Characteristics of cytokines in the sciatic nerve stumps and DRG 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(DRG) 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 SN 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 DRG 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 SN and DRG 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.

Biological characteristics of dynamic expression of nerve regeneration related growth factors in dorsal root ganglia after peripheral nerve injury

The regenerative capacity of peripheral nerves is limited after nerve injury.A number of growth factors modulate many cellular behaviors,such as proliferation and migration,and may contribute to nerve repair and regeneration.Our previous study observed the dynamic changes of genes in L4–6 dorsal root ganglion after rat sciatic nerve crush using transcriptome sequencing.Our current study focused on upstream growth factors and found that a total of 19 upstream growth factors were dysregulated in dorsal root ganglions at 3,9 hours,1,4,or 7 days after nerve crush,compared with the 0 hour control.Thirty-six rat models of sciatic nerve crush injury were prepared as described previously.Then,they were divided into six groups to measure the expression changes of representative genes at 0,3,9 hours,1,4 or 7 days post crush.Our current study measured the expression levels of representative upstream growth factors,including nerve growth factor,brain-derived neurotrophic factor,fibroblast growth factor 2 and amphiregulin genes,and explored critical signaling pathways and biological process through bioinformatic analysis.Our data revealed that many of these dysregulated upstream growth factors,including nerve growth factor,brain-derived neurotrophic factor,fibroblast growth factor 2 and amphiregulin,participated in tissue remodeling and axon growth-related biological processes Therefore,the experiment described the expression pattern of upstream growth factors in the dorsal root ganglia after peripheral nerve injury.Bioinformatic analysis revealed growth factors that may promote repair and regeneration of damaged peripheral nerves.All animal surgery procedures were performed in accordance with Institutional Animal Care Guidelines of Nantong University and ethically approved by the Administration Committee of Experimental Animals,China(approval No.20170302-017)on March 2,2017.

Blockade of transient receptor potential cation channel subfamily V member 1 promotes regeneration after sciatic nerve injury

The transient receptor potential cation channel subfamily V member 1（TRPV1） provides the sensation of pain（nociception）. However, it remains unknown whether TRPV1 is activated after peripheral nerve injury, or whether activation of TRPV1 affects neural regeneration. In the present study, we established rat models of unilateral sciatic nerve crush injury, with or without pretreatment with AMG517（300 mg/kg）, a TRPV1 antagonist, injected subcutaneously into the ipsilateral paw 60 minutes before injury. At 1 and 2 weeks after injury, we performed immunofluorescence staining of the sciatic nerve at the center of injury, at 0.3 cm proximal and distal to the injury site, and in the dorsal root ganglia. Our results showed that Wallerian degeneration occurred distal to the injury site, and neurite outgrowth and Schwann cell regeneration occurred proximal to the injury. The number of regenerating myelinated and unmyelinated nerve clusters was greater in the AMG517-pretreated rats than in the vehicle-treated group, most notably 2 weeks after injury. TRPV1 expression in the injured sciatic nerve and ipsilateral dorsal root ganglia was markedly greater than on the contralateral side. Pretreatment with AMG517 blocked this effect. These data indicate that TRPV1 is activated or overexpressed after sciatic nerve crush injury, and that blockade of TRPV1 may accelerate regeneration of the injured sciatic nerve.

Acetyl-11-keto-β-boswellic acid extracted from Boswellia serrata promotes Schwann cell proliferation and sciatic nerve function recovery

Frankincense can promote blood circulation. Acetyl-11-keto-β-boswellic acid （AKBA） is a small molecule with anti-inflammatory properties that is derived from Boswellia serrata. Here, we hypothesized that it may promote regeneration of injured sciatic nerve. To address this hypothesis, we established a rat model of sciatic nerve injury using a nerve clamping method. Rats were administered AKBA once every 2 days at doses of 1.5, 3, and 6 mg/kg by intraperitoneal injection for 30 days from the 1st day after injury. Sciatic nerve function was evaluated using the sciatic functional index. Degree of muscle atrophy was measured using the triceps surae muscle Cuadros index.Neuropathological changes were observed by hematoxylin-eosin staining. Western blot analysis was used to detect expression of phospho-extracellular signal-regulated kinase 1 and 2 （p-ERK1/2） in injured nerve. S100 immunoreactivity in injured nerve was detected by immunohistochemistry. In vivo experiments showed that 3 and 6 mg/kg AKBA significantly increased sciatic nerve index, Cuadros index of triceps muscle, p-ERK1/2 expression, and S100 immunoreactivity in injured sciatic nerve of sciatic nerve injury model rats. Furthermore,for in vitro experiments, Schwann cells were treated with AKBA at 0–20 μg/mL. Proliferation of Schwann cells was detected by Cell Counting Kit-8 colorimetry assay. The results showed that 2 μg/mL AKBA is the optimal therapeutic concentration. In addition, ERK phosphorylation levels increased following 2 μg/mL AKBA treatment. In the presence of the ERK1/2 inhibitor, PD98059 （2.5 μL/mL）, the AKBA-induced increase in p-ERK1/2 protein expression was partially abrogated. In conclusion, our study shows that AKBA promotes peripheral nerve regeneration with ERK protein phosphorylation playing a key role in this process.