrane proteins, agrin and SPARC, and the blood–brain barrier disruption after global cerebral ischemia

Vascular basement membrane (BM) stabilizes brain vessels and inhibits endothelial cell cycle. Cerebral ischemia causes BM breakdown with the loss of structural BM components including collagens and laminins. In this study, the expression changes of the BM proteoglycan agrin, and the non-structural BM constituent SPARC (BM-40, osteonectin), were studied in brain vessels after global cerebral ischemia. A transient 20-min forebrain ischemia followed by 1, 6 or 24 h of reperfusion was induced in adult Sprague–Dawley rats by combined bilateral common carotid artery occlusion and hypotension (42–45 mm Hg). In a separate group of animals, a mild (32 °C) post-ischemic hypothermia was induced for 6 h, starting immediately after ischemia. RNA from ∼ 500 brain vessels (20–100 μm) extracted by laser-capture microdissection (LCM) microscopy was used to determine the expression of proteoglycans agrin and SPARC mRNAs by quantitative PCR (Q-PCR). Protein expression was determined by immunohistochemistry in adjacent tissue sections. The BBB permeability was assessed using 3H-sucrose as an in vivo tracer and by examining fibrinogen immunoreactivity in tissue sections. A transient global brain ischemia resulted in a significant (ANOVA, p < 0.05; 6 animals/group) reduction in agrin and SPARC mRNAs in LCM-captured brain vessels 24 h after reperfusion. A time-dependent loss of agrin and SPARC from the BM during reperfusion was also observed by immunochemistry. A 6-h post-ischemic hypothermia reduced SPARC and agrin mRNA and protein losses, BBB transfer constant for 3H-sucrose as well as fibrinogen extravasation 24 h after reperfusion. It is conluded that a transient post-ischemic hypothermia stabilizes brain vessels and reduces BBB disruption in part by preventing proteolytic degradation of regulatory BM constituents, SPARC and agrin.

Neuroprotection with delayed calpain inhibition after transient forebrain ischemia

Delayed neurodegeneration after transient global brain ischemia offers a therapeutic window for inhibiting molecular injury mechanisms. One such mechanism is calpain-mediated proteolysis, which peaks 24 to 48 hrs after transient forebrain ischemia in rats. This study tests the hypothesis that delayed calpain inhibitor therapy can reduce brain calpain activity and neurodegeneration after transient forebrain ischemia. Prospective randomized placebo-controlled animal trial. University research laboratory. Adult male Long-Evans rats. Rats subjected to 10-min transient forebrain ischemia were randomized to intravenous infusion of calpain inhibitor CEP-3453 or vehicle beginning 22 hrs after injury. In a dose-response study, a 60 mg/kg bolus followed by 30 mg/kg infusion was required to reduce postischemic brain calpain activity measured by Western blot of hippocampal homogenates at 48 hrs after injury. The same dosing protocol decreased degeneration of CA1 pyramidal neurons measured at 72 hrs after injury. These results suggest a causal role for calpains in delayed postischemic neurodegeneration, and demonstrate a broad therapeutic window for calpain inhibition in this model.

Insulin blocks cytochrome c release in the reperfused brain through PI3‐K signaling and by promoting Bax/Bcl‐XL binding

The critical event of the intrinsic pathway of apoptosis following transient global brain ischemia is the release of cytochrome c from the mitochondria. In vitro studies have shown that insulin can signal specifically via phosphatidylinositol-3-OH-kinase (PI3-K) and Akt to prevent cytochrome c release. Therefore, insulin may exert its neuroprotective effects during brain reperfusion by blocking cytochrome c release. We hypothesized that insulin acts through PI3-K, Akt, and Bcl-2 family proteins to inhibit cytochrome c release following transient global brain ischemia. We found that a single bolus of insulin given immediately upon reperfusion inhibited cytochrome c release for at least 24 h, and produced a fivefold improvement in neuronal survival at 14 days. Moreover, insulin's ability to inhibit cytochrome c release was completely dependent on PI3-K signaling and insulin induces phosphorylation of Akt through PI3-K. In untreated animals, there was an increase in mitochondrial Bax at 6 h of reperfusion, and Bax binding to Bcl-X(L) was disrupted at the mitochondria. Insulin prevented both these events in a PI3-K-dependent manner. In summary, insulin regulates cytochrome c release through PI3-K likely by activating Akt, promoting the binding between Bax and Bcl-X(L), and by preventing Bax translocation to the mitochondria.

