Injuries to the peripheral nervous system rank among the most common causes of chronic neuropathic pain. Afflicting millions of people for months or even years, symptoms of this condition have proven difficult to treat clinically. A thorough understanding of the pathophysiological changes induced by such nerve lesions is essential to the development of more efficient therapeutic options. Peripheral nerve injury induces a robust and tightly regulated innate immune response in the dorsal horn of the spinal cord. The precise molecular mechanisms regulating the spatiotemporal dynamics and functional impact of the response remain incompletely understood. Preclinical evidence suggests mitigating this immune response can have a significant therapeutic benefit in the treatment of neuropathic pain, however these findings have yet to be clinically validated. To elucidate the mechanisms regulating the spinal immune response, we used a mouse model of partial sciatic nerve injury exclusively in male adult (2-3-month-old) mice. The spared nerve injury (SNI) model employed throughout our studies induces robust, persistent neuropathic pain-like behavior. We established a time course for the spinal immune response to SNI and used mRNA extracted from the ipsilateral dorsal horn of lumbar spinal cord segments L4 and L5 to analyze changes in the transcriptome at the peak of the immune reaction 7 days after nerve lesion. We discovered upregulation of multiple elements of the triggering receptor expressed on myeloid cells 2 (Trem2) pathway. Trem2 is considered a regulator of toll-like receptor signaling in innate immune cells. It also promotes microglia-mediated phagocytosis in the central nervous system. Recent work from our lab has established neuronal apoptosis in the ipsilateral dorsal horn after SNI as an essential mechanism leading to the development of chronic neuropathic pain-like behavior. We used TUNEL staining of L4 spinal cord sections to compare the clearance of apoptotic cell profiles in Trem2-/- mice to wild-type littermates and discovered a key role for Trem2 in the clearance of apoptotic cells after SNI. We further used genetic deletion of Trem2 as well as administration of a Trem2 agonist in C57Bl/6 mice to assess the impact of Trem2 signaling on both the spinal immune response and neuropathic pain-like behavior after SNI. Neither removal nor augmentation of Trem2 signaling significantly affected the development of neuropathic pain-like behavior. Utilizing flow cytometry, we also evaluated the cellular composition of the spinal immune response. We found no evidence that monocytes from the peripheral circulation invade the spinal cord after SNI, as has been previously suggested. These findings were corroborated by immunohistochemical analysis of spinal cord sections from transgenic mice that express distinct fluorescent proteins in their monocyte and microglia cell populations. To better understand the different mechanisms modulating the spinal immune response, we further examined several transcriptionally regulated signaling pathways. We achieved the greatest reduction of mechanical allodynia in nerve-lesioned mice treated with a P2x4r antagonist. Surprisingly, the removal of fractalkine (Cx3cl1) signaling, another prominent chemokine signaling pathway in microglia, had no significant impact on either the spinal immune response or mechanical allodynia after SNI. Reducing the number of spinal microglia by blocking Csf1r activation did not prevent the development of mechanical allodynia after SNI either. Our findings reveal a more nuanced concept of microglial activation after nerve injury. The impact on neuropathic pain-like behavior and phagocytosis appear to be regulated by pathways that differ from those controlling immune cell recruitment and global activation. These findings provide a greater understanding of the complex mechanisms governing microglial function and offer new insight into molecular targets essential to the development of more efficient treatment options for neuropathic pain.

No description available.

