Gostaríamos de agradecer a Andrea Tedeschi para ajudar na definição das experiências ChIP inicial em laboratório e Lindner Ricco por sua contribuição para afinar as condições para ChIP. Este trabalho foi financiado pela Fundação Hertie, o Grant Fortune, Universidade de Tuebingen, ea DFG concede DI 1497/1-1 (todos concedida a Simone Di Giovanni).

1. Lesão do nervo ciático e coluna dorsal <ol><li> O animal é colocado sobre uma toalha cirúrgica e debaixo dela um thermopad está presente em todo o processo de manter a temperatura corporal do rato a 37 ° C. Todos os animais são anestesiados para a cirurgia com uma contínua isoflurano / O 2 administração. Os instrumentos cirúrgicos são autoclavados antes do procedimento. </li><li> De lesão ciática, ambos posteriores são cuidadosamente raspada, e depilação completa-se com removedor de pêlos genéricas antes limpeza da pele com álcool seguido por Betadyne 3 vezes. </li><li> O nervo ciático é exposto por uma incisão 4 milímetros posterior e paralela ao fêmur no meio da coxa através da pele e espalhando os músculos além do bíceps femoral com uma pinça fina. </li><li> O nervo ciático é exposto em seguida, ferido, ea pele é fechada com dois clipes de sutura. </li></ol> NOTA: A mesma incisão é feita para expor o nervo ciático, mas a ferida é fechada com sutura deixando o nervo intacto para a lesão farsa. <ol start="5"><li> De lesão da coluna dorsal, a parte traseira do mouse é cuidadosamente raspado, e depilação completa-se com removedor de pêlos genéricas antes limpeza da pele com álcool spray / furto. </li><li> A medula espinhal é exposto por uma incisão de 2,5 cm de cerca de T7 a T13. A pele é afastados, e do tecido conjuntivo é removida ao longo da vértebra de T9 a T11. </li><li> A laminectomia é realizada no nível T10 </li></ol> NOTA: Para a maioria dos experimentos, a laminectomia é considerada a lesão Sham. <ol start="8"><li> Algumas gotas de Xylocain são adicionadas ao tecido para anestesiar o cabo, e dura-máter é removido, prestando atenção para não tocar na medula espinhal. </li><li> As colunas dorsal são feridos eo músculo é suturada perto, mais uma vez prestando atenção para não tocar na medula espinhal. Finalmente, a pele é fechada com sutura com grampos de sutura. Buprenorfina (0,1 peso corporal mg / kg) deve ser administrado duas vezes ao dia por 48 horas ou até que deixem de ser necessários. </li></ol> 2. Cross-linking <ol><li> Dissecar DRG L4 e L5 de ratos feridos após a eutanásia. Coletar um total de 16 DRG em gelada HBSS + inibidores da protease do cocktail (2 feridos e 2 farsa DRG de cada animal, para um total de 4 animais). </li><li> Brevemente centrifugar o DRG, em seguida, remova cuidadosamente o HBSS e adicionar 500 mL de formaldeído a 1% em PBS + cocktail inibidores da protease e incubar a amostra durante 30 minutos a 37 ° C. </li></ol> Etapa crítica. Use fresca, de formaldeído de grau de biologia molecular. <ol start="3"><li> Adicionar 125 mM de glicina para parar a fixação e incubar por 5 minutos em temperatura ambiente. </li><li> Brevemente centrífuga, aspirado off buffer, e lavar o tecido duas vezes com 500 gelada mL PBS + protease cocktail inibidores. </li></ol> 3. Preparação núcleos e cisalhamento cromatina <ol><li> Aspirar off PBS, adicionar 400 mL de tampão de lise SDS, transferir para um tubo de microcentrífuga pré-refrigeradas, e interromper o tecido com aproximadamente 30 golpes com a micropistilo. </li></ol> Etapa crítica. Lise e destruição do tecido são vitais para um bom rendimento da cromatina reticulada. <ol start="2"><li> Sonicate a amostra com 8 pulsos, 10 segundo cada um com saída de 70% (Bandelin, Sonoplus GM70). </li></ol> CUIDADO. O corte da cromatina deve ser otimizado para o seu sonicação especial configurar e fragmentação da cromatina adequada deve ser verificado antes de executar o experimento IP real (ver secção 3). NOTA fragmentação. Da cromatina também pode ser realizada por micrococcal nuclease digestão (MNase). Como regra geral, a fragmentação por sonicação é o preferido para tecido fixado, enquanto que a digestão é favorecida MNase para imunoprecipitação nativa tecido cromatina. Cruzada, cortado cromatina pode ser armazenado a -80 ° C por até 2 meses, mas evitar repetidos de congelamento / descongelamento. 4. Análise da digestão da cromatina (recomendado) <ol><li> Retire 10 ml da sua amostra cromatina cortado para análise. Adicionar 200 mM de NaCl para a amostra e reverter o cross-link, incubando a 65 ° C por 2h. </li><li> Purificar DNA (ver secção 8) e executar um gel 1% agarose. DNA deve ser fragmentado para um comprimento de cerca de 200-1000 pb (Figura 1). </li></ol> CUIDADO. Excessiva fragmentação da cromatina pode levar à diminuição do sinal, enquanto que a fragmentação incompleta levará à resolução diminuída e aumento de fundo. 