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

这项工作是部分由美国社会的试点手奖和美国国立卫生研究院/ NIAMS 1K01AR068386-01（到AEL）和NIAMS / NIH P30AR061307外科支持。

大学委员会的动物研究在罗切斯特大学批准的所有动物实验。十-12周龄雌性C57BL /使用了6J小鼠。 1.屈肌腱手术动物的制备（约15分钟） <ol><li>高压灭菌手术器械消毒，戴整个无菌手套，并保持无菌操作领域。 </li><li>麻醉通过腹膜内注射（IP）用氯胺酮对应于体重的体积（80毫克/千克）和赛拉嗪（10毫克/千克）鼠标。通过没有脚趾捏反射确认镇静深度。 </li><li>通过皮下注射施...

<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>

为完整横断和屈指长肌腱修复的鼠类模型中的外科手术过程，提出了这项研究。除了聚光用细胞学离心机小细胞群体的一种新颖的应用表明，允许屈肌腱愈合期间蜂窝环境的定量免疫细胞化学分析。屈肌腱修复的此模型表明可再现愈合反应，这可以被用来评估使用基因敲除模型或在治疗过程中的变化与药理干预。手术后，炎性反应开始24小时，在此期间，细胞被招募到损伤部位和纤维组织的愈伤?...

在音乐会的手工作与前臂的屈肌和数字鞘屈肌腱启用的位弯曲和手的抓握功能​​。屈肌腱沿手的掌面运行;这种相对位置表浅创伤手时常常导致伤及肌腱。肌腱愈合通过瘢痕组织反应，而不是正常的肌腱组织1再生。虽然这种疤痕组织提供连续性肌腱，功能显着相对健康的肌腱下降。肌腱瘢痕组织复合材料的特点是受损的机械性能1，使修复后的肌腱更容易破裂。此外，疤痕组织缺乏天然腱胶原纤维结构的组织，从而增加在肌腱尺寸和体积。由于肌腱鞘单元的解剖限制，即使是适度增加肌腱的大小可以大大的红UCE肌腱的滑翔功能，因此，运动和手功能的数字范围。 在此之前的1960年的伤及屈肌腱，特别是那些在手的II区，没有定期修理由于在愈合严重并发症，与这些维修2产生的。手的这个区域被称为“无人区”3。然而，在外科技术，缝合模式和理疗康复协议的改善大大提高了屈肌腱修复2的结果。尽管有这些进展，修理的高达40％造成足够的粘合形成阻碍手功能4。因此，需要一种生物的方法，以改善愈合。遗憾的是，很知之甚少在细胞和分子水平上的腱愈合过程。因此，我们的目标是开发可用于提高基本相互理解鼠模型克屈肌腱愈合和瘢痕形成反应的细胞和分子成分，以识别新的治疗目标，以提高愈合的手段。 更大的动物模型已经在促进屈肌腱愈合过程的认识工具。犬和兔的研究已经证明既屈肌腱5,6的内在和外在的愈合能力，早期控制被动运动的相对粘连形成最小化的固定化7的重要性，以及不同的缝合图形的对愈合过程8的影响，9。此外，犬模型已在测试平移组织工程方法来改善愈合10有用。但是，也有在利用鼠相对于大型动物模型的模型，其中包括的相对成本，鼠特异性试剂的可用性几个重要的优点，以及便于产生全球KNOC的K-切口或组织特异性缺失/过表达构建体。此外，人类和老鼠之间的功能类似相对于肌腱11表明在开发的小鼠模型的潜在效用。 屈肌腱切断术和修复模拟临床愈合的许多方面，包括丰富的疤痕组织和受损的力学性能形成的小鼠模型的建立。这里描述的模型不是临床实践的真实概括由于在肌腱交界处FDL的横断为了保护维修站点。此外，该模型没有考虑滑膜鞘细胞对愈合响应的贡献，因为没有滑膜鞘覆盖，其中修复发生肌腱的中间部分。尽管有这些限制，这种模式有运动限制性粘连发生范围，这还没有的优点在小鼠模型更CLOS待证实伊利近似临床情况。该模型已被用于评估敲除小鼠模型12,13，并以测试不同的药理学方法来改善愈合14-17。这种模式的组织学分析，采用免疫组化和原位杂交，可以愈合期间提供关键基因和蛋白质的本地化的重要见解。然而，组织学仅提供一剖空间分析和不允许在整个组织定量。流式细胞仪代表更加定量的方法，但只是细胞的数量非常有限可在小鼠模型中的愈合腱组织中分离，并且这个数字期间固定，透化和洗涤步骤进一步降低。采取这一到帐户，流式细胞术成为不可行的方法由于是将需要的动物的数量。另一种方法是需要保留的大多数这种小细胞群，以便以进一步表征愈合环境。用于实现此目的的方法，在这里显示的，涉及经由细胞学离心分离的细胞的浓度到载玻片，随后通过免疫细胞化学。在本研究的EdU（5-乙炔基-2&#39;脱氧尿苷，胸苷类似物）掺入和随后的标记被用来确定在愈合部位细胞的相对增殖状态。这种方法可以应用到测试对细胞增殖，基因敲除或过表达的药物治疗的功效，或确定和量化不同的细胞群。

