Changes of Functional, Morphological, and Inflammatory Reactions in Spontaneous Peripheral Nerve Reinnervation after Thermal Injury.

In recent decades, the use of energy-based devices has substantially increased the incidence of iatrogenic thermal injury to nerves (cauterization, etc.). While recovery of the nerve after thermal injury is important, the changes in neural structure, function, and peripheral inflammatory reactions postinjury remain unclear. This study is aimed at demonstrating the changes mentioned above during the acute, subacute, and chronic stages of nerve reinnervation after thermal injury. Spontaneous reinnervation was evaluated, including the neural structures, nerve conduction abilities, and muscle regeneration. These effects vary depending on the severity of thermal injury (slight, moderate, and severe). Peripheral inflammatory reactions, as impediments to reinnervation, were found in significant numbers 3 days after thermal injury, exhibiting high expression of IL-1ß and TNF-α, but low expression of IL-10. Our findings reveal the pathogenesis of peripheral nerve reinnervation after thermal injury, which will assist in selecting appropriate treatments in further research.

Exosome-Mediated miR-21 Was Involved in the Promotion of Structural and Functional Recovery Effect Produced by Electroacupuncture in Sciatic Nerve Injury.

PURPOSE: Our study is aimed at investigating the mechanism by which electroacupuncture (EA) promoted nerve regeneration by regulating the release of exosomes and exosome-mediated miRNA-21 (miR-21) transmission. Furthermore, the effects of Schwann cells- (SC-) derived exosomes on the overexpression of miR-21 for the treatment of PNI were investigated. METHODS: A sciatic nerve injury model of rat was constructed, and the expression of miR-21 in serum exosomes and damaged local nerves was detected using RT-qPCR after EA treatment. The exosomes were identified under a transmission electron microscope and using western blotting analysis. Then, the exosome release inhibitor, GW4869, and the miR-21-5p-sponge used for the knockdown of miR-21 were used to clarify the effects of exosomal miR-21 on nerve regeneration promoted by EA. The nerve conduction velocity recovery rate, sciatic nerve function index, and wet weight ratio of gastrocnemius muscle were determined to evaluate sciatic nerve function recovery. SC proliferation and the level of neurotrophic factors were assessed using immunofluorescence staining, and the expression levels of SPRY2 and miR-21 were detected using RT-qPCR analysis. Subsequently, the transmission of exosomal miR-21 from SC to the axon was verified in vitro. Finally, the exosomes derived from the SC infected with the miR-21 overexpression lentivirus were collected and used to treat the rat SNI model to explore the therapeutic role of SC-derived exosomes overexpressing miR-21. RESULTS: We found that EA inhibited the release of serum exosomal miR-21 in a PNI model of rats during the early stage of PNI, while it promoted its release during later stages. EA enhanced the accumulation of miR-21 in the injured nerve and effectively promoted the recovery of nerve function after PNI. The treatment effect of EA was attenuated when the release of circulating exosomes was inhibited or when miR-21 was downregulated in local injury tissue via the miR-21-5p-sponge. Normal exosomes secreted by SC exhibited the ability to promote the recovery of nerve function, while the overexpression of miR-21 enhanced the effects of the exosomes. In addition, exosomal miR-21 secreted by SC could promote neurite outgrowth in vitro. CONCLUSION: Our results demonstrated the mechanism of EA on PNI from the perspective of exosome-mediated miR-21 transport and provided a theoretical basis for the use of exosomal miR-21 as a novel strategy for the treatment of PNI.

Neuroprotective Effects of Ozone Therapy After Sciatic Nerve Cut Injury.

A number of antioxidants have been used to treat peripheral nerve injury. However, there are few definitive experimental studies of ozone therapy for peripheral nerve cut injury. We aimed to examine the effects of mild level ozone therapy on sciatic nerve regeneration. One hundred adult male Wistar albino rats were randomly divided into four groups: group 1 (n=20) no cut injury or therapy; group 2 (n=20) sham; group 3 (n=30) nerve cut injury, no therapy; group 4 (n=30) nerve cut injury and ozone therapy. Sciatic functional index (SFI) and withdrawal reflex (WDR) were measured for all groups before nerve cut, at postoperative day 1, and at weeks 2, 4, 6 and 8. More myelinated (M) nerve fibers were observed after nerve cut injury in the ozone-therapy group. Significant differences were seen in plasma SOD (superoxide dismutase), CAT (catalase) and GPx (glutathione peroxidase) activities (p<0.05), and significant functional improvement was observed at postoperative weeks 2 and 4 (p<0.05) after ozone treatment. This is the first study conducted for the purpose of examining the effects of ozone therapy on sciatic nerve cut injury.

Frontline Science: Blood-circulating leukocytes fail to infiltrate the spinal cord parenchyma after spared nerve injury.

