Mid-cerebral Artery Occlusion Research Articles

Stroke Alters the Function of Enteric Neurons to Impair Smooth Muscle Relaxation and Dysregulates Gut Transit.

Gut dysmotility is common after ischemic stroke, but the mechanism underlying this response is unknown. Under homeostasis, gut motility is regulated by the neurons of the enteric nervous system that control contractile/relaxation activity of muscle cells in the gut wall. More recently, studies of gut inflammation revealed interactions of macrophages with enteric neurons are also involved in modulating gut motility. However, whether poststroke gut dysmotility is mediated by direct signaling to the enteric nervous system or indirectly via inflammatory macrophages is unknown. We examined these hypotheses by using a clinically relevant permanent intraluminal midcerebral artery occlusion experimental model of stroke. At 24 hours after stroke, we performed invivo and exvivo gut motility assays, flow cytometry, immunofluorescence, and transcriptomic analysis. Stroke-induced gut dysmotility was associated with recruitment of muscularis macrophages into the gastrointestinal tract and redistribution of muscularis macrophages away from myenteric ganglia. The permanent intraluminal midcerebral artery occlusion model caused changes in gene expression in muscularis macrophages consistent with an altered phenotype. While the size of myenteric ganglia after stroke was not altered, myenteric neurons from post-permanent intraluminal midcerebral artery occlusion mice showed a reduction in neuronal nitric oxide synthase expression, and this response was associated with enhanced intestinal smooth muscle contraction exvivo. Finally, chemical sympathectomy with 6-hydroxydopamine prevented the loss of myenteric neuronal nitric oxide synthase expression and stroke-induced slowed gut transit. Our findings demonstrate that activation of the sympathetic nervous system after stroke is associated with reduced neuronal nitric oxide synthase expression in myenteric neurons, resulting in impaired smooth muscle relaxation and dysregulation of gut transit.

The role of β2 adrenergic receptor on infection development after ischaemic stroke

Mechanisms underlying post-stroke immune impairments and subsequent development of fatal lung infection have been suggested to involve multiple pathways, including hyperactivation of the sympathetic nervous system (SNS), which results in the excessive release of catecholamines and activation of β-adrenergic receptors (βARs). Indeed, previous reports from experimental studies demonstrated that post-stroke infection can be inhibited with treatment of β-blockers. However, the effectiveness of β-blockers in reducing post-stroke infection has yielded mixed results in retrospective clinical trials and its use remain controversial. In this study, we performed mid-cerebral artery occlusion in mice either genetically deficient in β2-adrenergic receptor (β2AR) or treated with non-selective and selective βAR antagonists to explore the contributions of the SNS in the development of post-stroke lung infection. Stroke induced a systemic activation of the SNS as indicated by elevated levels of plasma catecholamines and UCP-1 activity. However, β2AR deficient mice showed similar degrees of post-stroke immune impairment and infection rate compared to wildtype counterparts, potentially due to compensatory mechanisms common in transgenic animals. To overcome this, we treated post-stroke wildtype mice with pharmacological inhibitors of the βARs, including the non-selective antagonist propranolol (PPL) and selective β2AR antagonist ICI-118551. Both pharmacological strategies to block the action of SNS signalling were unable to reduce infection in mice that underwent ischaemic stroke. Overall, our data suggests that other mechanisms independent or in combination with β2AR activation contribute to the development of post-stroke infection.

