| Background: The treatment of peripheral nerve injury is a tough problem clinically, in which two crucial problems should be solved: the repair of nerve defects and neurotropic reinnervation of the nerve fibers. The standard clinical approach for bridging nerve defects is an autologous nerve graft, or autograft. Disadvantages of the autograft include limitation of the source of autologous nerve and loss of function at the donor site. Therefore, many researches are focused on developing proper biomaterials as an alternate to autograft. Chemically extracted acellular nerve allograft is a promising material. To remove immunogenicity such as Schwann cells and myelin while restore the nerve structures such as basal lamina tubes and extracellular matrix, we improved the preparation of allograft. We use the optimized allografts to bridge rat sciatic nerve defects and evaluate the outcomes of nerve regeneration. To combine neurotropic reinervation with promotion of nerve regeneration and ultimately to improve the effects of regeneration, we established the dorsal root ganglion(DRG) resection animal model. The model can provide an effective method for quantitative evaluation of selective regeneration of peripheral nerve. The neurotropic reinervation effects of acellular nerve allograft were quantitatively evaluated.Methods: (1) The improved method group, with the detergents of Triton X 200, sulfobetaine-10 (SB-10) and sulfobetaine-16 (SB-16) was compared with the Sondell method group, in which the nerve segments were chemically extractedwith the detergents of Triton X-100 and deoxycholate. The quality of the acellular nerve allograft was revealed by HE staining, fast-blue staining, laminin immunohistochemistry, and scanning electron microscopy. (2) Rat sciatic nerve defects were bridged by the allografts and autografts. The outcomes of nerve regeneration were evaluated by sciatic nerve function index (SFI), nerve electrophysiology, motor end-plate staining and computer-assisted image analysis. (3)The rat sciatic nerve was dissected to identify the nerve roots of sciatic nerve and to localize the DRG. 3 weeks after removal of DRGs, histology staining was performed to observe the degeneration of sensory nerve fibers. Compute-assisted image analysis was to measure the number of motor nerve fibers. Semithin section, toluidine blue staining and computer-assisted image analysis were performed to evaluate reliability of the animal model. (4) 3 months after rat sciatic nerve defects were bridged by the allografts and autografts, part of the experimental animals were undergone DRG resection operation. Pathological staining and compute-assisted image analysis were performed to calculate the misdirecton rate of nerve fibers and to quantitatively evaluate the neurotropic reinervation effects of acellular nerve allograft. The experimental animals that were not undergo the DRG resection were performed cholinesterase staining and carbonic anhydrase staining to qualitatively or semi-quantitatively evaluate the neurotropic reinnervation effects.Results: (1) HE staining demonstrated that the Schwann cells were completely removed from the nerves in two groups and there were no significant difference in the effect of cell removal between the groups. There were better effect of removal of myelin and preservation of laminin and nerve structures in the improved method group through fast-blue staining, laminin immunohistochemistry, and scanning electron microscopy. The comprehensive quality evaluation of the effect of removal of cells and myelin and preservation of laminin and nerve structures showed that the quality of acelluar nerve allografts in the improved group exceeded that in the Sondell method group. (2) After being grafted, moresatisfactory effects of nerve regeneration were obtained in improved method group compared with Sondell method group, as demonstrated in the aspects of sciatic nerve function index (SFI), motor nerve conduction velocity (MNCV), atrophy of gastrocnemius, mean diameter of nerve fibers and mean thickness of myelin sheath. And the effects of nerve regeneration in improved group were similar to that in autograft group. (3) 3 weeks after the DRGs resection, the myelinated nerve fibers in the sural nerve were completely degenerated indicated by fast-blue staining. The myelinated motor nerve fibers accounts for 58.4%, 54.6%, 45.4% and 0 in sciatic nerve, tibial nerve, peroneal nerve and sural nerve respectively showed by compute image analysis. (4) Compared with autograft group, brown stains were less in allograft group, as shown by cholinesterase staining while black stain were more, as by carbonic anhydrase staining. After DRGs resection in allograft group , more degenerated fibers can be seen in sural nerve. The misdirection rate(MDR) was 2.21% and 7.65% in allograft and autograft group respectively.Conclusion:(1) This optimized chemical extraction procedure with the detergents of Triton X 200, SB-10 and SB-16 is an ideal paradigm to develop high-quality acellular nerve allograft with the immunologic substances being basically removed while the laminin and nerve structured were well preserved.(2) The DRG resection animal model can effectively distinguish the sensory and motor nerve fibers and be used for quantitative evaluation of neurotropic regeneration of peripheral nerve. The model is very reliable. Effectiveness of selective nerve regeneration in different grafting materials and repair methods can be evaluated quantitatively in terms of its regeneration misdirection rate.(3) Chemically extracted acellular allograft can evidently promote neurotropic regeneration.(4) Chemically extracted acellular allograft can not only act as a scaffold in the period of nerve defects repair, but markedly enhance the effects of neurotropic reinervation, which artificial synthetic material can not. Chemically extracted acellular allograft could be an ideal nerve graft and may provide an alternate to the autologous nerve transplantation.
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