Regeneración de las lesiones críticas del nervio periférico con factores de crecimiento. Estudio experimental

Abstract

ENGLISH ABSTRACT (INTERNATIONAL THESIS): INTRODUCTION The purpose of a nerve graft procedure is to substitute a defect in a severed nerve trunk to allow regeneration of a maximal number of regenerated nerve fibers to their target organs. Millesi et al., 1972, obtained successful results by using autografts of the sural nerve, a technique that became the gold standard, but the limited availability of nerve tissue, the incomplete obtained functional recovery and problems with the donor, have motivated the search of other alternatives. A large number of studies have been undertaken to develop alternatives and to improve results. Keynes et al., 1984, discovered that the basement membrane of the muscle cells was useful as a way for the growth of regenerating axons, through the basal lamina. Glasby et al., 1986, obtained a useful step of axons through endomysium channels, as they are similar to those of endoneurium. Pereira et al., 1990, began their studies on denatured muscle graft substitute for mycobacterial granulomatous lesions in peripheral nerves of leprosy. Brunelli, Battiston et al., 1993, developed an experimental model in rats with a vein graft filled with fresh skeletal muscle, leading to similar results as the traditional nerve grafts. DeFranzo et al., 1994, examined the use of freeze-thawed muscle and nerve graft in rats, analyzing histologically the density of axons and myelination. Whitworth et al., 1995, incorporated a deposit of Schwann cells and other essential components in a nerve conduit, with a marked positive effect on axonal regeneration. Martín Ferrero et al., 1996, used freeze dried denatured skeletal muscle, with passage of axons to the distal segment in more than 60% of oligofascicular and more than 45% in the polyfascicular nerve. Battiston et al., 2000 and 2005, described how the muscle fibers represents a good support for advancing axons through adhesion molecules present in the basal lamina of muscle fibers increasing neurite extension. Lundborg, Dahlin et al., 2001, demonstrated that axons and Schwann cells grew more rapidly in the chemically acellular muscle grafts than in freezing-thawing used for denaturation. Other substances could promote regeneration of nerve tissue, such as nerve growth factor (NGF), epidermal growth factor (EGF) and platelet-derived growth factors (PDGF). Yu et al., 2009, looked at the histological effect of NGF trated acellular grafts, revealing an axonal diameter, total number and thickness of myelin better after NGF treatment than in nerves repaired with acellular grafts alone and acellular grafts with fibrin sealant. The aim of our study is to discover the best method of muscle grafts denaturation, trying to understand whether the use of an added growth factor (IGF-1) improves axonal regeneration. MATERIAL AND METHODS This study is a controlled experimental study, level I evidence. In twenty male Wistar rats (average weight 278 g), a critical defect of 15 mm was created in the sciatic nerve, which was bridged by an acellular muscle graft. In 10 of the animals the entire protocol was used but also, adding growth factors to their grafts. All muscle grafts were obtained from the gluteus medius muscle of two donor rats. The chemically cellular extraction protocol consisted of 7h in distilled water changed three times every 2-3h, one night in Triton detergent, and 24h in deoxycholate (Baptista el at., 2009). After intraperitoneal preoperative anesthesia, and by posterolateral approach, the sciatic nerve was exposed. After location of the trunk, the nerve was transected generating a 15 mm defect. An acellular muscle block was added, where the epimisium-epinerium were sutured with polypropylene 7/0 at each end. Within half of the grafts, we injected 2 ml of Increlex® (10 mg/ml mecasermin), thereby providing the IGF-1 factors. Sutures were sealed with fibrin (Tissucol®, Baxter, Valencia, Spain). Recovery of motor function was analyzed using the gait pattern and the Sciatic Functional Index (SFI). The value 0 represents normal function or absence of dysfunction, and value -100, the total loss of function. The Grasping Test consists of raising the rat from the tail, allowing it to grip with its legs to the grid, in order to control the motility of the limbs. It allows visualize if missing any finger and if it is able to stretch its joints. Rats were sacrificed at 90-100 days post-surgery (body-weight of 450-500g), and the segment of the sciatic nerve including the muscle graft was dissected. Samples were obtained for macro and microscopic preparations. The explanted pieces of sciatic nerve with grafts were carved and cut into seven pieces equally in all animals; pieces were processed in resin (toluidine blue staining) and also for paraffin embedding: hematoxylin-eosin