5. Imunoprecipitação <ol><li> Determinar o número de reações de imunoprecipitação (ver nota abaixo), e dividir amostras igualmente em novos tubos. Reduzir o volume de cadaaté 500 mL com tampão ChIP + cocktail inibidores da protease. </li><li> Retire 5 mL da sua amostra diluída e transferência para um novo tubo. Esta é a sua amostra de entrada de 1% e vai ser armazenadas a -20 ° C até ser necessário (Seção 7). </li><li> Para cada imunoprecipitação, adicione anticorpos IgG controle apropriado ou normal e incubar a 4 ° C durante a noite com rotação. </li></ol> NOTA. Ao determinar o número de imunoprecipitação, um controlo positivo (por exemplo, anticorpos Histone H3) e um controle negativo (IgG Normal) deve ser considerada. A quantidade de anticorpo utilizado para cada IP varia entre anticorpos e deve ser determinado empiricamente. No entanto, é geralmente dentro de uma escala de 2-10? G / imunoprecipitação. <ol start="4"><li> Para immunoprecipitate seu complexo anticorpo / proteína / DNA, adicionar 30 mL de chip de Contas da proteína G Grade Magnética e incubar por 2 horas a 4 ° C com rotação. </li></ol> 6. Lavagem <ol><li> Para suspenso ligado complexo cromatina talão, colocar o tubo na cremalheira magnética. Aguarde até que a solução é clara e, em seguida, remova cuidadosamente o seu sobrenadante. </li><li> Adicionar 1 ml de lavar baixo teor de sal (buffer ChIP) para as contas e incubar a 4 ° C por 3-5 minutos, com rotação, em seguida, puxe para baixo e aspire contas off lavar como no passo 6.1. Repita esta etapa para um total de 3 lavagens. </li><li> Adicionar 1 ml de tampão de lavagem de alta sal (buffer ChIP + 350 mM NaCl) para as contas e incubar por 3-5 minutos, com rotação, em seguida, puxe para baixo e aspire contas off lavar como no passo 6.1. </li></ol> 7. Eluição e reversão de cross-linking <ol><li> Colher amostras a sua entrada fora do -20 ° C, adicionar 150 mL de tampão de eluição ChIP e reserve em temperatura ambiente até Passo 7.5. </li><li> Adicionar 150 mL de tampão 1x ChIP eluição de cada amostra IP a partir do Passo 6.3. </li><li> Colocar os tubos em um Thermomixer e eluir a cromatina dos grânulos, incubando amostras a 65 ° C por 30 minutos com vortex gentil. </li><li> Suspenso contas no rack magnético, e transferir cuidadosamente a cromatina eluída (sobrenadante) em tubos de novo. </li><li> Em todos os tubos, incluindo amostras de sua entrada, adicione 200mM de NaCl e 40 g / reação de Proteinase K e incubar a 65 ° C por 2 horas. </li></ol> NOTA. A etapa de eluição pode ser realizado à temperatura ambiente, também, mas pode não ser tão eficiente. 8. Recuperação de DNA <ol><li> Adicionar 1 volume de clorofórmio Fenol / com suas amostras a partir do Passo 7.5 e vortex por 30 segundos. </li><li> Centrifugar a velocidade máxima em microcentrífuga a temperatura ambiente por 5 minutos. </li><li> Cuidadosamente a fase de recuperação (superior) aquosa para novos tubos, adicionar 1 volume de clorofórmio para a fase aquosa e vórtice por 30 segundos. </li><li> Centrifugar a velocidade máxima em microcentrífuga a temperatura ambiente por 5 minutos. </li><li> Cuidadosamente a fase de recuperação (superior) aquosa para novos tubos, em seguida, adicione 300mM de NaOAc, 20 mg / reação de glicogênio, e 2,5 volumes de etanol gelado de 100%. </li></ol> CUIDADO. A adição de uma transportadora, como o glicogênio, é necessária para aumentar a precipitação de relativamente pequenos fragmentos de DNA de tamanho. <ol start="6"><li> Incubar a -80 ° C por 2 horas para DNA precipitado. </li></ol> NOTA. A etapa de precipitação também pode ser realizada durante a noite a -20 ° C. <ol start="7"><li> Centrifugar as amostras a velocidade máxima em microcentrífuga por 20 minutos a 4 ° C. </li><li> Remova cuidadosamente o sobrenadante e lavar os pellets de DNA precipitado com 300 l de etanol gelado 70%. </li><li> Centrifugar as amostras a velocidade máxima em microcentrífuga por 10 minutos a 4 ° C. </li><li> Cuidadosamente remover o máximo possível sobrenadante e deixar o pellets de ar seco. </li><li> Ressuspender em 20 l de H 2 O. estéril Amostras estão agora prontos para PCR, ou o DNA pode ser armazenado a -20 ° C. </li></ol> 9. Resultados representante Um resultado representativo de uma experiência de chip em DRGs após lesão ciática é mostrado (Figuras 1 e 2). Primeiro, vamos mostrar DNA fragmentado para um comprimento de cerca de 200-1000 pb (Figura 1). Segundo, nós demonstramos um Chip sinal PCR seguintes do promotor proximal do crescimento associado a proteína-43 (GAP-43) somente após lesão do nervo ciático (faixa 4, Figura 2, 5 &#39;). Não há sinal de PCR está presente quando o animal recebe uma lesão farsa só (pista 3), e quando se usa soro normal de IgG para o IP (faixa 5 e 6). Para testar a especificidade do anticorpo, foram analisadas as amostras de DNA mesmo, mas usou um conjunto de primers de controle que detecta uma região no 3&#39;-UTR do nosso gene de interesse onde não há ocupação é esperado. Sinais de PCR estão ausentes de todas as pistas (pistas 3-6, Figura 2, 3 &#39;), exceto para o sinal de entrada, representando amostras de DNA não-IP como padrão PCR controles (faixa 1-2, FIGUre 2). Este procedimento chip pode ser realizada tanto após a lesão de coluna vertebral dorsal ciático ou conforme resumido em um esquema (Figura 3). <img src="/files/ftp_upload/2803/2803fig1.jpg" alt="Figura 1" /> Figura 1. Gel Agarose de DNA invertido cruzado que foi cortado por sonicação para a faixa adequada de comprimentos de fragmentos. <img src="/files/ftp_upload/2803/2803fig2.jpg" alt="Figura 2" /> Figura 2. Resultados Semi-PCR quantitativo do tecido DRG seguintes lesão do nervo ciático mostrando que p53 acetilada se liga ao GAP-43 região promotora proximal 48 horas após a lesão do nervo ciático. Controle de PCR a partir do mesmo show DRG tecido que p53 acetilada não se liga a uma região controle do DNA localizado na região 3&#39;-não traduzida (UTR) do gene GAP-43 (S = Sham, I = lesionados). <img src="/files/ftp_upload/2803/2803fig3.jpg" alt="Figura 3" /> Figura 3. Um diagrama geral mostrando a localização dos locais de lesão no nervo ciático ea coluna dorsal.

