Forebrain Ischemia-reperfusion Research Articles

The Effect of Cerebrolysin in an Animal Model of Forebrain Ischemic-Reperfusion Injury: New Insights into the Activation of the Keap1/Nrf2/Antioxidant Signaling Pathway.

Forebrain ischemia-reperfusion (IR) injury causes neurological impairments due to decreased cerebral autoregulation, hypoperfusion, and edema in the hours to days following the restoration of spontaneous circulation. This study aimed to examine the protective and/or therapeutic effects of cerebrolysin (CBL) in managing forebrain IR injury and any probable underlying mechanisms. To study the contribution of reperfusion to forebrain injury, we developed a transient dual carotid artery ligation (tDCAL/IR) mouse model. Five equal groups of six BLC57 mice were created: Group 1: control group (no surgery was performed); Group 2: sham surgery (surgery was performed without IR); Group 3: tDCAL/IR (surgery with IR via permanently ligating the left CA and temporarily closing the right CA for 30 min, followed by reperfusion for 72 h); Group 4: CBL + tDCAL/IR (CBL was given intravenously at a 60 mg/kg BW dose 30 min before IR); and Group 5: tDCAL/IR + CBL (CBL was administered i.v. at 60 mg/kg BW three hours after IR). At 72 h following IR, the mice were euthanized. CBL administration 3 h after IR improved neurological functional recovery, enhanced anti-inflammatory and antioxidant activities, alleviated apoptotic neuronal death, and inhibited reactive microglial and astrocyte activation, resulting in neuroprotection after IR injury in the tDCAL/IR + CBL mice group as compared to the other groups. Furthermore, CBL reduced the TLRs/NF-kB/cytokines while activating the Keap1/Nrf2/antioxidant signaling pathway. These results indicate that CBL may improve neurologic function in mice following IR.

Aucubin Exerts Neuroprotection against Forebrain Ischemia and Reperfusion Injury in Gerbils through Antioxidative and Neurotrophic Effects.

Aucubin is an iridoid glycoside that displays various pharmacological actions including antioxidant activity. However, there are few reports available on the neuroprotective effects of aucubin against ischemic brain injury. Thus, the aim of this study was to investigate whether aucubin protected against damage to hippocampal function induced by forebrain ischemia-reperfusion injury (fIRI) in gerbils, and to examine whether aucubin produced neuroprotection in the hippocampus against fIRI and to explore its mechanisms by histopathology, immunohistochemistry, and Western analysis. Gerbils were given intraperitoneal injections of aucubin at doses of 1, 5, and 10 mg/kg, respectively, once a day for seven days before fIRI. As assessed by the passive avoidance test, short-term memory function following fIRI significantly declined, whereas the decline in short-term memory function due to fIRI was ameliorated by pretreatment with 10 mg/kg, but not 1 or 5 mg/kg, of aucubin. Most of the pyramidal cells (principal cells) of the hippocampus died in the Cornu Ammonis 1 (CA1) area four days after fIRI. Treatment with 10 mg/kg, but not 1 or 5 mg/kg, of aucubin protected the pyramidal cells from IRI. The treatment with 10 mg/kg of aucubin significantly reduced IRI-induced superoxide anion production, oxidative DNA damage, and lipid peroxidation in the CA1 pyramidal cells. In addition, the aucubin treatment significantly increased the expressions of superoxide dismutases (SOD1 and SOD2) in the pyramidal cells before and after fIRI. Furthermore, the aucubin treatment significantly enhanced the protein expression levels of neurotrophic factors, such as brain-derived neurotrophic factor and insulin-like growth factor-I, in the hippocampal CA1 area before and after IRI. Collectively, in this experiment, pretreatment with aucubin protected CA1 pyramidal cells from forebrain IRI by attenuating oxidative stress and increasing neurotrophic factors. Thus, pretreatment with aucubin can be a promising candidate for preventing brain IRI.

