Os autores agradecem o apoio da Sra Sharon Spana no monitoramento roedor, manipulação e experimentação. PhD apoio financeiro foi fornecido pela Universidade de Sydney e do Centro Australiano de Investigação de Excelência em Vision.

Este protocolo segue as diretrizes de cuidados de animais de University of Sydney. 1. Os eléctrodos Factura <ol><li> Construir o eléctrodo positivo (o qual entrará em contacto com a córnea) a partir de uma (5 cm) de comprimento curto de fio de platina de 1-2 mm de diâmetro. Moldá-lo em um loop de alguns mm de diâmetro. Conecte-se este ciclo com uma vantagem convencional, o tempo suficiente para chegar à fase de entrada do seu amplificador (ver Figura 4). </li><li> Construir o eléctrodo negativo (o que vai na boca do animal), utilizando um de Ag / AgCl sedimentar 1-2 mm de diâmetro, também ligada a uma guia convenção (ver Figura 4). </li><li> Como um eletrodo de referência (que entrará em garupa do animal), use uma agulha hipodérmica limpo (23 G), também ligado a uma vantagem de comprimento adequado (consulte a Figura 4). </li><li> O ideal é usar cabos de três chumbo fornecidos por fabricantes de instrumentos, para conectar os três eletrodos (U positivo94; córnea, negativo → boca, referência → traseiro) para o amplificador. </li></ol> 2. Conexão e Calibração do estímulo luminoso e ERG Set-up <ol><li> Criar (ou localizar) um pequeno laboratório de gravação, o que pode ser feito escuro. Equipar com uma ou ambas de uma luz sobre-o-banco de vermelho ou uma cabeça-lâmpada vermelha. </li><li> Use um luxímetro para confirmar que a luz vermelha iluminância atingir os olhos do rato durante a instalação não exceda 1 lux. Nota: Um filtro de densidade neutro pode ser usado para reduzir o brilho da lâmpada e a fonte de luz da lâmpada deve especificamente emitem luz vermelha. Adaptação das Trevas vai ser comprometida se fontes de luz emitem comprimentos de onda (visíveis) mais baixos. </li><li> Lacrar todos a luz difusa que entra no laboratório de gravação (isso muitas vezes exige persistência com fita opaco) e preparar um filtro de densidade neutra (este pode ser comprado em folhas) grande o suficiente para caber mais, e assim fraca, qualquer tela de computador que você terá na laboratório. Nota: luz e Strayà luz de uma tela são suficientes para prejudicar a adaptação ao escuro do olho de rato. </li><li> Ligue o amplificador para hardware de aquisição de dados. Conecte-se, leads negativos e positivos de referência para o amplificador. Verifique se o computador ea unidade de alimentação Ganzfeld LED estão bem conectados a uma fonte de terra. Nota: Alguns laboratórios têm-se especializado pontos de aterramento, conectado a um terreno de construção; uma tubulação de água é uma alternativa eficaz. </li><li> Calibrar a fonte de luz LED com um radiómetro de investigação de qualidade. Corrigir o sensor do medidor na posição em que o olho do animal será localizado durante uma experiência. </li><li> Programe os LEDs Ganzfeld para executar um protocolo de campo total ERG com aumentos passo a passo em energia flash, duração do flash, flash e repetição tempo entre flashes, denominado intervalo interstimuls (ISI), configurações. Por exemplo um protocolo de campo total ver Tabela 1. Nota: O ERG de campo total flashes aumentará de flashes ofuscantes repetitivas para bdireito pisca em uma forma sábia passo. O programa de flash duplo surge na sequência do protocolo de campo total e permite o isolamento dos bastonetes e cones respostas. </li></ol> 3. Dia Antes do ERG Experimentação <ol><li> Escuro adaptar ratos Sprague-Dawley, durante 12 h antes da gravação. É conveniente fazer isso no laboratório de gravação, uma vez que a luz difusa foi eliminada. </li></ol> 4. Dia do ERG Experimentação <ol><li> Organizar para que o animal delicadamente ser aquecido durante a gravação. Nós usamos uma plataforma de metal leve construído de modo que a cabeça do animal pode descansar no ponto correto no momento da entrada para o Ganzfeld. A plataforma tem embutido tubagem através da qual se bombeia a água pré-aquecida a 40 ° C num banho de água. Nota: A experiência mostra que este mantém a temperatura do núcleo do animal a 37 ° C. </li><li> Pesar o rato sob condições de pouca luz. Peso recorde e make up cetamina correta (60 mg / kg) e xilazina (5 mg / kg) dose. Contenha o rato gently e injetar anestésico por via intraperitoneal. </li><li> Nota momento da injecção. Uma vez que o animal está inconsciente (geralmente dentro de 5 min) verificar profundidade da anestesia levemente beliscar uma almofada do pé, para ver se uma resposta reflexa está presente. É melhor esperar até que esse reflexo é ausente ou fraco, antes de prosseguir. </li><li> Aplicar uma única gota de atropina e outro de proxmethacaine a córnea. </li><li> Corte um comprimento de 10 centímetros da linha preta. Faça um laço com um nó simples e deslizar o laço sobre o equador do olho. Aperte-o ligeiramente; o efeito é chamar o globo ocular ligeiramente para a frente, com o mínimo de pressão. Isso mantém a córnea resulta das pálpebras. </li><li> Aplicar olho carbómero gotas à superfície da córnea. Certifique-se de carbómero permanece sobre a superfície da córnea e não derramar sobre as pálpebras ou o rosto. </li><li> Coloque roupas de cama absorvente em cima da plataforma aquecida. </li><li> Posição rato na cama, com a cabeça no lugar recomendado na abertura do Ganzfeld. </li><li> Inserir intErnal sonda de temperatura no recto. Sonda de temperatura segura na posição gravando cabo de sonda para a cauda. </li><li> Insira o eletrodo de referência (a agulha 23 G) por via subcutânea na perna de trás, e se conectar