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.

このプロトコルで説明されているすべての手順は、実験動物医学のためにミシガン大学の単位によって確立された規則に従って行われました。 1.マウスの遺伝的組換えを誘導します注：Gli1の TM3（CRE / ERT2）ALJ / Jマウス系統（Gli1の -CreERT2）13は、上皮をTDためにタモキシフェン誘導性の遺伝子組換えの標的化を可能にします。 Gli1のを生成するために22（ のLacZ）B6.129S4-GT（ROSA）26Sor tm1Sor / Jレポーターマウスとこの株を渡り、LacZを動物以下の染色ホールマウントにより、TD細胞を可視化します。 <ol><li>コー​​ン油中の12.5mg / mlの濃度にタモキシフェン溶液を調製します。 <ol><li> 1.5ミリリットルチューブでは、結晶性タモキシフェンを20mg、及びコーン油の後、1ミリリットルまで追加。タモキシフェンは、完全に（2-4時間）に溶解するまでのように、しっかりとボルテックスミキサーにチュ​​ーブをテープ、そしてRTで最高の設定で連続渦タモキシフェン微粒子の欠如のために解剖顕微鏡下で管を検査することによって確認されました。 </li><li> 15ミリリットルチューブにソリューションを転送し、追加のトウモロコシ油とミリリットルの12.5mg /の最終濃度にタモキシフェンを希釈します。さらに30秒間、粘性溶液をボルテックスして混合します。暗所で4℃で1週間までこの溶液を保管してください。 </li></ol></li><li> Gli1の腹腔内にタモキシフェンソリューションを注入し、LacZのマウスを、マウス体重の20グラムあたり200マイクロリットルの容量で、20グラムのマウスの重量当たり2.5ミリグラムの有効タモキシフェン投与量のために。 </li></ol> 2.収穫皮膚生検注：タモキシフェン誘導後数週間の実験に応じて、収穫の皮膚生検数日。すべての手術のために、滅菌手袋を使用して、外科手術用マスクやヘアネットを身に着けている、と手順の間、滅菌外科用ドレープで動物をカバーするなどの齧歯類の手術のための標準プロトコルに従います。 <ol><li>水に90 mg / mlでケタミンおよび6.5 mg / mlとキシラジンを混合することにより、10倍の株式麻酔薬溶液を調製します。使用直前に無菌PBSにこの原液を1:10に希釈し、最大8ヶ月間、暗所で室温で保管してください。 <ol><li>また、完全に動物を麻酔するために酸素と4％のガス濃度で始まる、イソフルラン吸入によってマウスを麻酔し、その後手続きの期間中、一から二パーセントにこれを下げます。 </li></ol></li><li> 20グラムマウス体重あたり200μlの用量で腹腔内麻酔液を注入します。動物はつま先のピンチアッセイによる鎮静の適切な面に到達したことを確認し、その心を確認し、呼吸率は（それぞれ、約600拍毎分160回の呼吸）正常です。 </li><li> 、背部背部皮膚に生検部位から髪を削除しないニックように注意してか、下にある皮膚を損傷する電気バリカンを使用してください。 </li><li>剃りを拭いて手術部位を準備ベタジンとアルコールワイプを使用して、前方対後方方向でD領域。すべての髪の切り抜きがサイトから削除されていることを確認します。 </li><li>無菌手術領域に加温パッドの上に動物を配置し、黒のマーカーを用いて、生検部位を概説し、（デモの目的のために、滅菌ドレープが視認性を高めるために省略されました）滅菌外科用ドレープでカバー。 注：毛包の長手方向部分を取得するには、生検（エッジが組織学のために切片するために、約1センチ）の長辺はにparasagital、前後方向（毛包の方向に平行）で実行する必要があります背側正中線（ 図1A）。 </li><li>根本的な筋肉の筋膜に損傷を与えることなく、マークされた領域に沿って全層切除を行うために、無菌＃11メスを使用してください。 注：切除した皮膚組織は、表皮、真皮、皮下脂肪と皮筋層を含んでいます。出血は通常、最小です。 </li><li>トンをフラット化彼は乾いたペーパータオルの上皮膚試料を切除し、真皮は、ダウンサイド過剰ペーパータオルを離れてトリム、および他のサンプルを収集する必要がある場合、最大1時間の冷PBSでサンプルを保存します。準備ができたら、組織学、または全載βガラクトシダーゼ（LacZを）染色のためのステップ4のためのサンプルを処理するためにステップ3.1または3.2に進みます。 </li><li>縫合糸は、およそ3ミリメートル離間シンプル中断したパターンで、6-0ナイロン縫合糸を使用して、生検部位を閉じます。 </li><li>完全回復後まで他の動物と同じケージに手術を受けたマウスを返さないでください。 </li><li>手術領域は、典型的には、1週間以内に、治癒するまで、彼らは毎日も意識を取り戻す、とまでは手術直後に動物を監視します。マウスは、痛みや苦痛の徴候を示す場合、指定機関の動物の管理と使用ガイドラインに従って鎮痛薬を使用してください。手術後7-10日以内に縫合糸を取り外します。 注：必要に応じて、（50 mg / mlで株式solutカルプロフェンを希釈することにより、鎮痛剤溶液を調製イオン）1：滅菌水で100。 20gの体重あたり200μlの（5ミリグラム/ kgのマウス体重）の用量で、首筋の近くに肩甲骨の間の溶液の皮下注射。 </li></ol>組織学のため3.プロセスのサンプル注：切除した組織を修復し、処理するには、アプリケーションに応じて、以下のいずれかの方法を使用します。 <ol><li>パラフィン包埋組織学的サンプルを生成するには、2週間まで70％エタノール中で、室温でPBS O / Nで3.7％ホルマリンで皮膚や店舗を修正。パラフィンに包埋する前にペーパータオルを削除します。 </li><li>凍結した組織学的サンプルを生成するために、PBS中の冷4％パラホルムアルデヒド中で組織を沈め、ゆっくりと1時間振とうします。溶液を除去し、PBSを3回交換し、約5分ごとにサンプルを洗います。次に、組織（「ショ糖沈没」）をcryoprotectするPBS中30％スクロース中にサンプルを沈めます。 </li><li> 4℃で穏やかに振盪しながら、O / Nでインキュベートします。翌日、ペーパータオルを削除エルと皮膚の真皮側から過剰な脂肪組織を切り落とします。 10月に直接組織を埋め込み、-80℃で凍結したブロックを格納します。 注意：以前に5,19に記載のように切 ​​片した後、パラフィンまたは凍結サンプルのいずれか、のTD、メルケル細胞とそれぞれケラチン17、ケラチン8およびニューロフィラメントに対する抗体を使用して神経を識別するために、免疫組織化学によって染色することができます。 </li></ol>ホールマウントのLacZ染色4.視覚化サンプル<ol><li> X-gal染色溶液を調製します。 <ol><li>滅菌水の250ミリリットルで0.94グラムの一塩基性リン酸ナトリウム、および2.6グラムのリン酸ナトリウム二塩基性を兼ね備えています。 7.3にpHを調整します。このために、1 M塩化マグネシウム、フェロシアン化カリウムの0.528グラム、およびフェリシアン化カリウムの0.412グラムの0.5ミリリットルを追加します。オクチルフェニルポリエチレングリコールの250μlを添加して、デオキシコール酸125mgの。塩基溶液は暗所で6ヶ月まで4℃で保存することができます。 </li><li> 50倍株式のX-galのsolutiを準備50 mg / mlで解決策を生成するためのX-galストックボトルにジメチルホルムアミドを添加することにより、上。暗所で-20℃でこの溶液を保管してください。 </li><li>使用直前に、染色溶液を生成するために、X-GALのベース溶液に在庫のX-gal溶液を1:50に希釈します。小さ ​​な生検（&lt;1 cm 2）を、サンプル当たりの染色溶液のアリコート1-2 mlのため。 </li></ol></li><li>静かに氷上で揺れ、30分間冷PBS中2％パラホルムアルデヒド/ 0.2％グルタルアルデヒドを含む溶液中で、ステップ2.7で採取した皮膚のサンプルを修正しました。小さ ​​い生検のために（&lt;1 cm 2）を、サンプルあたりの固定液1〜2mlのを使用します。 注：また、のみ、またはわずか0.5％グルタルアルデヒド中で2〜4％パラホルムアルデヒドでサンプルを固定します。最適な固定条件は、組織、LacZ発現およびアプリケーションの度合いに依存します。 </li><li>固定剤溶液を除去し、室温でシェーカー上で、PBSを3回交換し、5分ごとにサンプルをすすいでください。 </li><li>サンプルの下にペーパータオルを取り外し、excesを切り取ります鈍鉗子で脂肪を把持し、解剖ハサミでトリミングすることにより、皮膚の真皮側から脂肪組織をね。 </li><li> X-gal染色溶液中に試料を浸し、37°C​​O / Nでインキュベートします。 LacZ発現は、解剖顕微鏡（ 図1B）の下でブルー染色として表示されます。 