The authors thank the staff of the Center for Comparative Medicine and Research (CCMR) at Dartmouth for their expert care of the mice used for these studies. The authors also acknowledge Emily Clough for her excellent administrative support.

Todo o trabalho de animais utiliza protocolos revistos e aprovados pelo Comitê Institucional de Animal Care and Use (IACUC) no Geisel Faculdade de Medicina de Dartmouth. 1. O Modelo rato <ol><li> A indução da doença desmielinizante TMEV Induzida <ol><li> Mover as gaiolas contendo SJL feminina ratos de 4 a 6 semanas de idade / jhan do rack para um espaço de trabalho confortável. Marque a ratos (por exemplo, com uma etiqueta de orelha ou punção da orelha) para permitir a avaliação individual de doença clínica e histológica. </li><li> Desenhar 30 ul de estoque TMEV infectando (unidades formadoras de 2 x 10 6 de placa; UFP) em PBS para uma seringa de insulina 29-gauge e agulha. </li><li> Prepare a máquina de gás de anestesia: verificar o sistema para garantir a presença de quantidades adequadas de oxigênio e isoflurano para a duração do procedimento. </li><li> Ligar o medidor de fluxo de 1 L / min. Colocar o animal na câmara de indução e selar o topo. Ligue o vaporizer para 3,5% e monitorar o animal até reclinada. </li><li> Remover o animal da câmara e testar o mouse por beliscar a pata para assegurar a anestesia adequada. A falta de resposta a um beliscão forte indica anestesia adequada. </li><li> Limpar o local de injecção com álcool isopropílico a 70%. </li><li> Injectar 30 ul de estoque infectar TMEV no hemisfério cerebral direito por injecção à mão livre (Figura 1). O local da injecção é de aproximadamente meio caminho entre a linha do olho e da orelha e apenas fora da linha média. </li><li> Devolver o mouse para sua gaiola de retenção, uma vez totalmente alerta e móveis (geralmente 3-5 min). </li><li> Eutanásia ratinhos por sangria ou perfusão cardíaca 3 a 6 meses após a infecção TMEV, dependendo da rapidez de desenvolvimento da doença. </li></ol></li></ol> 2. Análise Rotarod <ol><li> O Aparelho Rotarod <ol><li> ratinhos de teste antes da infecção TMEV a familiarizarem-se com omáquina e para avaliar o seu comando normal coordenação equilíbrio linha de base e motor. </li><li> Iniciar o protocolo de adaptação nos dias -5 infecção pós (dpi, ou seja, 5 dias antes da infecção TMEV). </li><li> Permitir que os ratinhos se adapte à sala de teste durante pelo menos 30 min antes do teste de rotarod, a fim de lhes permitir adaptar ao ambiente. </li><li> Certifique-se de que tanto a fonte Rotarod eo computador estão ligados e conectados entre si (Figura 2). </li><li> Pré-definir o rotarod com as -5 dpi parâmetros do protocolo de treinamento, conforme descrito na Tabela 1. </li><li> Salve o arquivo de trabalho com as informações de data e identificação. </li><li> Mover a gaiola contendo o time a ser testada a partir do rack para uma mesa ao lado do rotarod. Ratos são geralmente testados em esquadrões de 4. </li><li> Pegar um rato pela cauda e colocá-lo sobre a haste, de costas para o operador. Repita o procedimento para o segundo através da quarta mouse. Se um rato falls ou saltos, colocá-lo de volta em sua pista sobre o rotarod até que todos os ratos estão em posição. Ignorar se quaisquer