Neither injury induced macrophages within the nerve, nor the environment created by Wallerian degeneration is necessary for enhanced in vivo axon regeneration after peripheral nerve injury.

BACKGROUND: Since the 1990s, evidence has accumulated that macrophages promote peripheral nerve regeneration and are required for enhancing regeneration in the conditioning lesion (CL) response. After a sciatic nerve injury, macrophages accumulate in the injury site, the nerve distal to that site, and the axotomized dorsal root ganglia (DRGs). In the peripheral nervous system, as in other tissues, the macrophage response is derived from both resident macrophages and recruited monocyte-derived macrophages (MDMs). Unresolved questions are: at which sites do macrophages enhance nerve regeneration, and is a particular population needed. METHODS: Ccr2 knock-out (KO) and Ccr2gfp/gfp knock-in/KO mice were used to prevent MDM recruitment. Using these strains in a sciatic CL paradigm, we examined the necessity of MDMs and residents for CL-enhanced regeneration in vivo and characterized injury-induced nerve inflammation. CL paradigm variants, including the addition of pharmacological macrophage depletion methods, tested the role of various macrophage populations in initiating or sustaining the CL response. In vivo regeneration, measured from bilateral proximal test lesions (TLs) after 2 d, and macrophages were quantified by immunofluorescent staining. RESULTS: Peripheral CL-enhanced regeneration was equivalent between crush and transection CLs and was sustained for 28 days in both Ccr2 KO and WT mice despite MDM depletion. Similarly, the central CL response measured in dorsal roots was unchanged in Ccr2 KO mice. Macrophages at both the TL and CL, but not between them, stained for the pro-regenerative marker, arginase 1. TL macrophages were primarily CCR2-dependent MDMs and nearly absent in Ccr2 KO and Ccr2gfp/gfp KO mice. However, there were only slightly fewer Arg1+ macrophages in CCR2 null CLs than controls due to resident macrophage compensation. Zymosan injection into an intact WT sciatic nerve recruited Arg1+ macrophages but did not enhance regeneration. Finally, clodronate injection into Ccr2gfp KO CLs dramatically reduced CL macrophages. Combined with the Ccr2gfp KO background, depleting MDMs and TL macrophages, and a transection CL, physically removing the distal nerve environment, nearly all macrophages in the nerve were removed, yet CL-enhanced regeneration was not impaired. CONCLUSIONS: Macrophages in the sciatic nerve are neither necessary nor sufficient to produce a CL response.

The Pulsed Nd:YAG Laser Therapy Enhanced Nerve Regeneration via Apoptosis Inhibition in a Rat Crushed Sciatic Nerve Model.

The study was aimed to validate the efficacy of the pulsed Nd:YAG laser on nerve regeneration in a rat sciatic nerve crushed model. 54 Wistar rats were randomly assigned into three groups: shame control, crush control, and laser treated group. For the laser treated group, the pulsed Nd:YAG laser (10 Hz) with 350 mJ per pulse in energy density and 50 J/cm2 in fluence was applied extracorporeally at the lesion site for 12 min to daily deliver 500 J immediately and consecutive 9 days following the crush injury. At week 1, the apoptosis-related activities in the injured nerve were examined (n = 8/each group). The sciatic functional index (SFI) was measured preoperatively and weekly until 4 weeks after the index procedure. The injured nerve and the innervated gastrocnemius muscle histology were assessed at week 4 (n = 10/each group). At week 1, the laser group showed the significant less TUNEL-positive ratio (P < 0.05), and the lower expression of cleaved caspase3/procaspase-3 and beclin-2/beclin-2-associated protein X ratios compared with the crush control. Furthermore, the laser group revealed significantly better SFI since week 1 and throughout the study (P < 0.05, all) compared with the crush control. At week 4, the laser group showed significantly higher axon density, lower myelin g-ratio, and the corresponding higher glycogen expression (P < 0.05, all) in the gastrocnemius muscle compared with those in the crush control. The pulsed Nd:YAG might enhance the injured nerve regeneration via apoptosis inhibition.

Electrical stimulation accelerates Wallerian degeneration and promotes nerve regeneration after sciatic nerve injury.

