Vinte e seis pacientes com paralisia facial unilateral de longa duração foram submetidos à reanimação facial em único estágio com transplante do músculo grácil inervado pelo ramo massetérico do nervo trigêmeo. Foram divididos em 2 grupos não-randomizados de acordo com a técnica de fixação: Grupo I (19 pacientes), fixação do músculo com uso do tendão palmar longo inserido no músculo orbicular nos lábios superior e inferior do lado não paralisado (além da linha média); Grupo II (7 pacientes), fixação do retalho apenas com pontos separados no músculo orbicular dos lábios superior e inferior no lado paralisado. A avaliação qualitativa demonstrou melhores resultados no Grupo I (94,1% vs 66,6%). Na comparação do posicionamento do arco de cúpido em repouso, no pré e pós-operatório, observamos melhora estatisticamente significante (p<0,05) em ambos os grupos. Durante o sorriso, entretanto, houve melhora significativamente maior na centralização do arco de cúpido nos pacientes submetidos à fixação com tendão palmar longo (Grupo I) / Twenty-six patients with unilateral long-stading facial palsy underwent 1-stage reanimation with free gracilis muscle transplant innervated by the masseteric branch of the trigeminal nerve. They were divided into 2 nonrandomized groups according to insertion technique: group I (19 patients), palmaris longus tendon graft placed between the gracilis free flap and the orbicularis oris of the upper and lower lip on the nonparalyzed side; group II (7 patients), interrupted suture between the free flap and the orbicularis oris of the upper and lower lip on the paralyzed side. Qualitative evaluation of the smile demonstrated better results in patients from group I (94,1% vs 66,6%). Comparing the position of the Cupid`s bow at rest, pre- and postoperatively in each patient, we observed significant improvement of facial symmetry in both groups. During smile, however, there was significantly higher rate of centralization of the Cupid`s bow in patients submitted to reanimation with the use of the palmaris longus tendon (group I)

Dor neuropática e neuropatia causadas pelo quimioterápico oxaliplatina são comuns e causam restrições às atividades funcionais e qualidade de vida. Muitos estudos têm quantificado e qualificado esses sintomas, porém raramente utilizando amostra expressiva e instrumentos validados e específicos para este fim. Neste estudo é proposta uma análise ampla, por instrumentos que quantificam e qualificam a dor e neuropatia relacionadas à oxaliplatina e suas características associadas. Objetivos: Identificar a prevalência de dor neuropática e neuropatia periférica em doentes com câncer colorretal recebendo tratamento com oxaliplatina durante os seis meses de tratamento quimioterápico e após seguimento (de 3 a 6 meses); avaliar, comparar e descrever as características da dor e neuropatia nesta população e avaliar seu impacto nas atividades de vida diária e qualidade de vida. Métodos: Foram incluídos 110 doentes (média 55,6 anos) com câncer colorretal durante o tratamento quimioterápico com oxaliplatina e seguidos por 3 a 6 meses após quimioterapia. Os doentes responderam ao questionário sócio-demográfico e a questionários específicos para dor e neuropatia antes da quimioterapia e bimestralmente por até seis meses durante a quimioterapia e em avaliação de seguimento realizada de 3 a 6 meses após o término da quimioterapia. Os instrumentos utilizados foram: Questionário de Qualidade de Vida da Organização Européia para Pesquisa e Tratamento do Câncer - C30 (EORTC QLQ-C30); Questionário de Dor McGill reduzido (QDMR), Inventário de Sintomas de Dor Neuropática (ISDN), Inventário Breve de Dor (BPI-Brief Pain Inventory) Questionário de Dor Neuropática 4 (DN4), Escala Hospitalar de Ansiedade e Depressão (HADS), Escore Total de Neuropatia (TNS - Total Neuropathy Score). Resultados: Em geral, a dor e neuropatia aumentaram durante o período de quimioterapia e diminuíram após fim do tratamento, permanecendo em níveis significativamente mais elevados após o fim do tratamento quimioterápico. A média de intensidade de dor (dor mais intensa) segundo o IBD foi 2,54 na V3 (após 4 meses de tratamento com oxaliplatina). A dor foi do tipo neuropática em 21,67% dos doentes ao fim da quimioterapia e em 10,00% após fim do seguimento. As médias segundo o ISDN