[Effect of short-term low-frequency electrical stimulation on nerve regeneration of delayed nerve defect during operation]

Abstract

Objective: To explore the effect of short-term low-frequency electrical stimulation (SLES) during operation on nerve regeneration in delayed peripheral nerve injury with long gap.

Methods: Thirty female adult Sprague Dawley rats, weighing 160-180 g, were used to prepare 13-mm defect model by trimming the nerve stumps. Then all rats were randomly divided into 2 groups, 15 rats in each group. After nerve defect was bridged by the contralateral normal sciatic nerve, SLES was applied in the experimental group, but was not in the control group. The spinal cords and dorsal root ganglions (DRGs) were harvested to carry out immunofluorescence histochemistry double staining for growth-associated proteins 43 (GAP-43) and brain-derived neurotrophic factor (BDNF) at 1, 2, and 7 days after repair. Fluorogold (FG) retrograde tracing was performed at 3 months after repair. The mid-portion regenerated segments were harvested to perform Meyer's trichrome staining, immunofluorescence double staining for neurofilament (NF) and soluble protein 100 (S-100) on the transversely or longitudinal sections at 3 months after repair. The segment of the distal sciatic nerve trunk was harvested for electron microscopy and morphometric analyses to measure the diameter of the myelinated axons, thickness of myelin sheaths, the G ratio, and the density of the myelinated nerve fibers. The gastrocnemius muscles of the operated sides were harvested to measure the relative wet weight ratios. Karnovsky-Root cholinesterase staining of the motor endplate was carried out.

Results: In the experimental group, the expressions of GAP-43 and BDNF were higher than those in the control group at 1 and 2 days after repair. The number of labeled neurons in the anterior horn of gray matter in the spinal cord and DRGs at the operated side from the experimental group was more than that from the control group. Meyer's trichrome staining, immunofluorescence double staining, and the electron microscopy observation showed that the regenerated nerves were observed to develop better in the experimental group than the control group. The relative wet weight ratio of experimental group was significantly higher than that of the control group ( t=4.633, P=0.000). The size and the shape of the motor endplates in the experimental group were better than those in the control group.

Conclusion: SLES can promote the regeneration ability of the short-term (1 month) delayed nerve injury with long gap to a certain extent.

Keywords: Short-time low-frequency electrical stimulation; chronic injury; nerve defect repair; peripheral nerve; rat.

图 1

Immunofluorescence double staining for GAP-43 and BDNF on the transversely sectioned spinal cord in 2 groups after repair (Fluorescence microscope×160)　From left to right was GAP-43 staining, BDNF staining, and merge a. Experimental group at 1 day; b. Experimental group at 2 days; c. Experimental group at 7 days; d. Control group at 1 day; e. Control group at 2 days; f. Control group at 7 days 两组修复术后各时间点脊髓横切面抗 GAP-43 及 BDNF 双重免疫荧光染色观察（倒置荧光显微镜×160） 从左至右依次为 GAP-43 染色、BDNF 染色及合图 a. 实验组 1 d；b. 实验组 2 d；c. 实验组 7 d；d. 对照组 1 d；e. 对照组 2 d；f. 对照组 7 d

图 2

Immunofluorescence double staining for GAP-43 and BDNF on the transversely sectioned DRGs in 2 groups after repair (Fluorescence microscope×160)　From left to right was GAP-43 staining, BDNF staining, and merge　 a. Experimental group at 1 day; b. Experimental group at 2 days; c. Experimental group at 7 days; d. Control group at 1 day; e. Control group at 2 days; f. Control group at 7 days 两组修复术后各时间点背根神经节横切面抗 GAP-43 和 BDNF 双重免疫荧光染色观察（倒置荧光显微镜×160） 从左至右依次为 GAP-43 染色、BDNF 染色及合图　a. 实验组 1 d；b. 实验组 2 d；c. 实验组 7 d；d. 对照组 1 d；e. 对照组 2 d；f. 对照组 7 d

图 3

Fluorescence microscope observation at 1 week after FG retrograde tracing in 2 groups (×160)　a. Spinal cord of experimental group; b. DRGs of experimental group; c. Spinal cord of control group; b. DRGs of control group 两组注射荧光金后 1 周倒置荧光显微镜观察（×160）　a. 实验组脊髓；b. 实验组背根神经节；c. 对照组脊髓；d. 对照组背根神经节

图 4

Gross view of regenerated nerve at 3 months after repair in 2 groups　a. Experimental group; b. Control group 两组修复术后 3 个月再生神经大体观察　a. 实验组；b. 对照组

图 5

Meyer’s trichrome staining observation on transversely sectioned mid-portion of the regenerated segment at 3 months after repair in 2 groups　a. Experimental group (×160); b. Experimental group (×640); c. Control group (×160); d. Control group (×640) 两组修复术后 3 个月再生神经中段横切面 Meyer 神经三色染色观察　a. 实验组（×160）；b. 实验组（×640）；c. 对照组（×160）；d. 对照组（×640）

图 6

Immunofluorescence double staining for NF and S-100 on the longitudinal sectioned mid-portion of the regenerated segment at 3 months after repair in 2 groups (Fluorescence microscope×160)　From left to right was NF staining, S-100 staining, and merge　 a. Experimental group; b. Control group 两组修复术后 3 个月再生神经中段纵切面抗 NF 与 S-100 双重免疫荧光染色（倒置荧光显微镜×160）　从左至右依次为 NF 染色、S-100 染色及合图　a. 实验组；b. 对照组

图 7

Transmission electron microscope observation of distal sciatic nerve at 3 months after repair in 2 groups　a. Experimental group (×6 000); b. Experimental group (×50 k); c. Control group (×6 000) 两组修复术后 3 个月神经组织透射电镜观察　a. 实验组（×6 000）；b. 实验组（×50 k）；c. 对照组（×6 000）

图 8

Karnovsky-Root cholinesterase staining observation of the motor endplate on transverse sections of gastrocnemius muscle at the operated side at 3 months after repair in 2 groups　a. Experimental group (×160); b. Experimental group (×640); c. Control group (×160); d. Control group (×640) 两组修复术后 3 个月术侧腓肠肌 Karnovsky-Root 运动终板胆碱酯酶组织学染色　a. 实验组（×160）；b. 实验组（×640）；c. 对照组（×160）；d. 对照组（×640）