Myelinating cocultures of rodent stem cell line‐derived neurons and immortalized Schwann cells

Myelination is one of the most remarkable biological events in the neuron–glia interactions for the development of the mammalian nervous system. To elucidate molecular mechanisms of cell‐to‐cell interactions in myelin synthesis in vitro, establishment of the myelinating system in cocultures of continuous neuronal and glial cell lines are desirable. In the present study, we performed co‐culture experiments using rat neural stem cell‐derived neurons or mouse embryonic stem (ES) cell‐derived motoneurons with immortalized rat IFRS1 Schwann cells to establish myelinating cultures between these cell lines. Differentiated neurons derived from an adult rat neural stem cell line 1464R or motoneurons derived from a mouse ES cell line NCH4.3, were mixed with IFRS1 Schwann cells, plated, and maintained in serum‐free F12 medium with B27 supplement, ascorbic acid, and glial cell line‐derived neurotrophic factor. Myelin formation was demonstrated by electron microscopy at 4 weeks in cocultures of 1464R‐derived neurons or NCH4.3‐derived motoneurons with IFRS1 Schwann cells. These in vitro coculture systems utilizing the rodent stable stem and Schwann cell lines can be useful in studies of peripheral nerve development and regeneration.     

Morphological characteristics of p75 neurotrophin receptor-positive cells define a new type of glial cell in the rat dorsal root ganglia

In the dorsal root ganglia (DRG), two types of glial cells (Schwann cells and satellite glial cells) have been identified based on cell morphology and expression of specific markers. In the present study, we observed unknown glial cells that were positive for p75 neurotrophin receptor (p75NTR), and therefore were immunohistochemically and ultrastructurally characterized for the first time. These cells exhibited stronger immunoreactivity against an anti-p75NTR antibody than the DRG neurons (hereafter referred to as p75NTR++ cells). Moreover, these cells covered the glial cells surrounding proximal process of the large-diameter DRG neurons. The proximal process is called “dendro-axon.” The p75NTR++ cells were predominantly distributed where the first myelinating Schwann cells appear. The p75NTR++ cells were also positive for the pan-glial cell markers S100, nestin, and Sox10, but negative for fibroblast and macrophage markers. Moreover, they were negative for a satellite glial cell marker, inwardly rectifying potassium channel Kir4.1, as well as a nonmyelinating Schwann cell marker, glial fibrillary acidic protein. In addition, their morphological features were distinct from those of the myelinating Schwann cells. To investigate the three-dimensional ultrastructure of the p75NTR++ cells, we used array tomography combined with correlative light and electron microscopic observation. Three-dimensional ultrastructural observation revealed that the p75NTR++ cells loosely covered glial cells around the dendro-axons with highly ramified processes. Glial cells with these morphological features have not been reported before, indicating that the p75NTR++ glial cells are a new glial cell type in the DRG. Our results will give new insights into cell–cell relationships.     

Effects of aging and axotomy on the expression of neurotrophin receptors in primary sensory neurons.

