Name: murine flexor tendon injury and repair surgery
Uploaded: 2016-09-19T14:00:00
Description: Watch this Scientific Journal Video about Murine Flexor Tendon Injury and Repair Surgery at JoVE.com

この作品は、部分的に手パイロット賞、NIH / NIAMS 1K01AR068386-01（AEL）とNIAMS / NIH P30AR061307の手術のためのアメリカの社会によってサポートされていました。

ロチェスター大学の動物研究に関する大学委員会は、すべての動物実験を承認しました。十12週齢の雌のC57BL / 6Jマウスを使用しました。 屈筋腱の手術のための動物の調製（〜15分） <ol><li>オートクレーブ手術器具は、滅菌を通して滅菌手袋を着用し、無菌手術野を維持します。 </li><li>体重に応じケタミンの量（80mg / kg）およびキシラジン（10mg / kg）を腹腔内注射（IP）?...

<ol>
	<li>Lin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanics+of+tendon+inury+and+repair.">Biomechanics of tendon inury and repair.</a> J Biomech. 37, 865-877 (2004).</li><li>Strickland, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+flexor+tendon+surgery:+twenty-five+years+of+progress.">Development of flexor tendon surgery: twenty-five years of progress.</a> J Hand Surg [Am]. 25, 214-235 (2000).</li><li>Bunnell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repair+of+tendons+in+the+fingers+and+description+of+two+new+instruments.">Repair of tendons in the fingers and description of two new instruments.</a> Surg Gynecol Obstet. 26, 103-110 (1918).</li><li>Aydin, 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=Single-stage+flexor+tendoplasty+in+the+treatment+of+flexor+tendon+injuries.">Single-stage flexor tendoplasty in the treatment of flexor tendon injuries.</a> Acta Orthop Traumatol Turc. 38, 54-59 (2004).</li><li>Gelberman, R. H., Steinberg, D., Amiel, D., Akeson, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+chemotaxis+after+tendon+repair.">Fibroblast chemotaxis after tendon repair.</a> J Hand Surg Am. 16, 686-693 (1991).</li><li>Lundborg, G., Rank, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+intrinsic+healing+of+flexor+tendons+based+upon+synovial+fluid+nutrition.">Experimental intrinsic healing of flexor tendons based upon synovial fluid nutrition.</a> J Hand Surg Am. 3, 21-31 (1978).</li><li>Aoki, M., Kubota, H., Pruitt, D. L., Manske, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+and+histologic+characteristics+of+canine+flexor+tendon+repair+using+early+postoperative+mobilization.">Biomechanical and histologic characteristics of canine flexor tendon repair using early postoperative mobilization.</a> J Hand Surg Am. 22, 107-114 (1997).</li><li>Kim, H. 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=Technical+and+biological+modifications+for+enhanced+flexor+tendon+repair.">Technical and biological modifications for enhanced flexor tendon repair.</a> J Hand Surg Am. 35, 1031-1037 (2010).</li><li>Aoki, M., Manske, P. R., Pruitt, D. L., Kubota, H., Larson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Work+of+flexion+after+flexor+tendon+repair+according+to+the+placement+of+sutures.">Work of flexion after flexor tendon repair according to the placement of sutures.</a> Clin Orthop Relat Res. , 205-210 (1995).</li><li>Zhao, 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=Award+for+Outstanding+Orthopaedic+Research:+Engineering+flexor+tendon+repair+with+lubricant,+cells,+and+cytokines+in+a+canine+model.">Award for Outstanding Orthopaedic Research: Engineering flexor tendon repair with lubricant, cells, and cytokines in a canine model.</a> Clin Orthop Relat Res. 472, 2569-2578 (2014).</li><li>Wong, J., Bennett, W., Ferguson, M. W., McGrouther, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscopic+and+histological+examination+of+the+mouse+hindpaw+digit+and+flexor+tendon+arrangement+with+3D+reconstruction.">Microscopic and histological examination of the mouse hindpaw digit and flexor tendon arrangement with 3D reconstruction.