Amy M. Moore

Processed allografts and type I collagen conduits for repair of peripheral nerve gaps

Autografting is the gold standard in the repair of peripheral nerve injuries that are not amenable to end‐to‐end coaptation. However, because autografts result in donor‐site defects and are a limited resource, an effective substitute would be valuable. In a rat model, we compared isografts with Integra NeuraGen® (NG) nerve guides, which are a commercially available type I collagen conduit, with processed rat allografts comparable to AxoGens Avance® human decellularized allograft product. In a 14‐mm sciatic nerve gap model, isograft was superior to processed allograft, which was in turn superior to NG conduit at 6 weeks postoperatively (P < 0.05 for number of myelinated fibers both at midgraft and distal to the graft). At 12 weeks, these differences were no longer apparent. In a 28‐mm graft model, isografts again performed better than processed allografts at both 6 and 22 weeks; regeneration through the NG conduit was often insufficient for analysis in this long graft model. Functional tests confirmed the superiority of isografts, although processed allografts permitted successful reinnervation of distal targets not seen in the NG conduit groups. Processed allografts were inherently non‐immunogenic and maintained some internal laminin structure. We conclude that, particularly in a long gap model, nerve graft alternatives fail to confer the regenerative advantages of an isograft. However, AxoGen processed allografts are superior to a currently available conduit‐style nerve guide, the Integra NeuraGen®. They provide an alternative for reconstruction of short nerve gaps where a conduit might otherwise be used. Muscle Nerve, 2008

Affinity-based release of glial-derived neurotrophic factor from fibrin matrices enhances sciatic nerve regeneration.

Glial-derived neurotrophic factor (GDNF) promotes both sensory and motor neuron survival. The delivery of GDNF to the peripheral nervous system has been shown to enhance regeneration following injury. In this study, we evaluated the effect of affinity-based delivery of GDNF from a fibrin matrix in a nerve guidance conduit on nerve regeneration in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated which received GDNF or nerve growth factor (NGF) with the delivery system within the conduit, control groups excluding one or more components of the delivery system, and nerve isografts. Nerves were harvested 6 weeks after treatment for analysis by histomorphometry and electron microscopy. The use of the delivery system (DS) with either GDNF or NGF resulted in a higher frequency of nerve regeneration vs. control groups, as evidenced by a neural structure spanning the 13 mm gap. The GDNF DS and NGF DS groups were also similar to the nerve isograft group in measures of nerve fiber density, percent neural tissue and myelinated area measurements, but not in terms of total fiber counts. In addition, both groups contained a significantly greater percentage of larger diameter fibers, with GDNF DS having the largest in comparison to all groups, suggesting more mature neural content. The delivery of GDNF via the affinity-based delivery system can enhance peripheral nerve regeneration through a silicone conduit across a critical nerve gap and offers insight into potential future alternatives to the treatment of peripheral nerve injuries.

Acellular nerve allografts in peripheral nerve regeneration: A comparative study

Introduction: Processed nerve allografts offer a promising alternative to nerve autografts in the surgical management of peripheral nerve injuries where short deficits exist. Methods: Three established models of acellular nerve allograft (cold‐preserved, detergent‐processed, and AxoGen‐processed nerve allografts) were compared with nerve isografts and silicone nerve guidance conduits in a 14‐mm rat sciatic nerve defect. Results: All acellular nerve grafts were superior to silicone nerve conduits in support of nerve regeneration. Detergent‐processed allografts were similar to isografts at 6 weeks postoperatively, whereas AxoGen‐processed and cold‐preserved allografts supported significantly fewer regenerating nerve fibers. Measurement of muscle force confirmed that detergent‐processed allografts promoted isograft‐equivalent levels of motor recovery 16 weeks postoperatively. All acellular allografts promoted greater amounts of motor recovery compared with silicone conduits. Conclusion: These findings provide evidence that differential processing for removal of cellular constituents in preparing acellular nerve allografts affects recovery in vivo. Muscle Nerve, 2011

Fibrin Matrices With Affinity-Based Delivery Systems and Neurotrophic Factors Promote Functional Nerve Regeneration