Following peripheral nerve injury (PNI), Wallerian degeneration (WD) in the distal stump can generate a microenvironment favorable for nerve regeneration. Brief low-frequency electrical stimulation (ES) is an effective treatment for PNI, but the mechanism underlying its effect on WD remains unclear. Therefore, we hypothesized that ES could enhance nerve regeneration by accelerating WD. To verify this hypothesis, we used a rat model of sciatic nerve transection and provided ES at the distal stump of the injured nerve. The injured nerve was then evaluated after 1, 4, 7, 14 and 21 days post injury (dpi). The results showed that ES significantly promoted the degeneration and clearance of axons and myelin, and the dedifferentiation of Schwann cells. It upregulated the expression of BDNF and NGF and increased the number of monocytes and macrophages. Through transcriptome sequencing, we systematically investigated the effect of ES on the molecular processes involved in WD at 4 dpi. Evaluation of nerves bridged using silicone tubing after transection showed that ES accelerated early axonal and vascular regeneration while delaying gastrocnemius atrophy. These results demonstrate that ES promotes nerve regeneration by accelerating WD and upregulating the expression of neurotrophic factors.

Evaluation of Stem Cell Laden Collagen + Polycaprolactone + Multi-Walled Carbon Nano-Tubes Nano-Neural Scaffold with and Without Insulin Like Growth Factor-I For Sciatic Nerve Regeneration Post Crush Injury in Wistar Rats.

BACKGROUND/AIMS: All body functions are activated, synchronized and controlled by a substantial, complex network, the nervous system. Upon injury, pathophysiology of the nerve injury proceeds through different paths. The axon may undergo a degenerative retraction from the site of injury for a short distance unless the injury is near to the cell body, in which case it continues to the soma and undergoes retrograde neuronal degeneration. Otherwise, the distal section suffers from Wallerian degeneration, which is marked by axonal swelling, spheroids, and cytoskeleton degeneration. The objective of the study was to evaluate the potential of mesenchymal stem cell laden neural scaffold and insulin-like growth factor I (IGF-I) in nerve regeneration following sciatic nerve injury in a rat model. METHODS: The animals were anaesthetized and a cranio-lateral incision over left thigh was made. Sciatic nerve was exposed and crush injury was introduced for 90 seconds using haemostat at second locking position. The muscle and skin were sutured in routine fashion and thus the rat model of sciatic crush injury was prepared. The animal models were equally distributed into 5 different groups namely A, B, C, D and E and treated with phosphate buffer saline (PBS), carbon nanotubes based neural scaffold only, scaffold with IGF-I, stem cell laden scaffold and stem cell laden scaffold with IGF-I respectively. In vitro scaffold testing was performed. The nerve regeneration was assessed based on physico-neuronal, biochemical, histopathological examination, and relative expression of NRP-1, NRP-2 and GAP-43 and scanning electron microscopy. RESULTS: Sciatic nerve injury model with crush injury produced for 90 seconds was standardized and successfully used in this study. All the biochemical parameters were in normal range in all the groups indicating no scaffold related changes. Physico-neuronal, histopathological, relative gene expression and scanning electron microscopy observations revealed appreciable nerve regeneration in groups E and D, followed by C and B. Restricted to no regeneration was observed in group A. CONCLUSION: Carbon nanotubes based scaffold provided electro-conductivity for proper neuronal regeneration while rat bone marrow-derived mesenchymal stem cells were found to induce axonal sprouting, cellular transformation; whereas IGF-I induced stem cell differentiation, myelin synthesis, angiogenesis and muscle differentiation.

Effect of Azithromycin on Sciatic Nerve Injury in the Wistar Rats.

