The authors would like to thank Autumn Peterson for assistance with mouse photography, Daniel Thoresen for assistance with mice, and Drs. Nicole Ward and Abdelmadjid Belkadi for assistance with surgical denervation. These studies were supported by funding from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (grants R00AR059796 and R01AR065409); the University of Michigan Department of Dermatology; the Biological Sciences Scholars Program; the Center for Organogenesis; the University of Michigan Comprehensive Cancer Center; and the John S. and Suzanne C. Munn Cancer Fund. S.C.P. was supported by funding from the National Institute of General Medical Sciences (grant T32 GM007315). This work was also supported by the NIH Intramural Research Program, Center for Cancer Research, National Cancer Institute.

Todos os procedimentos descritos neste protocolo foram realizados de acordo com os regulamentos estabelecidos pela Universidade de Michigan Unidade de Medicina Laboratorial Animal. 1. Induzir recombinação genética em ratos Nota: O Gli1 tm3 (cre / ERT2) ALJ / J estirpe de ratinhos (Gli1-CreERT2) 13 permite a segmentação de recombinação genética induzida pelo tamoxifeno para TD epitélios. Atravesse esta estirpe com B6.129S4-Gt (ROSA) ratos repórter 26Sor tm1Sor / J (LacZ) 22 para gerar Gli1; ​​animais LacZ para visualizar as células TD por whole-mount coloração abaixo. <ol><li> Preparar a solução de tamoxifeno a uma concentração de 12,5 mg / ml em óleo de milho. <ol><li> Em um tubo de 1,5 ml, adiciona-se a 20 mg de tamoxifen cristalinos, e, em seguida, 1 ml de óleo de milho. Firmemente fita do tubo para um misturador vortex e vortex continuamente ao mais alto ajuste à temperatura ambiente até que o tamoxifeno foi totalmente dissolvido (2-4 hr), comoconfirmado por exame do tubo sob um microscópio de dissecação para a ausência de partículas de tamoxifeno. </li><li> Transferir a solução para um tubo de 15 ml e diluir o tamoxifeno para uma concentração final de 12,5 mg / ml, com o óleo de milho adicional. Misturar em vortex a solução viscosa durante mais 30 segundos adicionais. Conserva-se a solução durante até 1 semana a 4 ° C no escuro. </li></ol></li><li> Injectar a solução de tamoxifeno por via intraperitoneal em Gli1; ​​ratinhos LacZ, a um volume de 200 mL por 20 g de peso corporal do rato, para uma dose eficaz de tamoxifeno 2,5 mg por 20 g de peso do rato. </li></ol> 2. biópsias de pele de colheita Nota: Dependendo do experimento, biópsias de pele colheita vários dias ou semanas após a indução tamoxifeno. Para todas as cirurgias, siga protocolos padrão para a cirurgia de roedores, incluindo o uso de luvas estéreis, vestindo uma máscara ou cirúrgica do cabelo net, e cobrindo o animal com um campo cirúrgico esterilizado durante o procedimento. <ol><li> Prepare 10x solução de anestésico através da mistura de 90 mg / ml de cetamina e 6,5 mg / ml de xilazina na água. Dilui-se esta solução de 1:10 em PBS estéril imediatamente antes da utilização, e armazenar à TA no escuro durante até 8 meses. <ol><li> Alternativamente, anestesiar os ratos por inalação de isoflurano, começando com uma concentração de gás de 4% com oxigénio para anestesiar completamente o animal, e, em seguida, subsequentemente, reduzindo isto a 1-2% para a duração do processo. </li></ol></li><li> Injectar a solução de anestésico por via intraperitoneal numa dose de 200 ul por 20 g de peso corporal do rato. Verifique se o animal atingiu o plano adequado de sedação por ensaio toe pitada, e confirmar que freqüências cardíaca e respiratória são normais (aproximadamente 600 batidas e 160 respirações por minuto, respectivamente). </li><li> Use um aparador elétrico para remover os pêlos do local da biópsia na pele dorsal de volta, tomando cuidado para não nick ou danificar a pele subjacente. </li><li> Prepare o local da cirurgia, limpando o barbearárea de d em um sentido ântero-a-posterior usando Betadine e álcool toalhetes. Assegurar