camundongos virar-se para enfrentar o operador. </li><li> Depois de colocar todos os ratos, pressione o botão &quot;Enter&quot; para iniciar o experimento. Observe os temporizadores são iniciados automaticamente e as rotações por minuto (rpm) em exposição para cada pista. <ol><li> Como cada animal cai da haste, registar a velocidade da haste no momento da queda, bem como a duração do tempo que o animal permaneceu sobre a haste. A haste vai continuar a rodar até que o último animal caiu do conjunto da haste. </li></ol></li><li> Depois de todos os ratinhos tenham caído, usar um lenço de papel para remover qualquer boli fecais e de urina a partir da haste. A presença de urina e material fecal podem afectar a capacidade dos ratinhos para agarrar a haste. <ol><li> Depois de um descanso de 3 min, dar os ratos um segundo e depois um terceiro ensaio. O tempo máximo por único ensaio é de 240 segundos. Administrar um total de 3 ensaios durante cada dia de teste. </li></ol></li><li> Devolver os ratos para sua gaiola e devolvê-los de volta para o rack. No final da sessão experimental, limpar o Rotarod com sabão e água, para remover toda a matéria fecal para fora da máquina. </li><li> Limpe a placa de base com etanol 70%. Spray para baixo toda a máquina com dióxido de cloro para desinfectar. </li><li> No dia - 4, - 3, - 2, e - uma pi, pré-definir o Rotarod com os parâmetros do protocolo de formação adequadas, como descrito na Tabela 1, e repetir os passos 2.1.2 a 2.1.12. </li><li> Após a obtenção das medidas de linha de base, infectar os ratinhos com TMEV. Permitir um período de recuperação pi 6 dias. </li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="2"> Protocolo </td><td rowspan="2"> Dia Testing </td><td rowspan="2"> Freqüência </td><td rowspan="2"> Velocidade de partida (rpm) </td><td rowspan="2"> MaxSpeed ​​(rpm) </td><td> Aceleração </td><td> Ensaios </td><td> ITI </td></tr><tr><td> (rpm / seg) </td><td> (N x seg) </td><td> (min) </td></tr><tr><td rowspan="4"> Treinamento </td><td> - 5 dpi </td><td> 1 dia </td><td> 1 </td><td> 12 </td><td> 01/03 </td><td> 3x240 sec </td><td> 3 </td></tr><tr><td> - 4 dpi </td><td> 1 dia </td><td> 1 </td><td> 13 </td><td> 01/03 </td><td> 3x240 sec </td><td> 3 </td></tr><tr><td> - 3 dpi </td><td> 1 dia </td><td> 1 </td><td> 14 </td><td> 01/03 </td><td> 3x240 sec </td><td> 3 </td></tr><tr><td> - 2; - 1 dpi </td><td> 1 dia </td><td> 5 </td><td> 40 </td><td> 01/03 </td><td> 3x240 sec </td><td> 3 </td></tr><tr><td rowspan="2"> Experimental </td><td> De 7-50 dpi </td><td> 2 / semana </td><td>5 </td><td> 40 </td><td> 30/5 </td><td> 3x240 sec </td><td> 3 </td></tr><tr><td> De 51-150 dpi </td><td> 1 / Semana </td><td> 5 </td><td> 40 </td><td> 30/5 </td><td> 3x240 sec </td><td> 3 </td></tr></tbody></table> Tabela 1: Parâmetros rotarod em treinamento e protocolos experimentais. <ol start="2"><li> O protocolo experimental Rotarod <ol><li> Em 7 dpi, pré-definir o Rotarod com os parâmetros do protocolo experimentais adequadas, como descrito na Tabela 1. Repita os passos 2.1.2 a 2.1.10. </li><li> No final do julgamento # 3, pesar cada rato e fazer uma nota do peso do corpo na folha de dados. Limpar e desinfectar o rotarod como por etapas 2.1.11 e 2.1.12. </li><li> Testar os ratinhos duas vezes por