foram 0,67 no basal, 18,67 na V2, 17,77 na V3, 17,44 após quimioterapia e 11,03 após seguimento. A característica da dor mais frequente foi em choque elétrico, enjoada e incômoda segundo o QDMR e segundo o ISDN foram choque elétrico, frio doloroso e dormência. A qualidade de vida sofreu impacto negativo. Conclusões: Dor neuropática foi prevalente após a quimioterapia e após seguimento e se associou com interferência negativa sobre as atividades diárias. Estes dados poderão auxiliar o desenvolvimento de tratamentos individualizados da neuropatia relacionada à oxaliplatina / Neuropathic pain and neuropathy caused by oxaliplatin chemotherapy are common and cause restrictions in activities of daily living and quality of life. Many studies have quantified and qualified these symptoms but only rarely used a comprehensive sample of patients and employed validated and specific instruments for pain assessment. In this study we proposed a comprehensive analysis by instruments that quantify and qualify the pain and neuropathy and its characteristics. Aim of Investigation: To identify the prevalence of neuropathic pain and peripheral neuropathy in patients with colorectal cancer receiving treatment with oxaliplatin during the six months of chemotherapeutic treatment and after follow-up (3 to 6 months); to evaluate, compare and describe pain and peripheral neuropathy characteristics in this population and evaluate their impact on activities of daily living. Methods:110 patients (mean 55.6 years) with colorectal cancer were included during the six months of chemotherapy with oxaliplatin and follow-up (3 to 6 months) after chemotherapeutic treatment. Patients answered socio-demographic questionnaires and specific assessment tools for pain and neuropathy evaluation at the baseline visit and every 2 months during chemotherapy and after follow-up (3-6 months). The instruments used were: The European Organization for Research and Treatment of Cancer QLQ-C30 (EORTC QLQ-C30); Reduced McGill Pain Questionnaire (MPQ), Neuropathic Pain Symptom Inventory (NPSI), Brief Pain Inventory (BPI) Neuropathic Pain Diagnostic Questionnaire (DN4), Hospital Anxiety and Depression Scale (HADS), Total Neuropathy Score (TNS), Results: In general, pain and neuropathy intensity increased during chemotherapy and decreased after the end of treatment (follow-up). The most severe pain according to the BPI was 2.54 in V3 (after 4 months treatment with oxaliplatin). Pain was neuropathic in 21.67% right after chemotherapy and in 10.00% after follow-up according to the DN4. The average sum of neuropathic pain symptoms according to the NPSI were 0.67 in V1, 18.67 in V2, 17.77 in V3, 17.44 after chemotherapy and 11.03 after follow-up. The most common characteristics of the pain was electric shocks, nauseating and fearful, according to MPQ and according to NPSI were electric shock, evoked by cold stimuli and tingling. Conclusions: Patients treated with oxaliplatin had significant pain and neuropathy that negatively interfeared with daily activities. These data may help tailor individualized treatment of chemotherapy related neuropathic pain

It is of interest to understand how new neurons incorporate themselves into the existing circuitry of certain neuronal populations. One such population of neurons is that which are born in the subventricular zone (SVZ) and migrate to the olfactory bulb where they differentiate into granule cells. Another area of interest is the role of brain-derived neurotrophic factor (BDNF) on the survival and overall health of these neurons. This study aimed to test whether or not BDNF is a survival factor for adult-born granule cells. Here were utilized a transgenic mouse model over-expressing BDNF under the α- calcium/calmodulin-dependent protein kinase II (CAMKIIα) promoter, and tested its effect on olfactory granule cells under sensory deprived conditions. Results from this experiment indicated that there was no significant difference in cell death or cell survival when comparing transgenic and wild type animals. We concluded that BDNF is not a survival factor for adult-born granule cells. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

