This work was supported in part by National Institutes of Health (NIH) Grants R01 GM-41514 (to M.D.C.), R39 GM-048071 (to I.P.), and R01 NS-074987 (to T.E.L.) and the National Multiple Sclerosis Society (NMSS) Collaborative Center Research Award CA1058-A-8 (to C.M.W., T.E.L. and M.D.C.), NMSS Grant RG4925, NIH Training Grant T32-AI-060573 (to M.L.G.), NMSS Postdoctoral Fellowship FG 1960-A-1 (to J.G.W.), and funding from the George E. Hewitt Foundation for Medical Research (M.P.M.).

Declaração de Ética: NOTA: O protocolo para manejo dos animais foi aprovado pelo Comitê de Cuidados e Uso do animal Institucional (IACUC) da Universidade da Califórnia, em Irvine, protocolo nº 2010-2943. 1. A remoção da Medula Espinhal <ol><li> Coloque folhas de papel umedecido com ~ 100% de isoflurano líquido, USP em câmara de eutanásia e coloque toalhas de papel seco em cima. Coloque o mouse na câmara em cima de toalhas de papel secas tão mouse não está tocando o isoflurano e certifique-se da câmara é coberto. Espere pelo menos um minuto após a cessação da respiração para garantir que o rato é sacrificado. </li><li> Realize uma transection espinhal do pescoço para garantir o rato é sacrificado. Não realize uma luxação cervical, pois pode danificar a medula espinhal. </li><li> Pulverizar o rato com etanol a 70% para molhar o cabelo. </li><li> Use tesouras finas afiadas para remover o cabelo e pele do dorso do rato para expor a coluna vertebral cervical lâmina de aproximadamente 1 (C1) Para lamina sacral 4 (S4). </li><li> Usando um bisturi com lâmina # 10, fazer incisões para a esquerda e direita da coluna vertebral, para separá-lo de músculo e gordura. Isso fará com que a remoção da medula espinhal muito mais fácil. </li><li> opcional: a curvas rongeurs Luer pode ser usado para colher longe carne adicional. </li><li> Enquanto mantém o topo da coluna vertebral com uma pinça Graefe serrilhadas, insira titânio curvadas tesoura Vannas com o lado curvo-se na coluna vertebral exposta na C1, tomando cuidado para não tocar na medula espinhal. </li><li> Deslize o titânio curvadas tesoura Vannas todo o caminho para a direita e fazer um pequeno corte. Um pequeno som crise deve ser ouvida e sentida como a tesoura cortar o vértebras. Repita este corte no lado esquerdo do cordão. Tenha cuidado para não ir demasiado depressa ou tornar muito grande de um corte como este corre o risco de danificar o cabo com a tesoura. </li><li> A única lâmina deve ser capaz de ser levantado com a pinça uma vez que os lados das vértebras são cortadas. REPEAT esta etapa para cada lâmina até S4, continuando a segurar e puxar para trás as vértebras da medula espinhal. NOTA: as vértebras acima do local de transplante provavelmente vai sair uma vez que é cortada para o site de transplante. Continuar o processo de arranque logo abaixo do local de transplante. </li><li> opcional: lançar as vértebras para baixo para ver onde o site e transplante é medida e cortar ~ 20 milímetros rostral e caudal para o site de transplante. NOTA: Quando o transplante NPCs mais não serão necessários, como os NPCs transplantados geralmente não migram mais longe do que 15 mm. A quantidade de medula espinhal necessária será dependente das características da experiência de migração e de tipo de célula transplantada. Fazendo cortes rostral e caudal, os quais são equidistantes ao local de transplante permitirá que o local de transplante a ser localizado mais facilmente durante o