<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

肌腱连接骨骼肌肉和骨骼，便利几乎整个身体的运动。在手，屈肌腱（FTS）使手指和一般手功能的屈曲。受伤的罚球是常见的，满意的治疗往往是受损由于过度的疤痕组织和肌腱与周围组织粘连之间。然而，知之甚少FT修复的分子和细胞成分。为此目的，该概括在人类愈合的许多方面，包括范围受损的运动和减小的机械性能的FT修复的鼠类模型，已开发和如前所述。这里这种手术过程的深入示范设置，涉及横断和在小鼠的后爪的趾长屈肌（FDL）腱的后续修复。这种技术可以用来进行不同类型的细胞的谱系分析，评估基因增益或功能失的影响，并以测试艾菲在愈合过程中的药理学干预cacy。不过，也有这种模型的两个主要限制：i）如鼠后爪，其中，发生在横断和修理的中间部分的FDL肌腱，不是由滑膜鞘包围。因此，该模型没有考虑护套的给瘢痕形成过程中的潜在贡献。 ⅱ）为了保护修复部位的完整性，对FT被释放在腱交界处，减少腱的机械力，可能有助于瘢痕形成增加。从期间流式细胞分析愈合过程中的FT的肉芽组织足够细胞分离已证明具有挑战性;细胞学离心浓缩这些细胞是用另一种方法，并且允许进行一代细胞制剂的可以在其上进行免疫荧光标记。用这种方法，FT愈合期间的细胞或感兴趣的蛋白质的定量成为可能。

在趾长屈肌（FDL）肌肉，位于小腿，行为经屈肌腱弯曲鼠标后爪的数字（蓝色轮廓图1A和图2A组织学显示），它从肌腱近端运行结并在远端指骨终止。在这个模型中屈肌腱愈合，所述FDL腱中的后爪的数字（红色箭头， 图1A）横切，并在脚中间修复，近端分叉。以防止修复，这将排除实验分析的破裂，肌腱（和相邻肌肉）被切断，或释放...

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

Murine Flexor Tendon Injury and Repair Surgery

在手屈肌腱通常受伤，导致受损的手部功能。然而，疤痕组织愈合反应不能很好地表征。屈肌腱愈合的小鼠模型在这里展示。该模型可以增强愈合过程的全面了解和评估治疗方法，以改善愈合。

鼠屈肌腱损伤修复手术

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.

在大鼠脊髓损伤后的急性和慢性触觉测试

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

科研

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.

单侧颈脊髓损伤的挫伤模型，利用无限的地平线冲击

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.

一种新的小鼠模型主动脉瘤腔内修复

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.

控制皮层影响模型创伤性脑损伤

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.

对无瘢痕修复鼠标胎儿皮肤模型

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

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.

动脉再狭窄小鼠模型：股导线损伤的技术方面

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.

肠缺血再灌注损伤的小鼠模型

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.

重复性的动脉剥脱损伤腹主动脉阻断在小鼠模型

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.

大鼠双侧髌腱损伤模型中干细胞治疗的评价

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.

小鼠原位转移性乳腺癌模型的建立及小鼠根治性乳房切除术

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