INTRODUÇÃO: A formação do hematoma intraneural com comprometimento dos nervos periféricos pode ocorrer após traumas ou em associação com distúrbios de coagulação. A opção por conduta conservadora (expectante) ou descompressão cirúrgica ainda é controversa. Essas duas condutas foram analisadas comparativamente por meio de modelo experimental em ratos submetidos a hematoma intraneural associado à compressão extrínseca. MATERIAL E MÉTODOS: Cinqüenta ratos Wistar foram divididos em 5 grupos. Em 4 grupos (A, B, C e D) o nervo ciático direito foi envolvido por tubo de silicone de diâmetro interno maior que o do nervo, simulando o trajeto do nervo periférico através de regiões inextensíveis (exemplo: túnel do carpo). No grupo B, foi realizada injeção intraneural no segmento envolvido pelo tubo de 0,2 ml de sangue autógeno. No grupo C, após produção do hematoma, foi feita a imediata remoção do tubo de silicone simulando-se a descompressão túnel do carpo, e epineurotomia longitudinal complementar. No grupo D, após produção do hematoma, foi realizada apenas a remoção do tubo de silicone. No grupo E (CONTROLE) o nervo foi apenas exposto sem a presença de hematoma ou compressão extrínseca. A avaliação funcional foi feita periodicamente durante 61 dias através do Índice de Função Ciática (IFC) de Bain-Mackinnon-Hunter, e a análise histomorfométrica realizada ao término deste período. RESULTADOS: O grupo A (compressão extrínseca) apresentou IFC inicial de -26,29±2,89 com retorno aos valores pré-operatórios no 5º dia de pós-operatório. O grupo B (hematoma e compressão extrínseca) foi aquele com pior função ciática inicial (IFC -85,23±3,51) com recuperação da função no 23º dia. O grupo C apresentou IFC inicial de -32,78±7,45 com normalização no 5º dia. O grupo D apresentou IFC inicial de -45,13±6,84 com normalização da função ciática no 5º pós-operatório. A análise estatística do IFC identificou diferença significativa (p<0,0001) entre a conduta expectante (grupo B) e o tratamento cirúrgico descompressivo (grupos C e D) até o 19° dia. O número e a densidade de fibras mielínicas em degeneração foi significativamente maior no grupo B quando comparado aos outros grupos. CONCLUSÃO: Neste estudo experimental, descompressão cirúrgica e epineurotomia reduziram a perda de fibras mielínicas e determinaram recuperação funcional mais rápida. / INTRODUCTION: Intraneural hematoma can result in the median nerve in the carpal tunnel after trauma or coagulation disorders. The decision for expectant management or descompressive surgical techniques is still controversial. MATERIAL AND METHODS: Fifty male Wistar rats were divided into 5 groups. The sciatic nerve was wrapped around with a silastic device in 4 groups. Group A the sciatic nerve was just wrapped by the silastic tube. In group B an intraneural injection of autologous blood was added. In group C, after the hematoma creation the silastic device was removed and a longitudinal epineurotomy was performed. In group D, we removed the silastic device after the hematoma but the nerve was not opened. In the group E (sham-operated) sciatic nerve was exposed without hematoma or compression. Nerve function recovery was assessed periodically over 61 days using the Bain-Mackinnon-Hunter Sciatic Function Index (SFI). RESULTS: Group A (extrinsic compression) presented initial SFI of -26.29±2.89, with return to baseline values on the 5th postoperative day. Group B (hematoma and extrinsic compression) exhibited the poorest function (SFI - 85.23±3,51) after surgery and recovery in 23 days. Group C (liberation of silastic and hematoma drainage through epineurotomy) and Group D (only removal of the silastic tube) presented similar initial SFI values of - 32.78±7.45 and - 45.13±6.84, respectively. In both groups SFI values returned to baseline level on 5th postoperative day. The statistical analysis of SFI identified a significant difference (p<0.0001) between the expectant management (group B) and the descompressive surgery approach (groups C & D) by 1st to 19th postoperative day. The number of degenerative fibers and density of degenerative fibers were statistically significant bigger in the group B when compared to the other groups. There was no statistical difference between the other groups when these parameters were analysed. CONCLUSION: Thus, immediate descompressive procedures of the intraneural hematoma provide a faster functional recovery and reduce the damage to the axon fibers.