Aging is accompanied by declined sensory perception, paralleled by widespread dystrophic and degenerative changes in both central and peripheral sensory pathways. Several lines of evidence indicate that neurotrophic interactions are of importance for a maintained plasticity in the adult and aging nervous system, and that changes in the expression of neurotrophins and/or their receptors may underpin senile neurodegeneration. We have here examined the expression of neurotrophin receptor (p75NTR, trkA, trkB, and trkC) mRNA and protein in intact and axotomized primary sensory neurons of young adult (3 months) and aged (30 months) rats. To examine possible differences among primary sensory neuron populations, we have studied trigeminal ganglia (TG) as well as cervical and lumbar dorsal root ganglia (DRG). In intact aged rats, a decrease in trk (A/B/C) mRNA labeling densities and protein-like immunoreactivities was observed. The decrease was most pronounced in lumbar DRG. In contrast, a small, not statistically significant, increase of p75NTR expression was observed in aged DRG neuron profiles. After axotomy, a down-regulation of mRNA and protein levels was observed for all neurotrophin receptors (p75NTR, trkA, trkB and trkC) in both young adult and aged rats. Consistent with the higher expression levels of neurotrophin receptors in unlesioned young adult primary sensory neurons, the relative effect of axotomy was more pronounced in the young adult than aged rats. Although a decrease in mean cell profile cross-sectional areas was found during aging and after axotomy, the characteristic distribution of neurotrophin receptor expression in different populations of NRG neurons was conserved. The present findings suggest an attenuation of neurotrophic signaling in primary sensory neurons with advancing age and that the expression of p75NTR and trks is regulated differently during aging. A similar dissociation of p75NTR and trk regulation has previously been reported in other neuronal systems during aging, suggesting that there may be a common underlying mechanism. Decreased access to ligands, disturbed axon function and systemic changes in androgen/estrogen levels are discussed as inducing and/or contributing factors.     

Metabolic Control of Sensory Neuron Survival by the p75 Neurotrophin Receptor in Schwann Cells

We report that the neurotrophin receptor p75 contributes to sensory neuron survival through the regulation of cholesterol metabolism in Schwann cells. Selective deletion of p75 in mouse Schwann cells of either sex resulted in a 30% loss of dorsal root ganglia (DRG) neurons and diminished thermal sensitivity. P75 regulates Schwann cell cholesterol biosynthesis in response to BDNF, forming a co-receptor complex with ErbB2 and activating ErbB2-mediated stimulation of sterol regulatory element binding protein 2 (SREBP2), a master regulator of cholesterol synthesis. Schwann cells lacking p75 exhibited decreased activation of SREBP2 and a reduction in 7-dehydrocholesterol (7-DHC) reductase (DHCR7) expression, resulting in accumulation of the neurotoxic intermediate, 7-dehyrocholesterol in the sciatic nerve. Restoration of DHCR7 in p75 null Schwann cells in mice significantly attenuated DRG neuron loss. Together, these results reveal a mechanism by which the disruption of lipid metabolism in glial cells negatively influences sensory neuron survival, which has implications for a wide range of peripheral neuropathies.SIGNIFICANCE STATEMENT Although expressed in Schwann cells, the role of p75 in myelination has remained unresolved in part because of its dual expression in sensory neurons that Schwann cells myelinate. When p75 was deleted selectively among Schwann cells, myelination was minimally affected, while sensory neuron survival was reduced by 30%. The phenotype is mainly due to dysregulation of cholesterol biosynthesis in p75-deficient Schwann cells, leading to an accumulation of neurotoxic cholesterol precursor, 7-dehydrocholesterol (7-DHC). Mechanism-wise, we discovered that in response to BDNF, p75 recruits and activates ErbB2 independently of ErbB3, thereby stimulating the master regulator, sterol regulatory element binding protein 2 (SREBP2). These results together highlight a novel role of p75 in Schwann cells in regulating DRG neuron survival by orchestrating proper cholesterol metabolism.     

Involvement of oxidative stress and impaired lysosomal degradation in amiodarone‐induced schwannopathy

Amiodarone hydrochloride (AMD), an anti‐arrhythmic agent, has been shown to cause peripheral neuropathy; however, its pathogenesis remains unknown. We examined the toxic effects of AMD on an immortalized adult rat Schwann cell line, IFRS1, and cocultures of IFRS1 cells and adult rat dorsal root ganglion neurons or nerve growth factor‐primed PC12 cells. Treatment with AMD (1, 5, and 10 μm ) induced time‐ and dose‐dependent cell death, accumulation of phospholipids and neutral lipids, upregulation of the expression of gangliosides, and oxidative stress (increased nuclear factor E2‐related factor in nuclear extracts and reduced GSH/GSSG ratios) in IFRS1 cells. It also induced the upregulation of LC3‐II and p62 expression, with phosphorylation of p62, suggesting that deficient autolysosomal degradation is involved in AMD‐induced IFRS1 cell death. Furthermore, treatment of the cocultures with AMD induced detachment of IFRS1 cells from neurite networks in a time‐ and dose‐dependent manner. These findings suggest that AMD‐induced lysosomal storage accompanied by enhanced oxidative stress and impaired lysosomal degradation in Schwann cells might be a cause of demyelination in the peripheral nervous system.     