研究目的：脊髓損傷是中樞神經系統的嚴重創傷，致殘率高。脊髓損傷後的再生修復一直是當前醫學的難題。迄今為止，脊髓損傷依然缺乏一種有效地治療方法。既往研究證明，胰島素樣生長因子-1對鼠和兔脊髓損傷有保護作用，為了進一步把這些發現應用到臨床方面，我們採用與人類生理更相近的豬只作為實驗動物，構建與臨床相似的脊髓損傷動物模型，并以此為基礎，系統性研究胰島素樣生長因子-1的脊髓保護作用，評估該治療的功效。 / 研究方法：以運動誘發電位為指導，通過直接壓迫和牽拉造成脊髓損傷。18頭猪只隨機分為3組：胰島素樣生長因子-1治療組、生長激素治療組及生理鹽水對照組。脊髓損傷后1小時、24小時及48小時經鞘內注射給藥。于術後第1天、第3天及第21天收集腦脊液檢測胰島素樣生長因子-1和生長激素濃度。連續21天使用修正的 Tarlov 評分標準對動物的運動功能進行評估。第21天處死動物並取材，檢測脊髓中NeuN, GFAP, caspase-3 的活性，并通過TUNEL染色觀察細胞凋亡情況，比較各組之間有無差別。 / 研究結果：通過這種方法建立的脊髓損傷動物模型穩定可靠，各組之間無明顯差異。鞘內給藥24小時及48小時后，腦脊液中胰島素樣生長因子-1和生長激素濃度明顯升高，術後21天檢測，其濃度恢復至基礎值。胰島素樣生長因子-1治療組的運動功能的恢復優於其它各組。與生理鹽水對照組比較，胰島素樣生長因子-1治療組可以明顯提高脊髓損傷后神經元的存活數量，抑制星形膠質細胞增生，減少細胞凋亡。而生長激素治療組僅抑制星形膠質細胞增生，其它方面與生理鹽水對照組無明顯差別。 / 結論：胰島素樣生長因子-1通過提高神經元存活數量，抑制星形膠質細胞增生，以及減少細胞凋亡促進脊髓損傷的恢復。 / Objective: Spinal cord injury is a devastating condition that leads to long-term disabilities. Currently, there is no effective treatment that minimizes spinal cord damage or enhances neurological recovery. Recent studies in rats or rabbits suggested that neurologic recovery after spinal cord injury could be improved with the administration of neurotropic hormones, such as insulin-like growth factor-1 (IGF-1). In order to apply such bench-side discovery to clinical practice, we conducted a study in a higher animal model, akin to human physiology, to evaluate the effectiveness of intrathecal injections of IGF-1to improve neurological recovery in a porcine model of acute traumatic spinal cord injury. / Methods: Traumatic spinal cord injury model was produced by controlled compression and distraction of the exposed T12 segment of the spinal cord. Eighteen pigs were randomly assigned to receive intrathecal injections of either IGF-1, growth hormone or saline at 1, 24 and 48 hours after spinal cord injury. Locomotor function was assessed daily using the validated modified Tarlov’s scale for 21 days. Spinal cord segments were then harvested and the survival of neurons, reactive astrogliosis and apoptosis were determined using neuronal-specific nuclear protein (NeuN), glial fibrillary acidic protein (GFAP), cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays. / Results: Intrathecal injections of IGF-1 and growth hormone significantly increase the concentrations of the neurotropic hormones in the cerebrospinal fluid after injury (p < 0.01). These concentrations returned to baseline by 21 days after drug delivery. Motor deficits on the first day after injury were comparable between animals in the treatment and control groups. By the end of the third week, neurologic recovery was better in animals receiving IGF-1 treatment (p < 0.05). Immunohistological and western blot studies of the injured segments of spinal cord showed that treatment with both IGF-1 and growth hormone prevented reactive astrogliosis (p < 0.05) while only IGF-1 improved the survival of mature neurons (p < 0.05). IGF-1 also inhibited apoptosis after spinal cord injury (p < 0.05). / Conclusions: In our clinically relevant model of traumatic spinal cord injury in pigs, intrathecal injection of IGF-1 demonstrated beneficial effects on neurological and histological recovery. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Qinzhou. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 105-122). / Abstract also in Chinese. / Declaration of origination --- p.I / Abstract --- p.II / Acknowledgements --- p.VI / Table of Contents --- p.VIII / List of Tables --- p.XII / List of Figures --- p.XIII / Abbreviations --- p.XVIII / Chapter Part 1 --- Spinal Cord Injury: A Review --- p.1 / Chapter Chapter 1-1 --- Acute Spinal Cord Injury: Epidemiology, Socioeconomic Impact --- p.2 / Chapter 1.1.1 --- Epidemiology of Spinal Cord Injury --- p.2 / Chapter 1.1.2 --- Socioeconomic Impact of Acute Spinal Cord Injury --- p.5 / Chapter Chapter 1-2 --- Mechanisms of Spinal Cord Injury --- p.6 / Chapter Chapter 1-3 --- Putative Treatments for Spinal Cord Injury --- p.8 / Chapter 1.3.1 --- Methylprednisolone --- p.8 / Chapter 1.3.2 --- Stem Cell Therapy --- p.11 / Chapter 1.3.3 --- Riluzole --- p.11 / Chapter 1.3.4 --- Other Pharmacological Therapies for Spinal Cord Injury --- p.12 / Chapter Chapter 1-4 --- Insulin-like Growth Factor-1 for the Treatment of Spinal Cord Injury --- p.13 / Chapter Chapter 1-5 --- Summary --- p.17 / Chapter Part 2 --- Insulin-like Growth Factor-1 