Intranasal Insulin Decreases Autophagic and Apoptotic Death of Neurons in the Rat Hippocampal C1 Region and Frontal Cortex under Forebrain Ischemia–Reperfusion

Intranasal Insulin Decreases Autophagic and Apoptotic Death of Neurons in the Rat Hippocampal C1 Region and Frontal Cortex under Forebrain Ischemia–Reperfusion

Neuroprotective effects of fasudil, a Rho-kinase inhibitor,in a rat transient forebrain ischemia-reperfusion model

Neuroprotective effects of fasudil, a Rho-kinase inhibitor,in a rat transient forebrain ischemia-reperfusion model

Abstract W MP78: Small Changes in Intracellular pH Block Ischemic and Excitotoxic Cell Death by Suppressing Neuronal Superoxide Production

Tissue acidosis has long been recognized as a crucial variable affecting outcome after brain ischemia, with mild acidosis (pH 7.0 - 6.6) providing robust neuroprotection. pH in ischemic tissues is reduced by anaerobic glucose metabolism, and normalized by reperfusion. Superoxide produced by neuronal NADPH oxidase (NOX2) has recently been identified as a major source of oxidative stress in ischemia and excitotoxicity. Given that NOX2 in other cell types is highly sensitive to intracellular pH, we evaluated the possibility that pH effects on neuronal NOX2 activity mediate the neuroprotective effects of mild acidosis. In cultured neurons, reducing medium pH from 7.2 to 7.0 produced a comparable 0.2 pH-unit intracellular acidification and completely prevented NMDA-induced NOX2 activity, superoxide production, and cell death. These effects were observed even when extracellular calcium was elevated to overcompensate for reduced NMDA-induced calcium influx at the lowered pH. Intracellular H + produced by NOX2 is be extruded to permit continued NOX2 activity. Neurons treated with an inhibitor of Na + /H + exchange, cariporide showed a 0.2- 0.3 unit intracellular acidification (with no change in extracellular pH). Cariporide- treated neurons also showed no superoxide formation and no cell death in response to NMDA, with no change in NMDA-induced calcium influx. To evaluate this effect in vivo, mice were given stereotaxic striatal injections NMDA. NMDA induced superoxide production and subsequent neuronal death, both of which were blocked by co-injection with cariporide. Superoxide production and neuronal death were similarly blocked in mice with reduced expression of Na + /H + exchanger isoform 1 protein (NHE-1 +/- mice). NHE-1 +/- mice also exhibited much less superoxide production and neuronal death after forebrain ischemia-reperfusion. These findings identify a mechanism by which mild acidosis improves outcomes in ischemia and excitotoxicity. The findings may also help explain why superoxide production is greater in reperfused than non-reperfused brain, and why both NOX2 inhibitors and NMDA receptor antagonists are more neuroprotective in reperfused than non-reperfused brain.

Activation of BKCa Channels Mediates Hippocampal Neuronal Death After Reoxygenation and Reperfusion

Excessive K(+) efflux promotes central neuronal apoptosis; however, the type of potassium channel that mediates K(+) efflux in response to different apoptosis-inducing stimuli is still unknown. It is hypothesized that the activation of large-conductance Ca(2+)-activated K(+) channels (BKCa) mediates hypoxia/reoxygenation (H/R)- and ischemia/reperfusion (I/R)-induced neuronal apoptosis. Rat hippocampal neuronal cultures underwent apoptosis after reoxygenation, as assessed by morphologic observation, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and caspase-3 activation. Single-channel recordings revealed upregulation of BKCa channel activity 6 h after reoxygenation, which might be caused by elevated cytosolic Ca(2+). The K(+) ionophore valinomycin and the BKCa channel opener NS1619 induced neuronal apoptosis. Transfection of the BKCa channel α subunit into Chinese hamster ovary (CHO-K1) cells, which do not express endogenous K(+) channels, or into neurons will induce cell apoptosis, indicating that the opening of the BKCa channel serves as a pivotal event in mediating cell apoptosis. The specific BKCa channel blockers charybdotoxin and iberiotoxin and the nonselective K(+) channel blocker tetraethylammonium at concentrations more specific to the BKCa channel were neuroprotective. The A-type potassium channel blocker 4-aminopyridine and apamin, a small-conductance Ca(2+)-activated K(+) channel blocker, were not protective. This result suggests the involvement of the BKCa channel in H/R-induced apoptosis. Similarly, specific BKCa channel blockers also showed neuroprotection in neurons subjected to oxygen-glucose deprivation/reoxygenation or animals subjected to forebrain ischemia-reperfusion. These results demonstrate that the over-activity of BKCa channels mediates hippocampal neuronal damage induced by H/R in vitro and I/R in vivo.

