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     

Peripheral nerve regeneration is delayed in neuropilin 2-deficient mice

Peripheral nerve transection or crush induces expression of class 3 semaphorins by epineurial and perineurial cells at the injury site and of the neuropilins neuropilin-1 and neuropilin-2 by Schwann and perineurial cells in the nerve segment distal to the injury. Neuropilin-dependent class 3 semaphorin signaling guides axons during neural development, but the significance of this signaling system for regeneration of adult peripheral nerves is not known. To test the hypothesis that neuropilin-2 facilitates peripheral-nerve axonal regeneration, we crushed sciatic nerves of adult neuropilin-2-deficient and littermate control mice. Axonal regeneration through the crush site and into the distal nerve segment, repression by the regenerating axons of Schwann cell p75 neurotrophin receptor expression, remyelination of the regenerating axons, and recovery of normal gait were all significantly slower in the neuropilin-2-deficient mice than in the control mice. Thus, neuropilin-2 facilitates peripheral-nerve axonal regeneration.     

Human umbilical cord mesenchymal stem cells promote peripheral nerve repair via paracrine mechanisms

Human umbilical cord-derived mesenchymal stem cells(h UCMSCs) represent a promising young-state stem cell source for cell-based therapy. h UCMSC transplantation into the transected sciatic nerve promotes axonal regeneration and functional recovery. To further clarify the paracrine effects of h UCMSCs on nerve regeneration, we performed human cytokine antibody array analysis, which revealed that h UCMSCs express 14 important neurotrophic factors. Enzyme-linked immunosorbent assay and immunohistochemistry showed that brain-derived neurotrophic factor, glial-derived neurotrophic factor, hepatocyte growth factor, neurotrophin-3, basic fibroblast growth factor, type I collagen, fibronectin and laminin were highly expressed. Treatment with h UCMSC-conditioned medium enhanced Schwann cell viability and proliferation, increased nerve growth factor and brain-derived neurotrophic factor expression in Schwann cells, and enhanced neurite growth from dorsal root ganglion explants. These findings suggest that paracrine action may be a key mechanism underlying the effects of h UCMSCs in peripheral nerve repair.     

Craniocerebral injury promotes the repair of peripheral nerve injury

The increase in neurotrophic factors after craniocerebral injury has been shown to promote fracture healing. Moreover, neurotrophic factors play a key role in the regeneration and repair of peripheral nerve. However, whether craniocerebral injury alters the repair of peripheral nerve injuries remains poorly understood. Rat injury models were established by transecting the left sciatic nerve and using a free-fall device to induce craniocerebral injury. Compared with sciatic nerve injury alone after 6–12 weeks, rats with combined sciatic and craniocerebral injuries showed decreased sciatic functional index, increased recovery of gastrocnemius muscle wet weight, recovery of sciatic nerve ganglia and corresponding spinal cord segment neuron morphologies, and increased numbers of horseradish peroxidase-labeled cells. These results indicate that craniocerebral injury promotes the repair of peripheral nerve injury.     

Role of neuregulin-1 in peripheral nerve injury

Peripheral nerve injury in humans often leads to permanent functional deficits. Schwann cells play an important role in the recovery of peripheral nerve injury by ensheathing axons and providing various neurotrophic factors. Neuregulin-1 (NRG-1) provides axonal signals, which allow dedifferentiation and rapid proliferation of Schwann cells. Subsequently, NRG-1 promotes axonal myelination and influences myelin thickness. Moreover, NRG-1 plays a critical role in synapse formation in the neuromuscular junction. These effects, together, suggest that NRG-1 promotes recovery of peripheral nerve injury.     