</a> J Anat. 209, 533-545 (2006).</li><li>Katzel, E. 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=Impact+of+Smad3+loss+of+function+on+scarring+and+adhesion+formation+during+tendon+healing.">Impact of Smad3 loss of function on scarring and adhesion formation during tendon healing.</a> J. Orthop. Res. 29, 684-693 (2011).</li><li>Loiselle, A. E., 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=Bone+marrow-derived+matrix+metalloproteinase-9+is+associated+with+fibrous+adhesion+formation+after+murine+flexor+tendon+injury.">Bone marrow-derived matrix metalloproteinase-9 is associated with fibrous adhesion formation after murine flexor tendon injury.</a> PloS one. 7, e40602 (2012).</li><li>Lee, D. 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=Parathyroid+hormone+1-34+enhances+extracellular+matrix+deposition+and+organization+during+flexor+tendon+repair.">Parathyroid hormone 1-34 enhances extracellular matrix deposition and organization during flexor tendon repair.</a> J Orthop Res. 33, 17-24 (2015).</li><li>Geary, M. 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=Systemic+EP4+Inhibition+Increases+Adhesion+Formation+in+a+Murine+Model+of+Flexor+Tendon+Repair.">Systemic EP4 Inhibition Increases Adhesion Formation in a Murine Model of Flexor Tendon Repair.</a> PloS one. 10, e0136351 (2015).</li><li>Loiselle, A. E., 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=Development+of+antisense+oligonucleotide+(ASO)+technology+against+Tgf-beta+signaling+to+prevent+scarring+during+flexor+tendon+repair.">Development of antisense oligonucleotide (ASO) technology against Tgf-beta signaling to prevent scarring during flexor tendon repair.</a> J Orthop Res. 33, 859-866 (2015).</li><li>Orner, C. A., Geary, M. B., Hammert, W. C., O'Keefe, R. J., Loiselle, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+and+short-duration+Matrix+Metalloproteinase+9+Inhibition+does+not+affect+adhesion+formation+during+murine+flexor+tendon+healing.">Low-dose and short-duration Matrix Metalloproteinase 9 Inhibition does not affect adhesion formation during murine flexor tendon healing.</a> Plast Reconstr Surg. , (2016).</li><li>Loiselle, A. E., 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=Remodeling+of+murine+intrasynovial+tendon+adhesions+following+injury:+MMP+and+neotendon+gene+expression.">Remodeling of murine intrasynovial tendon adhesions following injury: MMP and neotendon gene expression.</a> J Orthop Res. 27, 833-840 (2009).</li><li>Tsubone, T., 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=Effect+of+TGF-beta+inducible+early+gene+deficiency+on+flexor+tendon+healing.">Effect of TGF-beta inducible early gene deficiency on flexor tendon healing.</a> J Orthop Res. 24, 569-575 (2006).</li><li>Beason, D. P., Kuntz, A. F., Hsu, J. E., Miller, K. S., Soslowsky, 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=Development+and+evaluation+of+multiple+tendon+injury+models+in+the+mouse.">Development and evaluation of multiple tendon injury models in the mouse.</a> J Biomech. 45, 1550-1553 (2012).</li><li>David, M. 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=Tendon+repair+is+compromised+in+a+high+fat+diet-induced+mouse+model+of+obesity+and+type+2+diabetes.">Tendon repair is compromised in a high fat diet-induced mouse model of obesity and type 2 diabetes.</a> PloS one. 9, e91234 (2014).</li><li>Wong, J. K., 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+cellular+biology+of+flexor+tendon+adhesion+formation:+an+old+problem+in+a+new+paradigm.">The cellular biology of flexor tendon adhesion formation: an old problem in a new paradigm.</a> Am J Pathol. 175, 1938-1951 (2009).</li></ol>

