Vagus Nerve Stimulation During Rehabilitative Training Improves Functional Recovery After Intracerebral Hemorrhage
Background and Purpose—Vagus nerve stimulation (VNS) delivered during rehabilitative training enhances neuroplasticity and improves recovery in models of cortical ischemic stroke. However, VNS therapy has not been applied in a model of subcortical intracerebral hemorrhage (ICH). We hypothesized that VNS paired with rehabilitative training after ICH would enhance recovery of forelimb motor function beyond rehabilitative training alone.
Methods—Rats were trained to perform an automated, quantitative measure of forelimb function. Once proficient, rats received an intrastriatal injection of bacterial collagenase to induce ICH. Rats then underwent VNS paired with rehabilitative training (VNS+Rehab; n=14) or rehabilitative training without VNS (Rehab; n=12). Rehabilitative training began ≥9 days after ICH and continued for 6 weeks.
Results—VNS paired with rehabilitative training significantly improved recovery of forelimb function when compared with rehabilitative training without VNS. The VNS+Rehab group displayed a 77% recovery of function, whereas the Rehab group only exhibited 29% recovery. Recovery was sustained after cessation of stimulation. Both groups performed similar amounts of trials during rehabilitative, and lesion size was not different between groups.
Conclusions—VNS paired with rehabilitative training confers significantly improved forelimb recovery after ICH compared to rehabilitative training without VNS.
Spontaneous intracerebral hemorrhage (ICH) is a devastating subtype of stroke and often leaves survivors with significant disability.1 There is no consistently effective poststroke rehabilitative intervention; therefore, methods to improve recovery of motor function represent a significant clinical need.
Neuroplasticity is thought to support recovery of function after stroke, so methods that enhance plasticity may promote greater recovery after ICH. Stimulation of the vagus nerve releases neuromodulators associated with plasticity.2–4 Consequently, vagus nerve stimulation (VNS) paired with forelimb training drives robust neuroplasticity.5 On the basis of this enhancement of plasticity, we found that VNS paired with rehabilitative training represents a potential method to improve recovery after stroke. Studies in models of ischemic stroke demonstrate that VNS paired with rehabilitation results in significantly greater recovery of forelimb strength and movement speed than extensive rehabilitative training without VNS.6–8 VNS paired with rehabilitation is currently being investigated in patients with ischemic stroke.9
Despite the efficacy of VNS paired with rehabilitation after cortical ischemic stroke, ICH bears different pathological features that may interfere with the beneficial effects of VNS. In this study, we evaluate whether VNS paired with rehabilitative training can improve recovery of motor function beyond rehabilitative training without VNS in a rat model of ICH.
All procedures were approved by the University of Texas Institutional Animal Care and Use Committee. Fifty-eight female Sprague–Dawley rats (Charles River), weighing ≈250 g at the beginning of the experiment, were used. The rats were individually housed in a 12:12 hours reversed light cycle environment and were food deprived to no <85% of their normal body weight during training.
The bradykinesia assessment task (Vulintus Inc, Dallas, TX) was performed as previously described10 (online-only Data Supplement). Once proficient at the task, rats received a lesion and VNS implant. After 7 days of recovery, rats were returned for postlesion testing and were then assigned to groups (online-only Data Supplement). Rehabilitative training continued for the following 6 weeks.
ICH and VNS Implant Surgery
ICH was performed similar to previous descriptions.11 Rats were anesthetized with ketamine hydrochloride (80 mg/kg, IP) and xylazine (10 mg/kg, IP). Body temperature was maintained at 37°C throughout the surgery. Bacterial collagenase type IV-S (Sigma-Aldrich Corp, St Louis, MO) of 0.18 U in 1.0-µL saline was injected into the left hemisphere at 3.0 mm lateral and 6.0 mm ventral relative to bregma using a 26-gauge Hamilton syringe. Injections took place over a 2-minute period, and the syringe remained in place for additional 3 minutes. A 2-channel connector was then affixed to the skull, and a bipolar stimulating cuff with platinum-iridium leads (5 kΩ impedance) was implanted around the left cervical vagus nerve, as previously described.5–8,12 Amoxicillin (5 mg) and carprofen (1 mg) tablets were provided for 3 days after surgery.
