<p>Leber´s hereditary optic neuropathy (LHON), a disease affecting vision, is caused by several point mutations in mitochondrial DNA. Mutations leading to a defect NADH ubiquinone oxidoreductase protein will affect the respiratory chain and cause a disturbed ATP production. It is still unknown why this defect leads to the degeneration of retinal ganglion cells and cells in the opticus nerve as well as demyelination of axons in these areas. Analysis of mitochondrial DNA is an important tool in the diagnosis of the disease. At the present time analysis is based on cleavage by restriction enzymes, which only detects two of the most frequent mutations: m.3460G>A and m.11778G>A. This is far too few considering that more than 30 mutations are known to be associated with LHON. Therefore a new analysis method is requested. Here we describe a method based on the sequencing of the mitochondrial genes MT-ND1, MT-ND4 and MT-ND6, which will detect more than 15 different point mutations associated with the disease. To validate the analysis, DNA from 31 patients with LHON symptoms were sequenced; of these 10 were found to be positive for a LHON mutation. This result indicates that the sequencing analysis will be more effective in diagnosis of LHON than restriction enzymes.</p> / <p>Lebers hereditära optikus neuropati (LHON) är en sjukdom som beror på genetiska förändringar i arvsmassan som leder till att cellens energiomsättning rubbas. Detta gör att nervceller i ögat och synnerven bryts ned vilket leder till en synnedsättning. En patient som drabbas av LHON har inga symptom fram till dess att synen börjar försämras. Sjukdomsförloppet går snabbt och på bara några veckor är patienten ofta helt blind. Diagnostik av LHON idag utgörs av flera undersökningar av öga och synfält. Diagnosen bekräftas av en analys av arvsmassan som finns i mitokondrien, cellens energifabrik. Här beskriver vi en ny förbättrad analysmetod baserad på DNA sekvensering, dvs. bestämning av baserna i mitokondriella arvsmassan. För att utvärdera analysen har vi undersökt 31 patienter med misstänkt LHON - av dessa visade sig 10 bära på en sjuklig förändring. Resultatet visar att sekvensering med fördel kan ersätta den tidigare analysmetoden då fler sjukliga förändringar kan påvisas och utförandet av analysen är mer användarvänligt.</p>

Leber´s hereditary optic neuropathy (LHON), a disease affecting vision, is caused by several point mutations in mitochondrial DNA. Mutations leading to a defect NADH ubiquinone oxidoreductase protein will affect the respiratory chain and cause a disturbed ATP production. It is still unknown why this defect leads to the degeneration of retinal ganglion cells and cells in the opticus nerve as well as demyelination of axons in these areas. Analysis of mitochondrial DNA is an important tool in the diagnosis of the disease. At the present time analysis is based on cleavage by restriction enzymes, which only detects two of the most frequent mutations: m.3460G&gt;A and m.11778G&gt;A. This is far too few considering that more than 30 mutations are known to be associated with LHON. Therefore a new analysis method is requested. Here we describe a method based on the sequencing of the mitochondrial genes MT-ND1, MT-ND4 and MT-ND6, which will detect more than 15 different point mutations associated with the disease. To validate the analysis, DNA from 31 patients with LHON symptoms were sequenced; of these 10 were found to be positive for a LHON mutation. This result indicates that the sequencing analysis will be more effective in diagnosis of LHON than restriction enzymes. / Lebers hereditära optikus neuropati (LHON) är en sjukdom som beror på genetiska förändringar i arvsmassan som leder till att cellens energiomsättning rubbas. Detta gör att nervceller i ögat och synnerven bryts ned vilket leder till en synnedsättning. En patient som drabbas av LHON har inga symptom fram till dess att synen börjar försämras. Sjukdomsförloppet går snabbt och på bara några veckor är patienten ofta helt blind. Diagnostik av LHON idag utgörs av flera undersökningar av öga och synfält. Diagnosen bekräftas av en analys av arvsmassan som finns i mitokondrien, cellens energifabrik. Här beskriver vi en ny förbättrad analysmetod baserad på DNA sekvensering, dvs. bestämning av baserna i mitokondriella arvsmassan. För att utvärdera analysen har vi undersökt 31 patienter med misstänkt LHON - av dessa visade sig 10 bära på en sjuklig förändring. Resultatet visar att sekvensering med fördel kan ersätta den tidigare analysmetoden då fler sjukliga förändringar kan påvisas och utförandet av analysen är mer användarvänligt.

