Down‐regulation of long non‐coding RNA MEG3 promotes Schwann cell proliferation and migration and repairs sciatic nerve injury in rats

Peripheral nerve injury and regeneration are complex processes and involve multiple molecular and signalling components. However, the involvement of long non‐coding RNA (lncRNA) in this process is not fully clarified. In this study, we evaluated the expression of the lncRNA maternally expressed gene 3 (MEG3) in rats after sciatic nerve transection and explored its potential mechanisms. The expression of lncRNA MEG3 was up‐regulated following sciatic nerve injury and observed in Schwann cells (SCs). The down‐regulation of lncRNA MEG3 in SCs enhanced the proliferation and migration of SCs via the PTEN/PI3K/AKT pathway. The silencing of lncRNA MEG3 promoted the migration of SCs and axon outgrowth in rats after sciatic nerve transection and facilitated rat nerve regeneration and functional recovery. Our findings indicated that lncRNA MEG3 may be involved in nerve injury and injured nerve regeneration in rats with sciatic nerve defects by regulating the proliferation and migration of SCs. This gene may provide a potential therapeutic target for improving peripheral nerve injury.     

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.     

ErbB transmembrane tyrosine kinase receptors are expressed by sensory and motor neurons projecting into sciatic nerve.

Adult spinal cord motor and dorsal root ganglion (DRG) sensory neurons express multiple neuregulin-1 (NRG-1) isoforms that act as axon-associated factors promoting neuromuscular junction formation and Schwann cell proliferation and differentiation. NRG-1 isoforms are also expressed by muscle and Schwann cells, suggesting that motor and sensory neurons are themselves acted on by NRG-1 isoforms produced by their peripheral targets. To test this hypothesis, we examined the expression of the NRG-1 receptor subunits erbB2, erbB3, and erbB4 in rat lumbar DRG and spinal cord. All three erbB receptors are expressed in these tissues. Sciatic nerve transection, an injury that induces Schwann cell expression of NRG-1, alters erbB expression in DRG and cord. Virtually all DRG neurons are erbB2- and erbB3-immunoreactive, with erbB4 also detectable in many neurons. In spinal cord white matter, erbB2 and erbB4 antibodies produce dense punctate staining, whereas the erbB3 antibody primarily labels glial cell bodies. Spinal cord dorsal and ventral horn neurons, including alpha-motor neurons, exhibit erbB2, erbB3, and erbB4 immunoreactivity. Spinal cord ventral horn also contains a population of small erbB3+/S100beta+/GFAP- cells (GFAP-negative astrocytes or oligodendrocytes). We conclude that sensory and motor neurons projecting into sciatic nerve express multiple erbB receptors and are potentially NRG-1 responsive.     

FGF-2 Up-regulation and proliferation of neural progenitors in the regenerating amphibian spinal cord in vivo

Regeneration of the spinal cord occurs spontaneously in adult urodele amphibians. The key cells in this regenerative process appear to be the ependymal cells that following injury migrate and proliferate to form the ependymal tube from which the spinal cord regenerates. Very little is known about the signal(s) that initiates and maintains the proliferative response of these cells. Fibroblast growth factor 2 (FGF-2) has been shown to play a role in maintaining neural progenitor cell cycling in vitro and may be important for neuronal survival and axonal growth after injury. We have investigated its role in regeneration of the spinal cord in vivo following tail amputation in the adult salamander, Pleurodeles waltl. We show that only the low-molecular-weight form of FGF-2 is found in Pleurodeles and that in the normal cord it is expressed in a subset of neurons, but is hardly detectable in ependymal cells. Tail amputation results in induction of FGF-2 in the ependymal cells of the regenerating structure, and later in regeneration FGF-2 is up-regulated in some newborn neurons. FGF-2 pattern of expression in the ependymal tube parallels that of proliferation. Furthermore, exogenous FGF-2 significantly increases ependymal cell proliferation in vivo. Overall our results strongly support the view that one important role of FGF-2 during spinal cord regeneration in Pleurodeles is to induce proliferation of neural progenitor cells.     

