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

This work was partially supported by the American Society for Surgery of the Hand Pilot Award and NIH/NIAMS 1K01AR068386-01&#160;(to AEL) and NIAMS/NIH P30AR061307.

The University Committee on Animal Research at the University of Rochester approved all animal experiments. Ten-12 week old female C57BL/6J mice were used.
1. Preparation of Animals for Flexor Tendon Surgery (~15 min)
<ol>
	<li>Autoclave surgical instruments to sterilize, wear sterile gloves throughout, and maintain a sterile operating field.</li>
	<li>Anesthetize the mouse via intraperitoneal injection (i.p.) with a volume of ketamine (80 mg/kg) and xylazine (10 mg/kg) corresponding to body...

<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. 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The surgical procedure for a murine model of complete transection and repair of the flexor digitorum longus tendon is presented in this study. In addition a novel application of concentrating small cell populations with cytology centrifuge is demonstrated, allowing for quantitative immunocytochemical analysis of the cellular environment during flexor tendon healing. This model of flexor tendon repair demonstrates a reproducible healing response, which can be used to assess changes in the healing process using knockout mo...

Flexor tendons in the hand work in concert with the flexor muscles of the forearm and digital sheaths to enable flexion of the digits and grasping function of the hand. Flexor tendons run along the palmar aspect of the hand; this relatively superficial location often results in injuries to the flexor tendons during trauma to the hand. Tendons heal through a scar tissue response rather than regeneration of normal tendon tissue 1. While this scar tissue provides continuity to the tendon, function is dramatically decreased relative to healthy tendon. Tendon-scar tissue composites are characterized by impaired mechanical properties 1, rendering the repaired tendons more likely to rupture. In addition, scar tissue lacks the organization of the native tendon collagen fiber structure, resulting in an increase in tendon size and bulk. Given the anatomic constraints of the tendon-sheath unit, even a modest increase in tendon size can drastically reduce the gliding function of the tendon, and therefore digit range of motion and hand function.
Prior to the 1960&#39;s injuries to the flexor tendons, particularly those in Zone II of the hand, were not routinely repaired due to the severe complications in healing that arose with these repairs 2. This area of the hand was referred to as &#39;no man&#39;s land&#39; 3. However, improvements in surgical techniques, suture patterns and physical therapy rehabilitation protocols have dramatically improved outcomes of flexor tendon repairs 2. Despite these advances, up to 40% of repairs result in sufficient adhesion formation to impede hand function 4. Therefore, a biological approach is required to improve healing. Unfortunately, very little is known about the tendon healing process at the cellular and molecular level. Thus, the goal was to develop a murine model that could be used to improve the fundamental understanding of the cellular and molecular components of flexor tendon healing and the scar formation response, as a means to identify novel therapeutic targets to improve healing.
Larger animal models have been instrumental in furthering understanding of the flexor tendon healing process. Canine and rabbit studies have demonstrated both the intrinsic and extrinsic healing ability of flexor tendons 5,6, the importance of early controlled passive motion in minimizing adhesion formation relative to immobilization 7, as well as the effects of different suture patterns on the healing process 8,9. In addition, the canine model has been useful in testing translational tissue-engineering approaches to improve healing 10. However, there are several important advantages in using a murine model relative to a large animal model, including the relative cost, availability of murine specific reagents, and the ease of generating global knock-outs or tissue-specific deletion/overexpression constructs. Moreover, the functional similarities between human and mice with respect to flexor tendons 11 indicate the potential utility in developing a murine model.
Development of a murine model of flexor tendon transection and repair mimics many aspects of clinical healing, including the formation of abundant scar tissue and impaired mechanical properties. The model described here is not a true recapitulation of clinical practice due to transection of the FDL at the myotendinous junction in order to protect the repair site. Furthermore, this model does not account for the contribution of synovial sheath cells to the healing response, as there is no synovial sheath covering the mid-portion of the tendon where the repair occurs. Despite these limitations, this model has the advantage of generating range of motion-limiting adhesions, which has yet to be demonstrated in murine models that more closely approximate the clinical scenario. This model has been used to assess knock-out mouse models 12,13, and to test different pharmacological approaches to improve healing 14-17. Histological analyses of this model, using immunohistochemistry and in situ hybridization, can provide important insights in to the localization of key genes and proteins during healing. However, histology provides only a cross-sectional spatial analysis and does not permit quantification throughout the entire tissue. Flow cytometry represents a more quantitative approach, but only a very limited number of cells can be isolated from the healing tendon tissue in the mouse model, and this number is further decreased during fixation, permeabilization, and washing steps. Taking this in to account, flow cytometry becomes an unfeasible approach due to the number of animals that would be required. An alternative method is necessary to preserve the majority of this small cell population in order to further characterize the healing milieu. The method used to accomplish this, shown here, involves concentration of the isolated cells via cytology centrifugation onto a glass slide, followed by immunocytochemistry. In the present study EdU (5-ethynyl-2&#39;deoxyuridine, a thymidine analog) incorporation and subsequent labeling was used to determine the relative proliferative state of cells at the healing site. This approach can be applied to test the efficacy of pharmacological treatments on cell proliferation, gene knock-out or overexpression, or to identify and quantify different cell populations.