Group Assignment and Exclusion Criteria
The Rehab group (n=12) underwent rehabilitative training for 6 weeks, which consisted of freely performing the task during training sessions. The VNS+Rehab group (n=14) underwent identical rehabilitative training but received VNS during training based on 1 of 2 paradigms (online-only Data Supplement). One group (n=8) received VNS on successful trials, similar to previous studies.6–8 Another group (n=6) received VNS on all trials. VNS was delivered using identical parameters to previous studies: 500 ms train, 15 biphasic 0.8 mA pulses, 100 µs each, 30 Hz.5–8 No stimulation was delivered on the sixth week in any group to allow assessment of effects persisting after VNS cessation. Estrous phase was not monitored during the study because behavioral testing and stimulation occur over multiple cycles. Experimenters were blind to treatment group during testing, and automated data analysis eliminated any bias.10 Thirty-two rats were excluded from the main text because of (1) death, (2) failure to demonstrate a postlesion impairment, (3) impairment too severe to perform task, or (4) stimulation device failure (online-only Data Supplement). Data for all subjects are included in the online-only Data Supplement. Exclusion had minimal effects on statistical comparisons.
After behavioral testing, subjects were perfused with 4% paraformaldehyde. Cresyl violet staining and analysis were performed as previously described.6,7 Histology could not be performed on 3 of the 26 included subjects because of technical difficulties.
All data are expressed as mean±SEM. Significant differences between groups were determined using 2-way ANOVA or 2-tailed t tests where appropriate. α level was set at 0.05 for all comparisons.
Before ICH, all rats were highly proficient at the task (Movie I in the online-only Data Supplement). No significant difference in hit rate, second press latency, or number of trials was observed between groups (Figure 1, PRE; Rehab versus VNS+Rehab, unpaired t test, all P>0.05). ICH significantly worsened multiple measures of forelimb performance in both groups (Movie II in the online-only Data Supplement). No differences were observed in postlesion performance metrics between groups (Figure 1, POST; unpaired t test, all P>0.05).
VNS paired with rehabilitative training (VNS+Rehab; Movie III in the online-only Data Supplement) significantly enhances recovery when compared with rehabilitative training without VNS (Rehab; Movie IV in the online-only Data Supplement). ANOVA comparing hit rate for Rehab and VNS+Rehab groups during the course of therapy (weeks 1–6) revealed a significant effect of treatment (Figure 1A, 2-way ANOVA, F[1,144]=39.59; P=3.54×10–9). Enhanced recovery is maintained on week 6 after the cessation of stimulation. At the conclusion of therapy, VNS+Rehab group demonstrated significantly greater recovery of initial impairment when compared with Rehab (Rehab, 29.4±12.0% recovery; VNS+Rehab, 76.8±11.3% recovery; unpaired t test, P=0.0062). ANOVA on second press latency during the course of therapy reveals a significant effect of treatment, indicating that recovery of forelimb movement speed is enhanced by VNS+Rehab (Figure 1B; 2-way ANOVA, F[1,144]=57.67; P=3.58×10–12).
Total number of trials per day during therapy failed to demonstrate a significant effect of treatment (Figure 1C; 2-way ANOVA, F[1,144]=0.23; P=0.633), indicating that VNS does not affect training intensity. No differences in tissue loss were observed across groups (Figure 2; Rehab, 13.55±2.56 mm3; VNS+Rehab, 11.09±1.58 mm3; unpaired t test, P=0.38). No lesion metrics were correlated with impairment in individual subjects (online-only Data Supplement).
Previous studies show that VNS paired with rehabilitative training improves recovery of forelimb speed and strength after cortical ischemic lesion.6–8 The results from the present study extend the efficacy of VNS to a model of ICH that includes subcortical damage to both white and gray matter.13 VNS therapy, therefore, may be useful in patients with stroke bearing similar pathology and could potentially generalize to other mechanisms of brain injury.