<ol>
	<li>Beirowski, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+qualitative+analysis+of+Wallerian+degeneration+using+restricted+axonal+labelling+in+YFP-H+mice.">Quantitative and qualitative analysis of Wallerian degeneration using restricted axonal labelling in YFP-H mice.</a> Journal of Neuroscience Methods. 134, 23-35 (2004).</li><li>Carey, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Chromatin immunoprecipitation (ChIP). , (2009).</li><li>Dahl, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+micro+chromatin+immunoprecipitation+assay+(&amp;mu;ChIP.">A rapid micro chromatin immunoprecipitation assay (&amp;mu;ChIP.</a> Nat. Protoc. 3, 1032-1045 (2008).</li><li>Haring, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation:+optimization,+quantitative+analysis+and+data+normalization.+Plant+Methods.">Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods.</a> 11, 3-11 (2007).</li><li>Jiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neuronal+chromatin+from+brain+tissue.">Isolation of neuronal chromatin from brain tissue.</a> BMC Neurosci. 9, 42-42 (2008).</li><li>Lee, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neural+stem+cells+from+the+postnatal+cerebellum.">Isolation of neural stem cells from the postnatal cerebellum.</a> Nat Neurosci. 8, 723-729 (2005).</li><li>Makwana, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+in+successful+peripheral+regeneration.">Molecular mechanisms in successful peripheral regeneration.</a> Febs J. 272, 2628-2638 (2005).</li><li>Tedeschi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+p53-CBP/p300+transcription+module+is+required+for+GAP-43+expression,+axon+outgrowth,+and+regeneration.">A p53-CBP/p300 transcription module is required for GAP-43 expression, axon outgrowth, and regeneration.</a> Cell Death and Differentiation. 16, 543-554 (2009).</li><li>Teng, F. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+regeneration+in+adult+CNS+neurons--signaling+molecules+and+pathways.">Axonal regeneration in adult CNS neurons--signaling molecules and pathways.</a> J Neurochem. 96, 1501-1508 (2006).</li></ol>

