nNOS downregulation attenuates neuronal apoptosis by inhibiting nNOS-GluR6 interaction and GluR6 nitrosylation in cerebral ischemic reperfusion

Glutamate receptor 6 (GluR6) is well documented to play a pivotal role in ischemic brain injury, which is mediated by the GluR6·PSD95·MLK3 signaling module and subsequent c-Jun N-terminal kinase (JNK) activation. Our recent studies show that GluR6 is S-nitrosylated in the early stages of ischemia-reperfusion. NO (Nitric Oxide) is mainly generated from neuronal nitric oxide synthase (nNOS) in cerebral neurons during the early stages of reperfusion. Here, the effect of nNOS downregulation on GluR6 S-nitrosylation and GluR6-mediated signaling was investigated in cerebral ischemia and reperfusion. Administration of nNOS oligonucleotides confirmed that GluR6 nitrosylation is induced by nNOS-derived endogenous NO and further activates the GluR6·PSD95·MLK3 signaling module and JNK signaling pathway. Moreover, this study revealed for the first time that nNOS can bind with GluR6 during ischemic reperfusion, and PSD95 is involved in this interaction. In summary, our results suggest that nNOS binds with GluR6 via PSD95 and then produces endogenous NO to S-nitrosylate GluR6 in cerebral ischemia-reperfusion, which provides a new approach for stroke therapy.     

Reduced neuronal nitric oxide synthase is involved in ischemia-induced hippocampal neurogenesis by up-regulating inducible nitric oxide synthase expression

Nitric oxide (NO), a free radical with signaling functions in the CNS, is implicated in some developmental processes, including neuronal survival, precursor proliferation, and differentiation. However, neuronal nitric oxide synthase (nNOS) -derived NO and inducible nitric oxide synthase (iNOS) -derived NO play opposite role in regulating neurogenesis in the dentate gyrus after cerebral ischemia. In this study, we show that focal cerebral ischemia reduced nNOS expression and enzymatic activity in the hippocampus. Ischemia-induced cell proliferation in the dentate gyrus was augmented in the null mutant mice lacking nNOS gene (nNOS−/−) and in the rats receiving 7-nitroindazole, a selective nNOS inhibitor, after stroke. Inhibition of nNOS ameliorated ischemic injury, up-regulated iNOS expression, and enzymatic activity in the ischemic hippocampus. Inhibition of nNOS increased and iNOS inhibitor decreased cAMP response element-binding protein phosphorylation in the ipsilateral hippocampus in the late stage of stroke. Moreover, the effects of 7-nitroindazole on neurogenesis after ischemia disappeared in the null mutant mice lacking iNOS gene (iNOS−/−). These results suggest that reduced nNOS is involved in ischemia-induced hippocampal neurogenesis by up-regulating iNOS expression and cAMP response element-binding protein phosphorylation.     

Ginsenoside Rd attenuates early oxidative damage and sequential inflammatory response after transient focal ischemia in rats

We previously found that ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, attenuates neuronal oxidative damage in vitro induced by hydrogen peroxide and oxygen-glucose deprivation. In this study, we sought to investigate the potential protective effects and associated mechanisms of Rd in a rat model of focal cerebral ischemia. Rats administered with Rd (0.1-200mg/kg) or vehicle was subjected to transient middle cerebral artery occlusion. Rd at the dose of 10-50mg/kg significantly reduced the infarct volume and improved the long-term neurological outcome up to 6 weeks after ischemia. To evaluate the underlying mechanisms, in vivo free radical generation was monitored using microdialysis, oxidative DNA damage was identified by 8-hydroxy-deoxyguanosine immunostaining, oxidative protein damage was identified by the assessment of protein carbonyl and advanced glycosylation end products, and lipid peroxidation was estimated by determining the malondialdehyde and 4-hydroxynonenal formations. Microdialysis results displayed a prominent inhibitory effect of Rd on the hydroxy radical formation trapped as 2,3- and 2,5-DHBA. Early accumulations of DNA, protein and lipid peroxidation products were also suppressed by Rd treatment. Although Rd partly preserved endogenous antioxidant activities in the ischemic penumbra, in sham rats without stroke, endogenous antioxidant activities were not affected by Rd. Furthermore, we assayed sequential inflammatory response in a later phase after ischemia. Rd significantly eliminated inflammatory injury as indicated by the suppression of microglial activation, inducible nitric oxide synthase and cyclooxygenase-2 expression. Collectively, these findings demonstrated that Rd exerts neuroprotection in transient focal ischemia, which may involve early free radicals scavenging pathway and a late anti-inflammatory effect.     

