Spider silk constructs enhance axonal regeneration and remyelination in long nerve defects in sheep

Background

Surgical reapposition of peripheral nerve results in some axonal regeneration and functional recovery, but the clinical outcome in long distance nerve defects is disappointing and research continues to utilize further interventional approaches to optimize functional recovery. We describe the use of nerve constructs consisting of decellularized vein grafts filled with spider silk fibers as a guiding material to bridge a 6.0 cm tibial nerve defect in adult sheep.

Methodology/Principal Findings

The nerve constructs were compared to autologous nerve grafts. Regeneration was evaluated for clinical, electrophysiological and histological outcome. Electrophysiological recordings were obtained at 6 months and 10 months post surgery in each group. Ten months later, the nerves were removed and prepared for immunostaining, electrophysiological and electron microscopy. Immunostaining for sodium channel (NaV 1.6) was used to define nodes of Ranvier on regenerated axons in combination with anti-S100 and neurofilament. Anti-S100 was used to identify Schwann cells. Axons regenerated through the constructs and were myelinated indicating migration of Schwann cells into the constructs. Nodes of Ranvier between myelin segments were observed and identified by intense sodium channel (NaV 1.6) staining on the regenerated axons. There was no significant difference in electrophysiological results between control autologous experimental and construct implantation indicating that our construct are an effective alternative to autologous nerve transplantation.

Conclusions/Significance

This study demonstrates that spider silk enhances Schwann cell migration, axonal regrowth and remyelination including electrophysiological recovery in a long-distance peripheral nerve gap model resulting in functional recovery. This improvement in nerve regeneration could have significant clinical implications for reconstructive nerve surgery.     

Acidic fibroblast growth factor enhances peripheral nerve regeneration in vivo

When added to a collagen-filled nerve guide, purified acidic fibroblast growth factor (aFGF) increased the number of myelinated axons that regenerated across a 5-mm nerve gap distance. In addition, a greater number of primary sensory and motor neurons extended axons through the nerve guide in animals treated with aFGF. Thus the effect of aFGF on peripheral nerve regeneration is not simply an increase in axonal branching within the nerve guide tube. This is the first highly purified growth factor since nerve growth factor that has been shown to promote nerve regeneration in vivo. This experimental model provides a convenient and quantitative means to assess the effects of putative neuronotropic factors on peripheral nerve regeneration in vivo.     

Functional Recovery of Denervated Skeletal Muscle with Sensory or Mixed Nerve Protection: A Pilot Study

Functional recovery is usually poor following peripheral nerve injury when reinnervation is delayed. Early innervation by sensory nerve has been indicated to prevent atrophy of the denervated muscle. It is hypothesized that early protection with sensory axons is adequate to improve functional recovery of skeletal muscle following prolonged denervation of mixed nerve injury. In this study, four groups of rats received surgical denervation of the tibial nerve. The proximal and distal stumps of the tibial nerve were ligated in all animals except for those in the immediate repair group. The experimental groups underwent denervation with nerve protection of peroneal nerve (mixed protection) or sural nerve (sensory protection). The experimental and unprotected groups had a stage II surgery in which the trimmed proximal and distal tibial nerve stumps were sutured together. After 3 months of recovery, electrophysiological, histological and morphometric parameters were assessed. It was detected that the significant muscle atrophy and a good preserved structure of the muscle were observed in the unprotected and protective experimental groups, respectively. Significantly fewer numbers of regenerated myelinated axons were observed in the sensory-protected group. Enhanced recovery in the mixed protection group was indicated by the results of the muscle contraction force tests, regenerated myelinated fiber, and the results of the histological analysis. Our results suggest that early axons protection by mixed nerve may complement sensory axons which are required for promoting functional recovery of the denervated muscle natively innervated by mixed nerve.     

