Brief Electrical Stimulation Promotes Nerve Regeneration Following Experimental In-Continuity Nerve Injury.

BACKGROUND: Brief electrical stimulation (ES) therapy to the nerve may improve outcome in lacerated, repaired nerves. However, most human nerve injuries leave the nerve in continuity with variable and often poor functional recovery from incomplete axon regeneration and reinnervation. OBJECTIVE: To evaluate the effect of brief ES in an experimental model for neuroma-in-continuity (NIC) injuries in rodents. METHODS: Lewis rats were randomly assigned to 1 of 4 groups: NIC injury immediately followed by brief (1 h) ES; NIC injury without ES; sham-operated controls; sciatic nerve transection without repair. Outcome measures included serial behavioral evaluation and electrophysiology together with terminal retrograde spinal cord motor neuron labeling and histomorphological analysis for axonal regeneration. RESULTS: Applying brief ES immediately after in-continuity nerve injury resulted in earlier recovery and significantly improved locomotion function at 4 and 6 wk. At 8 wk, brief ES resulted in higher compound action potential amplitude. By 12 wk there was no significant difference between the 2 groups in behavior or electrophysiology. Histomorphological analysis demonstrated a significantly higher percentage of neural tissue in the brief ES group. Spinal cord motor neuron pool cell counts revealed a preference for regeneration into a motor over a sensory nerve, for the group receiving ES. CONCLUSION: The application of brief ES for in-continuity nerve injury promotes faster recovery, although in a rat model where regeneration distances are short the control group ultimately recovers to a similar degree. Brief EF requires further evaluation as a promising therapy for in-continuity nerve injuries in humans.

Testing the effectiveness and the contribution of experimental supercharge (reversed) end-to-side nerve transfer.

OBJECTIVE: Supercharge end-to-side (SETS) transfer, also referred to as reverse end-to-side transfer, distal to severe nerve compression neuropathy or in-continuity nerve injury is gaining clinical popularity despite questions about its effectiveness. Here, the authors examined SETS distal to experimental neuroma in-continuity (NIC) injuries for efficacy in enhancing neuronal regeneration and functional outcome, and, for the first time, they definitively evaluated the degree of contribution of the native and donor motor neuron pools. METHODS: This study was conducted in 2 phases. In phase I, rats (n = 35) were assigned to one of 5 groups for unilateral sciatic nerve surgeries: group 1, tibial NIC with distal peroneal-tibial SETS; group 2, tibial NIC without SETS; group 3, intact tibial and severed peroneal nerves; group 4, tibial transection with SETS; and group 5, severed tibial and peroneal nerves. Recovery was evaluated biweekly using electrophysiology and locomotion tasks. At the phase I end point, after retrograde labeling, the spinal cords were analyzed to assess the degree of neuronal regeneration. In phase II, 20 new animals underwent primary retrograde labeling of the tibial nerve, following which they were assigned to one of the following 3 groups: group 1, group 2, and group 4. Then, secondary retrograde labeling from the tibial nerve was performed at the study end point to quantify the native versus donor regenerated neuronal pool. RESULTS: In phase I studies, a significantly increased neuronal regeneration in group 1 (SETS) compared with all other groups was observed, but with modest (nonsignificant) improvement in electrophysiological and behavioral outcomes. In phase II experiments, the authors discovered that secondary labeling in group 1 was predominantly contributed from the donor (peroneal) pool. Double-labeling counts were dramatically higher in group 2 than in group 1, suggestive of hampered regeneration from the native tibial motor neuron pool across the NIC segment in the presence of SETS. CONCLUSIONS: SETS is indeed an effective strategy to enhance axonal regeneration, which is mainly contributed by the donor neuronal pool. Moreover, the presence of a distal SETS coaptation appears to negatively influence neuronal regeneration across the NIC segment. The clinical significance is that SETS should only employ synergistic donors, as the use of antagonistic donors can downgrade recovery.

Improved method to track and precisely count Schwann cells post-transplantation in a peripheral nerve injury model.

BACKGROUND: To optimize survival evaluation of Schwann cells (SCs) in vivo, we tested fluorescent labeling of the nucleus as an improved method of tracking and counting the transplanted SCs at sciatic nerve injury sites in rodents. We also investigated if co-administering cells with the glial growth factor Neuregulin-1 ß (NRG1ß) improves in vivo survival. NEW METHOD: We transduced SCs using a Lentiviral vector with a nuclear localization signal (NLS) fused with mCherry and transplanted them in the sciatic nerve of rat post-crush injury (bilateral) either in the presence or absence of NRG1ß in the injectate media. For comparison, in a separate group of similar injury, GFP-labeled cells were transplanted. After 10 days, nerves were harvested and sections (14µm) were counterstained with Hoechst and imaged. Cells showing co-localization with Hoechst and GFP or mCherry were exhaustively counted and data analyzed. RESULTS: Percentage cells counted in with- and without-NRG condition in both the groups were 0.83±0.13% and 0.06±0.04% (Group 1) & 2.83*±1.95% and 0.23*±0.29% (Group 2). COMPARISON TO EXISTING METHOD: We are introducing fluorescent labeling of the nucleus as a reliable and efficient technique to perform survival assessments in Schwann cell based treatment studies in animal model. This method can overcome the challenges and limitations of the existing method that could result in underestimation of the therapeutic outcome. CONCLUSIONS: Nucleus-restricted fluorescent labeling technique offer improved method of tracking as well as accurately counting transplanted SCs in vivo while NRG1ß in the injectate media can improve survival.