Glial‐derived neurotrophic factor (GDNF) and nerve growth factor (NGF) have both been shown to enhance peripheral nerve regeneration following injury and target different neuronal populations. The delivery of either growth factor at the site of injury may, therefore, result in quantitative differences in motor nerve regeneration and functional recovery. In this study we evaluated the effect of affinity‐based delivery of GDNF or NGF from fibrin‐filled nerve guidance conduits (NGCs) on motor nerve regeneration and functional recovery in a 13 mm rat sciatic nerve defect. Seven experimental groups were evaluated consisting of GDNF or NGF and the affinity‐based delivery system (DS) within NGCs, control groups excluding the DS and/or growth factor, and nerve isografts. Groups with growth factor in the conduit demonstrated equivalent or superior performance in behavioral tests and relative muscle mass measurements compared to isografts at 12 weeks. Additionally, groups with GDNF demonstrated greater specific twitch and tetanic force production in extensor digitorum longus (EDL) muscle than the isograft control, while groups with NGF produced demonstrated similar force production compared to the isograft control. Assessment of motor axon regeneration by retrograde labeling further revealed that the number of ventral horn neurons regenerating across NGCs containing GDNF and NGF DS was similar to the isograft group and these counts were greater than the groups without growth factor. Overall, the GDNF DS group demonstrated superior functional recovery and equivalent motor nerve regeneration compared to the isograft control, suggesting it has potential as a treatment for motor nerve injury. Biotechnol. Bioeng. 2010;106: 970–979.

Controlled Delivery of Glial Cell Line–Derived Neurotrophic Factor Enhances Motor Nerve Regeneration

PURPOSE To determine the effect of a motor-specific neurotrophic factor, glial-derived neurotrophic factor (GDNF) on motor nerve regeneration. METHODS We used a nerve conduit filled with a fibrin-based delivery system that provided controlled release of GDNF during nerve regeneration. The motor branch of the rat femoral nerve was used to assess motor nerve regeneration across a 5-mm gap. Four experimental groups (n = 4 to n = 8) were evaluated. These included GDNF with the fibrin-based delivery system (GDNF-DS), fibrin alone, empty conduit (negative control), and nerve isograft (positive control). Nerves were harvested at 5 weeks for analysis by histomorphometry and electron microscopy. RESULTS At 5 mm distal to the conduit or isografts, the GDNF-DS group was not significantly different from the nerve isograft group in the following histomorphometric measures: total nerve fibers, percentage of neural tissue, and nerve density. Both the GDNF-DS and isograft groups had significantly more fibers and a higher percentage of neural tissue than fibrin alone and empty conduit groups. There were no differences in fiber width among all groups. By electron microscopy, the GDNF-DS and isograft groups also demonstrated more organized nerve architecture than the fibrin alone and empty conduit groups. CONCLUSIONS The delivery of GDNF from the fibrin-based delivery system promotes motor nerve regeneration at a level similar to an isograft in the femoral motor nerve model. This study gives insight into the potential beneficial role of GDNF in the treatment of motor nerve injuries.

A transgenic rat expressing green fluorescent protein (GFP) in peripheral nerves provides a new hindlimb model for the study of nerve injury and regeneration

BACKGROUND In order to evaluate nerve regeneration in clinically relevant hindlimb surgical paradigms not feasible in fluorescent mice models, we developed a rat that expresses green fluorescent protein (GFP) in neural tissue. METHODS Transgenic Sprague-Dawley rat lines were created using pronuclear injection of a transgene expressing GFP under the control of the thy1 gene. Nerves were imaged under fluorescence microscopy and muscles were imaged with confocal microscopy to determine GFP expression following sciatic nerve crush, transection and direct suturing, and transection followed by repair with a nerve isograft from nonexpressing littermates. RESULTS In each surgical paradigm, fluorescence microscopy demonstrated the loss and reappearance of fluorescence with regeneration of axons following injury. Nerve regeneration was confirmed with imaging of Wallerian degeneration followed by reinnervation of extensor digitorum longus (EDL) muscle motor endplates using confocal microscopy. CONCLUSION The generation of a novel transgenic rat model expressing GFP in neural tissue allows in vivo imaging of nerve regeneration and visualization of motor endplate reinnervation. This rat provides a new model for studying peripheral nerve injury and regeneration over surgically relevant distances.

