Experimental Study Of The End-to-side Bypassed Nerve Grafting To A Peroneal Neuroma-in-continuity

Part1 The model of neuroma-in-continuity in rabbitsObjective: In view of being short of the mammalian model in neuroma-in-continuity, the experiment injured the part of peroneal nerve to the formation of the neuroma-in- continuity and was applied to the foundation of farther research.Methods: A longitudinal incision was made on the posterior aspect of the lower limb of sixteen Newzeland rabbits extending from mid-thigh to mid-calf. The biceps femoris was retracted to fully expose the peroneal, tibial, and sural branches of the sciatic nerve at the popliteal fossa. The peroneal nerve was found crossing the posterior tibial vessels, the posterior tibial vasculars that was used as a landmark for separating the peroneal nerve to the distal part into the medial and lateral fasciculus. It was used the vasculars as a landmark for partial neurectomy. A 15-mm lateral fascicule of the peroneal nerve was then removed using microsurgical scissors. At week 3, 4, 5, and 6 postoperatively, two rabbits of each time interval were re-anesthetized, and the peroneal nerves at the partial neurectomy sites were exposed for gross examination, and the nerve were then harvested for histological analysis. Slices were stained with hematoxylin-eosin. The slices with typical changes of neuroma were selected for further luxol fast blue and Van-Gieson staining. At week 6 postoperatively, the both peroneal nerves of remained eight rabbits were exposed under anesthesia and underwent electrophysiological test. Results: 1. Histological Analysis : When the wound was re-opened for gross examination at week four after surgery, the peroneal nerve with partial neurectomy were moderately adhered to the adjacent muscle, the neuroma-like scar tissue was found at the proximal neurectomy in the nerve trunk.From the fourth postoperative week, the nerve fasciculus was found dispersed into the matrix of mucus form the proximal stump of neurectomy. The fasciculus included the mixed regenerated and distorted axons, the proliferated schwann' s cells and fibroblast cells and fibers. The schwann' s cells were arranged to the shape of the mesh, whirlpool, and water drop. At the sixth postoperative week, the histology showed increase of hyaline degeneration and formation of scar tissue. Under luxol fast blue staining, the collagenous fiber of the neuromas was shown to red, and nervous tissue was shown blue . The collagenous fiber was red, and nervous tissue was yellow under Van-Gieson staining. 2. Electromyography test:The mean conduction velocity of the normal peroneal nerve was 59. 77 ± 9. 35 m/s. However, MNCV was 32. 74±7. 54 m/s in the nerve with neuroma formation at sixth postoperative week, which was significantly slower than that of the normal nerve (p<0.001). The waveform of CMAP was in regular pattern in the normal peroneal nerve, and the amplitude was 19. 40 ± 6. 89 mv. In the nerve with neuroma formation, the waveform was showed scattered, and the amplitude was significantly reduced when compared to the normal (6.95 ± 2.48 mv, p<0. 01). The denervated changes with sharp wave and fibrillation potential were recorded from the tibialis anterior muscle at the site of neuroma.Conclusion: The method of partly injuring the peroneal nerve can effectively set up the model of the neuroma-in-continuity, as an ideal model to investigate the effect of the treatment in neuroma-in-continuity, it is stable and practical.Part2 Expression changes of molecular makers in the rabbits'model of neuroma-in-continuityObject: The peroneal nerves were injuried and formed the neuroma-in-continuity. The expression amount of Ciliary neurotrophic factor (CNTF ) in schwann' s cells of injured nerve is synchronized with the nerve functional state. At spinal cord level, Calcitonin gene related peptide (CGRP) can promote the transmission of the pain signal and represent the substance relative to the neuropathic pain. We selected the molecular markers to determine their changes in correlate with formation of neuroma-in-continuity.Methods: One lateral faciculus of the six Newzland rabbits' peroneal nerves were resected,At the six week.it formed the model of the neuroma-in-continuity . RNA from the injured nerves and dorsal root ganglions of 2mg was isolated .After treatment with DNase I, one-tenth of each RNA sample was reverse-transcribed with random hexamers by superscript II. Real-time QPCR amplifications were performed using the Taqman probes specific for the target genes. GAPDH mRNA from the same tissue lysate was used as an internal control. The size for the PCR amplified CNTF, CGRP, and GAPDH fragments were 80, 101, and 103 bp, respectively. The real-time PCR signals were analyzed using iCycler software. The relative expression level of the gene of interest was computed with respect to the mRNA expression level of the internal standard GAPDH. Protein samples from rabbit nerves were separated on 10% sodium dodecyl- sulphate-polyacry-lamide gels and transferred onto polyvinylidene difluoride membrane, which were then blocked with 5% nonfat milk and incubated separately with goat anti-CGRP and anti-CNTF polyclonal antibody overnight. The proteins were then probed with a rabbit peroxidase-conjugated anti-goat secondary antibody, detected by enhanced chemiluminescence with the Supersignal Substrate, and exposed to X-ray film. The CNTF and CGRP protein expressions were presented with respect to the β— actinexpression.Results: 1. Real-time QPCR:The relative mRNA content of CNTF in the normal peroneal nerve was 1.28E+06 ± 2.08E+05. At the nerve with neuroma formation, the CNTF was 7.04E+05 ± 8. 