信号強度が弱い場合、翌日染色液を交換し、O / Nインキュベーションを繰り返します注意してください。バックグラウンド染色が強すぎる場合には、染色の時間を短縮し、またはその代わりに、37℃の室温でサンプルをインキュベートします。 </li><li>染色液を外し、ゆっくりと室温で振盪しながら、約5分間、3％のDMSOを含有するPBSの3変化でサンプルを洗います。 </li><li> 5分間、70％エタノールの2-3の変化で洗浄サンプル。 70％エタノールに保管サンプル。 </li></ol> 5.外科神経切除<ol><li>ステップ2.2のように動物を麻酔し、全体の背部皮膚を剃ります。 </li><li> O手術領域を準備しますベタジンとアルコール綿を使用して、背中の皮膚fおよび滅菌外科用ドレープ（デモンストレーション目的のために、滅菌ドレープが視認性を高めるために省略された）で動物をカバーしています。無菌手術領域で動作している間に加熱パッドを用いて動物を暖かく保ちます。 </li><li>尾以上のおよそ0.5センチメートルに首の付け根から背側正中線に沿って滅菌＃11メスを用いて切開を行います。 </li><li>鈍鉗子を使用して、静かにちょうど後肢上記に首の近くに肩甲骨脂肪パッドから下にある組織を可視化するために離れてサイドから左サイドの肌を反映しています。 注：背側皮膚神経は急カーブを作り、皮膚（ 図2）の下の疎性結合組織に入る前に、トランク壁の半透明の筋膜を介して尾側に移動する白いストランドとして表示されます。 </li><li>解剖光顕微鏡下で超微細鉗子を使用して、アニマの左側から独占的に神経を取り除きますどこから摘採によって解剖学的部位T3-12に位置リットル皮膚への参入サイトへのトランク壁におけるセグメントの曲がり（ 図2）。 <ol><li>オリエント鉗子垂直とその曲げサイトの下約0.5cmに把握し、上向きに引っ張り、伸ばし、周囲の組織（ 図2C - E）から分離するために神経を引き起こすことによって神経を取り除きます。隣接する血管を破裂しないように注意してください。 </li><li>皮膚へのトランク壁から延びているすべての神経が除去されるまで続けます。トランク壁の密な筋膜内の神経に影響を与えることはありません。定期的に0.9％滅菌生理食塩水の滴を適用することにより、作業中はしっとり組織を保管してください。 </li><li>また、鉗子でトランク壁の近く、その近位端を把握し、細かいハサミでつむことにより、神経を取り除きます。その後、皮膚に近い先端部を切断する（ 図2F - H）。最後に、int型を削除しますervening神経セグメント（ 図2I）。 </li></ol></li><li>ステップ5.4で露出した皮膚フラップから任意の神経を削除します。これらの繊維は、背側皮膚神経の遠位枝を含み、散発的に皮弁（ 図2J - K）の真皮側の結合組織内に配置された白の分岐ストランドとして表示されます。 <ol><li>これらの細かい枝を削除するには、微細な鉗子は、大きく、皮膚表面に平行神経を把握し、血管を破壊し、皮膚を穿刺避けるために、上向きに摘み取る位置付けます。すべての可視神経が削除されるまで続けます。 </li></ol></li><li>鈍的切開を使用して、背側正中切開の右側の皮膚を反映するが、神経を削除しないでください。これは、反対側の偽手術対照として機能します。 </li><li>切開部を閉鎖するための簡単​​な中断されたパターンで背側正中線に沿って縫合。回復とポストoperの中に動物を監視しますativelyとして以前に（ステップ2.8から10）を実証しました。手術後7-10日以内に縫合糸を取り外します。 </li><li>機能的に手術後数週間に安定した除神経アップを評価するために、電気バリカンを使用して背部皮膚から毛を取り除きます。 </li><li>静かに皮下注射針を使用して、除神経皮膚領域を刺すと、動物は、通常、身震いまたはその頭を回すことにより、応答するかどうかを注意してください。皮膚領域を安定的除神経された場合、動物は、ほとんどまたは全く応答を示すであろう。 </li><li>黒のマーカーを使用して、反対側の偽側の無応答の面積だけでなく、同​​様のサイズと位置の領域の輪郭を描きます。 </li><li>分析のための手順2.1から2.9のように、これらのサイトからの生検を収集します。 注：別の方法として、除神経および偽手術領域を含む全体の背背中の皮膚は、ステップ4で説明したように似たホールマウント染色のための単一のシートとして除去することができます。 </li></ol>

