Proliferating immature Schwann cells contribute to nerve regeneration after ischemic peripheral nerve injury

Schwann cells exhibit a high degree of plasticity in adult peripheral nerves after mechanical injury; they have, therefore, been implicated in promoting nerve regeneration. However, Schwann cell behavior after ischemic injury has not yet been elucidated. To determine how Schwann cell plasticity may contribute to recovery from ischemic neuropathy, we used a rat model in which ischemia was induced in the tibial nerve by a 5-hour occlusion of the supplying arteries. Proliferation of immature Schwann cells that emerged in the injured nerve was evaluated by double immunostaining for the p75 neurotrophin receptor and proliferating cell nuclear antigen. The number of proliferating cell nuclear antigen/p75 neurotrophin receptor double-positive cells increased significantly in 1 to 2 weeks after ischemia and subsequently decreased by 4 weeks. During this time, the postmitotic Schwann cells differentiated into mature cells, as demonstrated with bromodeoxyuridine incorporation, which facilitated axon guidance and subsequent axon remyelination. These results suggest the emergence and proliferation of immature Schwann cells that contribute to nerve regeneration after ischemic injury. The manipulation of this population of proliferating immature Schwann cells may be a useful strategy for treating ischemic peripheral neuropathy.     

Isolation and functional characterization of Schwann cells derived from adult peripheral nerve

To facilitate the development of autologous transplantation techniques with which to test the ability of Schwann cell (ScC) implantations to treat nervous system injury, we have developed a method for procuring large, essentially pure populations of ScCs from adult peripheral nerve. By allowing small explants of peripheral nerve trunk to undergo axonal and myelin breakdown in vitro, rather than dissociating the nerve immediately after harvest, we are able to (1) rid the explant of nearly all fibroblasts and (2) capitalize on the intrinsic ScC mitogenic response to peripheral nerve degeneration. Here, we describe a method that yields up to 98% pure ScC populations from adult rat sciatic nerve (based on cell soma and nuclear morphology, S100 staining, and behavior of dissociated cells on neurites) at cell yields of greater than 2 x 10(4) cells/mg of starting nerve weight. The purification technique was successfully applied to human tissue; human phrenic nerve yielded 98% pure ScC populations at cell yields of 2 x 10(4) cells/mg of initial nerve weight. Similar to neonatally derived ScCs, adult rat cells can be expanded in coculture with dorsal root ganglion (DRG) neurons or in isolation in the presence of glial growth factor and forskolin. Cells expanded indefinitely on DRG neurons, or up to 10 weeks on chemical mitogens, return to quiescence following removal of the mitogenic stimulus. Expanded adult-derived rat ScCs retain functional capacity, as evidenced by their ability to myelinate DRG neurites and to support regeneration of processes from embryonic rat retinal explants.     

FK506 enhances regeneration of axons across long peripheral nerve gaps repaired with collagen guides seeded with allogeneic Schwann cells

We assessed the effects of FK506 administration on regeneration after a 6-mm gap repair with a collagen guide seeded with allogeneic Schwann cells (SCs) in the mouse sciatic nerve. SCs were isolated from predegenerated adult sciatic nerves and expanded in culture using a defined medium, before being seeded in the collagen guide embedded in Matrigel. Functional reinnervation was evaluated by noninvasive methods to determine recovery of motor, sensory, and autonomic functions in the hindpaw over 4 months postoperation. Histological analysis of the regenerated nerves was performed at the end of the study. Using simple collagen guides for tubulization repair, treatment with an immunosuppressant dose of FK506 (5 mg/kg/day) resulted in significant improvement of the onset and the degree of reinnervation. While the introduction of allogeneic SCs did not improve regeneration versus a collagen guide filled only with Matrigel, treatment with FK506 allowed for successful regeneration in all the mice and for significant improvement in the levels of functional recovery. Compared with the untreated group, there was greater survival of transplanted pre-labeled SCs in the FK506-treated animals. Morphologically, the best nerve regeneration (in terms of nerve caliber and numbers of myelinated axons) was obtained with SC-seeded guides from FK506-treated animals. Thus, FK506 should be considered as adjunct therapy for various types of tubulization repair.     

Separation of functional Schwann cells and neurons from normal peripheral nerve tissue.

A method has been devised for obtaining viable cultures of normal sensory neurons and normal Schwann cells from rat dorsal root ganglia, using cytosine arabinoside and fluorodeoxyuridine for control of non-neuronal cell proliferation. These cultures were used to demonstrate (a) that Schwann cells could be generated and maintained in culture free of fibroblast contamination, (b) that Schwann cells could exist in either a quiescent or proliferative state, depending on the absence of neurons, (c) that sensory ganglia could be obtained that would provide an outgrowth of axons entirely free of non-neuronal cells even in the absence of antimitotic agents, and (d) that quiescent Schwann cells, induced to proliferate by 'bare' axons, could ensheath and, in time, myelinate some of these axons.     