exame. </li><li> Usando um bisturi com uma lâmina de # 11 de corte cuidadosamente o gânglio à direita e à esquerda da face ventral da medula espinal começando na extremidade rostral. Este wdoente permitir a medula espinal a ser removido a partir das vértebras ventral mais facilmente. NOTA: Feito rapidamente (passos 1,7-1,11), a medula espinhal não vai secar. No entanto, 1x PBS pode ser administrado para o cabo para mantê-lo húmido. </li><li> Inverta o mouse e segure o mouse para que a medula espinhal é voltado para baixo. Retire cuidadosamente para fora da medula espinhal usando uma pinça Graefe serrilhadas fechadas. Sem entalhamento da medula espinal, gânglios corte cuidadosamente quaisquer restantes para permitir a medula espinal a ser levantado para fora de uma peça única intacta. </li><li> Certifique-se a medula espinhal está em RPMI-1640 e colocá-lo em gelo durante o transporte para o microscópio 2P. </li></ol> 2. Preparação de Medula Espinhal for Imaging <ol><li> Incorporação da medula espinhal em agarose <ol><li> Manter o isolado da medula espinal em arrefecida RPMI-1640 em gelo antes da imagiologia. </li><li> Pesar de agarose (baixa temperatura de gelificação) e preparar uma solução de 5% em 5 ml de PBS 1x. Microondas A solução de agarose a 5% durante 15 segundos para dissolver o aglevantou-se e deixar a solução legal para 37 ° C. </li><li> Preparar a medula espinal, colocando-o sobre uma folha de Parafilm com o lado ventral para cima. </li><li> Pipetar cerca de 5 ml de solução de agarose a 5% sobre o lado ventral da medula espinal. Deixe a agarose solidificar por arrefecimento até à temperatura ambiente. Solidificação completa demora cerca de 5 min. </li><li> Aplique uma leve camada de cola de tecido para um quadrado deslizamento de cobertura 22 mm. </li><li> Inverta a medula espinhal incorporado, colocando o lado ventral em Parafilm. O lado dorsal será agora voltado para cima. Aderem a lamínula no lado dorsal, e mergulhe a preparação deslizamento agarose incorporado medula espinhal / tampa em RMPI para solidificar o adesivo. Retire o excesso de solidificou agarose usando uma lâmina de barbear. </li></ol></li><li> Montagem da medula espinal na platina do microscópio <ol><li> Aplique vaselina para a parte inferior da tampa de deslizamento. Isto irá estabilizar a preparação durante a perfusão. </li><li> Coloque a preparação da medula espinhal em umimagiologia bem na platina do microscópio, com o lado ventral para cima para alcançar o objectivo de imersão 25X. O poço de imagem custom-built é de aproximadamente 20 mm de profundidade, a 50 mm de comprimento, e 50 mm de diâmetro. </li><li> Imagem enquanto superfusing a preparação com aquecido (37 ° C), oxigénio (95: 5 de oxigénio: carbono em dióxido de carbogénio) meio RPMI-1640 sem soro. <ol><li> Mídia Pré-aquecer a 37 ° C em banho-maria e conectá-lo à bomba tubulação por inserir o tubo dentro da garrafa de mídia. </li><li> Manter a temperatura do meio a 37 ° C, um dispositivo de aquecimento no ponto de ligação da tubagem para a câmara. Meios Superfuse através bem a 3 ml / min, utilizando tubagem ligada ao tubo da bomba (Figura 1B). </li></ol></li></ol></li></ol> 3. 