Juxtacrine signalling via Notch and ErbB receptors in the switch to fate commitment of bone marrow‐derived Schwann cells

The phenotypic instability of adult tissue‐derived Schwann cell‐like cells (SCLCs) as revealed upon withdrawal of glia‐inducing culture supplements limits their clinical utility for cell therapy and disease modelling. We previously overcame this limitation by co‐culturing bone marrow‐derived SCLCs with neurons purified from developing rat and subsequently human sensory neurons such that direct contact between cell types accomplished the cell‐intrinsic switch to the Schwann cell fate. Here, our search for juxtacrine instructive signals found both Notch ligands and neuregulin‐1 type III localized on the surface of DRG neurons via live cell immunocytochemistry. Bypassing ligand‐induced release of the Notch intracellular domain (NICD) by transient transfection of SCLCs with the pAdlox/V5‐His‐NICD construct was shown to upregulate ErbB2/3. Interaction of ErbB2/3 with neuregulin‐1 type III (NRG1 type III) as presented on neurons then mediated the switch to the Schwann cell fate as demonstrated by expression of S100β/p75/ Sox10/Krox20. In contrast, treatment of cocultures with γ‐secretase inhibitor perturbed Notch signalling in SCLCs and consequently deterred both upregulation of ErbB2/3 and the transition to the Schwann cell fate. Taken together, juxtacrine signalling via Notch is key to the upregulation of ErbB receptors for neuregulin‐driven commitment of SCLCs to the Schwann cell fate.     

Differential expression of HNK-1 and p75(NTR) in adult canine Schwann cells and olfactory ensheathing cells in situ but not in vitro

Olfactory ensheathing cells (OECs) are promising candidates for autologous cell transplantation therapies of nervous system injury and disease. Large animal models are relevant for transferring experimental data into clinical practice. In vivo studies have suggested that adult canine OECs may display similar regenerating capacities as their rodent counterpart. However, data on their molecular phenotype required for generating pure cell preparations are still scarce. In the present study, we comparatively analyzed expression of the carbohydrate HNK-1 epitope and the neurotrophin receptor p75(NTR) in adult canine Schwann cells and olfactory ensheathing cells in situ and in vitro. Myelinating and nonmyelinating Schwann cells in situ exclusively expressed HNK-1 and p75(NTR), respectively, whereas OECs were negative for both markers. In vitro, OECs and Schwann cells shared cell surface expression of p75(NTR) but not of HNK-1, which could be detected transiently in intracellular vesicles. This suggests that Schwann cells and OECs in vitro phagozytose HNK-1+ cellular debris. The cultivation-induced downregulation of HNK-1 expression in Schwann cells and upregulation of p75(NTR) in OECs argues for the possibility that axonal signals control the expression of both markers in situ. Whereas HNK-1 expression in Schwann cells is most likely controlled by signals inducing myelination, e.g., neuregulin, the mechanisms that may suppress p75(NTR) expression in OECs in situ remain to be elucidated. Interestingly, HNK-1 expression in the adult dog was found in both sensory and motor nerve myelinating Schwann cells. This is reminiscent of humans and differs from rodents; it also underscores the importance of large animal models for translational research.     