Orientador: Edi Lucia Sartorato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T07:24:23Z (GMT). No. of bitstreams: 1 AmaralFernandes_MarcelaScabello_D.pdf: 2548021 bytes, checksum: 4a1e103fa460381f99df5a845fdfec67 (MD5) Previous issue date: 2013 / Resumo: A neuropatia óptica hereditária de Leber (LHON) é uma doença mitocondrial, com herança materna, caracterizada pela perda (sub) aguda, indolor e bilateral da visão, escotoma central ou cecocentral e discromatopsia, devido à degeneração do nervo óptico por apoptose das células parvo ganglionares da retina. As três mutações primárias G11778A, T14484C e G3460A são responsáveis por 90 a 95% dos casos da LHON e acometem subunidades dos genes MT-ND4, MT-ND6 e MT-ND1, respectivamente, que codificam proteínas para o complexo I da cadeia respiratória. Somente 5% dos pacientes possuem uma das demais mutações secundárias. A presença da mutação é fundamental para que LHON ocorra, no entanto, a penetrância incompleta e predileção pelo gênero masculino sugerem que fatores genéticos, epigenéticos e ambientais possam modular a expressão fenotípica da doença. O objetivo deste estudo foi analisar clínica e molecularmente para LHON 63 pacientes com neuropatia óptica, sendo 25 com quadro clínico típico de Leber (grupo I) e 38 com neuropatia óptica de etiologia a esclarecer (grupo II), assim como verificar a relação entre os agentes tóxicos tabaco e álcool e uma possível suscetibilidade genética entre os pacientes que faziam uso abusivo destes agentes. Estes pacientes foram submetidos à avaliação oftalmológica completa no ambulatório de neuroftalmologia do HC-UNICAMP e tiveram suas amostras de sangue coletadas e analisadas no CBMEG. A pesquisa das três mutações primárias foi realizada pelas técnicas de restrição enzimática e sequenciamento direto, e confirmada pelo PCR-multiplex e Plataforma Sequenom. Os pacientes que não apresentaram uma das mutações primárias foram rastreados pelo sequenciamento direto e pela Plataforma Sequenom, para oito principais mutações secundárias: G3733A e C4171A (MT-ND1), T10663C (MT-ND4L) e G14459A, C14482G, C14482A, A14495G e C14568T (MT-ND6). Os haplogrupos dos pacientes mutantes foram pesquisados pela Plataforma Sequenom. Dos 63 pacientes com neuropatia óptica foram encontrados 18 pacientes mutantes, sendo 14 do grupo I (11 com G11778A e 3 com T14484C) e 4 do grupo II (3 com G11778A e 1 com T14484C). Os haplogrupos encontrados nestes pacientes mutantes foram: C, D, M, U, e, principalmente L1/L2 e L3, que mostra a presença de ancestral comum de origem asiática, européia e, predominantemente, africana. Nenhum dos pacientes apresentou a mutação primária G3460A, assim como não foi encontrada nenhuma das 8 mutações secundárias rastreadas. Na análise estatística das variáveis estudadas houve diferença significativa para recorrência familiar materna, campo visual e presença de mutação, dentre os 63 pacientes com neuropatia óptica, sendo que achados mostraram que o quadro clínico clássico da doença descrito por Leber há mais de um século tem boa confiabilidade. Ao comparar as mesmas variáveis entre os 14 mutantes do grupo I com os 4 mutantes do grupo II, não houve diferença estatisticamente significativa para nenhuma das variáveis, evidenciando que o diagnóstico de LHON é molecular, através do rastreamento das mutações (inicialmente as primárias). Não foi possível estabelecer relação entre o uso abusivo do tabaco e álcool e uma suscetibilidade genética de base, isto é, a mutação da LHON, entre os pacientes com neuropatia óptica de etiologia a esclarecer e com consumo abusivo destes agentes / Abstract: Leber hereditary optic neuropathy (LHON) is maternally inherited mitochondrial disease, characterized by painless, bilateral, (sub) acute loss of vision, central or cecocentral scotoma and dyschromatopsia, due to the degeneration of optic nerve by the apoptosis of the p-retinal ganglion cells. The three primary mutations G11778A, T14484C and G3460A account for 90 to 95% of the cases of LHON and affect subunits of genes MT-ND4, MT-ND6 and MT-ND1, respectively, which encode proteins of the complex I of the respiratory chain. Only 5% of patients have one of the other secondary mutations. The mutation in mtDNA is essential for LHON occurs, however, the incomplete penetrance and the male predominance of the disease suggests that genetic, epigenetic and environmental factors may modulate the phenotypic expression of LHON. The aim of this study was to analyze clinical and molecularly for LHON 63 patients with optic neuropathy, 25 with classical clinical symptoms of Leber (group I) and 38 with optic neuropathy of unknown etiology (group II), as well as to investigate the relationship between toxic agents tobacco and alcohol and a possible genetic susceptibility among patients who were abusing these agents. These patients underwent complete ophthalmologic evaluation in the Neuro-Ophthalmoloy