Evaluation of the regeneration process of the sciatic nerve of a mouse animal model after application of freezing, crushing or electrocoagulation damage / Ocena procesu regeneracji nerwu kulszowego mysiego modelu zwierzecego po zastosowaniu uszkodzenia metoda mrozenia, zmiazdzenia i elektrokoagulacji.

The problem of regeneration of damaged peripheral nerves is an ongoing topic and has long been the subject of intensive research worldwide. This study examined the morphological and functional evaluation of the regeneration process within the damaged sciatic nerve, a mouse animal model. The effect of impaired expression of the TSC-1 gene on the process of nerve regeneration was evaluated, depending on the mode of damage. The research object consisted of 48, 2-month-old male TSC lines. The test group consisted of animals that underwent damage to the sciatic nerve by crushing, freezing and electrocoagulation, while the control group includes mice whose sciatic nerve was not damaged. Behavioral tests were conducted to evaluate the functional return of the limb, after 3,5,7 and 14 days. The first changes in the regeneration process of the damaged neurite are observed as early as day 3 after the injury, while on day 14 after the injury the functional return of the damaged limb was noted.

Effect of repeated sciatic nerve crush on the conditioning lesion response: Generating an experimental animal model to prolong the denervation period while maintaining peripheral nerve continuity.

Peripheral nerves exhibit long-term residual motor dysfunction following injury. The length of the denervation period before nerve and muscle reconnection is an important factor in motor function recovery. We aimed to investigate whether repeated nerve crush injuries to the same site every 7 days would preserve the conditioning lesion (CL) response and to determine the number of nerve crush injuries required to create an experimental animal model that would prolong the denervation period while maintaining peripheral nerve continuity. Rats were grouped according to the number of sciatic nerve crushes. A significant decrease in the soleus muscle fiber cross-sectional area was observed with increased crushes. After a single crush, macrophage accumulation and macrophage chemotaxis factor CCL2 expression in dorsal root ganglia were markedly increased, which aligned with the gene expression of Ccl2 and its receptor Ccr2. Macrophage numbers, histological CCL2 expression, and Ccl2 and Ccr2 gene expression levels decreased, depending on the number of repeated crushes. Histological analysis and gene expression analysis in the group with four repeated crushes did not differ significantly when compared with uninjured animals. Our findings indicated that repeated nerve crushes at the same site every 7 days sustained innervation loss and caused a loss of the CL response. The experimental model did not require nerve stump suturing and is useful for exploring factors causing prolonged denervation-induced motor dysfunction. SIGNIFICANCE STATEMENT: This study elucidates the effects of repeated nerve crush injury to the same site on innervation and conditioning lesion responses and demonstrates the utility of an experimental animal model that recapitulates the persistent residual motor deficits owing to prolonged denervation without requiring nerve transection and transection suturing.

Neuroinflammation and gliosis in the injured and contralateral retinas after unilateral optic nerve crush.

The main purpose of this study is to analyze the effects of unilateral optic nerve crush in the gene expression of pro- and anti-inflammatory mediators, and gliosis markers in injured and contralateral retinas. Retinas from intact, unilaterally optic nerve injured or sham-operated C57BL/6J mice were analyzed 1, 3, 9 and 30 days after the surgery (n = 5/group and time point) and the relative expression of TGF-ß1, IL-1ß, TNF-α, Iba1, AQP4, GFAP, MHCII, and TSPO was analyzed in injured and contralateral using qPCR. The results indicated that compared with intact retinas, sham-operated animals showed an early (day 1) upregulation of IL-1ß, TNF-α and TSPO and a late (day 30) upregulation of TNF-α. In sham-contralateral retinas, TNF-α and TSPO mRNA expression were upregulated and day 30 while GFAP, Iba1, AQP4 and MHCII downregulated at day 9. Compared with sham-operated animals, in retinas affected by optic nerve crush GFAP and TSPO upregulated at day 1 and TNF-α, Iba1, AQP4 and MHCII at day 3. In the crushed-contralateral retinas, TGF-ß1, TNF-α, Iba1 and MHCII were upregulated at day 1. TSPO was upregulated up to day 30 whereas TGF-ß1 and Iba1 downregulated after day 9. In conclusion, both sham surgery and optic nerve crush changed the profile of inflammatory and gliosis markers in the injured and contralateral retinas, changes that were more pronounced for optic nerve crush when compared to sham.