Faster nerve regeneration after sciatic nerve injury in mice over-expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF-2) is expressed in the peripheral nervous system and is up-regulated after nerve lesion. It has been demonstrated that administration of FGF-2 protects neurons from injury-induced cell death and promotes axonal regrowth. Using transgenic mice over-expressing FGF-2 (TgFGF-2), we addressed the importance of endogenously generated FGF-2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild-type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF-2. Morphometric evaluation of intact nerves from TgFGF-2 mice revealed no difference in number and size of myelinated fibers compared to wild-type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF-2 over-expression on Schwann cell proliferation during the early regeneration process, we used BrdU-labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild-types. We propose that endogenously synthesized FGF-2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination.     

Faster nerve regeneration after sciatic nerve injury in mice over‐expressing basic fibroblast growth factor

Basic fibroblast growth factor (FGF‐2) is expressed in the peripheral nervous system and is up‐regulated after nerve lesion. It has been demonstrated that administration of FGF‐2 protects neurons from injury‐induced cell death and promotes axonal regrowth. Using transgenic mice over‐expressing FGF‐2 (TgFGF‐2), we addressed the importance of endogenously generated FGF‐2 on sensory neuron loss and sciatic nerve regeneration. After sciatic nerve transection, wild‐type and transgenic mice showed the same degree of cell death in L5 spinal ganglia. Also, the number of chromatolytic, eccentric, and pyknotic sensory neurons was not changed under elevated levels of FGF‐2. Morphometric evaluation of intact nerves from TgFGF‐2 mice revealed no difference in number and size of myelinated fibers compared to wild‐type mice. One week after crush injury, the number of regenerated axons was doubled and the myelin thickness was significantly smaller in transgenic mice. After 2 and 4 weeks, morphometric analysis and functional tests revealed no differences in recovery of sensory and motor nerve fibers. To study the role of FGF‐2 over‐expression on Schwann cell proliferation during the early regeneration process, we used BrdU‐labeling to mark dividing cells. In transgenic mice, the number of proliferating cells was significantly increased distal to the crush site compared to wild‐types. We propose that endogenously synthesized FGF‐2 influences early peripheral nerve regeneration by regulating Schwann cell proliferation, axonal regrowth, and remyelination. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Polysialyltransferase overexpression in Schwann cells mediates different effects during peripheral nerve regeneration

The polysialic acid (PSA) moiety of the neural cell adhesion molecule (NCAM) has been shown to support dynamic changes underlying peripheral nerve regeneration. Using transgenic mice expressing polysialyltransferase ST8SiaIV under control of a glial-specific (proteolipid protein, PLP) promoter (PLP-ST8SiaIV-transgenic mice), we tested the hypothesis that permanent synthesis of PSA in Schwann cells impairs functional recovery of lesioned peripheral nerves. After sciatic nerve crush, histomorphometric analyses demonstrated impaired remyelination of regenerated axons at the lesion site and in target tissue of PLP-ST8SiaIV-transgenic mice, though the number and size of regenerating unmyelinated axons were not changed. This was accompanied by slower mechanosensory recovery in PLP-ST8SiaIV-transgenic mice. However, the proportion of successfully mono-(re)innervated motor endplates in the foot pad muscle was significantly increased in PLP-ST8SiaIV-transgenic mice when compared with wild-type littermates, suggesting that long-term increase in PSA levels in regenerating nerves may favor selective motor target reinnervation. The combined negative and positive effects of a continuous polysialyltransferase overexpression observed during peripheral nerve regeneration suggest that an optimized time- and differentiation-dependent control of polysialyltransferase expression in Schwann cells may further improve recovery after peripheral nerves injury.     

Immunohistochemical,Ultrastructural and Functional Analysis of Axonal Regeneration through Peripheral Nerve Grafts Containing Schwann Cells Expressing BDNF,CNTF or NT3