Outpatient HC-UNICAMP, had their blood samples collected and analyzed in CBMEG. The research of the three primary mutations was performed by restriction analysis and direct sequencing and confirmed by multiplex-PCR and Sequenom Platform. Patients who did not have one of the primary mutations were screened by direct sequencing and by Sequenom Platform for 8 major secondary mutations: G3733A and C4171A (MT-ND1), T10663C (MT-ND4L) and G14459A, C14482G, C14482A, A14495G and C14568T (MT -ND6). The haplogroups of mutant patients were screened by Sequenom Platform. Of 63 patients with optic neuropathy 18 patients were found to be mutants, 14 in group I (11 with G11778A and 3 with T14484C) and 4 in group II (3 with G11778A and 1 with T14484C). The haplogroups found in these mutants patients were: C, D, M, U, and especially L1/L2 and L3, which shows the presence of the common ancestor of Asian, European and, predominantly, African. None of the patients had a primary mutation G3460A, and nor it was found any of the eight secondary mutations screened. Statistical analysis of the variables studied showed significant differences for maternal familial recurrence, visual field and the presence of mutation among the 63 patients with optic neuropathy, demonstrating a good reliability to the classical clinical picture of the disease described by Leber over a century ago. When comparing the same variables among 14 mutants of group I with 4 mutants of group II, there was no statistically significant difference for any of the variables, indicating that the diagnosis of LHON is molecular, by tracking the mutations (initially the primaries ones). No relationship between abusive use of tobacco and alcohol and a genetic-based susceptibility, that is, the mutation for LHON could be correlated in patients with optic neuropathy of unknown etiology and history of heavy consumption of these agents / Doutorado / Oftalmologia / Doutora em Ciências Médicas

OBJETIVO: avaliar a capacidade de índices de preenchimento muscular da órbita, calculados a partir de medidas na tomografia computadorizada de multidetectores, detectar a neuropatia óptica distireoidiana em pacientes com orbitopatia de Graves. MÉTODOS: noventa e três pacientes com diagnóstico de orbitopatia de Graves foram prospectivamente estudados. Todos os pacientes foram submetidos a um exame oftalmológico completo e à tomografia computadorizada de multidetectores. Na estação de trabalho do tomógrafo foram calculados índices baseados em medidas lineares, de área e de volume de preenchimento muscular da órbita. O índice linear de Barrett, foi calculado a partir da relação entre a medida dos músculos extra-oculares em cortes coronais no ponto médio do nervo óptico orbitário e o diâmetro da órbita na mesma posição. Nesse mesmo corte coronal uma escala de 0 a 3 foi utilizada para categorizar o grau do preenchimento muscular e de compressão do nervo óptico segundo método descrito por Nugent e colaboradores. Medidas de área foram realizadas de cortes coronais a 12, 18 e 24 mm da linha interzigomática. Três índices de área foram calculados estimando-se a razão entre a área dos músculos extraoculares e a do continente ósseo medidas. Herniação de gordura orbitária pela fissura orbitária superior foi registrada como presente ou ausente conforme método descrito por Birchall e colaboradores. Dois índices volumétricos foram calculados, o primeiro baseado em cortes axiais de todo o continente orbitário (do ápice à rima orbitária anterior) e o segundo baseado em cortes coronais da órbita a partir de um ponto médio do segmento orbitário do nervo óptico até o limite anterior do canal óptico. As órbitas foram divididas em dois grupos, um com e outro sem neuropatia óptica distireoidiana e esses grupos foram comparados entre si. RESULTADOS: cento e duas órbitas de 61 pacientes atenderam aos critérios de inclusão e foram analisadas. Quarenta e uma órbitas eram do grupo com neuropatia e 60 daquele sem a neuropatia óptica distireoidiana. Para todos os índices, exceto para o de herniação de gordura, houve diferença significativa entre os dois grupos (<0,001). A melhor combinação de sensibilidade e especificidade para o índice linear de Barrett foi de 79% e 72% respectivamente, com razão de chances de 9.2. A escala categórica de Nugent apresentou sensibilidade de 58,3% e especificidade de 84,7%, acurácia de 74,7% e razão de chances de 7,8. Todos os índices de área e de volume apresentaram boa capacidade na detecção da neuropatia óptica distireoidiana. Os índices com melhor desempenho diagnóstico foram o de área a 18mm com sensibilidade de 91,7%, especificidade de 89,9%, acurácia de 90,5% e razão de chance de 97,2, e o de volume do ápice orbitário com sensibilidade de 92%, especificidade de 86%, acurácia de 88% e razão de chance de 97,2. CONCLUSÕES: os novos índices calculados baseados em área e volume foram preditores da neuropatia