The development of neuropathic pain after peripheral nerve injury involves neuroimmune-glial interactions in the spinal cord. However, whether the development of neuropathic pain depends on the infiltration of peripheral immune cells, such as monocytes, into the spinal cord parenchyma after peripheral nerve damage remains unclear. Here, we used a combination of different techniques such as transgenic reporter mouse (Cx3cr1GFP/+ and Ccr2RFP/+ mice), bone marrow chimeric mice, and parabiosis to investigate this issue in spared nerve injury (SNI) model. Herein, we provided robust evidence that, although microglial cells are activated/proliferate at the dorsal horn of the spinal cord after SNI, peripheral hematopoietic cells (including monocytes) are not able to infiltrate into the spinal cord parenchyma. Furthermore, there was no evidence of CCR2 expression in intrinsic cells of the spinal cord. However, microglial cells activation/proliferation in the spinal cord and mechanical allodynia after SNI were reduced in Ccr2-deficient mice. These results suggest that blood-circulating leukocytes cells are not able to infiltrate the spinal cord parenchyma after distal peripheral nerve injury. Nevertheless, they indicate that CCR2-expressing cells might be indirectly regulating microglia activation/proliferation in the spinal cord after SNI. In conclusion, our study supports that CCR2 inhibition could be explored as an interventional approach to reduce microglia activation and consequently neuropathic pain development after peripheral nerve injury.

Circulating messenger for neuroprotection induced by molecular hydrogen.

Molecular hydrogen (H2) showed protection against various kinds of oxidative-stress-related diseases. First, it was reported that the mechanism of therapeutic effects of H2 was antioxidative effect due to inhibition of the most cytotoxic reactive oxygen species, hydroxy radical (•OH). However, after chronic administration of H2 in drinking water, oxidative-stress-induced nerve injury is significantly attenuated even in the absence of H2. It suggests indirect signaling of H2 and gastrointestinal tract is involved. Indirect effects of H2 could be tested by giving H2 water only before nerve injury, as preconditioning. For example, preconditioning of H2 for certain a period (∼7 days) in Parkinson's disease model mice shows significant neuroprotection. As the mechanism of indirect effect, H2 in drinking water induces ghrelin production and release from the stomach via ß1-adrenergic receptor stimulation. Released ghrelin circulates in the body, being transported across the blood-brain barrier, activates its receptor, growth-hormone secretagogue receptor. H2-induced upregulation of ghrelin mRNA is also shown in ghrelin-producing cell line, SG-1. These observations help with understanding the chronic effects of H2 and raise intriguing preventive and therapeutic options using H2.

Supplementation of Cannabis sativa L. leaf powder accelerates functional recovery and ameliorates haemoglobin level following an induced injury to sciatic nerve in mouse model.

Peripheral nerve injury is a common condition with a multitude of signs and symptoms. The major consequence of injury is limited physical activity. Presently, we are lacking effective therapies for PNI and it is need of the hour is to explore potential remedies for the recovery of functional loss. Here, we have investigated the role of crude Cannabis sativa L. leaf powder in promoting functions recovery, in mouse model subjected to a traumatic sciatic nerve injury. A dose of 200mg/kg of the body weight per day was administered orally from the day of nerve crush till the end of the experiment. The motor functions were evaluated by measuring sciatic functional index, muscle grip strength and muscle mass; whereas the sensory functions were assessed by hotplate test. The haematology and serum analyses were carried out to estimate the effect of treatment on the systemic index and oxidative stress. The gain of motor functions was significantly improved and was early noticed in the treated mice. Restoration of muscle mass and elevated haemoglobin level were statistically significant in the treatment group. This study indicates that Cannabis sativa L. supplementation accelerates the motor functions recovery after nerve compression injury.

Sesame oil improves functional recovery by attenuating nerve oxidative stress in a mouse model of acute peripheral nerve injury: role of Nrf-2.

Peripheral nervous injury (PNI) is a common form of trauma in modern society, especially in sport players. Despite the advance of therapy for PNI, the recovery of function can never reach the preinjury level after treatments. Recently, inhibiting neural oxidative stress shows a beneficial effect in improving functional recovery after PNI. In addition, sesame oil has been reported to possess the excellent antioxidative properties. However, whether sesame oil can improve the functional recovery after PNI by its antioxidative effect has never been investigated. Thirty mice were randomly divided into five groups of six: group I mice received sham operation; group II mice received sciatic nerve crush; and groups III-V mice daily ingested 0.5, 1 and 2 ml/kg of sesame oil for 6 days, respectively, after sciatic nerve crush. Oxidative stress, GAP43 and nuclear Nrf2 levels as well as spinal somatosensory evoked potentials were assessed on day 6, while paw withdrawal latency and sciatic function index were assessed on days 0, 3, and 6. Sesame oil significantly decreased lipid peroxidation and increased nuclear factor erythroid 2-related factor 2 and GAP43 expression in sciatic nerve. Furthermore, sesame oil improved electrophysiological and functional assessments in mice with sciatic nerve crush. In conclusion, sesame oil may improve nerve functional recovery by attenuating nerve oxidative stress in mouse acute peripheral nerve injury. Further, application of natural product sesame oil may be an alternative approach for improving nerve functional recovery in the clinical setting.

Sciatic nerve regeneration in rats subjected to ketogenic diet.

