| Background and ObjectiveAt present, acute ischemic stroke has became the leading cause of the mortality associated with deseases in china. Thus, to restore the blood flow of the obstructed cerebral vessel timely should be the most direct and effective treatment. However, amounts of ROS, excessive inflammatory response, neurosis and apoptosis etc, which interact with each other, commonly occur after cerebral ischemia and reperfusion and lead to a second brain injury. As a results, it is necessary to develop an new treatment impacting on various molecular mechanisms following reperfusion. VNS (vagus nerve stimulation,VNS) was approved by USA to treat the partial refractory epilepsy patients by FDA (Food and Drug Administration, FDA) in 1997, then subsequently other countries continuely approved. Recently, some researches showed that VNS could also exert neuroprotective effect in acute cerebral ischemic stroke rat, however, the exactly mechanism was still unknown. The aim of our study was to test the neuroprotection induced by VNS following the ischemia and reperfusion and further detected the possible molecular mechanisms involved in the oxidative stress, apoptoic and the inflammatory responses.These findings might provide the theory basis for its clinical application.MethodsPart one:The expressions of miR210 in rats were silenced with miR210 antagomir preconditioning and transient middle cerebral artery occlusion models were made using the intraluminal suture technique. After 30 min of ischemia, the animals were received the electrical stimulation of the right vagus nerve. During the surgery and the stimulation period, the heart rate, the blood pressure, the blood gases and the cerebral blood flow of the right middle cerebral artery were continually monitored. At 24h after reperfusion, the miR210 expressions were measured by the qRT-PCR. The neurological deficit scores was evaluated using a scoring system that ranged from 0 to 4 and the infarct volume was assessed using 2,3,5-tripheny ltetrazolium chloride staining. The number of the neuronal apoptosis around the ischemia was stained using an in situ cell death detection kit; The levels of SOD (Superoxide Dismutase, SOD) MDA(Maleic Dialdehyde assay, MDA)、GSH(Glutathione,GSH) in the ischemic penumbra cortex tissue homogenates were analyzed using commercially available kits;The protein expressions of the p-Akt and cleaved caspase 3 were detected using the Western Blot technology. The number of the caspase 3 positive cells in the ischemic penumbra was examined using the immunofluorescence staining.Part Two:The gene expression of Peroxisome proliferator-activated receptor γ(PPARr) were silenced with PPARrsiRNA and transient middle cerebral artery occlusion models were made using the intraluminal suture technique. After 30 min of ischemia, the animals were received the electrical stimulation of the right vagus nerve. At 24h after reperfusion,the PPARr expressions were detected by the qRT-PCR and the Western Blot technology. The neurological deficit scores were evaluated using a scoring system that ranged from 1 to 12 and the infarct volume was assessed using 2,3,5-tripheny ltetrazolium chloride staining. The brain tissue pathology was evaluated using the HE staining; The expressions of the a7nAchR(a7 nicotinic acetylcholine receptor, a7nAchR) on the surface of the microglia and astrocytes were detected using the double immunofluorescence staining and the levels of TNF-a (Tumor necrosis factor a, TNF-a)、IL-1 β (Interleukin 1β, IL-1β) in the ischemia penumbra were assessed by ELISA.ResultsPart One:(1)Vagus nerve stimulation has no significant effection on the heart rate、the blood pressure、the blood gases and the cerebral blood flow of the right cerebral middle artery. (2) Compared with ischemia group (I/R), vagus nerve stimulation could further induce the expression of miR210(p<0.05), improve the neurological deficit scores and reduce the infarct volume during the acute ischemic stroke, also, reduce the number of the neuronal apoptosis in the ischemia penumbra (p< 0.05). Following miR210 knockdown, the brain injuries became more serious and the neuroprotection triggered byVNS was reduced (p< 0.05). (3) Compared with the sham I/R group, cerebral ischemia could decrease the SOD activity, reduce the level of GSH and increase the level of the MDA in the ischemia cortex; Compared with I/R group, VNS remarkably decreased the oxidative stress response in the ischemia penumbra (p< 0.05); Compared with I/R group, the oxidative stress response was more intense in the I/R+A group ((p<0.05), and the protective effect induced by VNS was reduced in the I/R+VNS+A group. (4) Compared with the sham I/R group, the level of P-Akt protein was decreased in the ischemia penumbra after cerebral ischemia (p< 0.05),while the reduction was reversed by the VNS treatment and the changes of P-Akt level was no significantly associated with antagomiR210 preconditioning (p>0.05). (5) Compared with the sham I/R group, the caspase 3 activity was significantly increased after ischemia/reperfusion, (p< 0.05) while the up-regulation was suppressed after VNS treatment (p< 0.05). After miR210 blockade, the caspase3 activity was remarkably up-regulated in the I/R+A group (p< 0.05) and increased in the I/R+VNS+A group.(p< 0.05).The trends of the number of the caspase 3 positive cells in all group were mostly consistent with the protein expression.Part Two:(1) QR-PCR and Western Blot technologies detect the PPARr expression following PPARrsiRNA in all group. Compared with the I/R+LV-control group, PPARr expression was increased in I/R+ VNS+LV-control group (p< 0.05).After PPARrRNAi preconditing, the gene and protein expression were decreased in the I/R+LV-shPPARr group and the I/R+VNS+LV-shPPARr group.(2) At 24h after reperfusion, VNS significantly improved the neurological deficit scores, reduced the infarct volume, decreased the brain pathological damage, suppressed the levels of inflammatory cytokines (TNF-α、IL-1β) in the ischemia penumbra. After PPARrRNAi preconditing, the neuroprotection induced by VNS was significantly decreased (p<0.05), the inflammatory cytokines increased as well (p<0.05). (3) Double immunofluorescence staining showed that VNS could activate the a7nAchR expression on the surface of the microglia and astrocytes in the ischemia cortex, regulate the morphology and function of the neural immune cells to exert the anti-inflammation effect.Conclusion(1) In the acute ischemia/reperfusion rats, VNS could significantly improve the neurological deficits scores and decrease the infarct volume. (2) VNS reduces the number of the neuronal apoptosis in the ischemia penumbra in the acute ischemia/reperfusion rats. (3) VNS suppresses the oxidative stress and apoptosis responses in rat brain at post-ischemia/reperfusion. (4) VNS suppresses the inflammatory response in rat brain after ischemia/reperfusion. (5) The expression of miR210 affects the oxidative stress and the apoptosis responses, also play a role in the neuroprotection induced by VNS. (6) The expression of PPAR y regulates the inflammatory response in rats after ischemia/reperfusion, also participates the neuroprotection induced by VNS. |
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