Effect of bone marrow-derived mononuclear cells on nerve regeneration in the transection model of the rat sciatic nerve

Bone marrow-derived stem cells enhance the rate of regeneration and clinical improvement in nerve injury, spinal cord injury and brain infarction. Recent experiments in rat spinal cord demyelination showed that remyelination was specific to intravenous delivery of the bone marrow-derived mononuclear cell (BM-MNC) fraction, although the specific role of this fraction in peripheral nerve regeneration has not been examined. Therefore we evaluated the role of BM-MNCs in peripheral nerve regeneration in the rat sciatic nerve transection model. After anesthesia, the right sciatic nerve of 20 adult-male Wistar rats was transected under an operating microscope. In the test group, the cut ends of the nerve were approximated with two epineural microsutures, the gap was filled with rat BM-MNCs and the approximated nerve ends were covered with fibrin glue. In the control group, the transected nerve ends were repaired with two epineural microsutures and fibrin sealant only. Histological assessment of the nerve was performed 30 days and 60 days after the operation and regenerative changes were compared between the two groups. The recovery after nerve anastamosis was far better in the test group at both 30 days and 60 days. There was a statistically significant difference in axonal regeneration, remyelination and myelin thickness at sites 5 mm and 10 mm from the site of repair of the nerve. Schwann cell proliferation and degenerative changes were more prevalent in the controls. This study demonstrates that local delivery of BM-MNCs (which can be isolated easily from bone marrow aspirates) into injured peripheral nerve increases the rate and degree of nerve regeneration. The present study highlights the role of BM-MNCs in peripheral nerve regeneration.     

Neuregulin-1在外周神经再生中的作用

人类外周神经再生通常导致永久性的功能丧失。在外周神经再生过程中,雪旺细胞发挥重要的作用,它可以包绕神经轴突形成髓鞘,并分泌各种神经营养因子。在这个过程中,NRG-1起着重要的作用。NRG-1通过信号传导,使雪旺细胞去分化,增殖。而且NRG-1调节神经轴突的髓鞘化,并决定髓鞘的厚度。最后,在神经肌肉接头形成的过程中,NRG-1 也起着重要的作用。总之,NRG-1促进了外周神经损伤后的再生。     

Nanoparticles carrying neurotrophin-3-modified Schwann cells promote repair of sciatic nerve defects

Schwann cells and neurotrophin-3 play an important role in neural regeneration,but the secretion of neurotrophin-3 from Schwann cells is limited,and exogenous neurotrophin-3 is inactived easily in vivo.In this study,we have transfected neurotrophin-3 into Schwann cells cultured in vitro using nanoparticle liposomes.Results showed that neurotrophin-3 was successfully transfected into Schwann cells,where it was expressed effectively and steadily.A composite of Schwann cells transfected with neurotrophin-3 and poly(lactic-co-glycolic acid) biodegradable conduits was transplanted into rats to repair 10-mm sciatic nerve defects.Transplantation of the composite scaffold could restore the myoelectricity and wave amplitude of the sciatic nerve by electrophysiological examination,promote nerve axonal and myelin regeneration,and delay apoptosis of spinal motor neurons.Experimental findings indicate that neurotrophin-3 transfected Schwann cells combined with bridge grafting can promote neural regeneration and functional recovery after nerve injury.     

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.     

Influence of aging on peripheral nerve function and regeneration

Aging deeply influences several morphologic and functional features of the peripheral nervous system (PNS). Morphologic studies have reported a loss of myelinated and unmyelinated nerve fibers in elderly subjects, and several abnormalities involving myelinated fibers, such as demyelination, remyelination and myelin balloon figures. The deterioration of myelin sheaths during aging may be due to a decrease in the expression of the major myelin proteins (P0, PMP22, MBP). Axonal atrophy, frequently seen in aged nerves, may be explained by a reduction in the expression and axonal transport of cytoskeletal proteins in the peripheral nerve. Aging also affects functional and electrophysiologic properties of the PNS, including a decline in nerve conduction velocity, muscle strength, sensory discrimination, autonomic responses, and endoneurial blood flow. The age-related decline in nerve regeneration after injury may be attributed to changes in neuronal, axonal, Schwann cell and macrophage responses. After injury, Wallerian degeneration is delayed in aged animals, with myelin remnants accumulated in the macrophages being larger than in young animals. The interaction between Schwann cells and regenerative axons takes longer, and the amount of trophic and tropic factors secreted by reactive Schwann cells and target organs are lower in older subjects than they are in younger subjects. The rate of axonal regeneration becomes slower and the density of regenerating axons decrease in aged animals. Aging also determines a reduction in terminal and collateral sprouting of regenerated fibers, further limiting the capabilities for target reinnervation and functional restitution. These age-related changes are not linearly progressive with age; the capabilities for axonal regeneration and reinnervation are maintained throughout life, but tend to be delayed and less effective with aging.     