神经桥接与导管套接修复猫动眼神经形态学观察

目的探求导管修复颅内段动眼神经的可行性.方法 20只健康家猫随机分为2组.将右侧动眼神经于脑池段切断后,分组采用自体神经桥接和导管套接的方法修复.术后14周末光镜、电镜观察神经纤维的连续性、再生纤维数目和直径.结果神经修复14周后,神经桥接组67%、导管套接组75%的动物其动眼神经功能均有一定程度的恢复.形态学显示两组均取得较好的神经再生效果,两组间再生纤维直径差异无显著性(P>0.05),但导管套接组再生神经纤维数目较多,差异有显著性(P<0.05).结论导管套接法可作为颅内段动眼神经损伤后的一种修复方法,其疗效近似或稍优于神经桥接法.     

An electrophysiological and histological study of myelinated axon regeneration after peripheral nerve injury and repair in the cat

Electrophysiological and histological methods have been combined to obtain quantitative measures of the success of regeneration of myelinated axons in a cutaneous nerve after injury and repair by a variety of procedures. Following a simple transection injury more axons regenerated successfully when the nerve was repaired by epineurial suturing or stump suturing than when it was left unrepaired; both types of repair gave similar results. After loss of a 10-mm piece of the nerve trunk, repair with an autograft produced more regeneration than when the nerve was left untouched, but repair by stump mobilization with epineurial suturing made matters worse. On the whole, the regenerated afferents had receptive field properties similar to those found in control animals but there was a higher incidence of units that could not be typed using conventional criteria. A small proportion of them had split receptive fields. Fibre diameters and conduction velocities were reduced compared with controls; this was particularly so through the neuroma and in the distal stump. There was also evidence of abnormal interactions, possibly ephaptic, between some regenerated axons.     

Schwann cell delivery of neurotrophic factors for peripheral nerve regeneration

Current treatments of injured peripheral nerves often fail to mediate satisfactory functional recovery. For axonal regeneration, neurotrophic factors (NTFs) play a crucial role. Multiple NTFs and other growth‐promoting factors are secreted, amongst others, by Schwann cells (SCs), which also provide cellular guidance for regenerating axons. Therefore, delivery of NTFs and transplantation of autologous or genetically modified SCs with therapeutic protein expression have been proposed. This article reviews polymer‐based and cellular approaches for NTF delivery, with a focus on SCs and strategies to modulate SC gene expression. Polymer‐based NTF delivery has mostly resided on nerve conduits (NC). While NC have generally provided prolonged NTF release, their therapeutic effect has remained significantly below that achieved with autologous nerve grafts. Several studies demonstrated enhanced nerve regeneration using NC seeded with SCs. The SCs have sometimes been modified genetically using non‐viral or viral vectors. Whereas non‐viral vectors produced poor transgene delivery, adenoviral vectors mediated high transgene transduction efficiency of SCs. Further improvements of safety and transgene expression of adenoviral vector may lead to rapid translation of pre‐clinical research to clinical trials.     

Effect of allogeneic Schwann cell transplantation on peripheral nerve regeneration

Transplantation of allogeneic Schwann cells (SC) would make it feasible to reconstruct immediately peripheral nerve defects, compared to using autologous SC; however, this treatment modality has not been adequately evaluated. The aim of this study was to characterize and compare the effects of allogeneic versus syngeneic SC transplantation following peripheral nerve injury. Polyhydroxybutyrate conduits were used to bridge a 10-mm gap in the rat sciatic nerve. The conduits were filled with alginate hydrogel with or without cultured allogeneic or syngeneic genetically labeled SC, without the use of immunosuppressive therapy, and examined after 2, 3, and 6 weeks with 5-bromo-4-chloro-3-indoyl-beta-D-galactosidase chemical staining and immunohistochemistry to quantify SC migration into the conduit, axonal regeneration, the state of SC differentiation, and the expression of major histocompatibility complexes (MHC) I and II, as well as to quantify macrophage and B- and T-lymphocyte infiltration. Allogeneic SC were rejected by 6 weeks, whereas syngeneic SC could still be identified. Allogeneic and syngeneic SC equally enhanced the axonal regeneration distance but the quantity of axons was greater using syngeneic SC. The ingrowth of SC into the conduits containing allogeneic SC was similar to that observed in the presence of syngeneic SC, indicating the absence of deleterious immune response. SC continued to express phenotypic markers of nonmyelination and these were highest in conduits with allogeneic SC. Expression of MHC I and II was higher in the conduits with allogeneic SC at 3 weeks and without significant difference in the number of macrophages and lymphocytes, except at 6 weeks, when there was a larger number of lymphocytes using syngeneic SC. In conclusion, allogeneic SC enhanced axonal regeneration distance and did not induce a deleterious immune response. In a clinical setting the immediate availability of allogeneic SC for transplantation may compensate for the better outcome achieved by the use of autologous SC that require a longer preparation time in culture.     