After a severe peripheral nerve injury, complete functional recovery is rare. Modulating the inflammatory response could be an effective way to enhance peripheral nerve regeneration. The present study aimed to determine the effect of azithromycin on functional recovery following sciatic nerve crush in Wistar rats. 40 male Wistar rats were used in four groups, including: the negative control, sham, and two groups of azithromycin (15 and 150 mg/kg/day) (n = 10).The rats' right sciatic nerve was crushed using a non-serrated clamp. In experimental groups, animals were treated with azithromycin (15 and 150 mg/kg/day) for 7 days. Then, sensory-motor functions were evaluated over eight weeks. Real-time PCR was used to measure the expression of NGF and BDNF genes. At the end of the 4th week, the sensory recovery accelerated in the azithromycin-treated rats so that the reaction times in the groups treated with 15 mg/kg and 150 mg/kg doses of azithromycin reached 5.14 s and 6.61 s, respectively, which were significantly lower than the 12 s in the negative control group (P < 0.05).Eventually, the mean SFI values in the negative control and both azithromycin-treated groups recovered to preoperative levels in the 8th week, with no significant difference between the sciatic lesion groups. Findings showed a seven-day course of azithromycin administered immediately after a sciatic nerve crush could accelerate regeneration and improve motor and sensory function recovery compared to negative controls. These significant effects were observed in both the azithromycin 15 mg/kg and the azithromycin 150 mg/kg treatment groups. Azithromycin treatment upregulated the expression of NGF and BDNF genes in crushed sciatic nerve. Our findings suggest that a seven-day treatment of azithromycin after a sciatic nerve injury could accelerate the regeneration process and improve functional recovery.

Interleukin-4 Induces the Release of Opioid Peptides from M1 Macrophages in Pathological Pain.

Interleukin-4 (IL-4) is an anti-inflammatory cytokine, which can be protective in inflammatory and neurologic disorders, and can alleviate pain. Classically, IL-4 diminishes pain by blocking the production of proinflammatory cytokines. Here, we uncovered that IL-4 induces acute antinociception by IL-4 receptor α (IL-4Rα)-dependent release of opioid peptides from M1 macrophages at injured nerves. As a model of pathologic pain, we used a chronic constriction injury (CCI) of the sciatic nerve in male mice. A single application of IL-4 at the injured nerves (14 d following CCI) attenuated mechanical hypersensitivity evaluated by von Frey filaments, which was reversed by co-injected antibody to IL-4Rα, antibodies to opioid peptides such as Met-enkephalin (ENK), ß-endorphin and dynorphin A 1-17, and selective antagonists of δ-opioid, µ-opioid, and κ-opioid receptors. Injured nerves were predominately infiltrated by proinflammatory M1 macrophages and IL-4 did not change their numbers or the phenotype, assessed by flow cytometry and qRT-PCR, respectively. Macrophages isolated from damaged nerves by immunomagnetic separation (IMS) and stimulated with IL-4 dose dependently secreted all three opioid peptides measured by immunoassays. The IL-4-induced release of ENK was diminished by IL-4Rα antibody, intracellular Ca2+ chelator, and inhibitors of protein kinase A (PKA), phosphoinositide 3-kinase (PI3K), and ryanodine receptors. Together, we identified a new opioid mechanism underlying the IL-4-induced antinociception that involves PKA-mediated, PI3K-mediated, ryanodine receptor-mediated, and intracellular Ca2+-mediated release from M1 macrophages of opioid peptides, which activate peripheral opioid receptors in injured tissue.SIGNIFICANCE STATEMENT Interleukin-4 (IL-4) is an anti-inflammatory cytokine, which can ameliorate pain. The IL-4-mediated effects are considered to mostly result from the inhibition of the production of proinflammatory mediators (e.g., IL-1ß, tumor necrosis factor, prostaglandin E2). Here, we found that IL-4 injected at the injured nerves attenuates pain by releasing opioid peptides from the infiltrating macrophages in mice. The opioids were secreted by IL-4 in the intracellular Ca2+-dependent manner and activated local peripheral opioid receptors. These actions represent a novel mode of IL-4 action, since its releasing properties have not been so far reported. Importantly, our findings suggest that the IL-4-opioid system should be targeted in the peripheral damaged tissue, since this can be devoid of central and systemic side effects.

Raman spectroscopy and sciatic functional index (SFI) after low-level laser therapy (LLLT) in a rat sciatic nerve crush injury model.