semana durante as 6 semanas seguintes, como descrito acima. Após 6 semanas (em que os ratos propensos atingiram uma fase de planalto) 8,9, teste a ratos uma vez por semana com o mesmo experimental protocolo. O protocolo de teste real dura uma média de 150 dias, dependendo do curso da doença específica. </li></ol></li><li> Índice Funcional neurológica <ol><li> Exportar os dados brutos em um arquivo de planilha e analisar os resultados. </li><li> Dados expressos como tempo de execução (Figura 3A): este é o tempo de execução normal mais o tempo de rotação passiva menos o tempo de atraso de rotação (Tabela 2) 10. Calcula-se a média de tempo de execução os três ensaios por dia. </li><li> Expressar os dados como um índice funcional neurológica (IFN; Figura 3B). <ol><li> Calcule o limite de desempenho de linha de base de cada rato individual. O limiar de desempenho da linha de base é determinada como a média de todos os tempos de execução de dias + 15 e + 45 pi 6,7. </li><li> Calcula-se a IFN-se a média dos três tempos de execução médios mais recentes dividido pelo limiar de 6,7 de ratinho específico que o desempenho da linha de base &lt;/ Sup&gt;. NOTA: Se a tempos de execução testado para um rato no dia + 72, + 76 e + 79 pi são 55 seg, 45 seg e 50 seg, eo tempo de linha de base para o mesmo rato foi de 135 segundos, o NFI para esse rato no 79 dpi será [(45 + 50 + 55) / 3] / 135 ou 0,37. </li></ol></li><li> Expressar os dados como um NFI ajustado (adjNFI; Figura 3C): ajustar os dados NFI por um valor de população para o único experimento. <ol><li> Calcula-se a adjNFI pela divisão do valor da média IFN por IFN obtidos pelo grupo tratado com sham naquele dia específico. </li></ol></li></ol></li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Prazo </td><td> Definição </td></tr><tr><td> tempo de funcionamento normal </td><td> O tempo total que o rato gasta funcionando ativamente sobre a haste rotativa, ou seja, a latência a cair. </td></tr><tr><td> tempo de rotação passiva </td><td> a ummontagem de tempo que o rato tem-se mantido na haste no modo de rotação passiva. </td></tr><tr><td> tempo de atraso Rotação </td><td> A quantidade de tempo que o rato permanece na vara durante o modo de rotação passiva </td></tr><tr><td> modo de rotação passiva </td><td> Quando o rato agarra a vara e roda sem ter de deambular. </td></tr><tr><td> O tempo total da sessão </td><td> A quantidade total de tempo que o rato permanece na haste rotativa durante a sessão. </td></tr><tr><td> desempenho da linha de base </td><td> desempenho motor pré-danos avaliados para determinar o limite mínimo de desempenho. </td></tr><tr><td> Índice função neurológica (NFI) </td><td> Índice clínico, o que compara cada desempenho motor do mouse, ou seja, tempo de execução, a qualquer momento para seu desempenho máximo. </td></tr><tr><td> Ajustado Índice de função neurológica (adjNFI) </td><td> Quando um processo de normalização é aplicado para ajustar os dados IFN por um valor da população paraa única experiência. </td></tr><tr><td> valor da população </td><td> valor médio IFN obtidos pelo grupo submetida a tratamento simulado a um dia específico. </td></tr></tbody></table> Tabela 2: Definições de rotarod parâmetros adotados para quantificar Neurological Impairment.