A role of migratory Schwann cells in a conditioning effect of peripheral nerve regeneration

The common peroneal nerve in mice was conditioned by axotomy around the head of the fibula. At various intervals from 1 day to 2, 3, 5, 15, and 25 days, a test lesion was made by axotomy 15 mm proximal to the conditioning lesion site. The proximal stump of the transected nerve was sandwiched between two sheets of thin plastic film and remained in vivo for various intervals from 3 h to 6, 9, 12, 24, 48, 72 and 96 h. The regenerating axons were visualized on the film with silver nitrate impregnation. Schwann cells were visualized migrating onto the film using immunohistochemistry with anti-S-100. To determine the effects of migratory Schwann cells on axonal outgrowth, a film model was established on one limb. After the nerve stump was removed from the film, the treated film was transferred to a new lesion on the contralateral limb and 2 days later the film was harvested for histological examination. Conditioned by a prior axotomy more than 3 days earlier, regenerating axons sprouted within less than 1 h after the test lesion was established and grew naked at five times higher rate: The growth rate was similar to that observed during regeneration in the presence of migratory Schwann cells (ordinary type). After a short interval, the axons, which had been ensheathed by migratory Schwann cells (reactive type), continued growing at a significantly (P < 0.01) higher rate. The reactive type of cells had fewer numbers of branches and higher activity in promoting axonal outgrowth than the ordinary type. Thus, both ordinary and reactive types of cells played key roles in initiating and maintaining a conditioning effect, respectively.     

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.     

Differentially promoted peripheral nerve regeneration by grafted Schwann cells over-expressing different FGF-2 isoforms

Artificial nerve grafts are needed to reconstruct massive defects in the peripheral nervous system when autologous nerve grafts are not available in sufficient amounts. Nerve grafts containing Schwann cells display a suitable substrate for long-distance regeneration. We present here a comprehensive analysis of the in vivo effects of different isoforms of fibroblast growth factor-2 (FGF-2) on peripheral nerve regeneration across long gaps. FGF-2 isoforms were provided by grafted, genetically modified Schwann cells over-expressing 18-kDa-FGF-2 and 21-/23-kDa-FGF-2, respectively. Functional tests evaluated motor and sensory recovery. Additionally, morphometrical analyses of regenerated nerves were performed 3 and 6 months after grafting. Distinct regeneration promoting effects of the different FGF-2 isoforms were found. 18-kDa-FGF-2 mediated inhibitory effects on the grade of myelination of regenerating axons, whereas 21-/23-kDa-FGF-2 mediated early recovery of sensory functions and stimulation of long-distance myelination of regenerating axons. The results contribute to the development of new therapeutic strategies in peripheral nerve repair.     

NOS-mediated differences in peripheral nerve graft revascularization and regeneration

Dependence of peripheral nerve revascularization on endogenous nitric oxide supply and consequences for nerve regeneration were investigated using a sciatic nerve graft model in mice lacking one of the nitric oxide synthase (NOS) isoforms. Wild type mice and mice lacking neuronal or inducible NOS exhibited similar revascularization patterns. Perfusion was consistently established 3 days after nerve reconstruction. Mice lacking endothelial NOS showed a delay in revascularization of about 2 days. The regeneration outcome did not reflect these differences. In mice lacking endothelial NOS, axon counts, myelination and recovery of sensory and motor function were comparable to wild type mice, whereas in mice lacking neuronal or inducible NOS a disturbed regeneration was found. Present results demonstrate, that the disturbance of nerve revascularization as result of reduced endothelial NOS-mediated NO supply can be tolerated, possibly by enhanced phagocytic capacity of Schwann cells and/or resident endoneurial macrophages.     

