Several molecular changes occur following axotomy, such as gene up-regulation and down-regulation. In our previous study using Affymetrix arrays, it was found that after the axotomy of sciatic nerve, there were many novel genes with significant expression changes. Among such genes was neuronatin, whose expression was significantly up-regulated. Neuronatin was identified as a gene selectively expressed in the neonatal brain and is involved in neuronal differentiation during brain development, and markedly reduced in adult brains. The present study investigated whether the expression of neuronatin correlates with symptoms of neuropathic pain in adult rats with transected sciatic nerve. Adult male Sprague-Dawley rats weighting 230 to 280 g were used. The rats were grouped into two: those that were sham operated and those that had sciatic nerve axotomy. The specimens-L4,5 dorsal root ganglians(DRG) and their corresponding spinal cords-were collected at post-axotomy day 1, day 3, and day 5. The neuronatin protein contents were analyzed by western blotting and immunohisto- chemistry. Changes in the mRNA levels were evaluated using RT-PCR. Randall and Selitto test was performed to reveal changes in the animal behaviors. The subcellular co-localization of neuronatin with neuronal cell type specific markers were also investigated in correlation with pain-related animal behavior. It was found that after sciatic nerve injury, the expression of neuronatin in dorsal root ganglians was increased in protein extracts. Furthermore, the results of immunohistochemistry revealed that the cell numbers of DRGs were relatively increased. Unmyelinated C-fiber and thinly myelinated A-£_ fiber in adult DRGs were also among the principal sub-population of primary afferent neurons with distributed neuronatin. The increased expression of neuronatin and its subcellular localization were related to mechanical hyperalgesia. The results indicated that there was a following significant correlation between mechanical allodynia axotomy of sciatic nerve and the increased expression of neuronatin in C-fiber and A-£_ fiber of DRG neurons.

Orientador: Fausto Viterbo / Banca: Valéria Paula Sassoli Fazan / Banca: Alexandre Leite Rodrigues de Oliveira / Resumo: A neurorrafia término-lateral é técnica consagrada em casos onde não há coto proximal após lesão de nervo periférico. O coto distal pode ser suturado na lateral de qualquer nervo ou receber enxerto de nervo unindo a sua face lateral à face lateral de outro nervo, atuando como uma ponte. O número de enxertos em ponte poderia aumentar o resultado da reinervação muscular? Para responder esta pergunta realizou-se este trabalho. Foram operados 100 ratos Wistar divididos em cinco grupos experimentais com 20 animais cada. O GN (grupo controle de normalidade) não sofreu nenhum procedimento. No GD (grupo controle de desnervação) o nervo fibular foi seccionado e ambos os cotos foram suturados em músculos adjacentes e distantes entre si. O G1 recebeu um enxerto (ponte de nervo sural) tendo uma de suas extremidades suturadas na face lateral do nervo tibial (NT) e a outra na face lateral do segmento distal do nervo fibular (NF). No G2 e no G3 foram realizados os mesmos procedimentos que no G1. O G3 com 3 e o G2 com 2 enxertos. Em todos os casos foram utilizadas neurorrafias término-laterais (NTL) tipo "embracing" sem retirada de janela de epineuro. Os animais foram sacrificados após 120 dias. Foram realizados testes de marcha ("Walking-tracks") e testes eletrofisiológicos além de aferição da massa dos animais e dos músculos tibiais craniais direitos (MTCD). Segmentos de nervo de interesse também foram coletados. Foram realizadas análises histomorfométricas das fibras musculares e nervosas. Após análise global dos atributos do MTCD como massa, IMM (índice de massa muscular), área, menor e maior diâmetro das fibras musculares, número de fibras por campo, análise funcional (IFC -índice funcional do ciático), análise eletrofisiológica e análise morfométrica dos segmentos nervosos mais importantes, concluímos que não houve benefício com o aumento do número de pontes / Abstract: The end-to-side neurorrhaphy technique is enshrined in cases where there is no proximal stump after peripheral nerve injury. The distal end can be sutured to the side of any nerve. We also can use a nerve graft joining the lateral face