Impairment and restoration of the blood-nerve barrier and its correlation with pain following gradual nerve elongation of the rat sciatic nerve.

Purpose: This study aimed to evaluate the time course of impairment and restoration of the blood-nerve barrier (BNB) following gradual elongation of the sciatic nerve and to clarify its association with nociception.Materials and Methods: The right femur was lengthened at a rate of 1.5 mm/day for 10 days. Von Frey tests were performed until 50 days after lengthening. Compound muscle action potentials (CMAPs) were measured to assess gross dysfunction of the elongated nerve. Evans blue-albumin tracing and immunohistochemistry for endothelial barrier antigen (EBA), rat endothelial cell antigen-1 (RECA-1), and CD68 for qualitative and quantitative analysis of the BNB and macrophage infiltration were performed for up to 50 days after cessation of lengthening in three segments of the sciatic nerves.Results: Paw-withdrawal threshold was significantly decreased at 7 days from initiation and began to recover from day 25 after lengthening. CMAPs showed delayed latency and attenuated amplitude but recovered at day 30 after cessation. On days 10 and 30 after cessation, spotted leakage of Evans blue-albumin in the endoneurium was observed, and the ratio of EBA/RECA-1-positive microvessels was significantly decreased, which subsequently recovered simultaneously in all segments on day 50 after cessation. Macrophages did not infiltrate the BNB at any time point.Conclusion: The restoration of BNB function following gradual nerve elongation was associated with the resolution of mechanical allodynia. Our findings provide insight into the association between nerve stretch injury and chronic nociception in adult male rats, which are potentially relevant to human orthopedic procedures and chronic neuropathic pain.

Inhibition of mite-induced dermatitis, pruritus, and nerve sprouting in mice by the endothelin receptor antagonist bosentan.

BACKGROUND: Endothelin-1 (EDN1) can evoke histamine-independent pruritus in mammals and is upregulated in the lesional epidermis of atopic dermatitis (AD). EDN1 increases the production of interleukin 25 (IL-25) from keratinocytes to accelerate T helper type 2 immune deviation. Plasma EDN1 levels are positively correlated with the clinical severity and itch intensity of AD. Therefore, we hypothesized that the inhibition of EDN1 might be useful for treating atopic inflammation and itch and investigated the effects of the topical application of the EDN1 receptor antagonist bosentan on the skin inflammation and itch in a murine AD model. METHODS: We analyzed the mite-induced AD-like NC/Nga murine model, which was topically applied with bosentan or ethanol control every day for 3 weeks. We also subjected in vitro primary sensory neuron culture systems to nerve elongation and branching assays after EDN1 stimulation. RESULTS: Topical application of bosentan significantly attenuated the development of mite-induced AD-like skin inflammation, dermatitis scores, ear thickness, scratching bouts, and serum level of thymus and activation-regulated chemokine in NC/Nga mice. Bosentan application also significantly reduced the gene expression of Il13, Il17, and Ifng in the treated lesions. Histologically, the number of infiltrated dermal cells, the epidermal EDN1 expression, and the number of intraepidermal nerve fibers were significantly inhibited upon bosentan application. While EDN1 significantly elongated the neurites of dorsal root ganglion cells in a dose- and time-dependent manner, bosentan treatment attenuated this. CONCLUSIONS: EDN1 plays a significant role in mite-induced inflammation and itch. Topical bosentan is a potential protective candidate for AD.

[Experimental study of the effect of the sciatic nerve elongation on pain in rats].