完全離断および長趾屈筋腱の修復のマウスモデルのための外科的処置は、本研究で提示されています。また細胞診遠心機で小細胞集団を濃縮する新たなアプリケーションは、屈筋腱の治癒中の細胞環境の定量的免疫細胞化学分析を可能にする、実証されています。屈筋腱修復のこのモデルは、ノックアウトモデルを用いて、治癒過程の変化または薬理学的介入を評価するために使用すること?...

屈筋前腕の筋肉やデジタル鞘と協調して手作業では屈筋腱は、数字の屈曲と手の把握機能を有効にします。屈筋腱は手の掌側面に沿って実行します。この比較的表面的な場所は、多くの場合、手の外傷の間に屈筋腱の損傷につながります。腱は瘢痕組織の応答ではなく、正常な腱組織1の再生を通じて癒します。この瘢痕組織は、腱への連続性を提供するが、機能は劇的に健康な腱に比べて減少します。腱-瘢痕組織の複合材料は、破裂する可能性が高く、修復腱をレンダリングする、損なわれた機械的性質1を特徴とします。また、瘢痕組織は、腱のサイズおよびバルクの増加をもたらす、天然の腱のコラーゲン線維構造の組織化を欠いています。腱鞘部の解剖学的制約、腱サイズであっても適度な増加を考えることができます大幅に赤滑走腱の機能、動作および手の機能の、したがって数字範囲をUCE。 屈筋腱に1960年の怪我、手のゾーンIIで特に以前には、日常的に起因するこれらの修理2で発生した治癒における重篤な合併症に修復されませんでした。手のこの領域は、「だれの土地3 &#39;と呼ばれていました。しかし、外科技術、縫合糸パターンと物理療法リハビリテーションプロトコールの改善は劇的に屈筋腱の修理2の成果を改善しています。これらの進歩にもかかわらず、修理の40％までは手の機能4を阻害するために十分な接着形成をもたらします。したがって、生物学的アプローチは、治癒を改善するために必要とされます。残念ながら、非常に少ないが、細胞および分子レベルでの腱の治癒過程について知られています。したがって、目標は、基本understandinを改善するために使用することができるマウスモデルを開発することでした治癒を改善するために新規な治療標的を同定するための手段としての屈筋腱の治癒および瘢痕形成の処置の細胞および分子成分のgでした。 より大きな動物モデルは、屈筋腱の治癒過程の理解を促進に尽力してきました。犬とウサギの研究は屈筋腱5,6の内因性および外因性の治癒能力、固定化7に癒着形成の相対を最小限に抑えることで、早期に制御受動運動の重要性だけでなく、治癒過程8上の異なる縫合パターンの両方の効果を実証しています、9。また、イヌのモデルは、治癒10を改善するために翻訳組織工学的ア ​​プローチをテストする際に有用でした。しかし、相対的なコスト、マウス特異的試薬の利用可能性、およびグローバルKNOCの生成を容易にするなどの大型動物モデルにマウスモデルの相対的なを使用していくつかの重要な利点は、ありますK-アウトまたは組織特異的欠失/過剰発現コンストラクト。また、屈筋腱11に対して人間とマウスとの間の機能的類似性はマウスモデルの開発に潜在的有用性を示しています。 屈筋腱の切断と修復を模倣豊富な瘢痕組織および減損の機械的特性の形成を含む臨床治癒の多くの側面のマウスモデルの開発。ここで説明したモデルは、修復部位を保護するためにmyotendinous接合部におけるFDLの切断に起因する臨床実践の真の要約ではありません。修理が発生した腱の中間部分を覆う全く滑膜シースがないのでさらに、このモデルは、治癒反応に滑膜鞘細胞の寄与を考慮していません。これらの制限にもかかわらず、このモデルは、よりクロことをマウスモデルにおいて実証されるように、まだ持っている、運動制限癒着の発生範囲の利点を有していますエリーの臨床シナリオに近似しています。このモデルは、ノックアウトマウスモデル12,13を評価するため、および治癒14-17を改善するための別の薬理学的ア ​​プローチを試験するために使用されてきました。組織学的、免疫組織化学を使用して、このモデルの解析およびin situハイブリダイゼーションでは、治癒の間に重要な遺伝子やタンパク質の局在化に重要な洞察を提供することができます。しかし、組織学のみ断面空間分析を提供し、組織全体を通じて定量化を許可しません。フローサイトメトリー、より定量的なアプローチであるが、細胞のごく限られた数のマウスモデルにおける治癒腱組織から単離することができ、その数はさらに固定、透過処理、および洗浄工程中に減少します。アカウントにこれを取って、フローサイトメトリーをすることは、必要とされる動物の数に実現不可能なアプローチになります。別の方法は、順番にこの小細胞集団の大部分を維持することが必要ですさらに癒しの環境を特徴づけます。ここに示され、これを達成するために使用される方法は、免疫細胞化学、続いてスライドガラス上に細胞学遠心分離によって単離された細胞の濃度を含みます。本研究EDU（5-エチニル2&#39;deoxyuridine、チミジンアナログ）に取り込みおよびその後の標識化は、治癒部位での細胞の相対的な増殖状態を決定するために使用しました。このアプローチは、細胞増殖、遺伝子ノックアウトまたは過剰発現の薬理学的治療の有効性を試験するために、または異なる細胞集団を同定し、定量化するために適用することができます。

