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Vagus nerve stimulation does not improve recovery of forelimb motor or somatosensory function in a model of neuropathic pain


Subjects

Seventy-eight adult female Sprague–Dawley rats, each weighing approximately 250 g when they entered the study, were used. Sample size was selected based on previous studies employing a similar design6,7. All animals were housed in a 12:12 h reversed light–dark cycle and were food deprived during motor training. Twenty-five animals were excluded from the study based on pre-determined criteria: VNS device failure (n = 4), mortality (n = 7), failure to display a motor deficit after lesion, as defined by an average post-lesion baseline performance with at least 30% of trials exceeding 60 degrees on the supination task (n = 14). All protocols were approved by The University of Texas at Dallas Institutional Animal Care and Use Committee (Protocol #14–10). All experiments described here conform to the ARRIVE guidelines.

Forepaw mechanical sensory testing

Forelimb mechanical withdraw thresholds were assessed before injury, 10 weeks after injury (‘After Injury’), and 16 weeks after injury (‘After Therapy’). Testing procedures were performed as described previously6,7. Assessment was performed in an acrylic chamber on a wire mesh floor. Mechanical sensitivity was tested on the right and left forepaws using a Dynamic Plantar Aesthesiometer (Catalog Number: 37550, Ugo Basile, Italy), which automatically detects and records force at the time of paw withdrawal5,6,10. The actuator filament (0.5 mm diameter) was placed on the plantar (ventral) surface of the forepaw, and a linearly increasing force was applied (20 s ramp time, 50 g maximal force). The force at which paw withdrawal occurred was recorded for analysis. The left and right paw were alternately tested, with a minimum 1 min interval between consecutive tests. The average force at withdrawal over 5 trials was calculated for each paw. Experimenters were blinded to group throughout assessment.

Forelimb motor assessment

To assess skilled forelimb motor function, a subset of animals underwent training on the supination task, as previously described11,12,13. The behavioral training apparatus consisted of an acrylic cage with a slot through which animals reach, grasp, and supinate their forelimb to rotate a spherical manipulandum. The manipulandum was affixed to a rotary encoder to measure turn angle. If an animal rotated the manipulandum past a predetermined angle within 2 s of initiating contact, the trial was recorded as a success and a food reward was delivered to a hopper in the cage (45 mg dustless precision pellet, BioServ, Frenchtown, NJ). If the turn angle did not exceed the threshold within the 2 s, the trial was recorded as a failure and no food reward was given. Control software adaptively scaled the turn angle required to receive a reward for each trial based on the median of the preceding 10 trials to a maximum turn angle threshold of 60°.

Training sessions occurred twice a day for 30 min each, 5 days a week. Pre-injury training continued until animals achieved a 75% success rate, defined as trials in which the turn angle exceeded 60°, averaged across 6 consecutive training sessions. Data from these six sessions is reported as the ‘Before Injury’ time point. All animals then received a radial nerve injury, according to the procedures described below. After a 10 week recovery period, performance was reassessed on the supination task for 10 sessions with at least 50 trials each session, with this data being used for ‘After Injury’ point in all analyses. Animals continued training on the task for an additional 6 weeks, as detailed below. Experimenters were blinded to group throughout assessment.

Radial nerve injury induction

All subjects underwent complete transection of the radial nerve proximal to the elbow followed by tubular repair in the trained right forelimb, as previously described9. Animals were deeply anesthetized with ketamine hydrochloride (80 mg/kg, IP) and xylazine (10 mg/kg, IP), and given supplemental doses as needed to maintain areflexia. A small incision was made proximal to the elbow in the right forelimb, and the radial nerve carefully isolated, exposed, and completely transected with micro-scissors. Immediately following transection, the proximal and distal stumps of the nerve were sutured 1 mm inside the opposite ends of a 6 mm saline filled polyurethane tube (Micro-Renathane 0.095″ I.D 0.066″ O.D., Braintree Scientific, Inc., Braintree, MA), resulting in a 4 mm gap between nerve stumps. The skin incision was sutured and treated with antibiotic ointment. All animals were given a single injection of enrofloxacin (7.5 mg/kg, IP) and sustained release buprenorphine (1.2 mg/kg, SC) immediately following surgery.

Vagus nerve cuff implant

Nine weeks after nerve injury, vagus nerve stimulating cuffs were implanted in all treated animals as previously described12,14,15,16. Careful dissection of the neck exposed the left cervical vagus nerve. The nerve was isolated and placed in a bipolar cuff electrode, which was attached to a connector anchored to the skull. After VNS implant surgery, all animals were administered a single injection of enrofloxacin (10 mg/kg) and buprenorphine (0.03 mg/kg). Animals then remained in their home cage for 1 week, after which animals were randomized and underwent tactile or motor rehabilitation, as appropriate for their group.

Tactile rehabilitation and VNS delivery

In the appropriate groups, sessions of tactile rehabilitation occurred once daily, 4 days per week, with each session lasting approximately 1.5 h, as previously described6,7. Each session consisted of delivery of mechanical stimulation of the right (injured) forepaw with a variety of stimuli, including a 10 g von Frey filament (North Coast Medical, Gilroy, CA), a paintbrush (Kiss Products, Port Washington, NY), a 4 mm-diameter copper rod (Everbilt, Atlanta, GA), a surgical spear (Surgical Weck Cell Spear, Beaver-Visitec International, Waltham, MA), and puffs of air delivered with a handheld bulb (Innovo Medical, Stafford, TX). Mechanical stimuli were delivered to the dorsal or ventral surface of the forepaw, as specified for each group. Individual stimuli were presented in blocks of 10 with at least 10 s between each delivery, with a total of 200 mechanical stimuli delivered each session. Each tactile stimulus was applied for approximately 1 s. Rats in the VNS groups received nerve stimulation triggered by a button press to coincide with delivery of each mechanical stimulus. Each VNS pairing consisted of a 500 ms train of pulses at 30 Hz, and each biphasic pulse was 0.8 mA in amplitude and 100 µs in pulse duration.

Motor rehabilitation and VNS delivery

In the appropriate groups, sessions of motor rehabilitative training occurred in two 30 min sessions per day, 5 days per week, for six weeks, as previously described12. Each session consisted of freely performing the supination task. Rats in the Motor Rehab + VNS group received stimulation paired with successful trials during first five weeks of motor rehabilitation. The software monitoring the rotary encoder sent a trigger signal to the isolated pulse stimulator to administer VNS immediately when the rotary encoder crossed the adaptively scaled turn angle threshold. VNS parameters were equivalent to those used for tactile rehabilitation in Experiment 1 and in previous studies5,12. No VNS was delivered on the final week to assess effects lasting after the cessation of stimulation, which is presented as ‘After Therapy’ in the figures. All rats in the Motor Rehab group were similarly connected to the stimulator, but no stimulation was delivered during training.

Statistical analysis and data availability

Statistical analysis was performed with MATLAB software. Forelimb withdrawal threshold data and motor performance data were normally distributed (Kolmogrov-Smirnov test, all p > 0.05). Paired t-tests were used to determine differences before and after nerve injury, and two-way repeated measures ANOVA was used to analyze the effect of time and treatment over therapy. Motor function data did not meet criteria for sphericity (Fig. 2a, Mauchly’s test, p < 0.05), so a Greenhouse–Geisser correction was applied. All data is reported as mean ± standard error of the mean (SEM) in the text and figures. All data is available upon request from the authors.



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