of the distal end to the lateral face of another nerve, serving as a bridge. The number of bridges would increase the outcome of muscle reinnervation? To answer this question took place this work. Were operated on 100 rats divided into five groups with 20 animals each. GN (normal control group) received no further procedure. For GD (control group denervation) the peroneal nerve was transected and both stumps were sutured to adjacent muscles and far between. G1 received one graft (sural nerve bridge) having one end stitched on the side of the tibial nerve (NT) and the other on the side of the distal segment of the peroneal nerve (NF). In G2 and G3 were performed the same procedures as in G1. G3 with three and G2 with two grafts. In all cases were used end-to-side neurorraphies (NTL) performed as "embracing" without removing epineural window. The animals were sacrificed after 120 days. We performed tests of gait ("Walking-tracks") and electrophysiological tests in addition to measuring the mass of animals and also the mass of cranial tibial muscles (MTCD). Nerve segments of interest were also collected. We performed histomorphometric analysis of muscle fibers and nerves. After comprehensive analysis of attributes such as mass MTCD, IMM (body mass index), area, smaller and larger diameter of muscle fibers, number of fibers per field, functional analysis (IFC, sciatic functional index), electrophysiological and morphometric analysis of the most important nerve segments, we conclude that there was no benefit to increasing the number of bridges / Mestre

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21675号 / 医博第4481号 / 新制||医||1036(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 林 康紀, 教授 井上 治久 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The mechanism of body and organ size control is an unsolved puzzle. Initially characterized in Drosophila melanogaster, the Salvador/Warts/Hippo (Hippo) signalling pathway, highly conserved throughout evolution, defines a novel signalling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis, and cancer development in mammals. The upstream regulation of this pathway has been less well defined than the core kinase cassette. Previously Willin/FRMD6 has been proposed as the human orthologue of Expanded and, to date, little is known about the functional role of Willin in mammalian cells. My study elucidated the mechanism by which Willin antagonizes the transcriptional co-activator YAP. In MCF10A cells, Willin ectopic expression antagonizes YAP-induced epithelial-mesenchymal phenotypes via YAP Ser127 phosphorylation site. Loss of Willin expression attenuates MST1/2, LATS1, and YAP phosphorylation promoting YAP's oncogenic transformation activity in vitro, as analysed by its ability to display epithelial-to-mesenchymal transition (EMT) features. These biological outputs are YAP dependent. These data support the involvement of Willin in the regulation of the mammalian Hippo signalling activity by activating the core Hippo pathway kinase cassette. KIBRA has been shown to function as an upstream member of the Hippo pathway by influencing the phosphorylation of LATS and YAP, but the functional consequences of these biochemical changes have not been previously addressed. I showed that in MCF10A cells, loss of KIBRA expression displays EMT features, which are concomitant with decreased LATS and YAP phosphorylation, but not MST1/2. In addition, ectopic KIBRA expression antagonizes YAP via the Ser 127 phosphorylation site and I showed that KIBRA, Willin and Merlin differentially regulate genes controlled by YAP. Willin/FRMD6 was first identified in rat sciatic nerve, which is composed of Schwann cells and fibroblasts. To elucidate the function of Willin in the mammalian sciatic nerve, I showed that Willin is predominantly expressed in fibroblasts and that its expression activates the Hippo signalling cascade and induces YAP translocation from the nucleus to the cytoplasm. In addition within these cells, although it inhibits cellular proliferation, Willin expression induces a quicker directional migration towards scratch closure and an increased expression of factors linked to nerve regeneration. These evidence show that Willin modulates sciatic nerve fibroblast activity, indicating that Willin may have a potential role in the regeneration of the peripheral nervous system.