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The authors have nothing to disclose.

Despite some limitations, the Rotarod performance test represents an important tool for assessing motor function and dysfunction in TMEV-IDD as well as the effect of pharmacological interventions on disability progression in mice.
The Rotarod test was first described in 1957 as a tool for measuring neurological deficits in rodents11. Rodents have to walk on a rotating rod, with increasing rotating speed, and try to avoid falling to the ground. The latency to fall is recorded and used as a quantitative end point for motor function: the more neurological impairment the animals have, the sooner they fall off the rod. The clear advantage of this test over the traditional visual scoring systems is that it generates an objective variable that can be used for statistical purposes to quantify the effects of therapies and experimental procedures on disability progression3. Visual scoring systems such as the righting reflex test might be preferred as being a simpler and more rapid test to assess locomotor abilities in mice. However, these systems lack the precision of a more quantitative test like the Rotarod performance test, and they should therefore not be used to monitor the natural history or phenotypic changes of mice with mild and progressive neurological impairment. On the other hand, visual tests like the righting reflex test are particularly adapted to mice that are too young or too affected to be tested on the Rotarod.
In order to avoid distorted assessments of the Rotarod test, the testing parameters (i.e., size, speed, and acceleration of the rotating rod; amount of training given to the animal; and final data processing) must be carefully replicated3. Today, the Rotarod performance test is still the most commonly used motor behavioral test, yet there is little consensus on the ideal testing parameters to produce optimal results. We have found studies with TMEV-IDD mice that have used rod diameters from 1.18 to 1.6 in. (3 to 4 cm)12,13, fixed-speed rates of 5 to 10 rpm12,13, and acceleration rates of 5 to 10 rpm/min12-14. However, it is unknown whether the conclusions about neurological differences would generalize to other test conditions. The present protocol offers recommendations of specific testing parameters that were optimized and are thus suitable for studying progressive neurological disability in the TMEV-IDD mouse model of MS using the Rotarod test. Particularly, before embarking on a Rotarod motor function study in mice, it is recommended that a number of potentially important factors-among others, baseline performance and passive rotation-be considered.
First, regarding baseline performance: exercised and uninfected mice improve their motor performance over time, eventually reaching a stable plateau over several days or weeks8,9. This is the result of the normal process of motor learning. However, this improvement is not permanent, and if mice are left untrained for several days, their motor performance on the rotating rod will drop rapidly9. Therefore, a baseline performance determination is crucial to differentiate between motor learning and actual recovery after a therapeutic intervention, as well as between a physiological motor performance drop and worsening due to an experimental procedure.
Our optimized Rotarod protocol expects to train TMEV-IDD mice from + 15 to + 45 dpi (i.e., in between the acute encephalitis, which occurs within + 3 to + 12 dpi, and the late chronic demyelinating disease, which develops from + 30 to + 40 dpi)15. This allows for the evaluation of motor performance as a result of progressive demyelination and thus excludes any contributing deficits due to the early encephalitis. Ideally, the Rotarod training should be performed prior to disease induction. However, in order to develop a chronic infection of the CNS, SJL mice have to be injected with the virus before their 7th week of age16. Since it is generally accepted that the ages of animals that the Rotarod test can be performed on is 8 to 26 weeks17, an effective training session cannot be completed in mice before TMEV infection.
Training and baseline performance evaluation are also important to exclude the effect of low motivation, low basal performance, and low learning skills. Each animal is different, and these differences need to be considered in a motor performance evaluation. This is best remedied by using a baseline performance normalized index, also known as an NFI. This index also accounts for performance fluctuations that might be observed in mice during different testing sessions. Performance variability is a common issue in behavioral testing, mainly due to factors outside the experimental protocol, such as low motivation and past experiences of the animal. The mean running times for the last three time points of each mouse provides an estimate of the central tendency of the distribution that is expected if performance remains stable. This accounts for day-to-day individual performance variability. Severe neurological disability is then defined by NFI values of less than or equal to 0.3, whereas relatively unaffected mice are scored with NFI values greater than or equal to 0.7. Unfortunately, this strategy does not completely remove the problem that motivational changes might affect motor performance. To further address this problem, mice may be trained with the addition of specific motivating features, such as mild food deprivation prior to training or increased fear of falling caused by adjustable falling heights of 17.5 to 23.5 in. (44.5 to 60 cm).
Second, regarding passive rotations or looping: perhaps the most important threat to the validity of Rotarod data is the occurrence of passive rotations that arise when the mouse can cling on and rotate passively as the rod turns18. Although passive rotation is observed on the Rotarod, especially in mice experiencing motor coordination problems, this coping behavior may result in late falling off the rod, which would incorrectly indicate an absence of motor deficits. Therefore, a secondary measure of disturbed motor coordination, which should be considered in the data analysis, is the count of the number of times the mouse turns around the rod without falling off. Also, passive rotation is influenced strongly by task parameters such as the diameter, speed and acceleration degree of the rod18. There are no perfect solutions for reducing the risk of passive rotation, but a possible resolution may be the use of a rod with a larger diameter (i.e., 1.18 to 3.15 in. (3 to 8 cm)) or less pronounced ridges so as to slightly reduce the grip of the mouse.
A limitation of the Rotarod performance test is that the motor assessment is limited to a short observation time. Moreover, testing at different time points in the light-dark cycle may alter the outcome of behavioral testing19. Thus, in order to overcome these issues, several companies are developing innovative home cage analysis systems that provide 24/7 monitoring of rodent behaviors. These monitoring systems surely represent the newest and most exciting technology available in rodent behavioral research, but are still extremely expensive and not always accessible to research laboratories.
In summary, the Rotarod performance test is a relatively simple, inexpensive, and well-characterized test for long-term evaluation of neurological disability in TMEV-IDD, a murine model of progressive MS. The advantage of this test over the traditional clinical symptoms score systems is that it generates an objective variable to quantify the effects of different drugs, conditions, and procedures on motor function. Given the robustness of the Rotarod performance test, by applying a few simple precautions, as described above, this procedure provides a baseline against which to assess (1) the relevance of the TMEV-IDD mouse model to progressive MS and (2) its usefulness for testing therapies aimed at treating progressive neurological conditions such as MS.