Biochemical,morphological, and functional changes during peripheral nerve regeneration

The success of axon regeneration after nerve injury should be judged by the extent to which the target organs regain their function. Recovery of muscle contraction involves axon regeneration, reestablishment of nerve-muscle connections, recovery of transmission, and muscle force. All these processes were investigated under the same experimental conditions and correlated in order to better understand their time-course and interdependence. The sciatic nerve of a rat was crushed in the thigh. The ingrowth of regenerating motor axons into the soleus (SOL) and extensor digitorum longus (EDL) muscles was monitored by measuring the activity of choline acetyltransferase (ChAT), a marker enzyme for cholinergic nerve terminals, in the muscles. The electron microscopic cytochemistry of acetylcholine esterase (AChE) was used to estimate the reestablishment of neuromuscular junctions in these two muscles. The recovery of muscle contraction was followed by measuring the force of isometric contraction in the triceps surae muscle in vivo. The pattern of ChAT recovery during reinnervation was similar in the EDL and SOL. The statistically significant increase of ChAT activity in these muscles, 14 d after the nerve crush, signified the entry of regenerating axons into the calf muscles. Electron microscopic cytochemistry revealed the first small nerve endings in contact with the denervated end plates 12 d after denervation. Subsequently, the number of reinnervated motor end plates and the surface area of the neuromuscular junctions steadily increased. The recovery of muscle force started between d 14 and 21 after the nerve crush. Thirty-five days after denervation, the difference between the muscle force of the reinnervated muscle and the control became statistically insignificant. Morphological normalization of the motor end plates was practically complete 33 d after denervation, concomitant with the normalization of the muscle force. At that time, however, ChAT activity in both muscles was still clearly subnormal (33.5% in EDL and 45% of the control in SOL) and therefore does not reflect the true extent of muscle force recovery. Yet, it seems that in spite of this, the regenerated nerve terminals contained sufficient amounts of acetylcholine (ACh) to trigger normal muscle contractions.     

Schwann cells direct peripheral nerve regeneration through the Netrin-1 receptors, DCC and Unc5H2

In the peripheral nervous system, Schwann cells (SCs) promote nerve regeneration by the secretion of trophic support molecules and the establishment of a supportive growth matrix. Elucidating factors that promote SC outgrowth following nerve injury is an important strategy for improving nerve regeneration. We identified the Netrin-1 receptors, Deleted in Colorectal Cancer (DCC) and Uncoordinated (Unc)5H2 as SC receptors that influence nerve regeneration by respectively promoting or inhibiting SC outgrowth. Significantly, we show both DCC and Unc5H2 receptors are distributed within SCs. In adult nerves, DCC is localized to the paranodes and Schmidt-Lantermann incisures of myelinating SCs, as well as along unmyelinated axons. After axotomy, DCC is prominently expressed in activated SCs at the regenerating nerve front. In contrast, Unc5H2 receptor is robustly distributed in myelinating SCs of the intact nerve and it is found at low levels in the SCs of the injury site. Local in vivo DCC siRNA mRNA knockdown at the growing tip of an injured nerve impaired SC activation and, in turn, significantly decreased axon regeneration. This forced DCC inhibition was associated with a dramatic reciprocal upregulation of Unc5H2 in the remaining SCs. Local Unc5H2 knockdown at the injury site, however, facilitated axon regrowth, indicating it has a role as an intrinsic brake to peripheral nerve regeneration. Our findings demonstrate that in adult peripheral nerves, SCs respond to DCC and Unc5H2 signaling, thereby promoting or hindering axon outgrowth and providing a novel mechanism for SC regulation during nerve regeneration.     

Long-term survival and axonal regeneration of retinal ganglion cells after optic nerve transection and a peripheral nerve graft

To investigate the effect of autologous peripheral nerve grafting on retinal ganglion cell survival and axonal regeneration after an injury, the optic nerve of adult Sprague-Dawley rats was transected and grafted with an autologous peripheral nerve from the peroneal branch of the left sciatic nerve. The numbers of both surviving and axon-regenerating retinal ganglion cells were determined at different times after surgery. The majority of retinal ganglion cells were rapidly lost within 3 weeks, followed by a slow and protracted phase of cell loss until the end of the 6-month study. FluoroGold-labelled axon-regenerating retinal ganglion cells were first detected by 2 weeks, followed by a period of high axonal regeneration that peaked at 8 weeks and accounted for over 35% of the total surviving retinal ganglion cells. However, retinal ganglion cells with regenerated axons eventually died. Our data thus indicate that axonal regeneration in the autologous peripheral nerve graft is insufficient to sustain the long-term survival of axotomized retinal ganglion cells.     

A newly synthesized neurotropic pyrimidine compound, MS-818, may activate migratory Schwann cells in peripheral nerve regeneration

Following transection of a peripheral nerve in mice, a newly synthesized neurotropic pyrimidine compound, MS-818 was administered intraperitoneally at a dose of 1 mg kg⁻¹ b.wt. day⁻¹. The film model experiments for analyzing the early growth of axonal regeneration suggested that MS-818 activated Schwann cells which migrate from the proximal stump, inducing axonal elongation in vivo.