que todos os recortes de cabelo são removidos do local. </li><li> Delinear o local da biópsia usando um marcador preto, colocar o animal em uma almofada de aquecimento em uma área cirúrgica asséptica, e cobrir com uma cortina cirúrgica estéril (para fins de demonstração, cortina estéril foi omitido para aumentar a visibilidade). Nota: Para obtenção de cortes longitudinais de folículos de cabelo, a mais borda da biópsia (a extremidade a ser cortada para a histologia, ~ 1 cm) deve ser executado numa direcção ântero-posterior (paralela à direcção dos folículos pilosos), parassagitais a a linha média dorsal (Figura 1A). </li><li> Use um estéril # 11 bisturi para fazer uma excisão espessura total ao longo da área marcada sem danificar a fáscia muscular subjacente. Nota: O tecido da pele excisada inclui a epiderme, a derme, e a gordura subcutânea panículo carnoso. Sangramento é normalmente mínimo. </li><li> achatar tele extirpou amostra de pele em uma toalha de papel seco, derme lado para baixo, aparar o excesso de toalha de papel, e armazenar a amostra em PBS frio por até 1 hora se outras amostras precisam ser coletadas. Quando estiver pronto, prossiga para Passos 3.1 ou 3.2 para processar amostras para histologia ou Passo 4 para β-galactosidase (LacZ) coloração todo-mount. </li><li> Sutura fechar o local da biópsia usando 6-0 nylon, em um padrão simples interrompido espaçadas a cerca de 3 mm. </li><li> Não devolva os ratos que foram submetidos a cirurgia na mesma gaiola como outros animais até após a recuperação completa. </li><li> Monitorar os animais imediatamente após a cirurgia até que recupere a consciência, e também diariamente até a área cirúrgica foi curado, geralmente dentro de uma semana. Use analgésicos de acordo com orientações de cuidados de animais e uso institucional designados se os ratos apresentam sinais de dor ou angústia. Remover suturas dentro de 7-10 dias após a cirurgia. Nota: Se for necessário, preparar a solução analgésica diluindo carprofeno (50 mg / ml da solutião) 1: 100 em água estéril. Injectar a solução por via subcutânea entre as omoplatas perto da nuca do pescoço, a uma dose de 200 ul por 20 g de peso corporal (5 mg / kg de peso corporal do rato). </li></ol> 3. As amostras de processo para histologia Nota: Para corrigir e processar o tecido excisado, use um método abaixo dependendo da aplicação. <ol><li> Para gerar amostras histológicas embebidos em parafina, corrigir a pele em 3,7% de formalina em PBS O / N à temperatura ambiente e armazenar em 70% de etanol por até 2 semanas. Retire o papel toalha antes de embutir em parafina. </li><li> Para a gerao de amostras histológicas congelados, submergir o tecido no frio paraformaldeído 4% em PBS e agitar suavemente durante 1 h. Remover a solução e lava-se a amostra com 3 mudas de PBS, cerca de 5 minutos cada. Em seguida, mergulhe a amostra em 30% de sacarose em PBS para cryoprotect o tecido ( &quot;afundamento sacarose&quot;). </li><li> Incubar com agitação suave O / N a 4 ° C. No dia seguinte, retire o papel de reboqueel e aparar o excesso de tecido adiposo do lado do dérmica da pele. Incorporar o tecido directamente para outubro e armazenar o bloco congelado a -80 ° C. Nota: Após o corte, quer parafina ou amostras congeladas pode ser manchado por imuno-histoquímica para identificar TDs, células de Merkel e nervos utilizando anticorpos contra queratina 17, queratina 8 e neurofilamento, respectivamente, como descrito anteriormente 5,19. </li></ol> 4. As amostras Visualize por Whole-mount LacZ Coloração <ol><li> Prepare solução de coloração X-gal. <ol><li> Combinar 0,94 g de fosfato de sódio monobásico, e 2,6 g de fosfato dibásico de sódio em 250 ml de água estéril. Ajustar o pH para 7,3. Para isso, adicionar 0,5 ml de cloreto de magnésio 1 M, 0,528 g de ferrocianeto de potássio, e 0,412 g de ferricianeto de potássio. Adicionar 250 ul de octilfenil-polietileno-glicol e 125 mg de desoxicolato. A solução de base