We used morphological, immunohistochemical and functional assessments to determine the impact of genetically-modified peripheral nerve (PN) grafts on axonal regeneration after injury. Grafts were assembled from acellular nerve sheaths repopulated ex vivo with Schwann cells (SCs) modified to express brain-derived neurotrophic factor (BDNF), a secretable form of ciliary neurotrophic factor (CNTF), or neurotrophin-3 (NT3). Grafts were used to repair unilateral 1 cm defects in rat peroneal nerves and 10 weeks later outcomes were compared to normal nerves and various controls: autografts, acellular grafts and grafts with unmodified SCs. The number of regenerated βIII-Tubulin positive axons was similar in all grafts with the exception of CNTF, which contained the fewest immunostained axons. There were significantly lower fiber counts in acellular, untransduced SC and NT3 groups using a PanNF antibody, suggesting a paucity of large caliber axons. In addition, NT3 grafts contained the greatest number of sensory fibres, identified with either IB₄ or CGRP markers. Examination of semi- and ultra-thin sections revealed heterogeneous graft morphologies, particularly in BDNF and NT3 grafts in which the fascicular organization was pronounced. Unmyelinated axons were loosely organized in numerous Remak bundles in NT3 grafts, while the BDNF graft group displayed the lowest ratio of umyelinated to myelinated axons. Gait analysis revealed that stance width was increased in rats with CNTF and NT3 grafts, and step length involving the injured left hindlimb was significantly greater in NT3 grafted rats, suggesting enhanced sensory sensitivity in these animals. In summary, the selective expression of BDNF, CNTF or NT3 by genetically modified SCs had differential effects on PN graft morphology, the number and type of regenerating axons, myelination, and locomotor function.     

Axonal Regeneration and Remyelination Evaluation of Chitosan/Gelatin-Based Nerve Guide Combined with Transforming Growth Factor-β1 and Schwann Cells

Despite efforts in peripheral nerve injury and regeneration, it is difficult to achieve a functional recovery following extended peripheral nerve lesions. Even if artificial nerve conduit, cell components and growth factors can enhance nerve regeneration, integration in peripheral nerve repair and regeneration remains yet to be explored. For this study, we used chitosan/gelatin nerve graft constructed with collagenous matrices as a vehicle for Schwann cells and transforming growth factor-β1 to bridge a 10-mm gap of the sciatic nerve and explored the feasibility of improving regeneration and reinnervation in rats. The nerve regeneration was assessed with functional recovery, electrophysiological test, retrograde labeling, and immunohistochemistry analysis during the post-operative period of 16 weeks. The results showed that the internal sides of the conduits were compact enough to prevent the connective tissues from ingrowth. Nerve conduction velocity, average regenerated myelin area, and myelinated axon count were similar to those treated with autograft (p > 0.05) but significantly higher than those bridged with chitosan/gelatin nerve graft alone (p < 0.05). Evidences from retrograde labeling and immunohistochemistry analysis are further provided in support of improving axonal regeneration and remyelination. A designed graft incorporating all of the tissue-engineering strategies for peripheral nerve regeneration may provide great progress in tissue engineering for nerve repair.     

Extracellular vesicles from human umbilical cord mesenchymal stem cells improve nerve regeneration after sciatic nerve transection in rats

Peripheral nerve injury results in limited nerve regeneration and severe functional impairment. Mesenchymal stem cells (MSCs) are a remarkable tool for peripheral nerve regeneration. The involvement of human umbilical cord MSC‐derived extracellular vesicles (hUCMSC‐EVs) in peripheral nerve regeneration, however, remains unknown. In this study, we evaluated functional recovery and nerve regeneration in rats that received hUCMSC‐EV treatment after nerve transection. We observed that hUCMSC‐EV treatment promoted the recovery of motor function and the regeneration of axons; increased the sciatic functional index; resulted in the generation of numerous axons and of several Schwann cells that surrounded individual axons; and attenuated the atrophy of the gastrocnemius muscle. hUCMSC‐EVs aggregated to rat nerve defects, down‐regulated interleukin (IL)‐6 and IL‐1β, up‐regulated IL‐10 and modulated inflammation in the injured nerve. These effects likely contributed to the promotion of nerve regeneration. Our findings indicate that hUCMSC‐EVs can improve functional recovery and nerve regeneration by providing a favourable microenvironment for nerve regeneration. Thus, hUCMSC‐EVs have considerable potential for application in the treatment of peripheral nerve injury.     