OBJECTIVES: Ketogenic diet (KD) is a high-fat-content diet with insufficiency of carbohydrates that induces ketogenesis. Besides its anticonvulsant properties, many studies have shown its neuroprotective effect in central nervous system, but its influence on peripheral nervous system has not been studied yet. We examined the influence of KD on regeneration of peripheral nerves in adult rats. METHODS: Fifty one rats were divided into three experimental (n = 15) and one control (n = 6) groups. Right sciatic nerve was crushed and animals were kept on standard (ST group) or ketogenic diet, the latter was introduced 3 weeks before (KDB group) or on the day of surgery (KDA group). Functional (CatWalk) tests were performed once a week, and morphometric (fiber density, axon diameter, and myelin thickness) analysis of the nerves was made after 6 weeks. Body weight and blood ketone bodies level were estimated at the beginning and the end of experiment. RESULTS: Functional analysis showed no differences between groups. Morphometric evaluation showed most similarities to the healthy (uncrushed) nerves in KDB group. Nerves in ST group differed mostly from all other groups. Ketone bodies were elevated in both KD groups, while post-surgery animals' body weight was lower as compared to ST group. DISCUSSION: Regeneration of sciatic nerves was improved in KD - preconditioned rats. These results suggest a neuroprotective effect of KD on peripheral nerves.

Anti-Ganglioside Antibodies Induce Nodal and Axonal Injury via Fcγ Receptor-Mediated Inflammation.

Guillain-Barré syndrome (GBS) is a postinfectious autoimmune neuropathy and anti-ganglioside antibodies (Abs) are strongly associated with this disorder. Several studies have implied that specific anti-ganglioside Abs induce neuropathy in patients with axonal forms of GBS. To study the mechanisms of anti-ganglioside Abs-induced neuropathy, we established a new passive transfer mouse model by L5 spinal nerve transection (L5SNT; modified Chung's model) and systemic administration of anti-ganglioside Abs. L5SNT causes degeneration of a small proportion of fibers that constitute sciatic nerve and its branches, but importantly breaks the blood-nerve barrier, which allows access to circulating Abs and inflammatory cells. Our studies indicate that, in this mouse model, anti-ganglioside Abs induce sequential nodal and axonal injury of intact myelinated nerve fibers, recapitulating pathologic features of human disease. Notably, our results showed that immune complex formation and the activating Fc gamma receptors (FcγRs) were involved in the anti-ganglioside Abs-mediated nodal and axonal injury in this model. These studies provide new evidence that the activating FcγRs-mediated inflammation plays a critical role in anti-ganglioside Abs-induced neuropathy (injury to intact nerve fibers) in GBS.

Ghrelin: a novel neuromuscular recovery promoting factor?

Promoting neuromuscular recovery after neural injury is a major clinical issue. While techniques for nerve reconstruction are continuously improving and most peripheral nerve lesions can be repaired today, recovery of the lost function is usually unsatisfactory. This evidence claims for innovative nonsurgical therapeutic strategies that can implement the outcome after neural repair. Although no pharmacological approach for improving posttraumatic neuromuscular recovery has still entered clinical practice, various molecules are explored in experimental models of neural repair. One of such molecules is the circulating peptide hormone ghrelin. This hormone has proved to have a positive effect on neural repair after central nervous system lesion, and very recently its effectiveness has also been demonstrated in preventing posttraumatic skeletal muscle atrophy. By contrast, no information is still available about its effectiveness on peripheral nerve regeneration although preliminary data from our laboratory suggest that this molecule can have an effect also in promoting axonal regeneration after nerve injury and repair. Should this be confirmed, ghrelin might represent an ideal candidate as a therapeutic agent for improving posttraumatic neuromuscular recovery because of its putative effects at all the various structural levels involved in this regeneration process, namely, the central nervous system, the peripheral nerve, and the target skeletal muscle.

Estradiol promotes neural stem cell differentiation into endothelial lineage and angiogenesis in injured peripheral nerve.

Neural stem cells (NSCs) differentiate into endothelial cells (ECs) and neuronal cells. Estradiol (E2) is known to exhibit proangiogenic effects on ischemic tissues via EC activation. Therefore, we hypothesized that E2 can promote the therapeutic potential of NSC transplantation for injured nerve repair via the differentiation of NSCs into ECs during neovascularization. NSCs isolated from newborn mouse brains were transplanted into injured sciatic nerves with (NSC/E2 group) or without E2-conjugated gelatin hydrogel (E2 group). The NSC/E2 group exhibited the greatest recovery in motor nerve conduction velocity, voltage amplitude, and exercise tolerance. Histological analyses revealed increased intraneural vascularity and blood perfusion as well as striking NSC recruitment to the neovasculature in the injured nerves in the NSC/E2 group. In vitro, E2 enhanced the NSC migration and proliferation inhibiting apoptosis. Fluorescence-activated cell sorting analysis also revealed that E2 significantly increased the percentage of CD31 in NSCs, and the effect of E2 was completely neutralized by the estrogen receptor antagonist ICI. The combination of E2 administration and NSC transplantation cooperatively improved the functional recovery of injured peripheral nerves, at least in part, via E2-associated NSC differentiation into ECs. These findings provide a novel mechanistic insight into both NSC biology and the biological effects of endogenous E2.