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.

在此协议中描述的所有程序按照由密歇根单位的大学实验动物医学既定法规的规定执行。 1.诱导小鼠基因重组注:TM3 中 Gli1（CRE / ERT2）ALJ / J小鼠品系（ 中Gli1-CreERT2）13启用定向三苯氧胺诱发基因重组到TD上皮。跨越这株B6.129S4-GT（ROSA）26Sor tm1Sor / J记者小鼠（LacZ的 ）22 中Gli1产生;动物LacZ的可视化通过整个安装下面染色TD细胞。 <ol><li>准备他莫昔芬溶液在玉米油中的12.5毫克/毫升的浓度。 <ol><li>在1.5ml管中，添加最多20毫克结晶他莫昔芬，并然后将1ml的玉米油。牢固胶带将管旋涡混合器，并在室温最高设置旋涡不断，直到他莫昔芬已完全溶​​解（2-4小时），如通过在解剖显微镜对没有他莫昔芬的微粒的下检查管证实。 </li><li>转移溶液到15毫升管中并稀释他莫昔芬，以12.5毫克/毫升用另外的玉米油的最终浓度。通过涡旋粘稠溶液另外30秒混合。存储在黑暗中在4℃下向该溶液长达1周。 </li></ol></li><li>腹膜内注射他莫昔芬溶液进入中Gli1; ​​LacZ的小鼠，在每个20克小鼠体重200μl的体积，对于每个20 G鼠标2.5重量毫克有效三苯氧胺的剂量。 </li></ol> 2.嘉实皮肤活检注:根据实验，收获皮肤活检几天到几周他莫昔芬诱导后。对于所有的手术，遵循标准协议灭鼠手术，包括使用无菌手套，戴口罩或头发网，覆盖在手术过程中，用无菌手术单的动物。 <ol><li>在水混合90毫克/毫升氯胺酮和6.5毫克/毫升的甲苯噻嗪准备10倍的股票麻醉剂的解决方案。稀释此储备溶液1:10比例无菌PBS只是在使用前，并在室温在黑暗中储存长达8个月。 <ol><li>可替代地，通过吸入异氟烷麻醉小鼠，用4％的气体浓度与氧气开始充分麻醉动物，并随后降低该至1-2％的程序的持续时间。 </li></ol></li><li>腹膜内注射麻醉剂溶液，剂量为每个20 G鼠标体重200微升。检查动物已经达到脚趾捏法镇静的正确平面，并确认心脏和呼吸率（分别约为600次每分钟160次呼吸，）是正常的。 </li><li>使用电剪从活检网站上的背背部皮肤取下毛发，小心不要缺口或损坏肌肤底层。 </li><li>经擦拭的剃须准备手术部位开发领域中使用聚维酮碘和酒精擦拭前 - 至 - 后方向。确保所有的头发都剪报从网站上删除。 </li><li>介绍了使用黑色标记活检部位，放置在暖垫动物在无菌手术区，并用无菌手术悬垂覆盖（用于演示目的，无菌的悬垂性被忽略了提高知名度）。 注意:要获得毛囊纵向部分，所述活检的长边（边缘被切片用于组织学，约1厘米）应在一个前 - 后方向延伸（平行于毛囊的方向），parasagital到背中线（ 图1A）。 </li><li>使用无菌＃11解剖刀使沿标记的区域的完整厚度切除而不损坏底层肌肉筋膜。 注意:将切下的皮肤组织包括表皮，真皮​​，皮下脂肪和肌层。出血通常是最小的。 </li><li>拼合ŧ他切除上干纸巾皮肤样品，真皮侧倒，修剪掉多余的纸巾，并将样品在冷PBS存储长达1小时，如果其它样品需要被收集。准备就绪后，前往步骤3.1或3.2来处理组织学样品，或整个安装β半乳糖苷酶（lacZ）染色步骤4。 </li><li>缝合关闭使用6-0尼龙缝线活检部位，在间隔开的大致为3毫米的简单中断模式。 </li><li>不要返回已接受手术到同一个笼子里的其他动物，直到完全恢复后的小鼠。 </li><li>监控动物手术后，直到他们每天回过神来，也是直到手术区已愈合，通常在1周。使用止痛剂按照指定的机构动物护理和使用指南，如果小鼠表现出疼痛或痛苦的迹象。手术后取出在7-10天内缝线。 注意:如果需要，通过稀释卡布洛芬（50毫克/毫升股票SOLUT准备镇痛液离子）1:在无菌水中100。注入肩胛骨之间的溶液皮下的颈背附近，在一个剂量的每个20克体重200微升（5毫克/千克小鼠体重）。 </li></ol> 3.处理样品的组织学注意:要解决和处理切除的组织，使用下面根据应用两种方法。 <ol><li>以产生石蜡包埋的组织学样品，固定在3.7％甲醛的PBS中O / N在室温在70％的乙醇对皮肤和存储长达2周。嵌入到石蜡之前取出纸巾。 </li><li>用于产生冷冻的组织学样品，浸入所述组织在PBS中冷的4％多聚甲醛和1小时轻轻摇动。除去溶液，并用PBS 3的变化，每个大约为5分钟，洗涤样品。接着，浸没在30％蔗糖的样品在PBS中cryoprotect组织（"蔗糖下沉"）。 </li><li>轻轻摇动O / N孵育在4℃下。次日，取出纸张束埃尔并从皮肤的真皮侧修剪掉多余的脂肪组织。嵌入组织直接进入OCT和冻块存储在-80℃。 注意:切片后，无论是石蜡或冷冻样品可通过免疫组织化学染色，以确定阵，默克尔细胞和使用抗角蛋白17，角蛋白8和神经丝，分别神经，如先前所描述5,19。 </li></ol> 4.可视化样品中全​​安装的LacZ染色<ol><li>制备的X-gal染色溶液。 <ol><li>结合0.94克磷酸二氢钠，和2.6加入磷酸氢二钠于250ml无菌水中。调节pH值至7.3。此，加入0.5毫升的1M氯化镁，0.528克亚铁氰化钾，和0.412克铁氰化钾。添加辛基苯基聚乙二醇250微升和125毫克脱氧胆。碱溶液可以储存在4℃下长达6个月在黑暗中。 </li><li>准备50X股票的X-gal soluti关于通过加入二甲基甲酰胺的X-gal的库存瓶以产生50毫克/毫升溶液。存储在黑暗中在-20℃下向该溶液。 </li><li>只是使用，稀释的库存的X-gal溶液1:50到的X-gal碱溶液，以产生染色溶液之前。对于较小的活检（&lt;1 平方厘米），每个样品染色溶液等分试样1-2毫升。 </li></ol></li><li>固定在含有2％多聚甲醛/ 0.2％戊二醛在冷PBS 30分钟，在冰上轻轻晃动的溶液步骤2.7中收集的皮肤样品。对于较小的活检（&lt;1 平方厘米），用1-2毫升，每样固定液。 注:另外，只有样品固定在2-4％多聚甲醛，或仅在0.5％的戊二醛。最佳固定条件取决于组织，LacZ的表达和应用的程度。 </li><li>除去固定液，用PBS 3的变化，每次5分钟，于室温在摇床上漂洗样品。 </li><li>取出纸巾样品下方，切去exces通过抓住钝钳脂肪和与解剖剪刀修整上来自皮肤的真皮侧的脂肪组织。 </li><li>浸没在X-gal的染色液的样品，并在37℃CO / N孵育。 LacZ的表达将作为一个解剖显微镜（ 图1B）下蓝染色可见。 