ao amplificador. </li><li> Inserir o eléctrodo negativo (o sedimento de Ag / AgCl) de forma segura na boca. Para evitar que isso escorregar para fora da boca, apor o cabo de ligação a uma superfície estável. </li><li> Posicionar o eléctrodo positivo ao longo do centro da córnea. Usando um micromanipulador, garantir que o eletrodo toca a córnea suavemente. </li><li> Verifique a temperatura do corpo é a 37,0-37,5 ° C. </li><li> Uma vez que o animal está devidamente posicionado e eletrodos estão no lugar, armar toda a configuração (Ganzfeld e animal) com um material opaco (para preservar a adaptação ao escuro). Nós usamos um pano preto macio. </li><li> No software de aquisição fixado em uma taxa de amostragem de 2 KHz com um tempo de coleta de 100-1000 ms com 5 ms de amostragem pré-coleção. Defina os filtros passa banda para 1-1,000Hz e garantir que a amostragem é acionado para provar o período de ~ 250 ms após um flash. </li><li> Verifique a linha de base a gravação. Deve ser livre de ruídos estranhos, mas mostrar algum ruído amplificador e uma oscilação respiratória. </li><li> Se a linha de base mostra ruídos estranhos, começar solução de problemas. A maioria dos problemas são relacionados a derrapagem na posição do eletrodo, ou aterramento. Use uma gaiola de Faraday para garantir gravações são livres de ruídos estranhos. </li><li> Executar um flash de teste, 0,4 log scot cd.sm -2. Uma forma de onda ERG semelhante à Figura 2A deve aparecer. Em nossas respostas típicas de laboratório para um flash cd.sm scot 0,4 log -2 são (a onda: -474 ± 39 mV e b-ondas: 1512 ± 160 mV, n = 11). </li><li> Permitir animal escuro re-adaptar para 10 min. É conveniente utilizar estes 10 min para verificar novamente a linha de base. </li><li> Após a confirmação do sinal estável começar a gravar. </li><li> No fim da sessão de gravação, que vá corpo temperature foi mantida. Remover eletrodos. Reaplicar carbï¿½ero polímero de córneas. Permitir que o animal se recuperar em uma almofada de calor até que esteja totalmente móvel e ativo, antes de retornar à habitação animal. </li></ol> 5. A isquemia remoto <ol><li> Execute isquemia remoto em ambos os roedores acordado ou anestesiados. </li><li> Se o animal é anestesiado, colocá-lo em uma plataforma aquecida (acima) e coloque o manguito do esfigmomanômetro sobre a parte superior da traseira-membro, claro do joelho. </li><li> Se os animais estão acostumados a serem tratadas, é possível executar esse procedimento sem anestesia; isto requer duas pessoas. Uma pessoa restringe o animal delicadamente eo segundo aplica-se o manguito do esfigmomanômetro e opera o esfigmomanômetro. </li><li> Para animais acordados, use um pedaço de toalha de ~ 15 centímetros x 30-50 cm para embrulhar cuidadosamente o animal, com um hindlimb livre. Coloque o animal em sua parte traseira em (digamos) o antebraço esquerdo, com a cabeça enfiada entre o braço do suporte e torso, e lugara braçadeira como acabado de descrever. </li><li> Esvaziar a manga e assegurar a válvula de pressão de ar é fechada. Bombear o manguito até 160 mmHg em animais anestesiados, e 180 mmHg em animais acordados. Isso excede a pressão sistólica (geralmente 140 mmHg e 160 mmHg, respectivamente). </li><li> Manter essas pressões, conforme necessário, usando a bomba de mão. </li><li> Após o tempo previsto para a isquemia (nós usamos dois períodos de 5 min separados por 5 min de reperfusão), esvazie a pressão do balonete, desapertando a válvula de pressão de ar. </li><li> Confirmar o efeito de isquemia remoto com uma sonda de temperatura ligada a pele da pata. A temperatura da pele cai tipicamente 32-30 ° C, durante 5 minutos e recupera por reperfusão. </li></ol> 6. Danos Luz <ol><li> Certifique-se de que os ratos estão em uma noite de adaptação ao escuro, antes do procedimento danos leves. </li><li> No momento adequado seguinte isquemia (em nossos experimentos sem atraso), cada animal é colocado isoladamente em um caixas de acrílico, with água e alimentos em recipientes à base de chão. Nota: O dano induzido por luz só pode ser realizado em animais albinos. </li><li> Ligue uma luz branca pré-calibrado 1.000 lux em um horário padrão (geralmente 09:00) e manter esta condição durante 24 horas. </li></ol> 7. ERG Extração de Dados e Análise <ol><li> Adquirir formas de onda médios do ERG. Se necessário, corrigir para uma linha de base diferente de zero, por subtracção. </li><li> Medir a amplitude da onda-um (apresentada em meados de altas intensidades de estímulo), como a diferença de tensão entre a linha de base e a primeira (&lt;30 ms latência) da calha (Figura 1). </li><li> Medir a amplitude da onda b como a diferença de tensão entre o pico da onda e um-a positiva da onda seguinte, que ocorre tipicamente a uma latência de 80-100 mseg (Figura 1). </li><li> Isolar potenciais oscilatórios usando uma transformada de Fourier para filtrar dados 60-235 Hz, com uma banda de transição 90 Hz <sup&gt; 7. Se necessário, o sinal de potencial oscilatório isolado pode então ser subtraída da forma de onda não filtrada para confirmar a identidade da calha de uma onda. </li><li> O tempo implícita (latência) dos a- e b-picos de onda também pode ser uma medida útil (Figura 1). Use os flashes individuais para isolar a resposta rod. Subtrair a resposta de cones (flash 2) a partir da resposta mista (flash 1) para isolar a resposta da haste (Figura 2). </li><li> Normalizar a intensidade da luz indivíduo uma onda e amplitudes da onda-b (pós-tratamento / pós-tratamento-base) ou a média para os grupos de tratamento. Curvas intensidade-resposta traçar as amplitudes de grupo e erro contra energia flash. </li></ol>