Novel experimental model for repeated forebrain ischemia-reperfusion

Background and Purpose: The lack of reliable rodent model for repeated ischemia-reperfusion (I-R) hampers experimental research on stroke. Therefore, the objective of the present study was to develop a mouse model for repeated I-R cycles in a single animal on different days. Methods and Results: The right common carotid artery (CCA) was ligated and both vertebral arteries were coagulated. A customized vascular occluder, with its actuating tubing glued to a microport, was cuffed around the isolated left CCA and secured. Inflating of the occluder diaphragm via the microport restricted the blood flow via the left CCA and reduced the cerebral blood flow (CBF) by 75% in both hemispheres, while deflating allowed for the CBF restoration. Two minutes of forebrain ischemia followed by a 24 h reperfusion period was tolerated by animals for 5 cycles. Importantly, repeated 2 min I-R cycles attenuated the infarct volume induced by occlusion of the middle cerebral artery. Conclusions: The described model is a reliable method to induce transient I-R events in the forebrain. The model mimics transient ischemic attacks and allows for controlling the ischemic durations, intervals, and numbers of I-R cycles.

Novel experimental model for repeated forebrain ischemia-reperfusion

Background and Purpose: The lack of reliable rodent model for repeated ischemia-reperfusion (I-R) hampers experimental research on stroke. Therefore, the objective of the present study was to develop a mouse model for repeated I-R cycles in a single animal on different days. Methods and Results: The right common carotid artery (CCA) was ligated and both vertebral arteries were coagulated. A customized vascular occluder, with its actuating tubing glued to a microport, was cuffed around the isolated left CCA and secured. Inflating of the occluder diaphragm via the microport restricted the blood flow via the left CCA and reduced the cerebral blood flow (CBF) by 75% in both hemispheres, while deflating allowed for the CBF restoration. Two minutes of forebrain ischemia followed by a 24 h reperfusion period was tolerated by animals for 5 cycles. Importantly, repeated 2 min I-R cycles attenuated the infarct volume induced by occlusion of the middle cerebral artery. Conclusions: The described model is a reliable method to induce transient I-R events in the forebrain. The model mimics transient ischemic attacks and allows for controlling the ischemic durations, intervals, and numbers of I-R cycles.

Urinary trypsin inhibitor suppresses excessive superoxide anion radical generation in blood, oxidative stress, early inflammation, and endothelial injury in forebrain ischemia/reperfusion rats

Objectives: To investigate the effects of ulinastatin, a urinary trypsin inhibitor (UTI), on jugular venous superoxide radical (O−2·) generation, oxidative stress, early inflammation, and endothelial activation in forebrain ischemia/reperfusion (FBI/R) rats.Methods: Fourteen Wistar rats were allocated to a control group (n = 7) and a UTI group (n = 7). Throughout the experiments, O−2· in the jugular vein was measured by the produced current using a novel electrochemical O−2· sensor. Forebrain ischemia was induced by occlusion of the bilateral common caroti darteries with hemorrhagic hypotension for 20 min, followed by reperfusion. In the UTI group, UTI (5 U/g) was administered intravenously immediately after reperfusion. At 60 min after reperfusion, plasma and brain were harvested, and malondialdehyde, high-mobility group box 1 (HMGB1) protein, and intercellular adhesion molecule-1 (ICAM-1) were measured.Results: O−2· current increased gradually during forebrain ischemia in both groups. The current increased markedly in the control group immediately after reperfusion but was significantly attenuated in the UTI group after reperfusion. Brain and plasma malondialdehyde, HMGB1, and ICAM-1 were significantly attenuated in the UTI group compared with those in the control group, except for brain HMGB1, which was associated with the amount of O−2· generated during FBI/R.Discussion: UTI suppressed jugular venous O−2· generation, oxidative stress, early inflammation, and endothelial activation in FBI/R rats. Therefore, UTI might be a useful agent for the therapy of the cerebral ischemia/reperfusion pathophysiology.