Axonotmesis causes sensorimotor and neurofunctional deficits, and its regeneration can occur slowly or not occur if not treated appropriately. Low-level laser therapy (LLLT) promotes nerve regeneration with the proliferation of myelinating Schwann cells to recover the myelin sheath and the production of glycoproteins for endoneurium reconstruction. This study aimed to evaluate the effects of LLLT on sciatic nerve regeneration after compression injury by means of the sciatic functional index (SFI) and Raman spectroscopy (RS). For this, 64 Wistar rats were divided into two groups according to the length of treatment: 14 days (n = 32) and 21 days (n = 32). These two groups were subdivided into four sub-groups of eight animals each (control 1; control 2; laser 660 nm; laser 808 nm). All animals had surgical exposure to the sciatic nerve, and only control 1 did not suffer nerve damage. To cause the lesion in the sciatic nerve, compression was applied with a Kelly clamp for 6 s. The evaluation of sensory deficit was performed by the painful exteroceptive sensitivity (PES) and neuromotor tests by the SFI. Laser 660 nm and laser 808 nm sub-groups were irradiated daily (100 mW, 40 s, energy density of 133 J/cm2). The sciatic nerve segment was removed for RS analysis. The animals showed accentuated sensory and neurofunctional deficit after injury and their rehabilitation occurred more effectively in the sub-groups treated with 660 nm laser. Control 2 sub-group did not obtain functional recovery of gait. The RS identified sphingolipids (718, 1065, and 1440 cm-1) and collagen (700, 852, 1004, 1270, and 1660 cm-1) as biomolecular characteristics of sciatic nerves. Principal component analysis revealed important differences among sub-groups and a directly proportional correlation with SFI, mainly in the sub-group laser 660 nm treated for 21 days. In the axonotmesis-type lesion model presented herein, the 660 nm laser was more efficient in neurofunctional recovery, and the Raman spectra of lipid and protein properties were attributed to the basic biochemical composition of the sciatic nerve.

Human Multipotent Mesenchymal Stromal Cell-Derived Extracellular Vesicles Enhance Neuroregeneration in a Rat Model of Sciatic Nerve Crush Injury.

Peripheral nerve injury remains a serious problem for medicine, with no effective method of treatment at the moment. The most prominent example of this problem is neonatal brachial plexus palsy, which results from the stretching of the brachial plexus nerves in the birth or perinatal period. Multipotent mesenchymal cells (MSCs) and the extracellular vesicles (EVs) they produce are known to have a marked neuroprotective effect in central nervous system injuries. We suggested that the use of MSCs-derived EVs may be an effective approach to the regeneration of peripheral nerves after injury. Sciatic nerve injury was modeled in rats via crushing, and then a gel containing MSCs-EVs was applied to the injured area. After 15 and 30 days, a histological, physiological, and functional assessment of nerve, dorsal root ganglia (DRG), and innervated muscles' recovery was performed. Transplantation of EVs to the area of sciatic nerve injury significantly reduced muscle atrophy as compared to the control group. Functional recovery of the innervated muscles, as measured by the extensor postural thrust test, was revealed 30 days after the surgery. We associate the obtained results with EVs-induced neuroprotective mechanisms, which were expressed in a decrease in apoptotic neuronal death and an increase in regeneration-associated proteins NF-200 and GAP-43, as well as in DRG and damaged nerve. We suggest that the therapeutic scheme we used is efficient for the treatment of acute peripheral nervous system injuries and can be transferred to the clinics. However, additional studies are required for a more detailed analysis of neuroprotection mechanisms.

Spinal Irisin Gene Delivery Attenuates Burn Injury-Induced Muscle Atrophy by Promoting Axonal Myelination and Innervation of Neuromuscular Junctions.

Muscle loss and weakness after a burn injury are typically the consequences of neuronal dysregulation and metabolic change. Hypermetabolism has been noted to cause muscle atrophy. However, the mechanism underlying the development of burn-induced motor neuropathy and its contribution to muscle atrophy warrant elucidation. Current therapeutic interventions for burn-induced motor neuropathy demonstrate moderate efficacy and have side effects, which limit their usage. We previously used a third-degree burn injury rodent model and found that irisin-an exercise-induced myokine-exerts a protective effect against burn injury-induced sensory and motor neuropathy by attenuating neuronal damage in the spinal cord. In the current study, spinal irisin gene delivery was noted to attenuate burn injury-induced sciatic nerve demyelination and reduction of neuromuscular junction innervation. Spinal overexpression of irisin leads to myelination rehabilitation and muscular innervation through the modulation of brain-derived neurotrophic factor and glial-cell-line-derived neurotrophic factor expression along the sciatic nerve to the muscle tissues and thereby modulates the Akt/mTOR pathway and metabolic derangement and prevents muscle atrophy.