Effect of hydrogen on mitochondrial membrane potential during focal cerebral ischemia-reperfusion in rats

Objective To investigate the effect of hydrogen on mitochondrial membrane potential during focal cerebral ischemia-reperfusion (I/R) in rats. Methods Eighty-four healthy adult male SPF Sprague-Dawley rats, weighing 220-240 g, were divided into 3 groups (n=28 each) using a random number table: sham operation group (Sham group), I/R group and hydrogen group (H2 group). Focal cerebral I/R was produced by mid-cerebral artery occlusion in I/R and H2 groups.Hydrogen 10 ml/kg was intraperitoneally injected immediately after onset of reperfusion and at 12 h of reperfusion in group H2.Neurological deficit was scored at 24 h of reperfusion.The rats were then sacrificed and brain tissues in cortex were removed for microscopic examination of the pathological changes and for determination of cerebral infarct size (by TTC staining), nerve cell apoptosis (by TUNEL), mitochondrial membrane potential(by JC-1 staining) and expression of Bcl-2 and Bax (by Western blot). Apoptotic index (AI) was calculated. Results Compared with Sham group, the neurological deficit score, percentage of cerebral infarct size and AI were significantly increased, the mitochondrial membrane potential was decreased, Bax expression in brain tissues was up-regulated, and Bcl-2 expression in brain tissues was down-regulated in I/R and H2 groups (P<0.05). Compared with I/R group, the neurological deficit score, percentage of cerebral infarct size and AI were significantly decreased, the mitochondrial membrane potential was increased, Bax expression in brain tissues was down-regulated, and Bcl-2 expression in brain tissues was up-regulated (P<0.05), and the pathological changes of brain tissues were significantly attenuated in H2 group. Conclusion The mechanism by which hydrogen reduces focal cerebral I/R injury is related to decreasing dissipation of mitochondrial membrane potential and inhibiting nerve cell apoptosis in rats. Key words: Hydrogen; Reperfusion injury; Brain; Membrane potential, mitochondrial

Activation of Endothelial Nitric Oxide Production by the Sigma Receptor Agonist Afobazole

Afobazole is a sigma‐1 receptor agonist that has been shown to enhance survival in a rat mid‐cerebral artery occlusion stroke model. The mechanism has been previously attributed to an inhibition of ischemia‐induced calcium dysregulation that lowers glial cell loss and infarct volume. A recently published finding is that afobazole can stimulate nitric oxide (NO) production from lymphatic endothelial cells. In the current study, we investigated the ability of afobazole to relax arterioles from the brain and potential interactions between the sigma‐1 receptor and endothelial NO synthase (eNOS). Parenchymal arterioles were isolated from brains of Sprague‐Dawley rats and mounted onto glass micropipettes in a 37 °C bath containing albumin‐physiological salt solution. Afobazole was applied to the bath at concentrations of 50, 100, 150, 200, and 250 μM. The ability of afobazole to elicit NO production from cultured human brain microvascular endothelial cells (HBMEC) was assessed using 4‐Amino‐5‐Methylamino‐2′,7′‐Difluorofluorescein Diacetate (DAF‐FM) loading and fluorescence microscopy. Phosphorylation of eNOS on Ser‐1177 in the absence or presence of afobazole was evaluated by Western blotting. Lastly, intracellular localization of eNOS and the sigma‐1 receptor were evaluated by immunofluorescence and laser confocal microscopy. The results show that 200 and 250 μM afobazole elicited significant dilation of parenchymal arterioles. Afobazole also elicited elevated NO production from HBMEC in association with elevated phosphorylation of eNOS on Ser‐177. Lastly, eNOS and the sigma‐1 receptor were found partially colocalized in what appears to be the Golgi complex in confocal image stacks of HBMEC. This study shows for the first time a novel mechanism by which afobazole can relax brain arterioles via eNOS‐mediated NO production. This mechanism is likely associated with previously reported improvements of outcomes following stroke induced brain injury.Support or Funding InformationSupported by NIH grant R01GM120774.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The role of argon in stroke