óptica e apresentaram desempenho superior aos métodos reproduzidos de estudos anteriores. A continuidade dos estudos na busca de métodos objetivos e capazes de identificar a neuropatia óptica distireoidiana com cada vez maior eficiência e praticidade é de grande relevância para a melhoria do diagnóstico e do tratamento da orbitopatia de Graves e suas complicações / PURPOSE: to evaluate the ability of orbital apex crowding indexes, calculated with multidetector-computed tomography (MDCT), to detect dysthyroid optic neuropathy (DON). METHODS: ninety-three patients with GO were studied prospectively with complete neuro-ophthalmic examination and MDCT scanning. All patients underwent a complete neuro-ophthalmic examination and scanning on a MDCT. In a dedicated workstation, linear, square and volumetric indexes of the orbital apex crowding were calculated. Barretts linear index was calculated as the percentage of the orbit occupied by muscles in a coronal scan halfway between the posterior globe and the orbital apex. At the same coronal scan, the severity of optic nerve crowding was also assessed by using the subjective grading scale (0, 1, 2 or 3) described by Nugent et al. Quantitative square area measurements were performed on coronal sections 12, 18 and 24 mm from the interzygomatic line. The muscular/bony area ratio was calculated as a crowding index. Intracranial fat prolapse through the superior orbital fissure was recorded as present or absent, according to a method described Birchall at al. Two volumetric indexes were calculated, one from axial scans of the entire orbit content (up to the orbital rim) and one from coronal scans of the orbital content from the entrance of the optic canal up to the middle point of the orbital segment of the optic nerve. Orbits were divided into two groups: with or without DON and comparison between groups was performed. RESULTS: one hundred and two orbits (41 orbits with DON, and 61 without DON) of 61 patients met the inclusion criteria and were included in the analysis. All indexes, except the intracranial fat prolapse, were significantly greater in orbits with DON (<0,001). There was no significant difference regarding intracranial fat prolapse. Area under the receiver operating characteristics curves was 0.91, 0.93 and 0.87 for CIs at 12, 18 and 24 mm, respectively. The best combination of sensitivity and specificity for the Barretts index was 79%/72% and odds ratio of 9.2. The Nugents subjective grading scale detected DON with 58.3% sensitivity, 84,7% specificity, 74.7% accuracy and odds ratio 7.8. All square area and volumetric indexes calculated presented good ability to detect DON. The best performance of the square area indexes was observed at 18 mm where a cutoff value of 57.5% showed sensitivity of 91.7% and a specificity of 89.8%, accuracy 90.5% and odds ratio of 97.2. The best sensitivity/specificity ratio of the volumetric indexes was found for the one calculated at the apex, with sensitivity of 93%, specificity of 84%, and accuracy of 88% for detecting DON. CONCLUSIONS: The new orbital crowding indexes based on area and volumetric measurements found to predict DON more reliably than Barretts linear index, subjective grading of orbital crowding or intracranial fat prolapse. Further imaging studies would be useful to validate and improve objective methods to predict DON, and thus contribute to early diagnosis treatment

OBJETIVO: avaliar a capacidade de índices de preenchimento muscular da órbita, calculados a partir de medidas na tomografia computadorizada de multidetectores, detectar a neuropatia óptica distireoidiana em pacientes com orbitopatia de Graves. MÉTODOS: noventa e três pacientes com diagnóstico de orbitopatia de Graves foram prospectivamente estudados. Todos os pacientes foram submetidos a um exame oftalmológico completo e à tomografia computadorizada de multidetectores. Na estação de trabalho do tomógrafo foram calculados índices baseados em medidas lineares, de área e de volume de preenchimento muscular da órbita. O índice linear de Barrett, foi calculado a partir da relação entre a medida dos músculos extra-oculares em cortes coronais no ponto médio do nervo óptico orbitário e o diâmetro da órbita na mesma posição. Nesse mesmo corte coronal uma escala de 0 a 3 foi utilizada para categorizar o grau do preenchimento muscular e de compressão do nervo óptico segundo método descrito por Nugent e colaboradores. Medidas de área foram realizadas de cortes coronais a 12, 18 e 24 mm da linha interzigomática. Três índices de área foram calculados estimando-se a razão entre a área dos músculos extraoculares e a do continente ósseo medidas. Herniação de gordura orbitária pela fissura orbitária superior foi registrada como presente ou ausente conforme método descrito por Birchall e colaboradores. Dois índices volumétricos foram calculados, o primeiro baseado em cortes axiais de todo