Inflammation and apoptosis play critical roles in the acute progression of ischemic injury pathology. Emerging evidence indicates that vagus nerve stimulation (VNS) following focal cerebral ischemia and reperfusion (I/R) may be neuroprotective by limiting infarct size. However, the underlying molecular mechanisms remain unclear. In this study, we investigated whether the protective effects of VNS in acute cerebral I/R injury were associated with anti-inflammatory and anti-apoptotic processes. Male Sprague-Dawley (SD) rats underwent VNS at 30 min after focal cerebral I/R surgery. Twenty-four h after reperfusion, neurological deficit scores, infarct volume, and neuronal apoptosis were evaluated. In addition, the levels of pro-inflammatory cytokines were detected using enzyme-linked immune sorbent assay (ELISA), and immunofluorescence staining for the endogenous “cholinergic anti-inflammatory pathway” was also performed. The protein expression of a7 nicotinic acetylcholine receptor (a7nAchR), phosphorylated Akt (p-Akt), and cleaved caspase 3 in ischemic penumbra were determined with Western blot analysis. I/R rats treated with VNS (I/R+VNS) had significantly better neurological deficit scores, reduced cerebral infarct volume, and decreased number of TdT mediated dUTP nick end labeling (TUNEL) positive cells. Furthermore, in the ischemic penumbra of the I/R+VNS group, the levels of pro-inflammatory cytokines and cleaved caspase 3 protein were significantly decreased, and the levels of a7nAchR and phosphorylated Akt were significantly increased relative to the I/R alone group. These results indicate that VNS is neuroprotective in acute cerebral I/R injury by suppressing inflammation and apoptosis via activation of cholinergic and a7nAchR/Akt pathways.

Vagus nerve stimulation (VNS) exerts neuroprotective effects against cerebral ischemia/reperfusion (I/R) injury and modulates redox status, potentially through the activity of miR-210, an important microRNA that is regulated by hypoxia-inducible factor and Akt-dependent pathways. The aim of this study was to determine the mechanisms of VNS-and miR-210-mediated hypoxic tolerance. Male Sprague-Dawley rats were preconditioned with a miR-210 antagomir (A) or with an antagomir control (AC), followed by middle cerebral artery occlusion and VNS treatment. The animals were divided into eight groups: sham I/R, I/R, I/R+AC, I/R+A, sham I/R+VNS, I/R+VNS, I/ R+VNS+AC, and I/R+VNS+A. Activation of the endogenous cholinergic a7 nicotinic acetylcholine receptor (a7nAchR) pathway was identified using double immunofluorescence staining. miR-210 expression was measured by PCR. Behavioral outcomes, infarct volume, and neuronal apoptosis were observed at 24 h following reperfusion. Markers of oxidative stress were detected using ELISA. Rats treated with VNS showed increased miR-210 expression as well as decreased apoptosis and antioxidant stress responses compared with the I/R group; these rats also showed increased p-Akt protein expression and significantly decreased levels of cleaved caspase 3 in the ischemic penumbra, as measured by western blot and immunofluorescence analyses, respectively. Strikingly, the beneficial effects of VNS were attenuated following miR-210 knockdown. In conclusion, our results indicate that miR-210 is a potential mediator of VNS-induced neuroprotection against I/R injury. Our study highlights the neuroprotective potential of VNS, which, to date, has been largely unexplored.

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Apoptosis plays an essential role in ischemic stroke pathogenesis. Research on the process of neuronal apoptosis in models of ischemic brain injury seems promising. The role of growth arrest and DNA-damage-inducible protein 45 beta (Gadd45b) in brain ischemia has not been fully examined to date. This study aims to investigate the function of Gadd45b in ischemia-induced apoptosis. Adult male Sprague-Dawley rats were subjected to brain ischemia by middle cerebral artery occlusion (MCAO). RNA interference (RNAi) system, which is mediated by a lentiviral vector (LV), was stereotaxically injected into the ipsilateral lateral ventricle to knockdown Gadd45b expression. Neurologic scores and infarct volumes were assessed 24 h after reperfusion. Apoptosis-related molecules were studied using immunohistochemistry and Western blot analysis. We found that Gadd45b-RNAi significantly increased infarct volumes and worsened the outcome of transient focal cerebral ischemia. Gadd45b-RNAi also significantly increased neuronal apoptosis as indicated by increased levels of Bax and active caspase-3, and decreased levels of Bcl-2. These results indicate that Gadd45b is a beneficial mediator of neuronal apoptosis.

BackgroundIt is well known that peroxisome proliferator-activated receptor gamma (PPARγ), a ligand-activated transcription factor, plays a protective role in anti-inflammatory responses in both acute and chronic central nerve system (CNS) insults. Emerging evidence in rats suggests that vagus nerve stimulation (VNS), while restraining inflammatory cytokine production in the peripheral nervous system, also exerts a significant CNS neuroprotective function against ischemic stroke injury. The aim of this study was to explore the role of PPARγ in VNS-mediated anti-inflammatory protection against ischemic stroke damage.Material/MethodsAdult male Sprague-Dawley rats (total n=160) preconditioned through transfection with either PPARγ small interfering RNA (siRNA) or lentiviral vector without siRNA and surgically subjected to middle cerebral artery occlusion and reperfusion subsequently received VNS treatment at 30 min post-occlusion. The expression of PPARγ after VNS treatment was measured by real-time PCR and Western blotting, also supported by immunofluorescence staining. Subsequently, the neurological deficits scores, the infarct volume, and the brain histopathology were all evaluated. Additionally, the influence on the pro-inflammatory cytokines expression and neuro-immune cells activation was determined by ELISA and immunofluorescence staining.ResultsWe found that VNS upregulated expression of PPARγ in ischemia penumbra, diminished the extent of ischemic infarct, alleviated neuronal injury, and suppressed pro-inflammatory cytokine expression and immune cell activation (P<0.05). However, rats with PPARγ silencing failed to manifest significant neuroprotection and anti-inflammatory effect induced by VNS treatment (p>0.05).ConclusionsPPARγ may participate in the process by which VNS modulates the neuro-inflammatory response following ischemia/reperfusion in rats.