Stroke, also known as “cerebrovascular accident”, is an acute cerebrovascular disease that is caused by a sudden rupture of blood vessels in the brain or obstruction of the blood supply by blockage of blood vessels, thus including hemorrhagic and ischemic strokes. The incidence of ischemic stroke is higher than that of hemorrhagic stroke, and accounts for 80% of the total number of strokes. However, the mortality rate of hemorrhagic stroke is relatively high. Internal carotid artery and vertebral artery occlusion and stenosis can cause ischemic stroke, and especially males over 40 years of age are at a high risk of morbidity. According to the survey, stroke in urban and rural areas has become the first cause of death in China. It is also the leading cause of disability in Chinese adults. In a word, stroke is characterized by high morbidity, high mortality and high disability rates. Studies have shown that many noble gases have the neuroprotective effects. For example, xenon has been extensively studied in various animal models of neurological injury including stroke, hypoxic-ischemic encephalopathy. Compared to xenon, Argon, as a noble gas, is abundant, cheap and widely applicable, and has been also demonstrated to be neuroprotective in many research studies. In a variety of models, ranging from oxygen-glucose deprivation in cell culture to complex models of mid-cerebral artery occlusion, subarachnoid hemorrhage or retinal ischemia-reperfusion injury in animals. Argon administration after individual injury demonstrated favorable effects, particularly increased cell survival and even improved neuronal function. Therefore the neuroprotective effects of argon may be of possible clinical use for opening a potential therapeutic window in stroke. It is important to illuminate the mechanisms of argon in nerve function and to explore the best use of this gas in stroke treatment.

Type-I interferon signalling through IFNAR1 plays a deleterious role in the outcome after stroke

Neuroinflammation contributes significantly to the pathophysiology of stroke. Here we test the hypothesis that the type I interferon receptor (IFNAR1) plays a critical role in neural injury after stroke by regulating the resultant pro-inflammatory environment. Wild-type and IFNAR1-/- primary murine neurons and glia were exposed to oxygen glucose deprivation (OGD) and cell viability was assessed. Transient cerebral ischemia/reperfusion injury was induced by mid-cerebral artery occlusion (MCAO) in wild-type and IFNAR1-/- and IFNAR2-/- mice in vivo, and infarct size, and molecular parameters measured. To block IFNAR1 signalling, wild-type mice were treated with a blocking monoclonal antibody directed to IFNAR1 (MAR-1) and MCAO was performed. Quantitative PCR confirmed MCAO in wild-type mice induced a robust type-I interferon gene regulatory signature. Primary cultured IFNAR1-deficient neurons were found to be protected from cell death when exposed to OGD in contrast to primary cultured IFNAR1-deficient glial cells. IFNAR1-/- mice demonstrated a decreased infarct size (24.9 ± 7.1 mm3 n = 8) compared to wild-type controls (65.1 ± 4.8 mm3 n = 8). Western blot and immunohistochemistry showed alterations in Akt and Stat-3 phosphorylation profiles in the IFNAR1-/- brain. MAR-1 injection into WT mice (i.v. 0.5 mg 60 min prior to MCAO) resulted in a 60% decrease in infarct size when compared to the IgG control. IFNAR2-/- mice failed to display the neuroprotective phenotype seen in IFNAR1-/- mice after MCAO. Our data proposes that central nervous system signalling through IFNAR1 is a previously unrecognised factor that is critical to neural injury after stroke.

The Molecular Pathway of Argon-Mediated Neuroprotection.

The noble gas argon has attracted increasing attention in recent years, especially because of its neuroprotective properties. In a variety of models, ranging from oxygen-glucose deprivation in cell culture to complex models of mid-cerebral artery occlusion, subarachnoid hemorrhage or retinal ischemia-reperfusion injury in animals, argon administration after individual injury demonstrated favorable effects, particularly increased cell survival and even improved neuronal function. As an inert molecule, argon did not show signs of adverse effects in the in vitro and in vivo model used, while being comparably cheap and easy to apply. However, the molecular mechanism by which argon is able to exert its protective and beneficial characteristics remains unclear. Although there are many pieces missing to complete the signaling pathway throughout the cell, it is the aim of this review to summarize the known parts of the molecular pathways and to combine them to provide a clear insight into the cellular pathway, starting with the receptors that may be involved in mediating argons effects and ending with the translational response.