Expression of matrix metalloproteinases in the neurogenic niche of the adult monkey hippocampus after ischemia

Matrix metalloproteinases (MMPs), zinc-dependent endopeptidases capable of remodeling extracellular matrix and regulating cellular signals, have been implicated in various neurological functions and diseases. However, the role of MMPs in the adult neurogenesis still remains to be clarified, particularly in the primate. Here, we studied differential expression of MMP9/2 in the neurogenic niche of the hippocampal dentate gyrus (DG) after transient global brain ischemia in young adult macaque monkeys. Zymography demonstrated biphasic upregulation of MMP9 in acute (Days 1-3) and delayed (Days 7-15) phases of postischemic reaction, whereas the level of MMP2 was elevated only in the delayed phase. Immunofluorescence histochemistry showed that MMP9 and MMP2 colabeled with markers of endothelial cells, astrocytes, and newborn neurons in the subgranular zone (SGZ) of the DG, and also that the percentage of coexpression significantly increased in the delayed postischemic phase, as compared with controls. However, colabeling with different cell selective markers reached its peak at different time points, i.e., with endothelial cells on Day 7, whereas with astrocytes and newborn neurons on Day 15, respectively. MMPs were localized both in the perikarya and dendrites in the newborn neurons. In conclusion, MMP9/2 expression was regulated in a cell- and time-specific manner in hippocampal neurogenic niche of adult primates.

Abstract 2837: Delayed Calpain Inhibition Is Neuroprotective After Transient Global Brain Ischemia

Introduction/Hypothesis: The delay in neurodegeneration after transient global brain ischemia offers a potentially broad therapeutic window for inhibiting molecular injury mechanisms. One such mechanism, calpain-mediated proteolysis, peaks between 24 and 72 hours after transient forebrain ischemia (TFI) in rats. This study tests the hypothesis that calpain inhibition is neuroprotective when initiated 22 hours after TFI. Methods: Male Long Evans rats (400 – 450 g) were anesthetized (halothane), mechanically ventilated, and instrumented for temperature and hemodynamic monitoring. Each underwent 10 minutes of TFI induced by reversible bilateral carotid occlusion and hypovolemic hypotension (MAP 30 mm Hg). Twenty-two hours after TFI, rats were block randomized (n=6/group) to receive either calpain inhibitor (CEP-3453 (Cephalon), 60 mg/kg IV bolus and 30 mg/kg IV infusion for 50 hours) or vehicle (normal saline IV bolus and infusion for 50 hours). Sham operated rats served as controls. Rats were euthanized 72 hours after injury and brains were processed for immunohistochemistry. Hippocampal CA1 sector calpain activity was analyzed by immunofluorescence using primary antibody that specifically detects calpain-cleaved alpha-spectrin (Ab38). Neurodegeneration was quantified by counting normal appearing Hoechst-stained neuronal nuclei in the hippocampal CA1 pyramidal layer. Ab38 immunofluorescence intensity and normal nuclei counts were compared between vehicle and CEP-3453 treatment groups using a two-tailed Student’s t-test (alpha error 0.05). Results: Baseline and post-injury hemodynamic parameters and temperature were not significantly different between vehicle and CEP-3453 treatment groups. Relative to sham operated controls, mean CA1 sector Ab38 immunofluorescence increased by 263 ± 281% in vehicle treated rats and 68 ± 147% in CEP-3453 treated rats (p=0.17). Normal CA1 pyramidal layer nuclei averaged 10 ± 12% of control in vehicle treated rats and 50 ± 42% of control in CEP-3453 treated rats (p=0.047). Conclusion: These results suggest a causal role for calpains in delayed post-ischemic neurodegeneration, and demonstrate a broad therapeutic window for calpain inhibition in this model of transient global brain ischemia.

Dynamics of Nitric Oxide and Peroxynitrite During Global Brain Ischemia/Reperfusion in Rat Hippocampus: NO-sensor Measurement and Modeling Study

The dynamics of nitric oxide (NO) and peroxynitrite concentration changes during brain ischemia/reperfusion are poorly understood. In this paper, a NO-selective sensor was used to measure NO concentration changes in the rat brain hippocampus during global brain ischemia/reperfusion. Four-vessel occlusion model of transient global brain ischemia was used. Global cerebral ischemia was induced by occluding both common carotid arteries with artery nips (for 20 min) and reperfusion was induced by loosening the artery nips. Results showed that the changes of NO concentration during global brain ischemia/reperfusion could be divided into different stages. Together with the effects of O2 tension changes and NO synthase (NOS) on nitric oxide levels, we determined five stages in the NO concentration profile: (1) acute O2-limited decrease stage; (2) O2-limited steady stage; (3) neuronal NOS activation stage; (4) acute O2-recovery elevation stage; and (5) O2-recovery steady stage. In addition, a chemical reaction network model was constructed to simulate the dynamics of peroxynitrite during the reperfusion stage, and the effects of a change in the NO formation rate on the dynamics of peroxynitrite were investigated specifically. Results show the rate of NO formation has a great influence on peroxynitrite dynamics.