Exosomes from human adipose-derived stem cells promote sciatic nerve regeneration via optimizing Schwann cell function

Human adipose-derived stem cells (ASCs) have a potential for the treatment of peripheral nerve injury. Recent studies demonstrated that stem cells can mediate therapeutic effect by secreting exosomes. We aimed to investigate the effect of human ASCs derived exosomes (ASC-Exos) on peripheral nerve regeneration in vitro and in vivo. Our results showed after being internalized by Schwann cells (SCs), ASC-Exos significantly promoted SC proliferation, migration, myelination, and secretion of neurotrophic factors by upregulating corresponding genes in vitro. We next evaluated the efficacy of ASC-Exo therapy in a rat sciatic nerve transection model with a 10-mm gap. Axon regeneration, myelination, and restoration of denervation muscle atrophy in ASC-Exos treated group was significantly improved compared to vehicle control. This study demonstrates that ASC-Exos effectively promote peripheral nerve regeneration via optimizing SC function and thereby represent a novel therapeutic strategy for regenerative medicine and nerve tissue engineering.     

Peripheral Nerve Lesion Induces an Up-regulation of Spy1 in Rat Spinal Cord

Spy1, as a member of the Speedy/RINGO family and a novel activator of cyclin-dependent kinases, was shown to promote cell cycle progression and cell survival in response to DNA damage. While its expression and roles in nervous system lesion and repair were still unknown. Here, we performed an acute sciatic nerve injury model in adult rats and studied the dynamic changes of Spy1 expression in lumbar spinal cord. Temporally, Spy1 expression was increased shortly after sciatic nerve crush and peaked at day 2. Spatially, Spy1 was widely expressed in the lumbar spinal cord including neurons and glial cells. While after injury, Spy1 expression was increased predominantly in astrocytes and microglia, which were largely proliferated. Moreover, there was a concomitant up-regulation of CDK2 activity and down-regulation of p27. Collectively, we hypothesized peripheral nerve injury induced an up-regulation of Spy1 in lumbar spinal cord, which was associated with glial proliferation. Ye Huang and Yonghua Liu contributed equally to this work.     

Evidence for a systemic regulation of neurotrophin synthesis in response to peripheral nerve injury

Up-regulation of neurotrophin synthesis is an important mechanism of peripheral nerve regeneration after injury. Neurotrophin expression is regulated by a complex series of events including cell interactions and multiple molecular stimuli. We have studied neurotrophin synthesis at 2?weeks time-point in a transvertebral model of unilateral or bilateral transection of sciatic nerve in rats. We have found that unilateral sciatic nerve transection results in the elevation of nerve growth factor (NGF) and NT-3, but not glial cell-line derived neurotrophic factor or brain-derived neural factor, in the uninjured nerve on the contralateral side, commonly considered as a control. Bilateral transection further increased NGF but not other neurotrophins in the nerve segment distal to the transection site, as compared to the unilateral injury. To further investigate the distinct role of NGF in regeneration and its potential for peripheral nerve repair, we transduced isogeneic Schwann cells with NGF-encoding lentivirus and transplanted the over-expressing cells into the distal segment of a transected nerve. Axonal regeneration was studied at 2?weeks time-point using pan-neuronal marker NF-200 and found to directly correlate with NGF levels in the regenerating nerve.     