and Growth Hormone for Spinal Cord Injury --- p.18 / Chapter Chapter 2-1 --- Hypothesis and Objectives --- p.19 / Chapter Chapter 2-2 --- Establishment of Animal Models for Acute Spinal Cord Injury --- p.22 / Chapter 2.2.1 --- Introduction --- p.22 / Chapter 2.2.2 --- Experimental Animals --- p.22 / Chapter 2.2.3 --- Anesthesia --- p.23 / Chapter 2.2.4 --- Transcranial Electrical Motor Evoked Potential --- p.26 / Chapter 2.2.5 --- Surgery --- p.28 / Chapter 2.2.6 --- Statistics --- p.34 / Chapter 2.2.7 --- Results --- p.34 / Chapter 2.2.8 --- Discussion --- p.38 / Chapter Chapter 2-3 --- Optimal Stimulation Protocols for Transcranial Electrical Motor Evoked Potential. --- p.42 / Chapter 2.3.1 --- Introduction --- p.42 / Chapter 2.3.2 --- Methods --- p.42 / Chapter 2.3.2.1 --- Experimental Animals and Anesthesia --- p.42 / Chapter 2.3.2.2 --- Transcranial Electrical Motor Evoked Potential Recording --- p.44 / Chapter 2.3.2.3 --- Stimulation Protocol --- p.44 / Chapter 2.3.3 --- Analyses --- p.44 / Chapter 2.3.4 --- Results --- p.45 / Chapter 2.3.5 --- Discussion --- p.52 / Chapter Chapter 2-4 --- Evaluation of the Efficacy of Insulin-like Growth Factor-1 and Growth Hormone in a Porcine Model --- p.54 / Chapter 2.4.1 --- Introduction --- p.54 / Chapter 2.4.2 --- Materials and Methods --- p.54 / Chapter 2.4.2.1 --- Study Design --- p.54 / Chapter 2.4.2.2 --- Intrathecal Injection and Collection of Cerebrospinal Fluid --- p.58 / Chapter 2.4.2.3 --- Measurements --- p.58 / Chapter 2.4.2.3.1 --- Clinical Evaluation --- p.58 / Chapter 2.4.2.3.2 --- Biochemical Assessments --- p.58 / Chapter 2.4.2.3.3 --- Spinal Cord Section, Histological and Immunochemical Staining --- p.63 / Chapter 2.4.2.3.4 --- Western Blot --- p.69 / Chapter 2.4.3 --- Statistical Analysis and Sample Size Calculation --- p.72 / Chapter 2.4.3.1 --- General Analysis --- p.72 / Chapter 2.4.3.2 --- Sample Size --- p.72 / Chapter 2.4.4 --- Results --- p.73 / Chapter 2.4.4.1 --- Changes of TceMEP --- p.73 / Chapter 2.4.4.2 --- Motor Deficit after Spinal Cord Injury at Baseline --- p.75 / Chapter 2.4.4.3 --- Insulin-like Growth Factor-1 and Growth Hormone in Cerebrospinal Fluid --- p.77 / Chapter 2.4.4.4 --- Clinical Assessment --- p.80 / Chapter 2.4.4.5 --- Demyelination, Neuron Survival and Astrocyte Reaction --- p.85 / Chapter 2.4.4.6 --- Apoptosis --- p.89 / Chapter 2.4.5 --- Discussion --- p.93 / Chapter 2.4.5.1 --- Principal Findings --- p.93 / Chapter 2.4.5.2 --- Insulin-like Growth Factor-1 and Neuroprotection after Spinal Cord Injury --- p.93 / Chapter 2.4.5.3 --- Growth Hormone and Neuroprotection after Spinal Cord Injury --- p.95 / Chapter 2.4.5.4 --- Strengths and Limitations of Our Study --- p.96 / Chapter 2.4.5.5 --- Summary --- p.97 / Chapter Part 3 --- Summary and Future Directions --- p.99 / Chapter Chapter 3-1 --- Summary --- p.100 / Chapter Chapter 3-2 --- Future Directions --- p.103 / Chapter Part 4 --- References and appendixes --- p.104 / References --- p.105 / Appendixes --- p.123

Resumo Acidentes e violências são responsáveis por elevadas taxas de mortalidade e morbidade causadas pelas lesões deles decorrentes. Entre essas lesões, os traumas da coluna vertebral, especificamente, e aquelas com comprometimento da medula espinal apresentam-se como um grande problema de Saúde Pública, uma vez que a maior parte dos lesados é composta de jovens e no auge da sua produtividade, tanto profissional quanto pessoalmente, além de as lesões serem responsáveis por seqüelas, geralmente, irreversíveis. Raros foram os estudos encontrados, que demonstrem a distribuição epidemiológica dessas lesões no Brasil. O trabalho objetiva conhecer o perfil das internações por traumas de coluna segundo características dos pacientes, lesões, causas externas que as produziram, bem como aspectos relativos às suas internações. O material de trabalho refere-se às internações SUS (dados do Banco do SIH/SUS) para traumas de coluna (S12, S14, S17, S19, S22, S24, S28, S29, S32, S34, S38, S39, T02.0, T02.1, T04.1, T04.2, T09.3 e T09.4 da CID-10*) As internações por traumas de coluna correspondem a cerca de 0,2% do total de internações no país e cerca de 2,7% do total de internações por lesões decorrentes de causas externas. Essa proporção, bem como a taxa de internação por traumas de coluna, foi