Dynamic Change of Prohibitin2 Expression in Rat Sciatic Nerve After Crush

As a novel cell cycle inhibitor, PHB2 controls the G1/S transition in cycling cells in a complex manner. Its aberrant expression is closely related to cell carcinogenesis. While its expression and role in peripheral nervous system lesion and repair were still unknown. Here, we performed an acute sciatic nerve crush (SNC) model in adult rats to examine the dynamic changes of PHB2. Temporally, PHB2 expression was sharply decreased after sciatic nerve crush and reached a valley at day 5. Spatially, PHB2 was widely expressed in the normal sciatic nerve including axons and Schwann cells. While after injury, PHB2 expression decreased predominantly in Schwann cells. The alteration was due to the decreased expression of PHB2 in Schwann cells after SNC. PHB2 expression correlated closely with Schwann cells proliferation in sciatic nerve post injury. Furthermore, PHB2 largely localized with GAP43 in axons in the crushed segment. Collectively, we suggested that PHB2 participated in the pathological process response to sciatic nerve injury and may be associated with Schwann cells proliferation and axons regeneration.     

Increased expression of Gem after rat sciatic nerve injury

Gem belongs to the Rad/Gem/Kir subfamily of Ras-related GTPases, whose expression is induced in several cell types upon activation by extracellular stimuli. Two functions of Gem have been demonstrated, including regulation of voltage-gated calcium channel activity and inhibition of Rho kinase-mediated cytoskeletal reorganization, such as stress fiber formation and neurite retraction. Because of the essential relationship between actin reorganization and peripheral nerve regeneration, we investigated the spatiotemporal expression of Gem in a rat sciatic nerve crush (SNC) model. After never injury, we observed that Gem had a significant up-regulation from 1 day, peaked at day 5 and then gradually decreased to the normal level. At its peak expression, Gem expressed mainly in Schwann cells (SCs) and macrophages of the distal sciatic nerve segment, but had few colocalization in axons. In addition, the peak expression of Gem was in parallel with PCNA, and numerous SCs expressing Gem were PCNA positive. Thus, all of our findings suggested that Gem may be involved in the pathophysiology of sciatic nerve after SNC.     

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.     

Spatiotemporal Expression of PSD-95 and nNOS After Rat Sciatic Nerve Injury

Neuronal nitric oxide synthase (nNOS) has been implicated to influence peripheral nerve lesion and regeneration. Post-synaptic density-95 (PSD-95) is one of nNOS-anchoring proteins and plays an important role in specifying the sites of reaction of NO in nervous system. Here we established a rat sciatic nerve crush (SNC) model to examine the spatiotemporal expression of PSD-95 and nNOS. At gene levels, PSD-95 mRNA diminished shortly after crush, and significantly elevated from 2 days to 2 weeks, whereas nNOS decreased progressively post-operation, reached the valley at 1 day, and markedly up-regulated from 1 to 2 weeks after SNC. The expression of both molecules returned to the control level at 4 weeks post-injury. At protein levels, PSD-95 and nNOS underwent the similar changes as their gene expression except for a time lag during up-regulating. At their peak expression, PSD-95 co-labeled with nNOS in Schwann cells (SCs) of sciatic nerve within 0.5 mm from the lesion site, but had few colocalization in axons. In addition, the interaction between PSD-95 and nNOS enhanced significantly at 2 weeks after SNC. These results suggest a correlation of PSD-95 up-regulation with nNOS in reactive SCs of crushed sciatic nerve, which may lead to understanding the function of PSD-95 during peripheral nerve regeneration. Shangfeng Gao and Min Fei contributed equally to this work.     

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

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

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.     

Changes in Ataxin-10 Expression after Sciatic Nerve Crush in Adult Rats

Ataxin-10 is a cytoplasmic protein that belongs to the family of armadillo repeat proteins and the ataxin proteins are ubiquitously expressed in nervous tissue. A loss of Ataxin-10 in primary neuronal cells causes increased apoptosis of cerebellar neurons. Knockdown of ATXN10 with siRNA in HeLa cells results in cytokinesis defects-multinucleation. Because of the essential role of Ataxin-10 in nervous system and cellular cytokinesis, we investigated the spatiotemporal expression of Ataxin-10 in a rat sciatic nerve crush (SNC) model. After never injury, we observed that Ataxin-10 had a significant up-regulation from 3d, peaked at day 5 and then gradually decreased to the normal level at 4 weeks. At its peak expression, Ataxin-10 expressed mainly in Schwann cells and macrophages of the distal sciatic nerve segment from injury, but had few co-localizations in axons. Besides, the peak expression of Ataxin-10 was in parallel with proliferating cell nuclear antigen (PCNA), and Ataxin-10 co-labeled with PCNA. Thus, all of our findings suggested that Ataxin-10 may be involved in the pathophysiology of sciatic nerve after SNC.     