Effect of propofol on rat forebrain ischemia reperfusion induced mitochondrial damage and uncoupling protein 2 expression

Objective To investigate the effect of propofol on rat forebrain ischemia reperfusion induced mitochondrial damage and uncoupling protein 2 (UCP 2)expression. Methods 45 male Wistar rats weighing 250 g-300 g were randomly divided into three groups (n= 15):control group (C) ;ischemia reperfusion group (I/R) ;propofol group (P). Forebrain cerebral ischemia reperfusion was produced by 2-vessel occlusion method. Bilateral carotid arteries were released after 10 min cerebral ischemia. Normal saline 1 mg/kg and propofol 1 mg/kg were separately injected into the lateral cerebral ventricle by micro syringe in group I/R and group P. The animals were decapitated at the end of 24 h reperfusion and the hippocampal were separated.The mitochondria of hippocampal in each group were isolated. The mitochondria in three groups were incubated by CaCl2for 5 min at 37℃. Morphological changes of mitoehondria were observed by using electron microscopy (n=3). Mitochondrial permeability transition pore (MPTP)opening were detected by ultravioletvisible absorption spectroscopy(n=6). The expression of UCP2 in each group were determined by western blotting method (n=6). Results Group C showed normal mitochondrial ultrastructure;Significant mitochondrial swelling, disrupted cristae and membrane rupture were showed in group I/R;Morphological changes of mitechondria in group P were between group C and group I/R. Absorbance values of mitochondrial decreased in group C, group I/R and group P. Compared with group C, absorbance values of mitochondrial were lower in group I/R and group P (P<0.05). The decrease of absorbance values of mitochondria was reduced in group P(0.017 ± 0.007)compared with that in group I/R (0.028 ± 0.007)(P<0.05). The expression of UCP2 in hippocampus was significantly up-regnlated in group I/R (0.88 ± 0.14) and group P (1.32 + 0. 10)(P<0.05). Compared with group I/R, the expression of UCP2 were higher in group P (P<0.05). Conclusion Propofol improve mitochondrial dysfunction induced by calcium overload after forebrain ischemia reperfusion, indicating by inhibition of MPTP opening and promotion of UCP2 protein expression. This maybe involved in the mechanisms of brain protection of propofol. Key words: Prupofol; Forebrain ischemia reperfusion; Mitochondria permeability transition pore; Uncoupling protein 2

The effects of propofol on hippocampal caspase-3 and Bcl-2 expression following forebrain ischemia–reperfusion in rats