Evidence that increased cholecystokinin (CCK) in the periaqueductal gray (PAG) facilitates changes in Resident-Intruder social interactions triggered by peripheral nerve injury.

Individual differences in the effects of a chronic neuropathic injury on social behaviours characterize both the human experience and pre-clinical animal models. The impacts of these changes to the well-being of the individual are often underappreciated. Earlier work from our laboratory using GeneChip® microarrays identified increased cholecystokinin (CCK) gene expression in the periaqueductal gray (PAG) of rats that showed persistent changes in social interactions during a Resident-Intruder encounter following sciatic nerve chronic constriction injury (CCI). In this study, we confirmed these gene regulation patterns using RT-PCR and identified the anatomical location of the CCK-mRNA as well as the translated CCK peptides in the midbrains of rats with a CCI. We found that rats with persistent CCI-induced changes in social behaviours had increased CCK-mRNA in neurons of the ventrolateral PAG and dorsal raphe nuclei, as well as increased CCK-8 peptide expression in terminal boutons located in the lateral and ventrolateral PAG. The functional significance of these changes was explored by microinjecting small volumes of CCK-8 into the PAG of uninjured rats and observing their Resident-Intruder social interactions. Disturbances to social interactions identical to those observed in CCI rats were evoked when injection sites were located in the rostral lateral and ventrolateral PAG. We suggest that CCI-induced changes in CCK expression in these PAG regions contributes to the disruptions to social behaviours experienced by a subset of individuals with neuropathic injury.

Delayed manipulation of regeneration within injured peripheral axons.

While several new translational strategies to enhance regrowth of peripheral axons have been identified, combined approaches with different targets are rare. Moreover, few have been studied after a significant delay when growth programs are already well established and regeneration-related protein expression has waned. Here we study two approaches, Rb1 (Retinoblastoma 1) knockdown that targets overall neuron plasticity, and near nerve insulin acting as a growth factor. Both are validated to boost regrowth only at the outset of regeneration. We show that local delivery of Rb1 siRNA alone, with electroporation to an area of prior sciatic nerve injury generated knockdown of Rb1 mRNA in ipsilateral lumbar dorsal root ganglia. While mice treated with Rb1-targeted siRNA, compared with scrambled control siRNA, starting 2 weeks after the onset of regeneration, had only limited behavioural or electrophysiological benefits, they had enhanced reinnervation of epidermal axons. We next confirmed that intrinsic Rb1 knockdown combined with exogenous insulin had dramatic synergistic impacts on the growth patterns of adult sensory neurons studied in vitro, prompting analysis of a combined approach in vivo. Using an identical delayed post-injury protocol, we noted that added insulin not only augmented epidermal reinnervation rendered by Rb1 knockdown alone but also improved indices of mechanical sensation and motor axon recovery. The findings illustrate that peripheral neurons that are well into attempted regrowth retain their responsiveness to both intrinsic and exogenous approaches that improve their recovery. We also identify a novel local approach to manipulate gene expression and outcome in regrowing axons.

The evolution of dystonia-like movements in TOR1A rats after transient nerve injury is accompanied by dopaminergic dysregulation and abnormal oscillatory activity of a central motor network.

