Transcutaneous Cervical Vagus Nerve Stimulation Ameliorates Acute Ischemic Injury in Rats

Abstract

Background: Direct stimulation of the vagus nerve in the neck via surgically implanted electrodes is protective in animal models of stroke. We sought to determine the safety and efficacy of a non-invasive cervical VNS (nVNS) method using surface electrodes applied to the skin overlying the vagus nerve in the neck in a model of middle cerebral artery occlusion (MCAO).

Methods: nVNS was initiated variable times after MCAO in rats (n = 33). Control animals received sham stimulation (n = 33). Infarct volume and functional outcome were assessed on day 7. Brains were processed by immunohistochemistry for microglial activation and cytokine levels. The ability of nVNS to activate the nucleus tractus solitarius (NTS) was assessed using c-Fos immunohistochemistry.

Results: Infarct volume was 43.15 ± 3.36 percent of the contralateral hemisphere (PCH) in control and 28.75 ± 4.22 PCH in nVNS-treated animals (p < 0.05). The effect of nVNS on infarct size was consistent when stimulation was initiated up to 4 hours after MCAO. There was no difference in heart rate and blood pressure between control and nVNS-treated animals. The number of c-Fos positive cells was 32.4 ± 10.6 and 6.2 ± 6.3 in the ipsilateral NTS (p < 0.05) and 30.4 ± 11.2 and 5.8 ± 4.3 in the contralateral NTS (p < 0.05) in nVNS-treated and control animals, respectively. nVNS reduced the number of Iba-1, CD68, and TNF-α 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: Cerebral ischemia; Electrical stimulation; Inflammation; Neuroprotection; Vagus nerve.

Figure 1

nVNS is neuroprotective in MCAO. (A) TTC-stained sections from representative control and nVNS-treated animals. Effect of nVNS on (B) infarct size, (C) neurological score, and (D) grip strength after MCAO. Treatment was initiated 30 minutes after MCAO. PCH: percentage hemispheric volume. *p<0.05 vs. corresponding control.

Figure 2

c-Fos immunoreactivity in the ipsilateral NTS. Representative images were taken from control (A) and ct-VNS-treated animals (B). Scale bar: 100μm

Figure 3

Microglia in the sensory cortex (S1FL) 24 hours after MCAO. Representative Iba-1 (A–B) and CD68 (C- immunopositive cells in the control and nVNS-treated animals. ScaleD) bar: 100μm

Figure 4

Cytokines in the sensory cortex (S1FL) 24 hours after MCAO. Representative TNF-α (A–B) and HMGB1 (C–D) immunopositive cells in the control and nVNS-treated animals. Scale bar: 100μm

Figure 5

Effect of nVNS on infarct size (A), neurological score (B) when the treatment was initiated 4 and 5 hours after MCAO. PCH: percentage hemispheric volume. *p<0.05 vs. corresponding control.