Agradecemos ao Dr. Alan Tessler para comentários e ajudar a editorial. Este trabalho foi financiado pelo NIH NS062320.

1. Microscópio de configurar e preparação de imagens <ol><li> Nossa imagem criada é constituída de um estereomicroscópio Leica MZ16 fluorescente com um disparo mais rápida e uma câmera CCD refrigerado controlado por software Metamorph. </li><li> Prepare uma almofada de aquecimento controlada por termostato e ajustar a saída de 32,5 ° C para manter a temperatura corporal do animal durante e após a cirurgia. </li><li> Solução de Ringer estéril morna ou líquido cefalorraquidiano artificial (ACSF) a 32,5 ° C com antecedência para a irrigação da medula espinhal durante a cirurgia. </li><li> Anestesiar o animal com uma injeção intraperitoneal de xilazina (8 mg / kg) e ketamina (120 mg / kg) cocktail. </li><li> Raspar a parte superior das costas com cortadores de pequenos animais e espalhe uma pequena gota de depilação loção sobre a área depilada com cotonetes. Minutos depois, remova a loção aplicada usando 70% de etanol esponjas gaze embebida. </li></ol> 2. Laminectomia e exposição cirúrgica da raiz dorsal L5 <ol><li> Colocar o animal em uma aquecida (32,5 ° C) pad e desinfectar a pele com compressas embebidas etanol 70%. </li><li> Sob iluminação de campo claro no estereomicroscópio, realizar uma incisão mediana (2 - 3 cm) na pele das costas. Se necessário, use cotonetes para parar o sangramento. </li><li> Refletem a musculatura da coluna vertebral para expor as vértebras lombares subjacente. </li><li> Expor os segmentos L3-S1 da coluna do lado direito por hemi-laminectomia usando fórceps de pequeno porte. A laminectomia parcial expondo 4-6 segmentos da medula lombar e sacral da coluna vertebral é criado por eliminar a parte direita dorsal das vértebras L5 ao nível da crista ilíaca do quadril (localização da DRG L5) rostralmente para as vértebras L2 (2 vértebras caudais para a última costela). Perfundir a cavidade com solução de Ringer estéril morna do. </li><li> A posição do animal em uma almofada de apoio (gaze de algodão laminado) para achatar a coluna vertebral. Use ganchos retração para ampliar a área exposta. </li><li> Neste ponto (aproximadamente 30 minutos após a primeira injeção ip de anestésico, um suplemento (0,5 X) deve ser administrado por via subcutânea, a fim de manter o animal anestesiado totalmente. Alternativamente anestesia gás, (2-4% isoflurano em oxigênio 0.5L/min ) pode ser usado para anestesiados repetida durante as sessões de imagem mais de 1 hora. </li></ol> 3. Rizotomia / esmagar raiz dorsal <ol><li> Mudar para a excitação de fluorescência para visualizar YFP rotulados (+) axônios. </li><li> Usando a ponta de uma Sub-Q agulha (26ga.), realizar uma pequena incisão na dura-máter que recobre a raiz dorsal L5 (DR). Perfundir repetidamente com solução de Ringer e limpa suavemente com cotonetes. </li><li> Identificar o local a ser esmagada e inserir um dos lados de uma pinça fina (Dumont # 5) subdurally. </li><li> Fechar a pinça delicadamente mas com firmeza, segurando a parte medial da raiz L5 por 10 segundos, e depois gentilmente liberamos o fórceps. </li><li> Lavar várias vezes com solução fisiológica e limpa suavemente com cotonetes. </li></ol> 4. Aquisição de imagem e pós-op procedimentos <ol><li> Obter várias imagens de toda a área exposta, incluindo o local do esmagamento ea DREZ antes e logo após o esmagamento em ambos ampliação baixa e alta. </li><li> Imagens são adquiridas tanto como snapshots único ou como múltiplos fluxos de 10 a 20 quadros adquiridos dentro de 30 - para 40 ms de tempo de exposição. Em foco imagens são então selecionados, e uma montagem panorâmica é criada posteriormente utilizando Photoshop. </li><li> Para minimizar a formação de cicatriz, bem aplicar um pedaço de membrana de matriz fina sintética (Biobrane), seguida pela dura artificial, no alto da medula espinal exposta. Certifique-se de peças cortadas para caber exatamente na janela medula espinal exposta de modo que sejam aderentes à medula espinhal. </li><li> Feche a musculatura com estéril 5-0 e fechar a incisão mediana com clipes ferida. </li><li> Injetar solução de Ringer (0,3 a 0,5 ml por via subcutânea), e administrar