TOR1A is the most common inherited form of dystonia with still unclear pathophysiology and reduced penetrance of 30-40%. ∆ETorA rats mimic the TOR1A disease by expression of the human TOR1A mutation without presenting a dystonic phenotype. We aimed to induce dystonia-like symptoms in male ∆ETorA rats by peripheral nerve injury and to identify central mechanism of dystonia development. Dystonia-like movements (DLM) were assessed using the tail suspension test and implementing a pipeline of deep learning applications. Neuron numbers of striatal parvalbumin+, nNOS+, calretinin+, ChAT+ interneurons and Nissl+ cells were estimated by unbiased stereology. Striatal dopaminergic metabolism was analyzed via in vivo microdialysis, qPCR and western blot. Local field potentials (LFP) were recorded from the central motor network. Deep brain stimulation (DBS) of the entopeduncular nucleus (EP) was performed. Nerve-injured ∆ETorA rats developed long-lasting DLM over 12 weeks. No changes in striatal structure were observed. Dystonic-like ∆ETorA rats presented a higher striatal dopaminergic turnover and stimulus-induced elevation of dopamine efflux compared to the control groups. Higher LFP theta power in the EP of dystonic-like ∆ETorA compared to wt rats was recorded. Chronic EP-DBS over 3 weeks led to improvement of DLM. Our data emphasizes the role of environmental factors in TOR1A symptomatogenesis. LFP analyses indicate that the pathologically enhanced theta power is a physiomarker of DLM. This TOR1A model replicates key features of the human TOR1A pathology on multiple biological levels and is therefore suited for further analysis of dystonia pathomechanism.

Differential effects of rat ADSCs encapsulation in fibrin matrix and combination delivery of BDNF and Gold nanoparticles on peripheral nerve regeneration.

BACKGROUND: Fibrin as an extracellular matrix feature like biocompatibility, creates a favorable environment for proliferation and migration of cells and it can act as a reservoir for storage and release of growth factors in tissue engineering. METHODS: In this study, the inner surface of electrospun poly (lactic-co-glycolic acid) (PLGA) nanofibrous conduit was biofunctionalized with laminin containing brain derived neurotrophic factor (BDNF) and gold nanoparticles in chitosan nanoparticle. The rats were randomly divided into five groups, including autograft group as the positive control, PLGA conduit coated by laminin and filled with DMEM/F12, PLGA conduit coated by laminin and filled with rat-adipose derived stem cells (r-ADSCs), PLGA conduit coated by laminin containing gold-chitosan nanoparticles (AuNPs-CNPs), BDNF-chitosan nanoparticles (BDNF-CNPs) and filled with r-ADSCs or filled with r-ADSCs suspended in fibrin matrix, and they were implanted into a 10 mm rat sciatic nerve gap. Eventually, axonal regeneration and functional recovery were assessed after 12 weeks. RESULTS: After 3 months post-surgery period, the results showed that in the PLGA conduit filled with r-ADSCs without fibrin matrix group, positive effects were obtained as compared to other implanted groups by increasing the sciatic functional index significantly (p < 0.05). In addition, the diameter nerve fibers had a significant difference mean in the PLGA conduit coated by laminin and conduit filled with r-ADSCs in fibrin matrix groups relative to the autograft group (p < 0.001). However, G-ratio and amplitude (AMP) results showed that fibrin matrix might have beneficial effects on nerve regeneration but, immunohistochemistry and real-time RT-PCR outcomes indicated that the implanted conduit which filled with r-ADSCs, with or without BDNF-CNPs and AuNPs-CNPs had significantly higher expression of S100 and MBP markers than other conduit implanted groups (p < 0.05). CONCLUSIONS: It seems, in this study differential effects of fibrin matrix, could be interfered it with other factors thereby and further studies are required to determine the distinctive effects of fibrin matrix combination with other exogenous factors in peripheral nerve regeneration.

Overexpression of miR-378 Alleviates Chronic Sciatic Nerve Injury by Targeting EZH2.

In numerous studies, microRNAs (miRNAs) have been authenticated to play vital roles in the pathophysiology of neuropathic pain and other neurological diseases. In our study, we focused on evaluating miR-378 and its potential effects in neuropathic pain development, as well as the underlying molecular mechanisms. Primarily, a chronic sciatic nerve injury (CCI) rat model was established. Next, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was employed to measure the expression levels of miR-378 and EZH2 mRNA; the EZH2 protein expression levels were detected by western blot. A luciferase activity assay monitored the interaction of miR-378 and EZH2. Mechanical and thermal hyperalgesia was also performed to quantitate the effects of overexpression of miR-378 or EZH2 on the CCI rats. We found that miR-378 was down-regulated in the CCI rats, and the overexpression of miR-378 produced significant relief in their pain management. EZH2 was the downstream gene of miR-378 and was negatively regulated by miR-378. The up-regulation of EZH2 reduced the inhibitory effects of miR-378 on the development of neuropathic pain in the CCI rats. miR-378 acts as an inhibitor in the progression of neuropathic pain via targeting EZH2; the miR-378/EZH2 axis may be a novel target for the diagnosis and therapy of neuropathic pain in clinical treatment.