注意:如果信号强度较弱，更换染色液第二天，重复O / N培养。如果背景染色过于激烈，减少染色的时间，或在室温下孵育样品，而不是37℃。 </li><li>除去染色液并在含有PBS中的3％DMSO的约5分钟，在RT轻轻摇动3变化洗样品。 </li><li>洗涤样品，每次5分钟，70％乙醇2-3变化。商店的样品在70％的乙醇。 </li></ol> 5.去神经外科<ol><li>麻醉动物如步骤2.2和刮整个背部皮肤。 </li><li>准备手术区○F使用聚维酮碘和酒精擦拭，并用无菌手术单动物的背部皮肤（用于演示目的，无菌的悬垂性被忽略了提高知名度）。用加热垫，而在无菌手术区经营保持动物温暖。 </li><li>使用沿着从颈部的底部背中线无菌＃11解剖刀大致0.5厘米尾部上方的切口。 </li><li>使用钝钳，轻轻反映在左侧的皮肤从侧翼远离形象化从肩胛脂肪垫下面的组织的颈部附近只后肢以上。 注意:背皮神经似乎锐弯并进入疏松结缔组织皮肤下方（ 图2）之前尾端行进穿过躯干壁的半透明面板，为白色链。 </li><li>使用解剖显微镜下超细镊子，从阴性特质的左侧只取出神经升位于通过从拔除解剖学部位T3-12在躯干壁到其进入位点到皮肤的分段弯曲（ 图2）。 <ol><li>东方镊子垂直和由下面的弯曲部位把持约为0.5厘米，向上拉，造成神经伸展和从周围组织（ - E 图2C）分离除去神经。小心避免破裂相邻血管。 </li><li>继续，直到从树干墙皮肤一直延伸神经都被删除。不要把后备箱壁上的密集筋膜内扰乱神经。通过周期性施加无菌的0.9％盐水溶液的液滴保持组织在整个过程湿润。 </li><li>另外，通过把握其近端主干墙钳附近，用细剪刀剪断取下的神经。随后，切断皮肤附近的远端（ 图2F - H）。最后，去掉INTervening神经节段（ 图2I）。 </li></ol></li><li>从步骤5.4暴露皮瓣取出所有的神经。这些纤维包括背皮神经的远端分支，并出现零星位于上皮瓣真皮侧（ 图2J - K）结缔组织内白色分支链。 <ol><li>要删除这些细的分支，定位细镊子大致平行于皮肤表面，抓住神经和勇敢向上，以避免破坏血管和穿刺皮肤。继续，直到所有可见的神经已被删除。 </li></ol></li><li>使用钝性分离，反映背中线切口右侧皮肤，但不删除神经。这将作为对侧假手术对照。 </li><li>沿着在一个简单的断续图案背中线缝合关闭切口。监视恢复和OPER后在动物atively如以前证明（步骤2.8-10）。手术后取出在7-10天内缝线。 </li><li>在功能上评估手术后稳定去神经长达数周，使用电动推剪去除背部皮肤的毛。 </li><li>轻轻挑破使用皮下注射针头失神经支配的皮肤区域，并注意是否动物的响应，通常由打了一个寒颤或转动它的头。如果皮肤面积已稳定失神经支配，动物会表现出很少或没有回应。 </li><li>使用黑色标记，轮廓没有反应的区域，以及类似的大小和位置的对侧假侧的区域。 </li><li>收集从这些站点活组织检查按照步骤2.1-2.9进行分析。 注意:可替换地，整个背侧背部皮肤，包括去神经和假手术的区域，可以作为一个单一的片材整个贴装染色，类似于如在步骤4中所描述的被移除。 </li></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.

