Stephen W.P. Kemp

Outcome measures of peripheral nerve regeneration.

Animal models of nerve compression, crush, and transection injuries of peripheral nerves have been subject to extensive study in order to understand the mechanisms of injury and axon regeneration and to investigate methods to promote axon regeneration and improve functional outcomes following nerve injury. Six outcome measures of regenerative success including axon and neuron counts, muscle and motor unit contractile forces, and behavior are reviewed in the context of nerve injury types, crush, transection and nerve repair by direct coaptation, or transection and repair via a nerve graft or conduit. The measures are evaluated for sciatic, tibial, common peroneal, femoral, single nerve branches such as the soleus nerve, and facial nerves. Their validity is discussed in the context of study objectives and the nerve branch. The case is made that outcome measures of axon counts and muscle contractile forces may be valid during the early phases of axon regeneration when regenerating sprouts emerge asynchronously from the proximal nerve stump and regenerate towards their denervated targets. However, care must be taken especially when experimental interventions differentially affect how many neurons regenerate axons and the number of axons per neuron that sprout from the proximal nerve stumps. Examples of erroneous conclusions are given to illustrate the need for researchers to ensure that the appropriate outcome measures are used in the evaluation of the success of peripheral nerve regeneration.

Fibrin gels containing GDNF microspheres increase axonal regeneration after delayed peripheral nerve repair

AIM Recovery following nerve transection declines when target reconnection is delayed for prolonged periods. GDNF has previously been shown to promote motor axon regeneration following delayed nerve repair. MATERIALS & METHODS We constructed delivery systems using fibrin gels containing free GDNF or poly(lactide-co-glycolide) microspheres with GDNF. The delivery systems were implanted with fluorescent fibrinogen surrounding the common fibular (CF; peroneal) nerve in transgenic Thy-1 GFP rats (whose axons express GFP) to track degradation of the system. A delayed nerve repair model was designed by transecting the rat CF nerve, where nerve regeneration was prevented by ligating the two stumps to surrounding muscle for 2 months prior to resuture. At resuture, either a delivery system with GDNF or an additional group consisting of fibrin gels with empty microspheres were implanted surrounding the repair site. In an additional positive control, the CF was transected and repaired immediately without delay. RESULTS ELISA assays demonstrated GDNF release in vitro for 2 weeks from fibrin gels with GDNF microspheres. Implanted delivery systems, including GDNF microspheres, remained surrounding the nerve for at least 10 days compared with 3 days for free GDNF. Four weeks after repair, histomorphometry of distal nerve cross-sections taken 20 mm from the repair site demonstrated increased fiber diameter and myelin thickness due to release of GDNF from microspheres compared with empty microspheres. Additionally, the number of motoneurons that regenerated their axons to the same site increased to comparable levels as immediate repair due to the extended delivery of GDNF from microspheres. CONCLUSION These findings demonstrate that early measures of nerve regeneration after delayed nerve repair is improved by GDNF microspheres implanted at the coaptation site.

Functional motor recovery is improved due to local placement of GDNF microspheres after delayed nerve repair

The majority of bioengineering strategies to promote peripheral nerve regeneration after injury have focused on therapies to bridge large nerve defects while fewer therapies are being developed to treat other nerve injuries, such as nerve transection. We constructed delivery systems using fibrin gels containing either free GDNF or polylactide–glycolic acid (PLGA) microspheres with GDNF to treat delayed nerve repair, where ELISA verified GDNF release. We determined the formulation of microspheres containing GDNF that optimized nerve regeneration and functional recovery in a rat model of delayed nerve repair. Experimental groups underwent delayed nerve repair and treatment with GDNF microspheres in fibrin glue at the repair site or control treatments (empty microspheres or free GDNF without microspheres). Contractile muscle force, muscle mass, and MUNE were measured 12 weeks following treatment, where GDNF microspheres (2 weeks formulation) were superior compared to either no GDNF or short‐term release of free GDNF to nerve. Nerve histology distal to the repair site demonstrated increased axon counts and fiber diameters due to GDNF microspheres (2 weeks formulation). GDNF microspheres partially reversed the deleterious effects of chronic nerve injury, and recovery was slightly favored with the 2 weeks formulation compared to the 4 weeks formulation. Biotechnol. Bioeng. 2013; 110: 1272–1281.

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.

α5GABAA receptor deficiency causes autism-like behaviors

The prevalence of autism spectrum disorders (ASDs), which affect over 1% of the population, has increased twofold in recent years. Reduced expression of GABAA receptors has been observed in postmortem brain tissue and neuroimaging of individuals with ASDs. We found that deletion of the gene for the α5 subunit of the GABAA receptor caused robust autism‐like behaviors in mice, including reduced social contacts and vocalizations. Screening of human exome sequencing data from 396 ASD subjects revealed potential missense mutations in GABRA5 and in RDX, the gene for the α5GABAA receptor‐anchoring protein radixin, further supporting a α5GABAA receptor deficiency in ASDs.

Functional recovery following peripheral nerve injury in the transgenic Thy1 -GFP rat

Transgenic mice have been previously used to assess nerve regeneration following peripheral nerve injury. However, mouse models are limited by their small caliber nerves, short nerve lengths, and their inability to fully participate during behavioral assessments. The transgenic Thy1 GFP rat is a novel transgenic rat model designed to assess regeneration following peripheral nerve injury. However, return of functional and behavioral recovery following nerve injury has not yet been evaluated in these rats. In this study, we ask whether differences in anatomy, recovery of locomotion, myological, and histomorphological measures exist between transgenic Thy1 GFP rats when compared to wild type (WT) Sprague Dawley rats following unilateral sciatic nerve injury. We found that both motor and sensory neuronal architecture, overground and skilled locomotion, muscle force, motor unit number estimation (MUNE) and wet muscle weights, and histomorphometric assessments are similar between both genetic phenotypes. Overall, these data support the use of the transgenic Thy1‐GFP rat in experiments assessing functional and behavioral recovery following nerve injury and repair.

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.