Upregulated Expression of TRIM32 Is Involved in Schwann Cell Differentiation,Migration and Neurite Outgrowth After Sciatic Nerve Crush

Tripartite motif containing 32 (TRIM32), a member of the tripartite motif (TRIM) family, plays an indispensable role in myoblast proliferation. It also regulates neuron and skeletal muscle stem cell differentiation. Although it is of great importance, we know little about the roles of TRIM32 during peripheral nervous system injury. Here, we examined the dynamic changes of TRIM32 in acute sciatic nerve crush (SNC) model. After crush, TRIM32 rapidly increased and reached the climax at 1 week but then gradually declined to the normal level at 4 weeks post-injury. Meanwhile, we observed similar changes of Oct-6. What is more, we found co-localization of TRIM32 with S100 and Oct-6 in 1-week-injured tissues using double immunofluorescent staining. In further vitro experiments, enhancive expression of TRIM32 was detected during the process of cyclic adenosine monophosphate (cAMP)-induced Schwann cell differentiation and nerve growth factor (NGF)-induced PC12 cell neurite outgrowth. More interestingly, specific si-TRIM32-transfected RSC96 cells exhibited obvious reduction in the ability of migration. Taken together, we inferred that upregulated TRIM32 was not only involved in the differentiation and migration of Schwann cells but the neurite elongation after SNC.     

Spatiotemporal Expression of Poly(rC)-Binding Protein PCBP2 Modulates Schwann Cell Proliferation After Sciatic Nerve Injury

Poly(C)-binding proteins (PCBPs), also known as RNA-binding proteins, interact in a sequence-specific fashion with single-stranded poly(C). It was reported that PCBP2 contributed to gastric cancer proliferation and survival through miR-34a, and knockdown of PCBP2 inhibited glioma proliferation through inhibition of cell cycle progression. In addition, PCBP2 might play a critical role in the regulation of cortical neurons apoptosis induced by hypoxia or ischemia. Because of the essential role of PCBP2 in nervous system and cell growth, we investigated the spatiotemporal expression of PCBP2 in a rat sciatic nerve crush (SNC) model. We detected the upregulated expression of PCBP2 in Schwann cell after SNC. Besides, the peak expression of PCBP2 was in parallel with proliferation cell nuclear antigen. In vitro, we observed increased expression of PCBP2 during the process of TNF-α-induced Schwann cell proliferation. Specially, PCBP2-specific siRNA-transfected Schwann cell showed significantly decreased ability for proliferation. Together, all these data indicated that the change of PCBP2 protein expression was associated with Schwann cell proliferation after the trauma of the peripheral nervous system.     

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1^-/- mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75^NTR) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.     

Changes in Aldose Reductase After Crush Injury of Normal Rat Sciatic Nerve

The response of aldose reductase (AR) to crush injury was studied in normal rat sciatic nerve. Enzyme activity and immunoreactivity of AR were determined at intervals of 1, 5, 14, 28, and 35 days after crush and correlated with histologic and immunocytochemical observations. During nerve degeneration in the distal segments of crushed nerves, a significant reduction in AR activity was detected. At 5 and 14 days, coincident with Schwann cell proliferation, enzyme activity decreased by nearly two- and fourfold, respectively. Although activity of AR increased by 28 days during nerve regeneration, it was not restored to normal levels at 35 days. Similar reductions were observed with the immunoblotting of the enzyme. Quantitative analysis of immunogold labelling on electron micrographs confirmed that proliferating as well as remyelinating Schwann cells contained reduced gold particle density compared to Schwann cells of noncrushed myelinated fibers. Immunoblots of P0, a marker for the degree of Schwann cell differentiation or myelination, showed that the temporal sequence of changes in P0 paralleled that of AR. Thus expression of AR is a function of differentiated or mature Schwann cells. The putative volume regulatory role of AR in Schwann cells may become superfluous during Wallerian degeneration.     

