Vagus nerve stimulation reduces spreading depolarization burden and cortical infarct volume in a rat model of stroke

Fig 1. Measuring SDs in a rat model of focal stroke. (A-B) Permanent middle cerebral artery occlusion (pMCAO) was induced in rats, and relative changes in cerebral blood flow (CBF) in peri-infarct cortex were measured through a cortical window using laser speckle contrast imaging (BPU, blood perfusion units; l, lateral; m, medial; r, rostral; c, caudal). Regions of interest (ROI) are depicted by circles. An intracortical electrode was inserted for direct current (DC) potential measurement. (C) CBF relative to baseline decreased in both ROIs following pMCAO induction (n = 32 animals), indicating that experiments were carried out in peri-infarct cortex. (D) Representative examples of an SD after MCAO. A transient negative deflection of the DC potential, measured using an intracortical electrode, is accompanied by propagating transient CBF changes (arrows show the delay of onset indicating propagation).

Fig 2. nVNS and iVNS reduce SD frequency during focal ischemia. (A) iVNS, administered 30 min after MCAO for 60 min, strongly reduced SD frequency and increased the latency to the first SD after iVNS initiation, whereas the cumulative area under the curve (AUC) as well as amplitude and SD velocity remained unchanged (n = 8 rats undergoing iVNS vs. n = 8 rats undergoing a sham VNS procedure; Mann-Whitney test for all comparisons). (B) nVNS, administered 30 and 47 min after MCAO for 2 min each, reduced SD frequency after VNS initiation as well. The latency to the first SD, SD amplitude, and velocity remained unchanged (n = 8 rats undergoing nVNS vs. n = 8 rats undergoing a sham VNS procedure; Mann-Whitney test for all comparisons). (C) Relative changes in cerebral blood flow (CBF) in peri-infarct cortex, measured using laser speckle contrast imaging, showed no significant effects of either iVNS or nVNS (n = 8 rats in each group; Mann-Whitney test for both comparisons).

Fig 3. Physiological parameters remain stable under VNS. (A) Heart rate, respiratory rate and oxygen saturation remained stable during the 4-hour time window after pMCAO induction in rats treated with iVNS or nVNS (n = 8 rats in all groups; Mann-Whitney test for all comparisons). (B) Heart rate, respiratory rate (n = 6 rats in each group) and oxygen saturation (n = 7 rats in each group) remained stable when measured immediately before and during nVNS application (Mann-Whitney test for all comparisons). (C) Systolic (sys) and diastolic (dias) blood pressure, measured at the initiation and end of pMCAO, were similar in rats treated with iVNS or nVNS and rats undergoing a sham VNS procedure (n = 8 control rats vs. n = 5 VNS-treated rats; Mann-Whitney test for all comparisons).

Fig 4. nVNS reduces cortical stroke volume. (A) Transient middle cerebral artery occlusion (tMCAO) was induced for 60 min. Sensorimotor function assessed before as well as 1, 2, and 3 days after tMCAO, followed by post-mortem infarct volumetry. (B) Cerebral blood flow (CBF) relative to baseline, measured through the thinned skull, verified successful MCAO induction and showed no differences between rats treated with nVNS and rats undergoing a sham nVNS procedure (n = 10 control vs. n = 11 VNS-treated rats; unpaired t-test). (C) nVNS led to a non-significant reduction of total infarct volume (n = 11 rats in each group; unpaired t-test; images show representative examples). (D) Separate analysis of cortical and subcortical stroke volume revealed a significant reduction of infarct volume in the cortex by nVNS, whereas infarct volume in the basal ganglia remained unchanged (n = 11 rats in each group; unpaired t-test for both comparisons).

Fig 5. Effects of nVNS on sensorimotor function after focal ischemia. (A) Rats after tMCAO and nVNS and rats after tMCAO and a sham nVNS procedure were compared to rats undergoing MCAO sham surgery using the Garcia neuroscore. Both tMCAO groups showed significantly worse sensorimotor function compared to the sham group (n = 11 rats in each group; two-way repeated measures ANOVA followed by Dunnett’s multiple comparisons test; * p < 0.05, ** p < 0.01, *** p < 0.0001). (B) When motor function of the contralateral forelimb was assessed using the grid walk test, motor performance in rats treated with VNS after tMCAO was similar to rats undergoing sham MCAO on day 2 after surgery, but significantly different in rats having received MCAO and a sham VNS procedure. However, on day 3 after MCAO, both post-MCAO groups performed significantly worse compared to control rats (Sham tMCAO and tMCAO/nVNS, n = 10 rats each; tMCAO/sham nVNS, n = 9 rats; two-way repeated measures ANOVA followed by Dunnett’s multiple comparisons test; * p < 0.05).