crescente no período analisado na pesquisa. O grupo do sexo masculino, na faixa de 20 a 29 anos, é o mais comumente afetado, embora, em casos específicos, como o das internações decorrentes de quedas, observe-se aumento da proporção de idosos e também da população feminina. O nível lombo-sacral agrega cerca de 60% do total de internações por trauma de coluna, seguido pelo nível cervical (cerca de 28%). Os traumas de nível cervical apresentam a maior proporção de traumas com comprometimento medular, com cerca de 45,8% do total de traumas no ano de 2005. Segundo o tipo de causa externa que ocasionou a lesão, cerca de 40% das internações são decorrentes de lesões causadas por quedas. Os acidentes de trânsito correspondem a cerca de 30% e as tentativas de homicídio, 7% do total de internações por traumas de coluna. O tempo médio de permanência dos pacientes vítimas de traumas de coluna variou de 8,2 a 9,2 dias nos anos analisados, com significativas diferenças segundo o nível da lesão e comprometimento medular. Quando comparados aos custos das internações por causas externas, de modo geral, as internações por trauma de coluna apresentaram valores maiores, variando também segundo o grau de comprometimento medular e nível da lesão. A análise epidemiológica das internações por traumas de coluna esclareceu aspectos interessantes referentes às principais causas externas envolvidas, qualidade das informações disponíveis, custos e gastos para o sistema de saúde, bem como um panorama do perfil das vítimas, permitindo o planejamento de ações específicas para promoção da saúde e cuidados dessa população. * Classificação Internacional de doenças 10ª Revisão, 1995 / Abstract Accidents and violence are responsible for high taxes of mortality and morbidity by the consequences of trauma. In this injuries the vertebral column traumas, specifically and with spinal injury are an important problem for Public Health, because the most of the victims are young persons in the top of her productive life. The most of injuries are responsible for irreversible consequences. Just a little number of epidemiologycals studies where found in Brazil. This research objective is describe the traumas´s hospitalizations by victims characteristics and external causes what was the basic cause for the hospitalization. The material reference are SUS hospitalizations (SIH/SUS) for column traumas (S12, S14, S17, S19, S22, S24, S28, S29, S32, S34, S38, S39, T02.0, T02.1, T04.1, T04.2, T09.3 e T09.4 da CID-10*). The column traumas are almost 0,2% by the total of Brazil´s hospitalizations and 2,7% by the injuries hospitalizations by external causes. This number was in elevation between the years f this research. The male group by 20-29 years old was most commun affected. The hospitalizations by consequence off falls show an elevation in elders group and female group. In the sacral level there is 60% by the total of hospitalizations follow by cervical level (28%). In the cervical level we can see the most causes of spinal injury with almost 45,8% by the total. Studying the external causes almost 40% by the hospitalizations in all years, are by traumas in consequence by falls. The traffic accidents are 30% and violence 7% by the total of hospitalizations. The middle time by the hospitalizations was between 8,2 to 9,2 days with important differences when analized in the level of injury. About costs, when compare with external causes general, the column traumas show higher values. This epidemiological analize could show interesting aspects about external causes with column traumas as consequence, information quality, costs, and victims by this kind of trauma, with important dates for actions for health promotion and care of this population. * CID 10ª Review, 1995