Utilizing mouse optic nerve crush to examine CNS remyelination.

In developing pro-myelination treatment, an important hurdle is the lack of reliable animal models for assessing de novo myelination in disease settings. We recently showed that regenerated axons in injured optic nerves fail to be myelinated, providing an animal model for this purpose. Here, we describe procedures to promote axonal regeneration, administer optic nerve crush, and assess oligodendrocyte differentiation and maturation into myelination-competent oligodendrocytes. This protocol allows for testing the efficacy of remyelination treatments in an in vivo central nervous system (CNS). For complete details on the use and execution of this protocol, please refer to Wang et al. (2020) and Bei et al. (2016).

Protective effects of intravitreal administration of mesenchymal stem cell-derived exosomes in an experimental model of optic nerve injury.

Traumatic optic neuropathy results in the loss of retinal ganglion cells (RGCs), leading to unavoidable visual impairment. However, there is no effective therapy by far. Accumulated studies support the perception that mesenchymal stem cells (MSCs) secrete exosomes that serve as a protective paracrine factor. The study aimed to explore and evaluate the potential therapeutic effects of intravitreal transplantation of MSC-derived exosomes (MSC-exos) in an experimental model of optic nerve crush (ONC). Exosomes were isolated from rat MSCs and characterized by transmission electron microscope and western blotting. At the onset of ONC, a single intravitreal injection of exosomes or PBS was administered to the rats. At day 30, hematoxylin and eosin staining, immunohistochemistry, and ßIII-tubulin staining were performed to evaluate the survival of RGCs. Moreover, TUNEL assay was used to examine the apoptosis of RGCs. Inflammation-relevant factors were identified via quantitative polymerase chain reaction. The expression levels of cell apoptosis-related molecules and key members of the PI3K/AKT signaling pathway were determined via western blot analysis. We found that MSC-exos exhibited typical characteristic morphologies (cup-shaped) and sizes (peak size of 93 nm). Furthermore, they exhibited substantial expression of the exosome markers CD63 and TSG101, but lacked the expression of the cellular marker GM130. Treatment with intravitreal MSC-exos notably promoted the survival of RGCs in ONC rats. The level of pro-inflammatory cytokines, including TNF-α, IL-1ß, IL-6, IL-8, and MCP-1, were reduced, whereas those of the anti-inflammatory factor IL-10 were increased. Moreover, the apoptosis induced by ONC was decreased by the administration of MSC-exos via upregulation of the Bcl-2/Bax ratio and downregulation of caspase-3 activity. Furthermore, MSC-exos significantly stimulated AKT phosphorylation, whereas LY294002 restored the apoptosis-preventing effects of MSC-exos. The results of our results demonstrated that intravitreal administration of MSC-exos ameliorates ONC-induced injury in a rat model. These findings might aid in the development of effective exosome-based therapeutic strategies for the treatment of optic nerve degeneration.

Sigma-1R Protects Retinal Ganglion Cells in Optic Nerve Crush Model for Glaucoma.

Purpose: The purpose of this study was to determine the effects of the Sigma-1R (σ-1r) on retinal ganglion cell (RGC) survival following optic nerve crush (ONC) and the signaling mechanism involved in the σ-1r protection. Methods: The overall strategy was to induce injury by ONC and mitigate RGC death by increasing σ-1r expression and/or activate σ-1r activity in σ-1r K/O mice and wild type (WT) mice. AAV2-σ-1r vector was used to increase σ-1r expression and σ-1r agonist used to activate the σ-1r and RGCs were counted. Immunohistochemical and Western blot analysis determined phosphorylated (p)-c-Jun, c-Jun, and Caspase-3. Pattern electroretinography (PERG) determined RGC activity. Results: RGC counts and function were similar in pentazocine-treated WT mice when compared to untreated mice and in WT mice when compared with σ-1r K/O mice. Pentazocine-induced effects and the effects of σ-1r K/O were only observable after ONC. ONC resulted in decreased RGC counts and activity in both WT and σ-1r K/O mice, with σ-1r K/O mice experiencing significant decreases compared with WT mice. The σ-1r transgenic expression resulted in increased RGC counts and activity following ONC. In WT mice, treatment with σ-1r agonist pentazocine resulted in increased RGC counts and increased activity when compared with untreated WT mice. There were time-dependent increases in c-jun, p-c-jun, and caspase-3 expression in ONC mice that were mitigated with pentazocine-treatment. Conclusions: These findings suggest that the apoptotic pathway is involved in RGC losses seen in an ONC model. The σ-1r offers neuroprotection, as activation and/or transgenic expression of σ-1r attenuated the apoptotic pathway and restored RGCs number and function following ONC.