Inhibition of nNOS reduces ischemic cell death through down-regulating calpain and caspase-3 after experimental stroke

In vitro nitric oxide (NO) regulates calpain and caspase-3 activation, and in vivo neuronal nitric oxide synthase (nNOS), calpain and caspase-3 participate in the ischemic brain injury. Our objective was to investigate whether nNOS was involved in the ischemic brain injury through activating calpain and caspase-3 during experimental stroke. Rats received 1-h ischemia by intraluminant filament, and then reperfused for 23 h (R 23 h). nNOS inhibitor 7-nitroindozale (7-NI, 50 mg/kg) was administrated intraperitoneally 5 min before ischemia. Our data showed that treatment with 7-NI markedly reduced neurological deficits, the brain swelling, and the infarct volume at R 23 h. Enzyme studies revealed significant suppression of the activities of m-calpain and caspase-3 in penumbra and core, and the activities of μ-calpain in penumbra, but not in core, in 7-NI-treated rats versus vehicle-treated rats. Western blot analysis demonstrated that 7-NI markedly increased the levels of MAP-2 and spectrin in penumbra and core compared with vehicle-treated rats. Histopathological studies displayed that 7-NI significantly reduced the necrotic cell death in penumbra and core, and apoptotic cell death in penumbra, but not in core. These data demonstrate the involvement of NO produced by nNOS in the ischemic neuronal injury through affecting the activation of calpain and caspase-3 in penumbra and core after experimental stroke, which provides a new perspective on possible mechanisms of action of nNOS inhibition in cerebral ischemia.     

Presence and activity of nitric oxide synthase isoforms in ischemia-reperfusion-injured flaps

Nitric oxide is produced from the amino acid L-arginine by nitric oxide synthase, which has three known isoforms: (1) endothelial nitric oxide synthase and (2) brain nitric oxide synthase, both of which are constitutive nitric oxide synthase; and (3) inducible nitric oxide synthase. The authors' hypothesis is that after reperfusion injury, endothelial cell dysfunction leads to disruption of nitric oxide synthase-mediated nitric oxide production and that this may in part explain the deleterious effects of ischemia-reperfusion injury on tissue survival and blood reflow in flaps. An experiment was designed to study the effects of ischemia-reperfusion injury on the bioactivity of all three isoforms of nitric oxide synthase. Buttock skin flaps and latissimus dorsi myocutaneous flaps were elevated in eight pigs. Flaps on one side of the animal were randomized to receive 6 hours of arterial ischemia, whereas flaps on the other side served as controls. At 6 hours of ischemia and at 1, 4, and 18 hours after reflow, tissue biopsy specimens were obtained and were processed for both constitutive nitric oxide synthase and inducible nitric oxide synthase enzyme activity on the basis of the L-citrulline assay. In addition, specimens were processed for Western blot analysis of the three isoforms. The authors' results revealed three key findings: first, there was a statistically significant (p < 0.001) decrease in constitutive nitric oxide synthase activity of ischemia-reperfusion-injured flaps as compared with controls in both skin and muscle for all time intervals measured. Second, Western blot analyses of endothelial nitric oxide synthase and brain nitric oxide synthase showed a significant decrease in the signal intensity in ischemic and reperfused tissue as compared with controls. Third, the inducible nitric oxide synthase isoform's activity and protein remained undetectable in both tissue types for all time points measured. The authors' data demonstrated that following ischemia-reperfusion injury in the pig flap model there was a disruption of constitutive nitric oxide synthase expression and activity, which may lead to decreased nitric oxide production. The significant decrease in nitric oxide synthase activity found in the current study may partly explain the mechanism of tissue damage in flaps subjected to ischemia-reperfusion injury. Knowledge of the kinetics of nitric oxide synthase activity under conditions of ischemia-reperfusion injury has important implications for the choice and timing of delivery of therapeutic agents whose goal is to increase the bioavailability of nitric oxide in reperfused tissue.     

