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.

Bu protokol açıklanan tüm işlemler Laboratuar Hayvan Tıp Michigan Birimi Üniversitesi tarafından belirlenen kurallara uygun olarak yapıldı. 1. Farelerde genetik rekombinasyonu azaltmak Not: Gli1 TM3 (CRE / ERT2) Alj / J fare suşu (Gli1-CreERT2) 13 epitel TD tamoksifen kaynaklı genetik rekombinasyon hedef mümkün kılar. Gli1 oluşturmak için B6.129S4-Gt (ROSA) 26Sor tm1Sor / J muhabiri fareler (LacZ) 22 Bu gerginlik çapraz; LacZ hayvanlar tüm montaj aşağıda boyanarak TD hücreleri görselleştirmek için. <ol><li> mısır yağı içinde 12.5 mg / ml&#39;lik bir konsantrasyona tamoksifen çözeltisi hazırlayın. <ol><li> 1.5 ml tüp içinde, kristalin tamoksifen 20 mg, ve mısır yağı, sonra 1 ml&#39;ye kadar ekleyin. tamoksifen tamamen (2-4 saat) çözülene kadar sıkıca gibi bir girdaplı karıştırıcı tüp, ve oda sıcaklığında en yüksek ayarda sürekli girdap banttamoksifen parçacık bulunmaması için bir mikroskop altında bir tüp incelenmesi ile teyit edilmiştir. </li><li> 15 ml&#39;lik bir tüpe transfer çözeltisi ve ek mısır yağı ile 12.5 ml mg / bir nihai konsantrasyona tamoksifen seyreltin. ilave bir 30 saniye süre ile yapışkan bir solüsyon vorteks karıştırın. Karanlıkta 4 ° C&#39;de 1 hafta kadar bu çözelti saklayın. </li></ol></li><li> Gli1 intraperitonal tamoksifen çözelti enjekte edilir; LacZ fareler, fare vücut ağırlığının her 20 gramı başına 200 ul bir hacimde, 20 g fare ağırlığı başına 2.5 mg etkin tamoksifen dozu. </li></ol> 2. Hasat Cilt biyopsiler Not: Tamoksifen indüksiyon sonrası hafta deneye bağlı olarak, hasat deri biyopsileri birkaç gün. Tüm ameliyatlar için, steril eldiven kullanarak cerrahi maske ya da saç net giyen ve işlem sırasında steril cerrahi örtü ile hayvan kapsayan dahil kemirgen cerrahi için standart protokoller, izleyin. <ol><li> Suda 90 mg / ml ketamin ve 6.5 mg / ml ksilazin karıştırarak 10x stok anestezik solüsyon hazırlanır. kullanımdan hemen önce, steril PBS içine Bu stok solüsyonu 1:10 seyreltilir ve 8 aya kadar karanlıkta oda sıcaklığında saklayın. <ol><li> Seçenek olarak ise, tam olarak hayvan anestetize oksijen ile% 4 arasında bir gaz konsantrasyonu ile başlayarak, izofluran teneffüs ile fareler anestezi ve daha sonra işlem sırasında% 1-2, bu düşürür. </li></ol></li><li> 20 g fare vücut ağırlığı başına 200 ul bir dozda intraperitonal anestezik enjekte edilir. hayvan ayak tutam testi ile sedasyon uygun düzlemine ulaştığını kontrol edin ve bu kalbi onaylamak ve solunum oranları (sırasıyla, yaklaşık 600 atım ve min başına 160 nefesler) normaldir. </li><li> Dorsal arka cilt üzerinde biyopsi sitesinden saç kaldırmak değil nick dikkatli olmak ya da altta yatan cilde zarar bir elektrik kesme makinesi kullanın. </li><li> tıraş silerek cerrahi sitesi hazırlamakBetadinede ve alkol mendil kullanarak ön-to-posterior yönde d alanı. Tüm saç kupürleri siteden kaldırılır emin olun. </li><li> Siyah kalem kullanarak biyopsi sitesi anahat aseptik cerrahi alanda bir ısınma pad üzerinde hayvan koyun ve bir steril cerrahi örtü ile kaplayın (gösteri amaçlı steril örtüyü görünürlüğünü artırmak için ihmal edilmiştir). Not: saç köklerinin uzunlamasına bölümleri elde etmek için, biyopsi uzun kenarının, bir ön-arka yönde parasagital (saç köklerinin yönüne paralel) çalıştırmanız gerekir (kenar ~ 1 cm histoloji için kesitli olmak üzere) dorsal orta hat (Şekil 1A). </li><li> Altta yatan kas fasya zarar vermeden işaretli alanı boyunca tam kalınlıkta eksizyonu yapmak için steril 11. neşter kullanın. Not: eksize cilt dokusu epidermis, dermis, deri altı yağ ve panniculus carnosus içerir. Kanama genellikle az. </li><li> t düzleştirmeko kuru bir kağıt havlu üzerine deri örneği eksize, dermis, aşağı yan aşırı kağıt havlu uzak trim ve diğer örnekleri toplandı gerekiyorsa kadar 1 saat boyunca soğuk PBS örnek saklayın. Hazır olduğunuzda, histoloji için numune veya tüm montaj β-galaktosidaz (LacZ) boyama Adım 4 işlemek için Adımlar 3.1 veya 3.2 geçin. </li><li> Sütür kabaca 3 mm aralıklı basit bir