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><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 1:1,000 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

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. 
The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn&#x2019;t require expert microsurgical skills.
The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. 
As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.

The Spared Nerve Injury SNI Model of Induced Mechanical Allodynia in Mice

The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

Poupado da lesão do nervo Model (SNI) de alodinia mecânica induzida em camundongos

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared ...

Medicina

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. ...

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

Axotomia do nervo facial em ratos: um modelo para estudo de resposta motoneuron para Injury

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 &#181;g/kg, respectively) and concluded that a critical dose of RTX (50 &#181;g/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

Estabelecer um modelo de Mouse de uma neuropatia de pura fibra pequena com o ultrapotente agonista do Receptor transitório potencial Vanilloid tipo 1

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to ...

Neurociência

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

Protocolo para criar feridas crônicas em camundongos diabéticos

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

<meta charset="utf8"></head><body>Modelo de cirurgia de lesão nervosa poupada modificada de dor neuropática em camundongos

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Modelo de cirurgia de lesão nervosa poupada modificada de dor neuropática em camundongos

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Ligadura parcial do nervo ciático: um modelo murino de dor neuropática crônica para estudar o efeito antinociceptivo de novas terapias

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...

Direcionamento de neurônios simpáticos de camundongos: uma abordagem de simpatectomia lombar

Surgical Lumbar Sympathectomy in Mice

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays many critical roles in maintaining bodily homeostasis, but it has rarely been studied in the context of peripheral nerve injury. The extent of postganglionic sympathetic neuronal functions in distal targets in the periphery is currently unclear. To better explore the role of sympathetic innervation of peripheral targets, a surgical &#34;knock-out&#34; model provides an alternative approach. Although this can be achieved chemically, chemical destruction of postganglionic sympathetic neurons can be nonspecific and dose-dependent. The use of a surgical lumbar sympathectomy in mice, once thought to be &#34;virtually not practicable&#34; in small animals, allows for specific targeting of postganglionic sympathetic neurons that innervate the hind limbs. This manuscript describes how to surgically remove the L2-L5 lumbar sympathetic ganglia from a mouse as a survival surgery, which reliably decreases the hind paw sweat response and the number of sympathetic axons in the sciatic nerve.

Autor em destaque: explorando a plasticidade dos neurônios simpáticos

This manuscript presents a protocol for surgically removing the postganglionic lumbar sympathetic neurons from a mouse. This procedure will facilitate a multitude of studies aimed at investigating the role of sympathetic innervation in distal tissue targets.

Simpatectomia Lombar Cirúrgica em Camundongos

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays ...

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

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