Up-Regulation of HDAC4 is Associated with Schwann Cell Proliferation After Sciatic Nerve Crush

Histone deacetylase 4 (HDAC4), a member of the class IIa HDACs subfamily, has emerged as a critical regulator of cell growth, differentiation, and migration in various cell types. It was reported that HDAC4 stimulated colon cell proliferation via repression of p21. Also, HDAC4 contributes to platelet-derived growth factor-BB-induced proliferation and migration of vascular smooth muscle cells. Furthermore, HDAC4 may play an important role in the regulation of neuronal differentiation and survival. However, the role of HDAC4 in the process of peripheral nervous system regeneration after injury remains virtually unknown. Herein, we investigated the spatiotemporal expression of HDAC4 in a rat sciatic nerve crush model. We found that sciatic nerve crush induced up-regulated expression of HDAC4 in Schwann cells. Moreover, the expression of the proliferation marker Ki-67 exhibited a similar tendency with that of HDAC4. In cell cultures, we observed increased expression of HDAC4 during the process of TNF-α-induced Schwann cell proliferation, whereas the protein level of p21 was down-regulated. Interference of HDAC4 led to enhanced expression of p21 and impaired proliferation of Schwan cells. Taken together, our findings implicated that HDAC4 was up-regulated in the sciatic nerve after crush, which was associated with proliferation of Schwann cells.     

Fibrin inhibits peripheral nerve remyelination by regulating Schwann cell differentiation

Remyelination is a critical step for functional nerve regeneration. Here we show that fibrin deposition in the peripheral nervous system after injury is a key regulator of remyelination. After sciatic nerve crush, fibrin is deposited and its clearance correlates with remyelination. Fibrin induces phosphorylation of ERK1/2 and production of p75 NGF low-affinity receptor in Schwann cells and maintains them in a nonmyelinating state, suppresses fibronectin production, and prevents synthesis of myelin proteins. In mice depleted of fibrin(ogen), remyelination of myelinated axons is accelerated due to the faster transition of the Schwann cells to a myelinating state. Regulation of fibrin clearance and/or deposition could be a key regulatory mechanism for Schwann differentiation after nerve damage.     

Extracellular ATP inhibits Schwann cell dedifferentiation and proliferation in an ex vivo model of Wallerian degeneration

After nerve injury, Schwann cells proliferate and revert to a phenotype that supports nerve regeneration. This phenotype-changing process can be viewed as Schwann cell dedifferentiation. Here, we investigated the role of extracellular ATP in Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Using several markers of Schwann cell dedifferentiation and proliferation in sciatic explants, we found that extracellular ATP inhibits Schwann cell dedifferentiation and proliferation during Wallerian degeneration. Furthermore, the blockage of lysosomal exocytosis in ATP-treated sciatic explants is sufficient to induce Schwann cell dedifferentiation. Together, these findings suggest that ATP-induced lysosomal exocytosis may be involved in Schwann cell dedifferentiation.     

Laminin gamma1 is critical for Schwann cell differentiation, axon myelination, and regeneration in the peripheral nerve

Laminins are heterotrimeric extracellular matrix proteins that regulate cell viability and function. Laminin-2, composed of alpha2, beta1, and gamma1 chains, is a major matrix component of the peripheral nervous system (PNS). To investigate the role of laminin in the PNS, we used the Cre-loxP system to disrupt the laminin gamma1 gene in Schwann cells. These mice have dramatically reduced expression of laminin gamma1 in Schwann cells, which results in a similar reduction in laminin alpha2 and beta1 chains. These mice exhibit motor defects which lead to hind leg paralysis and tremor. During development, Schwann cells that lack laminin gamma1 were present in peripheral nerves, and proliferated and underwent apoptosis similar to control mice. However, they were unable to differentiate and synthesize myelin proteins, and therefore unable to sort and myelinate axons. In mutant mice, after sciatic nerve crush, the axons showed impaired regeneration. These experiments demonstrate that laminin is an essential component for axon myelination and regeneration in the PNS.     

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.