pode ser armazenado a 4 ° C durante até 6 meses no escuro. </li><li> Prepare 50x banco de soluti X-galem dimetilformamida, adicionando ao frasco de estoque de X-gal para gerar uma solução a 50 mg / ml. Conserva-se a solução a -20 ° C no escuro. </li><li> Imediatamente antes do uso, dilui-se solução de reserva de X-gal 1:50 em solução de base de X-gal para gerar solução de coloração. Para biópsias mais pequenos (&lt;1 cm 2), alíquotas de 1-2 ml de solução de coloração por amostra. </li></ol></li><li> Fixar a amostra de pele recolhida no Passo 2.7 numa solução contendo 2% paraformaldeído / 0,2% de glutaraldeído em PBS frio durante 30 min, agitando suavemente em gelo. Para biópsias mais pequenos (&lt;1 cm 2), utilizar 1-2 ml de solução fixadora por amostra. Nota: Como alternativa, fixar amostras em 2-4% paraformaldeído apenas, ou apenas em glutaraldeído 0,5%. condições de fixação óptimas dependem do tecido, grau de expressão de LacZ e de aplicação. </li><li> Remover a solução fixadora, e lavar as amostras com 3 mudas de PBS, 5 minutos cada, num agitador à temperatura ambiente. </li><li> Remover a toalha de papel por baixo da amostra e cortar excess tecido adiposo a partir de lado dérmico da pele ao agarrar a gordura com uma pinça sem corte e de corte com uma tesoura de dissecação. </li><li> Submergir a amostra na solução de coloração com X-gal, e incuba-se a 37 ° CO / N. Expressão de LacZ será visível como uma mancha azul sob um microscópio de dissecação (Figura 1B). Nota: Se a intensidade do sinal é fraco, substitua a solução de coloração no dia seguinte e repetir o O / N incubação. Se a coloração de fundo é demasiado intensa, reduzir o tempo de coloração, ou incubar a amostra a temperatura ambiente em vez de 37 ° C. </li><li> Remover a solução de coloração e lavar as amostras em 3 mudas de PBS contendo 3% de DMSO durante aproximadamente 5 min, agitando à temperatura ambiente. </li><li> Lavar as amostras em 2-3 mudanças de etanol a 70% para 5 minutos cada. Armazene as amostras em 70% de etanol. </li></ol> 5. A desnervação cirúrgica <ol><li> Anestesiar o animal como no Passo 2.2 e raspar a pele dorsal inteiro. </li><li> Prepare a área cirúrgica of a pele de volta usando Betadine e toalhetes com álcool, e cobrir o animal com um campo cirúrgico estéril (para fins de demonstração, cortina estéril foi omitido para aumentar a visibilidade). Manter o animal aquecido usando uma almofada de aquecimento durante a operação em uma área cirúrgica asséptica. </li><li> Realizar uma incisão utilizando um bisturi estéril # 11 ao longo da linha média dorsal desde a base do pescoço até cerca de 0,5 centímetros acima da cauda. </li><li> Usando fórceps rombas, reflectir suavemente a pele sobre o lado esquerdo longe do flanco para visualizar o tecido subjacente das almofadas de gordura escapular perto do pescoço até imediatamente acima do membro posterior. Nota: os nervos dorsais cutâneas aparecem como fios brancos que viajam caudalmente translúcida através da faseia da parede do tronco antes de fazer curvas apertadas e entrar no tecido conjuntivo frouxo debaixo da pele (Figura 2). </li><li> Usando fórceps ultra-finos sob um microscópio de luz dissecando, remover os nervos exclusivamente a partir do lado esquerdo da animal localizados em locais anatômicos T3-12 por arrancar a partir de onde a segmentos de curva na parede do tronco para seus locais de entrada na pele (Figura 2). <ol><li> Fórceps orientar verticalmente e remover os nervos agarrando aproximadamente 0,5 cm abaixo dos seus locais de curvatura e puxando para cima, fazendo com que o nervo para esticar e separar a partir do tecido circundante (Figura 2C - E). Tenha cuidado para evitar a ruptura dos vasos sanguíneos adjacentes. </li><li> Continuar até que todos os nervos que se estendem a partir da parede do tronco para a pele são removidos. Não perturbar os nervos no interior da fáscia densa da parede do tronco. Manter o tecido húmido em todo o