Effect of ropivacaine on peripheral neuropathy in streptozocin diabetes-induced rats through TRPV1-CGRP pathway

Abstract

Objective To determine the effect of ropivacaine on peripheral neuropathy in diabetic rats and its possible mechanism. Methods Forty-eight Sprague-Dawley rats were randomly divided into six groups: nondiabetic control group, nondiabetic group A (0.25% ropivacaine), nondiabetic group B (0.75% ropivacaine), diabetic control group (diabetic peripheral neuropathy (DPN) +artificial cerebrospinal fluid), diabetic group A (DPN+0.25% ropivacaine), and diabetic group B (DPN + 0.75% ropivacaine), with eight rats in each group. Within an hour of the last administration, the sciatic motor nerve conduction velocity (MNCV) of each group was measured, and the morphological changes of rat sciatic nerve were observed by HE, Weil's staining and electron microscopy. The expression of transient receptor potential vanilloid (TRPV1) in the spinal cord dorsal horn of rats was analyzed by immunohistochemistry, and the expression of Calcitonin gene-related peptide (CGRP) protein in the spinal cord was analyzed by Western blot. Results Compared with the nondiabetic control group, elevated blood glucose, decreased weight and reduced average mechanical withdrawal threshold (MWT), additionally, the sciatic nerves showed significantly slowed conduction velocity (both P<0.001) and damaged pathological structure, the expression of TRPV1 and CGRP were decreased (both P<0.001) in the diabetic groups. Compared with the diabetic control group, down-regulation of TRPV1 and CGRP in spinal cord was significant for the diabetic groups A and B treated with 0.25 and 0.75% ropivacaine, the higher concentration of ropivacaine correlated with a greater change. Conclusion Ropivacaine can significantly block sciatic nerve conduction velocity in DPN rats in a concentration-dependent manner, which may be related to the expression of the TRPV1-CGRP pathway.

Keywords: calcitonin gene-related peptide; diabetes; peripheral nerve; rat; ropivacaine.

Figure 1. Experimental scheme to determine the effect of intrathecally injected ropivacaine at common dosages on nerve conduction velocity and CGRP expression in the spinal dorsal horn in STZ-induced DPN rats

Figure 2. Changes in diabetic group rats

(A) The change of weight during the modeling process. (B) The change of blood glucose during the modeling process. (C) The change of reflex pain threshold during the modeling process. Values are expressed as mean ± S.E.M. (n=24 in each group). Data were analyzed by one-way ANOVA followed by Tukey’s multiple test. Data were compared with data from the control group over the same period. **P<0.01, ***P<0.001.

Figure 3. Effect of ropivacaine on pathomorphological changes of the sciatic nerve in DPN rats

(A) Histopathology changes of sciatic nerve in rats (HE, 40×). (B) Histopathology changes in sciatic nerve in rats (Weil’s myelin staining, 40×). (C) Observation of sciatic nerves in rats by transmission electron microscope (10k×). (a) Nondiabetic control group; (b) nondiabetic group A; (c) nondiabetic group B; (d) diabetic control group; (e) diabetic group A; (f) diabetic group B.

Figure 4. Effects of ropivacaine on the TRPV1-CGRP pathway in the spinal dorsal root ganglion of DPN rats

(A–G) Effect of ropivacaine on the expression of TRPV-1 in spinal dorsal horn in rats (40×). (A) nondiabetic control group; (B) nondiabetic group A; (C) nondiabetic group B; (D) diabetic control group; (E) diabetic group A; (F) diabetic group B; (G) proportion of immuno-positive cells in each group. Values are expressed as mean values ± S.E.M, n=8 in each group. Compared with the nondiabetic control group, ***P<0.001; compared with the diabetic control group, ^#P<0.05. (H,I) Effect of ropivacaine on the expression of CGRP protein in spinal cord in rats. (H) Western blot images representing the expression of CGRP in each group; (I) comparison of gray value between each group. Values are expressed as mean ± S.E.M. (n=8 in each group). Compared with the nondiabetic control group, *P<0.05, ***P<0.001; compared with the diabetic control group, ^#P<0.05, ^##P<0.01.