Presence and activation of pro-inflammatory macrophages are associated with CRYAB expression in vitro and after peripheral nerve injury.

BACKGROUND: Inflammation constitutes both positive and negative aspects to recovery following peripheral nerve injury. Following damage to the peripheral nervous system (PNS), immune cells such as macrophages play a beneficial role in creating a supportive environment for regrowing axons by phagocytosing myelin and axonal debris. However, a prolonged inflammatory response after peripheral nerve injury has been implicated in the pathogenesis of negative symptoms like neuropathic pain. Therefore, the post-injury inflammation must be carefully controlled to prevent secondary damage while allowing for regeneration. CRYAB (also known as alphaB-crystallin/HSPB5) is a small heat shock protein that has many protective functions including an immunomodulatory role in mouse models of multiple sclerosis, spinal cord injury, and stroke. Because its expression wanes and rebounds in the early and late periods respectively after PNS damage, and CRYAB null mice with sciatic nerve crush injury display symptoms of pain, we investigated whether CRYAB is involved in the immune response following PNS injury. METHODS: Sciatic nerve crush injuries were performed in age-matched Cryab knockout (Cryab-/-) and wildtype (WT) female mice. Nerve segments distal to the injury site were processed by immunohistochemistry for macrophages and myelin while protein lysates of the nerves were analyzed for cytokines and chemokines using Luminex and enzyme-linked immunosorbent assay (ELISA). Peritoneal macrophages from the two genotypes were also cultured and polarized into pro-inflammatory or anti-inflammatory phenotypes where their supernatants were analyzed for cytokines and chemokines by ELISA and protein lysates for macrophage antigen presenting markers using western blotting. RESULTS: We report that (1) more pro-inflammatory CD16/32+ macrophages are present in the nerves of Cryab-/- mice at days 14 and 21 after sciatic nerve crush-injury compared to WT counterparts, and (2) CRYAB has an immunosuppressive effect on cytokine secretion [interleukin (IL)-ß, IL-6, IL-12p40, tumor necrosis factor (TNF)-α] from pro-inflammatory macrophages in vitro. CONCLUSIONS: CRYAB may play a role in curbing the potentially detrimental pro-inflammatory macrophage response during the late stages of peripheral nerve regeneration.

Characteristics of cytokines in the sciatic nerve stumps and DRGs after rat sciatic nerve crush injury.

BACKGROUND: Cytokines are essential cellular modulators of various physiological and pathological activities, including peripheral nerve repair and regeneration. However, the molecular changes of these cellular mediators after peripheral nerve injury are still unclear. This study aimed to identify cytokines critical for the regenerative process of injured peripheral nerves. METHODS: The sequencing data of the injured nerve stumps and the dorsal root ganglia (DRGs) of Sprague-Dawley (SD) rats subjected to sciatic nerve (SN) crush injury were analyzed to determine the expression patterns of genes coding for cytokines. PCR was used to validate the accuracy of the sequencing data. RESULTS: A total of 46, 52, and 54 upstream cytokines were differentially expressed in the SNs at 1 day, 4 days, and 7 days after nerve injury. A total of 25, 28, and 34 upstream cytokines were differentially expressed in the DRGs at these time points. The expression patterns of some essential upstream cytokines are displayed in a heatmap and were validated by PCR. Bioinformatic analysis of these differentially expressed upstream cytokines after nerve injury demonstrated that inflammatory and immune responses were significantly involved. CONCLUSIONS: In summary, these findings provide an overview of the dynamic changes in cytokines in the SNs and DRGs at different time points after nerve crush injury in rats, elucidate the biological processes of differentially expressed cytokines, especially the important roles in inflammatory and immune responses after peripheral nerve injury, and thus might contribute to the identification of potential treatments for peripheral nerve repair and regeneration.

Early phosphoproteomic changes in the retina following optic nerve crush.

