Foi estudada a ação do fator neurotrófico ciliar (CNTF) e da interleucina 6 (IL-6) exógenos na regeneração nervosa periférica. Aplicação de CNTF recombinante humano: o nervo ciático de 12 camundongos adultos C57BL/6J (3 grupos, n=4) foi seccionado e os cotos proximal e distal ancorados com ponto único de sutura no interior de tubo de polietileno (TP) com 6 mm d e comprimento e 0,76 mm de diâmetro interno, com intervalo de 4 mm entre os cotos. Os TP foram implantados vazios ou preenchidos com uma das seguintes soluções: (1) colágeno (col.) purificado (Vitrogen, 2,4 mg/ml) em tampão fosfato (0,2M), na proporção d e 1:1; (2) col.+CNTF (1:1, com 100 ng/ml de CNTF/tubo). Após 6 semanas os tubos contendo ao cabos nervosos regenerado s (CR) foram processados e incluídos em Epon. Fibras nervosas mielínicas foram contadas na porção média dos CR com um sistema controlado por computador (Biographics). Camundongos que receberam CNTF apresentaram número significativamente maior de axônios regenerados (3027±62, média±erro padrão) em relação aos animais implantados com tubos vazios (1384±128) ou preenchidos apenas com colágeno (1639±104). Outros 12 animais adicionais tiveram o nervo ciático seccionado e reparado da mesma maneira. Após 6 semanas, um tubo contendo solução do traçador neuronal HRP foi implantado no coto distal do nervo. Decorrido um período suplementar de 3 dias, neurônios marcados foram contados na medula espinhal e nos gânglios das raízes dorsais L4,5,6- Não houve diferença significante no número de motoneurônios entre os diferentes grupos experimentais (vazio=740±21; col.=749±44; CNTF=790±14) e o s animais não-operados (794±30). Não houve também diferença significante no número de neurônios sensitivos entre os diferentes grupos experimentais (vazio=1920±59; co!.=2262±152; CNTF=2124+96) e todos apresentaram número menor de neurônios sensitivos quando comparados com os animais não operados (4211+96) Aplicação de IL-6 recombinante murina: 12 camundongos C57BL/6J adultos foram divididos em dois grupos (n=6) e implantados com TP preenchidos com: (1) Vitrogen (2,4 mg/ml) e tampão fosfato (0,2 M), na proporção d e 1:1; (2) Vitrogen + IL-6 (1:1, com 100 |ig/ml de IL-6/tubo). Após tempo d e sobrevida de 6 semanas , os tubos com os CR foram processados da mesma forma que no experimento anterior. Fibras nervosas mielínicas foram contadas na porção média d o s CR. Os resultados mostraram que os animais implantados com col.+IL-6 tinham número significativamente maior de axônios mielínicos (2025+143) que os animais injetados apenas com col. (1542±122). O gânglio L5 foi também removido dos mesmos animais e cortado seriadamente (5|am), para posterior contagem do número de neurônios sensitivos. Não houve diferença significante no número de neurônios sensitivos entre os grupos experimentais (col.=528+42, col.+IL-6=554±37); todos, no entanto, apresentaram número significantemente menor de neurônios sensitivos quando comparados com os animais não operados (1112+63). Esses resultados indicam que a aplicação local de CNTF ou IL-6 estimula a regeneração de nervos seccionados e que este efeito é devido, provavelmente, a um aumento da taxa de brotamento dos axônios em regeneração / We studied the actions of exogenous ciliary neurotrophic factor (CNTF) and interleukin-6 (IL-6) on peripheral nerve regeneration. The sciatic nerve of 12 adult C57BL/6J mice (3 groups; n=4) w a s transected and both proximal and distal nerve stumps were secured by a single 10-0 suture into a 6-mm of a polyethylene tube (PT) (0,76 mm ID) to give a final gap length of 4mm. The PT were implanted empty or filled with one of the following solutions: (1) a purified preparation of collagen (Vitrogen, 2,4 mg/ml) plus phosphate buffer solution (0,2M), in 1:1 ratio; (2) Vitrogen + human recombinant CNTF (1:1, with 100 ng/ml of CNTF/tube). After 6 weeks the tubes containing the regenerated nerve cables (NC) were processed for Epon embedding. Myelinated nerve fibers were counted from the mid-portion of the cab les with a computer-controlled system (Biographics). CNTF injected mice regenerated significantly more myelinated axons (3027±62, mean±SEM) compared to the animals implanted with tubes left empty (1384±128) or filled with collagen alone (1639±104). Next, 12 additional animals had the sciatic nerve transected and repaired a s above. After 6 