de Theiler murinos Vírus da Encefalomielite (TMEV) é um vírus de ARN de cadeia simples que neurotrópico persistentemente infecta o sistema nervoso central de murino (SNC). Em ratinhos susceptíveis, a infecção com TMEV provoca, uma doença desmielinizante imunomediada-progressiva crónica, conhecida como doença desmielinizante induzida por TMEV (TMEV-DDI). A infecção experimental de ratinhos leva um curso da doença que se assemelha observada em formas progressivas de esclerose múltipla (EM). TMEV-DID é caracterizada por duas fases distintas: a fase aguda e a fase crónica. A fase aguda é, normalmente um 1,2 encefalite subclínica leve. A segunda fase, crónica, começando cerca de um mês após a infecção, é composto por uma deficiência lentamente progressiva caracterizada por desmielinização, inflamação e axonal 1,2 danos. A fraqueza observada em ratos está associada a espasticidade e, ocasionalmente, graves espasmos tônico. Porque há atualmente nenhuma medications para melhorar a incapacidade progressiva em pacientes, os investigadores estão particularmente atraídos por TMEV-IDD, o que representa um modelo animal ideal para o acompanhamento do impacto das drogas modificadoras da doença na progressão da doença. No entanto, em ratinhos, bem como em pacientes com esclerose múltipla, a monitorização da progressão da incapacidade requer uma observação clínica contínua ao longo de períodos de tempo prolongados. Em camundongos, monitorização a longo prazo para a progressão de incapacidade pode ser realizado com o teste de desempenho rotarod. O teste de desempenho Rotarod é um teste comportamental padrão que avalia funções associadas ao motor, como coordenação, equilíbrio e fadiga em roedores. Os ratos têm a manter o equilíbrio em uma haste de giro, que é rotativa em aceleração contínua; latência do tempo de queda a partir dessa haste é gravado. Os animais com disfunção neurológica é incapaz de permanecer na haste rotativa desde que controles, e eles normalmente caem quando a velocidade de rotação é superior à suacapacidade motora. O comprometimento mais neurológica os animais têm, quanto mais cedo eles cair a vara, e quanto menor a latência é o tempo. A vantagem do teste de rotarod ao longo dos sistemas de pontuação visuais tradicionais é que ele gera um objetivo, mensurável variável o tempo de latência que pode finalmente ser utilizada para análises estatísticas para quantificar os efeitos das terapias e procedimentos experimentais 3. No Laboratório de Neuroimunologia (LONI) em Dartmouth, os ratos são submetidos a um protocolo de adaptação, onde são testados antes da infecção TMEV, a fim de familiarizá-los com a máquina e para avaliar a sua &quot;base&quot; a coordenação normal de equilíbrio e controle motor 4, 5. Uma vez que a linha de base é estabelecida e os ratinhos são infectados com TMEV, eles são testados uma vez ou duas vezes por semana ao longo de um período de vários meses. O protocolo de teste real dura uma média de 150 dias, permitindo assim uma avaliação dao declínio do equilíbrio, coordenação e controle motor durante todo o curso da doença desmielinizante. Várias centenas de TMEV-DDI e tratados de forma simulada os ratos foram testados até agora para disfunção neurológica em Dartmouth. Estes ratinhos tinham recebido vários imunomoduladores, mas nenhum agente farmacológico foi encontrado para ser eficaz no melhoramento da progressão da incapacidade 6,7. O presente artigo e o protocolo relacionado descrevem como caracterizar o comprometimento neurológico progressivo apresentado por ratos TMEV-DDI. Em particular, o protocolo oferece recomendações de parâmetros de teste específicos acredita-se ser geralmente adequados para estudar incapacidade neurológica em ratinhos TMEV-DID utilizando o teste de rotarod. Este procedimento fornece uma linha de base para avaliar (1) a relevância deste modelo de rato para progressiva MS e (2) a sua utilidade para testar terapias destinadas a tratar condições neurológicas progressivas, tais como MS. Claramente, oteste de desempenho Rotarod e a corrente de protocolo de parâmetros e de teste optimizados não só são úteis na detecção de incapacidade neurológica progressiva no modelo de ratinho TMEV-DID, mas são também úteis na detecção de deficiências em outros modelos de ratinho genética induzida por vírus e / ou de doenças neurodegenerativas.