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / From the need for a specific instrument to evaluate the quality of life of people with spinal cord injury in the Brazilian language, this study was conducted and had the following objectives: to translate and culturally adapt to Portuguese Ferrans and Powers Quality of Life Index Spinal Cord Injury Version â III and characterise the sample in terms of sociodemographic and clinical aspects. Methodological procedure, in which the authorization from the author was obtained. First, there was a stage of translating with the participation of six translators. Then, there was a cultural adaptation taking into account the opinion of five judges. The pretest sample consisted on 30 patients with spinal cord injury, which were selected from the database of the Center for Research and Consulting in Neurological Nursing, and which also met the criteria for inclusion. Data was collected from August to November of 2013 by the final version in Portuguese called Ãndice de LesÃo Medular e Qualidade De Vida â VersÃo III and a questionnaire with social, demographic and clinical data. The study was approved by the Ethics in Research Committee, under the number: 344.927/2013. An index with 74 items was obtained, it was divided into two parts (satisfaction / importance ), on which some adjustments were made for a better understanding. These adjustments resulted in: the addition of four new pronouns or articles to change the genre; nine exclusions of words or articles; two changes from a word to a term; four additional expressions for the purpose of illustrating; four adjusts in expressions; nine changes of only one word. Therefore, there was 24 modifications. It was observed that, in the criteria of semantics equivalence of the index, ortography was rated as âa very appropriate translationâ by a number greater than 87 % ; vocabulary and grammar, by 86%; idiomatic equivalence was rated by a number higher than 74%; experimental equivalence, higher than 78% and conceptual equivalence, by a number greater than 70%. Statistical analysis on semantic, idiomatic, experimental and conceptual evaluations by kappa showed a slight to moderate conformity between the pairs of analysis, adopting (p &#8804; 0.05). It was concluded that this instrument, after being transculturally adapted, has proved to be appropriate under the semantic , idiomatic, experimental and conceptual views, as well as easy to use to evaluate the quality of life of people with spinal cord injury. The study enabled the expansion of scientific knowledge of nursing, especially for professionals in the neurological field, by making it possible to plan, intercede and evaluate the caring for the special needs of these patients. It is now possible to resort to a technology that takes into account a great number of subjective relationships that are not, generally, observed or disclosed. / A partir da necessidade de um instrumento especÃfico para avaliar a qualidade de vida de pessoas com lesÃo medular no idioma brasileiro, realizou-se este estudo que teve como objetivos: traduzir e adaptar culturalmente para a lÃngua portuguesa Ferrans and Powers Quality of Life Index Spinal Cord Injury Version â III e caracterizar a amostra quanto aos aspectos sociodemogrÃficos e clÃnicos. Estudo do tipo metodolÃgico, no qual obteve-se autorizaÃÃo da autora para utilizaÃÃo do instrumento. Inicialmente procedeu-se a etapa de traduÃÃo com a participaÃÃo de seis tradutores, em seguida a adaptaÃÃo cultural pela confrontaÃÃo de cinco juÃzes. A amostra do prÃ-teste constituiu-se de 30 pacientes com lesÃo medular selecionados a partir do banco de dados disponibilizados pelo NÃcleo de Pesquisa e ExtensÃo em Enfermagem NeurolÃgica, e que atenderam aos critÃrios de inclusÃo. A coleta de dados ocorreu nos meses de agosto a novembro/2013, por meio da utilizaÃÃo da versÃo final traduzida para o portuguÃs denominada de Ãndice de LesÃo Medular e Qualidade De Vida â VersÃo III e um questionÃrio com dados sociodemogrÃficos e clÃnicos. O estudo foi aprovado pelo Comità de Ãtica em Pesquisa, Parecer n 344.927/2013. Obteve-se um Ãndice com 74 itens, divididos em duas partes (satisfaÃÃo / importÃncia), com alguns ajustes para facilitar a compreensÃo, dos quais resultaram em quatro acrÃscimos de pronome ou artigo para variaÃÃo do gÃnero; nove retiradas de uma palavra ou artigo; duas mudanÃas de uma palavra para uma expressÃo; quatro expressÃes adicionais com o intuito de exemplificar; quatro ajustes em expressÃes; nove mudanÃas de apenas uma palavra. Desse modo, obteve-se ao todo 24 modificaÃÃes. Quanto aos critÃrios de equivalÃncia semÃntica do Ãndice observou-se que, em relaÃÃo a ortografia o percentual de itens avaliados como TraduÃÃo Muito Adequada foi superior a 87%, vocabulÃrio e gramÃtica a 86%. Na equivalÃncia