Impaired nerve regeneration in reeler mice after peripheral nerve injury

Reelin, an extracellular matrix protein, plays an important role in the regulation of neuronal migration and cortical lamination in the developing brain. Little is known, however, about the role of this protein in axonal regeneration. We have previously shown that Reelin is secreted by Schwann cells in the peripheral nerve compartment during postnatal development and that it is up-regulated following nerve injury in adult mice. In this work, we generated mice deficient in Reelin ( reeler ) that express yellow fluorescent protein (YFP) in a subset of neurons and examined the axonal regeneration following nerve crush. We found that axonal regeneration was significantly altered compared with wild-type mice. By contrast, retrograde tracing with Fluorogold dye after sciatic nerve crush was unaffected in these mutants, being comparable with normal axonal transport observed in wild-type. These results indicate that the absence of Reelin impairs axonal regeneration following injury and support a role for this protein in the process of peripheral nerve regeneration.     

重组腺病毒载体AxCA-BDNF基因转染修复坐骨神经损伤*☆

背景：如何促进周围神经损伤修复与再生一直是基础与临床研究的热点。基因治疗有可能成为今后解决该问题的主要手段之一。 目的：观察携带小鼠脑源性神经营养因子(brain-derived neurotrophic factor,BDNF) cDNA表达片段的重组腺病毒载体AxCA-BDNF转染大鼠损伤坐骨神经后BDNF的表达,以及脊髓前角运动神经元的存活和神经生长情况。 方法：切除成年Wistar大鼠股中部10 mm长的坐骨神经,AxCA-BDNF转染组、BDNF组和对照组分别用硅胶管内置AxCA-BDNF原液,BDNF溶液或空白病毒稀释液桥接坐骨神经两断端。术后3,7,14 d,1,2,4个月应用原位杂交和免疫组织化学方法检测损伤坐骨神经及相应脊髓节段BDNF mRNA和蛋白的表达,并观察损伤坐骨神经的组织学及超微结构改变,再生的神经元及有髓神经纤维数目和髓鞘厚度。 结果与结论：术后3,7,14 d及1个月时,AxCA-BDNF转染组损伤坐骨神经近、远端神经干及脊髓(L3~6)中BDNF mRNA和蛋白水平明显高于BDNF组和对照组(P < 0.01)。光、电镜病理组织学检查和图像分析证实,BDNF基因转染后,脊髓前角运动神经元存活数量、新生神经纤维数目及其髓鞘厚度、神经联接的再形成均明显优于对照组(P < 0.01)。说明经腺病毒介导转染的BDNF基因可在大鼠坐骨神经内有效表达,并通过轴突逆行转运到了相应的脊髓神经元,不仅能促进损伤神经纤维再生,也能保护损伤的脊髓神经元。 关键词：坐骨神经损伤;重组腺病毒;脑源性神经营养因子;基因转染;免疫组织化学;原位分子杂交;神经再生     

The neurotrophin receptor p75NTR in Schwann cells is implicated in remyelination and motor recovery after peripheral nerve injury

The function of the p75(NTR) neurotrophin receptor (p75(NTR)) in nervous system regeneration is still controversial. Part of that controversy may be due to the fact that p75(NTR) is expressed by both neuronal and glial cell types and may have very distinct and even contradictory roles in each population. In this study, to elucidate the in vivo function of p75(NTR) in Schwann cells during remyelination after peripheral nerve injury, we established a new animal model for p75(NTR)-deficient Schwann cell transplantation in nude mice. We performed quantitative assessments of the functional, histological, and electrophysiological recovery after sciatic nerve injury, and compared them with those of the p75(NTR)(+/+) Schwann cell transplanted animals. At 7-10 weeks after injury, the motor recovery in the p75(NTR)(-/-) Schwann cell transplanted animals was significantly impaired compared with that in the p75(NTR)(+/+) Schwann cell transplanted animals. The lower number of the retrogradely labeled motoneurons and the hypomyelination in the p75(NTR)(-/-) Schwann cell transplanted animals were evident at 6 and 10 weeks after injury. At 10 weeks after injury, the radial growth in the axon caliber was also impaired in the p75(NTR)(-/-) Schwann cell transplanted animals. Measurement of the amount of myelin proteins and the nerve conduction velocity at 10 weeks after injury reflected these results. In summary, the p75(NTR) expression in Schwann cells is important for remyelination process, and the motor recovery after injury is impaired due to impaired axonal growth, remyelination, and radial growth in the axon calibers.     