Retinal ganglion cell (RGC) death causes irreversible blindness in adult mammals. Death of RGC occurs in diseases including glaucoma or injuries to the optic nerve (ON). To investigate mechanisms involved in RGC degeneration, we evaluated the phosphoproteomic changes in the retina induced by ON injury. Intraorbital optic nerve crush (ONC) was performed in adult C57BL/6J mice. Retinas were collected at 0, 6, and 12 h following ONC. Retinal proteins labeled with CyDye-C2 were subject to 2D-PAGE, followed by phosphoprotein staining and in-gel/cross-gel image analysis. Proteins with significant changes in phosphorylation (ratios ≥1.2) in retinas of the injured eyes compared to the control eyes were spot-picked, tryptic digested, and peptide fragments were analyzed by MALDI-TOF (MS) and TOF/TOF (tandem MS/MS). Intraorbital ONC increased phosphorylation of many retinal proteins. Among them, 29 significantly phosphorylated proteins were identified. PANTHER analysis showed that these proteins are associated with a variety of protein classes, cellular components, biological processes and signaling pathways. One of the identified proteins, phosphoprotein enriched in astrocytes 15 (PEA15), was further validated by western blotting and immunofluorescence staining. Functions of PEA15 were determined in cultured astrocytes. PEA15 knockdown reduced astrocyte phagocytic activity but promoted cell migration. Long term PEA15 knockdown also decreased astrocyte ATP level. This study provides new insights into mechanisms of RGC degeneration after ON injury, as well as central nervous system (CNS) neurodegeneration, since the retina is an extension of the CNS. These new insights will lead to novel therapeutic targets for retinal and CNS neurodegeneration.

The pudendal nerve motor branch regenerates via a brain derived neurotrophic factor mediated mechanism.

Peripheral nerve injuries can significantly reduce quality of life. While some recover, most do not recover fully, resulting in neuropathic pain and loss of sensation and motor function. Research on the mechanisms of peripheral nerve regeneration could elucidate poor patient outcomes and potential treatments. This study was designed to determine if brain derived neurotrophic factor (BDNF) is necessary for pudendal nerve regeneration and functional recovery. Peripheral administration of tyrosine kinase B functional chimera (TrkB) was used to inhibit the BDNF regenerative pathway. Female Sprague-Dawley rats received tyrosine kinase B functional chimera (TrkB) or saline after a pudendal nerve crush (PNC) or Sham PNC and were divided into three groups: Sham PNC, PNC + Saline, and PNC + TrkB. Seven days after injury, relative ßII tubulin expression (1.0 ± 0.2) was significantly decreased after PNC + TrkB compared to PNC + saline (2.9 ± 1.0). Three weeks after injury, BDNF plasma concentration (1320.8 ± 278.1 pg/ml) was significantly reduced in PNC + TrkB compared to PNC + saline rats (2053.4 ± 211.0 pg/ml). Pudendal nerve motor branch firing rate (54.0 ± 9.5 Hz) was significantly decreased in the PNC + TrkB group compared to the PNC + saline group (120.4 ± 17.1 Hz); while nerve firing rate of the PNC + saline group was not significantly different from sham PNC rats (121.8 ± 26.6 Hz). This study demonstrated that peripheral administration of TrkB bound free BDNF and inhibited the regenerative response after PNC. BDNF is necessary for normal PN motor branch recovery after PNC.

Effectiveness of electrical stimulation on nerve regeneration after crush injury: Comparison between invasive and non-invasive stimulation.

Several studies have investigated the use of invasive and non-invasive stimulation methods to enhance nerve regeneration, and varying degrees of effectiveness have been reported. However, due to the use of different parameters in these studies, a fair comparison between the effectiveness of invasive and non-invasive stimulation methods is not possible. The present study compared the effectiveness of invasive and non-invasive stimulation using similar parameters. Eighteen Sprague Dawley rats were classified into three groups: the iES group stimulated with fully implantable device, the tES group stimulated with transcutaneous electrical nerve stimulation (TENS), and the injury group (no stimulation). The iES and tES groups received stimulation for 6 weeks starting immediately after the injury. Motor function was evaluated using the sciatic functional index (SFI) every week. The SFI values increased over time in all groups; faster and superior functional recovery was observed in the iES group than in the tES group. Histological evaluation of the nerve sections and gastrocnemius muscle sections were performed every other week. The axon diameter and muscle fiber area in the iES group were larger, and the g-ratio in the iES group was closer to 0.6 than those in the tES group. To assess the cause of the difference in efficiency, a 3D rat anatomical model was used to simulate the induced electric fields in each group. A significantly higher concentration and intensity around the sciatic nerve was observed in the iES group than in the tES group. Vector field distribution showed that the field was orthogonal to the sciatic nerve spread in the tES group, whereas it was parallel in the iES group; this suggested that the tES group was less effective in nerve stimulation. The results indicated that even though rats in the TENS group showed better recovery than those in the injury group, it cannot replace direct stimulation yet because rats stimulated with the invasive method showed faster recovery and superior outcomes. This was likely attributable to the greater concentration and parallel distribution of electric field with respect to target nerve.