processo por aplicação periódica de gotas de solução salina estéril a 0,9%. </li><li> Como alternativa, remova nervos, segurando suas extremidades proximais perto da parede do tronco com uma pinça e cortando com uma tesoura fina. Em seguida, cortar as extremidades distais perto da pele (Figuras 2F - H). Finalmente, remova o intervening segmentos de nervo (Figura 2I). </li></ol></li><li> Remova todos os nervos do retalho de pele exposta no Passo 5.4. Estas fibras compreendem os ramos distais dos nervos cutâneos dorsais e aparecem como fios de ramificação brancos localizados esporadicamente dentro do tecido conjuntivo no lado dérmico do retalho de pele (Figuras 2J - K). <ol><li> Para remover esses galhos finos, posicione a pinça fina mais ou menos paralelo à superfície da derme, segure os nervos e arrancar para cima, para evitar perturbar os vasos sanguíneos e perfurar a pele. Continuar até que todos os nervos visíveis tenham sido removidos. </li></ol></li><li> Utilizando uma dissecção romba, reflectir a pele do lado direito da linha média dorsal incisão, mas não remover os nervos. Isto irá servir como o controle sham-operados contralateral. </li><li> Sutura ao longo da linha média dorsal, em um teste padrão simples interrompido para fechar a incisão. Monitorar o animal durante a recuperação e pós-opertivamente como já foi demonstrado (Passos 2,8-10). Remover suturas dentro de 7-10 dias após a cirurgia. </li><li> Para avaliar funcionalmente estável desnervação até várias semanas após a cirurgia, remover os pêlos da pele dorsal usando um aparelho eléctrico. </li><li> Gentilmente picar a área da pele desnervado usando uma agulha hipodérmica, e observar se o animal responde, normalmente estremecendo ou virar a cabeça. Se a área da pele foi desnervado de forma estável, o animal vai exibem pouca ou nenhuma resposta. </li><li> Utilizando um marcador de preto, o contorno da área de não resposta, assim como uma área de tamanho e local semelhante no lado contralateral sham. </li><li> Recolha biópsias a partir destes sites que nos Passos 2.1-2.9 para análise. Observação: Como alternativa, toda a pele dorsal para trás, incluindo as regiões desnervados e operados por simulação, pode ser removido como uma única folha para montagem inteira-coloração, semelhante ao descrito no Passo 4. </li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+dependence+in+tissue,+organ,+and+appendage+regeneration.">Nerve dependence in tissue, organ, and appendage regeneration.</a> Trends Neurosci. 35, 691-699 (2012).</li><li>Rinkevich, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+reveals+nerve-dependent+and+independent+roles+on+mammalian+hind+limb+tissue+maintenance+and+regeneration.">Clonal analysis reveals nerve-dependent and independent roles on mammalian hind limb tissue maintenance and regeneration.</a> Proc Natl Acad Sci USA. 111, 9846-9851 (2014).</li><li>Ueno, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+corneal+stem/progenitor+cells+on+ocular+surface+innervation.">Dependence of corneal stem/progenitor cells on ocular surface innervation.</a> Invest Ophthalmol Vis Sci. 53, 867-872 (2012).</li><li>Westphalen, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lived+intestinal+tuft+cells+serve+as+colon+cancer-initiating+cells.">Long-lived intestinal tuft cells serve as colon cancer-initiating cells.</a> J Clin Invest. 124, 1283-1295 (2014).</li><li>Uhmann, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hedgehog+receptor+Patched+controls+lymphoid+lineage+commitment.">The Hedgehog receptor Patched controls lymphoid lineage commitment.</a> Blood. 110, 1814-1823 (2007).</li><li>Lau, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+control+of+gene+deletion+in+sensory+ganglia+using+a+tamoxifen-inducible+Advillin-Cre-ERT2+recombinase+mouse.">Temporal control of gene deletion in sensory ganglia using a tamoxifen-inducible Advillin-Cre-ERT2 recombinase mouse.</a> Mol Pain. 7, 100 (2011).</li></ol>