o continente orbitário (do ápice à rima orbitária anterior) e o segundo baseado em cortes coronais da órbita a partir de um ponto médio do segmento orbitário do nervo óptico até o limite anterior do canal óptico. As órbitas foram divididas em dois grupos, um com e outro sem neuropatia óptica distireoidiana e esses grupos foram comparados entre si. RESULTADOS: cento e duas órbitas de 61 pacientes atenderam aos critérios de inclusão e foram analisadas. Quarenta e uma órbitas eram do grupo com neuropatia e 60 daquele sem a neuropatia óptica distireoidiana. Para todos os índices, exceto para o de herniação de gordura, houve diferença significativa entre os dois grupos (<0,001). A melhor combinação de sensibilidade e especificidade para o índice linear de Barrett foi de 79% e 72% respectivamente, com razão de chances de 9.2. A escala categórica de Nugent apresentou sensibilidade de 58,3% e especificidade de 84,7%, acurácia de 74,7% e razão de chances de 7,8. Todos os índices de área e de volume apresentaram boa capacidade na detecção da neuropatia óptica distireoidiana. Os índices com melhor desempenho diagnóstico foram o de área a 18mm com sensibilidade de 91,7%, especificidade de 89,9%, acurácia de 90,5% e razão de chance de 97,2, e o de volume do ápice orbitário com sensibilidade de 92%, especificidade de 86%, acurácia de 88% e razão de chance de 97,2. CONCLUSÕES: os novos índices calculados baseados em área e volume foram preditores da neuropatia óptica e apresentaram desempenho superior aos métodos reproduzidos de estudos anteriores. A continuidade dos estudos na busca de métodos objetivos e capazes de identificar a neuropatia óptica distireoidiana com cada vez maior eficiência e praticidade é de grande relevância para a melhoria do diagnóstico e do tratamento da orbitopatia de Graves e suas complicações / PURPOSE: to evaluate the ability of orbital apex crowding indexes, calculated with multidetector-computed tomography (MDCT), to detect dysthyroid optic neuropathy (DON). METHODS: ninety-three patients with GO were studied prospectively with complete neuro-ophthalmic examination and MDCT scanning. All patients underwent a complete neuro-ophthalmic examination and scanning on a MDCT. In a dedicated workstation, linear, square and volumetric indexes of the orbital apex crowding were calculated. Barretts linear index was calculated as the percentage of the orbit occupied by muscles in a coronal scan halfway between the posterior globe and the orbital apex. At the same coronal scan, the severity of optic nerve crowding was also assessed by using the subjective grading scale (0, 1, 2 or 3) described by Nugent et al. Quantitative square area measurements were performed on coronal sections 12, 18 and 24 mm from the interzygomatic line. The muscular/bony area ratio was calculated as a crowding index. Intracranial fat prolapse through the superior orbital fissure was recorded as present or absent, according to a method described Birchall at al. Two volumetric indexes were calculated, one from axial scans of the entire orbit content (up to the orbital rim) and one from coronal scans of the orbital content from the entrance of the optic canal up to the middle point of the orbital segment of the optic nerve. Orbits were divided into two groups: with or without DON and comparison between groups was performed. RESULTS: one hundred and two orbits (41 orbits with DON, and 61 without DON) of 61 patients met the inclusion criteria and were included in the analysis. All indexes, except the intracranial fat prolapse, were significantly greater in orbits with DON (<0,001). There was no significant difference regarding intracranial fat prolapse. Area under the receiver operating characteristics curves was 0.91, 0.93 and 0.87 for CIs at 12, 18 and 24 mm, respectively. The best combination of sensitivity and specificity for the Barretts index was 79%/72% and odds ratio of 9.2. The Nugents subjective grading scale detected DON with 58.3% sensitivity, 84,7% specificity, 74.7% accuracy and odds ratio 7.8. All square area and volumetric indexes calculated presented good ability to detect DON. The best performance of the square area indexes was observed at 18 mm where a cutoff value of 57.5% showed sensitivity of 91.7% and a specificity of 89.8%, accuracy 90.5% and odds ratio of 97.2. The best sensitivity/specificity ratio of the volumetric indexes was found for the one calculated at the apex, with sensitivity of 93%, specificity of 84%, and accuracy of 88% for detecting DON. CONCLUSIONS: The new orbital crowding indexes based on area and volumetric measurements found to predict DON more reliably than Barretts linear index, subjective grading of orbital crowding or intracranial fat prolapse. Further imaging studies would be useful to validate and improve objective methods to predict DON, and thus contribute to early diagnosis treatment

No description available.