a buprenorfina como analgesia pós-operatória (0,05 mg / kg) por via subcutânea a cada 12 h durante 2 dias. </li><li> Manter o animal em uma almofada de aquecimento (34 ° -35 ° C) até que se recuperou. </li></ol> 5. Imagem repetida <ol><li> Anestesiar o animal e remover os clipes de feridas e suturas. </li><li> Remover dura artificial e finas manchas membrana sintética matriz e mantê-los em um tubo estéril contendo solução de Ringer para reutilização posterior. </li><li> Remova com cuidado o tecido cicatricial acumulado conjuntivo com a ponta de uma agulha dobrada Sub-Q e uma pinça fina. Perfundir freqüentemente com solução de Ringer quente é. </li><li> Re-expor o campo operatório, incluindo local do esmagamento e DREZ, relocar YFP + axônios fotografada em sessões anteriores, e repetir os procedimentos descritos na seção 4. </li></ol> 6. Resultados representativos: Temos observado que, enquanto eles não regenerar após uma lesão transecção, quase todos os axônios + YFP cresceu através do site daferimento por 3 dias após esmagar (Figura 1) 11. Tipicamente, no dia seguinte após esmagar, observou-se a morrer-back degeneração de axônios do coto proximal e fragmentação / degeneração dos axônios distais à mesma paixão, que confirmaram que tinham sido devidamente axônios danificados (por exemplo, a Figura 1; Dia 3 e 5) . Vários critérios adicionais são aplicadas de forma inequívoca distinguir axônios em regeneração de axônios que foram poupados ou tinha recuperado da lesão. Estes incluíram o seguinte: (1) axônios em regeneração mostram uma expansão da porção não-fluorescente do YFP + axônio no local do esmagamento devido à degeneração proximal e distal (em contraste com a redução da diferença unlabeled devido ao citoplasma fluorescentes recarga do local do esmagamento axônios se sobreviveu ao ferimento), (2) axônios em regeneração muito mais fino, menos brilhante fluorescente, e mais ondulado do que os axônios que sobreviveram à lesão; (3) neurites regeneração são mais finas e mais fracamente fluorescentes que os fragmentos de axônios em degeneração fluorescente através da qual eles estenderam; (4) em contraste com a sobrevivência ou poupado axônios, os axônios em regeneração parar no DREZ e (5) em contraste com a sobrevivência ou poupado axônios, os axônios em regeneração não apresentam nódulos de Ranvier. A Figura 1 mostra quatro axônios YFP superficial + imediatamente após esmagar (A1; setas coloridas). Três dias após o esmagamento, todos os quatro axônios estender uma neurite única que cresce através do site paixão (A2). Cinco dias após esmagar, neurites permanecer estável e não há um crescimento adicional de estes ou outros axônios proximal (A3). O neurites regeneração que atravessou o site esmagar alongada através muito mais espessa e mais brilhante fluorescente fragmentos de um axônio em degeneração (ou seja, tubos endoneuro), e chegar ao DREZ tão cedo quanto 4 dias após o esmagamento (cerca de 3 milímetros / 2 dias) 11. Imagem repetida destes axônios e suas pontas a cada dois ou três dias por mais duas semanas (Figura 2) revelou que eles não crescer ou retrair a frente, mas permaneceu imóvel. A única mudança visível foi o inchaço das pontas e eixos de alguns axônios. Estas observações mostram pois imobilização surpreendentemente rápida e crônica da axônios em regeneração na DREZ. <img src="/files/ftp_upload/3126/3126fig1.jpg" alt="Figura 1"/> Figura 1: imagem repetida de L5 DR YFP + axônios no local do esmagamento da raiz dorsal durante 5 dias. A porção medial da raiz L5 foi esmagado (vermelho ponta de seta) e fotografada nos dias 0, 3 e 5 após o esmagamento. A área de esmagamento é ampliada nos painéis à direita (A1-A3). <img src="/files/ftp_upload/3126/3126fig2.jpg" alt="Figura 2"/> Figura 2:. Imagem repetida de axônios chegou ao DREZ mais de 20 dias após o esmagamento da raiz L5 Dia 4, três axônios (setas coloridas) chegou ao DREZ. As pontas desses axônios permanecem no mesmo local e têm uma aparência semelhante nas sessões subseqüentes imagem nos dias 7, 9, 13, 15 e 20. Posições de uma dica em relação ao axônio axônio outras dicas e marcos (asteriscos) foram usados ​​para determinar a motilidade axônio entre as sessões de imagem.