Effect of vitamin E-deficiency on regeneration of the sciatic nerve

The regeneration of the sciatic nerve fibres was studied in both normal and vitamin E-deficient rats at 30 and 60 days after crush. The vitamin E is involved in one of the most important mechanisms of protection against peroxidation of plasma membrane lipids; the plasma membrane plays certainly a role in nerve regeneration. Both the diameter and the total number of myelinated nerve fibres was calculated at different times. The number of myelinated fibres in the undenervated deficient animals was lower than that found in the undenervated normals animals. Following the nerve crush, in normal animals after two months the number of myelinated fibres exceeded the number found in undenervated normal animals, whereas in the deficient rat nerves it was significantly lower than in the corresponding controls and moreover it did not even reach the number found in the nerves of undenervated deficient rats. Finally, the caliber distribution of myelinated fibres in undenervated and denervated deficient rats shows a relative percent increase in the number of greatest axons and a decrease in smaller axons. This result confirm the vitamin E to be an important factor of the normal process of nerve regeneration.     

Modulation of peripheral nerve regeneration: a tissue-engineering approach. The role of amnion tube nerve conduit across a 1-centimeter nerve gap

A new type of a biodegradable nerve graft conduit material, the amnion tube, has been developed in our laboratory. To test the tube in the peripheral nerve regeneration process, it was initially applied across a 1-cm sciatic nerve gap in rats and was compared with other nerve conduit materials. We used male Sprague-Dawley rats as our animal model. The experiment included 66 rats that were randomly assigned into five groups: autograft (n = 17), amnion tube (n = 19), silicone tube (n = 20), no repair (n = 7), and sham group (n = 3). The process of peripheral nerve regeneration was evaluated at 2, 4, 10, and 17 weeks following injury and repair by using morphologic and functional assessments of the outcome of nerve regeneration in each animal. Nerve regeneration across the amnion tube nerve conduit was comparable with that seen in autograft and superior to that of the silicone group. A uniform nerve tissue was seen filling and crossing the amnion conduit, and the regenerated nerve from the proximal stump reached the distal end and was undifferentiated from the normal nerve tissues. At 4 months, the amnion tube biodegraded and no longer could be identified and differentiated from the nerve tissues. The amnion tube animal group showed a number of axons very close to that in the nerve autograft group (37,157 versus 33,054). Functional recovery at a 2- to 4-week interval was significantly statistically higher only in the amnion tube animal group (p = 0.01). However, the improvement disappeared between 10 and 17 weeks. In conclusion, the amnion tube is a potential ideal nerve conduit material secondary to its unique characteristics: it contains important neurotropic factors, is biodegradable, provokes a very weak immune response, is semiflexible, is readily available, and is easily manufactured into different sizes and diameters.     

Knockout of p75(NTR) impairs re-myelination of injured sciatic nerve in mice

Remyelination is an important aspect of nerve regeneration after nerve injury but the underlying mechanisms are not fully understood. The neurotrophin receptor, p75(NTR), in activated Schwann cells in the Wallerian degenerated nerve is up-regulated and may play a role in the remyelination of regenerating peripheral nerves. In the present study, the role of p75(NTR) in remyelination of the sciatic nerve was investigated in p75(NTR) mutant mice. Histological results showed that the number of myelinated axons and thickness of myelin sheath in the injured sciatic nerves were reduced in mutant mice compared with wild-type mice. The myelin sheath of axons in the intact sciatic nerve of adult mutant mice is also thinner than that of wild-type mice. Real-time RT-PCR showed that mRNA levels for myelin basic protein and P0 in the injured sciatic nerves were significantly reduced in p75(NTR) mutant animals. Western blots also showed a significant reduction of P0 protein in the injured sciatic nerves of mutant animals. These results suggest that p75(NTR) is important for the myelinogenesis during the regeneration of peripheral nerves after injury.     

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.     