Direct transplantation of uncultured hair‐follicle pluripotent stem (hfPS) cells promotes the recovery of peripheral nerve injury

We previously showed that the stem cell marker nestin is expressed in hair follicle stem cells which suggested their pluripotency. We subsequently showed that the nestin‐expressing hair‐follicle pluripotent stem (hfPS) cells can differentiate in culture to neurons, glial cells, keratinocytes, and other cell types and can promote regeneration of peripheral nerve and spinal cord injuries upon injection to the injured nerve or spinal cord. The location of the hfPS cells has been termed the hfPS cell area (hfPSCA). Previously, hfPS cells were cultured for 1–2 months before transplantation to the injured nerve or spinal cord which would not be optimal for clinical application of these cells for nerve or spinal cord repair, since the patient should be treated soon after injury. In the present study, we addressed this issue by directly using the upper part of the hair follicle containing the hfPSCA, without culture, for injection into the severed sciatic nerve in mice. After injection of hfPSCA, the implanted hfPS cells grew and promoted joining of the severed nerve. The transplanted hfPS cells differentiated mostly to glial cells forming myelin sheaths, which promoted axonal growth and functional recovery of the severed nerve. These results suggest that the direct transplantation of the uncultured upper part of the hair follicle containing the hfPSA is an important method to promote the recovery of peripheral nerve injuries and has significant clinical potential. J. Cell. Biochem. 110: 272–277, 2010. © 2010 Wiley‐Liss, Inc.     

Myelosuppressive Conditioning Using Busulfan Enables Bone Marrow Cell Accumulation in the Spinal Cord of a Mouse Model of Amyotrophic Lateral Sclerosis

Myeloablative preconditioning using irradiation is the most commonly used technique to generate rodents having chimeric bone marrow, employed for the study of bone marrow-derived cell accumulation in the healthy and diseased central nervous system. However, irradiation has been shown to alter the blood-brain barrier, potentially creating confounding artefacts. To better study the potential of bone marrow-derived cells to function as treatment vehicles for neurodegenerative diseases alternative preconditioning regimens must be developed. We treated transgenic mice that over-express human mutant superoxide dismutase 1, a model of amyotrophic lateral sclerosis, with busulfan to determine whether this commonly used chemotherapeutic leads to stable chimerism and promotes the entry of bone marrow-derived cells into spinal cord. Intraperitoneal treatment with busulfan at 60 mg/kg or 80 mg/kg followed by intravenous injection of green fluorescent protein-expressing bone marrow resulted in sustained levels of chimerism (∼80%). Bone marrow-derived cells accumulated in the lumbar spinal cord of diseased mice at advanced stages of pathology at both doses, with limited numbers of bone marrow derived cells observed in the spinal cords of similarly treated, age-matched controls; the majority of bone marrow-derived cells in spinal cord immunolabelled for macrophage antigens. Comparatively, significantly greater numbers of bone marrow-derived cells were observed in lumbar spinal cord following irradiative myeloablation. These results demonstrate bone marrow-derived cell accumulation in diseased spinal cord is possible without irradiative preconditioning.     

Direct evidence for spinal cord microglia in the development of a neuropathic pain-like state in mice

The present study was undertaken to further investigate the role of glial cells in the development of the neuropathic pain-like state induced by sciatic nerve ligation in mice. At 7 days after sciatic nerve ligation, the immunoreactivities (IRs) of the specific astrocyte marker glial fibrillary acidic protein (GFAP) and the specific microglial marker OX-42, but not the specific oligodendrocyte marker O4, were increased on the ipsilateral side of the spinal cord dorsal horn in nerve-ligated mice compared with that on the contralateral side. Furthermore, a single intrathecal injection of activated spinal cord microglia, but not astrocytes, caused thermal hyperalgesia in naive mice. Furthermore, 5-bromo-2'-deoxyuridine (BrdU)-positive cells on the ipsilateral dorsal horn of the spinal cord were significantly increased at 7 days after nerve ligation and were highly co-localized with another microglia marker, ionized calcium-binding adaptor molecule 1 (Iba1), but neither with GFAP nor a specific neural nuclei marker, NeuN, in the spinal dorsal horn of nerve-ligated mice. The present data strongly support the idea that spinal cord astrocytes and microglia are activated under the neuropathic pain-like state, and that the proliferated and activated microglia directly contribute to the development of a neuropathic pain-like state in mice.     