The collagenase injection model of ICH results in protracted neuronal death, with lesion size evolving as long as 4 weeks after the initial injection.13 In the present study, VNS did not begin until ≥9 days after collagenase injection, after which the lesion is predicted to have reached >75% of its final size.13 As expected, we did not observe a difference in tissue loss between groups; therefore, the improved functional outcomes resulting from VNS cannot be attributed to reduced lesion size. The absence of neuroprotective effects when VNS is delivered on this timescale after lesion onset is consistent with previous studies.6,7 The lack of a difference in lesion size between groups suggests that VNS is not enhancing forelimb recovery through neuroprotection but rather acting through a different mechanism, such as enhancing neuroplasticity. The degree of forelimb impairment after ICH was not correlated with any of the anatomic measures in this study. This suggests that a feature not observed with gross anatomy, such as partial damage to projections or pathological plasticity, may underlie at least part of the functional impairment after ICH. VNS has been successfully used to reverse pathological plasticity and confer benefits in chronic tinnitus patients.14 Similarly, VNS paired with rehabilitative training may promote beneficial plasticity to drive functional recovery after ICH.
Neuroplasticity is thought to be a substrate for recovery after brain damage. Similar to ischemic stroke and traumatic brain injury, rehabilitative training after ICH likely supports recovery by promoting reorganization within motor circuitry. Previous studies correlate increased dendritic complexity, a morphological feature associated with plasticity, with improved motor recovery in subjects that receive rehabilitative training after ICH.15 Brain-derived neurotrophic factor is known to promote increased dendritic complexity, and VNS provides a potential direct link to plasticity by driving increased expression of brain-derived neurotrophic factor and activation of TrkB signaling.2,3 Despite links to plasticity,8 the mechanism by which VNS improves recovery after ICH remains unclear and should be addressed in future studies.
Unlike studies in models of ischemic stroke,6,7 VNS paired with rehabilitative training results in an incomplete recovery of forelimb function after ICH, which is likely accounted for by the differences in the lesion characteristics described above. To attempt to improve recovery, 2 different stimulation paradigms were used (online-only Data Supplement). One group received stimulation on successful trials similar to the design used in previous studies, and the second group received stimulation on all trials, resulting in ≈40% more stimulations during the course of the therapy. Consistent with previous reports, additional VNS does not result in greater recovery.8 No difference in recovery was observed between either VNS paradigm. Parameters, such as current intensity and timing of stimulation, modulate the effects of VNS.8 Therefore, optimizing these parameters is of key importance for clinical implementation.
VNS paired with physical rehabilitation represents a potentially attractive method to improve recovery after stroke and is currently under evaluation in patients with ischemic stroke.9 VNS is Food and Drug Administration approved to treat epilepsy and depression, and >60 000 patients are implanted with VNS devices. VNS is safe and well tolerated. The implementation of VNS in the present study uses 100-fold less daily stimulation than is approved for epilepsy, which may further reduce any occurrence of adverse effects. Along with the evidence of safety and preclinical efficacy of VNS paired with rehabilitation in models of ischemic and hemorrhagic stroke, this report strengthens the viability of VNS as a poststroke therapy.
Conclusions and Future Directions
This study demonstrates that VNS paired with rehabilitative training improves recovery of forelimb function after ICH compared with rehabilitative training without VNS. This extends the efficacy of VNS to models that include subcortical and white matter damage. Furthermore, the beneficial effects last after the cessation of VNS, suggesting that functional improvements may be lasting. Clinical investigation in patients may be warranted. Further preclinical studies should evaluate the cellular and molecular mechanisms underlying VNS-dependent enhancement of recovery.
We thank Iqra Qureshi, Xavier Carrier, Priyanka Das, and Meera Iyengar for help with behavioral training, Reema Casavant for help with surgical procedures, and Eric Meyers for engineering support.
Sources of Funding
This work was supported by grants from the Michael J. Fox Foundation, US National Institute for Deafness and Other Communicative Disorders, Texas Biomedical Device Center, and Vulintus.
Dr Kilgard is a consultant and has a financial interest in MicroTransponder, Inc. Dr Sloan is an employee of, and Dr Rennaker owns Vulintus, Inc. The other authors report no conflicts.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.114.006654/-/DC1.
- Received July 8, 2014.
- Revision received August 1, 2014.
- Accepted August 5, 2014.
- © 2014 American Heart Association, Inc.
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