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Medicine

Murine Flexor Tendon Injury and Repair Surgery

Published: September 19th, 2016

DOI:

10.3791/54433

1Center for Musculoskeletal Research, Department of Orthopaedics & Rehabilitation, University of Rochester Medical Center

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 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. 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.

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 r....

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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)

  1. Autoclave surgical instruments to sterilize, wear sterile gloves throughout, and maintain a sterile operating field.
  2. Anesthetize the mouse via intraperitoneal injection (i.p.) with a volume of ketamine (80 mg/kg) and xylazine (10 mg/kg) corresponding to body.......

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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.......

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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.......

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

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

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