Involvement of Upregulated P53-Induced Death Domain Protein in Retinal Ganglion Cells Apoptosis After Optic Nerve Crush.

PURPOSE: Retinal ganglion cells (RGCs) apoptosis is a common characteristic of optic neuropathies. p53-induced protein with a death domain (PIDD) is a well-known regulator of genotoxic stress-induced apoptosis, which is constitutively cleaved into three main fragments: PIDD-N, PIDD-C and PIDD-CC. Thus, we aim to determine the physiological relevance of PIDD in RGCs apoptosis in an optic nerve crush (ONC) model. METHODS: All animals were evenly randomized into four groups: sham-control group, con-siRNA group, ONC group, and PIDD-siRNA group (ONC +PIDD-siRNA). Expressions of PIDD, caspase-2, Brn3a and tBid in ONC model were analyzed by Western blot and immunofluorescence. Mean densities of RGCs/mm2 were calculated with Fluoro-Gold (FG). Moreover, we tested the effect of PIDD-siRNA on ONC-induced RGCs apoptosis using TUNEL staining. RESULTS: The level of full-length PIDD was weakly present and showed no significant differences at any time points. PIDD-CC and PIDD-C were significantly up-regulated in the retina at 3 days after ONC. Meanwhile, the expression of PIDD was significantly increased in Brn3a (a marker of RGCs) positive cells, indicating that the localization of PIDD appeared to be confined to RGCs. Furthermore, inhibition of PIDD prevented RGCs apoptosis by inhibiting caspase-2 and tBid activation. CONCLUSION: Taken together, PIDD may play a crucial role in RGCs apoptosis after ONC, and this process may be relevant to caspase-2 and tBid.

Intramuscular delivery of HGF-expressing recombinant AAV improves muscle integrity and alleviates neurological symptoms in the nerve crush and SOD1-G93A transgenic mouse models.

Hepatocyte growth factor (HGF) is a versatile neurotrophic factor that mediates a variety of cellular activities. In this study, we investigated the effects of intramuscularly injected recombinant AAV vectors expressing HGF in two pathologic conditions: the sciatic nerve crush and the SOD1-G93A transgenic mouse models. AAV serotype 6 (rAAV6) was chosen based on its expression levels in, and capability of moving to, the spinal cord from the injected muscle area. In the nerve crush model, rAAV6-HGF was shown to reduce the degree of mechanical allodynia, increase the cross-sectional area of muscle fibers, promote regrowth of peripheral axons, and improve motor functions. In the SOD1-G93A TG mouse model, rAAV6-HGF increased the mass of the tibialis anterior and gastrocnemius, alleviated disease symptoms, and prolonged survival. Improvements in integrity and functions of muscle in these models seemed to have come from the ability of HGF produced from rAAV6-HGF to regulate the expression of various atrogenes through the control of the FOXO signaling pathway. Our findings suggested that intramuscular injection of rAAV6-HGF might be used to relieve various symptoms associated with muscle atrophy and/or nerve damages observed in a majority of neuromuscular diseases.

Intrathecal delivery of recombinant AAV1 encoding hepatocyte growth factor improves motor functions and protects neuromuscular system in the nerve crush and SOD1-G93A transgenic mouse models.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease resulting from motor neuron degeneration that causes muscle weakness, paralysis, and eventually respiratory failure. We investigated whether recombinant adeno-associated virus encoding human hepatocyte growth factor (rAAV-HGF) could generate beneficial effects in two mouse models with neuromuscular problems when intrathecally delivered to the subarachnoid space. We chose AAV serotype 1 (rAAV1) based on the expression levels and distribution of HGF protein in the lumbar spinal cord (LSC). After a single intrathecal (IT) injection of rAAV1-HGF, the protein level of HGF in the LSC peaked on day 14 and thereafter gradually decreased over the next 14 weeks. rAAV1-HGF was initially tested in the mouse nerve crush model. IT injection of rAAV1-HGF improved mouse hindlimb strength and rotarod performance, while histological analyses showed that the length of regenerated axons was increased and the structure of the neuromuscular junction (NMJ) was restored. rAAV1-HGF was also evaluated in the SOD1-G93A transgenic (TG) mouse model. Again, rAAV1-HGF not only improved motor performance but also increased the survival rate. Moreover, the number and diameter of spinal motor neurons (SMNs) were increased, and the shape of the NMJs restored. Data from in vitro motor cortical culture experiments indicated that treatment with recombinant HGF protein (rHGF) increased the axon length of corticospinal motor neurons (CSMNs). When cultures were treated with an ERK inhibitor, the effects of HGF on axon elongation, protein aggregation, and oxidative stress were suppressed, indicating that ERK phosphorylation played an important role(s). Taken together, our results suggested that HGF might play an important role(s) in delaying disease progression in the SOD1-G93A TG mouse model by reducing oxidative stress through the control of ERK phosphorylation.

