Delay modulates the immune response to nerve repair.

Effective regeneration after peripheral nerve injury requires macrophage recruitment. We investigated the activation of remodeling pathways within the macrophage population when repair is delayed and identified alteration of key upstream regulators of the inflammatory response. We then targeted one of these regulators, using exogenous IL10 to manipulate the response to injury at the repair site. We demonstrate that this approach alters macrophage polarization, promotes macrophage recruitment, axon extension, neuromuscular junction formation, and increases the number of regenerating motor units reaching their target. We also demonstrate that this approach can rescue the effects of delayed nerve graft.

Functional electrical stimulation following nerve injury in a large animal model.

INTRODUCTION: Controversy exists over the effects of functional electrical stimulation (FES) on reinnervation. We hypothesized that intramuscular FES would not delay reinnervation after recurrent laryngeal nerve (RLn) axonotmesis. METHODS: RLn cryo-injury and electrode implantation in ipsilateral posterior cricoarytenoid muscle (PCA) were performed in horses. PCA was stimulated for 20 weeks in eight animals; seven served as controls. Reinnervation was monitored through muscle response to hypercapnia, electrical stimulation and exercise. Ultimately, muscle fiber type proportions and minimum fiber diameters, and RLn axon number and degree of myelination were determined. RESULTS: Laryngeal function returned to normal in both groups within 22 weeks. FES improved muscle strength and geometry, and induced increased type I:II fiber proportion (p = 0.038) in the stimulated PCA. FES showed no deleterious effects on reinnervation. DISCUSSION: Intramuscular electrical stimulation did not delay PCA reinnervation after axonotmesis. FES can represent a supportive treatment to promote laryngeal functional recovery after RLn injury. Muscle Nerve 59:717-725, 2019.

Targeted Muscle Reinnervation: Considerations for Future Implementation in Adolescents and Younger Children.

Prosthetic options for patients with proximal upper limb absence are limited. Current above-elbow prostheses may restore basic motor functions for crucial activities, but they are cumbersome to operate, lack sensory feedback, and are often abandoned. Targeted muscle reinnervation is a novel surgical procedure that enhances the ability of patients with above-elbow amputations to intuitively control a myoelectric prosthesis. By transferring multiple severed peripheral nerves to a robust target muscle, targeted muscle reinnervation restores physiologic continuity and enables more intuitive prosthetic control. Although reports have been limited to adults, targeted muscle reinnervation has great potential for application in a pediatric population with congenital or acquired proximal upper limb absence. In this review, the authors describe the rehabilitative challenges of proximal upper limb amputees and outline the objectives, techniques, and outcomes of targeted muscle reinnervation. The authors then discuss important considerations for adapting targeted muscle reinnervation to pediatric patients, including cause of upper limb absence, central plasticity, timing of prosthesis fitting, role of the family, surgical feasibility, and bioethical aspects. The authors believe that carefully screened school-age children and adolescents with bilateral proximal upper limb absence, and select adolescents with unilateral proximal upper limb absence, should be seriously considered for targeted muscle reinnervation performed by an experienced surgical and rehabilitation team.

Electrical muscle stimulation elevates intramuscular BDNF and GDNF mRNA following peripheral nerve injury and repair in rats.

Despite advances in surgery, patients with nerve injuries frequently have functional deficits. We previously demonstrated in a rat model that daily electrical muscle stimulation (EMS) following peripheral nerve injury and repair enhances reinnervation, detectable as early as two weeks post-injury. In this study, we explain the enhanced early reinnervation observed with electrical stimulation. In two groups of rats, the tibial nerve was transected and immediately repaired. Gastrocnemius muscles were implanted with intramuscular electrodes for sham or muscle stimulation. Muscles were stimulated daily, eliciting 600 contractions for one hour/day, repeated five days per week. Sixteen days following nerve injury, muscles were assessed for functional reinnervation by motor unit number estimation methods using electromyographic recording. In a separate cohort of rats, surgical and electrical stimulation procedures were identical but muscles and distal nerve stumps were harvested for molecular analysis. We observed that stimulated muscles had significantly higher motor unit number counts. Intramuscular levels of brain-derived and glial cell line-derived neurotrophic factor (BDNF and GDNF) mRNA were significantly upregulated in muscles that underwent daily electrical stimulation compared to those without stimulation. The corresponding levels of trophic factor mRNA within the distal stump were not different from one another, indicating that the intramuscular electrical stimulus does not modulate Schwann cell-derived trophic factor transcription. Stimulation over a three-month period maintained elevated muscle-derived GDNF but not BDNF mRNA. In conclusion, EMS elevates intramuscular trophic factor mRNA levels which may explain how EMS enhances neural regeneration following nerve injury.