<table border="0" cellpadding="0" cellspacing="0" width="1047">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6J mice&#160;</td>
			<td>Jackson Laboratories</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine</td>
			<td>Hospira</td>
			<td>NDC# 0409-2051-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine</td>
			<td>Lloyd Inc.</td>
			<td>NDC# 61311-482-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Buprenorphine</td>
			<td>Par Pharmaceutical Inc.</td>
			<td>NDC# 42023-179-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.9% sodium chloride irrigation</td>
			<td>Hospira</td>
			<td>NDC# 0409-6138-03</td>
			<td>For preparation of ketamine/xylazine and buprenorphine solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1ml syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30G needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone-Iodine solution</td>
			<td>Aplicare</td>
			<td>82-226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70% ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puralube vet opthalmic ointment</td>
			<td>Dechra Veterinary Products</td>
			<td>NDC# 17033-211-38</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Portable balance 200g</td>
			<td>Ohaus</td>
			<td>SP202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15124-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle holders</td>
			<td>Fine Science Tools</td>
			<td>91201-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro needle holders</td>
			<td>Fine Science Tools</td>
			<td>12061-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-0 nylon sutures</td>
			<td>Ethicon</td>
			<td>668G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8-0 microsurgery nylon sutures</td>
			<td>Ethicon</td>
			<td>2808G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab-Line histology slide warmer</td>
			<td>Barnstead International</td>
			<td>26025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytospin method</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase Type I, lyophilized</td>
			<td>Life Technologies&#160;</td>
			<td>1700-017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>Cell Signaling Technologies</td>
			<td>9998S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1X PBS</td>
			<td>Thermo Fisher</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytology funnels</td>
			<td>Fisher HealthCare</td>
			<td>10-354</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HistoBond+ microscope slides</td>
			<td>VWR</td>
			<td>16005-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cytospin 2 centrifuge</td>
			<td>Shandon</td>
			<td>SH-CYTO2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunocytochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide staining tray with black lid</td>
			<td>IHC World</td>
			<td>M920-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Click-iT Plus EdU Imaging Kit</td>
			<td>Life Technologies&#160;</td>
			<td>C10639</td>
			<td>Includes EdU and&#160; Hoeschst 33342</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immedge hydrophobic barrier pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Diamond mounting medium</td>
			<td>Thermo Fisher</td>
			<td>P36970</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass coverslips 24x50mm #1.5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clear nail polish</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

murine flexor tendon injury and repair surgery

腱は、ほぼ全身の動きを促進する、骨格筋と骨をつなぎます。手には、屈筋腱（FTS）は、指や一般的な手の機能の屈曲を可能にします。 FTへの傷害は一般的であり、十分な治癒は、多くの場合、過剰な瘢痕組織と腱と周囲の組織との間の癒着に損なわれています。しかし、少しはFT修復の分子と細胞成分について知られています。そのために、運動の障害範囲を含む、ヒトにおける治癒の多くの側面を再現すると、機械的特性を低下FT修復のマウスモデルを開発し、以前に記載されています。ここでは、この外科手術手順の詳細なデモが離断およびマウス後足における屈筋digitorumの長い（FDL）腱のその後の修復を含む、提供されます。この技術は、遺伝子の獲得または機能喪失の効果を評価し、EFFIをテストするために、異なる細胞型の系統解析を実施するために使用することができ治癒過程における薬理学的介入のcacy。 ⅰ）離断し、修復が発生し、マウスの後足の中間部におけるFDLの腱は、滑膜シースに囲まれていません。しかし、このモデルには2つの主要な制限があります。したがって、このモデルは、瘢痕形成プロセスへのシースの潜在的寄与を考慮していません。 ⅱ）修復部位の完全性を保護するために、FTは、おそらく増加した瘢痕形成に寄与し、腱の機械的な力を減少させること、myotendinous接合部で放出されます。フローサイトメトリー分析のために治癒過程中FTの肉芽組織から十分な細胞の単離は困難なことが証明されています。これらの細胞を濃縮するための細胞学遠心分離が使用される代替方法であり、免疫蛍光標識を行うことができるの細胞調製物の生成を可能にします。この方法では、FT治癒の間、細胞又は対象のタンパク質の定量化が可能となります。

ふくらはぎに位置屈筋digitorumの長い（FDL）筋肉は、myotendinousから近位に実行屈筋腱（ 図1Aに青色で概説され、 図2Aに組織学的に示される）を介して、マウスの後足の数字を曲げるように作用します接合部および遠位指節で終了。後足の桁（赤い矢印、 図1A）に分岐し、近位の屈筋腱の治癒のこのモデルでは、FDLの腱が離断され、?...