Increased angiogenesis by the rotational muscle flap is crucial for nerve regeneration.

BACKGROUND: The gold standard surgical treatment of nerve injury includes direct repair, nerve graft, and neurolysis. The underlying effects (either beneficial or detrimental) of angiogenesis during nerve regeneration by rotational muscle flap have not yet determined. We assess the neurological outcome and angiogenesis of nerve injury following a rotational muscle flap. METHODS: We retrospectively analyzed the outcome of the patients with severe radial nerve injury by neurolysis and rotational muscle flap; we also mimicked the clinical situation by nerve crush followed by rotational muscle flap in animals to assess associated angiogenesis factor expression. RESULTS: Twenty-three out of 25 (92%) cases of severe radial nerve injury underwent neurolysis assisted by muscle flap rotation and eventually reached their preinjury neurological outcome. In the animal study, both FITC-dextran and Dil infusion showed a remarkably increased vascular structure in the crushed nerve integrated by the muscle flap and abolished by Avastin injection. The rotational muscle flap significantly increased angiogenesis factor expression, and this was attenuated by Avastin injection. The increased angiogenesis factor expression paralleled the improvement seen in neurobehavioral and electrophysiological studies as well as the significant expression of nerve regeneration markers and the restoration of denervated muscle morphology. CONCLUSION: Based on the clinical and animal data analysis, we conclude that muscle flap rotation provides a platform for angiogenesis in the acceleration of nerve regeneration. It appears that the muscle flap rotation augmented the nerve regeneration process which may be beneficial for nerve repair in clinical application.

Transcriptional landscape of alternative splicing during peripheral nerve injury.

Alternative splicing (AS) regulates a variety of biological activities in numerous tissues and organs, including the nervous system. However, the existence and specific roles of AS events during peripheral nerve repair and regeneration remain largely undetermined. In the current study, by mapping splice-crossing sequence reads, we identified AS events and relevant spliced genes in rat sciatic nerve stumps following sciatic nerve crush. AS-related genes at 1, 4, 7, and 14 days post nerve crush were compared with those at 0 day to discover alternatively spliced genes induced by sciatic nerve crush. These injury-induced alternatively spliced genes were then categorized to diseases and biological functions, genetic networks, and canonical signaling pathways. Bioinformatic analysis indicated that these alternatively spliced genes were mainly correlated to immune response, cellular growth, and cellular function maintenance. Our study elucidated AS events following peripheral nerve injury and might help deepen our understanding of the molecular mechanisms underlying peripheral nerve regeneration.

c-Fos is necessary for HGF-mediated gene regulation and cell migration in Schwann cells.

We previously reported that the expression of hepatocyte growth factor (HGF) was highly induced after peripheral nerve damage, and that c-Fos is one of many cellular genes whose expressions are affected by the increased level of HGF[1]. c-Fos is an important component of AP-1 heterodimer, but its role has not been clearly understood in the context of HGF and Schwann cells (SCs). In this study, we investigated the relationship between HGF and c-Fos. First, it was confirmed that the c-Fos was increased in SCs after nerve injury, while this effect abrogated by PHA-665752, an inhibitor of c-met receptor. When primary SCs were treated with recombinant HGF protein, c-Fos expression was regulated in a typical quick, transient fashion at both RNA and proteins levels. HGF-mediated induction of c-Fos expression was highly suppressed by specific inhibitors of ERK and CREB, respectively. The knock down of c-Fos expression by siRNA almost completely blocked various HGF-mediated effects in SCs, such as induction of gene expression of GDNF, LIF, and c-Myc, and migration of SCs, indicating that c-Fos might play a key role in HGF effects. Taken together, our results suggested that c-Fos plays a key role(s) in HGF-mediated effects on neurotrophic genes and cell migration.

