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>
	<li>Magnon, 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=Autonomic+nerve+development+contributes+to+prostate+cancer+progression.">Autonomic nerve development contributes to prostate cancer progression.</a> Science. 341, 1236361 (2013).</li><li>Zhao, C. M., 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=Denervation+suppresses+gastric+tumorigenesis.">Denervation suppresses gastric tumorigenesis.</a> Sci Transl Med. 6, 115 (2014).</li><li>Chiu, I. M., von Hehn, C. A., Woolf, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenic+inflammation+and+the+peripheral+nervous+system+in+host+defense+and+immunopathology.">Neurogenic inflammation and the peripheral nervous system in host defense and immunopathology.</a> Nat Neurosci. 15, 1063-1067 (2012).</li><li>Gautron, L., Elmquist, J. K., Williams, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+control+of+energy+balance:+translating+circuits+to+therapies.">Neural control of energy balance: translating circuits to therapies.</a> Cell. 161, 133-145 (2015).</li><li>Peterson, S. 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=Basal+cell+carcinoma+preferentially+arises+from+stem+cells+within+the+hair+follicle+and+mechanosensory+niches.">Basal cell carcinoma preferentially arises from stem cells within the hair follicle and mechanosensory niches.</a> Cell Stem Cell. 16, 400-412 (2015).</li><li>Ostrowski, S. M., Belkadi, A., Loyd, C. M., Diaconu, D., Ward, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+denervation+of+psoriasiform+mouse+skin+improves+acanthosis+and+inflammation+in+a+sensory+neuropeptide-dependent+manner.">Cutaneous denervation of psoriasiform mouse skin improves acanthosis and inflammation in a sensory neuropeptide-dependent manner.</a> J Invest Dermatol. 131, 1530-1538 (2011).</li><li>Ward, N. L., 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=Botulinum+neurotoxin+A+decreases+infiltrating+cutaneous+lymphocytes+and+improves+acanthosis+in+the+KC-Tie2+mouse+model.">Botulinum neurotoxin A decreases infiltrating cutaneous lymphocytes and improves acanthosis in the KC-Tie2 mouse model.</a> J Invest Dermatol. 132, 1927-1930 (2012).</li><li>Roggenkamp, D., 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=Epidermal+nerve+fibers+modulate+keratinocyte+growth+via+neuropeptide+signaling+in+an+innervated+skin+model.">Epidermal nerve fibers modulate keratinocyte growth via neuropeptide signaling in an innervated skin model.</a> J Invest Dermatol. 133, 1620-1628 (2013).</li><li>Riol-Blanco, L., 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=Nociceptive+sensory+neurons+drive+interleukin-23-mediated+psoriasiform+skin+inflammation.">Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation.</a> Nature. 510, 157-161 (2014).</li><li>Zanchi, M., 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=Botulinum+toxin+type-A+for+the+treatment+of+inverse+psoriasis.">Botulinum toxin type-A for the treatment of inverse psoriasis.</a> J Eur Acad Dermatol Venereol. 22, 431-436 (2008).</li><li>Farber, E. M., Lanigan, S. W., Rein, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+psychoneuroimmunology+in+the+pathogenesis+of+psoriasis.">The role of psychoneuroimmunology in the pathogenesis of psoriasis.</a> Cutis. 46, 314-316 (1990).</li><li>Dewing, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remission+of+psoriasis+associated+with+cutaneous+nerve+section.">Remission of psoriasis associated with cutaneous nerve section.</a> Arch Dermatol. 104, 220-221 (1971).</li><li>Brownell, I., Guevara, E., Bai, C. B., Loomis, C. A., Joyner, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve-derived+sonic+hedgehog+defines+a+niche+for+hair+follicle+stem+cells+capable+of+becoming+epidermal+stem+cells.">Nerve-derived sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells.</a> Cell Stem Cell. 8, 552-565 (2011).</li><li>Liao, X. H., Nguyen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+expression+of+Lgr6+is+dependent+on+nerve+endings+and+Schwann+cells.">Epidermal expression of Lgr6 is dependent on nerve endings and Schwann cells.</a> Exp Dermatol. 23, 195-198 (2014).</li><li>Lumpkin, E. A., Marshall, K. L., Nelson, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+touch.">The cell biology of touch.</a> J Cell Biol. 191, 237-248 (2010).</li><li>Maricich, S. M., 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=Merkel+cells+are+essential+for+light-touch+responses.">Merkel cells are essential for light-touch responses.</a> Science. 324, 1580-1582 (2009).</li><li>Reinisch, C. M., Tschachler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+touch+dome+in+human+skin+is+supplied+by+different+types+of+nerve+fibers.">The touch dome in human skin is supplied by different types of nerve fibers.</a> Ann Neurol. 58, 88-95 (2005).</li><li>Doucet, Y. S., Woo, S. H., Ruiz, M. E., Owens, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+touch+dome+defines+an+epidermal+niche+specialized+for+mechanosensory+signaling.">The touch dome defines an epidermal niche specialized for mechanosensory signaling.</a> Cell Reports. 3, 1759-1765 (2013).</li><li>Xiao, 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=Neural+Hedgehog+signaling+maintains+stem+cell+renewal+in+the+sensory+touch+dome+epithelium.">Neural Hedgehog signaling maintains stem cell renewal in the sensory touch dome epithelium.</a> Proc Natl Acad Sci USA. 112, 7195-7200 (2015).</li><li>English, K. B., Van Norman, D. K. -. V., Horch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+chronic+denervation+in+type+I+cutaneous+mechanoreceptors+(Haarscheiben).">Effects of chronic denervation in type I cutaneous mechanoreceptors (Haarscheiben).</a> Anat Rec. 207, 79-88 (1983).</li><li>Burgess, P. R., English, K. B., Horch, K. W., Stensaas, L. 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 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.

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

Research

JoVE Journal

Medicine

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

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.

Tam Kan Stimülasyon Testi kullanarak Pro-inflamatuar sitokin IL-1β Doğru ve Basit Ölçme

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

Tıp

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.

Amyotrofik Lateral Skleroz için Fare Modeli (ALS) Klinik Test ve Omurilik Kaldırma

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.

Greft damar sertliği için fare modelleri

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.

Fare Akciğer Entübasyonun Basit Bir Yöntem

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;

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. ...

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.

Fare Böbrek Transplantasyonu: Allogreft Reddi Modelleri

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.

Scarless Yara Repair Fare Fetal Cilt Modeli

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.

Fare ile Gastroözofageal Reflü Cerrahi Modelleri

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.

Domuz uzun süreli Kardiyovasküler Egzersiz Testi için Kateter Cerrahi Yerleştirme

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.

Nöromusküler Hastalığının Fare Modelinde Solunum Kas Aktivitesi ve Havalandırma tekrarlanan ölçüm

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