Nerve Allotransplantation as it Pertains to Composite Tissue Transplantation

Nerve allografts provide a temporary scaffold for host nerve regeneration and allow for the repair of significant segmental nerve injuries. From rodent, large animal, and nonhuman primate studies, as well as clinical experience, nerve allografts, with the use of immunosuppression, have the capacity to provide equal regeneration and function to that of an autograft. In contrast to solid organ transplantation and composite tissue transfers, nerve allograft transplantation requires only temporary immunosuppression. Furthermore, nerve allograft rejection is difficult to assess, as the nerves are surgically buried and are without an immediate functional endpoint to monitor. In this article, we review what we know about peripheral nerve allograft transplantation from three decades of experience and apply our current understanding of nerve regeneration to the emerging field of composite tissue transplantation.

Schwann cells seeded in acellular nerve grafts improve functional recovery

Introduction: This study evaluated whether Schwann cells (SCs) from different nerve sources transplanted into cold‐preserved acellular nerve grafts (CP‐ANGs) would improve functional regeneration compared with nerve isografts. Methods: SCs isolated and expanded from motor and sensory branches of rat femoral and sciatic nerves were seeded into 14mm CP‐ANGs. Growth factor expression, axonal regeneration, and functional recovery were evaluated in a 14‐mm rat sciatic injury model and compared with isografts. Results: At 14 days, motor or sensory‐derived SCs increased expression of growth factors in CP‐ANGs versus isografts. After 42 days, histomorphometric analysis found CP‐ANGs with SCs and isografts had similar numbers of regenerating nerve fibers. At 84 days, muscle force generation was similar for CP‐ANGs with SCs and isografts. SC source did not affect nerve fiber counts or muscle force generation. Conclusions: SCs transplanted into CP‐ANGs increase functional regeneration to isograft levels; however SC nerve source did not have an effect. Muscle Nerve 49: 267–276, 2014

“Supercharge Nerve Transfer to Enhance Motor Recovery, a Laboratory Study”

PURPOSE To investigate the ability of a supercharge end-to-side (SETS) nerve transfer to augment the effect of regenerating native axons in an incomplete rodent sciatic nerve injury model. METHODS Fifty-four Lewis rats were randomized to 3 groups. The first group was an incomplete recovery model (IRM) of the tibial nerve complemented with an SETS transfer from the peroneal nerve (SETS-IRM). The IRM consisted of tibial nerve transection and immediate repair using a 10-mm fresh tibial isograft to provide some, but incomplete, nerve recovery. The 2 control groups were IRM alone and SETS alone. Nerve histomorphometry, electron microscopy, retrograde labeling, and muscle force testing were performed. RESULTS Histomorphometry of the distal tibial nerve showed significantly increased myelinated axonal counts in the SETS-IRM group compared with the IRM and SETS groups at 5 and 8 weeks. Retrograde labeling at 8 weeks confirmed increased motoneuron counts in the SETS-IRM group. Functional recovery at 8 weeks showed a significant increase in muscle-specific force in the SETS-IRM group compared with the IRM group. CONCLUSIONS An SETS transfer enhanced recovery from an incomplete nerve injury as determined by histomorphometry, motoneuron labeling within the spinal cord, and muscle force measurements. CLINICAL RELEVANCE An SETS distal nerve transfer may be useful in nerve injuries with incomplete regeneration such as proximal Sunderland II- or III-degree injuries, in which long regeneration distance yields prolonged time to muscle reinnervation and suboptimal functional recovery.

Differential Gene Expression in Motor and Sensory Schwann Cells in the Rat Femoral Nerve

Phenotypic differences in Schwann cells (SCs) may help to guide axonal regeneration down motor or sensory specific pathways following peripheral nerve injury. The goal of this study was to identify phenotypic markers for SCs harvested from the cutaneous (sensory) and quadriceps (motor) branches of the rat femoral nerve and to study the effects of expansion culture on the expression patterns of these motor or sensory phenotypic markers. RNA was extracted from SCs harvested from the motor and sensory branches of the rat femoral nerve and analyzed using Affymetrix Gene Chips (Rat Genome 230 v2.0 Array A). Genes that were upregulated in motor SCs compared with the sensory SCs or vice versa were identified, and the results were verified for a subset of genes using quantitative real‐time polymerase chain reaction (qRT‐PCR). The expression levels of the “phenotype‐specific” genes were then evaluated in SC expansion cultures at various time points over 30 days by qRT‐PCR to determine the effect of expansion on SC phenotype. Expression levels of the phenotype‐specific genes were significantly altered after expansion culture for both the motor and the sensory markers compared with fresh nerve tissue. These results indicate that both motor and sensory SC gene expression patterns are disrupted during expansion in vitro and may affect the ability of SCs to express phenotype‐specific genes after transplantation.