Protective Effects of Curcumin Against Paclitaxel-Induced Spinal Cord and Sciatic Nerve Injuries in Rats.

Paclitaxel (PTX) is an antineoplastic agent commonly used in the treatment of solid tumors and is known to cause dose-limiting peripheral neurotoxicity. This study was performed to evaluate the protective effect of curcumin (CUR) against PTX-induced spinal cord and sciatic nerve injuries in rats. The rats were administered PTX (2 mg/kg, BW) intraperitoneally for the first 5 consecutive days followed by administration of CUR (100 and 200 mg/kg, BW daily in corn oil) orally for 10 days. Our results showed that CUR significantly reduced mRNA expression levels of NF-κB, TNF-α, IL-6, iNOS and GFAP whereas caused an increase in levels of Nrf2, HO-1 and NQO1 in the spinal cord and sciatic nerve of PTX-induced rats. In addition, CUR suppressed the activation of apoptotic and autophagic pathways by increasing Bcl-2 and Bcl-xL, and decreasing p53, caspase-3, Apaf-1, LC3A, LC3B and beclin-1 mRNA expression levels. The results showed that CUR also maintained the spinal cord and sciatic nerve histological architecture and integrity by both LFB staining and H&E staining. Immunohistochemical expressions of 8-OHdG, caspase-3 and LC3B in the PTX-induced spinal cord tissue were decreased after administration of CUR. Taken together, our findings demonstrated that CUR has protective effects on PTX-induced spinal cord and sciatic nerve injuries in rats.

Whole-body vibration therapy does not improve the peripheral nerve regeneration in experimental model.

OBJECTIVES: Whole-body vibration (WBV) is commonly used to improve motor function, balance and functional performance, but its effects on the body are not fully understood. The main objective was to evaluate the morphometric and functional effects of WBV in an experimental nerve regeneration model. METHODS: Wistar rats were submitted to unilateral sciatic nerve crush and treated with WBV (4-5 weeks), started at 3 or 10 days after injury. Functional performances were weekly assessed by sciatic functional index, horizontal ladder rung walking and narrow beam tests. Nerve histomorphometry analysis was assessed at the end of the protocol. RESULTS: Injured groups, sedentary and WBV started at 3 days, had similar functional deficits. WBV, regardless of the start time, did not alter the histomorphometry parameters in the regeneration process. CONCLUSIONS: The earlier therapy did not change the expected and natural recovery after the nerve lesion, but when the WBV starts later it seems to impair function parameter of recovery.

Toll-Like Receptor 9-Mediated Neuronal Innate Immune Reaction Is Associated with Initiating a Pro-Regenerative State in Neurons of the Dorsal Root Ganglia Non-Associated with Sciatic Nerve Lesion.

One of the changes brought about by Wallerian degeneration distal to nerve injury is disintegration of axonal mitochondria and consequent leakage of mitochondrial DNA (mtDNA)-the natural ligand for the toll-like receptor 9 (TLR9). RT-PCR and immunohistochemical or Western blot analyses were used to detect TLR9 mRNA and protein respectively in the lumbar (L4-L5) and cervical (C7-C8) dorsal root ganglia (DRG) ipsilateral and contralateral to a sterile unilateral sciatic nerve compression or transection. The unilateral sciatic nerve lesions led to bilateral increases in levels of both TLR9 mRNA and protein not only in the lumbar but also in the remote cervical DRG compared with naive or sham-operated controls. This upregulation of TLR9 was linked to activation of the Nuclear Factor kappa B (NFκB) and nuclear translocation of the Signal Transducer and Activator of Transcription 3 (STAT3), implying innate neuronal immune reaction and a pro-regenerative state in uninjured primary sensory neurons of the cervical DRG. The relationship of TLR9 to the induction of a pro-regenerative state in the cervical DRG neurons was confirmed by the shorter lengths of regenerated axons distal to ulnar nerve crush following a previous sciatic nerve lesion and intrathecal chloroquine injection compared with control rats. The results suggest that a systemic innate immune reaction not only triggers the regenerative state of axotomized DRG neurons but also induces a pro-regenerative state further along the neural axis after unilateral nerve injury.