Every year over 2.5 million traumatic brain injuries (TBI) occur and are the leading cause of death and disability among adolescents. There are no approved treatments for TBI. Survivors suffer from persistent cognitive impairment due to posttraumatic tissue damage and disruption of neural networks which significantly detract from their quality of life. Posttraumatic cognitive impairment depends in part on the brain's limited ability to repair or replace damaged cells. Immature neurons in the hippocampus dentate gyrus, a brain region required for learning and memory, are particularly vulnerable to TBI. Insulin-like growth factor-1 (IGF1) is a potential therapeutic for TBI because it is a potent neurotrophic factor capable of mediating neuroprotection, neuro-repair, and neurogenesis. We hypothesized that conditional IGF1 overexpression in the mouse hippocampus following experimental controlled cortical impact injury (CCI) would enhance posttraumatic neurogenesis chronically. To this end, conditional astrocyte-specific IGF1 overexpressing mice (IGFtg) and wild-type (WT) mice received CCI or sham injury. The proliferation marker BrdU was used to label neurons born the first week after injury. Six weeks after injury, when surviving posttrauma-born neurons would be fully developed, we counted proliferated cells (BrdU+) and the subset expressing a mature neuronal marker (NeuN+/BrdU+) in the hippocampus. We also assessed cognitive performance during radial arm water-maze reversal (RAWM-R) testing, a neurogenesis-sensitive assay. IGF1 promoted end-stage maturity and decreased mis-migration of neurons born after trauma. These effects coincide with IGF1 induced improvements in performance on neurogenesis sensitive cognition following TBI. Mammalian target of rapamycin (mTOR), an early signaling molecule downstream of IGF1, has been identified as a potential target for TBI interventions because of its regulatory role in neuronal plasticity and neurogenesis. However, recent studies have also reported maladaptive plasticity and recovery associated with posttraumatic mTOR activation. It is imperative to elucidate the mechanism of action of IGF1 during pre-clinical evaluations. We hypothesized that IGF1 mediates posttraumatic neurogenic effects through IGF1 induction of mTOR activation. We injured cohorts of IGFtg and WT mice and harvested their brains for immunohistochemistry to assess IGF1 overexpression effects on posttraumatic mTOR activation at 1, 3, and 10 days post-injury (dpi). We found that IGF1 upregulated mTOR activation following TBI in a region-specific manner at 1 and 3dpi. To determine if IGF1 regulated differentiation and arborization through the mTOR pathway, injured WT and IGFtg mice received daily i.p. injections of rapamycin (10mg/kg), the inhibitor of mTOR, or its vehicle for 7 days. Vehicle and rapamycin administration began 3dpi, after the cells dividing at the peak of posttraumatic proliferation were labeled with BrdU. IGF1 enhancement of posttraumatic neurogenesis was not dependent on mTOR activation. In summary, IGF1 directs newborn neuron localization, promotes end-stage maturation, and chronically improves cognition. IGF1 can stimulate posttraumatic neurogenesis and plasticity independent of mTOR activation. These data suggest that IGF1 can stimulate neuron replacement following trauma-induced hippocampal neuron loss and cognitive improvement. Further studies should investigate IGF1 and mTOR inhibition as a combination therapy for neurorehabilitation.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron death and subsequent muscle atrophy. Approximately 15% of ALS cases are inheritable, and mutations in the Fused in Sarcoma (FUS) gene contribute to approximately 5% of these cases, as well as about 2% of sporadic cases. FUS performs a diverse set of cellular functions, including being a major regulator of RNA metabolism. FUS undergoes liquid- liquid phase transition in vitro, allowing for its participation in stress granules and RNA transport granules. Phase transition also contributes to the formation of cytoplasmic inclusions found in the cell bodies of FUS ALS patients motor neurons. The nature of these inclusions has remained elusive, as the proteins localized to them have not been identified. Additionally, the functional consequence of the accumulation of cytoplasmic FUS inclusions has not been established, nor is it understood how they contribute to selective motor neuron death. We carried out two related, but independent studies to characterize the proteins that may be included in FUS-positive inclusions. In this first study, we utilized immunoprecipitation of wild-type and mutant FUS in the presence and absence of RNase, followed by LC MS/MS. The identified proteins represent those that directly or indirectly interact with FUS, with relatively high affinity that can be pulled down with immunoprecipitation. A wide variety of interacting proteins were identified and they are involved in a multitude of pathways including: chromosomal organization, transcription, RNA splicing, RNA transport, localized translation, and stress response. Their interaction with FUS varied greatly in their requirements for RNA. Most notably, FUS interacted with hnRNPA1 and Matrin-3, proteins also known to cause familial ALS. Immunofluorescent staining of proteins interacting with mutant FUS were localized to cytoplasmic inclusions. We concluded that mis-localization of these proteins potentially lead to their dysregulation or loss of function, thus contributing to FUS pathogenesis. In the second study, we developed a protocol to isolate dynamic FUS inclusions and employed LC MS/MS to identify all proteins associated with FUS inclusions. We identified a cohort of proteins involved in translation, splicing, and RNA export to be associated with the FUS inclusions. Further pathway and disease association analysis suggested that proteins associated with translation and RNA quality control pathways may be the most significant. Protein translation assays using both N2A and ALS patient fibroblasts demonstrated suppression of protein biosynthesis in mutant FUS expressing cells. However, translation initiation was not impaired. To understand how protein synthesis is suppressed by mutant FUS mediated defects in RNA metabolism, we examined changes in a well conserved RNA turnover pathway namely: nonsense mediated decay (NMD). We found that NMD is hyperactivated in cells expressing mutant FUS, likely due to chronic suppression of protein translation shifting the pathways autoregulatory circuit to allow for hyperactivation. We concluded that mutant FUS suppresses protein biosynthesis and disrupts NMD regulation. These defects together likely contribute to motor neuron death.