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Investigative Ophthalmology &amp; Visual Science. 55 (4), 2088-2096 (2014).</li><li>Maccarone, R., Di Marco, S., Bisti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saffron+supplement+maintains+morphology+and+function+after+exposure+to+damaging+light+in+mammalian+retina.">Saffron supplement maintains morphology and function after exposure to damaging light in mammalian retina.</a> Investigative Ophthalmology &amp; Visual Science. 49 (3), 1254-1261 (2008).</li><li>Hood, D. C., Birch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+abnormal+rod+photoreceptor+activity+with+the+a-wave+of+the+electroretinogram:+Applications+and+methods.">Assessing abnormal rod photoreceptor activity with the a-wave of the electroretinogram: Applications and methods.</a> Documenta Ophthalmologica. 92 (4), 253-267 (1996).</li><li>Robson, J. G., Frishman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rod-driven+a-wave+of+the+dark-adapted+mammalian+electroretinogram.">The rod-driven a-wave of the dark-adapted mammalian electroretinogram.</a> Progress in Retinal and Eye Research. 39, 1-22 (2014).</li><li>Hood, D. C., Birch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+COMPUTATIONAL+MODEL+OF+THE+AMPLITUDE+AND+IMPLICIT+TIME+OF+THE+B-WAVE+OF+THE+HUMAN+ERG.">A COMPUTATIONAL MODEL OF THE AMPLITUDE AND IMPLICIT TIME OF THE B-WAVE OF THE HUMAN ERG.</a> Visual Neuroscience. 8 (2), 107-126 (1992).</li><li>Wachtmeister, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillatory+potentials+in+the+retina:+what+do+they+reveal.">Oscillatory potentials in the retina: what do they reveal.</a> Progress in Retinal and Eye Research. 17 (4), 485-521 (1998).</li></ol>