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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.

神経は感覚ではなく、哺乳動物の臓器の開発、保守及び再生13,24-27だけでなく重要な機能を果たします。神経が最近多様な皮膚疾患に関与しているように、ここで説明される技術は、動物疾患モデルの様々な神経支配の要件を研究するために使用することができます。確かに、一方的除神経の技術は、同じマウスから無傷または破壊神経のいずれかで皮膚の直接比較を可能にします。...

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.

タモキシフェン誘導性Gli1の-CreERT2およびLacZレポーター対立遺伝子を発現するマウスを生成することで、TD上皮を視覚化し、時間をかけてこれらの細胞の運命を追跡することが可能です。全体の除神経手順は、典型的にはマウスあたり1時間以内に完了することができ、動物に最小限の苦痛を引き起こす必要があります。 <p class="jove_content" fo:keep-together.wi...

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.

皮膚外科神経切除：皮膚病のマウスモデルにおける神経のための要件をテストする方法

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 ビュー.  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.

ビデオ: 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.

マウスで誘導機械的異痛症の倹約神経損傷（SNI）モデル

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

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;

脛骨神経切断 - マウスにおける脱神経誘発性骨格筋萎縮のために標準化されたモデル

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.

マウスにおける顔面神経軸索切断：傷害に運動ニューロン応答を研究するためのモデル

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.

一過性受容体電位バニロイド型 1 Ultrapotent アゴニストと純粋な小さな繊維の神経疾患のマウスモデルを確立します。

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

神経科学

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.

糖尿病マウスの慢性創傷を作成するためのプロトコル

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>マウスの神経因性疼痛の改良温存神経損傷手術モデル

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.

マウスの神経因性疼痛の改良温存神経損傷手術モデル

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.

坐骨神経部分結紮術:新規治療法の抗侵害受容効果を研究するための慢性神経因性疼痛のマウスモデル

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

マウス交感神経ニューロンの標的化:腰椎交感神経切除術アプローチ

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.

著者スポットライト:交感神経ニューロンの可塑性の探索

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.

マウスの外科的腰椎交感神経切除術

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

皮膚外科神経切除：皮膚病のマウスモデルにおける神経のための要件をテストする方法

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坐骨神経部分結紮術:新規治療法の抗侵害受容効果を研究するための慢性神経因性疼痛のマウスモデル

マウスの外科的腰椎交感神経切除術

マウスの外科的腰椎交感神経切除術