and Masson trichrome. For the histomorphometric study, myelinated axons were counted in crossections. Results were analyzed statistically. The differences with p-value <0.05 were considered significant. RESULTS Animals of the control group developed two ulcers by pressure and lost fingers were seen in three rats, unlike the animals with IGF-1, in which there were no signs of these findings. Digital flexion contracture in the affected limb was detected in 80% of controls, and in 45% of cases during the realization of Grasping Test. SFI index showed the same pattern between different experiemental groups studied over time. Values 0 to -20 in the preoperative period reflected a normal function in both groups; close to -100 at 7-30 days after surgery; thus, a complete loss of function, which indicated an absence of innervation in this period. From the 30th day these values were improving, reflecting a gradual recovery of function. On day 90 after surgery, results were close to normal levels, with a characteristic functional recovery possibly related to polyinnervation peak in all the animals. However. There was a statistically significant difference with a favorable development in IGF-1 cases. Macroscopically, a bead yellowish or tubular structure filled the gap between the two sutures of the graft. Microscopically, proximal ends appeared as a normal nerve, with proper, regular and orderly fascicular distribution, perfectly defined by its three wraps, with little connective tissue. At the graft, regenerated axons used the muscle as a bridge, with many small nerve fascicles separated by host tissue and plenty of blood vessels. Axons followed their basal lamina, defining small fascicles with large vessels and abundant connective tissue. At the distal end, there was no a clear division in fascicles. There was little connective tissue between the fibers and the epineurium was quite developed. The untidiness may be justified because the fibers are still looking for its way. There were a short number of myelinated fibers with a smaller diameter and more irregular arrangements in the untreated group (mean of axons in the distal stump of the animals with IGF-1 is 62% of proximal number, 56% in controls). We found a statistically significant difference in myelin thickness at the graft and the distal stump between both groups. DISCUSSION Peripheral nerve injuries represent one major cause of morbidity and disability in affected patients and generate high economic costs. Their repair is a challenge for current research, and by the need to discover economic and biologically feasible techniques. The results have to match the classical autologous nerve. With this study, we have tried to advance in search of different ways to present the muscle graft as a bridge in the critically injured nerve, and the options available for us to enrich it biologically, supporting our project in the current experiments with growth factors. According Varejão et al., 2004, SFI index represents the most reliable method of analysis of functional recovery, which allows integration between sensory and motor systems. The functional test results were positive in both experimental groups, although major differences appeared within 30 days of surgery in the group that was treated with IGF-1. That recovery was statistically significant until the 90th day, although values were not different at the end of the follow-up. In the microscopic analysis, results tended to be positive for IGF-1 as a promoter of axonal regeneration, with higher number of myelinated axons by field, higher density of axons and larger diameter of the nerve at the distal end. The myelin thickness in the graft and in the distal stump in the IGF-1 animals were significantly increased. From the analysis of the obtained results, we can say that the acellular muscle that we employed as a graft provides a bridge tool for the advancement of the regenerating axons, perhaps due to the structural proximity of laminin to nerve sheaths. With the use of growth factors we achieved more satisfactory clinical and functional results. We don¿t know the half-life of IGF-1 locally deposited in the muscle graft, but our attempt was to seal the growth factor inside by using fibrin, which was placed at the suture levels. Perhaps we can boost the regeneration in the early stage, but it is insufficient to keep the boost at that level. Several authors, (Rind et al., 2002; Rabinovsky et al., 2003; Apel et al., 2010) have described pulsed or continuous local IGF-1 through pumps, with empowerment of the functional and histological findings after the sacrifice of the animals. CONCLUSIONS The selected biological method for chemical treatment of skeletal muscle achieves acellular grafts. We successfully could repair a critical lesion of the sciatic nerve in rats using this acellular muscle graft. There were significant