Semaphorin3A Signaling Is Dispensable for Motor Axon Reinnervation of the Adult Neuromuscular Junction.

The neuromuscular junction (NMJ) is a specialized synapse that is formed by motor axon innervation of skeletal muscle fibers. The maintenance of motor-muscle connectivity is critical for the preservation of muscle tone and generation of movement. Injury can induce a robust regenerative response in motor axons, but severe trauma or chronic denervation resulting from neurodegenerative disease typically leads to inefficient repair and poor functional recovery. The axon guidance molecule Semaphorin3A (Sema3A) has been implicated as a negative regulator of motor innervation. Upon binding to a plexinA-neuropilin1 (Npn1) receptor complex, Sema3A initiates a downstream signaling cascade that results in axonal repulsion. Here, we established a reproducible nerve crush model to quantify motor nerve regeneration. We then used that model to investigate the role of Sema3A signaling at the adult NMJ. In contrast to previous findings, we found that Sema3A and Npn1 mRNA decrease in response to denervation, suggesting that Sema3A-Npn1 signaling may regulate NMJ reinnervation. To directly test that hypothesis, we used inducible knockout models to ubiquitously delete Sema3A or Npn1 from adult mice. Despite demonstrating that we could achieve highly efficient gene deletion, disruption of Sema3A-Npn1 signaling did not affect the normal maintenance of the NMJ or disrupt motor axon reinnervation after a denervating injury.

Neuroprotective effect of acute prior inflammation with lipopolysaccharide for adult male rat facial motoneurones.

Increases in inflammatory cytokines are reported to have both neuroprotective and neurotoxic effects depending on the type and age of neurones studied. This study aimed to determine the effect of experimental inflammation induced by Lipopolysaccharide (LPS) on the survival of injured male adult rat facial motoneurones. Time- and dose-response studies were done to optimise the LPS administration time and dose, to best correlate with inflammatory levels previously reported for aged rats. 12 cytokines were assayed through multiplex analysis. 24 h after intraperitoneal injection of 0.5 mg/kg Lipopolysaccharide in rats, IL-1ß, IL-5 and IL-12p70 levels were elevated, with no observed LPS-associated sickness behaviour. In other groups of 5-6 adult rats, the facial nerve was either crushed (as mild injury) or avulsed (as severe injury) after the LPS priming injection. Stereology revealed that most motoneurones survived 28 days after nerve crush only and LPS- or saline-priming preceding nerve crush. Most motoneurones died following nerve avulsion only, whereas over half survived when LPS-priming preceded nerve avulsion. We suggest that elevated levels of experimental inflammation are neuroprotective for severely injured adult male rat facial motoneurones.

Up-Regulation of Cdc37 Contributes to Schwann Cell Proliferation and Migration After Sciatic Nerve Crush.

Cell division cycle protein 37 (Cdc37), a molecular chaperone takes part in a series of cellular processes including cell signal transduction, cell cycle progression, cell proliferation, cell motility, oncogenesis and malignant progression. It can not only recruit immature protein kinases to HSP90 but also work alone. Cdc37 was reported to be associated with neurogenesis, neurite outgrowth, axon guidance and myelination. However, the roles of Cdc37 on Schwann cells (SC) after peripheral nerve injury (PNI) remain unknown. In this study, we found that the expression of Cdc37 increased and reached the peak at 1 week after sciatic nerve crush (SNC), which was consistent with that of proliferation cell nuclear antigen. Immunofluorescence verified that Cdc37 co-localized with SC in vivo and in vitro. Intriguingly, Cdc37 protein level was potentiated in the model of TNF-α-induced SC proliferation. Moreover, we found that Cdc37 silencing impaired proliferation of SC in vitro. Moreover, Cdc37 suppression attenuated kinase signaling pathways of Raf-ERK and PI3K/AKT which are crucial cell signaling for SC proliferation. Finally, we found that Cdc37 silencing inhibited SC migration in vitro. In conclusion, we demonstrated that the way Cdc37 contributed to SC proliferation is likely via activating kinase signaling pathways of Raf-ERK and PI3K/AKT, and CDC37 was also involved in SC migration after SNC.