weeks, HRP was applied to the distal stump of the regenerated nerves and labeled neurons were counted in the spinal cord an d L4.5.6 dorsal root ganglia (DRG). No significant difference in the number of motoneurons was detected between the experimental (empty=740±21; collagen=749±44; CNTF=790±14) and non-operated (794±30) animals. No difference was also found in the n umber of labeled DRG neurons among the experimental groups (empty=1920±59; collagen=2262±152; CNTF=2124±96) and all had fewer labeled sensory neurons compared to the non-operated group (4211+96). For the IL-6 experiments, 12 C57BL/6J mice were divided into 2 groups (n=6) and implanted with PT filled with one of the following solutions: (1) Vitrogem (2,4 mg/ml) plus phosphate buffer solution (0,2 M), in 1:1 ratio; (2) Vitrogen + murine recombinant IL-6 (1:1, with 100 |ig/ml of IL-6/tube). Following a survival time of 6 weeks, the tubes with the regenerating nerve cables were processed for Epon embedding and myelinated nerve fiber counting. The results showed a significant difference in the number of myelinated axons between the collagen+IL-6 group (2025±143) and the collagen alone group (1542±122). The L5 DRG was also removed from the same mice, and serially sectioned (5^m) for sensory neuron counts. No significant difference was found in the number of DRG neurons between the experimental groups (collagen=528±42; collagen+IL-6=554±37). However, all had significantly fewer sensory neurons compared to the non-operated group (1112+63). These results indicate that locally applied CNTF and IL-6 stimulate peripheral nerve regeneration in adult animals, and that the effects are due to a neurite-promoting activity on axotomized neurons

Objetivamos o estudo quantitativo e temporal do padrão de inervação do músculo extensor longo dos dedos (edl), após transecção do nervo ciático em camundongos e reparo imediato pela técnica de tubulização. Assim, o músculo edl apresentou-se desnervado na segunda semana após a tubulização. Todas as fibras musculares do edl estavam reinervados na sexta semana após o implante da protese tubular. Entre a sexta e a quadragesima semanas pós-implante a proporção de fibras musculares do edl com mono e poli-inervação aumentou de 3:1 para 4:1. Na quadragésima semana após o implante do tubo foi atingida a porcentagem máxima (80%) de inervação troncular das placas motoras do edl mono-inervadas. Mesmo com tempos prolongados de sobrevivencia após implante, não houve estabilização do padrão de origem das fibras nervosas que convergiam para as placas poli-inervadas do edl. A mono-inervação troncular foi readquirida por 65% das fibras musculares do edl dos animais tubulizados, padrão encontrado em 100% das fibras musculares do edl dos animais não operados. Os padrões morfometricos de reinervação do músculo edl foram idênticos, a partir do terceiro mes, entre o grupo de animais com secção e tubulização do nervo ciatico e o grupo com simples esmagamento do nervo, evidenciando padrão similar de reinervação muscular a longo prazo. / We aimed to study the quantitative and temporal pattern of muscle innervation extensor digitorum longus (EDL), following transection of the sciatic nerve in mice and immediate repair technique for tubing. Thus, the EDL muscle denervation presented in the second week after the tubing. All of the EDL muscle fibers were reinnervated in the sixth week after the implantation of prosthetic tube. Between the sixth and FORTY weeks post-implantation, the proportion of muscle fibers of the EDL with mono-and poly-innervation increased from 3:1 to 4:1. The forty weeks after the implantation of the tube was reached maximum percentage (80%) of trunk innervation of motor endplates of the EDL mono-innervated. Even with prolonged survival times after implantation, there was no stabilization of the pattern of origin of nerve fibers that converged on the plates of poly-innervated EDL. The mono-innervation trunk was regained by 65% of EDL muscle fibers of animals tubularized, pattern found in 100% of EDL muscle fibers of animals not operated. The morphometric patterns of reinnervation of the EDL muscle were identical, from the third month, the group of animals with tubing and resources section of the sciatic nerve and the group with simple nerve crush, showing similar pattern of muscle reinnervation in the long term.

