1C). the ipsilateral NTS (p <0. 05) and 30. 411. 2 and 5. 84. a few in the contralateral NTS (p <0. 05) in nVNS-treated and control animals, respectively. nVNS reduced the number of Iba-1, CD68, and TNF- positive cells and increased the number of HMGB1 positive cells. == Conclusions == nVNS inhibits ischemia-induced immune activation and reduces the extent of tissue injury and functional deficit in rats without causing cardiac or hemodynamic adverse effects when initiated up to 4 hours after MCAO. Keywords: vagus nerve, electrical stimulation, cerebral ischemia, neuroprotection, inflammation == Introduction == There is a surge of interest in brain stimulation to reduce tissue injury and to restore the lost function in a wide variety of neurological disorders. Experimental evidence indicates that stimulation of a group of anatomically connected areas such as the fastigial nucleus [1], periaqueductal gray matter [2], subthalamic vasodilator area [3], sphenopalatine ganglion [4], and cervical vagus nerve [58] leads to a reduction in infarct volume by up to 50% in pet models of cerebral ischemia. Unlike intracranial structures that require craniotomy for access, the vagus nerve is accessible in the neck and hence can 4-Aminobutyric acid be stimulated using surgically implanted electrodes following a small incision in the overlying skin. Neural impulses following cervical vagus nerve stimulation (cVNS) project to a wide variety of cortical and subcortical structures via the nucleus tractus solitarius (NTS)[9, 10] and can activate circuits that inhibit neuronal excitability [11] and block microglial response to ischemia-induced inflammation [12]. Although cVNS is protective in experimental models of cerebral ischemia, it is not feasible for human application in the setting of acute ischemic stroke because direct nerve stimulation requires a surgical procedure. This urges a search for less invasive or non-invasive techniques for cVNS. In this study, we explored the safety and efficacy of a non-invasive transcutaneous cervical vagus nerve stimulation (nVNS) approach using surface electrodes applied to the skin overlying the vagus nerve in the neck in a model of middle cerebral artery 4-Aminobutyric acid occlusion (MCAO) in rats. == Material and Methods == All experiments were performed in accordance with the United States Public Health Services Policy on Humane Care and Use of Laboratory Animals and were approved 4-Aminobutyric acid by the Massachusetts General Hospital Institutional Pet Care and Use Committee. Adult male spontaneously hypertensive rats (SHR, 326420g, n=66; 4-Aminobutyric acid Charles River Laboratories, Wilmington MA) were used. We used SHR in compliance with the Stroke Therapy Academic Industry Roundtable (STAIR) criteria which explicitly require the use of animals with co-morbid conditions in order to increase the quality of translational stroke research.[13] == Experimental Protocols == We studied the following four hypotheses: nVNS reduces infarct volume and improves neurological outcome after MCAO: Animals were randomly allocated into two experimental groups: treatment (351. 00 14. 41g; n=7) and control (336. 17 16. 46g; n=6). Electrical stimulation of the right cervical vagus nerve was initiated 30 minutes after occlusion of the right MCA and repeated every 10 minutes for a period of 1 hour. Control animals received sham stimulation delivered to the right quadriceps femoris muscle. Twenty minutes after the last stimulation animals were returned to their home cages. Functional assessments were performed daily after surgery for 7 days. Infarct volume was assessed on SPRY1 day 7. The following safety parameters were recorded in each pet: arterial blood pressure (ABP), heart rate (HR), arterial blood gases (ABGs) and pH, symptomatic brain hemorrhage, and mortality or development of any of the euthanasia criteria described by AVMA Guidelines for the Euthanasia of Animals 2013. nVNS activates NTS: We explored whether nVNS caused activation in the NTS after MCAO in rats. We performed the same surgical and stimulation procedures in Experiment-1 but animals were euthanized a few hours after MCAO and brains were processed for c-Fos immunohistochemistry. There were two experimental groups: treatment (390. 25 5. 32g; n=4) and control (394. 00 4. 83g; n=4). nVNS leads to inhibition of MCAO-induced immune.