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	<li>Feng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Lichtman, J. W., Sanes, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watching+the+neuromuscular+junction.">Watching the neuromuscular junction.</a> J Neurocytol. 32, 767-775 (2003).</li><li>Bishop, D. L., Misgeld, T., Walsh, M. K., Gan, W. B., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+branch+removal+at+developing+synapses+by+axosome+shedding.">Axon branch removal at developing synapses by axosome shedding.</a> Neuron. 44, 651-661 (2004).</li><li>Balice-Gordon, R. J., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+visualization+of+the+growth+of+pre-+and+postsynaptic+elements+of+neuromuscular+junctions+in+the+mouse.">in vivo visualization of the growth of pre- and postsynaptic elements of neuromuscular junctions in the mouse.</a> J Neurosci. 10, 894-908 (1990).</li><li>Trachtenberg, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+in+vivo+imaging+of+experience-dependent+synaptic+plasticity+in+adult+cortex.">Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex.</a> Nature. 420, 788-794 (2002).</li><li>Pan, F., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+dendritic+spine+development+in+the+mouse+cortex.">Two-photon imaging of dendritic spine development in the mouse cortex.</a> Dev Neurobiol. 68, 771-778 (2008).</li><li>Grutzendler, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+synaptic+plasticity+and+pathology+in+the+living+mouse+brain.">Two-photon imaging of synaptic plasticity and pathology in the living mouse brain.</a> NeuroRx. 3, 489-496 (2006).</li><li>Kerschensteiner, M., Schwab, M. E., Lichtman, J. W., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+axonal+degeneration+and+regeneration+in+the+injured+spinal+cord.">in vivo imaging of axonal degeneration and regeneration in the injured spinal cord.</a> Nat Med. 11, 572-577 (2005).</li><li>Misgeld, T., Nikic, I., Kerschensteiner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+single+axons+in+the+mouse+spinal+cord.">in vivo imaging of single axons in the mouse spinal cord.</a> Nat Protoc. 2, 263-268 (2007).</li><li>Davalos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+in+vivo+imaging+of+densely+populated+glia,+axons+and+blood+vessels+in+the+mouse+spinal+cord+using+two-photon+microscopy.">Stable in vivo imaging of densely populated glia, axons and blood vessels in the mouse spinal cord using two-photon microscopy.</a> J Neurosci Methods. 169, 1-7 (2008).</li><li>Maio, D. D. i. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=in+vivo+imaging+of+dorsal+root+regeneration:+Rapid+immobilization+and+presynaptic+differentiation+at+the+CNS/PNS+border.">in vivo imaging of dorsal root regeneration: Rapid immobilization and presynaptic differentiation at the CNS/PNS border.</a> Journal of Neuroscience. 31, 4569-4582 (2011).</li></ol>