Parkinson’s disease (PD) is a disorder of the nervous system that causes problems with movement (motor symptoms) as well as other problems such as mood disorders, cognitive changes, sleep disorders, constipation, pain, and other non-motor symptoms. The severity of PD symptoms worsens over time as the disease progresses, and while there are treatments for the motor and some non-motor symptoms there is no known cure for PD. Thus there is a high demand for therapies to slow the progressive neurodegeneration observed in PD. Two clinical trials at the University of Kentucky College of Medicine (NCT02369003, NCT01833364) are currently underway that aim to develop a disease-modifying therapy that slows the progression of PD. These clinical trials are evaluating the safety and feasibility of an autologous peripheral nerve graft to the substantia nigra in combination with Deep Brain Stimulation (DBS) for the treatment of PD. By grafting peripheral nerve tissue to the Substantia Nigra, the researchers aim to introduce peripheral nerve tissue, which is capable of functional regeneration after injury, to the degenerating Substantia Nigra of patients with PD. The central hypothesis of these clinical trials is that the grafted tissue will slow degeneration of the target brain region through neural repair actions of Schwann cells as well as other pro-regenerative features of the peripheral nerve tissue. This dissertation details analysis of the peripheral nerve tissue used in the above clinical trials with respect to tissue composition and gene expression, both of injury-naive human peripheral nerve as well as the post-conditioning injury nerve tissue used in the grafting procedure. RNA-seq analysis of sural nerve tissue pre and post-conditioning show significant changes in gene expression corresponding with transdifferentiation of Schwann cells from a myelinating to a repair phenotype, release of growth factors, activation of macrophages and other immune cells, and an increase in anti-apoptotic and neuroprotective gene transcripts. These results reveal in vivo gene expression changes involved in the human peripheral nerve injury repair process, which has relevance beyond this clinical trial to the fields of Schwann cell biology and peripheral nerve repair. To assess the neurobiology of the graft post-implantation we developed an animal model of the grafting procedure, termed Neuro-Avatars, which feature human graft tissue implanted into athymic nude rats. Survival and infiltration of human graft cells into the host brain were shown using immunohistochemistry of Human Nuclear Antigen. Surgical methods and outcomes from the ongoing development of this animal model are reported. To connect the results of these laboratory studies to the clinical trial we compared the severity of motor symptoms before surgery to one year post-surgery in patients who received the analyzed graft tissue. Motor symptom severity was assessed using the Unified Parkinson’s Disease Rating Scale Part III. Finally, the implications and future directions of this research is discussed. In summary, this dissertation advances the translational science cycle by using clinical trial findings and samples to answer basic science questions that will in turn guide future clinical trial design.