Introduction: Conventional suture repair of peripheral nerves following injury is associated with several limitations such as technical difficulty, intra- and extra-neural scar formation, axonal escape and the leakage of neurotrophic factors. These limitations are particularly relevant following nerve grafting when regenerating axons must traverse two coaptation sites. Outcomes following suture repair are notoriously poor, providing large impetus for the development of alternative methods. Photochemical tissue bonding (PTB) uses visible light to create sutureless, non-thermal bonds between two closely apposed tissue surfaces stained with a photoactive dye. When used with a human amnion nerve wrap for end-to end nerve repair, this technique results in superior functional and histological outcomes in comparison to conventional epineurial suture. When initially applied to large gap injury and nerve grafting, outcomes were unsuccessful due to proteolytic degradation of amnion and photochemical bonds during extended periods of recovery. Chemical crosslinking of nerve wraps prior to PTB may improve wrap durability and efficacy of technique. This thesis provides a comprehensive three-phase assessment of the efficacy of this novel approach when applied to the repair of large gap injuries with nerve grafts. Phase 1 assesses the ex vivo biomechanical properties of nerve wraps and light activated bonds in addition to the in vivo performance of photochemically sealed crosslinked nerve wraps against several other clinically relevant fixation methods in a rodent sciatic nerve isograft model. Following major multi-limb injury and amputation, demand for autogenous nerve graft may exceed that which can be supplied by the patient. Acellular nerve allograft (ANA) is an alternative option in these circumstances although outcomes are typically inferior to autograft. Phase 2 assesses the performance of the optimum repair strategy from phase 1 against conventional epineurial suture when applied to ANA. Most studies investigating the efficacy of novel repair techniques tend to perform repairs immediately following injury, a situation that rarely occurs clinically. Delays of weeks or months are not uncommon and have been shown to have a detrimental effect on regeneration and outcome. Phase 3 assesses the efficacy of PTB when applied to delayed nerve grafting. Additional work investigating a novel imaging technique for visualizing nerve revascularisation following injury and repair has been included. Optical frequency domain imaging (OFDI) uses low power infrared light to provide real time in vivo imaging of tissue microvasculature and flow characteristics. Originally applied to the study of tumour biology, this technique may prove useful for outcome assessment in preclinical research and eventually for the assessment of nerve viability in the clinical setting. Experiments investigating the early development of a brain body interface system (BBI) for upper limb reanimation following spinal cord injury (SCI) have also been included. The ultimate aim of this project is to restore autonomous motor control in a non-human primate (NHP) using cortically driven stimulation of peripheral nerves via implantable nerve cuffs. The experiments reported in this thesis detail the development of a selective, reversible paralysis model of elbow flexion in a NHP and demonstrate selective fascicular stimulation using acute and chronically implanted nerve cuffs in rodent and murine models. Methods: Phase 1: Three candidate nerve wraps (human amnion (HAM), crosslinked human amnion (xHAM), crosslinked swine intestinal sub-mucosa (xSIS)) and 3 fixation methods (suture, fibrin glue, PTB) were investigated. Crosslinking was performed using (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS). Biomechanical tests were performed using a tensiometer. Ex vivo wrap durability was assessed using a type- 2 collagenase degradation assay. Under isoflurane anaesthesia, 110 inbred male Lewis rats had 15mm left sciatic nerve defects created and repaired with reversed isografts. 9 groups (n=10) had isografts secured by one of the aforementioned wrap/fixation combinations. PTB repairs had nerve wraps and nerve ends stained with photoactive dye (Rose Bengal) and, once nerve ends were apposed and wrapped circumferentially, the interface was illuminated with a 532nm laser. Fibrin repairs had nerve ends apposed, wrapped circumferentially and secured with Tisseel fibrin glue. Suture repairs had nerve ends apposed, wrapped circumferentially and then secured with two 10-0 nylon sutures at each coaptation site (one either side of each repair). Positive and negative control groups (n=10) were repaired with graft+suture (10-0 nylon) and no repair respectively. Phase 2: 20 sciatic nerves were harvested from Sprague Dawley rats and sent to AxoGen Inc. for decellularisation. An additional 20 male inbred Lewis rats were randomized into 2 groups (n=10). All rats had 15mm left sciatic nerve defects created and repaired with processed ANA. 1 group had nerves secured using conventional epineurial suture. The remaining group had ANA secured using photochemically sealed amnion wraps. Phase 3: 40 inbred male Lewis rats were randomized into 4 groups (n=10). All 40 rats had 15mm left sciatic nerve gaps created and reconstructed with reversed isografts harvested from donor Sprague Dawley rats. In groups 1 and 2, nerve gaps were repaired immediately with either conventional epineurial suture or photochemically sealed amnion wraps, respectively. In groups 3 and 4, repair took place 30- days following