The authors have nothing to disclose.

Nervos desempenham funções cruciais não só na sensação, mas também no desenvolvimento de órgãos de mamíferos, manutenção e regeneração 13,24-27. Como nervos recentemente têm sido implicados em diversas doenças de pele, as técnicas descritas aqui podem ser usadas para estudar a exigência de inervação numa variedade de modelos de doenças animais. Na verdade, a técnica desnervação unilateral permite a comparação directa da pele quer com os nervos intactos ou interromperam a partir do me...

Over the past few years, there has been a widening appreciation for the influence of nerves on diseases not typically regarded as classical neuropathies1-4. In the skin, recent experimental evidence has suggested that sensory nerves can modulate diverse pathologies ranging from psoriasis to cancer5-9. This has been demonstrated using techniques such as surgical denervation and pharmacological inhibition of neural function in rodents. In the case of psoriasis, these studies have provided a mechanistic framework for understanding why human psoriatic plaques regress following loss of neural function7,10-12.
Cutaneous nerves can also affect gene expression13,14 and are critical for mechanosensing in normal skin15. In particular, touch dome (TD) epithelia are comprised of a patch of columnar epidermal cells in juxtaposition with neuroendocrine Merkel cells innervated by slowly adapting type 1 (SA1) nerve fibers16-18. TDs mediate light touch sensation and have been shown to display Hedgehog pathway activity5,19. TD maintenance depends on innervation20,21, as nerves secrete Hedgehog ligands to sustain normal TDs and their associated Merkel cells19. In addition, innervation promotes Hedgehog-dependent tumor formation from TD epithelia5. Together, these studies reinforce the notion that intricate molecular interactions occurring between nerves and the surrounding cells in their niche are crucial for normal TD physiology as well as disease.
To interrogate the nature of these interactions, we describe here a series of in vivo techniques for manipulating gene expression in the TD, as well as for harvesting skin biopsies for TD visualization after lineage tracing. Finally, we describe procedures for performing unilateral surgical denervation, wherein nerves are removed from one side of the mouse dorsal skin, while leaving the contralateral side intact as a sham internal control. Several weeks after surgery, denervated and sham control skin are compared to assess changes that occur when nerves are ablated. Although these techniques are described in the context of normal TDs, the denervation procedure has been used to examine the requirement for nerves in mouse models of psoriasis6, wound healing13 and tumorigenesis5. Finally, since the skin is amenable to repeated biopsies, this provides an opportunity to monitor the long-term fates of labeled cells or to assess disease progression over multiple time points.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alcohol prep pads</td>
			<td>PDI</td>
			<td>B339</td>
			<td></td>
		</tr>
		<tr>
			<td>AnaSed (Xylazine)</td>
			<td>Lloyd</td>
			<td>NADA 139-236</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody, anti-Keratin 8</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>TROMA-I</td>
			<td>rat antibody, use at 1:500 concentration</td>
		</tr>
		<tr>
			<td>Antibody, anti-Keratin 17</td>
			<td>Cell Signaling</td>
			<td>#4543</td>
			<td>rabbit antibody, use at 1:1,000 concentration</td>
		</tr>
		<tr>
			<td>Antibody, anti-Neurofilament</td>
			<td>Cell Signaling</td>
			<td>C28E10</td>
			<td>rabbit antibody, use at 1:500 concentration</td>
		</tr>
		<tr>
			<td>Betadine prep pads</td>
			<td>Medline</td>
			<td>MDS093917</td>
			<td></td>
		</tr>
		<tr>
			<td>Carprofen (Rimadyl)</td>
			<td>Zoetis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cordless rechargable clipper</td>
			<td>Wahl</td>
			<td>trimmer model 8900</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn Oil</td>
			<td>Sigma-Aldrich</td>
			<td>C8267</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1860</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ThermoFisher Scientific</td>
			<td>D1306</td>
			<td>use at 1:1000 concentration</td>
		</tr>
		<tr>
			<td>Deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory Cream</td>
			<td>Nair</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylforamide</td>
			<td>Sigma-Aldrich</td>
			<td>319937</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>G5882</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmEdge Pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine HCl</td>
			<td>Hospira</td>
			<td>NDC 0409-2051-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro cover glass</td>
			<td>VWR</td>
			<td>48404-454</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>VWR</td>
			<td>BDH0502-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 nylon sutures</td>
			<td>DemeTECH</td>
			<td>NL166012F4P</td>
			<td></td>
		</tr>
		<tr>
			<td>Octylphenyl-polyethylene glycol</td>
			<td>Sigma-Aldrich</td>
			<td>I8896</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. Compound</td>
			<td>Sakura Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Pottasium ferrocyanide</td>
			<td>Sigma-Aldrich</td>
			<td>P9387</td>
			<td></td>
		</tr>
		<tr>
			<td>Pottasium ferricyanide</td>
			<td>Sigma-Aldrich</td>
			<td>702587</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>S5136</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>84097</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma-Aldrich</td>
			<td>T5648-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra fine forceps</td>
			<td>Dumont</td>
			<td>0103-5-PO</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories</td>
			<td>H1000</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Roche</td>
			<td>10 651 745 001</td>
			<td>Disolve in dimethylforamide to create 50x stock prior to use</td>
		</tr>
	</tbody>
</table>

cutaneous surgical denervation:  a method for testing the requirement for nerves in mouse models of skin disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

Por ratinhos que expressam gerando tamoxifeno induzível Gli1-CreERT2 e um alelo repórter LacZ, é possível visualizar TD epitélios e rastrear o destino destas células ao longo do tempo. O procedimento de desnervação inteira normalmente pode ser concluída dentro de 1 hora por rato e deve causar desconforto mínimo para o animal. Os nossos estudos anteriores têm indicado que os nervos são cruciais par...