RIG-I Signaling in the Innate Immune Response After Stroke (INM1P.445)

Abstract Stroke is associated with an inflammatory response that contributes to deleterious outcomes. However, the molecular basis in the regulation of inflammation remains poorly understood. Here, we examine the activation of retinoic acid inducible gene-like (RIG) receptors (RLRs) and the involvement of RLR signaling in the regulation of type I IFNs following mid-cerebral artery occlusion (MCAO). In this study we show that in the rat hippocampus RIG-I and IFN-α expression are increased after MCAO. Similarly, we show that in astrocytes in culture, oxygen glucose deprivation (OGD) resulted in increased RIG-I and IFN-α expression. These data are consistent with immunohistochemical analysis of hippocampal sections, indicating that in glial fibrillary acidic protein (GFAP) positive cells there was an increase in RIG-I immunoreactivity after MCAO. Moreover, n-propyl gallate (n-PG) can be used to inhibit RIG-I/IFN-α signaling in astrocytes. In conclusion, these data indicate that RIG-I is involved in the inflammatory response after stroke.

Role of mitochondrial ATP-sensitive potassium channel in sevoflurane preconditioning-reduced focal cerebral ischemia-reperfusion injury in rats

Objective To investigate the role of the mitochondrial ATP-sensitive potassium (mito-K_(ATP)) channel in sevoflurane preconditioning-reduced focal cerebral ischemia-reperfnsion (I/R) injury in rats. Methods One hundred healthy 3-4 month old male SD rats 250-300 g were randomly assigned into 5 groups (n = 20 each) : group Ⅰ sham operatiun (group S); group Ⅱ I/R; group Ⅲ sevoflurane preconditioning (group Sevo); group Ⅳ 5-hydroxydecannate (5-HD) and group Ⅴ 5-HD + Sevo. Focal cerebral I/R was produced by mid-cerebral artery occlusion (MCAO) in group Ⅱ-Ⅴ . Cerebral ischemia was maintained for 2 h followed by 6 and 24 h reporfnsiun. In group Ⅲ and Ⅴ 2.4% sevoflurane in 97.6% O_2 was inhaled for 60 min at 24 h before MCAO. In group Ⅴ 5-HD (a selective mito-K_(ATP) channel antagonist) 40 mg/kg was given intraporitoneally (IP) at 30 min before sevoflurane preconditioning. In group Ⅳ 100% O_2 was inhaled instead of sevoflurune and 5-HD 40 mg/kg was given IP at 30 min before O_2 inhalation. Neurological deficit score (NDS, 0=no deficit, 7 = death) were measured at 6 and 24 h of reperfusion and the animals were then killed. Their brains were removed for determination of infaret size by TTC and cellular translocation of PKCe by Western blot analysis. Results NDS were significantly lower and brain infarct size was significantly smaller in group.Sevo (Ⅲ) than in I/R group (Ⅱ). The neuroprotection induced by sevoflurane preconditioning was reversed by 5-HD 40 mg/kg in group Ⅴ (5-HD + Sevo). There was no significant difference in infarct size and NDS among group Ⅱ , Ⅳ and Ⅴ . PKCe was activated and translocated to the membrane fraction at 6 h of reperfusion in group Sevo (Ⅲ) and this effect of sevoflurane was also abolished by 5-HD in group Ⅴ (5-HD + Sevo). Conclusion Scvoflurane preconditioning-induced neuroprotection is mediated by mito-K_(ATP) channel probably through regulating PKCe membrane translocation. Key words: Anesthetics,inhalation; Ischemic preconditioning; KATP channels; Brain; Reperfusion injury

NAP protects against cytochrome c release: Inhibition of the initiation of apoptosis