A quantitative electron microscope analysis of peripheral nerve in the urodele amphibian in relation to limb regenerative capacity

An approximate 1:1 ratio of myelinated to unmyelinated fibers was established in counts from electron micrograph montages in nerves of the newt, Triturus (Notophthalmus) viridescens. The number of myelinated fibers correspond to the number counted with the light microscope after osmium fixation. Light microscope counts of silver impregnated sections yielded a value slightly higher suggesting that, except for bundles of unmyelinated fibers, the silver technique revealed mainly myelinated fibers. The results were used to reassess previous quantitative studies on the relation between number of nerve fibers and the control which nerves exert on regeneration. For a truer estimate of the number of axons affecting regeneration, fiber values previously reported should now be doubled to include the large number of unmyelinated fibers. However, calculations show that the unmyelinated fibers contribute less than 3% of the total neuroplasm in the peripheral nerve. Finally, counts made of Schwann cells and fibroblasts show that the latter are few in number.     

Consequences of slow Wallerian degeneration for regenerating motor and sensory axons.

The time course of Wallerian degeneration in the tibial and saphenous nerves was compared in Balb/c mice and mice of the C57BL/Ola strain (Lunn et al., 1989). Axons, particularly myelinated ones, in nerves of C57BL/Ola mice are very slow to degenerate, many still being present 3 weeks after axotomy. Nuclear numbers in the distal stump peak much later and do not reach the levels found in Balb/c mice; debris removal is very slow, and Schwann cell numbers only rise slightly above normal levels in the long term. Regeneration was investigated electrophysiologically and by electron microscopy (EM). Myelinated sensory axons regenerated slowly and incompletely compared with motor ones which were only slightly slowed after nerve crush (although they were significantly hindered after nerve section). Total myelinated axon numbers were still some 20% less than normal even after 200 days in sensory nerves. Even after all axons had degenerated in C57BL/Ola mice, regeneration rates of neither myelinated nor unmyelinated sensory axons reached those achieved in Balb/c mice. It is concluded that while regeneration can eventually proceed slowly when Wallerian degeneration is much delayed, the usual rapid time course of Wallerian degeneration is necessary if axons, particularly sensory ones, are to regenerate at optimal rates and to maximum extent. While local obstruction to axon growth probably impedes the early phase of regeneration in C57BL/Ola mice, it seems possible that a lack of adequate early signals affects regeneration permanently by minimizing the cell body reaction to injury.     

Functional Recovery of Regenerating Motor Axons is Delayed in Mice Heterozygously Deficient for the Myelin Protein P0 Gene

Mice with a heterozygous knock-out of the myelin protein P₀ gene (P₀+/?) develop a neuropathy similar to human Charcot–Marie–Tooth disease. They are indistinguishable from wild-types (WT) at birth and develop a slowly progressing demyelinating neuropathy. The aim of this study was to investigate whether the regeneration capacity of early symptomatic P₀+/? is impaired as compared to age matched WT. Right sciatic nerves were lesioned at the thigh in 7–8 months old mice. Tibial motor axons at ankle were investigated by conventional motor conduction studies and axon excitability studies using threshold tracking. To evaluate regeneration we monitored the recovery of motor function after crush, and then compared the fiber distribution by histology. The overall motor performance was investigated using Rotor-Rod. P₀+/? had reduced compound motor action potential amplitudes and thinner myelinated axons with only a borderline impairment in conduction and Rotor-Rod. Plantar muscle reinnervation occurred within 21 days in all mice. Shortly after reinnervation the conduction of P₀+/? regenerated axons was markedly slower than WT, however, this difference decayed with time. Nevertheless, after 1 month, regenerated P₀+/? axons had longer strength-duration time constant, larger threshold changes during hyperpolarizing electrotonus and longer relative refractory period. Their performance at Rotor-Rod remained also markedly impaired. In contrast, the number and diameter distribution of regenerating myelinated fibers became similar to regenerated WT. Our data suggest that in the presence of heterozygously P₀ deficient Schwann cells, regenerating motor axons retain their ability to reinnervate their targets and remyelinate, though their functional recovery is delayed.     

Omentum-wrapped scaffold with longitudinally oriented micro-channels promotes axonal regeneration and motor functional recovery in rats

Background

Tissue-engineered nerve scaffolds hold great potential in bridging large peripheral nerve defects. However, insufficient vascularization of nerve scaffolds limited neural tissues survival and regeneration, which hampered the successful implantation and clinical application of nerve scaffolds. The omentum possesses a high vascularization capacity and enhances regeneration and maturation of tissues and constructs to which it is applied. However, combined application of nerve scaffolds and omentum on axonal regeneration and functional recovery in the treatment of large peripheral nerve defects has rarely been investigated thus far.