Roles of glial p75NTR in axonal regeneration

The neurotrophin receptor p75 (p75NTR) is expressed by both neurons and glia. Nerve injury triggers up-regulation of p75NTR in Schwann cells (SC) but not in central glia. In contrast to neuronal p75NTR, which mediates negative signals from myelin-associated proteins resulting in neurite collapse, glial p75NTR may play a positive role in nerve regeneration by forming neurotrophin chemoattractant gradients or by competitively antagonizing the NOGO/NgR/LINGO-1 signal through cell-cell contact or regulated intramembranous proteolysis (RIP) of p75NTR. This piece presents some recent evidence supporting this hypothesis.     

GDNF and NGF family members and receptors in human fetal and adult spinal cord and dorsal root ganglia.

We describe the expression of mRNA encoding ligands and receptors of members of the GDNF family and members of the neurotrophin family in the adult human spinal cord and dorsal root ganglia (DRG). Fetal human spinal cord and ganglia were investigated for the presence of ligands and receptors of the neurotrophin family. Tissues were collected from human organ donors and after routine elective abortions. Messenger RNA was found encoding RET, GFR alpha-1, BDNF, trkB, and trkC in the adult human spinal cord and BDNF, NT-3, p75, trkB, and trkC in the fetal human spinal cord. The percentage of adult human DRG cells expressing p75, trkA, trkB, or trkC was 57, 46, 29, and 24%, respectively, and that of DRG cells expressing RET, GFR alpha-1, GFR alpha-2, or GFR alpha-3 was 79, 20, 51, and 32%, respectively. GFR alpha-2 was expressed selectively in small, GFR alpha-3 principally in small and GFR alpha-1 and RET in both large and small adult human DRG neurons. p75 and trkB were expressed by a wide range of DRG neurons while trkA was expressed in most small diameter and trkC primarily in large DRG neurons. Fetal DRG cells were positive for the same probes as adult DRG cells except for NT-3, which was only found in fetal DRG cells. Messenger RNA species only expressed at detectable levels in fetal but not adult spinal cord tissues included GDNF, GFR alpha-2, NT-3, and p75. Notably, GFR alpha-2, which is expressed in the adult rat spinal cord, was not found in the adult human spinal cord.     

Schwann cell apoptosis in the postnatal axotomized sciatic nerve is mediated via NGF through the low-affinity neurotrophin receptor

Schwann cell death is a developmentally regulated phenomenon and is also induced after peripheral nerve axotomy in neonatal rodents. In this study, we explored whether ligand-induced activation of the low-affinity neurotrophin receptor (p75(NTR)) is responsible for inducing Schwann cell death in vivo. Administration of exogenous nerve growth factor (NGF) to the axotomized nerve site in wild-type animals resulted in a 2.6-fold increase in Schwann cell apoptosis in the distal nerve stumps compared to axotomy alone. No increase in apoptosis, above baseline levels, was seen in p75(NTR)-mutant mice either with or without NGF When anti-NGF antibodies were administered to the site of the peripheral nerve lesion in wild-type mice there was a reduction in the percentage of Schwann cell apoptosis to levels seen in both the quiescent state and in the axotomized nerves of the p75(NTR)-mutant mice. These results demonstrate that apoptosis of Schwann cells in axotomized peripheral nerve is mediated predominantly through p75(NTR) signaling and initiated via endogenously produced NGF.     

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.     

Distribution of central sensory axons in transgenic mice overexpressing nerve growth factor and lacking functional p75 neurotrophin receptor expression