injury using either conventional epineurial suture or photochemically sealed amnion wraps, respectively. All outcomes were assessed using walking track analysis and calculation of sciatic function index (SFI). Walking track analysis and SFI was performed pre-operatively, after the 30-day delay (phase 3) and at 30-day intervals following surgery. Following sacrifice after 5-months, left (experimental) and right (control) gastrocnemius muscles were excised and weighed for calculation of muscle mass retention. Nerves were excised for histomorphometric analysis including axon count, fiber diameter, axon diameter, myelin thickness and G-ratio. For all in vivo experiments, statistical analysis was performed using ANOVA, repeated measures ANOVA and the post hoc Bonferroni test. Optical Frequency Domain Imaging (OFDI) pilot study: eight rodents were randomized into 4 groups (n=2): (1) crush injury, (2) transection and end-to-end repair, (3) transection and repair of 10mm nerve gap using contralateral autograft, (4) transection and repair of 10mm nerve gap using ANA. Under ketamine/xylazine anaesthesia, all rodents had sciatic nerves exposed through hind limb dorsolateral incisions. Imaging was performed immediately pre-injury, immediately post-injury and on post-operative days 1, 3, 5 and 7. Rodents were secured firmly to polystyrene platforms in order to reduce movement artifact during imaging Brain-Body Interface (BBI) experiments: In the upper limb of a Rhesus macaque nonhuman primate, the median nerve branch to brachialis and radial nerve branch to brachioradialis were transected, leaving elbow flexion entirely reliant on the musculocutaneous nerve. The musculocutaneous nerve was transposed into a subcutaneous position. Ultrasound guided nerve block resulted in a highly selective, reversible paralysis of elbow flexion. Under ketamine/xylazine anaesthesia, Sprague Dawley rats (n=5) and C57 Black 6 mice (n=5) had sciatic nerves exposed through dorsolateral, muscle splitting incisions. 8-channel stimulating cuff electrodes were wrapped around sciatic nerves and connected to a Tucker14 Davies stimulation/recording system. Electromyography (EMG) needle electrodes were inserted into the tibialis anterior (TA) and gastrocnemius (G) muscles to record muscle activity.

The transplantation of primary Schwann cells (SC) has been shown to improve nerve regeneration. However, to monitor the survival of transplanted cells within the host, a stable labelling method is required. The in vitro characteristics of green fluorescent protein labelled SC (GFP SC) and their effects in an in vivo peripheral nerve injury model were investigated.   The GFP-SC were readily visualised ex vivo and stimulated significantly better axonal regeneration compared to controls. Clinical use of autologous SC for the treatment of nerve injuries is of limited use due to difficulty in obtaining clinically useful numbers. However, bone marrow mesenchymal stem cells (MSC) can trans-differentiate into SC like cells (dMSC). The in vitro and in vivo differentiation of MSC was explored, and the study extended to include the easily-accessible adipose stem cells (ASC).  In vitro, glial growth factor stimulated MSC express S100, a SC marker, and its expression is maintained following in vivo transplantation.  Similarly, untreated MSC transplanted in vivo also expressed S100, which indicates glial differentiation in response to local cytokines and growth factors. Using an in vitro model, comprising dMSC or dASC co-cultured with adult dorsal root ganglia (DRG) neurons, the capacity of the dMSC and SC like differentiated ASC (dASC) to promote axon myelination was verified: both cell types expressed transcripts for protein zero, peripheral myelin protein-22 and myelin basic protein. The potential of stem cells in nerve repair may be limited by innate cellular senescence or donor age affecting cell functionality thus it was essential to determine the effects of donor age on morphology and functionality of stem cells.  The proliferation rates, expression of senescence markers (p38 and p53) and the stimulation of neurite outgrowth from DRG neurons by stem cells isolated from neonatal, young or old rats were very similar. However, the distribution and ultrastructure of mitochondria in dMSC and dASC from young and old rats were quite different, and seem to indicate physiological senescence of the aged cells.  Given the wide-ranging influence of Notch signalling in cell differentiation, including the neural crest to a glial cell type switch, and self-renewal in mammals, its role in the differentiation of stem cells to SC was investigated. The mRNA for notch-1 and -2 receptors were expressed in the dASC, blockage of notch signaling did not affect the neurotrophic and myelination potential of dASC.  In conclusion, these findings show that GFP labelling has no deleterious effect on SC survival and function. MSC and ASC differentiated into glial-type cells acquire SC morphology, and express characteristic SC markers, and the differentiation process was independent of the Notch signaling pathway. Also, following transplantation into a nerve gap injury dMSC improve regeneration. This study established that following co-culture with DRG neurons, dMSC and dASC were able to express peripheral myelin proteins.  Also, the functional bioactivity of these cells is independent of the donor animal age. Finally, although the glial lineage differentiated aged cells characterized in this study expressed markers typical of senescence they retained the potential to support axon regeneration.