OBJECTIVE: To investigate the effect of the sciatic nerve elongation on pain in rats. METHODS: Thirty-six adult male Wistar rats of SPF grade, weighing 250-300 g. Eighteen of them were randomly divided into 3 groups, 6 rats in each group. They were sciatic nerve elongation group (group A), nerve no-elongation group (group B), and nerve ligation group (group C). The model of 10-mm sciatic nerve defect was established in all 3 groups. The sciatic nerve was extended at a speed of 1 mm/d for 14 days in group A. The group B was only installed with external fixation. The nerve stumps were ligated in the group C. At 3, 7, 10, and 14 days after operation, the foot injury was evaluated by the autotomy scoring scale. At 14 days after operation, the dorsal root ganglia (DRG) of L 4-S 1 spinal cord of rats in each group was observed by tumor necrosis factor α (TNF-α) immunohistochemical staining, and the primary antibodies were replaced by pure serum as negative control group. Another 18 rats were randomly divided into 3 groups, 6 rats in each group. They were sciatic nerve elongation group (group A1), nerve no-elongation group (group B1), positive control group (group C1). In groups A1 and B1, the 10-mm long sciatic nerve defect model was established by the same method as groups A and B, and then fixed with external fixation. Nerve elongation was done or not done without anesthesia at 3 days after operation. In group C1, no modeling was done and 20 µL 2.5% formaldehyde was injected into the toes. After 90 minutes, the dorsal horn of spinal cord of L 4-S 1 segment of rats was cutting for c-Fos immunohistochemical staining and the number of positive cells was counted. Primary antibodies were replaced with pure serum as negative control group. RESULTS: The autotomy scores of rats in groups B and C gradually increased postoperatively, and group A remained stable at 0.25±0.50. The scores of group C were significantly higher than those of group A and group B at each time point postoperatively ( P<0.05). The scores of group A were significantly lower than those of group B at 10 and 14 days postoperatively ( P<0.05). TNF-α immunohistochemical staining showed that the TNF-α expression in group A was weak, slightly positive (+/-); in group B was positive (+); in group C was strongly positive (++); and the negative control group had no TNF-α expression (-). c-Fos immunohistochemical staining showed that the c-Fos expressions in groups A1 and B1 were weak positive, in group C1 was strong positive, and negative control group had no c-Fos positive expression. The number of c-Fos positive cells in groups A1, B1, C1, and negative control group were (21.5±6.6), (19.3±8.1), (95.6±7.4), and 0 cells/field, respectively, and group C1 was significantly higher than groups A1 and B1 ( P<0.05), there was no significant difference between group A1 and group B1 ( P>0.05). CONCLUSION: Nerve elongation does not cause obvious pain neither during the operation of elongation nor throughout the whole elongation.

Functional, electrophysiological recoveries of rats with sciatic nerve lesions following transplantation of elongated DRG cells.

OBJECTIVES: Functional data are essential when confirming the efficacy of elongated dorsal root ganglia (DRG) cells as a substitute for autografting. We present the quantitative functional motor, electrophysiological findings of engineered DRG recipients for the first time. METHODS: Elongated DRG neurons and autografts were transplanted to bridge 1-cm sciatic nerve lesions of Sprague Dawley (SD) rats. Motor recoveries of elongated DRG recipients (n=9), autograft recipients (n=9), unrepaired rats (n=9) and intact rats (n=6) were investigated using the angle board challenge test following 16 weeks of recovery. Electrophysiology studies were conducted to assess the functional recovery at 16 weeks. In addition, elongated DRGs were subjected to histology assessments. RESULTS: At threshold levels (35° angle) of the angle board challenge test, the autograft recipients', DRG recipients' and unrepaired group's performances were equal to each other and were less than the intact group (p<0.05). However, during the subthreshold (30°) angle board challenge test, the elongated DRG recipients' performance was similar to both the intact group and the autograft nerve recipients, and was better (p<0.05) than the unrepaired group. The autograft recipients' performance was similar to the unrepaired group and was significantly different (p<0.05) compared with the performance of the intact group. During electrophysiological testing, the rats with transplanted engineered DRG constructs had intact signal transmission when recorded over the lesion, while the unrepaired rats did not. It was observed that elongated DRG neurons closely resembled an autograft during histological assessments. CONCLUSION: Performances of autograft and elongated DRG construct recipients were similar. Elongated DRG neurons should be further investigated as a substitute for autografting.

Role of sodium channels in recovery of sciatic nerve-stretch injury in rats.

INTRODUCTION: To elucidate the mechanism of functional recovery after gradual nerve-stretch injury, we used rats in which the femur length was increased by 15 mm at 1.5 mm/day. METHODS: We performed electrophysiology, mRNA analysis of tetrodotoxin-resistant voltage-gated sodium channels (TTX-R VGSCs) in dorsal root ganglia, and histology of unmyelinated sciatic nerve fibers and examined pain thresholds at 1, 10, 20, and 30 days after cessation of lengthening. RESULTS: Electrophysiology revealed conduction block after cessation that recovered after 30 days. TTX-R VGSC levels decreased immediately after cessation but were restored after 10 (Nav1.9) or 20 (Nav1.8) days. Histology revealed that injured unmyelinated nerve fibers regenerate 30 days after cessation. Pain threshold decreased gradually during lengthening but had not recovered to the control group level after 30 days. CONCLUSIONS: Early restoration of TTX-R VGSC mRNA in dorsal root ganglia preceded functional recovery of stretched nerves before regeneration of injured unmyelinated nerve fibers.