56E+04 (p<0. 05). The relative mRNA content of CGRP in the dorsal root ganglions at the healthy peroneal nerve side was 8. 76E+03 ± 7. 22E+02. The CGRP mRNA in the dorsal root ganglions was 2. 73E+04 ± 7. 30E+0 3 at the neuroma side (p<0. 05). 2. Western-blot:the relative ratio of CNTF in the normal peroneal nerve was 0.94 ± 0.06. At the nerve with neuroma formation, the ratio was 0. 66 ± 0. 05 (p<0. 01). The relative ratio of CGRP in the dorsal root ganglions at the healthy peroneal nerve side was 1.06 ± 0.09. The ratio of CGRP protein in the dorsal root ganglions was 1.86 ± 0.16 at the neuroma side (p<0. 01). Conclusion: The method of partly injuring the peroneal nerve could effectively set up the model of the neuroma-in-continuity, furthermore , resulted to the expression changes of the CNTF, CGRP mRNA and protein. Part3Exprimental study of the end-to-side bypassed nerve grafting to a peroneal neuroma-in-continuityObject: The present study tests the outcome of a neuroma-in-continuity bypassed with an end-to-side bridge graft, intend to increase the functional recovery and explore the experimental foundation for this disease' treatmnet.Methods: The 36 rabbits of the neuroma-in-continuity models were divided into three groups (A, B, C) randomly. At the four week, Group A was bypassed with an end-to-side bridge graft. At the six week, group B was treated by segmental resection with interposition grafting. Group C was used as control one. At the 20 weeks postoperatively, Each group of was equally divided into two groups . After the electrophysiological test, thespecimens of the nerves and the muscles were taken , stained by toluidine blue and HE respectively in group A₁ , B₁, C₁.The numbers of myelinated fibers and the areas of myelinated fibers were quantitated by image analysis system. The section areas of tibialis anterior muscles were determineded by the image analysis system. The histological and ultrastructural observation to the regenerated nerve and target organ were evaluated by the transmission electron microscopy. At the same time, the peroneal nerves and the dorsal root ganglions were biopsed for the examination of Real-time QPCR and western-blot in group A₂ , B₂, C₂. Results: 1. EMG: The motor nerve velocity and compound motor action potential of the tibialis anterior muscles were significantly increased in group A₁, B₁ as compared to controls (P<0. 05) . Group B₁ compared to group A₁, there were statistical difference in the motor nerve velocity and compound motor action potential of the tibialis anterior (P<0. 05) .The experimental groups (A₁ and B₁) could present low amplitude, polyphasic and late potentials. 2. Numbers of myelinated fibers: The myelinated fiber counts in group A₁ and B₁ were significantly higher than group C₁ (P<0.001) .There were statistical difference between A₁ and B₁ in the myelinated fiber counts (P<0. 001). 3. Areas of myelinated fibers :Compared with C₁, there were significant differences to the other groups respectively (P<0.05) . There were no statistical difference between A₁ and B₁ in areas of myelinated fibers.4. The section areas of tibialis anterior muscles: Compared with C₁,there were significant differences to the other groups respectively (P<0. 01). There were statistical difference between A, and B₁ in the section areas of muscles (P<0. 05) .5. Transmission electron microscopy: The motor end plates disappeared in group C₁. The myocyte of the tibialis anterior muscles atrophied seriously. The distal axons were destroied , at the same time, myelin degenerated and dissolved. There were a few new motor end plates in bypss grafting group and interposition grafting group. There were regenerated myelinated axons and unmyelinated axons in the distal axons of group A₁. The degenerative and regenerative never fibers coexisted in the bypass grafts. There were also regenerated myelinated axons and unmyelinated axons in the distal axons of group B₁ .Parts of the new myeline sheath achieved normal thicknessin group B₁. 6. Real-time QPCR: Compared with C₂, there were significant differences to the other groups respectively in the relative mRNA content of CNTF and CGRP (P<0. 001) . There were statistical differences between A₂ and B₂ in the relative protein content of CNTF and CGRP (P<0. 05) . 7. western-blot: Compared with C₂,there were significant differences to the other groups respectively in the relative mRNA content of CNTF and CGRP (P<0. 001) . There were statistical differences between A₂ and B₂ in the ratio of CNTF and CGRP protein (P<0. 05) . Conclusion: The end-to-side nerve bypass grafting could promote the motor nerve functional recovery to the lesion of the neuroma-in-con-tinuity, and decrease the expression of the substance relatived to the neuropathic pain .