We first sought to determine whether pairing VNS with tactile rehabilitation would improve somatosensory function in a current intensity-dependent manner. To do so, all rats underwent transection and repair of their median and ulnar nerves to induce chronic hyposensitivity in the ventral forepaw. As expected, withdrawal thresholds of the injured forepaw were significantly elevated 8 weeks after nerve injury (Before Injury v. After Injury; Paired t-test, t(58) = 1.67, p = 1.42 x 10–25).
Animals were then assigned to groups and underwent 4 weeks of daily tactile rehabilitation with VNS delivered at a fixed low, moderate, or high intensity or no stimulation, as appropriate for their group. Group analysis of withdrawal threshold recovery revealed a significant effect of group (1-way repeated measures ANOVA, No VNS v. Low VNS v. Moderate VNS v. High VNS; F[4, 228] = 7.75, p = 7.3 x 10− 3. No differences in withdrawal thresholds were observed across groups prior to beginning rehabilitation (Fig. 1A; Unpaired t-tests, No VNS v. Low VNS v. Moderate VNS v. High VNS; t(30) = 1.70, p = 0.84, t(30) = 1.70, p = 0.71, t(30) = 1.70, p = 0.99).Consistent with previous reports, Moderate VNS paired with tactile rehabilitation significantly decreased withdrawal thresholds compared to equivalent tactile rehabilitation without VNS (Fig. 1A; Unpaired t-test, No VNS After Therapy v. Moderate VNS After Therapy, t(28) = 1.70, p = 3.2 x 10− 3). These findings confirm that pairing moderate intensity VNS with tactile rehabilitation improves recovery of somatosensory function.
We next tested if VNS delivered at lower or higher intensities would similarly improve recovery. Low intensity VNS delivered at 0.4 mA failed to improve recovery compared to no stimulation (Fig. 1A; Unpaired t-test, No VNS After Therapy v. Low VNS After Therapy, t(28) = 1.70, p = 0.33). Similarly, High VNS did not improve recovery compared to no stimulation (Fig. 2A; Unpaired t-test, No VNS After Therapy v. High VNS After Therapy, t(29) = 1.70, p = 0.12). At the end of therapy, the Moderate VNS group was the only group to demonstrate a significant percent recovery over No VNS (Fig. 1B; One-way ANOVA, F[3, 55], = 4.45, p = 7.2 x 10− 3; Unpaired t-tests, Percent Recovery No VNS v. Low VNS, v. Moderate VNS, and High VNS; t(27) = 1.70, p = 0.12; t(28) = 1.70, p = 8.7 x 10− 3; t(28) = 1.70, p = 0.14). Additionally, when we stratified animals by the magnitude of somatosensory recovery, a chi-squared test of independence revealed that moderate intensity VNS resulted in a greater proportion of fully recovered animals (as defined by a > 90% restoration of pre-injury withdrawal threshold), with 93% of animals demonstrating full recovery compared to the No VNS group where only 7% of animals completely recovered (Fig. 1C; X2 Test for Independence, Degree of Recovery: No VNS v. Moderate VNS; X2 (2, N = 30) = 12.1, p = 2.3 x 10− 3). Low and high intensity VNS resulted in approximately 65% of animals with full recovery but the proportion of animals who made a complete recovery did not statistically differ from the No VNS group (Fig. 1C; X2 Test for Independence, Degree of Recovery: No VNS v. Low VNS and High VNS; X2 (2, N = 29) = 3.06, p = 0.22; X2 (2, N = 30) = 3.45, p = 0.18). These findings define an inverted-U relationship between stimulation intensity and recovery of somatosensory function.
We next tested if VNS-dependent improvements in somatosensory function would generalize to other measures of forelimb sensorimotor function. No improvements in forelimb use during spontaneous exploration or weight bearing were observed across groups (Fig. 2A, 1-way repeated measures ANOVA, F[1,49] = 0.12, p = 0.73; Fig. 2B, 1-way repeated measures ANOVA, F[1,51] = 2.1X10-4, p = 0.99). These findings suggest that VNS benefits do not generalize.
The finding that VNS-dependent recovery exhibits an inverted-U relationship with stimulation intensity provides circumstantial evidence that plasticity may underlie recovery. In our second and third experiments, we sought to probe this mechanism more directly. Prior studies show that VNS paired with other forms of training modulates expression of proteins associated with synaptic plasticity, including Activity-regulated cytoskeleton-associated protein (Arc) [35–37]. Our second experiment assessed whether VNS paired with tactile rehabilitation increased the expression of Arc transcript in cortex compared to tactile rehabilitation without VNS in a separate cohort of animals. Because moderate intensity VNS produced the largest improvement in recovery, we utilized this parameter set in the subsequent experiments. A separate cohort of animals underwent one week of tactile therapy with either moderate intensity VNS (n = 8) or no stimulation (n = 7). VNS paired tactile rehabilitation significantly increased Arc transcript levels in cortex compared to equivalent tactile training with no stimulation (Fig. 3A; Unpaired t-test, Arc No VNS v. Arc VNS; t(13) = 2.62, p = 0.021). Additionally, Arc transcript levels were weakly, but significantly, correlated with the degree of recovery (Fig. 3B; Pearson correlation, r2 = 0.27, p = 0.0492). These observations are consistent with a scenario in which VNS-directed plasticity enables recovery.
If indeed cortical plasticity underlies VNS-dependent recovery of somatosensory function after nerve injury, then manipulations that prevent this plasticity should block recovery. In our third experiment, we directly explored this in a separate cohort of animals using IgG-192-saporin to deplete forebrain acetylcholine, a common technique known to abrogate plasticity [4, 38]. Animals underwent nerve injury as in the initial experiment and were randomized to receive either IgG-192-SAP injections to deplete acetylcholine (ACh-; VNS, n = 6) or vehicle injections (ACh+; VNS, n = 4). All animals received equivalent moderate intensity VNS paired with tactile rehabilitation. ANOVA revealed a significant effect of treatment (1-way repeated measures ANOVA, VNS; ACh + v. VNS; ACh-, F[6,48] = 4.19; p = 2.9 x 10− 5). After VNS therapy, animals with depletion of acetylcholine demonstrated significantly worse recovery compared to animals with intact cholinergic signaling (Fig. 4; Unpaired t-test, After Therapy; ACh+; VNS v. ACh-; VNS, t(8) = 9.93, p = 8.93 x 10− 6). These findings are consistent with the hypothesis that VNS enhances somatosensory recovery by directing synaptic plasticity.