Jonathan Stone é o diretor do CSCM Pty Ltd

O flash método ERG de adaptação ao escuro descrito acima é um método confiável para avaliar a função da retina em ratos. Tanto a uma onda e onda-b foram reduzidas em danos leves. Remoto pré-condicionamento isquêmico mitigado reduções de luz induzida por danos no um-ondas e b-ondas. Esta preservação da função da retina sugere que o pré-condicionamento isquêmico remoto tem induzido neuroproteção, assemelhando-se outras formas de pré-condicionamento de proteção, tais como hipóxia, isquemia e exercí...

O ERG é um potencial eléctrico gerado pela retina em resposta a luz, e gravado a partir da superfície da córnea do olho. Quando as condições de gravação são geridos com cuidado, o ERG pode ser usada em uma variedade de maneiras de avaliar a função da retina. Aqui descrevemos como para gravar o &#39;flash ERG&#39;, o potencial gerado quando a retina é exposto a um breve flash, brilhante apresentada em um fundo Ganzfeld. O Ganzfeld dispersa a luz homogeneamente eo flash de luz atinge toda a retina aproximadamente uniformemente. Se a retina está adaptado escuro antes de gravar, e o de adaptação ao escuro é mantida como o animal é preparado para a gravação, o ERG obtido é gerado por ambos os bastonetes e cones fotorreceptores. O flash ERG adaptação ao escuro tem uma forma de onda característica, que foi analisada em duas maneiras. Em primeiro lugar, precoce e tardio componentes da forma de onda do ERG foram distinguidos, e relacionados com a sequência de neurónioactivação ai na retina. O primeiro componente é um de curta latência negativo ido potencial, a uma onda (Figura 1). Isto é seguido por um potencial positivo Ir, chamado a onda b. A fase ascendente da onda b mostra oscilações, que são considerados um componente separado (potenciais oscilatórios ou PO). A onda um é considerado ser gerado por fotorreceptores, o b-ondas por células da camada nuclear interna, e as células PO por amacrine 1. Com base na força do estímulo, as respostas a pisca muito fraca denominado o limiar de resposta escotópica são possíveis. O limiar de resposta escotópica é entendida para ser gerada a partir das células ganglionares da retina 2-4. Em segundo lugar, o flash ERG podem ser separados por adaptação à luz, ou por um protocolo de dois flashes descrito abaixo, em componentes rod- e orientada para o cone. Sob condições fotópicas, a uma onda não é detectável em ratazanas, pois a população cone é baixo, mas PO e uma onda b são5 claro. Nos primatas, cujas retinas têm populações superiores cone, tanto rod- e vias cone- gerar um detectável uma onda 6. Duas medidas úteis muitas vezes extraídos do flash ERG são as amplitudes de a- e b-ondas, medidos como na Figura 1, com as respostas de flash típicos mostrados na Figura 2. Quando a população de fotorreceptores é reduzido, por exemplo por exposição a prejudicialmente brilhante luz, todos os componentes do ERG são reduzidos. Intervenções neuroprotetores, como remota pré-condicionamento isquêmico (RIP), pode ser validado pela preservação das amplitudes do a- e b-ondas (Figura 3). Em resumo, a análise do ERG permite comparações entre saudável, luz e da retina danificado neuroprotected.