Breast cancer metastasizes to the brain in 15-30% of all breast cancer cases, and metastasis is the predominant cause of breast cancer-related deaths. Patients with HER2-enriched and triple-negative breast cancers (TNBCs) are more likely to develop brain metastases. While targeted therapies exist for HER2-enriched breast cancers, there are no effective treatments for TNBCs. Thus, a greater understanding of how these cancers spread to the brain is critical. In order to spread to the brain, disseminated breast cancer cells must overcome 2 major steps—crossing the blood-brain barrier (BBB) and survival and successful colonization of the distinctive and mostly cellular brain environment. Here, we report a novel role for breast cancer cell surface receptor, Syndecan-1 (Sdc1), a heparan sulfate proteoglycan, in promoting breast cancer cell transmigration across the BBB. We found that when we silenced Sdc1 expression in a highly metastatic TNBC cell line, MDA-MB-231, these cells exhibited reduced migration across an in vitro BBB model system. Further, in an in vivo experimental model of metastasis, mice injected with MDA-MB-231 Sdc1 KD (knock-down) cells developed less brain metastases than mice injected with control non-silencing (NS1) cells. Conversely, we found that overexpression of Sdc1 in a metastatic triple-negative mouse mammary carcinoma cell line, 4T1, led to an increase in brain metastases compared to empty vector control-treated mice. We predicted that a secreted factor(s) facilitated BBB disruption that allowed for Sdc1-mediated BBB transmigration, and found that silencing Sdc1 led to decreases in the production and/or release of various cytokines and chemokines implicated in BBB permeability and transmigration. In addition to supporting BBB transmigration, through an in vitro tissue section adhesion assay, we found that Sdc1 also facilitates adhesion of breast cancer cells to the brain, and not to the liver or lungs, revealing specificity for the brain. Further, we report that Sdc1 is expressed in 81% of breast cancer patient brain metastases in our tissue microarray study and that patients with TNBC and high Sdc1 expression have shorter disease-free survival based on a study performed using data from The Cancer Genome Atlas. Taken together, we predict that breast cancer cell Sdc1-regulated cytokines and chemokines promote BBB permeability and/or support transmigration to facilitate breast cancer metastasis to the brain. We also provide evidence for breast cancer-secreted extracellular vesicles, namely exosomes, in supporting the formation of a pro-metastatic brain environment. We compared exosomes derived from the metastatic 4T1 mouse mammary carcinoma cell line to a non-metastatic counterpart, the 67NR cell line, to assess their microRNA and protein composition and their effect(s) on recipient astrocytes, known mediators of brain metastasis. We found that there are inherent differences in both the microRNA and protein cargo from the metastatic 4T1 cells compared to the non-metastatic 67NR cells, whereby the metastatic 4T1 cells contained various tumor-promoting microRNAs and proteins, and also contained 4.5-fold more protein than the non-metastatic 67NR cells. Mouse astrocytes treated with the metastatic 4T1 exosomes exhibited a shift towards a pro-metastatic phenotype, characterized by upregulation of pro-inflammatory genes, and genes associated with astrocyte reactivity and cancer, whereby 67NR exosome-treated astrocytes exhibited a response profile that overlapped with untreated controls. Overall, these findings reveal an important role for exosomes in driving changes in the brain microenvironment to create a site conducive for cancer growth. Together, both studies help to elucidate how breast cancer cells can invade and colonize the unique brain environment.

BACKGROUND: Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by degeneration and loss of spinal cord motor neurons leading to denervation and muscular atrophy. It is caused by defects in the Survival Motor Neuron 1 gene (SMN1) and it is classified by age of onset and motor milestones into three main types which strongly correlate with the copy number of its homologous gene, SMN2. SMN2 expresses markedly less full‐length protein than SMN1, provoking disease manifestations. The essential neuropathological feature in SMA is motor neuron death. Previous studies in SMA foetal samples showed early pathological changes in spinal cord suggesting that the disease is a developmental disorder. Studies in mouse SMA models support that neuromuscular junctions (NMJs) may play a significant role in the disease, although this implication has not yet been addressed in humans. AIMS AND METHODOLOGIES: To better understand the mechanisms of SMA disease, a comprehensive histologic, immunohistochemical and ultrastructural analysis of the muscle and neuromuscular junctions in prenatal and postnatal SMA samples was carried out. To further correlate neuropathological findings with early developmental manifestations of the disease, foetal motility between 11‐14 gestational weeks was recorded and analyzed by 2D ultrasound in pregnancies predicted to develop SMA. RESULTS: At 12 weeks, most SMA myotubes were smaller than controls, indicating a delay in muscle maturation. At this stage, the presence of early acetylcholine receptor (AChR) clusters in developing SMA muscles suggested that