kesintiye desen, 6-0 naylon sütür ile biyopsi sitesini kapatın. </li><li> tam iyileşme sonrasına kadar diğer hayvanlar aynı kafesin içine ameliyat geçiren fareler iade etmeyin. </li><li> cerrahi alan tipik olarak 1 hafta içinde, iyileşinceye kadar her gün aynı zamanda bilinci kazanmak ve kadar ameliyattan hemen sonra hayvanların izleyin. Fareler ağrı veya sıkıntı belirtileri göstermesi durumunda belirlenen kurumsal hayvan bakımı ve kullanım yönergelerine uygun olarak analjezikler kullanın. Ameliyat sonrası 7-10 gün içinde sütür çıkarın. Not: Gerekirse, (50 mg / ml stok SOLUT karprofen seyreltilmesiyle analjezik bir çözelti hazırlamakiyon): 1, steril su içinde 100. 20 g vücut ağırlığı başına 200 ul (5 mg / kg fare vücut ağırlığı) bir dozda, Boynun ense yakınındaki kürek kemikleri arasında çözelti, deri altına enjekte edilir. </li></ol> Histoloji 3. İşlem Örnekleri düzeltmek için ve eksize doku işlemek, uygulamaya bağlı olarak aşağıdaki yöntemi kullanabilirsiniz: Not. <ol><li> parafine gömülmüş histolojik örnekleri oluşturmak için en fazla 2 hafta boyunca% 70 etanol içinde oda sıcaklığında PBS O / N% 3.7 formalin deri ve mağaza düzeltmek. parafin içine gömmeden önce kağıt havluyu çıkarın. </li><li> Dondurulmuş histolojik numune üretilmesi için, PBS içinde, soğuk% 4 paraformaldehid daldırın doku ve yavaşça 1 saat boyunca çalkalanır. çözelti çıkarın ve PBS 3 değişiklik, yaklaşık 5 dakika ile her numune yıkanır. Daha sonra, ( &quot;sükroz batma&quot;) doku cryoprotect PBS içinde% 30 sukroz içinde örnek daldırın. </li><li> 4 ° C&#39;de nazik bir çalkalama O / N kuluçkalayın. Ertesi gün, kağıt yedekte kaldırmakEl derinin dermal tarafında aşırı yağ dokusu uzak trim ve. doğrudan OCT içine doku embed ve -80 ° C&#39;de dondurulmuş blok saklayın. Not: Daha önce 5,19 açıklandığı gibi kesit sonra, parafin veya dondurulmuş numuneler ya TDS Merkel hücreleri ve sırasıyla Keratin 17, Keratin 8 ve Nörofilaman, karşı antikorlar kullanılarak sinirler tanımlamak için immünohistokimyasal olarak boyanmış olabilir. </li></ol> Tüm montaj LacZ Boyama tarafından 4. gözünüzde canlandırın Örnekleri <ol><li> X-gal boyama çözüm hazırlayın. <ol><li> steril su, 250 ml 0.94 g sodyum fosfat monobazik ve 2.6 g sodyum fosfat dibazik birleştirin. 7.3 pH ayarlayın. Buna, 1 M magnezyum klorid, 0.5 ml, potasyum ferrosiyanür 0.528 g, potasyum ferrisiyanür ve 0.412 g ilave edin. oktilfenil-polietilen glikol 250 ul ve deoksikolat 125 mg ekleyin. baz çözeltisi karanlıkta 6 ay için 4 ° C&#39;de saklanabilir. </li><li> 50x stok X-gal soluti hazırlayın50 mg / ml solüsyon oluşturmak için X-gal hazır şişeye dimetilformamid eklenerek ilgili. karanlıkta -20 ° C&#39;de bu çözüm saklayın. </li><li> boyama çözüm üretmek için X-gal baz çözeltisi içine stok X-gal çözüm 01:50 sulandırmak, kullanımdan hemen önce. Daha küçük biyopsi (&lt;1 cm2), örnek başına boyama çözeltisi alikosu 1-2 ml. </li></ol></li><li> yavaşça buz üzerinde sallayarak, 30 dakika boyunca soğuk PBS içinde% 2 paraformaldehit /% 0.2 glutaraldehit ihtiva eden bir çözelti içinde Aşama 2.7&#39;de elde deri örneği düzeltildi. Daha küçük biyopsi (&lt;1 cm2) için, numune başına sabitleyici çözeltisi 1-2 ml kullanın. Not: Alternatif olarak, sadece% 2-4 paraformaldehid örnekleri düzeltmek, ya da% 0.5 glutaraldehit sadece. Optimum sabitleme koşulları doku, lacZ ekspresyonu ve uygulama derecesine bağlıdır. </li><li> sabitleyici çözeltisini çıkarın ve oda sıcaklığında, bir çalkalayıcı üzerinde PBS 3 değişiklikler, her biri 5 dakika ile örnekleri yıkayın. </li><li> numunenin altında kağıt havlu çıkarın ve excès kesipkünt forseps ile yağ tutma ve kesme makas ile kırparak derinin dermal tarafında yağ dokusu s. </li><li> X-gal ile boyama çözeltisi içinde örnek daldırın ve 37 ° CO / H inkübe edin. LacZ ifadesi bir mikroskop (Şekil 1B) altında mavi bir leke olarak görülebilir. Not: sinyal yoğunluğu zayıfsa, boyama çözüme ertesi gün yerini ve O / N inkübasyon tekrarlayın. arka plan boyama çok yoğun ise, boyama süresini azaltmak, ya da C ° 37 oda sıcaklığında örnek inkübe yerine. </li><li> boyama çözeltisini çıkarın ve PBS yavaşça oda sıcaklığında çalkalanarak yaklaşık 5 dakika için% 3 DMSO içeren 3 değişiklikleri örnekleri yıkayın. </li><li> Her biri 5 dakika boyunca% 70 etanol içinde 2-3 değişiklikler yıkayın örnekler. % 70 etanol içinde mağazası örnekleri. </li></ol> 5. Cerrahi denervasyon <ol><li> Adım 2.2 gibi hayvan anestezisi ve tüm dorsal cilt tıraş. </li><li> Cerrahi alan o hazırlayınBetadine ve alkol mendil ve bir steril cerrahi örtü ile hayvan kapak kullanarak arka cilt f (gösteri amaçlı steril örtüyü görünürlüğünü artırmak için ihmal edilmiştir). aseptik cerrahi alanda çalışırken bir ısıtma yastığı kullanılarak hayvan sıcak tutun. </li><li> kuyruk yukarıda kabaca 0.5 cm boyun tabanından dorsal orta hat boyunca steril 11. neşter kullanılarak bir kesi yapmak. </li><li> Künt forseps kullanarak, hafifçe sadece arka bacak yukarıda boyun yakın skapular yağ pedleri altta yatan doku görselleştirmek için uzağa zayıf sol tarafta cilt yansıtmaktadır. Not: Dorsal deri sinirler (Şekil 2) keskin virajlı yapma ve deri altına gevşek bağ dokusu girmeden önce gövde duvarının saydam fasya ile kaudal seyahat gibi beyaz ipliklerini görünür. </li><li> Diseksiyon ışık mikroskobu altında ultra-ince forseps kullanarak, anima sol kanattan gelen sadece sinirleri çıkarmakyerden toplama anatomik sitelerde T3-12 bulunan l deri içine kendi giriş sitelerine gövde duvara bölümleri bend (Şekil 2). <ol><li> Orient forseps dikey ve streç ve çevre dokuya (- E Şekil 2C) ayrı sinir neden kendi viraj siteleri aşağıda yaklaşık olarak 0.5 cm açgözlü ve yukarı doğru çekerek sinirleri kaldırın. Bitişik kan damarlarının çatlaması önlemek için dikkatli olun. </li><li> Cilde gövde duvarının uzanan tüm sinirler kaldırılana kadar devam edin. gövde duvarının yoğun fasya içinde sinirleri bozmaya etmeyin. periyodik steril% 0.9 tuzlu su çözeltisi damla damlatılarak işlem boyunca nemli bir doku tutun. </li><li> Alternatif olarak, forseps ile gövde duvarının yakınında yakın kenarlarından tutarak ve ince makas ile kırpma yoluyla sinirleri kaldırın. Daha sonra, cilt yakın uzak uçları sever (Şekil 2F - H). Son olarak, int kaldırmakervening sinir segmentleri (Şekil 2I). </li></ol></li><li> Adım 5.4 maruz cilt flebi herhangi sinirleri çıkarın. Bu lifler dorsal deri sinirlerin distal dalları içerir ve cilt kapağının dermal tarafında (Şekil 2J - K) üzerinde bağ dokusu içinde düzensiz bulunan beyaz dallanma ipliklerini olarak görünür. <ol><li> Bu ince dalları kaldırmak için, ince forseps kabaca, dermal yüzeyine paralel sinirleri kavramak ve kan damarlarını bozarak ve cilt delinmesiyle önlemek için yukarı doğru koparmak yerleştirin. Görünen tüm sinirler kaldırılıncaya kadar devam edin. </li></ol></li><li> Künt diseksiyon kullanarak, dorsal orta hat kesi sağ tarafında cilt yansıtan, ama sinirleri çıkarmayın. Bu kontralateral sahte operasyona kontrol olarak görev yapacak. </li><li> Basit bir kesintiye desen dorsal orta hat boyunca sütür insizyon kapatmak için. kurtarma ve post-oper sırasında hayvan izleyinonları bir arada, daha önce (Adım 2,8-10) gösterdi. Ameliyat sonrası 7-10 gün içinde sütür çıkarın. </li><li> işlevsel ameliyattan sonra birkaç hafta istikrarlı denervasyon kadar değerlendirmek için, bir elektrik clipper kullanarak sırt derisine saç kaldırmak. </li><li> Yavaşça bir derialtı iğne kullanılarak Denerve deri alanını prick ve hayvan genellikle titreyerek ya da başını çevirerek, yanıt verip not edin. cilt alanı stabil sinirsiz olmuşsa, hayvan çok az veya hiç tepki gösterecektir. </li><li> siyah kalem kullanarak, herhangi bir yanıt alanı yanı sıra kontralateral sahte tarafında benzer boyut ve konumu bir alan belirtin. </li><li> Adımlar analiz için 2,1-2,9 gibi bu sitelerden biyopsi toplayın. Not: Alternatif olarak, Denerve ve sham operasyonu bölgeler dahil olmak üzere tüm dorsal arka deri, Adım 4&#39;te tarif edildiği gibi benzer tüm montaj boyama için tek bir sayfaya olarak kaldırılabilir. </li></ol>