2P por imagem da Medula Espinhal Ventral <ol><li> Setup Microscópio <ol><li> Adquirir imagens de Thy1-EYFP medulas espinhais usando um Chameleon Ultra laser de Ti: Safira sintonizado 900 nm. Atenuar potência do laser noespécime para &lt;5% para assegurar o mínimo de 16 de fototoxicidade. Manter a temperatura irrigando perfundido com oxigénio meio RPMI-1640 a uma temperatura constante de 37 ° C utilizando um aquecedor de solução única linha de imagem para garantir estável, e para impedir a dispersão do tecido e danos celulares. </li><li> Para separar o sinal fluorescente eGFP e EYFP, coloque um 520 nm dicróica de ponta única e um nm-edge único feixe dicróica divisor 560 em série para separar 2P de emissão em três canais, propositadamente dividir a emissão verde para melhorar a visibilidade. NOTA: Estes canais são referidos como azul-verde (emissão &lt;520 nm), verde-amarelo (520-560 nm) e vermelho (emissão&gt; 560 nm). Tubos fotomultiplicadores detectar a luz emitida em cada canal. </li></ol></li><li> Localizando áreas de imagem de interesse <ol><li> Usando a ocular e uma fonte de luz de campo claro, concentrar o objectivo de imersão no bordo ventral da medula espinal para definir um ponto de referência. </li><li> Faça fonte de luz ambiente certeza está fora e swcoceira para 2P excitação abrindo o obturador laser. </li><li> Se estiver disponível, na busca de tecido para áreas de interesse, use uma configuração de resolução mais baixa, maior volume sem zoom digital, e maior velocidade de varredura do que na aquisição de imagens finais. As configurações típicas com este sistema na busca de tecido para uma região de interesse são: Resolução: 256 pixels; volume de: x = 600 um, y = 600 um, z = 0-300 um. </li><li> Observar os axónios EYFP perto da borda ventral da medula espinal. Segundo sinal harmónico de colágeno (azul) será mais brilhante na borda do tecido da medula espinhal. Localize os axônios EYFP apenas dorsal para o segundo sinal harmônico do colágeno e sinal EYFP é visualizado no canal verde-amarelo. </li><li> Localizar o local de transplante no centro longitudinal da preparação da medula espinal 14. Para camundongos um dia após o transplante eGFP-NPC, localize o site de transplante, concentrando-se a caminho do feixe no plano z profundamente no tecido. O wi site de transplantell variam ligeiramente entre os animais, mas geralmente está localizado ~ 200 mm a partir da borda ventral. Observe os aglomerados de eGFP-NPCs nos tratos de substância branca, mais perto das bordas lateral e ventral em ratos após dia 1 pós-transplante. </li></ol></li><li> Obtenção de imagens finais <ol><li> Adquirir resolução de imagem de 512 pixels; volume de: x = 270 mm, y = 212 mm, e z = 100 um usando software Slidebook 6. Compilar z-stacks através da aquisição de planos focais sequenciais em incrementos de 2,5 mM. </li><li> Executar uma verificação bidirecional com deslocamento para garantir quantificação rápida de quaisquer objetos que migram da medula espinhal interlace adequada. Certifique-se de que a taxa ideal quadro aquisição para determinar a velocidade do celular dentro da medula espinhal é de aproximadamente 1 frame / sec. NOTA: As configurações de imagem pode ser alterado para as preferências do usuário individuais, tais como taxa de quadros de aquisição, resolução de imagem e volumes de imagem. Volumes de imagem maiores geralmente levam mais tempo para adquirir a um GIVresolução en. </li><li> Analisar, de culturas, liso, e pseudocoloridas arquivos de imagem Slidebook com software de análise de imagem, tais como Bitplane Imaris 7.7. Produza vídeos finais de lapso de tempo penteando volumes de imagens consecutivas usando um processo automatizado em Slidebook e Imaris. </li></ol></li></ol>