Neuroprotective Potential of Fasudil Mesylate in Brain Ischemia-Reperfusion Injury of Rats

We previously reported that inhibition of Rho-kinase (ROCK) by hydroxyl fasudil improves cognitive deficit and neuronal damage in rats with chronic cerebral ischemia (Huang et al., Cell Mol Neurobiol 28:757–768, 2008). In this study, fasudil mesylate (FM) was investigated for its neuroprotective potential in rats with ischemia following middle cerebral artery occlusion (MCAO) and reperfusion. The effect of fasudil mesylate was also studied in rat brain cortical and hippocampal slices treated with oxygen-glucose deprivation (OGD) injury. Gross anatomy showed that cerebral infarct size, measured with 2,3,5-triphenyltetrazolium chloride (TTC) staining, was significantly smaller in the FM-treated than in the non-FM-treated ischemic rats. In the brain regions vulnerable to ischemia of ischemic rats, fasudil mesylate was also found to significantly restore the enzyme protein expression level of endothelial nitric oxide synthase (eNOS), which was decreased in ischemia. However, it remarkably reduced the protein synthesis of inducible nitric oxide synthase (iNOS) that was induced by ischemia and reperfusion. In rat brain slices treated with OGD injury, fasudil mesylate increased the neuronal cell viability by 40% for cortex and by 61% for hippocampus, respectively. Finally, in the presence of OGD and fasudil mesylate, superoxide dismutase (SOD) activity was increased by 50% for cortex and by 58% for hippocampus, compared to OGD only group. In conclusion, our in vivo study showed that fasudil mesylate not only decreased neurological deficit but also reduced cerebral infarct size, possibly and at least partially by augmenting eNOS protein expression and inhibiting iNOS protein expression after ischemia-reperfusion. Xian-Ju Huang contributed equally to this article.     

缺血再灌注对小鼠肠神经丛nNOS 和iNOS表达的影响

目的观察缺血再灌注后小鼠回肠神经型一氧化氮合酶(neuron alnitric oxide synthase,nNOS)和诱导型一氧化氮合酶(induciblenitric oxide synthase,iNOS)的表达,探讨肠缺血再灌注损伤(ischemia-reperfusion injury,IRI)的发生机制。方法采用小鼠肠系膜上动脉缺血再灌注模型,根据不同再灌注时间对小鼠随机分1d组、3d组、5d组、7d组、对照组和假手术组,用SP法检测小鼠回肠nNOS和iNOS的表达情况。结果与对照组和假手术组相比较,nNOS在再灌注1d后开始在肌间神经丛持续高表达(P<0.01);而iNOS在再灌注3d后开始在肌间神经丛持续高表达(P<0.05)。结论nNOS和iNOS在肠缺血再灌注后的表达增强,提示一氧化氮及一氧化氮合酶与肠神经节细胞在缺血再灌注中的损伤有着密切关系。     