pre‐patterned and nerve‐independent AChR clustering would not be affected by the disease. However, as development proceeded in the following weeks, early degeneration of nerve terminals was present associated with a dispersion of AChRs and abnormal preterminal accumulation of vesicles. These findings pointed towards a defect in maintenance of the initial innervation in developmental SMA muscle. Furthermore, postnatal muscle of type I SMA patients showed persistence of the foetal subunit of acetylcholine receptors, suggesting a continuous delay in maturation of neuromuscular junctions. Therefore, if nerve terminals are unable to efficiently maintain functional NMJs, a dying‐back process leading to motor neuron degeneration and loss may appear, with the consequent increase in programmed cell death. Despite all these early neuropathological findings, we did not observe qualitative differences in foetal movements between unaffected and SMA prenatal cases. The synaptic defects in SMA at this stage of development, therefore, might be compensated by several mechanisms. During perinatal and postnatal periods compensation would no longer be present, resulting in the drastic SMA pathology and clinical manifestations. CONCLUSIONS: These developmental studies open new possibilities to improve our knowledge of presymptomatic SMA stages. Early therapeutic strategies should be investigated to reverse the process of denervation, maintain activity of the NMJ, and improve maturity of the motor endplates. / INTRODUCCIÓN: La atrofia muscular espinal (AME) es una enfermedad neuromuscular infantil caracterizada por la muerte de las neuronas motoras del asta anterior de la médula espinal. Como consecuencia de ello hay una degeneración y atrofia muscular, por lo que los pacientes mueren a menudo de insuficiencias respiratorias graves. La AME se clasifica en tres tipos principales según el grado de gravedad, la edad de aparición y las pautas motoras. Se trata de una enfermedad con patrón de herencia autosómico recesivo causada por ausencia o mutaciones en el gen Survival Motor Neuron 1 (SMN1). Existe un gen homólogo, SMN2, que está presente en todos los pacientes aunque a diferencia del SMN1, produce mucha menor proteína SMN completa y, por lo tanto, no evita la aparición de la enfermedad. Sin embargo se ha demostrado una importante correlación con el tipo de AME y el número de copias de SMN2. El rasgo patológico esencial de la AME es la muerte de las neuronas motoras. Estudios hechos en muestras fetales indican que ya existen hallazgos patológicos en el estadio prenatal lo que sugiere que la AME sería un trastorno del desarrollo. Además en modelos de ratón AME se ha determinado que la unión neuromuscular tendría un papel importante en la patogenia de la enfermedad aunque en humanos todavía no existen investigaciones al respecto. OBJETIVOS Y METODOLOGÍA: Con el fin de profundizar en los conocimientos de la patogenia de la AME, en esta tesis se ha realizado un análisis histológico, inmunohistoquímico y utraestructural del músculo y la unión neuromuscular en muestras prenatales y postnatales de controles y AME. Paralelamente, se han correlacionado los resultados neuropatológicos obtenidos con el estudio de la motilidad fetal por ecografía 2D entre las 11 y 14 semanas de gestación en embarazos diagnosticados genéticamente como AME. RESULTADOS: A partir de las 12 semanas, los miotubos AME son más pequeños que los controles lo que es compatible con un retraso en la maduración muscular. En esta etapa, la presencia de receptores de acetilcolina agrupados en músculo AME sugiere que éste es capaz de formar la placa neuromuscular. Sin embargo en semanas posteriores se observa una degeneración temprana de los terminales nerviosos asociados a una dispersión de los receptores de acetilcolina y acumulación anormal de vesículas presinápticas. Esto indica que en este período uno de los principales defectos sería la falta de mantenimiento de la unión neuromuscular. El músculo postnatal AME muestra persistencia de la expresión del receptor fetal de acetilcolina que refuerza la idea de una maduración retardada de la unión neuromuscular que persiste durante todo el desarrollo. La falta de mantenimiento de las uniones neuromusculares justificaría el inicio de un proceso de muerte retrograda (“dying back process”) dando lugar a una excesiva pérdida de neuronas motoras en la médula espinal. El estudio de los movimientos fetales, sin embargo, no demostró diferencias cualitativas entre los fetos normales y los AME. La falta de correlación entre la neuropatología descubierta en los fetos con AME tipo I y la presencia de movimientos fetales normales en ese mismo grupo indica que deben existir mecanismos compensatorios en el feto AME que enmascaran las posibles consecuencias funcionales de los defectos sinápticos hallados. Estos mecanismos compensatorios desaparecerían más tarde dando lugar a las graves manifestaciones de la enfermedad en las etapas perinatal y neonatal. CONCLUSIONES: Los resultados obtenidos contribuyen al mejor conocimiento de esta enfermedad en etapas presintomáticas, y abre nuevas perspectivas para investigar estrategias terapéuticas a fin de revertir los procesos de denervación, mantener la actividad de las uniones neuromusculares y mejorar la maduración de las placas motoras.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2010. / Härtill 4 uppsatser.