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The authors have no competing interests.

Real-time 2P imagiologia de tecido intacto é necessária para investigar a cinética da APN e interacções após o transplante para o rato demyelinated medula espinhal. Intravital 2P imagem é utilizada para determinar a dinâmica celular na face dorsal da medula espinal em ratos vivos, e tem sido utilizada para estudar a desmielinização dorsal em doenças desmielinizantes 17-19. No entanto, porque NPCs transplantadas migrar para a substância branca ventral, o qual se encontra muito profundo para imagem ...

Modelos de ratos de desmielinização, incluindo a encefalomielite autoimune experimental (EAE) e infecção intracraniana com neuroadapted vírus da hepatite do camundongo (MHV), são excelentes ferramentas para estudar vias moleculares e interações celulares associados à doença. Eles têm conduzido a e suportado a eficácia de terapias farmacêuticas aprovado pela FDA, principalmente contra a cessação de autoimunidade e inflamação 1. No entanto, uma vez que a remielinização endógeno falhou, as terapias actualmente aprovadas não eficaz para reparar lesões de desmielinização do sistema nervoso central. Portanto, terapias focadas no reparo nesta fase da doença são fundamentais para o alívio de sintomas crônicos e melhoria da qualidade de vida. Recentemente, as células precursoras neurais (NPCs) vieram à tona como um potencial modalidade terapêutica regenerativa para atingir áreas de inflamação e desmielinização. Vários estudos têm destacado a capacidade de NPCs para induzir endógenonos remielinização e participar diretamente remielinização 2-8. Porque NPCs estão envolvidos em remielinização direta, é imperativo compreender a sua cinética e interações com células endógenas após o transplante. Após o transplante, NPCs migrar ventralmente para áreas de lesões da substância branca, em seguida, rostral caudal e relativas ao local de transplante 5,9. A cinética da migração diferem em resposta aos estímulos ambientais; NPCs transplantadas em uma medula espinhal não danificado têm velocidades maiores que as NPCs transplantadas em uma medula espinhal danificada 6. Depois de um período migratório, transferido NPCs proliferam extensivamente, a uma taxa mais elevada num medular danificado em relação a uma medula espinhal intacta 6. Finalmente, a maioria dos NPCs diferenciar em oligodendrócitos e iniciar 4,6,9 remielinização directa. A lesão desmielinização é complexo e pode incluir uma população diversa de células em vários estágios de umctivation. Por exemplo, uma lesão activa esclerose múltipla (EM) podem incluir uma população significativa de células T activadas, a microglia e macrófagos M1 M1, mas uma lesão crónica MS silenciosa pode ser constituída essencialmente por astrócitos reactivos com poucas células inflamatórias 10-13. Devido à diversidade de células efectoras, de dois fotões (2P) de imagem em modelos de rato de desmielinização é uma ferramenta extremamente útil para ajudar a compreender as interacções celulares locais no interior da lesão. Em muitos modelos de MS e MS de investigação amplamente utilizadas, a maioria das lesões está localizada no lado ventral da medula espinhal, uma região inacessível para imagens intravital 2P devido à profundidade da lesão e o elevado teor de lípidos da medula espinhal. Para contornar esses problemas e as interações célula-célula estudo dentro de lesões ao longo da medula espinhal ventral, temos desenvolvido um simples ex vivo 2P preparação imaging 6. Este estudo segue-se uma publicação métodos anterior, que mostrouo procedimento para o transplante reforçada proteína verde fluorescente (eGFP) NPCs -expressing para a medula espinhal de ratos após JHMV estirpe de desmielinização induzida por MHV 14. Cinco semanas ratos velhos são infectadas com JHMV e transplantados com eGFP-NPCs ao nível torácico 9 no dia 14 pós-infecção. O protocolo apresentado aqui fornece etapas detalhadas sobre como extrair a medula espinhal, fazer uma preparação ex vivo agarose, e imagem transplantado interações eGFP-NPC com melhor proteína fluorescente amarela (EYFP) axônios -expressing. Ratos que expressam EYFP sob a Thy1 promotor específico neuronal foram usados ​​neste procedimento de 15. Apenas alguns dos axônios expressar EYFP, tornando-o útil para a imagem latente axônios individuais. Aqui nós mostramos medulas espinhais removidas em 7 dias pós-transplante; no entanto, a espinal medula pode ser extraída em qualquer momento após o transplante. Enquanto que mostram as interacções de NPCs com axónios danificados, o nosso protocolo pode ser utilizado em combinação com marcadores fluorescentes genéticosde outros tipos de células para investigar uma variedade de interacções celulares que ocorrem durante todo o rato medula espinhal.