Effect of Ischemic Preconditioning on Mitochondrial Dysfunction and Mitochondrial P53 Translocation after Transient Global Cerebral Ischemia in Rats

Transient global brain ischemia induces dysfunctions of mitochondria including disturbance in mitochondrial protein synthesis and inhibition of respiratory chain complexes. Due to capacity of mitochondria to release apoptogenic proteins, ischemia-induced mitochondrial dysfunction is considered to be a key event coupling cerebral blood flow arrest to neuronal cell death. Ischemic preconditioning (IPC) represents an important phenomenon of adaptation of central nervous system (CNS) to sub-lethal short-term ischemia, which results in increased tolerance of CNS to the lethal ischemia. In this study we have determined the effect of ischemic preconditioning on ischemia/reperfusion-associated inhibition of mitochondrial protein synthesis and activity of mitochondrial respiratory chain complexes I and IV in the hippocampus of rats. Global brain ischemia was induced by 4-vessel occlusion in duration of 15 min. Rats were preconditioned by 5 min of sub-lethal ischemia and 2 days later, 15 min of lethal ischemia was induced. Our results showed that IPC affects ischemia-induced dysfunction of hippocampal mitochondria in two different ways. Repression of mitochondrial translation induced during reperfusion of the ischemic brain is significantly attenuated by IPC. Slight protective effect of IPC was documented for complex IV, but not for complex I. Despite this, protective effect of IPC on ischemia/reperfusion-associated changes in integrity of mitochondrial membrane and membrane proteins were observed. Since IPC exhibited also inhibitory effect on translocation of p53 to mitochondria, our results indicate that IPC affects downstream processes connecting mitochondrial dysfunction to neuronal cell death.

Maternal Exposure to Low Levels of Corticosterone during Lactation Protects the Adult Offspring against Ischemic Brain Damage

A growing body of evidence underscores the importance of early life events as predictors of health in adulthood. Abnormalities in maternal care or other forms of early postnatal stress induce long-term changes in behavior and influence the vulnerability to illnesses throughout life. Some of these changes may be produced by the activation of the hypothalamic-pituitary-adrenal (HPA) axis, which is invariably associated with stress. We used a model in which neonate rats are fed by mothers drinking water supplemented with 0.2 mg/ml corticosterone, the main glucocorticoid hormone in rodents. Plasma corticosterone levels increased in the dams to an extent similar to that induced by a mild stress. Corticosterone-treated dams also showed an increase in maternal care. Remarkably, adult rats that had been nursed by corticosterone-treated mothers were protected against neuronal damage and cognitive impairment produced by transient global brain ischemia. Neuroprotection was associated with a reduced HPA response to ischemia and was primarily decreased when corticosterone was injected at a dose that eliminated any difference in endogenous corticosterone levels between rats raised by mothers supplemented with corticosterone and their matched controls. These data suggest that an increased maternal care protects the offspring against ischemic neuronal damage and that at least a component of neuroprotection is mediated by a reduced response of the HPA axis to ischemia.

Ketone bodies metabolism during ischemic and reperfusion brain injuries following bilateral occlusion of common carotid arteries in rats

To evaluate the in vivo alterations on ketone bodies metabolism after cerebral ischemia/reperfusion through an experimental model of brain ischemia induced by simple occlusion of common carotid arteries (CCAs) in Wistar rats. Forty-eight male Wistar rats were randomly distributed on two groups (S - Sham; T - Test) and further redistributed into four times sets of study. After bilateral occlusion of CCAs for 30 min, the animals of group T were allowed reperfusion for 0, 5, 10 and 15 min. Samples of cerebral tissue and systemic arterial blood were collected and the metabolites acetoacetate (ACT) and beta-hydroxybutyrate (BHB) were determined. Cerebral ACT and BHB levels increased significantly in Group T after 30 min of carotid occlusion (time 0). The highest brain ketone bodies (ACT+BHB) concentration was verified at 5 min of reperfusion, decreasing after 10 min of recirculation. Systemic ketone bodies levels increased similarly between test and sham groups. Group S demonstrated a significant increase in cerebral and systemic ACT and BHB concentrations mainly after 40-45 min of study. The partial transient acute global brain ischemia induced by the bilateral carotid occlusion in Wistar rats triggered ketogenesis probably due to a central stimulation of catecholamine secretion. There was an increased cerebral uptake of ketone bodies following brain ischemia, reaffirming these metabolites as alternative energy substrates under conditions of cerebral metabolic stress as well as its potential role on neuroprotection. The greatest changes in ketone bodies metabolism were verified at initial minutes of recirculation as a result of the reperfusion injury phenomenon.