Su Huan Xing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 79-89). / Abstracts in English and Chinese. / Abstract --- p.i / 中文摘要 --- p.iii / Acknowledgements --- p.iv / Abbreviations Frequently Used --- p.v / Table of contents --- p.vi / Chapter Chapter1 --- General Introduction --- p.1 / Chapter Chapter2 --- Effects of an intravitreal optic nerve graft on the sprouting and regeneration of axotomized retinal ganglion cells --- p.17 / Chapter Chapter3 --- Effects of an intravitreal pre-injured optic nerve graft on the sprouting and regeneration of axotomized retinal ganglion cells --- p.44 / Chapter Chapter4 --- Effects of co-transplantation of an optic nerve graft and a peripheral nerve graft into the vitreous body on the sprouting and regeneration of axotomized retinal ganglion cells --- p.60 / Chapter Chapter5 --- General discussion --- p.74 / References --- p.79 / Tables --- p.90

Regenerative medicine holds promises for many neurodegenerative diseases such as Traumatic Brain Injury (TBI), a disorder that occurs when a sudden trauma causes damage to the brain, leading to apoptosis or necrosis of brain neurons. More than 5 million Americans suffer from TBI as a result of inability to regenerate damaged neurons. The aim of this project was to develop a biocompatible and electrically conductive substrate to promote growth and regeneration of neurons and for our long-term goal as a probe to record intracellular and multisite signals from brain. The substrate was fabricated by pyrolyzing a polymeric precursor -SPR 220.7 at temperatures higher than 700 ºC. Human Neuroblastoma cells - SK-N-MC, SY5Y and mouse teratocarcinoma cells P-19 were found to attach and proliferate on photoresist derived carbon film. Growth and differentiation of rat pheochromocytoma cell-PC12 that serves as a model for primary neurons was demonstrated. Initial examination of cell growth and differentiation was done by observing cell shape and size, and measuring the length of neurites after the cells were differentiated by NGF. Further characterization of cells cultured on photoresist derived carbon substrate was achieved by testing mRNA genes- GADPH and Tau. Findings from this investigative work would possibly help to study new approaches to promote neuronal growth and differentiation in damaged brain regions of people with TBI or in patients with other neurodegenerative disorders, such as Alzheimer's disease in regaining memories.