idiomÃtica obteve-se valores superior a 74%, na equivalÃncia experimental superior a 78% e na equivalÃncia conceitual superior a 70%. A anÃlise estatÃstica para as avaliaÃÃes semÃntica, idiomÃtica, experimental e conceitual por meio do kappa apontou de ligeira a moderada a concordÃncia das anÃlises entre os pares, ao adotar (p &#8804; 0,05). Conclui-se que este instrumento apÃs ser adaptado transculturalmente demonstrou ser adequado do ponto de vista semÃntico, idiomÃtico, experimental e conceitual, alÃm de fÃcil aplicaÃÃo para avaliar a qualidade de vida de pessoas com lesÃo medular. O estudo possibilitou a ampliaÃÃo do saber cientÃfico da enfermagem, em especial aos profissionais que atuam na Ãrea neurolÃgica, por possibilitar o planejamento, as intervenÃÃes e avaliaÃÃo de cuidados direcionados Ãs necessidades tÃo peculiares destes pacientes, podendo agora recorrer a uma tecnologia que mensura muitas relaÃÃes subjetivas e que nÃo sÃo observadas ou reveladas.

Spinal muscular atrophy (SMA) is a severe genetic neuromuscular disorder characterised by lower motor neuron degeneration and paralysis. Although it is a leading genetic cause of childhood death no approved treatment options currently exist. As SMA is caused by low levels of the survival motor neuron (SMN) protein the majority of therapeutic strategies under development are therefore aimed at trying to elevate SMN levels. However, a number of limitations with these approaches exist demonstrating a need for the investigation of SMN-independent therapeutics. Of these non-classical pathways, the ubiquitin proteasome system (UPS) is an exciting new area of SMA research. The UPS is a system which degrades unwanted or damaged proteins and alterations in the UPS (including reduced levels of ubiquitin-like modifier activating enzyme 1 [Uba1] and increased levels of ubiquitin carboxyl-terminal esterase L1 [Uchl1] and β-catenin) have been recently identified in the neuromuscular system of SMA mice, providing promising new targets for therapy development. In this thesis I demonstrate that UPS perturbations are also present in other organ systems of severe ‘Taiwanese’ SMA mice and in other SMA models including intermediate Smn2B/− mice, zebrafish and patient derived iPSC motor neurons. Given the previously demonstrated improved neuromuscular phenotype in SMA mice treated with the β-catenin inhibitor quercetin I have been establishing whether other compounds with β-catenin inhibition offer similar or even better therapeutic options. Aspirin, indomethacin and iCRT-14 trials did not improve the SMA phenotype with likely off-target adverse effects meaning that quercetin remains the most tolerable β- catenin inhibitor in SMA mice to date. Another potential target of the UPS for SMA therapeutics is the deubiquitinating enzyme Uchl1, levels of which are increased in SMA. In this thesis I show that pharmacological inhibition of Uchl1 did not improve survival or motor performance in SMA mice and instead had a detrimental impact on the disease phenotype which could be explained by worsening SMA ubiquitin defects. Histological analysis revealed that there was no improvement in lower motor neuron count numbers, neuromuscular junction deficits or muscle fibre diameters. Mimicking the UPS phenotype in primary neuronal cells suggested that targeting UPS perturbations observed in SMA that are upstream of Uchl1, particularly the loss of Uba1, may therefore offer a more effective therapeutic option. Finally, I therefore examined whether increasing Uba1 levels in SMA mice using gene therapy technology was able to improve the SMA phenotype. My initial studies indicate that delivery of AAV9-UBA1 to SMA mice may be beneficial as intraperitoneal injection of AAV9-UBA1 was found to increase the weight and improve motor performance of SMA mice. Intravenous delivery of AAV9-UBA1 was found to further improve expression levels and biodistribution of AAV9-UBA1 in the central nervous system as well as systemically in all body organs and tissues. Western blot and proteomic analysis revealed that AAV9-UBA1 gene therapy is also able to correct downstream UPS perturbations found in SMA as well as increase SMN levels. Together, these results suggest that AAV9-UBA1 gene therapy is an exciting novel therapeutic approach for SMA.