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measuring progressive neurological disability in a mouse model of multiple sclerosis

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating disease, with clinical features similar to the progressive forms of multiple sclerosis (MS). The mice show progressive disability with loss of motor and sensory functions, which can be assessed with multiple apparatuses and protocols. Among them, the Rotarod performance test is a very common behavioral test, its advantage being that it provides objective measurements, but it is often used assuming that it is straightforward and simple. In contrast to visual scoring systems used in some models of MS, which are highly subjective, the Rotarod test generates an objective, measurable, continuous variable (i.e., length of time), allowing almost perfect inter-rater concordances. However, inter-laboratory reliability is only achieved if the various testing parameters are replicated. In this manuscript, recommendations of specific testing parameters, such as size, speed, and acceleration of the rod; amount of training given to the animals; and data processing, are presented for the Rotarod test.

O objectivo desta experiência representativa foi comparar a incapacidade neurológica induzida pela estirpe Daniels (DA) e tensão de feijão de TMEV. Para efeitos do presente estudo, um grupo de 32 ratinhos fêmea SJL foram infectados por via intracraniana com TMEV, quer da estirpe DA (n = 16) ou a estirpe de feijão (n = 16), e os seus sinais clínicos foram monitorizados ao longo do tempo. Um grupo adicional de 20 ratos foi tratado simulada (ou seja, solução salina foi injectado por via intracraniana) e serviu como grupo de controlo saudável. O teste de desempenho Rotarod foi usada para avaliar a progressão da doença crónica em ratinhos TMEV-infectadas e controlos simulados. Os ratos foram individualmente marcados orelha e submetidos ao protocolo de adaptação ao dia na semana antes da injecção de TMEV e, em seguida, para o protocolo de teste real que durou 125 dias. Para cada dia de teste, o tempo de funcionamento, definida como o comprimento de TIme o rato ficou sobre a haste, foi armazenada individualmente para cada rato como a média dos 3 ensaios para esse dia. rotarod dados também é expressa como IFN e como adjNFI sham-normalizada. Como mostrado na Figura 3A, a infecção com TMEV afectados negativamente os tempos de execução dos ratinhos. A medidas repetidas ANOVA de duas vias revelou um efeito significativo de infecção TMEV (F = 56,76, p &lt;0,0001), bem como um efeito significativo de tempo (F = 3,26, p &lt;0,0001) e uma interacção tratamento tempo (F = 8,065, p &lt;0,0001). Teste de comparações múltiplas de Dunnett a seguir revelou que ambos os grupos de ratinhos infectados TMEV-NFI tinham valores significativamente mais baixos do que os dos ratos sham (p ≤ 0,01; Figura 3B). A diferença foi estatisticamente significativa a partir de dia + 61 pi até o final do follow-up (Figura 3B). Não houve diferença comparing progressão de incapacidade em camundongos infectados com a estirpe de feijão e os infectados com a estirpe DA em todos os momentos (p&gt; 0,5; Figura 3C). <img alt="figura 1" src="/files/ftp_upload/54616/54616fig1.jpg" /> Figura 1:. Injeção intracraniana Para executar injeções intracranianos, o rato é e contido em uma superfície sólida anestesiados com gás. Até 30 ul são injectados com segurança no cérebro do rato. O local da injeção é aproximadamente a meio caminho entre a linha do olho e da orelha e apenas fora da linha média. Uma agulha 30 G penetra diretamente no crânio e uma seringa de insulina é convenientemente usado para evitar que a agulha se estende muito profundamente no cérebro. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54616/54616fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Figura 2:. O aparelho rotarod O teste rotarod é um ensaio neurocomportamental padrão em que os ratos são treinados para executar em uma haste rotativa e são cronometrados por quanto tempo eles podem ficar na haste, uma vez que lentamente se acelera. A haste é suspensa acima de um chão da gaiola, e os ratos naturalmente tentar permanecer na haste para evitar cair no chão. A queda a partir da haste para a base do instrumento não causa danos aos ratos. A) Um aparelho rotarod padrão consiste de uma haste accionada por um motor com velocidades constantes ou acelerando. B) Os painéis dividir a haste em pistas separadas, cada uma adequada para um animal individual. Um total de 4 ratinhos podem ser testados em cada ensaio. C) A haste é em 1,18. (Cerca de 3 cm) de diâmetro, e a sua superfície é serrilhada, permitindo que os ratinhos para obter uma melhor aderência. A queda do mouse é precisamente rastreados por um feixe de fotos quegrava automaticamente a quantidade de tempo gasto na haste. Os dados experimentais é registrado em um computador depois que os ratos têm todos caído da haste. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54616/54616fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/54616/54616fig3.jpg" /> Figura 3:. Rotarod Teste de Desempenho em Ratos TMEV infectados e sham-controls O teste rotarod foi realizada para medir a função motora da farsa, DA-infectados, e os ratos de feijão-infectados. Além do tempo (seg) (A) em execução, os dados rotarod foram também expressas como um índice da função neurológica (IFN) (B), e um simulado normalizada ajustado IFN (adjNFI) (C). A latência a cair a partir de um rotarod a acelerar foi medida em três ensaios por dia. Os dados são apresentados como média ± erro padrão da média (SEM). Blue círculo, n = 16 ratos feijão-infectados; quadrados vermelhos, n = 16 ratos DA-infectados; triângulos negros, n = 20 controles tratados com placebo. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54616/54616fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis at JoVE.com

Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice.

Medindo Progressive Neurological Disability em um modelo de rato com esclerose múltipla

After intracerebral infection with the Theiler's Murine Encephalomyelitis Virus (TMEV), susceptible SJL mice develop a chronic-progressive demyelinating ...

measuring-progressive-neurological-disability-mouse-model-multiple

Nanyang Technological University

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Neuroscience

10.9K visualizações.  Geisel School of Medicine at Dartmouth. An optimized testing protocol is presented in this paper for the Rotarod performance test, used for measuring progressive neurological disability in TMEV-infected mice. 

Vídeo: Measuring Progressive Neurological Disability in a Mouse Model of Multiple Sclerosis

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

Múltiplas mouse-Imagem por Ressonância Magnética neuroanatômicos

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Neurociência

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

Análise Quantitativa de Defeitos de escalada em um modelo de Drosophila de doenças neurodegenerativas

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Medindo a endocitose da vesícula sináptica nos neurônios Hippocampal culturas

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Establishing Mouse Models for Zika Virus-induced Neurological Disorders Using Intracerebral Injection Strategies: Embryonic, Neonatal, and Adult

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly and additional brain abnormalities. However, the mechanism underlying the pathogenesis of ZIKV in the developing brain remains unclear. Intracerebral surgical methods are frequently used in neuroscience research to address questions about both normal and abnormal brain development and brain function. This protocol utilizes classical surgical techniques and describes methods that allow one to model ZIKV-associated human neurological disease in the mouse nervous system. While direct brain inoculation does not model the normal mode of virus transmission, the method allows investigators to ask targeted questions concerning the consequence after ZIKV infection of the developing brain. This protocol describes embryonic, neonatal, and adult stages of intraventricular inoculation of ZIKV. Once mastered, this method can become a straightforward and reproducible technique that only takes a few hours to perform.