Abstract Human mitochondria contain a circular genome called mitochondrial DNA (mtDNA). It encodes subunits of the respiratory chain enzymes involved in energy conservation in oxidative phosphorylation and the necessary RNA needed for their expression. Errors in these genes have been shown to cause diseases, called mitochondrial diseases, which mainly affect tissues with high energy-demand, such as brain, heart, and skeletal muscle, or to lead to the production of harmful by-products in the form of reactive oxygen species (ROS) during cellular respiration. ROS damage lipids, proteins, and DNA, especially mtDNA. Accumulation of mtDNA mutations has also been associated with aging. Mitochondrial complex I is located in the inner mitochondrial membrane and catalyzes NADH-ubiquinone oxidoreduction coupled to the translocation of four protons from the inside of the mitochondrion to the intermembranous space. Bacteria contain a homologous but simpler enzyme, NDH-1, with the same catalytic mechanism and which is therefore considered the catalytical core of mitochondrial complex I. Seven of the conserved membranous subunits in complex I are encoded in the mtDNA and are targets for mutations causing mitochondrial diseases, like MELAS syndrome or Leber hereditary optic neuropathy (LHON). We used Paracoccus denitrificans and Escherichia coli NDH-1 enzymes to reveal the role of selected conserved charged residues and MELAS or LHON amino acid substitutions in enzyme catalysis. The growth phenotypes and NDH-1-dependent activities in mutant bacterial membranes were characterized, in addition to the sensitivity to selected complex I inhibitors. In order to enable ROS production measurements in the bacterial model of human mitochondrial diseases, we evaluated the reliability of two superoxide detecting probes, lucigenin and coelenterazine. Elimination of the acidic residue in ND1 (position E228) previously found to cause MELAS, was found detrimental for NDH-1 assembly and activity. Also, elimination of the acidic residue at position E36 in ND4L resulted in an inactive enzyme. ND1-E216A, ND4L-E72Q and -E36Q/I39D/A69D/E72Q substitutions decreased NDH-1 activity somewhat (normal activity in the last mutant), but displayed a negative growth phenotype under NDH-1 dependent conditions, suggestive of impaired energy conservation in these mutants. ND1-Y229, whose substitution causes MELAS, charged residues in loop five of ND1, and ND1-E157, whose substitution causes LHON, were also found important for the enzyme activity. Coelenterazine was found a reliable probe for quantitative superoxide production measurement in mitochondrial or bacterial membranes, and its sensitivity is not affected by the reduction level of the respiratory chain. Therefore, coelenterazine is suitable for quantitative superoxide production measurements.