Motor deficits are a hallmark of Down syndrome (DS), yet little is known about their exact cause. Despite the rich understanding of the neurobiology of DS, there is still a lack of targetable mechanisms for early intervention aimed at alleviating motor changes in people with DS. Therefore, we utilized a mouse model of DS known as Ts65Dn to characterize for the first time the effects of trisomy 21 on spinal cord (SC) development. A central molecular player in SC patterning and cell-type specification, Oligodendrocyte transcription factor 2 (Olig2), is located on human chromosome 21 (Hsa21) and is triplicated in both people with DS and in Ts65Dn mice. To observe the effects of the supernumerary Olig2, we used immunohistochemistry to visualize the OLIG2-derived cellular populations (i.e., motor neurons (MNs) and oligodendrocytes (OLs)), as well as adjacent and interacting cell populations (i.e., ventral spinal interneurons (INs)). We limited our analyses to two embryonic ages—embryonic days (E) 12.5 and 14.5. Our results indicate that there is no overall change in the numbers of OLs at either E12.5 or E14.5. However, there tend to be more OL-fated cells within the pMN domain, where they originate, and migrating cells tend to be clustered closer to the pMN domain at E12.5. IN populations show some changes in Ts65Dn mice at E12.5, with both total and abventricular PAX6+ cell numbers and abventricular NKX2.2+ cell numbers increased in Ts65Dn embryos compared to euploid mice. However, at E14.5 the number of NKX2.2+ cells is unchanged. No difference in the NKX6.1+ population was seen at either time-point. In contrast, there are significant changes in the MN population at both E12.5 and E14.5. Specifically, at E12.5, the total ISL1+ MN population is significantly increased and shows altered regional distribution in the ventral horn of Ts65Dn SCs. Conversely, the Ts65Dn spinal MN population is normalized to euploid levels at E14.5. Overall, our results suggest that neurogenesis, gliogenesis, and cell-type specification of OLIG2-lineage cells are altered in the developing SC of Ts65Dn mice. Thus, this work identifies a novel target for future therapeutic interventions aimed at ameliorating motor changes in DS.

Spinal muscular atrophy (SMA) is a debilitating neuromuscular disorder characterized by insufficient SMN protein, resulting in motoneuron death. Initially, it was thought that motoneuronal death is followed by muscle atrophy; however, recent research is beginning to reveal possible muscle intrinsic defects, independent of motoneuron defects, in SMA. Previous studies have elucidated the cooperative involvement of CARM1, HuD and SMN in motoneurons, revealing HuD as a possible key player in the SMA phenotype. In this study, we focus on HuR, a ubiquitous family member of HuD, and the possibility that it plays a similar key role with CARM1 and SMN in skeletal muscle. Through the use of an shCARM1 stable line of C2C12s, we show that CARM1 is necessary for HuR functionality during differentiation. We further show that the methylation of HuR is necessary for its capability to translocate cytoplasmically during differentiation. We confirm an interaction between HuR and SMN, suggestive of a similar mechanism as was shown previously with HuD. In light of these findings, we next progressed to determine whether HuR is misregulated in an SMA mouse model. We report increased CARM1 levels in skeletal muscles of these mice. We further discovered that a deficiency in SMN protein impairs HuR upregulation and cytoplasmic translocation in response to HuR activation through sciatic nerve denervation. These findings were correlated with aberrant mRNA expression of HuR targets upon denervation. Taken together, these results show that HuR methylation is essential for proper myogenesis, and that the mechanism by which it acts likely requires sufficient SMN protein levels. In a deficiency of SMN, HuR shows signs of misregulation that may play a role in the inability to maintain or repair muscle in SMA.