Investigation of Neuropathology after Nerve Release in Chronic Constriction Injury of Rat Sciatic Nerve.

Peripheral compressive neuropathy causes significant neuropathic pain, muscle weakness and prolong neuroinflammation. Surgical decompression remains the gold standard of treatment but the outcome is suboptimal with a high recurrence rate. From mechanical compression to chemical propagation of the local inflammatory signals, little is known about the distinct neuropathologic patterns and the genetic signatures after nerve decompression. In this study, controllable mechanical constriction forces over rat sciatic nerve induces irreversible sensorimotor dysfunction with sustained local neuroinflammation, even 4 weeks after nerve release. Significant gene upregulations are found in the dorsal root ganglia, regarding inflammatory, proapoptotic and neuropathic pain signals. Genetic profiling of neuroinflammation at the local injured nerve reveals persistent upregulation of multiple genes involving oxysterol metabolism, neuronal apoptosis, and proliferation after nerve release. Further validation of the independent roles of each signal pathway will contribute to molecular therapies for compressive neuropathy in the future.

The Cofilin/Limk1 Pathway Controls the Growth Rate of Both Developing and Regenerating Motor Axons.

Regenerating axons often have to grow considerable distances to reestablish circuits, making functional recovery a lengthy process. One solution to this problem would be to co-opt the "temporal" guidance mechanisms that control the rate of axon growth during development to accelerate the rate at which nerves regenerate in adults. We have previously found that the loss of Limk1, a negative regulator of cofilin, accelerates the rate of spinal commissural axon growth. Here, we use mouse models to show that spinal motor axon outgrowth is similarly promoted by the loss of Limk1, suggesting that temporal guidance mechanisms are widely used during development. Furthermore, we find that the regulation of cofilin activity is an acute response to nerve injury in the peripheral nervous system. Within hours of a sciatic nerve injury, the level of phosphorylated cofilin dramatically increases at the lesion site, in a Limk1-dependent manner. This response may be a major constraint on the rate of peripheral nerve regeneration. Proof-of-principle experiments show that elevating cofilin activity, through the loss of Limk1, results in faster sciatic nerve growth, and improved recovery of some sensory and motor function.SIGNIFICANCE STATEMENT The studies shed light on an endogenous, shared mechanism that controls the rate at which developing and regenerating axons grow. An understanding of these mechanisms is key for developing therapies to reduce painful recovery times for nerve-injury patients, by accelerating the rate at which damaged nerves reconnect with their synaptic targets.

Signatures of altered DNA methylation gene expression after central and peripheral nerve injury.

Nerve damage can lead to movement and sensory dysfunction, with high morbidity and disability rates causing severe burdens on patients, families, and society. DNA methylation is a kind of epigenetics, and a great number of previous studies have demonstrated that DNA methylation plays an important role in the process of nerve regeneration and remodeling. However, compared with the central nervous system, the peripheral nervous system shows stronger recovery after injury, which is related to the complex microenvironment and epigenetic changes occurring at the site of injury. Therefore, what common epigenetic changes between the central and peripheral nervous systems remain to be elucidated. We first screened differential methylation genes after spinal cord injury and sciatic nerve injury using whole-genome bisulfite sequencing and methylated DNA immunoprecipitation sequencing, respectively. Subsequently, a total of 16 genes had the same epigenetic changes after spinal cord injury and sciatic nerve injury. The Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed to identify the critical biological processes and pathways. Furthermore, a protein-protein interaction network analysis indicated that Dnm3, Ntrk3, Smurf1, Dpysl2, Kalrn, Shank1, Dlg2, Arsb, Reln, Bmp5, Numbl, Prickle2, Map6, and Htr7 were the core genes. These outcomes may provide novel insights into the molecular mechanism of the subacute phase of nerve injury. These verified genes can offer potential diagnostic and therapeutic targets for nerve injury.