Watch this Scientific Journal Video about Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease at JoVE.com

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

A desnervação cirúrgica cutânea: um método para testar o requisito para os nervos no mouse modelos de doença de pele

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...

cutaneous-surgical-denervation-method-for-testing-requirement-for

Research

JoVE Journal

Medicine

9.7K visualizações.  University of Michigan. This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount β-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

Vídeo: Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

Medição precisa e simples do pró-inflamatórias IL-1β citocinas usando um ensaio Estimulação Sangue Total

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

Medicina

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 years for the sporadic disease and around 50 years for the familial disease. Due to its progressive course, 50% of the patients die within 30 months of symptom onset. In order to evaluate novel treatment options for this disease, genetic mouse models of ALS have been generated based on human familial mutations in the SOD gene, such as the SOD1 (G93A) mutation. Most important aspects that have to be evaluated in the model are overall survival, clinical course and motor function. Here, we demonstrate the clinical evaluation, show the conduction of two behavioural motor tests and provide quantitative scoring systems for all parameters. Because an in depth analysis of the ALS mouse model usually requires an immunohistochemical examination of the spinal cord, we demonstrate its preparation in detail applying the dorsal laminectomy method. Exemplary histological findings are demonstrated. The comprehensive application of the depicted examination methods in studies on the mouse model of ALS will enable the researcher to reliably test future therapeutic options which can provide a basis for later human clinical trials.

Clinical Testing and Spinal Cord Removal in a Mouse Model for Amyotrophic Lateral Sclerosis ALS

A mouse model for amyotrophic lateral sclerosis (ALS) is examined clinically and behaviorally. As a prerequisite for an accompanying immunohistological analysis the preparation of the spinal cord is depicted in detail.

Testes clínicos e Remoção da Medula Espinhal em um modelo do rato para a Esclerose Lateral Amiotrófica (ELA)

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder resulting in progressive degeneration of motoneurons. Peak of onset is around 60 ...

Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-&gamma; in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-&gamma; followed by administration of mouse IFN-&gamma; (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.

We describe protocols for our mouse graft arteriosclerois (GA) models which involve interposition of a mouse vessel segment into a recipient of the same inbred strain. By backcrossing additional genetic changes into the vessel donor, the model can assess the effect of specific genes on GA.

Os modelos de mouse para Arteriosclerose Graft

Graft arteriosclerois (GA),  also called allograft vasculopathy, is a pathologic lesion that develops over  months to years in transplanted organs ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

Um método simples de intubação Lung Rato

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice. &#160;

Secção do nervo tibial - um modelo padronizado para denervação induzida por atrofia muscular em ratos

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rato Transplante Renal: Modelos de rejeição de enxertos

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

Um modelo de pele Rato Fetal de reparação de feridas Scarless

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Modelos cirúrgicos de refluxo gastroesofágico com Ratos

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

Cirurgia de colocação de cateteres para Cardiovascular Testes exercício de longo prazo em Suínos

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Repeated Measurement of Respiratory Muscle Activity and Ventilation in Mouse Models of Neuromuscular Disease

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated measurements over weeks or months of accessory respiratory muscle activity while simultaneously measuring ventilation in a non-anesthetized, freely behaving mouse. The technique includes the surgical implantation of a radio transmitter and the insertion of electrode leads into the scalene and trapezius muscles to measure the electromyogram activity of these inspiratory muscles. Ventilation is measured by whole-body plethysmography, and animal movement is assessed by video and is synchronized with electromyogram activity. Measurements of muscle activity and ventilation in a mouse model of amyotrophic lateral sclerosis are presented to show how this tool can be used to investigate how respiratory muscle activity changes over time and to assess the impact of muscle activity on ventilation. The described methods can easily be adapted to measure the activity of other muscles or to assess accessory respiratory muscle activity in additional mouse models of disease or injury.

This paper introduces a method for repeated measurements of ventilation and respiratory muscle activity in a freely behaving amyotrophic lateral sclerosis (ALS) mouse model throughout disease progression with whole-body plethysmography and electromyography via an implanted telemetry device.

Medida repetida de actividade muscular respiratória e Ventilação no rato modelos de doença neuromuscular

Accessory respiratory muscles help to maintain ventilation when diaphragm function is impaired. The following protocol describes a method for repeated ...