Bilateral activation of STAT3 by phosphorylation at the tyrosine-705 (Y705) and serine-727 (S727) positions and its nuclear translocation in primary sensory neurons following unilateral sciatic nerve injury

Unilateral sciatic nerve compression (SNC) or complete sciatic nerve transection (CSNT), both varying degrees of nerve injury, induced activation of STAT3 bilaterally in the dorsal root ganglia (DRG) neurons of lumbar (L4-L5) as well as cervical (C6–C8) spinal cord segments. STAT3 activation was by phosphorylation at the tyrosine-705 (Y705) and serine-727 (S727) positions and was followed by their nuclear translocation. This is the first evidence of STAT3(S727) activation together with the well-known activation of STAT3(Y705) in primary sensory neurons upon peripheral nerve injury. Bilateral activation of STAT3 in DRG neurons of spinal segments anatomically both associated as well as non-associated with the injured nerve indicates diffusion of STAT3 activation inducers along the spinal cord. Increased levels of IL-6 protein in the CSF following nerve injury as well as activation and nuclear translocation of STAT3 in DRG after intrathecal injection of IL-6 shows that this cytokine, released into the subarachnoid space can penetrate the DRG to activate STAT3. Previous results on increased bilateral IL-6 synthesis and the present manifestation of STAT3 activation in remote DRG following unilateral sciatic nerve injury may reflect a systemic reaction of the DRG neurons to nerve injury.     

The impact of physical,biochemical, and electrical signaling on Schwann cell plasticity

Peripheral nervous system (PNS) injuries are an ongoing health care concern. While autografts and allografts are regarded as the current clinical standard for traumatic injury, there are inherent limitations that suggest alternative remedies should be considered for therapeutic purposes. In recent years, nerve guidance conduits (NGCs) have become increasingly popular as surgical repair devices, with a multitude of various natural and synthetic biomaterials offering potential to enhance the design of conduits or supplant existing technologies entirely. From a cellular perspective, it has become increasingly evident that Schwann cells (SCs), the primary glia of the PNS, are a predominant factor mediating nerve regeneration. Thus, the development of severe nerve trauma therapies requires a deep understanding of how SCs interact with their environment, and how SC microenvironmental cues may be engineered to enhance regeneration. Here we review the most recent advancements in biomaterials development and cell stimulation strategies, with a specific focus on how the microenvironment influences the behavior of SCs and can potentially lead to functional repair. We focus on microenvironmental cues that modulate SC morphology, proliferation, migration, and differentiation to alternative phenotypes. Promotion of regenerative phenotypic responses in SCs and other non-neuronal cells that can augment the regenerative capacity of multiple biomaterials is considered along with innovations and technologies for traumatic injury.     

Axonal Regeneration after Sciatic Nerve Lesion Is Delayed but Complete in GFAP- and Vimentin-Deficient Mice

Peripheral axotomy of motoneurons triggers Wallerian degeneration of injured axons distal to the lesion, followed by axon regeneration. Centrally, axotomy induces loss of synapses (synaptic stripping) from the surface of lesioned motoneurons in the spinal cord. At the lesion site, reactive Schwann cells provide trophic support and guidance for outgrowing axons. The mechanisms of synaptic stripping remain elusive, but reactive astrocytes and microglia appear to be important in this process. We studied axonal regeneration and synaptic stripping of motoneurons after a sciatic nerve lesion in mice lacking the intermediate filament (nanofilament) proteins glial fibrillary acidic protein (GFAP) and vimentin, which are upregulated in reactive astrocytes and Schwann cells. Seven days after sciatic nerve transection, ultrastructural analysis of synaptic density on the somata of injured motoneurons revealed more remaining boutons covering injured somata in GFAP^–/–Vim^–/– mice. After sciatic nerve crush in GFAP^–/–Vim^–/– mice, the fraction of reinnervated motor endplates on muscle fibers of the gastrocnemius muscle was reduced 13 days after the injury, and axonal regeneration and functional recovery were delayed but complete. Thus, the absence of GFAP and vimentin in glial cells does not seem to affect the outcome after peripheral motoneuron injury but may have an important effect on the response dynamics.