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。 <p class=...

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

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

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

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

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</stro...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

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

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

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

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

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

幸免神经损伤（SNI）模型引起的机械触感小鼠

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

科研

医学

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. ...

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

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

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

面神经干切断小鼠:模型，研究运动神经元损伤反应

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

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

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

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

利用瞬态受体电位辣椒1型 Ultrapotent 激动剂建立单纯小纤维神经病变的小鼠模型

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

神经科学

Protocol to Create Chronic Wounds in Diabetic Mice

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

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

在糖尿病小鼠中创建慢性伤口的协议

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

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

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

<meta charset="utf8"></head><body>改良的小鼠神经性疼痛备用神经损伤手术模型

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

改良的小鼠神经性疼痛备用神经损伤手术模型

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

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

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

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

部分坐骨神经结扎术:慢性神经性疼痛的小鼠模型，用于研究新疗法的抗伤害感受作用

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

靶向小鼠交感神经元：一种腰交感神经切除术方法

Surgical Lumbar Sympathectomy in Mice

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

作者聚焦：探索交感神经元的可塑性

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

小鼠手术腰交感神经切除术

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

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

摘要

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此视频中的章节

幸免神经损伤（SNI）模型引起的机械触感小鼠

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