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+in+the+regeneration+of+type+I+cutaneous+receptors.">Patterning in the regeneration of type I cutaneous receptors.</a> J Physiol. 236, 57-82 (1974).</li><li>Soriano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+lacZ+expression+with+the+ROSA26+Cre+reporter+strain.">Generalized lacZ expression with the ROSA26 Cre reporter strain.</a> Nat Genet. 21, 70-71 (1999).</li><li>Wright, M. C., 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=Atoh1++progenitors+maintain+the+Merkel+cell+population+in+embryonic+and+adult+mice.">Atoh1+ progenitors maintain the Merkel cell population in embryonic and adult mice.</a> J Cell Biol. 208, 367-379 (2015).</li><li>Kumar, A., Brockes, J. 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.

Sinirler duyu değil, aynı zamanda, memeli organ gelişimine, bakımı ve rejenerasyonu 13,24-27 değil sadece önemli fonksiyona hizmet. sinirler en son çeşitli cilt bozuklukları işaret edildiği gibi, burada tarif edilen teknikler hayvan hastalık modellerinde çeşitli innervasyonundan gereksinimi çalışma için kullanılabilir. Nitekim, tek taraflı denervasyon tekniği aynı fare bozulmamış veya bozulmuş sinir ya cildin doğrudan karşılaştırma için izin verir. sonraki veri eşleştirilmiş...