In another study, hyperthermic treatment was applied to study whether HSP27 expression would be induced in un-injured RGCs, and whether this treatment performed after axotomy would have effects on HSP27 expression, RGC survival and/or regeneration into PN graft. Brief duration of heat shock that elevate the body temperature to 42°C did not up-regulate HSP27 in normal retina. About 8-10% increase in RGC survival in the hyperthermia group was observed compared to those received a 37°C treatment at one week post-axotomy and it depended on the number of post-injury heat treatments applied. At the same time, the number of HSP27-RGCs was also doubled, although the same increase occurred was irrespective of the number of hyperthermic treatments. Multiple heat shock application also significantly enhanced RGC regeneration into PN graft through increased the number of HSP27 regenerating RGCs. These results suggest that post-injury hyperthermic treatment enhance HSP27 induction in RGCs and lead to their successful regeneration into the PNG, whereas further studies are necessary to determine whether the protective effect on survival by heat shock is due to the increase in a subset of HSP27-RGCs. (Abstract shortened by UMI.) / In the second study, different neurotrophic factors were injected into the vitreous to enhance RGC survival and/or regeneration. Brain-derived neurotrophic factor (BDNF) significantly reduced RGC death transiently at 14 days after ON cut, but the expression of HSP27 was reduced compared to bovine serum albumin-injected controls. In peripheral nerve (PN)-grafted retina, BDNF suppressed RGC regeneration via reducing the number of HSP27-RGCs regenerating into the PN graft. In ciliary neurotrophic factor (CNTF)-injected group, although there was only a 10% increase in RGC survival, a 5-fold drastic increase in the number of RGCs which expressed HSP27 was observed, and some of these were found to undergo intra-retinal sprouting similar to VPN-transplanted retina. Combined treatment of intra-vitreal CNTF injection with PNG resulted in a 5 fold-increase in the number of regenerating RGCs as well as increasing the proportion of cells which expressed HSP27 from about 60% to about 80%. The data indicates that HSP27 participates in axonal regrowth especially under synergistic interaction of CNTF and PNG. Intra-vitreal injection of hepatocyte growth factor (HGF) significantly sustained RGC survival compared to BDNF at 28 days after axotomy, but the HSP27 expression in RGCs did not change correspondingly. In the PN-ON grafted retina, HGF promoted more RGCs regenerate without altering the number of HSP27-RGCs regrowing into the PNG. Such results indicate that some trophic factors can specifically enhance or suppress RGC regeneration by modulating HSP27 expression, while other trophic factors promote regeneration which is independent to HSP27. Therefore, it suggests that RGCs may regenerate through at least two different mechanisms. / In this study, the detailed in vivo expression of HSP27 in retinal ganglion cells (RGCs) of golden hamster following axotomy and regeneration stimulated by peripheral nerve grafting and neurotrophic factors have been examined. / In whole-mount normal retinas, HSP27 was constitutively expressed in astrocytes and blood vessels, but not in RGCs. Three days after optic nerve (ON) transection, a small subset of surviving RGC began to express HSP27, the number of which peaked at 7 days and dropped to a minimal level at two weeks post-axotomy. When axotomy was done more proximally to their cell bodies, RGCs survival was significantly decreased but HSP27 expression did not change. This suggests the HSP27 expression does not correlate with cell survival after axonal injury. When a viable peripheral nerve (VPN) was transplanted intravitreally into the eye after ON cut, it induced intra-retinal sprouting of RGCs. Although it did not promote RGC survival, VPN prolonged HSP27 expression up to 56 days after surgery and significantly increased the number of HSP27-RGCs. This protein was localized in the cell body, and especially, in dendritic sprouts and growth cones, indicating that it was transported to active growing sites where it may have a functional role associated with regenerative sprouting. / Wong, Wai Kai. / Adviser: Eric Cho. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3270. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 159-198). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Despite that the RGC axons closely associated with astrocytes, the role of astrocytes in RGC regeneration was uncertain. In view of this, the effect of cultured adult astrocytes on RGC regeneration through an extensive ON lesion segment was studied. Adult ON astrocytes were prepared by sub-culturing of cells migrating out of ON explants. A small hole in the ON was punctured by 27G needle and about 0.5 to 1.0mul (1000 cells) cultured astrocytes was injected into the extensive ON lesion segment. We found that cultured adult astrocytes promoted significant RGC axon regeneration in the extensive ON lesion. / Finally, co-transplantation of intravitreal PN followed by transplantation of astrocytes into the extensive lesion has a synergistic effect on the regrowth of RGC axons, as indicated by the maximum distance achieved by regenerating axons and integrated intensity of staining of the CTB-labeled axons. Transplanatation of VPN+AST, VPN+NAST and NPN+AST as 3.9, 2.5 and l.9 times more potent in inducing regeneration than that of NPN+NAST as shown by integrated intensity measurement. However, co-transplantation of PN and astrocytes could not enhance RGC survival. (Abstract shortened by UMI.) / In this study, we have established an extensive lesion paradigm to study the behavior of injured retinal ganglion cell (RGC) axons after ON crush in adult golden hamster. We found that RGC axons regenerated in the extensive lesion for 406.8mum at 1 week post-crush to 1174.0mum at 4 weeks post-crush. RGC axons were able to regenerate the entire lesion segment but they terminated precisely at the interface between the lesion and the distal segment of the ON. Regrowing axons were intimately associated with astrocytes which repopulated the lesion segment. Repopulated oligodendrocytes were scattered in the lesion segment and myelin debris was significantly decreased in the lesion segment with time. / It is commonly believed that central nervous system (CNS) neurons are unable to regenerate after injury. Recently, there have been several lines of evidence showing that damaged CNS neurons can undergo axonal regeneration under appropriate conditions. Since the retina and optic nerve (ON) are regarded as part of the CNS, therefore, they are used as a model to study CNS regeneration. / Kong Wai Chi. / "July 2005." / Adviser: Y.P. Cho. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3616. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 96-115). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Tse, Chi Lok. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 116-131). / Abstracts in English and Chinese. / Declaration --- p.i / Abstract --- p.iii / Abstract in Chinese --- p.v / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Figures --- p.X / List of Tables --- p.xii / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- General Introduction / Chapter 1.1 --- Regenerative Medicine --- p.1 / Chapter 1.2 --- Embryonic Stem Cells and Reprogramming --- p.3 / Chapter 1.3 --- Transdifferentiation --- p.6 / Chapter 1.4 --- The Cerebellum --- p.7 / Chapter 1.4.1 --- Functions of the cerebellum --- p.7 / Chapter 1.4.2 --- Structure and organization of the cerebellum --- p.8 / Chapter 1.4.3 --- Principle cellular components in the cerebellum --- p.12 / Chapter 1.4.3.1 --- Purkinje cells --- p.12 / Chapter 1.4.3.2 --- Granule cells --- p.12 / Chapter 1.4.3.3 --- Mossy fibres --- p.13 / Chapter 1.4.3.4 --- Climbing fibres --- p.13 / Chapter 1.4.3.5 --- Deep cerebellar nuclei --- p.13 / Chapter 1.4.3.6 --- Other cerebellar neurons --- p.14 / Chapter 1.4.3.7 --- Neuroglia of the cerebellum --- p.16 / Chapter 1.4.4 --- Circuitry of the cerebellum --- p.17 / Chapter 1.5 --- Development of the Cerebellum --- p.21 / Chapter 1.5.1 --- Anatomical changes during cerebellar development --- p.21 / Chapter 1.5.2 --- Molecular control of cerebellar development --- p.25 / Chapter 1.5.2.1 --- Specification of the cerebellar region --- p.25 / Chapter 1.5.2.2 --- Neurogenesis from the ventricular zone --- p.26 / Chapter 1.5.2.3 --- Neurogenesis from rhombic lip --- p.29 / Chapter 1.6 --- Scope of the Thesis --- p.33 / Chapter CHAPTER 2 --- Materials and General Methods / Chapter 2.1 --- Materials for Molecular Biological Work --- p.35 / Chapter 2.1.1 --- Enzymes --- p.35 / Chapter 2.1.2 --- Chemicals and others --- p.35 / Chapter 2.1.3 --- Plasmid vectors and plasmid --- p.36 / Chapter 2.1.4 --- Solutions and media --- p.36 / Chapter 2.2 --- Materials for Tissue/Cell Culture --- p.38 / Chapter 2.2.1 --- Chemicals --- p.38 / Chapter 2.2.2 --- Culture media and solutions --- p.38 / Chapter 2.2.3 --- Culture cells --- p.39 / Chapter 2.3 --- Animals --- p.40 / Chapter 2.4 --- Materials for Immunocytochemistry --- p.40 / Chapter 2.5 --- Oligonucleotide Primers --- p.41 / Chapter 2.6 --- RNA Extraction --- p.44 / Chapter 2.7 --- Generation of cDNA from mRNA --- p.44 / Chapter 2.8 --- Preparation of Recombinant Plasmid DNA --- p.45 / Chapter 2.8.1 --- Small scale preparation of DNA --- p.45 / Chapter 2.8.2 --- QLAGEN plasmid midiprep kit method --- p.46 / Chapter 2.9 --- Preparation of Specific DNA Fragment from Agarose Gel --- p.46 / Chapter 2.10 --- Subcloning of DNA Fragments --- p.47 / Chapter 2.10.1 --- Preparation of cloning vectors --- p.47 / Chapter 2.10.2 --- Subcloning of DNA fragment --- p.48 / Chapter 2.10.3 --- Transformation of DNA into competent cells --- p.48 / Chapter 2.11 --- Preparation of Competent Cells --- p.48 / Chapter CHAPTER 3 --- Generation and Characterization of Induced Neurons / Chapter 3.1 --- Introduction --- p.50 / Chapter 3.2 --- Experimental Procedures --- p.51 / Chapter 3.2.1 --- Construction of expression vector --- p.51 / Chapter 3.2.1.1 --- Preparation of insert DNA --- p.51 / Chapter 3.2.1.2 --- Construction of entry vector --- p.52 / Chapter 3.2.1.3 --- Construction of destination vector --- p.52 / Chapter 3.2.1.4 --- Construction of expression vector --- p.52 / Chapter 3.2.2 --- Generation of induced neural cells --- p.57 / Chapter 3.2.2.1 --- Culture of mouse embryonic fibroblasts (MEF) --- p.57 / Chapter 3.2.2.2 --- Production of expression vector containing retroviruses --- p.57 / Chapter 3.2.2.3 --- Transfection and induction of neural fate of MEF --- p.57 / Chapter 3.2.3 --- Immunocytochemcial analysis --- p.58 / Chapter 3.2.4 --- Efficiency calculation --- p.59 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- A screen for cerebellar Purkinje and granule cell fate-inducing factors --- p.62 / Chapter 3.3.2 --- Characterization of the induced neurons --- p.67 / Chapter 3.3.2.1 --- Granule cell induction --- p.67 / Chapter 3.3.2.2 --- Purkinje cell induction --- p.71 / Chapter 3.4 --- Discussion --- p.102 / Chapter 3.4.1 --- Roles of inducing factors in Purkinje cells and granule cells development --- p.102 / Chapter 3.4.2 --- Mechanism of neural transdifferentiation --- p.107 / Chapter CHAPTER 4 --- Future Directions / Chapter 4.1 --- Complete Induction of Purkinje Cell Fate --- p.111 / Chapter 4.2 --- Induced Neurons of Different Subtypes --- p.112 / Chapter 4.3 --- Mechanism of Transdifferentiation --- p.114 / Chapter 4.4 --- Transdifferentiation and Regenerative Medicine --- p.114 / Bibliography --- p.116