PP2, a potent inhibitor of Src family kinases, protects against hippocampal CA1 pyramidal cell death after transient global brain ischemia

It has been indicated that Src family protein tyrosine kinases (SrcPTKs) potentiate N-methyl-D-aspartate (NMDA) receptor function by phosphorylating NR2A subunits and that postsynaptic density protein 95 (PSD-95) facilitates this regulation. In this paper, we define the role of SrcPTKs in delayed neuronal damage following transient brain ischemia and explore the underlying mechanisms involved in this event. Transient global brain ischemia was induced by the four-vessel occlusion method. A specific Src family kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine) and a PP2 negative control PP3 (4-amino-7-phenylpyrazolo[3,4-d]pyramidine) were infused into rat cerebroventricule 30 min before occlusion. Hematoxylin and eosine staining showed that the number of surviving pyramidal neurons in rat hippocampal CA1 subfield increased markedly in PP2-treated rats comparing to PP3-treated groups after 5 days of reperfusion following ischemia. Additionally, immunoprecipitation and immunoblot analysis revealed that preadministration of PP2, but not PP3, attenuated not only the increased tyrosine phosphorylation of NR2A but also the enhanced interactions among Src, NR2A and PSD-95 induced by ischemia/reperfusion. In conclusion, SrcPTKs promote binding of the kinases and their substrate NR2A attributed to the scaffolding effect of PSD-95 during transient brain ischemia and reperfusion, which are responsible for the elevation of NR2A tyrosine phosphorylation and consequent delayed neuronal cell death.

Neuroprotection of selenite against ischemic brain injury through negatively regulating early activation of ASK1/JNK cascade via activation of PI3K/AKT pathway

To investigate whether selenite, a known antioxidant, could decrease the activation of apoptosis signal regulating kinase 1/c-jun N-terminal kinase (ASK1/JNK) signaling cascade in cerebral ischemia/reperfusion (I/R) by activating the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in rat hippocampi, and the neuroprotective effect of selenite against ischemic injury after 15 min of transient brain ischemia. Transient global brain ischemia was induced by 4-vessel occlusion into adult male Sprague-Dawley rats weighing 250–300 g. The rats were pretreated only with selenite (0.3 mg/kg dissolved in 0.9% saline) every 24 h for 7 d by means of intravenous injection of the tail or combined with LY294002 from d 5 by left cerebral ventricle injection before surgery. Selenite significantly increased AKT1 activation and decreased the activation of ASK1/JNK cascade via phosphorylating ASK1 at Ser-83 residue by AKT1 during early reperfusion after 15 min transient global brain ischemia. On the contrary, combined pretreatment of the rats with LY294002 (a specific PI3K inhibitor) and selenite significantly inhibited the effects solely with selenite. The activation of the pro-apoptotic ASK1/JNK cascade, which is closely associated with oxidative stress, could be suppressed by selenite through activating the anti-apoptotic PI3K/AKT pathway during early reperfusion after cerebral ischemia in rat hippocampi.

Neuroprotection of selenite against ischemic brain injury through negatively regulating early activation of ASK1/JNK cascade via activation of PI3K/AKT pathway

To investigate whether selenite, a known antioxidant, could decrease the activation of apoptosis signal regulating kinase 1/c-jun N-terminal kinase (ASK1/ JNK) signaling cascade in cerebral ischemia/reperfusion (I/R) by activating the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in rat hippocampi, and the neuroprotective effect of selenite against ischemic injury after 15 min of transient brain ischemia. Transient global brain ischemia was induced by 4-vessel occlusion into adult male Sprague-Dawley rats weighing 250-300 g. The rats were pretreated only with selenite (0.3 mg/kg dissolved in 0.9% saline) every 24 h for 7 d by means of intravenous injection of the tail or combined with LY294002 from d 5 by left cerebral ventricle injection before surgery. Selenite significantly increased AKT1 activation and decreased the activation of ASK1/ JNK cascade via phosphorylating ASK1 at Ser-83 residue by AKT1 during early reperfusion after 15 min transient global brain ischemia. On the contrary, combined pretreatment of the rats with LY294002 (a specific PI3K inhibitor) and selenite significantly inhibited the effects solely with selenite. The activation of the pro-apoptotic ASK1/JNK cascade, which is closely associated with oxidative stress, could be suppressed by selenite through activating the antiapoptotic PI3K/AKT pathway during early reperfusion after cerebral ischemia in rat hippocampi.