Transient cerebral ischemia may result in neuronal apoptosis. During this process, several apoptosis-regulatory genes are induced in apoptotic cells. Among these genes, cysteinyl aspartate-specific protease-3 (caspase-3) and B-cell leukemia-2 (Bcl-2) are the most effective apoptotic regulators because they play a decisive role in the occurrence of apoptosis. Research has shown that propofol, which is an intravenous anesthetic agent, exhibits neuroprotective effects against cerebral ischemia–reperfusion injury, although the neuroprotective mechanism is still unclear. In this study, we examined the effects of propofol in rats after forebrain ischemia–reperfusion. We assessed the expression of hippocampal caspase-3, which acts as an apoptotic activator, and Bcl-2, which acts as an apoptotic suppressor. Forebrain ischemia was induced in hypotensive rats by clamping the bilateral common carotid arteries for 10 min. Propofol was administered via a lateral cerebral ventricle injection using a microsyringe after the induction of ischemia. Neuronal damage was determined by histological examination of brain sections at the level of the dorsal hippocampus. Caspase-3 and Bcl-2 expression in the hippocampus were detected using semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) and Western blot analysis. We also used an immunohistochemical method after ischemia–reperfusion. In the hippocampus, caspase-3 and Bcl-2 mRNA were dramatically increased at 24 h after forebrain ischemia. Following 6–24 h of reperfusion, forebrain ischemia for 10 min induced a gradual increase in the expression of caspase-3 and Bcl-2 protein in the rat hippocampus, which peaked at 24 h. In the propofol (1.0 mg/kg) intervention group, the hippocampal expression of caspase-3 mRNA decreased significantly in rats 24 h after ischemia; Bcl-2 mRNA was increased at the same time point. During the 24-h reperfusion period and after treatment with propofol, the level of caspase-3 protein expression was low, while the level of Bcl-2 was high. Thus, our results suggest that the neuroprotective effects of propofol against neuronal apoptosis may be mediated by the inhibition of caspase-3 expression and an increase in Bcl-2 expression.

Effect of ischemic preconditioning on HSP70 expression and learning/memory after cerebral ischemia-reperfusion in gerbils

Objective To study the effect of ischemic preconditioning on heat-shock protein 70 ( HSP70) expression and the learning,memory functions after forebrain ischemia-reperfusion injury in gerbils. Methods Gerbils ( n = 100) were evenly randomized into four groups:Sham group,ischemia -reperfusion( I/R) group,ischemia preconditioning( IP) group,and Cycloheximide + IP group ( Cycloheximide was administered 30 min before IP). Transient forebrain ischemia-reperfusion model was established by bilateral common carotid artery occlusion according to the method described previously,and ischemia preconditioning model was established as described by Kitagawa. Changes of neuron morphous in hippocampus CA1 region were observed by H-E staining 1,2,and 3 days after reperfusion. The expression of HSP70 was examined by immunohistochemistry and Western blotting assay. Neuron apoptosis was detected by TUNEL method. The learning/memory functions of gerbils were examined using 4-PTT dry path maze 3,4,5,6,and 7 days after reperfusion. Results Compared with Sham group,I/R group had significantly decreased survival neurons( P 0. 05) ,increased neuron apoptosis( P 0. 05) ,increased expression of HSP70 ( P 0. 05) ,and decreased learning/memory functions( P 0. 05). Compared with I/R group,IP group had significantly increased survival neurons( P 0. 05) ,decreased neuron apoptosis ( P 0. 05) ,and increased expression of HSP70 ( P 0. 05) ,and improved learning/memory functions( P 0. 05). However,cycloheximide almost totally abolished the effect of ischemia preconditioning,with the neuron morphology,density,apoptosis,HSP70 expression,and learning/memory functions similar to those of I/R group. Conclusion Ischemic preconditioning can protect against cerebral ischemia injury and improve the learning/memory functions after forebrain ischemia-reperfusion damage,which is possibly through promoting HSP70 expression and starting endogenous neuroprotective mechanism,subsequently reinforcing the protective effect against ischemia.

Effects of one-day reperfusion after transient forebrain ischemia on circulatory system in the rat

Although ischemia/reperfusion injury remains incompletely understood, it appears that reactive oxygen species produced mainly during postischemic recirculation play a critical role. The present study examined the impact of forebrain ischemia and subsequent one-day reperfusion on several blood parameters. We determined glutamate concentration in whole blood, measured Cu/Zn- and Mn-SOD (superoxide dismutase) activity in blood cells as well as plasma, and investigated the prevalence of single and double strand breaks of lymphocyte DNA. The results of our experiment showed that the concentration of glutamic acid in whole blood was increased by about 25%. Antioxidant activity of total SOD and Cu/Zn-SOD was reduced in blood cells and plasma. Mn-SOD activity in blood cells was not affected by ischemic insult and one-day reperfusion, but we detected its significantly lower activity in samples of plasma. We observed a weakly reduced level of double and a significantly elevated level of single strand breaks of lymphocyte DNA. In conclusion, one day of recovery after the ischemic attack failed to return peripheral circulatory system to physiological conditions. Reduced antioxidant capacity in the blood and an elevated level of excitotoxic amino acid glutamate may cause lymphocyte DNA damage, and probably contribute to insufficient postischemic recovery of brain tissue.