Nitroxyl exacerbates ischemic cerebral injury and oxidative neurotoxicity

Nitroxyl (HNO) donor compounds function as potent vasorelaxants, improve myocardial contractility and reduce ischemia-reperfusion injury in the cardiovascular system. With respect to the nervous system, HNO donors have been shown to attenuate NMDA receptor activity and neuronal injury, suggesting that its production may be protective against cerebral ischemic damage. Hence, we studied the effect of the classical HNO-donor, Angeli's salt (AS), on a cerebral ischemia/reperfusion injury in a mouse model of experimental stroke and on related in vitro paradigms of neurotoxicity. I.p. injection of AS (40 μmol/kg) in mice prior to middle cerebral artery occlusion exacerbated cortical infarct size and worsened the persistent neurological deficit. AS not only decreased systolic blood pressure, but also induced systemic oxidative stress in vivo indicated by increased isoprostane levels in urine and serum. In vitro , neuronal damage induced by oxygen-glucose-deprivation of mature neuronal cultures was exacerbated by AS, although there was no direct effect on glutamate excitotoxicity. Finally, AS exacerbated oxidative glutamate toxicity – that is, cell death propagated via oxidative stress in immature neurons devoid of ionotropic glutamate receptors. Taken together, our data indicate that HNO might worsen cerebral ischemia-reperfusion injury by increasing oxidative stress and decreasing brain perfusion at concentrations shown to be cardioprotective in vivo .     

The potential role of carbon dioxide in the neuroimmunoendocrine changes following cerebral ischemia

Carbon dioxide (CO(2)) interacts in complex ways with the brain and the endocrine and immune systems. Arterial CO(2) may be elevated or decreased following cerebral ischemia-reperfusion injury or stroke. The aim of the present review is to delineate potential changes in the neuroimmunoendocrine system following cerebral ischemia-reperfusion injury and to provide evidence for the modulatory role of carbon dioxide in this setting. It appears that lesions of the right and left cerebral hemispheres are associated with different patterns of immune activation and cytokine release. Changes in arterial CO(2) can profoundly alter the neuroimmunoendocrine system, especially the hypothalamic-pituitary-adrenal (HPA) axis and the production of pro-inflammatory cytokines. Hypercapnia activates the HPA axis, exerts antiinflammatory and antioxidant effects, and can alter the secretion and function of various brain neurotransmitters. There is conflicting evidence surrounding arterial CO(2): its effects on the ischemic brain may be either beneficial or deleterious. Mild hypercapnia may exert some neuroprotection following cerebral ischemia, but severe hypercapnia may aggravate neuronal injury by extra- and intra-cellular acidification and/or impairment of cellular calcium hemostasis. Future studies are required to delineate the potential relationship between arterial CO(2) and prognosis and long-term survival following cerebral ischemia-reperfusion injury. "Therapeutic hypercapnia" seems to be a promising approach to the treatment of stroke patients, and its use should be justified by further experimental and clinical studies.     

The double-edged role of nitric oxide in brain function and superoxide-mediated injury.

Fetal ischemia or hypoxia can lead to cerebral palsy, mental retardation and epilepsy. We propose that the production of nitric oxide and oxygen radicals by neurons when ischemic or hypoxic brain is reperfused may contribute to cerebral injury. Ischemia will depolarize neuronal membranes causing the synaptic discharge of the excitatory neurotransmitter glutamate, which in turn opens the voltage-dependent, N-methyl-D-aspartic acid-specific glutamate receptor/ionophore, allowing calcium to accumulate in the neuron. Calcium in turn activates an oxygen-dependent neuronal nitric oxide synthetase, which oxidizes arginine to produce nitric oxide (.NO) when oxygen is readmitted to brain by reperfusion. Nitric oxide reacts with the oxygen radical superoxide (O2-), also produced by reperfusion, to form peroxynitrite (ONOO-). Peroxynitrite can diffuse for several micrometers before decomposing to form the powerful and cytotoxic oxidants hydroxyl radical and nitrogen dioxide. The hypothesis is consistent with available evidence on the protective action of glutamate antagonists and of oxygen radical scavengers for limiting cerebral infarction following focal ischemia.     