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DR regeneração imagem diretamente em ratos vivos é particularmente desafiador, porque exige uma laminectomia dorsal substancial para monitorar o crescimento do axônio em uma ampla área seguido por vários procedimentos cirúrgicos invasivos e anestésico em sessões de imagem subseqüentes. Várias estratégias ajudaram a superar esses desafios. De imagem, primeiro sucesso requerida a redução da mortalidade mouse (aproximadamente 25%), minimizando a duração da anestesia e sangramento, e pelo cuidado pós-operat...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários </th></tr><tr><td> H linha thy1-YFP (2-4 meses de idade, ambos os sexos) </td><td> Jackson Laboratory (Bar Harbor, ME) </td><td> 003782 </td><td></td></tr><tr><td> Xilazina (injeção AnaSed, solução estéril) </td><td> Lloyd Laboratories, (Shenandoah, LA) </td><td> 4811 </td><td> 8 mg / kg </td></tr><tr><td> Ketamina (injeção de cloridrato de ketamina, USP) </td><td> Hospira, Inc. (Lake Forest, IL) </td><td> 2051 </td><td> 120 mg / kg </td></tr><tr><td> Buprenorfina (Buprenex injetável) (0,05 mg / kg) </td><td> Reckitt Benckiser Pharmaceuticals Inc. (Richmond, VA) </td><td> 7571 </td><td></td></tr><tr><td> Cortar cabelo de pequenos animais </td><td> Oster Professional, (McMinnville, TN) </td><td> 76059-030 </td><td></td></tr><tr><td> Loção depilação </td><td> Church &amp; Dwight Co. (Princeton, NJ) </td><td> NAIR com Baby Oil </td><td></td></tr><tr><td> Esponjas de gaze </td><td> Fisher Scientific, (Pittsburgh, PA) </td><td> 22-362-173 </td><td></td></tr><tr><td> Cotonetes </td><td> Fisher Scientific, (Pittsburgh, PA) </td><td> 14-960-3T </td><td></td></tr><tr><td> Seringas de 1 mL </td><td> Becton, Dickson and Company Franklin Lakes, NJ) </td><td> 309602 </td><td></td></tr><tr><td> Subcutânea (Sub-Q) agulhas, 26ga. </td><td> Becton, Dickson and Company (Franklin Lakes, NJ) </td><td> 305115 </td><td></td></tr><tr><td> Tesouras e pinças de primavera </td><td> Multa Ferramentas Science, (Foster City, CA) </td><td></td><td></td></tr><tr><td> De 2,5 mm fórceps curvos </td><td> Multa Ferramentas Science, (Foster City, CA) </td><td> 16221-14 </td><td></td></tr><tr><td> Injeção de Ringer com lactato USP </td><td> B. Braun Medical Inc., (Irvine, CA) </td><td> BBR-L7502 </td><td></td></tr><tr><td> Solução salina estéril </td><td> APP Pharmaceuticals, (Schaumburg, IL) </td><td> 918610 </td><td></td></tr><tr><td> Membrana fina matriz sintética (Biobrane) </td><td> Bertek Pharmaceuticals, (Morgantown, WV) </td><td> 62794-096-251 </td><td></td></tr><tr><td> Artificial dura </td><td> Gore Impedir MVP Substituto Dura, WL Gore and Associates, (Flagstaff, AZ) </td><td> 1MVP40 </td><td></td></tr><tr><td> 5-0 de seda </td><td> Ethicon, Inc. (Somerville, NJ) </td><td> K-580 </td><td></td></tr><tr><td> Grampos de sutura </td><td> Perfeito - Ets Bruneau, (Burnea, França) </td><td> A75 </td><td></td></tr><tr><td> Estereomicroscópio fluorescentes </td><td> Leica Microsystems, (Wetzlar, Alemanha) </td><td> MZ16 </td><td></td></tr><tr><td> CCD da câmera </td><td> Hamamatsu, (Bridgewater, NJ) </td><td> ORCA-Rx2 </td><td></td></tr><tr><td> Controlador de temperatura </td><td> Mundial Precision Instruments (Sarasota, FL) </td><td> ATC 1000 </td><td></td></tr><tr><td> Metamorph software </td><td> Os dispositivos moleculares, (Sunnyvale, CA) </td><td></td><td></td></tr><tr><td> Photoshop </td><td> Adobe Systems, San Jose, CA </td><td></td><td></td></tr></tbody></table>