Cholinergic agonist physostigmine suppresses excessive superoxide anion radical generation in blood, oxidative stress, early inflammation, and endothelial injury in rats with forebrain ischemia/reperfusion

The cholinergic anti-inflammatory pathway is reportedly important in modulating the inflammatory response in local and systemic diseases, including ischemia/reperfusion pathophysiology. In this study, we investigated the effects of the cholinergic agonist, physostigmine, on jugular venous superoxide radical (O 2 − ) generation, oxidative stress, early inflammation, and endothelial activation during forebrain ischemia/reperfusion (FBI/R) in rats. Fourteen male Wistar rat were allocated to the control group ( n = 7) or physostigmine group ( n = 7). The physostigmine group received 80 ng/g physostigmine intraperitoneally 24 h and 1 h before forebrain ischemia was established. The jugular venous O 2 − current was measured for 10 min during forebrain ischemia and for 120 min after reperfusion. The O 2 − current increased gradually during forebrain ischemia in both groups. The current increased markedly immediately after reperfusion in the control group but was significantly attenuated in the physostigmine group after reperfusion. Brain and plasma malondialdehyde, high-mobility group box 1 (HMGB1) protein, and intercellular adhesion molecule 1 (ICAM1) were significantly attenuated in the physostigmine group compared with the control group, except for brain HMGB1. The amount of O 2 − generated during FBI/R correlated with malondialdehyde, HMGB1, and ICAM1 in both the brain and plasma. In conclusion, the cholinergic agonist physostigmine suppressed jugular venous O 2 − generation, oxidative stress, early inflammation, and endothelial activation in the brain and plasma in the acute phase of cerebral ischemia/reperfusion. Therefore, the suppression of O 2 − is a key mechanism of the cholinergic anti-inflammatory pathway in the pathophysiology of cerebral ischemia/reperfusion.

Moderate hypothermia suppresses jugular venous superoxide anion radical, oxidative stress, early inflammation, and endothelial injury in forebrain ischemia/reperfusion rats

The aim of this study was to assess the effect of moderate hypothermia (MH) on generation of jugular venous superoxide radical (O 2 − ·), oxidative stress, early inflammation, and endothelial injury in forebrain ischemia/reperfusion (FBI/R) rats. Twenty-one Wistar rats were allocated to a control group ( n = 7, 37 °C), a pre-MH group ( n = 7, 32 °C before ischemia), and a post-MH group ( n = 7, 32 °C after reperfusion). MH was induced before induction of ischemia in the pre-MH group and just after reperfusion in the post-MH group. Forebrain ischemia was induced by occlusion of bilateral common carotid arteries with hemorrhagic hypotension for 10 min, followed by reperfusion. O 2 − · in the jugular vein was measured from the produced current using a novel O 2 − · sensor. The O 2 − · current showed a gradual increase during forebrain ischemia in the control and post-MH groups but was attenuated in the pre-MH group. Following reperfusion, the current showed a marked increase in the control group but was strongly attenuated in the pre- and post-MH groups. Concentrations of malondialdehyde, high-mobility group box 1 (HMGB1) protein, and intercellular adhesion molecule-1 (ICAM-1) in the brain and plasma 120 min after reperfusion in the pre- and post-MH groups were significantly lower than those in the control group, except for plasma HMGB1 in the post-MH group. In conclusion, MH suppressed O 2 − · measured in the jugular vein, oxidative stress, early inflammation, and endothelial injury in FBI/R rats.