<table><tbody><tr><td>Isoflurane, USP</td><td>Piramal Critical Care, Inc</td><td>N/A</td><td></td></tr><tr><td>Fine scissors</td><td>Fine Science Tools</td><td>14060-09</td><td>sharp</td></tr><tr><td>scalpel blade #10</td><td>Fine Science Tools</td><td>10010-00</td><td></td></tr><tr><td>scalpel handle</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Luer rongeurs</td><td>Fine Science Tools</td><td>16001-15</td><td></td></tr><tr><td>Graefe forceps</td><td>Fine Science Tools</td><td>11052-10</td><td></td></tr><tr><td>Vannas scissors</td><td>Fine Science Tools</td><td>15615-08</td><td></td></tr><tr><td>scalpel blade #11</td><td>Fine Science Tools</td><td>10011-00</td><td></td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>12-115F</td><td></td></tr><tr><td>agarose, low gelling temperature</td><td>Sigma</td><td>A9414-25G</td><td></td></tr><tr><td>Parafilm</td><td>Fisher Scientific</td><td>13-374-12</td><td></td></tr><tr><td>Vetbond (tissue adhesive)</td><td>3M</td><td>1469SB</td><td></td></tr><tr><td>22 mm square cover slip</td><td>Fisher Scientific</td><td>12-547</td><td></td></tr><tr><td>25X dipping objective, 1.1 NA</td><td>Nikon</td><td>CFI Apo LWD 25XW</td><td></td></tr><tr><td>Single inline solution heater</td><td>Warner Instruments</td><td>64-0102</td><td></td></tr><tr><td>520 nm single-edge dichroic beam splitter</td><td>Semrock</td><td>FF520-Di02-25x36</td><td>Brightline</td></tr><tr><td>560 nm single-edge dichroic beam splitter</td><td>Semrock</td><td>FF560-FDi01-25x36</td><td>Brightline</td></tr><tr><td>photomultiplier tubes</td><td>Hamamatsu</td><td>R928</td><td></td></tr><tr><td>C/L variable-speed tubing pump</td><td>Masterflex</td><td>77122-22</td><td></td></tr><tr><td>digital thermometer</td><td>Comar Instruments</td><td>3501</td><td></td></tr><tr><td>Chameleon Ultra Ti:Sapphire laser&amp;nbsp;</td><td>Coherent</td><td>N/A</td><td></td></tr><tr><td>Slidebook 6 software</td><td>3i</td><td>N/A</td><td></td></tr><tr><td>Imaris 7.7 software</td><td>Bitplane</td><td>N/A</td><td></td></tr></tbody></table>

two-photon imaging of cellular dynamics in the mouse spinal cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

<html> Embora o protocolo explantado imagiologia medula espinal pode ser utilizado para visualizar qualquer fluorescência na medula espinhal, os nossos resultados demonstram representativos interacções eGFP-NPC com EYFP-axónios. Em primeiro lugar, vamos mostrar a preparação cabo embutido ventral espinhal na Figura 1A. A seguir, mostram a configuração de 2P microscópio e componentes principais na Figura 1B. A Figura 2 demonstra eGFP e EYFP fluorescência em um único z-pilha no i...

Watch this Scientific Journal Video about Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord at JoVE.com

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Uma nova ex vivo preparação para a imagem latente o mouse medula espinhal. Este protocolo permite a dois fotões imagiologia de interacções celulares vivos ao longo da medula espinal.

Dois fótons de imagem da Cellular Dynamics na Medula Espinhal Rato

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

two-photon-imaging-of-cellular-dynamics-in-the-mouse-spinal-cord

Research

JoVE Journal

Neuroscience

10.3K visualizações.  University of California, Irvine.  Uma nova ex vivo preparação para a imagem latente o mouse medula espinhal. Este protocolo permite a dois fotões imagiologia de interacções celulares vivos ao longo da medula espinal. 

Vídeo: Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

Em Imaging vivo do Cordão do rato Spinal Usando Two-photon Microscopia

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

Neurociência

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

Two-Photon<em&gt; In vivo</em&gt; Imagem de espinhas dendríticas no Rato Cortex Usando um desbaste-crânio Preparação

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by repeated, time-lapse in vivo imaging in live rodents. These studies were made possible by straightforward surgical procedures, which enabled optical access for a prolonged period of time without repeat surgical procedures. In contrast, analogous studies of the spinal cord have been previously limited to only a few imaging sessions, each of which required an invasive surgery. As previously described, we have developed a spinal chamber that enables continuous optical access for upwards of 8 weeks, preserves mechanical stability of the spinal column, is easily stabilized externally during imaging, and requires only a single surgery. Here, the design of the spinal chamber with its associated surgical implements is reviewed and the surgical procedure is demonstrated in detail. Briefly, this video will demonstrate the preparation of the surgical area and mouse for surgery, exposure of the spinal vertebra and appropriate tissue debridement, the delivery of the implant and vertebral clamping, the completion of the chamber, the removal of the delivery system, sealing of the skin, and finally, post-operative care. The procedure for chronic in vivo imaging using nonlinear microscopy will also be demonstrated. Finally, outcomes, limitations, typical variability, and a guide for troubleshooting are discussed.