O tratamento cirúrgico mais utilizado para a reparação das lesões de nervos periféricos com perda de substância é através de auto-enxertia de nervo. Essa técnica produz alterações na área doadora e muitas vezes não se dispõe de tecido suficiente para grandes perdas de tecido neural. Desta forma é necessária a busca de novas técnicas, menos traumáticas e mais simples, visando eliminar a morbidade na área doadora e prover a quantidade necessária de tecido para a regeneração neural. O objetivo desse trabalho foi comparar, em ratos Wistar, o grau de regeneração neural, utilizando-se de análise histológica e análise funcional, através de interposição de enxerto autógeno de nervo (Grupo A), veia autógena conservada em glicerol (Grupo B) e enxerto alógeno de nervo conservado em glicerol (Grupo C) para correção de defeito de 5 mm de nervo fibular. Quinze ratos machos da raça Wistar, com peso variando entre 200 e 300g, e idade ao redor de oito semanas foram divididos em três grupos de cinco animais de acordo com o tratamento empregado. Para a coleta de segmentos de nervos alógenos, foram escolhidos cinco ratos da Raça Sprague-Dawley, com sexo, idade e peso semelhantes ao da raça Wistar. Os animais foram submetidos à avaliação funcional (\"walking track analysis\") imediata, com três semanas e com seis semanas e sacrificados para realização dos estudos histológicos com a coloração de tetróxido de ósmio. Na análise microscópica das lâminas, realizada, em todos os grupos foram visualizados pequenos fascículos contendo axônios mielinizados de tamanhos variados e degeneração Walleriana em pequeno número de axônios. Nos grupos B (Veia autógena conservada em glicerol) e C(Nervo alógeno conservado em glicerol) o escape de fibras axonais mielinizadas para fora dos limites do epineuro e o processo inflamatório local foram menores que o do grupo A (Auto-enxertia). Em relação à avaliação funcional, onde foi utilizada a análise estatística por meio do modelo de análise de variância com medidas repetidas e o de comparações múltiplas de Bonferroni (p<0.05), não houve diferença estatisticamente significativa entre as recuperações funcionais do nervo fibular independente do tipo de reparação utilizada em nenhum período avaliado. / The most used surgical treatment of peripheral nerves for the repair of injuries with loss of substance is using nerve autograft. This technique produces alterations in the donor area and often not sufficient tissue is available for great losses of neural tissue. Thus the need for new less traumatic and simpler techniques is needed in order to eliminate morbidity in the donor area and to provide the necessary amount of tissue for neural regeneration. The objective of this study was to compare, in Wistar rats, the degree of neural regeneration, using histological and functional analysis through interposition of autogenous nerve graft (Group A), autogenous vein preserved in glycerol (Group B) and allogenous graft preserved in glycerol (Group C) for the correction of a 5-mm defect of fibular nerve. Fifteen male Wistar rats, weighing between 200 and 300 g and approximately 8 weeks old were divided into 3 groups of five animals according to the used treatment. For the collection of autogenous nerve segments 5 Sprague-Dawley rats were chosen, with similar sex and age as the Wistar rats. The animals were submitted to immediate functional evaluation (walking track analysis), at 3 and 6 weeks and sacrificed for histologic studies using osmium tetroxide stain. On microscopic analysis of the slides, performed in all groups, small fascicles containing myelinated axons of varied sizes and wallerian degeneration in a small number of axons were visualized. In Groups B (autogenous vein preserved in glycerol) and C (allogenous nerve preserved in glycerol) escape of axonal myelinated fibers outside the limits of the epineurium and the local inflammatory process were less than in Group A (autograft). Regarding functional evaluation, where statistical analysis by variance analysis using the models of repeated measures and that of Bonferroni\'s multiple comparisons were used (p<0.05) there was no statistically significant difference between functional recoveries of the fibular nerve independent of the type of repair used in any evaluated period.