Watch this Scientific Journal Video about Murine Flexor Tendon Injury and Repair Surgery at JoVE.com

Murine Flexor Tendon Injury and Repair Surgery

手に屈筋腱は、一般的に損なわれた手の機能につながる、負傷しています。しかし、瘢痕組織の治癒反応は十分に特徴付けされていません。屈筋腱の治癒のマウスモデルは、ここに示されています。このモデルは、治癒過程の全体的な理解を高め、治癒を改善するための治療法を評価することができます。

マウス屈筋腱損傷および修復手術

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers ...

The over all goal of this surgical procedure is to enhance the understanding of the cellular and molecular processes of scar and adhesion formation during flexor tendon healing. This method can help answer key questions about the flexor tendon healing process, such as which cell types contribute to scar formation and repair. The main advantage of this technique is that transgenic and knock out mice can be used to better understand the healing process.This technique may identify potential biological targets for improving the outcomes of flexor tendon healing as it facilitates the investigation of these poorly designed cell populations that contribute to scarring. After confirming a lack of response to toe pinch, apply a ophthalmic ointment to the animals eyes, and clip the fur from the entire hind limb. Sterilize the surgical site with sequential povedone iodine and 70%ethanol scrubs, and locate the flexor digitorum longus, or FDL tendon within the medial aspect of the calf.Next, make a 0.5 to once centimeter incision in the skin to expose the tendon. Use the forceps to separate the FDL tendon from the surrounding tissue and trace the tendon up to the miotendinous junction. Using spring scissors, release the tendon at the junction, taking care to avoid the posterior tibial artery.Then close the skin with 5-0 nylon sutures. Now transfer the mouse under a stereo microscope and use spring micro scissors to make a three millimeter incision over the posterolateral aspect of the hind paw. Use the forceps to gently retract the surrounding soft tissue and muscle, taking care to minimize the tissue damage, and identify the FDL tendon.Then gently raise the tendon and use micro scissors to completely transect the tissue. Using 8.0 sutures in a modified Kessler pattern, ligate the ends of the FDL tendon. Periodically hydrating the tendon with saline throughout the procedure if necessary.When the ends have been sutures, return the soft tissue and muscle over the tendon and use 5.0 nylon sutures to close the incision. Then place the mouse on a 37 degrees Celsius slide warmer with monitoring until fully recovered. 24 hours before the experimental end point, inject the mice with 100 micro liters of EDU.The next day, locate the repaired tendon as just demonstrated, and harvest the transected tissue. Isolating the granulated tissue surrounding the healing tendon is a critical step for allowing the eventual aminocytochemical analysis. And care must be taken to minimize the inclusion of peripheral tissues.Place the tendon in a petri dish containing one milliliter of collagenase. Then, mince the tissues with the scalpel followed by trituration of the fragments with several passes through an 18 gauge needle. Transfer the slurry into 1.5 milliliter centrifuge tubes and digest the tendon pieces for one hour and 37 degrees Celsius with shaking.At the end of the digestion, centrifuge the tissue suspensions and resuspend the pellets in 500 micro liters of three percent BSA in PBS. Next, filter the cells through a 70 micron strainer to remove the debris and large pieces of undigested tendon. And place the cells in a 37 degrees Celsius incubator.Transfer the undigested tendon pieces to new, 1.5 milliliter micro centrifuge tubes containing one milliliter of fresh collagenase, and mix the pieces by pipe heading. After another hour of digestion with shaking, spin down the tissue pieces again, and resuspend the pellets in 500 microliters of BSA supplemented PBS. Then filter the suspension through a new 70 micron cell strainer into the tube of cells collected after the first digestion.Count the cells and spin them down by centrifugation. Then resuspend the pellet at a three to six times 10 to the fourth cells per 100 microliters of PBS supplemented with BSA concentration. To detect EDU incorporation by immunocytochemistry, first use a hydrophobic barrier pen to circle the cells on the slides and immediately fix the samples with 150 microliters of three percent paraformaldehyde and PBS at room temperature.After 15 minutes, rinse the cells with two five minute washes in 150 microliters of BSA supplemented PBS. Then permeableize the samples with 0.5%triton X 100 in PBS for 20m minutes. After washing as just demonstrated, add 150 microliters of freshly prepared EDU reaction cocktail to the cells for a 30 minute incubation at room temperature.Then wash the cells again and counter stain the samples with 150 microliters of Hoechst. After 30 minutes at room temperature, add one to two drops of antifade mounting medium to the cells, and carefully cover each sample with the cover slip, taking care to avoid bubbles. Cure the mounting medium over night in the dark at room temperature.Then seal the cover slips with clear nail polish. Finally, image the slides on a fluorescent microscope using 40X magnification. In a successful repair, the granulation tissue displays a greatly increased cellularity compared to the uninjured tendon.In this image, a histological example of a ruptured flexor tendon repair is shown. EDU labeling highlights only those cells that actively proliferate during the 24 hours following the EDU pulse in life mice. Facilitating a quantitative assessment of the proliferation within the granulation tissue of the healing tendon.After watching this video you should have a good understanding of how to perform the flexor tendon injury and repair surgery in mice, to better understand the cell populations that contribute to scar formation in the flexor tendon during healing.