live imaging of dorsal root axons after rhizotomy

Os axônios sensoriais primárias feridos por lesões de raízes nervosas não conseguem regenerar na medula espinhal, levando à dor crônica e perda sensorial permanente. Regeneração da raiz dorsal (DR) em axônios da medula espinhal é impedido na raiz dorsal entrada de zona (DREZ), a interface entre o CNS e PNS. Nossa compreensão dos eventos moleculares e celulares que impedem a regeneração em DREZ está incompleto, em parte porque as mudanças complexas associadas a lesão do nervo ter sido deduzida a partir de análises pós-morte. Dinâmicos processos celulares, tais como a regeneração de axônios, são melhor estudadas com técnicas que capturam eventos em tempo real com múltiplas observações de cada animal vivo. Nossa capacidade de acompanhar os neurônios em série in vivo aumentou dramaticamente devido a inovações revolucionárias em óptica e transgênicos mouse. Várias linhas de camundongos transgênicos thy1-GFP, na qual subconjuntos de neurônios são geneticamente distintos rotulados em cores fluorescentes, permitem que os neurônios individuais a ser trabalhada in vivo 1. Esses camundongos têm sido amplamente utilizadas para in vivo de imagens de músculo 2-4 e 5-7 cérebro, e forneceram introspecções novas nos mecanismos fisiológicos que análises estáticas não poderia ter resolvido. Estudos de imagem de neurônios da medula espinhal em viver só recentemente começaram. Lichtman e seus colegas demonstraram a sua viabilidade através do rastreamento feridos coluna dorsal (DC) com axônios de grande campo de microscopia de 8,9. Multi-fotão in vivo de imagens de axônios profundamente posicionado DC, microglia e os vasos sanguíneos também tem sido realizados 10. Ao longo dos últimos anos, somos pioneiros na aplicação de imagem in vivo para monitorar a regeneração de axônios DR usando gama microscopia de campo e linha de H thy1-YFP camundongos. Esses estudos nos levaram a uma nova hipótese sobre por que os axônios DR são impedidos de regeneração dentro da medula espinhal 11. H em linha de thy1-YFP ratos, distinto axônios YFP + são superficialmente posicionado, o que permite que vários axônios ser monitorados simultaneamente. Aprendemos que os axônios DR chegar a DREZ são melhores visualizados na lombar do que na medula espinhal cervical. No presente relatório, nós descrevemos várias estratégias que temos encontrado útil para garantir a imagem de longo prazo e repetiu sucesso dos axônios em regeneração DR. Estes incluem métodos que eliminam intubação repetidas e interrupção respiratória, minimizar a cirurgia associada à tensão e formação de cicatrizes, e adquirir imagens estáveis ​​em alta resolução sem fototoxicidade.

Watch this Scientific Journal Video about Live Imaging of Dorsal Root Axons after Rhizotomy at JoVE.com

Live Imaging of Dorsal Root Axons after Rhizotomy

Um<em&gt; In vivo</em&gt; Imagem de protocolo para monitor principal axônios sensitivos seguintes esmagar raiz dorsal é descrito. Utilizar os procedimentos de campo amplo de microscopia de fluorescência e thy1-YFP camundongos transgênicos, e permitir que imagens repetidas de regeneração de axônios mais de 4 cm no PNS e interações com a interface do axônio do SNC.

Imagens ao vivo de axônios da raiz dorsal após rizotomia

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

live-imaging-of-dorsal-root-axons-after-rhizotomy

Research

JoVE Journal

Neuroscience

13.8K visualizações.  Shriners Hospitals Pediatric Research Center and Department of Anatomy and Cell Biology. Um In vivo Imagem de protocolo para monitor principal axônios sensitivos seguintes esmagar raiz dorsal é descrito. Utilizar os procedimentos de campo amplo de microscopia de fluorescência e thy1-YFP camundongos transgênicos, e permitir que imagens repetidas de regeneração de axônios mais de 4 cm no PNS e interações com a interface do axônio do SNC.

Vídeo: Live Imaging of Dorsal Root Axons after Rhizotomy

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

Cromatina imunoprecipitação de Tissue Dorsal Root Gânglios seguintes Lesão Axonal

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

Neurociência

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Estudo genético da regeneração do axônio com cultivadas Adulto neurônios do gânglio da raiz dorsal

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

Imagens ao vivo de nicotina Induzida Sinalização de Cálcio e Neurotransmitter lançamento Junto ventrais do hipocampo Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These neurotropic cancer cells have a unique ability to migrate unidirectionally along nerves towards the central nervous system (CNS). The dorsal root ganglia (DRG)/cancer cell model is a three dimensional (3D) in vitro model frequently used for studying the interaction between neural stroma and cancer cells. In this model, mouse or human cancer cell lines are grown in ECM adjacent to preparations of freshly dissociated cultured DRG. In this article, the DRG isolation protocol from mice, and implantation in petri dishes for co-culturing with pancreatic cancer cells are demonstrated. Five days after implantation, the cancer cells made contact with the DRG neurites. Later, these cells formed bridgeheads to facilitate more extensive polarized, neurotropic migration of cancer cells.

In Vitro Modeling of Cancerous Neural Invasion: The Dorsal Root Ganglion Model

This video article shows the use of the dorsal root ganglia (DRG)/cancer cell model in pancreatic ductal adenocarcinoma.