Abstract not available

Transplants of olfactory ensheathing cells (OECs) from olfactory bulbs have recently been shown to support regrowth and reinnervation of damaged spinal cord, which has led to improved functional recovery. Using complete transection in adult rat, the studies presented in this thesis examine the role of peripherally derived olfactory tissue in promoting axonal regeneration and functional recovery. Chapter One and Two provide the background to the area of spinal cord regeneration and the methods used in this thesis. Chapter Three shows that transplants of OECs from rat olfactory lamina propria (OLP) are able to support axon regrowth in the lesioned spinal cord. The BBB score was significantly higher in experimental rats (5.4???0.84) compared with control animals (1.9???0.33) (P&lt0.001). These dissociated OECs from OLP can promote axonal regrowth through the lesion. Histological assessment showed that: 1) axons labelled with Fluororuby grew into the injury site in OECs-transplanted rats, with occasional fibres extending into the rostral cord; 2) brainstem neurons in the raphe nucleus were retrogradely labeled with Fluororuby; and 3) serotonergic axons were detectable distal to the lesion in OECs-transplanted rats. No fibres grew into the injured region and no retrograde labeling or serotonergic fibres were seen in control animals. The role of regenerated serotonergic fibres in OECs-transplanted rats is discussed. Chapter Four demonstrates that solid pieces of OLP dissected from the nose can re-establish the continuity of the transected cord and supply the OECs that can migrate to the cord stumps to support the axon regeneration. Experimental rats which received OLP from olfactory mucosa showed significantly greater locomotive recovery (BBB scores: OLP, 5.0???1.9; control, 1.5???0.5, p&lt0.0001). In animals with OLP transplants, histological analysis indicated that nerve fibres, expressing neurofilament and serotonin were present at the transection site. Locomotive recovery of the hindlimbs occurred, similar to that seen after OECs transplantation. Retrograde labeling of medullary raphe neurons and gigantocellular reticular nucleus occurred following Fluororuby injection in the cord distal to the lesion, further supporting the supraspinal origin of the 5-HT innervation in the present studies. These results indicate that OLP is effective in promoting partial spinal cord repair. Chapter Five examines functional recovery of spinal reflex circuitry, ie., H-reflex excitability using paired stimuli, in OLP-transplanted rats compared with normal and respiratory lamina propria (RLP) transplanted animals. H-reflex amplitude of the conditioned response was significantly reduced in OLP transplanted rats compared to RLP transplanted animals (p&lt 0.05). Therefore, hindlimb reflex excitability can be modulated by OLP transplants after transection of the spinal cord in adult rats. Chapter Six examines whether functional recovery can occur if transplantation of OLP tissue is delayed by 1 month after the spinal cord transection. The BBB score was significantly higher in experimental rats (4.3???0.8 for OLP) compared with control animals (1.0???0.3, P&lt 0.001), but recovery was less than after acute transplantation. Asx before, histological assessment of OLP animals showed: a) serotonergic axons were present in the cord below the transection site; b) brainstem raphe nuclei was retrogradely labeled; c) bisbenzimide pre-labeled cells from OLP transplants migrated in host spinal cord. These changes were not seen in control animals. These results indicate that OLP has the ability to promote axonal regeneration in chronically injured cord of adult rats. Chapter Seven compares the results from these three types of intervention. In conclusion, these studies show that peripherally derived OECs or solid pieces of OLP can promote partial spinal cord repair in acute or chronic transection injuries. Such tissue might provide a potential source for autologous grafting in human paraplegia.