Serial estimation of motor unit numbers using an implantable system following nerve injury and repair in rats.

Motor unit number estimation (MUNE) is an established technique to assess recovery following peripheral nerve injury. In rats, where the vast majority of peripheral nerve research is conducted, assessing motor units at various time points requires a terminal procedure due to the invasive nature of current techniques. Here, we present an implanted system that was used to serially assess MUNE after peripheral nerve injury and repair in rats. This system significantly increases the efficiency of peripheral nerve research by negating the need for terminal procedures, allowing for serial MUNE assessment over time in the same rat. Our system utilizes a commercial implantable stimulator, custom designed cuff electrode, and corresponding custom software with automatic M-wave classification to quickly assess functional reinnervation up to 8 weeks following nerve injury and repair. The concepts presented in this paper are applicable to any implanted device with a transcutaneous radio frequency or inductive link that can be used to trigger nerve stimulation. The methodology is also applicable to researchers without access to implantable devices.

Electrical Stimulation to Promote Peripheral Nerve Regeneration.

Peripheral nerve injury afflicts individuals from all walks of life. Despite the peripheral nervous system's intrinsic ability to regenerate, many patients experience incomplete functional recovery. Surgical repair aims to expedite this recovery process in the most thorough manner possible. However, full recovery is still rarely seen especially when nerve injury is compounded with polytrauma where surgical repair is delayed. Pharmaceutical strategies supplementary to nerve microsurgery have been investigated but surgery remains the only viable option. Brief low-frequency electrical stimulation of the proximal nerve stump after primary repair has been widely investigated. This article aims to review the currently known biological basis for the regenerative effects of acute brief low-frequency electrical stimulation on axonal regeneration and outline the recent clinical applications of the electrical stimulation protocol to demonstrate the significant translational potential of this modality for repairing peripheral nerve injuries. The review concludes with a discussion of emerging new advancements in this exciting area of research. The current literature indicates the imminent clinical applicability of acute brief low-frequency electrical stimulation after surgical repair to effectively promote axonal regeneration as the stimulation has yielded promising evidence to maximize functional recovery in diverse types of peripheral nerve injuries.

Retrograde labeling of regenerating motor and sensory neurons using silicone caps.

BACKGROUND: Retrograde labeling permits the investigation of the number, distribution and axonal projections of neurons in the peripheral nervous system. The well technique for labeling peripheral nerves consists of incubating the exposed peripheral nerve in a well for one hour, a time intensive technique. However, other techniques that inject tracers directly into the nerve or muscle may result in variable labeling depending on nerve preparation and location of injection. NEW METHOD: We describe a method of retrograde labeling peripheral nerves that increases tracer uptake and improves labeling efficiency. This technique utilizes a silicone cap over the nerve that is kept in place with fibrin glue, permitting closure of the incision with the cap in place, mitigating the need to wait one hour for back-labeling as with the standard well technique. RESULTS: In the rat common peroneal nerve, the new silicone cap technique, compared to the standard well technique, labeled 405±11 (SEM) vs. 378±21 motoneurons and 953±40 vs. 948±57 sensory neurons. These counts were not statistically different. Labeling intensity was greater in DRG neurons with the silicone cap technique, but this difference was not evident in motoneurons. COMPARISON WITH EXISTING METHOD: Retrograde-labeling with silicone caps labels an equal number of motor and sensory neurons in comparison with the standard well technique and labels sensory neurons with greater intensity. CONCLUSIONS: Retrograde-labeling with silicone caps reliably labels neurons and significantly decreases the time required for labeling, reducing anesthetic exposure and improving the efficiency of the technique.

Characterization of neuronal death and functional deficits following nerve injury during the early postnatal developmental period in rats.

In contrast to adult rat nerve injury models, neonatal sciatic nerve crush leads to massive motor and sensory neuron death. Death of these neurons results from both the loss of functional contact between the nerve terminals and their targets, and the inability of immature Schwann cells in the distal stump of the injured nerve to sustain regeneration. However, current dogma holds that little to no motoneuron death occurs in response to nerve crush at postnatal day 5 (P5). The purpose of the current study was to fully characterize the extent of motor and sensory neuronal death and functional recovery following sciatic nerve crush at mid-thigh level in rats at postnatal days 3-30 (P3-P30), and then compare this to adult injured animals. Following nerve crush at P3, motoneuron numbers were reduced to 35% of that of naïve uninjured animals. Animals in the P5 and P7 group also displayed statistically fewer motoneurons than naïve animals. Animals that were injured at P30 or earlier displayed statistically lower sensory neuron counts in the dorsal root ganglion than naïve controls. Surprisingly, complete behavioral recovery was observed exclusively in the P30 and adult injured groups. Similar results were observed in muscle twitch/tetanic force analysis, motor unit number estimation and wet muscle weights. Rats in both the P5 and P7 injury groups displayed significant neuronal death and impaired functional recovery following injury, challenging current dogma and suggesting that severe deficits persist following nerve injury during this early postnatal developmental period. These findings have important implications concerning the timing of neonatal nerve injury in rats.