Following microsurgical reconstruction of injured peripheral nerves, severed axons are able to undergo spontaneous regeneration. However, the functional result is always unsatisfactory with poor sensory recovery and reduced motor function. One contributing factor is the retrograde neuronal death, which occurs in the dorsal root ganglia (DRG) and in the spinal cord. An additional clinical problem is the loss of nerve tissue that often occurs in the trauma zone and which requires “bridges” to reconnect separated nerve ends. The present thesis investigates the extent of retrograde degeneration in spinal motoneurons and cutaneous and muscular afferent DRG neurons after permanent axotomy and following treatment with N-acetyl-cysteine (NAC). In addition, it examines the survival and growth-promoting effects of nerve reconstructions performed by primary repair and peripheral nerve grafting in combination with NAC treatment. In adult rats, cutaneous sural and muscular medial gastrocnemius DRG neurons and spinal motoneurons were retrogradely labeled with fluorescent tracers from the homonymous transected nerves. Survival of labeled neurons was assessed at different time points after nerve transection, ventral root avulsion and ventral rhizotomy. Axonal regeneration was evaluated using fluorescent tracers after sciatic axotomy and immediate nerve repair. Intraperitoneal or intrathecal treatment with NAC was initiated immediately after nerve injury or was delayed for 1-2 weeks. Counts of labeled gastrocnemius DRG neurons did not reveal any significant retrograde cell death after nerve transection. Sural axotomy induced a delayed loss of DRG cells, which amounted to 43- 48% at 8-24 weeks postoperatively. Proximal transection of the sciatic nerve at 1 week after initial axonal injury did not further increase retrograde DRG degeneration, nor did it affect survival of corresponding motoneurons. In contrast, rhizotomy and ventral root avulsion induced marked 26- 53% cell loss among spinal motoneurons. Primary repair or peripheral nerve grafting supported regeneration of 53-60% of the motoneurons and 47-49% of the muscular gastrocnemius DRG neurons at 13 weeks postoperatively. For the cutaneous sural DRG neurons, primary repair or peripheral nerve grafting increased survival by 19-30% and promoted regeneration of 46-66% of the cells. Regenerating sural and medial gastrocnemius DRG neurons upregulate transcription of peripherin and activating transcription factor 3. The gene expression of the structural neurofilament proteins of high molecular weight was significantly downregulated following injury in both regenerating and non-regenerating sensory neurons. Treatment with NAC was neuroprotective for spinal motoneurons after ventral rhizotomy and avulsion, and sural DRG neurons after sciatic nerve injury. However, combined treatment with nerve graft and NAC had significant additive effect on neuronal survival and also increased the number of sensory neurons regenerating across the graft. In contrast, NAC treatment neither affected the number of regenerating motoneurons nor the number of myelinated axons in the nerve graft and in the distal nerve stump. In summary, the present results demonstrate that cutaneous sural sensory neurons are more sensitive to peripheral nerve injury than muscular gastrocnemius DRG cells. Moreover, the retrograde loss of cutaneous DRG cells taking place despite immediate nerve repair would still limit recovery of cutaneous sensory functions. Experimental data also show that NAC provides a highly significant degree of neuroprotection in animal models of adult nerve injury and could be combined with nerve grafting to further attenuate retrograde neuronal death and to promote functional regeneration.

Regrowth in the lesioned central nervous system is impeded by inhibitory molecules including myelin-associated inhibitors (MAIs) and chondroitin sulfate proteoglycans (CSPGs). Inhibitory molecules engage neuronal cell surface receptors and activate the small GTPase RhoA in injured neurons to mediate neurite outgrowth inhibition through targeted modifications to the cytoskeleton. Inhibition of RhoA with the ribosyltransferase C3 attenuates neurite outgrowth inhibition in vitro and in vivo but the ubiquitous expression and multifunctionality of RhoA may limit the specificity of therapeutic RhoA antagonists. The hypothesis of the thesis is that molecules that functionally interact with RhoA to mediate myelin-dependent inhibition may represent more specific targets for therapeutic intervention. We have explored the contribution of two RhoA interacting proteins to the neurite outgrowth inhibitory effects of MAIs. In Chapter 2 we describe the contribution of the rho effector, Rho kinase (ROCK) to MAI responses in neurons. In Chapter 3 we identify the cytosolic phosphoprotein CRMP4b (Collapsin Response Mediator Protein 4b) as a novel RhoA binding partner that mediates neuronal responses to CNS inhibitors. By structure function analysis we have developed a molecular antagonist of CRMP4b-RhoA binding that promotes neurite outgrowth on inhibitory substrates in vitro and has the potential to be a potent and specific molecular therapeutic for spinal cord injury. In Chapter 4 we identify glycogen sythase kinase 3b (GSK3b) as an important kinase in the MAI pathway that regulates protein interactions with RhoA. This thesis provides insights into the signal transduction machinery that is engaged in response to CNS inhibitors and suggests several novel therapeutic targets to promote axon regeneration following CNS injury.