murine-flexor-tendon-injury-and-repair-surgery

Research

JoVE Journal

Medicine

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

ラット脊髄損傷後の急性および慢性の触覚官能検査

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

医学

A Contusive Model of Unilateral Cervical Spinal Cord Injury Using the Infinite Horizon Impactor

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of spinal cord injury (SCI) in which the thoracic spinal cord is injured. Additionally, because most human cord injuries occur as the result of blunt, non-penetrating trauma (e.g. motor vehicle accident, sporting injury) where the spinal cord is violently struck by displaced bone or soft tissues, the majority of SCI researchers are of the opinion that the most clinically relevant injury models are those in which the spinal cord is rapidly contused.1 Therefore, an important step in the preclinical evaluation of novel treatments on their way to human translation is an assessment of their efficacy in a model of contusion SCI within the cervical spinal cord. Here, we describe the technical aspects and resultant anatomical and behavioral outcomes of an unilateral contusive model of cervical SCI that employs the Infinite Horizon spinal cord injury impactor.
Sprague Dawley rats underwent a left-sided unilateral laminectomy at C5. To optimize the reproducibility of the biomechanical, functional, and histological outcomes of the injury model, we contused the spinal cords using an impact force of 150 kdyn, an impact trajectory of 22.5&deg; (animals rotated at 22.5&deg;), and an impact location off of midline of 1.4 mm. Functional recovery was assessed using the cylinder rearing test, horizontal ladder test, grooming test and modified Montoya's staircase test for up to 6 weeks, after which the spinal cords were evaluated histologically for white and grey matter sparing.
The injury model presented here imparts consistent and reproducible biomechanical forces to the spinal cord, an important feature of any experimental SCI model. This results in discrete histological damage to the lateral half of the spinal cord which is largely contained to the ipsilateral side of injury. The injury is well tolerated by the animals, but does result in functional deficits of the forelimb that are significant and sustained in the weeks following injury. The cervical unilateral injury model presented here may be a resource to researchers who wish to evaluate potentially promising therapies prior to human translation.

A reliable and repeatable way to produce a cervical unilateral spinal cord injury using the Infinite Horizon impactor is described. The method takes advantage of a custom designed frame and clamp to stabilize the spine. The standardized procedure and biomechanical injury parameters result in sufficient and sustained injuries.

無限の地平線インパクターを用いた片側頚髄損傷の打撲のモデル

While the majority of human spinal cord injuries occur in the cervical spinal cord, the vast majority of laboratory research employs animal models of ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

血管内大動脈瘤の修復の新しいマウスモデル

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

外傷性脳損傷のための制御皮質衝撃モデル

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

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.

瘢痕創傷修復のマウス胎児皮膚モデル

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

動脈再狭窄のマウスモデル：大腿ワイヤー傷害の技術的な側面

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

腸管虚血 - 再灌流傷害のマウスモデル

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

マウスモデルにおける下腹部大動脈クランプすることにより、再現性動脈侵食に起因けが

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

ラットの両側膝蓋腱損傷モデルの幹細胞療法の評価

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

マウス実験転移性乳癌癌モデルを生成、およびマウス乳癌根治術を実行

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...