NAPVSIPQ (NAP), an 8 amino acid peptide derived from activity-dependent neuroprotective protein (ADNP), provides neuroprotection through interaction with microtubules. Previous results have demonstrated NAP protection against oxygen-glucose deprivation in hippocampal cells in culture. Furthermore, in vivo studies have shown that NAP reduces caspase 3 activation in rats subjected to permanent mid-cerebral artery occlusion (a rat model of stroke). Oxygen-glucose deprivation (ischemia) has been associated with microtubule breakdown and cytochrome c release from mitochondria leading to apoptosis. Here, NAP in concentrations ranging from 10 − 14 M to 10 − 8 M completely blocked cytochrome c release in cortical neurons subjected to oxygen-glucose deprivation. Furthermore, quantitative microscopy coupled to microtubule immunocytochemistry suggested that NAP prevented microtubule degradation under oxidative stress. As cytochrome c release is a known initiator of the apoptotic pathway, it is suggested that NAP inhibits the early events of apoptosis.

Absence of glutathione peroxidase‐1 exacerbates cerebral ischemia‐reperfusion injury by reducing post‐ischemic microvascular perfusion

Mice deficient in the anti-oxidant enzyme glutathione peroxidase-1 (Gpx1) have a greater susceptibility to cerebral injury following a localized ischemic event. Much of the response to ischemia-reperfusion is caused by aberrant responses within the microvasculature, including inflammation, diminished endothelial barrier function (increased vascular permeability), endothelial activation, and reduced microvascular perfusion. However, the role of Gpx1 in regulating these responses has not been investigated. Wild-type and Gpx1-/- mice underwent focal cerebral ischemia via mid-cerebral artery occlusion followed by measurement of cerebral perfusion via laser Doppler and intravital microscopy. Post-ischemic brains in wild-type mice displayed significant deficit in microvascular perfusion. However, in Gpx1-/- mice, the deficit in cerebral blood flow was significantly greater than that in wild-type mice, and this was associated with significant increase in infarct size and increased vascular permeability. Ischemia-reperfusion also resulted in expression of matrix metalloproteinase-9 (MMP-9) in endothelial cells. The absence of Gpx1 was associated with marked increase in pro-MMP-9 expression as well as potentiated MMP-9 activity. Pre-treatment of Gpx1-/- mice with the anti-oxidant ebselen restored microvascular perfusion, limited the induction and activation of MMP-9, and attenuated the increases in infarct size and vascular permeability. These findings demonstrate that the anti-oxidant function of Gpx1 plays a critical role in protecting the cerebral microvasculature against ischemia-reperfusion injury by preserving microvascular perfusion and inhibiting MMP-9 expression.

1-Hydroxy-2-pyridinone-based MMP inhibitors: Synthesis and biological evaluation for the treatment of ischemic stroke

Matrix metalloproteinase-9 (MMP-9) has been implicated in the breakdown of the blood-brain barrier during cerebral ischemia. As a result, inhibition of MMP-9 may have utility as a therapeutic intervention in stroke. Towards this end, we have synthesized a series of 1-hydroxy-2-pyridinones that have excellent in vitro potency in inhibiting MMP-9 in addition to MMP-2. Representative compounds also demonstrate good efficacy in the mouse transient mid-cerebral artery occlusion (tMCAO) model of cerebral ischemia.

Mesenchymal stem cells (MSC) as therapeutic cytoreagents for gene therapy

We developed human mesenchymal stem cell (MSC) lines that could differentiate into various tissue cells including bone, neural cells, bone marrow (BM) stromal cells supporting the growth of hematopoietic stem cell (HSC), and so-called 'tumor stromal cells' mixing with tumor cells. We investigated the applicability of MSC as therapeutic cell transplanting reagents (cytoreagents). Telomerized human BM derived stromal cells exhibited a prolonged lifespan and supported the growth of hematopoietic clonogenic cells. The gene transfer of Indian hedgehog (Ihh) remarkably enhanced the HSC expansion supported by the human BM stromal cells. Gene-modified MSC are useful as therapeutic tools for brain tissue damage (e.g. brain infarction) and malignant brain neoplasms. MSC transplantation protected the brain tissue from acute ischemic damage in the midcerebral artery occlusion (MCAO) animal model. Brain-derived neurotrophic factor (BDNF)-gene transduction further enhanced the protective efficacy against the ischemic damage. MSC possessed excellent migratory ability and exerted inhibitory effects on the proliferation of glioma cells. Gene-modification of MSC with therapeutic cytokines clearly augmented the antitumor effect and prolonged the survival of tumor-bearing animals. Gene therapy employing MSC as a tissue-protecting and targeting cytoreagent would be a promising approach.