Skin derived precursor Schwann cells improve behavioral recovery for acute and delayed nerve repair.

Previous work has shown that infusion of skin-derived precursors pre-differentiated into Schwann cells (SKP-SCs) can remyelinate injured and regenerating axons, and improve indices of axonal regeneration and electrophysiological parameters in rodents. We hypothesized that SKP-SC therapy would improve behavioral outcomes following nerve injury repair and tested this in a pre-clinical trial in 90 rats. A model of sciatic nerve injury and acellular graft repair was used to compare injected SKP-SCs to nerve-derived Schwann cells or media, and each was compared to the gold standard nerve isograft repair. In a second experiment, rats underwent right tibial nerve transection and received either acute or delayed direct nerve repair, with injections of either 1) SKP-SCs distal to the repair site, 2) carrier medium alone, or 3) dead SKP-SCs, and were followed for 4, 8 or 17weeks. For delayed repairs, both transected nerve ends were capped and repaired 11weeks later, along with injections of cells or media as above, and followed for 9 additional weeks (total of 20weeks). Rats were serially tested for skilled locomotion and a slip ratio was calculated for the horizontal ladder-rung and tapered beam tasks. Immediately after nerve injury and with chronic denervation, slip ratios were dramatically elevated. In the GRAFT repair study, the SKP-SC treated rats showed statistically significant improvement in ladder rung as compared to all other groups, and exhibited the greatest similarity to the sham controls on the tapered beam by study termination. In the ACUTE repair arm, the SKP-SC group showed marked improvement in ladder rung slip ratio as early as 5weeks after surgery, which was sustained for the duration of the experiment. Groups that received media and dead SKP-SCs improved with significantly slower progression. In the DELAYED repair arm, the SKP-SC group became significantly better than other groups 7weeks after the repair, while the media and the dead SKP-SCs showed no significant improvement in slip ratios. On histomorphometrical analysis, SKP-SC group showed significantly increased mean axon counts while the percent myelin debris was significantly lower at both 4 and 8weeks, suggesting that a less inhibitory micro-environment may have contributed to accelerated axonal regeneration. For delayed repair, mean axon counts were significantly higher in the SKP-SC group. Compound action potential amplitudes and muscle weights were also improved by cell therapy. In conclusion, SKP-SC therapy improves behavioral recovery after acute, chronic and nerve graft repair beyond the current standard of microsurgical nerve repair.

The impact of motor axon misdirection and attrition on behavioral deficit following experimental nerve injuries.

Peripheral nerve transection and neuroma-in-continuity injuries are associated with permanent functional deficits, often despite successful end-organ reinnervation. Axonal misdirection with non-specific reinnervation, frustrated regeneration and axonal attrition are believed to be among the anatomical substrates that underlie the poor functional recovery associated with these devastating injuries. Yet, functional deficits associated with axonal misdirection in experimental neuroma-in-continuity injuries have not yet been studied. We hypothesized that experimental neuroma-in-continuity injuries would result in motor axon misdirection and attrition with proportional persistent functional deficits. The femoral nerve misdirection model was exploited to assess major motor pathway misdirection and axonal attrition over a spectrum of experimental nerve injuries, with neuroma-in-continuity injuries simulated by the combination of compression and traction forces in 42 male rats. Sciatic nerve injuries were employed in an additional 42 rats, to evaluate the contribution of axonal misdirection to locomotor deficits by a ladder rung task up to 12 weeks. Retrograde motor neuron labeling techniques were utilized to determine the degree of axonal misdirection and attrition. Characteristic histological neuroma-in-continuity features were demonstrated in the neuroma-in-continuity groups and poor functional recovery was seen despite successful nerve regeneration and muscle reinnervation. Good positive and negative correlations were observed respectively between axonal misdirection (p<.0001, r(2)=.67), motor neuron counts (attrition) (p<.0001, r(2)=.69) and final functional deficits. We demonstrate prominent motor axon misdirection and attrition in neuroma-in-continuity and transection injuries of mixed motor nerves that contribute to the long-term functional deficits. Although widely accepted in theory, to our knowledge, this is the first experimental evidence to convincingly demonstrate these correlations with data inclusive of the neuroma-in-continuity spectrum. This work emphasizes the need to focus on strategies that promote both robust and accurate nerve regeneration to optimize functional recovery. It also demonstrates that clinically relevant neuroma-in-continuity injuries can now also be subjected to experimental investigation.