This study examined the roles of nerve growth factor (NGF) and the p75 neurotrophin receptor (p75NTR) in the growth of dorsal root ganglion (DRG) central processes in the dorsal horn. Two genetically modified mouse strains were used: transgenic mice that overexpress NGF in the CNS under the control of the glial fibrillary acidic protein promoter, and p75NTR exon III null mutant mice that express a hypomorphic form of this receptor. In both NGF transgenic and nontransgenic mice with hypomorphic expression of p75NTR, there is a significant loss of DRG neurons compared to mice with normal p75NTR expression. This reduction in neuron number has been shown to underlie a corresponding decrease in peripheral nociceptive sensory innervation. Within the CNS, however, nociceptive innervation of the dorsal horn appears to be unaffected by hypomorphic expression of p75NTR. Comparisons of calcitonin gene-related peptide immunoreactivity in the dorsal horn revealed that the area occupied by DRG central processes was not significantly different between p75NTR hypomorphic mice and wild-type siblings, or between NGF transgenic mice with either hypomorphic or normal expression of p75NTR. We propose that DRG central processes arborize extensively in both NGF-transgenic and nontransgenic p75NTR hypomorphic mice in order to compensate for the loss of DRG neurons and restore dorsal horn innervation to normal levels. We also present evidence suggesting that NGF plays only a minor role in the growth of DRG central processes.     

Attenuation of a caspase-3 dependent cell death in NT4- and p75-deficient embryonic sensory neurons

Neuronal survival during the developmental period of naturally occurring cell death is mediated through a successful competition for limiting concentrations of neurotrophic factors, and the deprived neurons will die. New results show that induced death through the p75 neurotrophin receptor (p75(NTR)), a member of the p55TNF/Fas family of cell death receptors, may also influence survival during development. We find that eliminating p75(NTR) or neurotrophin 4 (NT4) in mice leads to a marked attenuation of apoptosis during the programmed cell death period of the trigeminal ganglion neurons, suggesting that NT4 can induce the death of these neurons through the p75(NTR). These in vivo findings were reproduced in primary cell cultures, where NT4 was found to induce death in a p75(NTR)-dependent pathway. Analysis of p75 deficient and wild-type cells revealed two separate cell death pathways, a p75(NTR)- and caspase-3-independent pathway activated by trophic factor deprivation, and a p75(NTR)- and caspase-3-dependent pathway initiated by NT4. Crossing in the NT4 null alleles in brain-derived neurotrophic factor (BDNF) null mutant mice led to a rescue of a large proportion of BDNF-dependent neurons from excessive cell death, indicating that trophic factor deprivation is not sufficient for the death of many neurons and that additional death inducing signals might be required. Our results suggest that NT4 competitively signals survival and death of sensory neurons through trkB and p75(NTR), respectively.     

NGF ligand alters NGF signaling via p75(NTR) and trkA

Nerve growth factor (NGF) binds to two neurotrophin receptors: p75(NTR) and p140(Trk) (TrkA). Both receptors dimerize in response to NGF binding. TrkA homodimers and heteromers of TrkA and p75(NTR) promote cell survival whereas homodimers of p75(NTR) mediate apoptosis upon binding of NGF. The interaction between receptor and NGF can be inhibited either on the level of the ligand by altering NGF conformation so that NGF is no longer recognized by the receptor or on the level of the receptor by blocking the binding site of p75(NTR) or TrkA. The effect of altering NGF conformation on NGF signaling was investigated in two neuron-like cell lines: in human SK-N-MC cells that express only p75(NTR) and in rat PC12 cells that express both p75(NTR) and TrkA. In the present study we demonstrate that Ro 08-2750 binds to the NGF dimer thereby probably inducing a change in its conformation such that NGF cannot bind to p75(NTR) anymore. In SK-N-MC cells this leads to inhibition of NGF-induced programmed cell death. In PC12 cells enhanced signaling through TrkA was observed.     