Les dommages au nerf optique (neuropathie optique) peuvent entraîner la perte permanente de la vision ou la cécité causée par la mort des cellules ganglionnaires de la rétine (CGR). Nous avons identifié qu’une surproduction de l'anion superoxyde constitue un événement moléculaire critique précédant la mort cellulaire induite par des lésions. Récemment, Suarez-Moreira et al (JACS 131:15078, 2009) ont démontré que la vitamine B12 peut capter l’anion superoxyde aussi efficacement que l’enzyme superoxyde dismutase. La carence en vitamine B12 peut conduire à une neuropathie optique causée par des mécanismes inconnus. Nous avons étudié la relation entre la captation de superoxyde par la cyanocobalamine (forme de vitamine B12 la plus abondante) et ses propriétés neuroprotectrices dans les cellules neuronales. La cyanocobalamine aux concentrations de 10 μM et 100 μM a réduit le taux de production de superoxyde respectivement par 34% et 79% dans les essais sans-cellule. Dans les cellules RGC-5 traités avec la ménadione, les concentrations de cyanocobalamine supérieures à 10 nM ont diminué l’anion superoxyde à des valeurs similaires à celles traitées par PEG-SOD. La cyanocobalamine aux concentrations de 100 μM et 1 μM a réduit la mort des cellules RGC-5 exposées à la ménadione par 20% et 32%, respectivement. Chez les rats avec section du nerf optique unilatérale, une dose intravitréenne de 667 μM de cyanocobalamine a réduit le nombre de CGRs exposées au superoxyde. Cette dose a également augmenté le taux de survie des CGRs comparativement aux rats injectés avec la solution témoin. Ces données suggèrent que la vitamine B12 peut être un neuroprotecteur important, et sa carence nutritionnelle pourrait causer la mort de CGRs. La vitamine B12 pourrait aussi potentiellement être utilisée comme une thérapie pour ralentir la progression de la mort CGR chez les patients avec les neuropathies optiques caractérisés par une surproduction de superoxyde. / Damage to the optic nerve (optic neuropathy) can result in permanent loss of vision or blindness through retinal ganglion cell (RGC) death. Our prior work identified a burst of superoxide anion as a critical molecular event in RGCs prior to injury-induced cell death. Recently, Suarez-Moreira et al (JACS 131:15078, 2009) demonstrated that vitamin B12 scavenges superoxide as effectively as superoxide dismutase. Vitamin B12 deficiency can lead to optic neuropathy through unknown mechanisms. We investigated the relationship between superoxide scavenging by cyanocobalamin, the most abundant vitamin B12¬¬ vitamer, and its neuroprotective properties in neuronal cells. Cyanocobalamin at concentrations of 10 μM and 100 μM reduced the rate of superoxide generation by 34% and 79% in cell free assays, respectively. In menadione-treated RGC-5 cells, cyanocobalamin concentrations above 10 nM scavenged superoxide anion similar to those treated with pegylated-SOD. Cyanocobalamin at concentrations of 100 μM and 1 mM reduced RGC-5 cell death from menadione by 20% and 32%, respectively. In rats with unilateral optic nerve transection, a single intravitreal dose of 667 μM cyanocobalamin significantly reduced the number of RGCs with superoxide. This dose also increased RGC survival rate compared to rats injected with saline control. These data suggest that vitamin B¬¬12 may be an important neuroprotectant, which could cause death of RGCs when depleted in nutritional deficiency. Vitamin B12 could also potentially be used as a therapy to slow progression of RGC death in patients with optic neuropathies characterized by overproduction of superoxide.

Les manifestations neuro-ophtalmologiques, observées dans la sclérose en plaques sont parfaitement déterminées à l’heure actuelle. Cependant, l’aspect chronique des troubles visuels résultants n’est pas toujours précisément évalué, or de telles lacunes sont un frein à leur prise en charge. Dans une première partie, les caractéristiques des troubles visuels chroniques ont été mesurées. Il s’agissait de quantifier le pourcentage de plaintes visuelles chroniques chez des patients atteints de sclérose en plaques puis de mesurer le degré d’intensité des troubles visuels chroniques, de déterminer leurs origines physiopathologiques et de rendre compte de leur retentissement sur la qualité de vie des patients se plaignant de troubles chroniques. Les voies visuelles afférentes étaient altérées dans 68% des cas. Des troubles oculomoteurs étaient fréquemment observés (89%) dont le nystagmus pendulaire (28%), source de gêne visuelle. Dans une seconde partie, nous nous sommes donc intéressés au nystagmus pendulaire et à sa conséquence fonctionnelle, l’oscillopsie, afin de proposer une prise en charge spécifique. Néanmoins, les mécanismes de ce nystagmus ne sont pas encore bien définis. Il était donc important de développer une hypothèse explicative à partir de l’observation de deux cas particuliers de nystagmus monoculaire et de démontrer que le nystagmus pendulaire de la sclérose en plaques est à distinguer du nystagmus pendulaire du tremblement oculopalatin, souvent confondus. Enfin, nous proposons une méthode évaluant la détection du mouvement (par