BD™ PuraMatrix™ peptide hydrogel seeded with Schwann cells for peripheral nerve regeneration

This study investigated the effects of a membrane conduit filled with a synthetic matrix BD™ PuraMatrix™ peptide (BD) hydrogel and cultured Schwann cells on regeneration after peripheral nerve injury in adult rats.After sciatic axotomy, a 10 mm gap between the nerve stumps was bridged using ultrafiltration membrane conduits filled with BD hydrogel or BD hydrogel containing Schwann cells. In control experiments, the nerve defect was bridged using either membrane conduits with alginate/fibronectin hydrogel or autologous nerve graft. Axonal regeneration within the conduit was assessed at 3 weeks and regeneration of spinal motoneurons and recovery of muscle weight evaluated at 16 weeks postoperatively.Schwann cells survived in the BD hydrogel both in culture and after transplantation into the nerve defect. Regenerating axons grew significantly longer distances within the conduits filled with BD hydrogel when compared with the alginate/fibronectin hydrogel and alginate/fibronectin with Schwann cells. Addition of Schwann cells to the BD hydrogel considerably increased regeneration distance with axons crossing the injury gap and entering into the distal nerve stump. The conduits with BD hydrogel showed a linear alignment of nerve fibers and Schwann cells.The number of regenerating motoneurons and recovery of the weight of the gastrocnemius muscle was inferior in BD hydrogel and alginate/fibronectin groups compared with nerve grafting. Addition of Schwann cells did not improve regeneration of motoneurons or muscle recovery.The present results suggest that BD hydrogel with Schwann cells could be used within biosynthetic conduits to increase the rate of axonal regeneration across a nerve defect.     

Schwann cells transduced with a lentiviral vector encoding Fgf‐2 promote motor neuron regeneration following sciatic nerve injury

Fibroblast growth factor 2 (FGF‐2) is a trophic factor expressed by glial cells and different neuronal populations. Addition of FGF‐2 to spinal cord and dorsal root ganglia (DRG) explants demonstrated that FGF‐2 specifically increases motor neuron axonal growth. To further explore the potential capability of FGF‐2 to promote axon regeneration, we produced a lentiviral vector (LV) to overexpress FGF‐2 (LV‐FGF2) in the injured rat peripheral nerve. Cultured Schwann cells transduced with FGF‐2 and added to collagen matrix embedding spinal cord or DRG explants significantly increased motor but not sensory neurite outgrowth. LV‐FGF2 was as effective as direct addition of the trophic factor to promote motor axon growth in vitro. Direct injection of LV‐FGF2 into the rat sciatic nerve resulted in increased expression of FGF‐2, which was localized in the basal lamina of Schwann cells. To investigate the in vivo effect of FGF‐2 overexpression on axonal regeneration after nerve injury, Schwann cells transduced with LV‐FGF2 were grafted in a silicone tube used to repair the resected rat sciatic nerve. Electrophysiological tests conducted for up to 2 months after injury revealed accelerated and more marked reinnervation of hindlimb muscles in the animals treated with LV‐FGF2, with an increase in the number of motor and sensory neurons that reached the distal tibial nerve at the end of follow‐up. GLIA 2014;62:1736–1746     

Vascularized nerve grafts: an experimental study

Objective: The aim of this study is to define an experimental model in order to promote the functional recovery of the nerves using grafts with vascular support (Vascular Nerve Grafts – VNG). The aim of this study is to define, on an experimental model in normal recipient bed, whether the functional recovery with VNG is superior to that obtained non-vascularized graft (NNG).