differences in the repair of a critical defect of rat sciatic nerve by acellular allogeneic striated muscle graft enriched with growth factors IGF-1. The evolution of treatment of sciatic nerve injuries with and without the addition of a concentrate of IGF-1, supports the role of growth factors as a promoter of nerve regeneration. Functional and histological study of treated nerves confirms the success of the evaluated technique. REFERENCES: Bain JR, Mackinnon SE, Hunter DA. Functional evaluation of complete sciatic, peroneal, and posterior tibial nerve lesions in the rat. Plast Reconstr Surg. 1989;83:129-38. Baptista PM, Orlando G, Mirmalek-Sani SH, Siddiqui M, Atala A, Soker S. Whole organ decellularization-a tool for bioscaffold fabrication and organ bioengineering. Conf Proc IEEE Eng Med Biol Soc. 2009;2009:6526-9. Battiston B, Geuna S, Ferrero M, Tos P. Nerve repair by means of tubulization: literature review and personal clinical experience comparing biological and synthetic conduits for sensory nerve repair. Microsurgery. 2005;25(4):258-67. Battiston B, Tos P, Geuna S, Giacobini-Robecchi MG, Guglielmone R. Nerve repair by means of vein filled with muscle grafts. II. Morphological analysis of regeneration. Microsurgery. 2000;20(1):37-41. Brunelli GA, Battiston B, Vigasio A, Brunelli G, Marocolo D. Bridging nerve defects with combined skeletal muscle and vein conduits. Microsurgery. 1993;14(4):247-51. De Medinacelli L, Freed WJ, Wiatt RJ. An index of the functional condition of rat sciatic nerve based on measurements made from walking tracks. Exp Neurol. 1982;77(3):634-43. DeFranzo AJ, Morykwas MJ, LaRosse JR, Jennings DA, Challa V, Argenta LC. Autologous denatured muscle as a nerve graft. J Reconstr Microsurg. 1994;10(3):145-9. Glasby MA, Hitchcock RJI, Huang CLH. The effect of muscle basement membrane on the regeneration of rat sciatic nerve. J Bone Joint Surg. 1986;68B:829-33. Hall S. Axonal regeneration through acellular muscle grafts. J Anat. 1997;190:57-71. Kanje M, Skottner A, Lundborg G, Sjöberg J. Does insulin-like growth factor I (IGF-1) trigger the cell body reaction in the rat sciatic nerve?. Brain Res. 1991;563(1-2):285-7. Keynes, RJ, Hopkins WG, Huang CHL. Regeneration of mouse peripheral nerves in degenerating skeletal muscle. Guidance by residual muscle fibre basement membrane. Brain Res. 1984;295:275-81. Liu XL, Arai T, Sondell M, Lundborg G, Kanje M, Dahlin LB. Use of chemically extracted muscle grafts to repair extended nerve defects in rats. Scand J Plast Reconstr Surg Hand Surg. 2001;35(4):337-45. Martín Ferrero MA, Agudo Bernal J, Vaquero Puerta C, García Cantera M, Sánchez Martín MM. Autoinjerto de músculo estriado desnaturalizado en las pérdidas de sustancia del nervio periférico. Estudio experimental. Rev Esp Cir Ortop Traumatol. 1996;40:77-83. Millesi H, Meissl G, Berger A. The interfascicular nerve grafting of the median and ulnar nerve. J Bone Joint Surg (Am). 1972;54A:727-50. Noble J, Munro CA, Prasad VS, Midha R. Analysis of upper and lower extremity peripheral nerve injuries in a population of patients with multiple injuries. J Trauma. 1998;45(1):116-22. Oliveira EF, Mazzer N, Barbieri CH, DelBel EA. The use of a muscle graft to repair a segmentary nerve defect. An experimental study using the sciatic nerve of rats as model. J Neurosci Methods. 2004;133(1-2):19-26. Pereira JH, Cowley SA, Gschmeissner SE, Bowden RE, Turk JL. Denatured muscle grafts for nerve repair. An experimental model of nerve damage in leprosy. J Bone Joint Surg Br. 1990;72(5):874-80. Peter J. Apel, MD1,2, Jian Jun, MA, MD, PhD1, Michael Callahan, PhD1, Casey N. Northam. Effect of locally delivered igf-1 on nerve regeneration during aging: an experimental study in rats. Muscle Nerve. 2010;41(3):335-41. Sjöberg J, Kanje M. Insulin-like growth factor (IGF-1) as a stimulator of regeneration in the freeze-injured rat sciatic nerve. Brain Res. 1989;485(1):102-8. Varejão ASP, Melo-Pinto P, Meek MF, Filipe VM, Bulas-Cruz J. Methods for the experimental functional assessment of rat sciatic nerve regeneration. Neurol Res. 2004; 26:186-94. Welch JA, Kraus KH, Wells MR, Blunt DG, Weremowitz. Effect of combined administration of insulin-like growth factor and platelet-derived growth factor on the regeneration of transected and anastomosed sciatic nerve in rats. J Am J Vet Res. 1997;58(9):1033-7. Whitworth IH, Doré C, Hall S, Green CJ, Terenghi G. Different muscle graft denaturing methods and their use for nerve repair. Br J Plast Surg. 1995;48(7):492-9. Whitworth IH, Doré CJ, Green CJ, Terenghi G. Increased axonal regeneration over long nerve gaps using autologous nerve-muscle sandwich grafts. Microsurgery. 1995;16(12):772-8. Yu H, Peng J, Guo Q, Zhang L, Li Z, Zhao B, Sui X, Wang Y, Xu W, Lu S. Improvement of peripheral nerve regeneration in acellular nerve grafts with local release of nerve growth factor. Microsurgery. 2009;29(4):330-6.