stimuli de contraste asservis au regard) ainsi qu’un protocole de stimulation optocinétique tentant de réduire ce symptôme / Neuro-ophthalmic manifestations observed in multiple sclerosis are well-known. However, the chronic feature of visual dysfunctions is not always precisely determinated. These imprecision impede the development of specific therapeutic approach. In a first part, the chronic characteristics of visual dysfunctions were assessed. The percentage of chronic visual complaints in multiple sclerosis patients was quantified and then the intensity of chronic visual deficits was measured, their pathophysiologic origins determined and finally their impact on quality of life was taken into account. Visual pathways were impaired in 68% of patients. Ocular motor disorders were frequently observed (89%) including pendular nystagmus (28%), accounted for visual discomfort. In a second part, we focused on pendular nystagmus and its functional consequence, oscillopsia, to propose a specific treatment. First of all, the mechanisms of this nystagmus are not yet well defined. Therefore, we developed some hypothesis from the observation of two patients with monocular nystagmus and demonstrated in a second part that the pendular nystagmus in multiple sclerosis is distinct from the pendular nystagmus of oculopalatal tremor. Finally, we proposed a method evaluating oscillopsia (motion detection by contrast stimuli moving synchronically with gaze) that was tested before and after an optokinetic stimulation protocol aimed to reduce this symptom

Les dommages au nerf optique (neuropathie optique) peuvent entraîner la perte permanente de la vision ou la cécité causée par la mort des cellules ganglionnaires de la rétine (CGR). Nous avons identifié qu’une surproduction de l'anion superoxyde constitue un événement moléculaire critique précédant la mort cellulaire induite par des lésions. Récemment, Suarez-Moreira et al (JACS 131:15078, 2009) ont démontré que la vitamine B12 peut capter l’anion superoxyde aussi efficacement que l’enzyme superoxyde dismutase. La carence en vitamine B12 peut conduire à une neuropathie optique causée par des mécanismes inconnus. Nous avons étudié la relation entre la captation de superoxyde par la cyanocobalamine (forme de vitamine B12 la plus abondante) et ses propriétés neuroprotectrices dans les cellules neuronales. La cyanocobalamine aux concentrations de 10 μM et 100 μM a réduit le taux de production de superoxyde respectivement par 34% et 79% dans les essais sans-cellule. Dans les cellules RGC-5 traités avec la ménadione, les concentrations de cyanocobalamine supérieures à 10 nM ont diminué l’anion superoxyde à des valeurs similaires à celles traitées par PEG-SOD. La cyanocobalamine aux concentrations de 100 μM et 1 μM a réduit la mort des cellules RGC-5 exposées à la ménadione par 20% et 32%, respectivement. Chez les rats avec section du nerf optique unilatérale, une dose intravitréenne de 667 μM de cyanocobalamine a réduit le nombre de CGRs exposées au superoxyde. Cette dose a également augmenté le taux de survie des CGRs comparativement aux rats injectés avec la solution témoin. Ces données suggèrent que la vitamine B12 peut être un neuroprotecteur important, et sa carence nutritionnelle pourrait causer la mort de CGRs. La vitamine B12 pourrait aussi potentiellement être utilisée comme une thérapie pour ralentir la progression de la mort CGR chez les patients avec les neuropathies optiques caractérisés par une surproduction de superoxyde. / Damage to the optic nerve (optic neuropathy) can result in permanent loss of vision or blindness through retinal ganglion cell (RGC) death. Our prior work identified a burst of superoxide anion as a critical molecular event in RGCs prior to injury-induced cell death. Recently, Suarez-Moreira et al (JACS 131:15078, 2009) demonstrated that vitamin B12 scavenges superoxide as effectively as superoxide dismutase. Vitamin B12 deficiency can lead to optic neuropathy through unknown mechanisms. We investigated the relationship between superoxide scavenging by cyanocobalamin, the most abundant vitamin B12¬¬ vitamer, and its neuroprotective properties in neuronal cells. Cyanocobalamin at concentrations of 10 μM and 100 μM reduced the rate of superoxide generation by 34% and 79% in cell free assays, respectively. In menadione-treated RGC-5 cells, cyanocobalamin concentrations above 10 nM scavenged superoxide anion similar to those treated with pegylated-SOD. Cyanocobalamin at concentrations of 100 μM and 1 mM reduced RGC-5 cell death from menadione by 20% and 32%, respectively. In rats with unilateral optic nerve transection, a single intravitreal dose of 667 μM cyanocobalamin significantly reduced the number of RGCs with superoxide. This dose also increased RGC survival rate compared to rats injected with saline control. These data suggest that vitamin B¬¬12 may be an important neuroprotectant, which could cause death of RGCs when depleted in nutritional deficiency. Vitamin B12 could also potentially be used as a therapy to slow progression of RGC death in patients with optic neuropathies characterized by overproduction of superoxide.