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Este protocolo fornece um método para solicitar directamente sobre o meio ambiente cromatina durante a regeneração axonal no sistema nervoso adulto após lesão axonal. Ele incorpora o modelo de lesão DRG com imunoprecipitação da cromatina para sondar o ambiente de transcrição e epigenéticos após a lesão ou o PNS ou CNS. É particularmente útil para pesquisadores que gostariam de caracterizar suposta sítios de ligação para o seu fator de transcrição favorito, e para determinar se a ocupação destes loc...

<table border="1"><tbody><tr><th> Reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> Tampão ChIP 10x </td><td> Sinalização celular </td><td> 7008 </td></tr><tr><td> Tampão eluição 2x ChIP </td><td> Sinalização celular </td><td> 7009 </td></tr><tr><td> ChIP Beads proteína G Grade Magnética </td><td> Sinalização celular </td><td> 9006 </td></tr><tr><td> Rack Punho Magna (8 também) </td><td> Millipore </td><td> 2-40 </td></tr><tr><td> Clorofórmio </td><td> MERCK </td><td> ONU 1888 </td></tr><tr><td> O formaldeído 37% </td><td> ROTH </td><td> CP10.1 </td></tr><tr><td> Solução Glycine 10x </td><td> Sinalização celular </td><td> 7005 </td></tr><tr><td> Glicogênio </td><td> Sigma </td><td> G1767 </td></tr><tr><td> HBSS 10x </td><td> Gibco </td><td> 14185 </td></tr><tr><td> Anticorpo histona H3 (coelho) </td><td> Sinalização celular </td><td> 2650 </td></tr><tr><td> Normais coelho IgG </td><td> Sinalização celular </td><td> 2729 </td></tr><tr><td> Fenol / clorofórmio / álcool isoamílico </td><td> ROTH </td><td> A156.1 </td></tr><tr><td> Cocktail de protease Comprimidos Inibidores </td><td> Roche </td><td> 04 693 116 001 </td></tr><tr><td> Proteinase K (20 mg / ml) </td><td> Sinalização celular </td><td> 10012 </td></tr><tr><td> SDS tampão de lise </td><td> Norte do estado </td><td> 20-163 </td></tr></tbody></table><table border="1"><tbody><tr><th> Equipamentos necessários </th></tr><tr><td> Sonicador </td></tr><tr><td> Micropistilo </td></tr><tr><td> Microcentrífuga </td></tr><tr><td> Thermomixer </td></tr></tbody></table>

chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury

Axônios no sistema nervoso central (SNC) não se regeneram, enquanto as do sistema nervoso periférico (SNP) se regeneram de forma limitada após a lesão (Teng et al., 2006). Reconhece-se que os programas de transcrição essencial para crescimento de neuritos e axonal são reativados após lesão no PNS (Makwana et al., 2005). No entanto as ferramentas disponíveis para analisar regulação gênica neuronal in vivo são limitados e muitas vezes desafiador. Os gânglios da raiz dorsal (DRG) oferecem um sistema excelente modelo prejuízo, porque tanto o CNS e PNS são inervados por um axônio bifurcada proveniente da soma mesma. Os gânglios representar uma coleção discreta de corpos celulares, onde todos os eventos ocorrem de transcrição, e assim fornecer uma região claramente definida de atividade transcricional que pode ser facilmente reproduzível e retirado do animal. Lesão de fibras nervosas no PNS (por exemplo, nervo ciático), onde a regeneração axonal ocorre, deve revelar um conjunto de programas de transcrição que são distintas das respondendo a uma lesão similar no sistema nervoso central, onde a regeneração não ocorre (por exemplo, na medula espinhal ). Sites para o fator de transcrição de ligação, histona e modificação de DNA resultante da lesão a qualquer PNS ou SNC podem ser caracterizadas usando cromatina imunoprecipitação (chip). Aqui, nós descrevemos um protocolo chip usando tecido DRG fixo rato após lesão axonal. Esta poderosa combinação oferece um meio para caracterizar o ambiente cromatina pró-regeneração necessária para promover a regeneração axonal.

Watch this Scientific Journal Video about Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury at JoVE.com

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Nós apresentamos um método para imunoprecipitação da cromatina do tecido da raiz dorsal após lesão axonal gânglios. A abordagem pode ser usada para identificar locais de transcrição específicos fator determinante e importante modificação epigenética de histonas e DNA para a regeneração de axônios lesados ​​tanto no sistema nervoso periférico e central.

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 ...

chromatin-immunoprecipitation-from-dorsal-root-ganglia-tissue

Research

JoVE Journal

Neuroscience

16.0K visualizações.  University of Tuebingen. Nós apresentamos um método para imunoprecipitação da cromatina do tecido da raiz dorsal após lesão axonal gânglios. A abordagem pode ser usada para identificar locais de transcrição específicos fator determinante e importante modificação epigenética de histonas e DNA para a regeneração de axônios lesados ​​tanto no sistema nervoso periférico e central.

Vídeo: Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

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. ...

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 ...

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. ...

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) are structures containing the sensory neurons of the peripheral nervous system. When dissociated, they can be co-cultured with SC-like adipose-derived stem cells (ASC), providing a valuable model to study in vitro nerve regeneration and myelination, mimicking the in vivo environment at the injury site.

Dorsal Root Ganglia neurônios e células-tronco diferenciadas adiposo-derivados: An<em&gt; In Vitro</em&gt; Co-cultura modelo para estudar a regeneração do nervo periférico

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

Extrusão hidráulico da medula espinhal e Isolamento de gânglios das raízes dorsais em roedores

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

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 ...

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 ...

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

Purificação de Fibroblastos e Células Schwann de nervos sensoriais e motores in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

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 ...