In Vitro Modelagem de Cancerous Neural Invasion: o gânglio Modelo de raiz dorsal

One way that solid tumors disseminate is through neural invasion. This route is well-known in cancers of the head and neck, prostate, and pancreas. These ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

Dorsal Root Injection gânglio da raiz dorsal e lesão por esmagamento como um modelo para Sensorial Axon Regeneração

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Dorsal root ganglia (DRG) primary cultures are frequently used to study physiological functions or pathology-related events in sensory neurons. Here, we demonstrate the use of lumbar DRG cultures to detect the release of neurotransmitters after neuropeptide FF receptor type 2 stimulation with a selective agonist.

Isolamento de gânglios das raízes dorsais e cultura primária para estudar a liberação de neurotransmissores

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

Microscopia de reflectométrica espectral em dendritos In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

Isolação rápida de macrófagos dorsal do gânglio da raiz

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...

In Vitro Myelination of Peripheral Axons in a Coculture of Rat Dorsal Root Ganglion Explants and Schwann Cells

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, neurons and Schwann cells engage in a complex interaction to control the myelination of axons. Disturbances of this interaction and breakdown of the myelin sheath are hallmarks of inflammatory neuropathies and occur secondarily in neurodegenerative disorders. Here, we present a coculture model of dorsal root ganglion explants and Schwann cells, which develops a robust myelination of peripheral axons to investigate the process of myelination in the peripheral nervous system, study axon-Schwann cell interactions, and evaluate the potential effects of therapeutic agents on each cell type separately. Methodologically, dorsal root ganglions of embryonic rats (E13.5) were harvested, dissociated from their surrounding tissue, and cultured as whole explants for 3 days. Schwann cells were isolated from 3-week-old adult rats, and sciatic nerves were enzymatically digested. The resulting Schwann cells were purified by magnetic-activated cell sorting and cultured under neuregulin and forskolin-enriched conditions. After 3 days of dorsal root ganglion explant culture, 30,000 Schwann cells were added to one dorsal root ganglion explant in a medium containing ascorbic acid. The first signs of myelination were detected on day 10 of coculture, through scattered signals for myelin basic protein in immunocytochemical staining. From day 14 onward, myelin sheaths were formed and propagated along the axons. Myelination can be quantified by myelin basic protein staining as a ratio of the myelination area and axon area, to account for the differences in axonal density. This model provides experimental opportunities to study various aspects of peripheral myelination in vitro, which is crucial for understanding the pathology of and possible treatment opportunities for demyelination and neurodegeneration in inflammatory and neurodegenerative diseases of the peripheral nervous system.

In the coculture system of dorsal root ganglia and Schwann cells, myelination of the peripheral nervous system can be studied. This model provides experimental opportunities to observe and quantify peripheral myelination and to study the effects of compounds of interest on the myelin sheath.

Mielinização in vitro de axônios periféricos em cocultura de explantes de gânglios dorsais de rato e células de Schwann

The process of myelination is essential to enable rapid and sufficient signal transduction in the nervous system. In the peripheral nervous system, ...

Enrichment of Adult Mouse Dorsal Root Ganglia Neuron Cultures by Immunopanning

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as well as various other cell types. Published culture protocols often refer to whole dissociated DRG cultures as being neuronal, despite the presence of fibroblasts, Schwann cells, macrophages, and lymphocytes. While these whole DRG cultures are sufficient for imaging applications where neurons can be discerned based on morphology or staining, protein or RNA homogenates collected from these cultures are not primarily neuronal in origin. Here, we describe an immunopanning sequence for cultured mouse DRGs. The goal of this method is to enrich DRG cultures for neurons by removing other cell types. Immunopanning refers to a method of removing cell types by adhering antibodies to cell culture dishes. Using these dishes, we can negatively select against and reduce the number of fibroblasts, immune cells, and Schwann cells in culture. This method allows us to increase the percentage of neurons in cultures.

This paper describes an immunopanning protocol for adult mouse dorsal root ganglia. By adhering antibodies to culture plates, we can negatively select and remove non-neuronal cells. We show that the cultures are enriched for neurons using this protocol, allowing for an in-depth study of neuronal responses to manipulation.

Enriquecimento de Culturas de Neurônios de Gânglios da Raiz Dorsal de Camundongos Adultos por Immunopanning

Dorsal root ganglia (DRGs) are peripheral structures adjacent to the dorsal horn of the spinal cord, which house the cell bodies of sensory neurons as ...