Here we describe a method for establishing a model of Zika virus-induced microcephaly in mouse. This protocol includes methods for embryonic, neonatal, and adult-stage intracerebral inoculation of the Zika virus.

Estabelecer modelos de Mouse para doenças neurológicas induzida por vírus Zika usando estratégias de injeção Intracerebral: embrionárias, Neonatal e adulto

The Zika virus (ZIKV) is a flavivirus currently endemic in North, Central, and South America. It is now established that the ZIKV can cause microcephaly ...

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Cryosectioning simultânea de vários cérebros de roedores

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

Lipidomics and Transcriptomics in Neurological Diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Lipidomics e Transcriptomics em Doenças Neurológicas

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

Programa de autópsia abrangente para indivíduos com esclerose múltipla

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Applying the RatWalker System for Gait Analysis in a Genetic Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta. Gait abnormalities, including decreased arm swing, slower walking speed, and shorter steps are common in PD patients and appear early in the course of disease. Thus, the quantification of motor patterns in animal models of PD will be important for phenotypic characterization during disease course and upon therapeutic treatment. Most cases of PD are idiopathic; however, the identification of hereditary forms of PD uncovered gene mutations and variants, such as loss-of-function mutations in Pink1 and Parkin, two proteins involved in mitochondrial quality control that could be harnessed to create animal models. While mice are resistant to neurodegeneration upon loss of Pink1 and Parkin (single and combined deletion), in rats, Pink1 but not Parkin deficiency leads to nigral DA neuron loss and motor impairment. Here, we report the utility of FTIR imaging to uncover gait changes in freely walking young (2 months of age) male rats with combined loss of Pink1 and Parkin prior to the development of gross visually apparent motor abnormality as these rats age (observed at 4-6 months), characterized by hindlimb dragging as previously reported in Pink1 knockout (KO) rats.

Here we describe the RatWalker system, built by redesigning the MouseWalker apparatus to accommodate for the increased size and weight of rats. This system uses frustrated total internal reflection (FTIR), high-speed video capture, and open-access analysis software to track and quantify gait parameters.

Aplicando o Sistema RatWalker para Análise de Marcha em um Modelo de Rato Genético da Doença de Parkinson

Parkinson's disease (PD) is a progressive neurodegenerative disorder caused by the loss of dopaminergic (DA) neurons in the substantia nigra pars ...

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, leading to motor dysfunctions, psychical disability, and cognitive impairment during MS progression. Positron emission tomography (PET) is an imaging technique able to quantify in vivo cellular and molecular alterations.
Radiotracers with affinity to intact myelin can be used for in vivo imaging of myelin content changes over time. It is possible to detect either an increase or decrease in myelin content, what means this imaging technique can detect demyelination and remyelination processes of the central nervous system. In this protocol we demonstrate how to use PET imaging to detect myelin changes in the lysolecithin rat model, which is a model of focal demyelination lesion (induced by stereotactic injection) (i.e., a model of multiple sclerosis disease). 11C-PIB PET imaging was performed at baseline, and 1 week and 4 weeks after stereotaxic injection of lysolecithin 1% in the right striatum (4 &#181;L) and corpus callosum (3 &#181;L) of the rat brain, allowing quantification of focal demyelination (injection site after 1 week) and the remyelination process (injection site at 4 weeks).
Myelin PET imaging is an interesting tool for monitoring in vivo changes in myelin content which could be useful for monitoring demyelinating disease progression and therapeutic response.

Positron Emission Tomography Imaging for In Vivo Measuring of Myelin Content in the Lysolecithin Rat Model of Multiple Sclerosis

This protocol has the aim of monitoring in vivo myelin changes (demyelination and remyelination) by positron emission tomography (PET) imaging in an animal model of multiple sclerosis.

Tomografia de emissão de pósitrons para medição in vivo de conteúdo de mielina no modelo de rato de lysolecithin de esclerose múltipla

Multiple sclerosis (MS) is a neuroinflammatory disease with expanding axonal and neuronal degeneration and demyelination in the central nervous system, ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Análise citométrica de fluxo de múltiplos parâmetros mitocondriais em células-tronco pluripotentes induzidas por humanos e seus derivados neurais e gliais

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...