神经起到至关重要的作用不仅轰动，而且在哺乳动物器官发育，维持和再生13,24-27。作为神经最近已在不同的皮肤疾病有牵连，此处所描述的技术可以被用来研究在多种动物疾病模型的为支配的要求。的确，单方面去神经技术允许的皮肤与来自相同小鼠完整的或破坏的神经直接比较。这提供了一个理想的内部控制，以补偿从动物到动物的差异，与随后的数据分析利用配对t检验。 而这里描述的过程在很大程度上利用LacZ报告基因，这些实验可以适于使得中Gli1-CreERT2等位基因与其它荧光报道或有条件的等位基因组合以修改在TD基因表达。例如， 中Gli1-CreERT2小鼠可以越过与动物窝藏Patched1（B6N.129-PTCH1 tm1Hahn / J）28的条件基因产生形成诱导他莫昔芬后5 TD-源性肿瘤的小鼠。值得注意的是， 中Gli1-CreERT2应变也诱导重组在物理上不同于在TD 13分离中Gli1 +毛囊干细胞的一个子集是重要的。 下面去神经，皮肤神经保持数月（ 图3C）5,19稳定地烧蚀。在其他研究中，但是，一些重新支配已报道过时间6时发生。去神经表型的perdurance可取决于神经切除的完整性，因为它是绝对关键的切除从胸壁到接近于部位插入到皮肤的真皮层的点的出口之间的神经段。 完全removi纳克从活检之前的皮肤毛发可以增强随后形象化通过整个贴染色（ 图1B）阵的能力。这是通过将脱毛霜到限幅皮肤持续2分钟，然后用棉球擦拭头发远在一个前 - 至 - 后方向来实现的。请注意，脱毛可以通过促进进入生长期生长期影响毛发周期动力学。可替代地，毛发可以完全用刀片除去。此外，整个装入可进行免疫组织化学来可视化上从真皮分离上皮片阵和梅克尔细胞，如先前已描述23。 的可能性仍然是手术去神经可能在手术部位引起炎症，潜在混杂任何观察到的表型。根据我们的经验，我们还没有发现失神经后显著的炎症，可能是因为抵押品组织损伤的发生SK在该过程是否得当是轻微的。的可能性降到最低炎症可能影响结果，额外的控制可以掺入实验。例如，我们观察到去神经特异性抑制的TD源性肿瘤，但不相邻毛囊相关的相同皮肤样品中的病变，认为去神经和不一般的伤口诱导的炎症反应，有可能抑制肿瘤发生在TD 5。 要注意的是外科去神经烧蚀所有皮神经，包括感觉和交感神经纤维5是很重要的，并且因此提供了这些神经上或者是正常的或患病的皮肤的影响的一般整体评估。其他的实验方法，例如使用pharmacologics如肉毒杆菌神经毒素阻断神经传递，可能会产生更详细的机理的见解7，虽然目前还不清楚这些药剂是否抑制逆行细胞因子如刺猬配体的分泌。可替代地，已经使用诸如6-羟基化合物中的皮肤9以烧蚀交感神经。此外，对于神经源性因素如降钙素基因相关肽和P物质靶向受体可以是用于其利基6内询问神经和周围的细胞之间的特异性相互作用有用的。最终，多个策略可以组合用于识别，或至少排除，潜在的信号的机制。 最后，使用或者WNT1-Cre重组酶或Advillin-Cre重组酶可能代表阐明那些神经和自己的优势19之间交换信号的金标准有针对性的神经源性因子基因缺失的神经系。既不这些菌株是他莫昔芬诱导，然而，一些警告需要注意确保这些信号的该中断不损害神经的发育或适当た神经传入rgeting。一个他莫昔芬诱导酶Cre如Advillin-CreERT2的使用可能会有助于规避这些问题29。 总体而言，技术描述这里 - 一个谱系追踪，细胞可视化和去神经外科手术，提供了强有力的办法结合，为研究神经对正常和病变皮肤的影响。随着经验的积累，这些程序可以定期和可靠地执行，而造成最少困扰的动物或调查。