The main objective of my dissertation was to evaluate the biomedical and psychosocial factors associated with pain and disability in patients following traumatic upper extremity nerve injuries. This was approached by conducting 3 studies. The first study surveyed peripheral nerve surgeons regarding the assessment of pain in patients with nerve injury. The results showed that only 52% of surgeons always evaluate pain in patients referred for motor/sensory dysfunction. Pain assessment frequently includes verbal response and assessment of psychosocial factors is infrequent. The second study was a retrospective review to assess disability, as measured by the Disabilities of the Arm, Shoulder and Hand (DASH), in patients with chronic nerve injury. Results showed substantial disability (mean DASH 52 + 22) and a significantly lower health status (p < 0.001) compared with well-established norms. In the regression model, the factors associated with the DASH (R2 = 44.5%) were pain, older age and nerve injured. The third study was a cross-sectional evaluation of the biomedical and psychosocial factors associated with pain and disability after upper extremity nerve injury in 158 patients. DASH scores were significantly higher in patients with workers’ compensation or litigation (p = 0.03), brachial plexus injuries (p < 0.001) and unemployed patients (p < 0.001). In the multivariable regression analysis, the final model explained 52.7% of the variance with these predictors; pain intensity (Beta = .230, p = 0.006), nerve injured (Beta = -.220, p = 0.000), time since injury (Beta = -.198, p = 0.002), pain catastrophizing (Beta = .192, p = 0.025), age (Beta = .187, p = 0.002), work status (Beta = .179, p = 0.008), cold sensitivity (Beta = .171, p = 0.015), depression score (Beta = .133, p = 0.066), workers’ compensation/litigation (Beta = .116, p = 0.049) and gender (Beta = -.104, p = 0.09). Future investigation regarding treatments of the factors that are associated with disability and chronic pain will assist to improve health related quality of life in patients with traumatic nerve injury.

The main objective of my dissertation was to evaluate the biomedical and psychosocial factors associated with pain and disability in patients following traumatic upper extremity nerve injuries. This was approached by conducting 3 studies. The first study surveyed peripheral nerve surgeons regarding the assessment of pain in patients with nerve injury. The results showed that only 52% of surgeons always evaluate pain in patients referred for motor/sensory dysfunction. Pain assessment frequently includes verbal response and assessment of psychosocial factors is infrequent. The second study was a retrospective review to assess disability, as measured by the Disabilities of the Arm, Shoulder and Hand (DASH), in patients with chronic nerve injury. Results showed substantial disability (mean DASH 52 + 22) and a significantly lower health status (p < 0.001) compared with well-established norms. In the regression model, the factors associated with the DASH (R2 = 44.5%) were pain, older age and nerve injured. The third study was a cross-sectional evaluation of the biomedical and psychosocial factors associated with pain and disability after upper extremity nerve injury in 158 patients. DASH scores were significantly higher in patients with workers’ compensation or litigation (p = 0.03), brachial plexus injuries (p < 0.001) and unemployed patients (p < 0.001). In the multivariable regression analysis, the final model explained 52.7% of the variance with these predictors; pain intensity (Beta = .230, p = 0.006), nerve injured (Beta = -.220, p = 0.000), time since injury (Beta = -.198, p = 0.002), pain catastrophizing (Beta = .192, p = 0.025), age (Beta = .187, p = 0.002), work status (Beta = .179, p = 0.008), cold sensitivity (Beta = .171, p = 0.015), depression score (Beta = .133, p = 0.066), workers’ compensation/litigation (Beta = .116, p = 0.049) and gender (Beta = -.104, p = 0.09). Future investigation regarding treatments of the factors that are associated with disability and chronic pain will assist to improve health related quality of life in patients with traumatic nerve injury.