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.

Üretilmesi tamoksifen indüklenebilir Gli1-CreERT2 ifade eden farelerde ve LacZ haberci alel tarafından, TD epitel görselleştirmek ve zaman içinde bu hücrelerin kaderini izlemek mümkündür. Tüm denervasyon işlemi genellikle fare başına 1 saat içinde tamamlanabilir ve hayvana en az sıkıntıya neden gerekir. 5,19 - Daha önceki çalışmalar sinirler, normal TDS yanı sıra bunların i...

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.

Deri Cerrahi denervasyon: Cilt Hastalıkları Fare modelleri Sinirler için Gereği Test Bir Yöntem

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

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9.7K Görüntüleme.  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.

Video: 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.

Fare İndüklenmiş Mekanik allodini ayrılan sinir hasarı (SNI) Model

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

Tıp

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;

Tibial Sinir transeksiyonu - Farelerde denervasyon bağlı İskelet Kas Atrofi için bir Standart Model

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

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

Farelerde Nöropatik Ağrı Siyatik Sinir cuffing Modeli

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Fare Siyatik Sinir Vivo elektrofizyolojik ölçümlerde

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Nörobilim

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.

Farelerde Yüz Sinir Aksotomi: Sakatlık Motonöron Yanıtı Eğitim için Bir Model

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.

Saf küçük Fiber nöropati bir fare modeli ile geçici reseptör potansiyel Vanilloid tip 1 Ultrapotent Agonist kurulması

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

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>Farelerde Nöropatik Ağrının Modifiye Edilmiş Korunmuş Sinir Yaralanması Cerrahisi Modeli

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.

Farelerde Nöropatik Ağrının Modifiye Edilmiş Korunmuş Sinir Yaralanması Cerrahisi Modeli

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

Traumatic Peripheral Nerve Injury in Mice

Traumatic peripheral nerve injury (TPNI) is a common cause of morbidity following orthopedic trauma. Reproducible and precise methods of injuring nerve and denervating muscle have long been a goal in musculoskeletal research. Many traumatically injured limbs have nerve trauma that defines the long-term patient outcome. Over several years, precise methods of producing microsurgical nerve injuries have been developed, including crush, lacerations, and nerve-gap grafting, allowing for reproducible outcome assessments. Moreover, newer methods are created for calibrated crush injuries that offer clinically relevant correlations with outcomes used to assess human patients. The principles of minimal manipulation to ensure low variability in nerve injury allow for adding still more associated tissue injuries into these models. This includes direct muscle crush and other components of limb injury. Finally, atrophy assessment and precise analysis of behavioral outcomes make these methods a complete package for studying musculoskeletal trauma that realistically incorporates all the elements of human traumatic limb injury.

<meta charset="utf8"></head><body>Farelerde Travmatik Periferik Sinir Yaralanması

The present protocol describes traumatic peripheral nerve injuries (TPNIs), including precisely calibrated crush, strictly aligned and misaligned laceration, as well as grafted and non-grafted gaps of the sciatic nerve in mice. Custom-designed sensors are developed to gauge nerve trauma, induced with commonly available tools to ensure reproducible post-TPNI outcomes.

Farelerde Travmatik Periferik Sinir Yaralanması

Traumatic peripheral nerve injury (TPNI) is a common cause of morbidity following orthopedic trauma. Reproducible and precise methods of injuring nerve ...

Deri Cerrahi denervasyon: Cilt Hastalıkları Fare modelleri Sinirler için Gereği Test Bir Yöntem

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Fare İndüklenmiş Mekanik allodini ayrılan sinir hasarı (SNI) Model

Tibial Sinir transeksiyonu - Farelerde denervasyon bağlı İskelet Kas Atrofi için bir Standart Model

Farelerde Nöropatik Ağrı Siyatik Sinir cuffing Modeli

Fare Siyatik Sinir Vivo elektrofizyolojik ölçümlerde

Farelerde Yüz Sinir Aksotomi: Sakatlık Motonöron Yanıtı Eğitim için Bir Model

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