Caffeic acid phenethyl ester reduces neurovascular inflammation and protects rat brain following transient focal cerebral ischemia

Ischemic stroke is a neurovascular disease treatable by thrombolytic therapy, but the therapy has to be initiated within 3 h of the incident. This therapeutic limitation stems from the secondary injury which results mainly from oxidative stress and inflammation. A potent antioxidant/anti-inflammatory agent, caffeic acid phenethyl ester (CAPE) has potential to mitigate stroke's secondary injury, and thereby widening the therapeutic window. We observed that CAPE protected the brain in a dose-dependent manner (1-10 mg/kg body weight) and showed a wide therapeutic window (about 18 h) in a rat model of transient focal cerebral ischemia and reperfusion. The treatment also increased nitric oxide and glutathione levels, decreased lipid peroxidation and nitrotyrosine levels, and enhanced cerebral blood flow. CAPE down-regulated inflammation by blocking nuclear factor kappa B activity. The affected mediators included adhesion molecules (intercellular adhesion molecule-1 and E-selectin), cytokines (tumor necrosis factor-alpha and interleukin-1beta) and inducible nitric oxide synthase. Anti-inflammatory action of CAPE was further documented through reduction of ED1 (marker of activated macrophage/microglia) expression. The treatment inhibited apoptotic cell death by down-regulating caspase 3 and up-regulating anti-apoptotic protein Bcl-xL. Conclusively, CAPE is a promising drug candidate for ischemic stroke treatment due to its inhibition of oxidative stress and inflammation, and its clinically relevant wide therapeutic window.     

Effects of L-NAME and L-arginine on ischemia-reperfusion injury in rat skeletal muscle

The involvement of nitric oxide in ischemia-reperfusion injury remains controversial and has been reported to be both beneficial and deleterious, depending on the tissue and model used. This study evaluated the effects of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine-methyl ester (L-NAME) and the substrate for nitric oxide synthase, L-arginine on skeletal muscle necrosis in a rat model of ischemia-reperfusion injury. The rectus femoris muscle in male Wistar rats (250 to 500 g) was isolated on its vascular pedicle and subjected to 4 hours of complete arteriovenous occlusion. The animals were divided into five groups: (1) sham-raised control, no ischemia, no treatment (n = 6); (2) 4 hours of ischemia (n = 6); (3) vehicle control, 4 hours of ischemia + saline (n = 6); (4) 4 hours of ischemia + L-arginine infusion (n = 6); and (5) 4 hours of ischemia + L-NAME infusion (n = 6). The infusions (10 mg/kg) were administered into the contralateral femoral vein beginning 5 minutes before reperfusion and during the following 30 to 45 minutes. Upon reperfusion, the muscle was sutured in its anatomic position and all wounds were closed. The percentage of muscle necrosis was assessed after 24 hours of reperfusion by serial transections, nitroblue tetrazolium staining, digital photography, and computerized planimetry. Sham (group 1) animals sustained baseline necrosis of 11.9 +/- 3.0 (percentage necrosis +/- SEM). Four hours of ischemia (group 2) significantly increased necrosis to 79.2 +/- 1.4 (p < 0.01). Vehicle control (group 3) had no significant difference in necrosis (81.17 +/- 5.0) versus untreated animals subjected to 4 hours of ischemia (group 2). Animals treated with L-arginine (group 4) had significantly reduced necrosis to 34.6 +/- 7.5 versus untreated (group 2) animals (p < 0.01). Animals infused with L-NAME (group 5) had no significant difference in necrosis (68.2 +/- 6.7) versus untreated (group 2) animals. L-Arginine (nitric oxide donor) significantly decreased the severity of muscle necrosis in this rat model of ischemia-reperfusion injury. L-arginine is known to increase the amount of nitric oxide through the action of nitric oxide synthase, whereas L-NAME, known to inhibit nitric oxide synthase and decrease nitric oxide production, had comparable results to the untreated 4-hour ischemia group. These results suggest that L-arginine, presumably through nitric oxide mediation, appears beneficial to rat skeletal muscle subjected to ischemia-reperfusion injury.     

Mitochondrial respiratory chain and free radical generation in stroke

Being the second most common cause of death in the industrial countries and one of the major causes of death and disability, stroke has a great effect on public health and is the neurological disease which accounts for the largest number of hospitalizations. In order to develop new treatments, biochemical mechanisms involved in brain damage have been investigated. Among them, oxidant species generated during stroke have been implicated as critical mediators of neuronal injury in this condition, although neuroprotective roles have also been demonstrated. This review is focused on the role of the mitochondrial respiratory chain as both source and target of reactive oxygen and nitrogen species such as nitric oxide, superoxide and peroxynitrite produced in cerebral ischemia. The neuroprotective role of antioxidants or other molecules acting on the mitochondrial respiratory chain and ATP synthesis in the setting of cerebral ischemia is discussed.     

Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95

Stroke is a major public health problem leading to high rates of death and disability in adults. Excessive stimulation of N-methyl-D-aspartate receptors (NMDARs) and the resulting neuronal nitric oxide synthase (nNOS) activation are crucial for neuronal injury after stroke insult. However, directly inhibiting NMDARs or nNOS can cause severe side effects because they have key physiological functions in the CNS. Here we show that cerebral ischemia induces the interaction of nNOS with postsynaptic density protein-95 (PSD-95). Disrupting nNOS-PSD-95 interaction via overexpressing the N-terminal amino acid residues 1-133 of nNOS (nNOS-N(1-133)) prevented glutamate-induced excitotoxicity and cerebral ischemic damage. Given the mechanism of nNOS-PSD-95 interaction, we developed a series of compounds and discovered a small-molecular inhibitor of the nNOS-PSD-95 interaction, ZL006. This drug blocked the ischemia-induced nNOS-PSD-95 association selectively, had potent neuroprotective activity in vitro and ameliorated focal cerebral ischemic damage in mice and rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion. Moreover, it readily crossed the blood-brain barrier, did not inhibit NMDAR function, catalytic activity of nNOS or spatial memory, and had no effect on aggressive behaviors. Thus, this new drug may serve as a treatment for stroke, perhaps without major side effects.     

Neuroprotective mechanisms of cerium oxide nanoparticles in a mouse hippocampal brain slice model of ischemia

Cerium oxide nanoparticles (nanoceria) are widely used as catalysts in industrial applications because of their potent free radical-scavenging properties. Given that free radicals play a prominent role in the pathology of many neurological diseases, we explored the use of nanoceria as a potential therapeutic agent for stroke. Using a mouse hippocampal brain slice model of cerebral ischemia, we show here that ceria nanoparticles reduce ischemic cell death by approximately 50%. The neuroprotective effects of nanoceria were due to a modest reduction in reactive oxygen species, in general, and ~ 15% reductions in the concentrations of superoxide (O₂^•−) and nitric oxide, specifically. Moreover, treatment with nanoceria markedly decreased (~ 70% reduction) the levels of ischemia-induced 3-nitrotyrosine, a modification to tyrosine residues in proteins induced by the peroxynitrite radical. These findings suggest that scavenging of peroxynitrite may be an important mechanism by which cerium oxide nanoparticles mitigate ischemic brain injury. Peroxynitrite plays a pivotal role in the dissemination of oxidative injury in biological tissues. Therefore, nanoceria may be useful as a therapeutic intervention to reduce oxidative and nitrosative damage after a stroke.     

The role of free radicals in cerebral hypoxia and ischemia

This review focuses on the effects that ischemia and hypoxia have on the cerebral cortex and the cerebellum during different periods of life. The acute interruption or reduction of cerebral blood flow, that can be induced by several factors and clinical pathologies, reduces available oxygen to the nervous system and this causes either focal or global brain damage, with characteristic biochemical and molecular alterations that can result in permanent or transitory neurological sequelae or even death. Under these circumstances, an increase in the activity of different isoforms of nitric oxide synthase occurs and nitric oxide is produced. This excess of nitric oxide reacts with cellular proteins yielding nitrotyrosine, thus contributing to cerebral damage. This phenomenon has been studied at different stages of perinatal and postnatal development, including aging animals. Both the duration and the intensity of the ischemic injury were evaluated. In all cases there is overproduction of nitric oxide in ischemia, which may represent an effort to reestablish normal blood flow. Unfortunately, in many cases this response becomes excessive and it triggers a cascade of free-radical reactions, leading to modifications of cerebral plasticity and overt injury.