Methods

In the present study, an omentum-wrapped collagen-chitosan scaffold was used to bridge a 15-mm-long sciatic nerve defect in rats. Rats that received nerve autografts or scaffolds alone were served as positive control or negative control, respectively. The axonal regeneration and functional recovery were examined by a combination of walking track analysis, electrophysiological assessment, Fluoro-Gold (FG) retrograde tracing, as well as morphometric analyses to both regenerated nerves and target muscles.

Findings

The results demonstrated that axonal regeneration and functional recovery were in the similar range between the omentum-wrapping group and the autograft group, which were significantly better than those in the scaffold alone group. Further investigation showed that the protein levels of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were significantly higher in the omentum-wrapping group than those in the scaffold alone group in the early weeks after surgery.

Conclusion

These findings indicate that the omentum-wrapped scaffold is capable of enhancing axonal regeneration and functional recovery, which might be served as a potent alternative to nerve autografts. The beneficial effect of omentum-wrapping on nerve regeneration might be related with the proteins produced by omentum.     

Nogo-A expressed in Schwann cells impairs axonal regeneration after peripheral nerve injury

Injured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.     

Effects of high fat diet induced obesity on peripheral nerve regeneration and levels of GAP 43 and TGF-β in rats

The increasing frequency of obesity is important because of its accompanying related health problems. The effects of obesity on peripheral nerves have not been elucidated. We investigated the effects of obesity on sciatic nerve regeneration using electrophysiology, stereology, immunohistochemistry, histopathology and functional tests. We used control, obese, control injured and obese injured groups of rats. Electrophysiological results showed that nerve conduction velocity and EMG were same in the experimental groups, but the amplitude of the compound action potential of the control group was significantly higher than that of the obese group. Examination of the nerves showed that the control and obese groups had both larger axon diameters and thicker myelin sheaths. The number of myelinated axons was decreased in both of the injured groups. Axon diameters and myelin sheath thicknesses of the control injured group were significantly greater those of the obese injured group. There were no significant differences in functional tests among the groups. Although growth associated protein 43 immunostaining in the control injured group was significantly greater than that of the obese injured group, no significant difference was observed between the control and obese groups. There was no significant difference in immunohistochemical staining for transforming growth factor beta 3 between the control injured and obese injured groups. Our results suggest that obesity may affect peripheral nerve regeneration negatively after crush injury.     

ErbB2 Receptor Over-Expression Improves Post-Traumatic Peripheral Nerve Regeneration in Adult Mice

In a transgenic mice (BALB-neuT) over-expressing ErbB2 receptor, we investigated the adult mouse median nerve in physiological and pathological conditions. Results showed that, in physiological conditions, the grip function controlled by the median nerve in BALB-neuT mice was similar to wild-type (BALB/c). Stereological assessment of ErbB2-overexpressing intact nerves revealed no difference in number and size of myelinated fibers compared to wild-type mice. By contrast, after a nerve crush injury, the motor recovery was significantly faster in BALB-neuT compared to BALB/c mice. Moreover, stereological assessment revealed a significant higher number of regenerated myelinated fibers with a thinner axon and fiber diameter and myelin thickness in BALB-neuT mice. At day-2 post-injury, the level of the mRNAs coding for all the ErbB receptors and for the transmembrane (type III) Neuregulin 1 (NRG1) isoforms significantly decreased in both BALB/c and BALB-neuT mice, as shown by quantitative real time PCR. On the other hand, the level of the mRNAs coding for soluble NRG1 isoforms (type I/II, alpha and beta) increased at the same post-traumatic time point though, intriguingly, this response was significantly higher in BALB-neuT mice with respect to BALB/c mice. Altogether, these results suggest that constitutive ErbB2 receptor over-expression does not influence the physiological development of peripheral nerves, while it improves nerve regeneration following traumatic injury, possibly through the up-regulation of soluble NRG1 isoforms.