Methods: Twenty male rabbits, which underwent dissection of sciatic nerve, were later treated by reinnervation through an autograft. In 10 animals the reconstruction of sciatic nerve was realized with VNG; in 10 control animals the reconstruction of sciatic nerve was realized with NNG.

Results: The VNG group showed a better axonal organization and a significantly higher number of regenerated axons in the early phases (after 30 days) than the NNG group, whereas the difference in the axonal number at day 90 was less significant; besides, the axon diameter and the myelin thickness were not significantly improved by VNG group.

Discussion: Our data suggests that the use of VNG leads to a faster regeneration process and a better functional recovery, although the final results are comparable to those of the NNG. VNG improve the quality of the axonal regeneration (axonal diameter and Schwann cells), although the increase in the axonal number is not significant and does not improve the long-term functional outcome.     

Decellularized sciatic nerve matrix as a biodegradable conduit for peripheral nerve regeneration

The use of autologous nerve grafts remains the gold standard for treating nerve defects, but current nerve repair techniques are limited by donor tissue availability and morbidity associated with tissue loss. Recently, the use of conduits in nerve injury repair, made possible by tissue engineering, has shown therapeutic potential. We manufactured a biodegradable, collagen-based nerve conduit containing decellularized sciatic nerve matrix and compared this with a silicone conduit for peripheral nerve regeneration using a rat model. The collagen-based conduit contains nerve growth factor, brain-derived neurotrophic factor, and laminin, as demonstrated by enzyme-linked immunosorbent assay. Scanning electron microscopy images showed that the collagen-based conduit had an outer wall to prevent scar tissue infiltration and a porous inner structure to allow axonal growth. Rats that were implanted with the collagen-based conduit to bridge a sciatic nerve defect experienced significantly improved motor and sensory nerve functions and greatly enhanced nerve regeneration compared with rats in the sham control group and the silicone conduit group. Our results suggest that the biodegradable collagen-based nerve conduit is more effective for peripheral nerve regeneration than the silicone conduit.     

Sustained release of collagen VI potentiates sciatic nerve regeneration by modulating macrophage phenotype

Although the peripheral nervous system has an intrinsic ability for repair and regeneration after injury, bridging long peripheral nerve defects remains a challenge. Functional nerve regeneration depends on interactions among axons, Schwann cells, fibroblasts and immune cells. Macrophages, as immune cells recruited early in this process, show polarization toward phenotypes that are detrimental or beneficial to tissue remodeling depending on the microenvironment of the scaffolds. In this study, we investigated the effects of macrophage phenotypes modulated by collagen VI on axonal regeneration and functional recovery by bridging a 15‐mm‐long sciatic nerve defect in rats. Our results showed that local delivery of collagen VI within a polycaprolactone (PCL) electrospun conduit increased the recruitment of macrophages and their polarization toward the pro‐healing (M2) phenotype. In addition, the axonal regeneration and neurologic functional recovery in the PCL/collagen VI conduit group are equivalent to that of an autograft. In conclusion, the present study confirmed that PCL/collagen VI conduits with sustained release of collagen VI in the local microenvironment may, through triggering macrophage M2 polarization to enhance the nerve regeneration, suggest that our combined biomaterial‐immunomodulatory system may be an attractive candidate for peripheral nerve regeneration.     

Glial cell line-derived neurotrophic factor enhances axonal regeneration following sciatic nerve transection in adult rats

Adult rat sciatic nerve was transected and sutured with an entubulation technique. The nerve interstump gap was filled with either collagen gel (COL) or collagen gel mixed with glial cell line-derived neurotrophic factor (COL/GDNF). Four weeks after nerve transection, horseradish peroxidase (HRP)-labelled spinal cord motoneurons and the myelinated distal stump axons were quantified. Compared with the COL group, the percentages of labeled spinal somas and axon number were significantly increased after topically applied glial cell line-derived neurotrophic factor (GDNF). The functional recovery of the transected nerve was improved in COL/GDNF group. GAP-43 expression was also significantly higher in COL/GDNF group 1 and 2 weeks after sciatic nerve axotomy vs. COL group. These data provide strong evidence that GDNF could promote axonal regeneration in adult rats, suggesting the potential use of GDNF in therapeutic approaches to peripheral nerve injury and neuropathies.