Electrical Stimulation Enhances Reinnervation After Nerve Injury.

Electrical muscle stimulation following peripheral nerve injury has been a controversial method of treatment due primarily to the inconsistent literature surrounding it. In this presentation transcript I outline ongoing experiments investigating a clinically translatable daily muscle stimulation paradigm in rats following nerve injury. Results show that reinnervation of muscle and functional behavioural metrics are enhanced with daily stimulation with upregulation of intramuscular neurotrophic factors as a potential mechanism. In addition, the impact of stimulation on terminal sprouting, a mentioned negative aspect of electrical muscle stimulation, was a minor contributor to long term functional reinnervation of stimulated muscles in our studies.

Daily Electrical Muscle Stimulation Enhances Functional Recovery Following Nerve Transection and Repair in Rats.

BACKGROUND: Incomplete recovery following surgical reconstruction of damaged peripheral nerves is common. Electrical muscle stimulation (EMS) to improve functional outcomes has not been effective in previous studies. OBJECTIVE: To evaluate the efficacy of a new, clinically translatable EMS paradigm over a 3-month period following nerve transection and immediate repair. METHODS: Rats were divided into 6 groups based on treatment (EMS or no treatment) and duration (1, 2, or 3 months). A tibial nerve transection injury was immediately repaired with 2 epineurial sutures. The right gastrocnemius muscle in all rats was implanted with intramuscular electrodes. In the EMS group, the muscle was electrically stimulated with 600 contractions per day, 5 days a week. Terminal measurements were made after 1, 2, or 3 months. Rats in the 3-month group were assessed weekly using skilled and overground locomotion tests. Neuromuscular junction reinnervation patterns were also examined. RESULTS: Muscles that received daily EMS had significantly greater numbers of reinnervated motor units with smaller average motor unit sizes. The majority of muscle endplates were reinnervated by a single axon arising from a nerve trunk with significantly fewer numbers of terminal sprouts in the EMS group, the numbers being small. Muscle mass and force were unchanged but EMS improved behavioral outcomes. CONCLUSIONS: Our results demonstrated that EMS using a moderate stimulation paradigm immediately following nerve transection and repair enhances electrophysiological and behavioral recovery.

Sensory nerve cross-anastomosis and electrical muscle stimulation synergistically enhance functional recovery of chronically denervated muscle.

BACKGROUND: Long-term muscle denervation leads to severe and irreversible atrophy coupled with loss of force and motor function. These factors contribute to poor functional recovery following delayed reinnervation. The authors' previous work demonstrated that temporarily suturing a sensory nerve to the distal motor stump (called sensory protection) significantly reduces muscle atrophy and improves function following reinnervation. The authors have also shown that 1 month of electrical stimulation of denervated muscle significantly improves function and reduces atrophy. In this study, the authors tested whether a combination of sensory protection and electrical stimulation would enhance functional recovery more than either treatment alone. METHODS: Rat gastrocnemius muscles were denervated by cutting the tibial nerve. The peroneal nerve was then sutured to the distal tibial stump following 3 months of treatment (i.e., electrical stimulation, sensory protection, or both). Three months after peroneal repair, functional and histologic measurements were taken. RESULTS: All treatment groups had significantly higher muscle weight (p<0.05) and twitch force (p<0.001) compared with the untreated group (denervated), but fiber type composition did not differ between groups. Importantly, muscle weight and force were significantly greater in the combined treatment group (p<0.05) compared with stimulation or sensory protection alone. The combined treatment also produced motor unit counts significantly greater than sensory protection alone (p<0.05). CONCLUSIONS: The combination treatment synergistically reduces atrophy and improves reinnervation and functional measures following delayed nerve repair, suggesting that these approaches work through different mechanisms. The authors' research supports the clinical use of both modalities together following peripheral nerve injury.

Electrical muscle stimulation after immediate nerve repair reduces muscle atrophy without affecting reinnervation.