A Procedure for Implanting a Spinal Chamber for Longitudinal In Vivo Imaging of the Mouse Spinal Cord

In this video, we describe a procedure for implanting a chronic optical imaging chamber over the dorsal spinal cord of a live mouse. The chamber, surgical procedure, and chronic imaging are reviewed and demonstrated.

Um Processo de Implantação de uma Câmara Spinal para Longitudinal<em&gt; In Vivo</em&gt; Criação de Imagens do Cordão do rato Spinal

Studies in the mammalian neocortex have enabled unprecedented resolution of cortical structure, activity, and response to neurodegenerative insults by ...

Intravital Imaging of Axonal Interactions with Microglia and Macrophages in a Mouse Dorsal Column Crush Injury

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. Intra-vital two-photon microscopy enables the study of macrophages and microglia in the spinal cord lesion in the living animal. This can be performed in adult animals with a traumatic injury to the dorsal column. Here, we describe methods for distinguishing macrophages from microglia in the CNS using an irradiation bone marrow chimera to obtain animals in which only macrophages or microglia are labeled with a genetically encoded green fluorescent protein. We also describe a injury model that crushes the dorsal column of the spinal cord, thereby producing a simple, easily accessible, rectangular lesion that is easily visualized in an animal through a laminectomy. Furthermore, we will outline procedures to sequentially image the animals at the anatomical site of injury for the study of cellular interactions during the first few days to weeks after injury.

Two-photon intravital imaging can be used to investigate interactions among different cell types in the spinal cord in their native tissue environment in a bone marrow chimeric animal with a dorsal column traumatic spinal cord crush injury.

Imagem Intravital de axonais Interações com Microglia e macrófagos em um mouse Dorsal Coluna lesão por esmagamento

Traumatic spinal cord injury causes an inflammatory reaction involving blood-derived macrophages and central nervous system (CNS)-resident microglia. ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Simultânea de dois fótons<em&gt; In Vivo</em&gt; Imagem de Synaptic Entradas e metas pós-sinápticos no córtex do rato retrosplï¿½ico

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues throughout the acute injury phase. In fact, endothelial cells appear to be the first to die after a contusive SCI. The early vascular events, including increased permeability of the blood-spinal cord barrier (BSCB), induce vasogenic edema and contribute to detrimental secondary injury events caused by complex injury mechanisms. Targeting the vascular disruption, therefore, could be a key strategy to reduce secondary injury cascades that contribute to histological and functional impairments after SCI. Previous studies were mostly performed on postmortem samples and were unable to capture the dynamic changes of the vascular network. In this study, we have developed an in vivo duo-color two-photon imaging method to monitor acute vascular dynamic changes following contusive SCI. This approach allows detecting blood flow, vessel diameter, and other vascular pathologies at various sites of the same rat pre- and post-injury. Overall, this method provides an excellent venue for investigating vascular dynamics.

An In Vivo Duo-color Method for Imaging Vascular Dynamics Following Contusive Spinal Cord Injury

We introduce an in vivo imaging method using two different fluorescent dyes to track dynamic spinal vascular changes following a contusive spinal cord injury in adult Sprague-Dawley rats.

Um Vivo em Método de dupla-cor de imagem dinâmica Vascular após lesão da medula espinhal Contusive

Spinal cord injury (SCI) causes significant vascular disruption at the site of injury. Vascular pathology occurs immediately after SCI and continues ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

Implante de janela de laminectomia e medula espinhal no mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

In vivo Imagem de tecidos biológicos com fluorescência combinada de dois fótons e microscopia de dispersão de Raman estimulada

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Bioengenharia

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Imagem Simultânea da Dinâmica Microglial e Atividade Neuronal em Camundongos Acordados

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in an Awake Mouse

Dois fótons de imagem da Cellular Dynamics na Medula Espinhal Rato

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