Synthesis, localization and externalization of galectin-1 in mature dorsal root ganglion neurons and Schwann cells

We recently confirmed that oxidized galectin-1 is a novel factor enhancing axonal growth in peripheral nerves after axotomy, but the process of extracellular release and oxidization of endogenous galectin-1 in the injured nervous tissue remains unknown. In the present study, we examined the distribution of galectin-1 in adult rat dorsal root ganglia (DRG) in vivo and in vitro. By RT-PCR analysis and in situ hybridization histochemistry, galectin-1 mRNA was detected in both DRG neurons and non-neuronal cells. Immunohistochemical analyses revealed that galectin-1 was distributed diffusely throughout the cytoplasm in smaller diameter neurons and Schwann cells in DRG sections. In contrast, the immunoreactivity for galectin-1 was detected in almost all DRG neurons from an early stage in culture (3 h after seeding) and was restricted to the surface and/or extracellular region of neurons and Schwann cells at later stages in culture. In a manner similar to the primary cultured cells, we also observed the surface and extracellular expression of this molecule in immortalized adult mouse Schwann cells (IMS32). Western blot analysis has revealed that both reduced and oxidized forms of galectin-1 were detected in culture media of DRG neurons and IMS32. These findings suggest that galectin-1 is externalized from DRG neurons and Schwann cells upon axonal injury. Some of the molecules in the extracellular milieu may be converted to the oxidized form, which lacks lectin activity but could act on neural tissue as a cytokine.     

Distinct effects of p75 in mediating actions of neurotrophins on basal forebrain oligodendrocytes

Previous studies indicate that brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) increase myelin basic protein, (MBP) in differentiating basal forebrain (BF) oligodendrocytes (OLGs) (Du, Y., Fischer, T.Z., Lee, L.N., Lercher, L.D., Dreyfus, C. F., 2003. Regionally specific effects of BDNF on oligodendrocytes. Dev. Neurosci. 25, 116-126). While receptors, trk and p75, are expressed by subsets of oligodendrocytes (Du, Y., Fischer, T.Z., Lee, L.N., Lercher, L.D., Dreyfus, C. F., 2003. Regionally specific effects of BDNF on oligodendrocytes. Dev. Neurosci. 25, 116-126), those responsible for affecting differentiation have not been defined. In contrast, studies of peripheral Schwann cells reported that myelination is enhanced by BDNF working through p75, and diminished by trkC mediated processes (Cosgaya, J.M., Chan, J.R., Shooter, E.M., 2002. The neurotrophin receptor p75NTR as a positive modulator of myelination. Science 298, 1245-1248). To define receptors affecting central oligodendrocyte MBP, p75 knockout animals, p75 blocking antibodies, and an inhibitor of neurotrophin binding to p75, PD90780, were utilized. While p75 was implicated in the actions of NGF and NT-3, it did not affect actions of BDNF. On the other hand, K252a, an inhibitor of trk receptors, abolished the effects of the neurotrophins, including BDNF. All neurotrophins activated their respective trk receptors.     

Differential effects of pro-BDNF on sensory neurons after sciatic nerve transection in neonatal rats

Brain-derived neurotrophic factor (BDNF) plays a critical role in the development of the central and peripheral nervous systems, and also in neuronal survival after injury. The actions of BDNF are mediated by its high-affinity receptors TrkB and p75NTR. Recent studies have shown that proneurotrophins bind p75NTR and sortilin with high affinity, and trigger apoptosis of neurons in vitro . As proneurotrophins are a dominant form of gene products in developing and adult animals, it is imperative to understand their physiological functions in animals. Here, we showed differential roles of proBDNF in injured and uninjured sensory neurons. proBDNF, p75NTR and sortilin are highly expressed in dorsal root ganglia (DRG) neurons. Recombinant proBDNF induced a dose-dependent death of PC12 cells and the death activity was completely abolished in the presence of antibodies against the prodomain of BDNF. The exogenous proBDNF enhanced the death of axotomized sensory neurons and the neutralizing antibodies to the prodomain or exogenous sortilin-extracellular domain-Fc fusion molecule reduced the death of axotomized sensory neurons. Interestingly, the treatment of neutralizing antibody in vivo increased the number of sensory neurons in the contralateral DRG. We conclude that proBDNF may induce the death of axotomized sensory neurons and suppress neuronal addition in the intact DRG in neonatal rats, and the suppression of endogenous proBDNF may protect neurons after neurotrauma.     

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.