Elevation of jugular venous superoxide anion radical is associated with early inflammation, oxidative stress, and endothelial injury in forebrain ischemia–reperfusion rats

A novel electrochemical sensor was used in this study to determine the correlations between jugular venous O2− and HMGB1, malondialdehyde (MDA), and intercellular adhesion molecule-1 (ICAM-1) in rats with forebrain ischemia/reperfusion (FBI/R). Twenty-one male rats were divided into a Sham group, a hemorrhagic shock/reperfusion (HS/R) group, and a forebrain ischemia/reperfusion (FBI/R) group. The O2− sensor in the jugular vein detected the current derived from O2− generation (abbreviated as “O2− current”), which was integrated as the partial value of quantified electricity during ischemia (QI) and after reperfusion (QR). The plasma O2− current showed a gradual increase during forebrain ischemia in the HS/R and the FBI/R groups. The current showed a marked increase immediately after reperfusion and continued for more than 60 min in the FBI/R group. In the HS/R group, the current was gradually attenuated to the baseline level. Brain and plasma HMGB1 increased significantly in the FBI/R group compared with those in the Sham and the HS/R groups, and both brain and plasma HMGB1 correlated significantly with the sum of QI and QR (total Q). Brain and plasma MDA and plasma soluble ICAM-1 also correlated significantly with total Q. Here, we report the correlation between O2− and HMGB1, MDA, and sICAM-1 in rats with cerebral ischemia–reperfusion, using a novel electrochemical sensor. These data indicated that excessive production of O2− after ischemia–reperfusion was associated with early inflammation, oxidative stress, and endothelial activation in the brain and plasma, which might enhance the ischemia–reperfusion injury.

Effects of Estrogen on Postischemic Pial Artery Reactivity to ADP

The aims of this work were to determine if 1) ischemia alters pial artery responsiveness to the partially nitric oxide (NO)-dependent dilator, ADP, 2) the alteration depends on 17beta-estradial (E2), and 3) NO contributes to E2 protective effects. Response to ADP and the non-NO-dependent dilator, PGE(2), were examined through closed cranial windows. Ovariectomized (OVX) and E2-replaced (E25, 0.025 mg; or E50, 0.05 mg) rats were subjected to 15-minute forebrain ischemia and one-hour reperfusion. Endothelial NO synthase (eNOS) expression was determined in pre- and postischemic isolated cortical microvessels. In OVX rats, ischemia depressed pial responses to ADP, but not to PGE(2). Both doses of E2 maintained responses to ADP and had no effect on the response to PGE(2). eNOS inhibition decreased the ADP response by 60% in the E25 rats and 50% in the E50 rats, but had no effect in the OVX rats. Compared to the OVX group, microvessel expression of eNOS was increased by E2, but postischemic eNOS was unchanged in both groups. The nearly complete loss of postischemic dilation to ADP suggests that normal non-NO-mediated dilatory mechanisms may be acutely impaired after ischemic injury. Estrogen's protective action on ADP dilation may involve both NO- and non-NO-mediated mechanisms.

Glucose and NADPH oxidase drive neuronal superoxide formation in stroke

Hyperglycemia has been recognized for decades to be an exacerbating factor in ischemic stroke, but the mechanism of this effect remains unresolved. Here, we evaluated superoxide production by neuronal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase as a possible link between glucose metabolism and neuronal death in ischemia-reperfusion. Superoxide production was measured by the ethidium method in cultured neurons treated with oxygen-glucose deprivation and in mice treated with forebrain ischemia-reperfusion. The role of NADPH oxidase was examined using genetic disruption of its p47(phox) subunit and with the pharmacological inhibitor apocynin. In neuron cultures, postischemic superoxide production and cell death were completely prevented by removing glucose from the medium, by inactivating NADPH oxidase, or by inhibiting the hexose monophosphate shunt that generates NADPH from glucose. In murine stroke, neuronal superoxide production and death were decreased by the glucose antimetabolite 2-deoxyglucose and increased by high blood glucose concentrations. Inactivating NADPH oxidase with either apocynin or deletion of the p47(phox) subunit blocked neuronal superoxide production and negated the deleterious effects of hyperglycemia. These findings identify glucose as the requisite electron donor for reperfusion-induced neuronal superoxide production and establish a previously unrecognized mechanism by which hyperglycemia can exacerbate ischemic brain injury.