Fate of stem cell transplants in peripheral nerves.

While damaged peripheral nerves demonstrate some potential to regenerate, complete functional recovery remains infrequent, owing to a functional loss of supportive Schwann cells distal to the injury. An emerging solution to improve upon this intrinsic regenerative capacity is to supplement injured nerves with stem cells derived from various tissues. While many of these strategies have proven successful in animal models, few studies have examined the behavior of transplanted stem cells in vivo, including whether they survive and differentiate. In previous work, we demonstrated that cells derived from neonatal rodent dermis (skin-derived precursor cells, or SKPs) could improve regenerative parameters when transplanted distal to both acute and chronic nerve injuries in Lewis rats. The aim of this work was to track the fate of these cells in various nerve injury paradigms and determine the response of these cells to a known glial growth factor. Here, we report that SKPs survive, respond to local cues, differentiate into myelinating Schwann cells, and avoid complete clearance by the host's immune defenses for a minimum of 10weeks. Moreover, the ultimate fate of SKPs in vivo depends on the nerve environment into which they are injected and can be modified by inclusion of heregulin-1ß.

A long peripheral nerve autograft model in the sheep forelimb.

BACKGROUND: Autologous nerve grafts remain the only proven means of bridging lengthy gaps in peripheral nerve. However, there is very little literature on a reliable long (> 5 cm) nerve autograft animal model. OBJECTIVE: To establish a reproducible long nerve gap and autograft animal model that is clinically relevant but not cost prohibitive. METHODS: The extent of nerve regeneration and electrophysiological recovery after segmental repair of a long nerve defect was evaluated with a sheep model. Thirteen Suffolk sheep were used. An 18-cm segment of radial sensory nerve was harvested from the forelimb, trimmed, divided into 2 equal segments of 7 cm each, and microsurgically repaired to a surgically created defect of 5 cm in the median nerve within the same forelimb. Electrophysiological studies were performed on 6 sheep at 6 months and 6 sheep at 9 months. Samples of the grafted segments were obtained for histology, immunohistochemistry, and morphometric analyses. Electric studies were also performed on an uninjured median nerve of a control animal in tissue that was similarly harvested and processed. RESULTS: At 6 and 9 months, all sheep had recordable robust nerve action potentials. Nerve conduction velocity and amplitude were slightly decreased compared with control, but the difference was statistically insignificant. Histomorphometric assessment demonstrated that the autografts contained a large number of regenerating axons through graft fascicles in all animals. CONCLUSION: The median nerve in the sheep forelimb is a reproducible and reliable model for assessing regeneration through long peripheral nerve grafts.

Motoneuron survival after chronic and sequential peripheral nerve injuries in the rat.

OBJECT: Surgical repair of peripheral nerves following chronic nerve injury is associated with poor axonal regeneration and outcome. An underlying possibility is that chronic injuries may increase motoneuron cell death. The hypothesis that substantial motoneuron death follows chronic and sequential nerve injuries was tested in adult rats in this study. METHODS: Thirty adult male Lewis rats underwent bilateral multistage surgeries. At initial surgery, Fast Blue (FB) tracer was injected at a nerve-crush injury site in the right control femoral motor nerve. The left femoral motor nerve was transected at the same level and either capped to prevent regeneration (Group 1), or repaired to allow axonal regeneration and reinnervation of the target quadriceps muscle (Group 2) (15 rats in each group). After 8 weeks in 6 rats/group, the left femoral nerve was cut and exposed to FB just proximal to prior nerve capping or repair and the rats were evaluated for FB-labeled motoneuron counts bilaterally in the spinal cord (this was considered survival after initial injury). In the remaining 9 animals/group, the left nerve was recut (sequential injury), exposed to FB, and repaired to a fresh distal saphenous nerve stump to permit axonal regeneration. Following another 6 weeks, Fluoro-Gold, a second retrograde tracer, was applied to the cut distal saphenous nerve. This allowed us to evaluate the number of motoneurons that survived (maintained FB labeling) and the number of motoneurons that survived but that also regenerated axons (double labeled with FB and Fluoro-Gold). RESULTS: A mean number of 350 and 392 FB-labeled motoneurons were found after 8 weeks of nerve injury on the right and the left sides, respectively. This indicated no significant cell death due to initial nerve injury alone. A similar number (mean 390) of motoneurons were counted at final end point at 14 weeks, indicating no significant cell death after sequential and chronic nerve injury. However, only 50% (mean 180) of the surviving motoneurons were double labeled, indicating that only half of the population regenerated their axons. CONCLUSIONS: The hypothesis that significant motoneuron cell death occurs after chronic and or sequential nerve injury was rejected. Despite cell survival, only 50% of motoneurons are capable of exhibiting a regenerative response, consistent with our previous findings of reduced regeneration after chronic axotomy.