Diminished Akt phosphorylation in neurons lacking glutathione peroxidase‐1 (Gpx1) leads to increased susceptibility to oxidative stress‐induced cell death

We have previously identified an increased susceptibility of glutathione peroxidase-1 (Gpx1)-/- mice to neuronal apoptosis following mid-cerebral artery (MCA) occlusion. This study was designed to elucidate the mechanisms involved in elevated neuronal cell death arising from an altered endogenous oxidant state. This was addressed in both an in vitro and in vivo model of oxidative stress in the form of exogenous H2O2 and cerebral ischaemia, respectively. Increased levels of cell death were detected in primary neurons lacking Gpx1 following the addition of exogenous H2O2. This increased apoptosis correlated with a down-regulation in the activation of the phospho-inositide 3-kinase [PI3K]-Akt survival pathway. The importance of this pathway in protecting against H2O2-induced cell death was highlighted by the increased susceptibility of wildtype neurons to apoptosis when treated with the PI3K inhibitor, LY294002. The Gpx1-/- mice also demonstrated elevated neuronal cell death following MCA occlusion. Although Akt phosphorylation was detected in the Gpx1-/- brains, activation was not seen in later reperfusion events, as demonstrated in wildtype brains. Previous studies have highlighted the importance of Akt phosphorylation in protecting against neuronal cell death following cerebral ischaemia-reperfusion. Our results suggest that the increased susceptibility of Gpx1-/- neurons to H2O2-induced apoptosis and neuronal cell death in vivo following cerebral ischaemia-reperfusion injury can be attributed in part to diminished activation of Akt. Perturbations in key anti-apoptotic mechanisms as a result of an altered redox state may have implications in the study of oxidative stress-mediated neuropathologies.

CONTRIBUTION OF CEREBRAL NITRIC OXIDE TO BLADDER OVERACTIVITY AFTER CEREBRAL INFARCTION IN RATS

We investigated the contribution of cerebral nitric oxide to neurogenic voiding dysfunction after cerebral infarction. The left mid cerebral artery in female Sprague-Dawley rats was occluded with 4-zero monofilament nylon thread. Bladder activity was monitored during infusion cystometrography. Time or dose dependent effects of intracerebral ventricular administration of the nonselective nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), were investigated in conscious, sham operated and cerebral infarcted rats. The selective neuronal nitric oxide synthase inhibitor 1-(2-trifluoromethylphenyl) imidazole was also administered to determine the participation of nitric oxide synthase subtypes. Cross-sectional infarct area was measured and infarct volume was calculated 12 hours after mid cerebral artery occlusion. Bladder capacity was reduced by 54% 30 minutes after mid cerebral artery occlusion. L-NAME significantly increased bladder capacity in a dose and time dependent manner in cerebral infarcted rats but had no effect on sham operated rats. L-NAME (50 microg./kg.) administered 3 or 5 hours after occlusion significantly increased bladder capacity. This effect of L-NAME was reversed by injecting 250 microg. L-arginine per rat, which alone did not produce any significant change in bladder capacity in cerebral infarcted rats. Administration of 1-(2-trifluoromethylphenyl) imidazole also significantly increased bladder capacity in these rats. On the other hand, 5 microg. of the nitric oxide donor FK-409 per rat reduced bladder capacity for 10 to 15 minutes. None of the drugs affected infarct volume. These results indicate that supraspinal nitric oxide has an important role in bladder overactivity after cerebral infarction but it does not affect normal micturition in rats. This finding suggests a central mechanism sensitive to nitric oxide for bladder overactivity after cerebral infarction.