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			<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>
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			<td height="21" style="height:21px;">Ketamine</td>
			<td>Hospira</td>
			<td>NDC# 0409-2051-05</td>
			<td></td>
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			<td height="21" style="height:21px;">Xylazine</td>
			<td>Lloyd Inc.</td>
			<td>NDC# 61311-482-10</td>
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			<td>Par Pharmaceutical Inc.</td>
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			<td>BD</td>
			<td>305106</td>
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			<td>Aplicare</td>
			<td>82-226</td>
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			<td height="21" style="height:21px;">70% ethanol</td>
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			<td>Dechra Veterinary Products</td>
			<td>NDC# 17033-211-38</td>
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			<td>Fine Science Tools</td>
			<td>91201-13</td>
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			<td height="21" style="height:21px;">Micro spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15003-08</td>
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			<td>Fine Science Tools</td>
			<td>12061-02</td>
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			<td height="21" style="height:21px;">5-0 nylon sutures</td>
			<td>Ethicon</td>
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			<td height="21" style="height:21px;">8-0 microsurgery nylon sutures</td>
			<td>Ethicon</td>
			<td>2808G</td>
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			<td height="21" style="height:21px;">Lab-Line histology slide warmer</td>
			<td>Barnstead International</td>
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			<td>Life Technologies&#160;</td>
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			<td>Cell Signaling Technologies</td>
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			<td>Thermo Fisher</td>
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			<td>Fisher HealthCare</td>
			<td>10-354</td>
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			<td height="21" style="height:21px;">HistoBond+ microscope slides</td>
			<td>VWR</td>
			<td>16005-110</td>
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			<td height="21" style="height:21px;">Cytospin 2 centrifuge</td>
			<td>Shandon</td>
			<td>SH-CYTO2</td>
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			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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			<td height="21" style="height:21px;">Immunocytochemistry</td>
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			<td>IHC World</td>
			<td>M920-2</td>
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			<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>
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			<td>Vector Laboratories</td>
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			<td height="21" style="height:21px;">Glass coverslips 24x50mm #1.5</td>
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			<td height="21" style="height:21px;">Clear nail polish</td>
			<td></td>
			<td></td>
			<td></td>
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murine flexor tendon injury and repair surgery

Tendon connects skeletal muscle and bone, facilitating movement of nearly the entire body. In the hand, flexor tendons (FTs) enable flexion of the fingers and general hand function. Injuries to the FTs are common, and satisfactory healing is often impaired due to excess scar tissue and adhesions between the tendon and surrounding tissue. However, little is known about the molecular and cellular components of FT repair. To that end, a murine model of FT repair that recapitulates many aspects of healing in humans, including impaired range of motion and decreased mechanical properties, has been developed and previously described. Here an in-depth demonstration of this surgical procedure is provided, involving transection and subsequent repair of the flexor digitorum longus (FDL) tendon in the murine hind paw.&#160;This technique can be used to conduct lineage analysis of different cell types, assess the effects of gene gain or loss-of-function, and to test the efficacy of pharmacological interventions in the healing process. However, there are two primary limitations to this model: i) the FDL tendon in the mid-portion of the murine hind paw, where the transection and repair occur, is not surrounded by a synovial sheath. Therefore this model does not account for the potential contribution of the sheath to the scar formation process. ii) To protect the integrity of the repair site, the FT is released at the myotendinous junction, decreasing the mechanical forces of the tendon, likely contributing to increased scar formation. Isolation of sufficient cells from the granulation tissue of the FT during the healing process for flow cytometric analysis has proved challenging; cytology centrifugation to concentrate these cells is an alternate method used, and allows for generation of cell preparations on which immunofluorescent labeling can be performed. With this method, quantification of cells or proteins of interest during FT healing becomes possible.

The flexor digitorum longus (FDL) muscle, located in the calf, acts to flex the digits of the mouse hind paw via the flexor tendon (outlined in blue in Figure 1A, and shown histologically in Figure 2A), which runs proximally from the myotendinous junction and terminates in the distal phalanges. In this model of flexor tendon healing, the FDL tendon is transected and repaired at the mid-foot, proximal to the bifurcation in to the digits of the hind paw (re...

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

Murine Flexor Tendon Injury and Repair Surgery

Flexor tendons in the hand are commonly injured, leading to impaired hand function. However, the scar-tissue healing response is not well characterized. A murine model of flexor tendon healing is demonstrated here. This model can enhance overall understanding of the healing process and assess therapeutic approaches to improve healing.

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

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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 ...

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 ...

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 ...

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, ...

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 ...

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 ...

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, ...

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 ...

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 ...