INTRODUCTION: Electrical stimulation of denervated muscle has been shown to minimize atrophy and fibrosis and increase force in animal and human models. However, electrical stimulation after nerve repair is controversial due to questions of efficacy. METHODS: Using a rat model, we investigated the efficacy of short-term electrical muscle stimulation for increasing reinnervation and preventing muscle atrophy. After tibial nerve transection and immediate repair with the fibular nerve, 1 month of electrical stimulation was applied 5 days/week for 1 hour to the gastrocnemius muscle via implanted electrodes. RESULTS: After 2 months of further recovery without stimulation, muscle weights, twitch forces, and type I fiber areas were significantly greater in stimulated animals than in repaired controls without stimulation. Motor unit size and numbers were not different between the 2 groups. CONCLUSIONS: Short-term electrical muscle stimulation after nerve repair significantly reduces muscle atrophy and does not affect motor reinnervation.

A New System and Paradigm for Chronic Stimulation of Denervated Rat Muscle.

Traditionally, animal studies employing electrical stimulation for conditioning denervated muscle rely on 24-hour-based stimulation paradigms, most employing implantable stimulators. While these stimulators provide the necessary current to cause muscular contraction, they have problems with battery life, programmability, and long-term robustness. Continuous 24-hour stimulation, while shown to be effective in animals, is not easily translatable to a clinical setting. It is also difficult to evaluate animal comfort and muscular contraction throughout a 24-hour period. We have developed a system and stimulation paradigm that can stimulate up to five animals at one time for one hour per day. The constant current stimulator is a USB-powered device that can, under computer control, output trains of pulses with selectable shapes, widths, durations and repetition rates. It is an external device with no implantable parts in the animal except for the stimulating electrodes. We tested the system on two groups of rats with denervated gastrocnemius muscles. One group was stimulated using a one-hour-per-day, 5-days-per-week stimulation paradigm for one month, while the other group had electrodes implanted but received no stimulation. Muscle weight and twitch force were significantly larger in the stimulated group than the non-stimulated group. Presently, we are using the stimulator to investigate electrical stimulation coupled with other therapeutic interventions that can minimize functional deficits after peripheral nerve injuries.

Determining the effects of electrical stimulation on functional recovery of denervated rat gastrocnemius muscle using motor unit number estimation.

The use of electrical muscle stimulation to treat denervated muscle prior to delayed reinnervation has been widely debated. There is evidence showing both positive and negative results following different protocols of electrical stimulation. In this study we investigated the role electrical stimulation has on muscle reinnervation following immediate and delayed nerve repair using motor unit estimation techniques. Rat gastrocnemius muscle was denervated and repaired using the peroneal nerve either immediately or following three-months with and without electrical stimulation. Motor unit counts, average motor unit sizes, and maximum compound action potentials were measured three-months following peroneal nerve repair. Motor unit counts in animals that were denervated and stimulated were significantly higher than those that were denervated and not stimulated. Both average motor unit sizes and maximum compound action potentials showed no significant differences between denervated and denervated-stimulated animals. These results provide evidence that electrical stimulation prior to delayed nerve repair increases muscle receptivity to regenerating axons and may be a worthwhile treatment for peripheral nerve injuries.

Design and testing of an instrumentation system to reduce stimulus pulse amplitude requirements during FES.

Functional Electrical Stimulation (FES) has been used for decades to restore muscle function following neural trauma. Another promising use has been to maintain or increase muscle strength following injury. Unfortunately in the latter case there is considerable stimulation pain for the sensorially intact subject during effective levels of stimulation using surface electrodes. Recent research [1][2] has suggested using a constant long (up to 10 ms) low amplitude or ramped conditioning pulse just prior to the high amplitude stimulus pulse (100 - 200 microsec) to reduce the excitably of sensory nerve fibers. However, commercial muscle stimulators cannot be easily modified to provide such complex pulse patterns and flexible pulse train control. We have designed and implemented a novel, very flexible LabVIEW based monophasic constant current muscle stimulator that provides pulse trains with long duration prepulses and high voltage stimulus pulses with selectable shapes, amplitudes, durations and frequencies. As well, the stimulator system includes an isolated EMG amplifier to record the evoked M-waves, which are used to estimate the fraction of muscle motor units being stimulated. We are presently testing this system, by stimulating the median nerve of human subjects and measuring the evoked M-waves for a range of stimulus pulse amplitudes, preceded by either ramped or rectangular low amplitude prepulses. The results indicate that, rather than inhibiting the activation of motor axons, as has been suggested [4], the prepulses at very low amplitudes excite these axons to subthreshold levels. The amplitudes of the stimulus pulses can then be significantly reduced while still achieving high levels of muscle activation.