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.

通过表达他莫昔芬诱导中Gli1-CreERT2产生小鼠和LacZ报道等位基因，这是可能的可视化的TD上皮和跟踪这些细胞的命运随时间。整个去神经过程通常可以每只​​小鼠1小时内完成，并应产生最少的困扰的动物。 我们先前的研究已经表明，神经是维持正常阵以及与其相关的角蛋白8 +默克尔细胞（ 图3A - C）的关键5,19。神经也可用于促进在TD（ 图3D） 中Gli1的表达是至关重要的。鉴于TD集群的整个皮肤（ 图1B）的相对较少的外观，当务之急是要多品尝冰冻切片准确定量TD频率。通常情况下，我们评估15个非利弊ecutive部分（各10微米厚，约1厘米长）来自假和失神经支配的皮肤从每个动物。下面去神经，神经的稳定损耗，无论是在TD和整个皮肤，可以通过没有在任一冷冻或石蜡切片（ 图3A和3C标准泛神经标记物如神经丝免疫组织化学染色进行确认，并如先前报道5）。可替代地，神经也可以通过β3微管蛋白或PGP9.5 6,9的表达鉴定。 通过使用中Gli1; ​​LacZ的小鼠中，也有可能在激活在TD Hedgehog信号并通过改变他莫昔芬诱导的重组和去神经的序列保持TD细胞的命运，以确认这两个对神经的要求。如果去神经被重组之前进行，例如，这将测试中激活刺猬通道法对神经的要求Y，如通过Cre重组酶的活性和水平，这与在这些动物中Gli1表达相关监视。另一方面，如果去神经被重组后进行，这将评估在TD保持已标记的细胞为神经的要求。 <img alt="图1" src="/files/ftp_upload/54050/54050fig1.jpg" /> 图1.皮肤活检和TD上皮全安装的LacZ染色。（A）活检后小鼠（顶部）和照片之前，缝合。 （下左）活检部位的放大图像。从活检与干纸巾摊平的真皮侧获得（右下）皮肤样品。 （B）从皮肤中Gli1的全 ​​安装的LacZ染色; LacZ的鼠标，他莫昔芬诱导后7天，脱毛活检，以改善皮肤的可视化前夕。 GLI1 + / LacZ的+ TD所标示为强烈的蓝色集群。比例尺= 1厘米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54050/54050fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54050/54050fig2.jpg" /> 图2.去神经背部皮肤的两种方法。（A）神经支配小鼠皮肤的卡通图。红色虚线表示沿背中线，暴露于躯干壁（紫色）以及真皮（灰色，星号）的反射皮下底层肌肉单个切除。背皮神经尾端旅游出现"弯曲"，因为他们离开体壁（黑色箭头指示了一个这样的弯曲）。待切除神经节段由黑色虚线划定。完整的神经（B）与照片显示的弯曲（箭头）的网站。蓝色箭头指向2神经节段，将REMOved的。 （C - D）照片显示失神经支配的技术。采用超细镊子，夹住神经0.5厘米其弯曲和向外拉的网站下方。 （E）照片显示去除2神经节段（蓝色箭头）后，体腔。剩余的神经也需要被除去。 （F - I）的神经除去的另一种方法被描述，其中神经在它们的近端剪断端略低于那里弯曲（箭头）（G）中，并且还向远侧，靠近其入口的部位进入皮肤（箭头） （H）。注意，在这些图像中的中线切口是比典型的为更好的可视化的目的更长。反射皮瓣真皮侧（J）的照片，以中线的一侧。 （K）神经概述（黑线），以红色表示的血管较大。位于滑雪真皮侧的神经n翼板也需要切除。星号，从反射皮瓣真皮底层。比例尺= 1毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54050/54050fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54050/54050fig3.jpg" /> 图3.神经失神经支配后，TD的恶化稳定的损失。（A）免疫组化显示，在假手术的皮肤角蛋白8 +默克尔细胞（K8，绿色）和神经丝+神经（NF，红色）TD上皮。 （B）卡通描绘，在绿色的紫色，默克尔细胞强调TD上皮细胞，并以蓝色感觉神经。 （C）免疫组化显示神经支配的皮肤（DEN）缺乏TD区内默克尔细胞突起物。虚线黄线，毛囊上皮。星号，背景和基础ð染色。 （ 四 ）背的全安装的LacZ染色背部皮肤从LacZ的 中Gli1 / +鼠标2周后皮肤单侧失神经支配。在框中愈合正中切口（箭头）的左侧，标有丰富TD上皮细胞在假手术皮肤观察。中线的右侧，阵不在去神经皮肤可见。比例尺=10μm的为（A），（C）; 1毫米（D）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54050/54050fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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.

皮肤去神经外科手术:一种用于皮肤病的小鼠模型测试要求的神经方法

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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Medicine

9.7K 次观看.  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.

视频: 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.

使用全血刺激试验的促炎性细胞因子IL -1β的精确和简单的测量

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

科研

医学

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.

临床试验和脊髓去除在肌萎缩性侧索硬化症的小鼠模型（ALS）

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.

嫁接动脉硬化的小鼠模型

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.

一个简单的方法小鼠肺插管

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;

胫骨神经切断术 - 一个标准化的模型失神经诱导小鼠骨骼肌

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.

鼠肾移植:移植排斥反应模型

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.

对无瘢痕修复鼠标胎儿皮肤模型

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.

胃食管反流与小鼠手术模型

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.

导管手术放置了长期的心血管运动试验猪

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.

在神经肌肉疾病的模型小鼠呼吸肌肉活动和通风的重复测量

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