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			<td>PC computer</td>
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			<td>PL 35044</td>
			<td>Acquistion of ERG</td>
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			<td>Photometric solutions</td>
			<td>FS-250A</td>
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			<td>Research Radiometer</td>
			<td>International light technologies</td>
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			<td>negative electode</td>
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			<td>Water bath</td>
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			<td>Tubing</td>
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			<td>Homeothermic blanket system with flexible probe</td>
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			<td>Bausch &#38; Lomb</td>
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			<td>topical anaesthetic</td>
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using the electroretinogram to assess function in the rodent retina and the protective effects of remote limb ischemic preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

O protocolo pode ser utilizado para medir a função visual da retina de roedores in vivo. A uma onda, uma medida da função de fotorreceptores, e da onda b, uma medida da função retina interna, são anotados na Figura 1. Os dominados-rod ERG sinal aumenta com o aumento do estímulo de luz, como mostrado na Figura 2A. A onda de um torna-se aparente a ~ 0,4 log Scot cd.sm -2 e a amplitude de um dos aumentos de onda até à saturação a...

Watch this Scientific Journal Video about Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning at JoVE.com

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

Usando o Eletrorretinograma na Avaliação da Função na Roedor Retina e os efeitos protetores do remoto Limb Pré-Condicionamento Isquêmico

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

using-electroretinogram-to-assess-function-rodent-retina-protective

Research

JoVE Journal

Neuroscience

16.0K visualizações.  University of Sydney. The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

Vídeo: Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

Strategies for Study of Neuroprotection from Cold-preconditioning

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-&alpha; to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.

We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.

Estratégias para Estudo da neuroproteção de frio pré-condicionamento

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

Neurociência

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Preparação do tecido e imunocoloração de Fibras nervosas sensoriais que inervam rato da Pele e Ossos Limb

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Entrega intraventricular direta de medicamentos para o Sistema Nervoso Central Rodent

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

Assessment of Vascular regeneração no SNC Usando o rato Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

Eletrorretinograma Análise da Visual Response no peixe-zebra Larvas

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Imagiologia Fibras Serotoninérgicos na medula espinhal mouse usando o CLARIDADE / Técnica CUBIC

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

Em Gene Vivo Transferência de células de Schwann no nervo ciático de roedores por Eletroporação

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Efeitos de Neurotrauma induzida por explosão em roedores pressurizado artérias cerebrais de médio

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Análise Cinética 3D para a Avaliação Funcional no modelo de rat da lesão ciática de esmagamento nervoso

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

Combinando Comportamento e EEG para Estudar os Efeitos da Meditação Mindfulness na Memória Episódica

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...