Desflurane Affords Greater Protection Than Halothane in the Function of Mitochondria Against Forebrain Ischemia Reperfusion Injury in Rats

Halothane and desflurane have been shown to attenuate neuronal injury; however, the effects of these anesthetics on mitochondria are unclear. We investigated whether halothane and desflurane affect the function of mitochondria after cerebral ischemia in rats. Forty male Wistar rats were randomly divided into four groups (n = 10 each): sham group; 1.5 minimal alveolar concentration (MAC) halothane group; 1.0 MAC desflurane; and 1.5 MAC desflurane group. Forebrain ischemia was induced after 40-min inhalation of 1.5 MAC halothane, 1.0 MAC or 1.5 MAC desflurane by clamping the bilateral common carotid arteries and decreasing arterial blood pressure. After isolation of the brain mitochondria, mitochondrial membrane permeability was assayed spectrophotometrically with 40-200 microM Ca(2+), and mitochondrial membrane potentials were measured by a fluorospectrophotometer with the addition of rhodamine 123. The activities of mitochondrial respiratory chain complexes were also assayed spectrophotometrically. The results showed obvious mitochondrial swelling, loss of membrane potential with the addition of Ca(2+), and inhibition of the activities of complexes I + III and IV after forebrain ischemia reperfusion injury. Compared with the 1.5 MAC halothane group, 1.0 and 1.5 MAC desflurane reduced mitochondrial swelling by 23.9% (P < 0.001) and 23.2% (P < 0.001), whereas membrane potential dissipation was suppressed by 22.4% (P = 0.013) and 20.4% (P = 0.027). The activities of complexes I + III and IV were better preserved in 1.0 MAC and 1.5 MAC desflurane groups than in the 1.5 MAC halothane group by 34.6% (P = 0.027), 38.7% (P = 0.011), 53.9% (P = 0.009), and 55.8% (P = 0.007), respectively. Desflurane shows better preservation of mitochondrial function at 4 h after cerebral ischemia reperfusion injury, indicated by inhibition of mitochondrial swelling, increase of membrane potential, and improvement of functions of mitochondria respiratory complexes I + III and IV when compared with halothane.

Contribution of Downregulation of L-type Calcium Currents to Delayed Neuronal Death in Rat Hippocampus after Global Cerebral Ischemia and Reperfusion

Transient forebrain ischemia induces delayed, selective neuronal death in the CA1 region of the hippocampus. The underlying molecular mechanisms are as yet unclear, but it is known that activation of L-type Ca2+ channels specifically increases the expression of a group of genes required for neuronal survival. Accordingly, we examined temporal changes in L-type calcium-channel activity in CA1 and CA3 pyramidal neurons of rat hippocampus after transient forebrain ischemia by patch-clamp techniques. In vulnerable CA1 neurons, L-type Ca2+-channel activity was persistently downregulated after ischemic insult, whereas in invulnerable CA3 neurons, no change occurred. Downregulation of L-type calcium channels was partially caused by oxidation modulation in postischemic channels. Furthermore, L-type but neither N-type nor P/Q-type Ca2+-channel antagonists alone significantly inhibited the survival of cultured hippocampal neurons. In contrast, specific L-type calcium-channel agonist remarkably reduced neuronal cell death and restored the inhibited channels induced by nitric oxide donor. More importantly, L-type calcium-channel agonist applied after reoxygenation or reperfusion significantly decreased neuronal injury in in vitro oxygen-glucose deprivation ischemic model and in animals subjected to forebrain ischemia-reperfusion. Together, the present results suggest that ischemia-induced inhibition of L-type calcium currents may give rise to delayed death of neurons in the CA1 region, possibly via oxidation mechanisms. Our findings may lead to a new perspective on neuronal death after ischemic insult and suggest that a novel therapeutic approach, activation of L-type calcium channels, could be tested at late stages of reperfusion for stroke treatment.