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Abstract

Introduction

Protocol

Representative Results

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Materials

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Immunology and Infection

Partial Heterotopic Hindlimb Transplantation Model in Rats

Published: June 9th, 2021

DOI:

10.3791/62586

1Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, 2Vascularized Composite Allotransplantation Laboratory, Center for Transplantation Sciences, Massachusetts General Hospital, Harvard Medical School, 3Shriners Hospital for Children, 4Service de Chirurgie Plastique, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris (APHP), Université de Paris
* These authors contributed equally

This paper presents a partial heterotopic osteomyocutaneous flap transplantation protocol in rats and its potential outcomes in the mid-term follow-up.

Vascularized composite allotransplantations (VCA) represent the most advanced reconstruction option for patients without autologous surgical possibilities after a complex tissue defect. Face and hand transplantations have changed disfigured patients' lives, giving them a new aesthetic and functional social organ. Despite promising outcomes, VCA is still underperformed due to life-long immunosuppression comorbidities and infectious complications. The rat is an ideal animal model for in vivo studies investigating immunological pathways and graft rejection mechanisms. Rats are also widely used in novel composite tissue graft preservation techniques, including perfusion and cryopreservation studies. Models used for VCA in rats must be reproducible, reliable, and efficient with low postoperative morbidity and mortality. Heterotopic limb transplantation procedures fulfill these criteria and are easier to perform than orthotopic limb transplants. Mastering rodent microsurgical models requires solid experience in microsurgery and animal care. Herein is reported a reliable and reproducible model of partial heterotopic osteomyocutaneous flap transplantation in rats, the postoperative outcomes, and the means of prevention of potential complications.

Over the past two decades, VCA has evolved as a revolutionary treatment for patients who suffer severe disfigurement including face1, upper limb amputations2, penile3, and other complex tissue defects4,5. However, the consequences of life-long immunosuppression still hinder a broader application of these complex reconstructive surgeries. Basic research is crucial to improve anti-rejection strategies. Increasing VCA preservation time is also essential to improve transplantation logistics and increase the donor pool (as VCA donors must fulfi....

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All animals received humane care in accordance with the NIH Guide for the Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee (IACUC-protocol 2017N000184) and Animal Care and Use Review Office (ACURO) approved all animal protocols. Inbred male Lewis rats (250-400 g) were used for all experiments.

1. Surgery

  1. Anesthetize the Lewis rats using isoflurane inhalation. Induce anesthesia with 5% isoflurane in the induction chamber, and maintain anesthesia wi.......

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In this single-operator study, 30 syngeneic heterotopic partial limb transplants were performed. Success was defined at postoperative day 21 as the absence of VCA failure or complications requiring euthanasia. The normal evolution of the graft is represented in Figure 3. The mean duration for partial limb procurement and graft inset in the recipient were 35 and 105 min, respectively; the mean ischemia time was 105 min. During follow-up, two types of complications occurred (Table 1

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Orthotopic limb transplantation models in rodents have been described in the literature16,17,18; however, they require a nerve repair, muscle reattachment, and a perfect osteosynthesis of the femur, which can be a very difficult step. These models are also associated with a higher morbidity and mortality rate in rodents14, especially in the short-term follow-up as the recovery of a normal function of a tr.......

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This work was supported by the Office of the Assistant Secretary of Defense for Health Affairs through the Congressionally Directed Medical Research Program under Award No. W81XWH-17-1-0680. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense.

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Name Company Catalog Number Comments
24 GA angiocatheter BD Insyte Autoguard 381412
4-0 suture Black monofilament non absorbable suture Ethicon 1667 Used to suture the E-collar to the back of the neck
4-0 suture Coated Vicryl Plus Antibacterial Ethicon VCP496
Adson Tissue Forceps, 11 cm, 1 x 2 Teeth with Tying Platform ASSI ASSI.ATK26426
Bipolar cords ASSI 228000C
Black Polyamide Monofilament USP 10-0, 4 mm 3/8c AROSurgical T04A10N07-13 Used to perform the microvascular anastomoses
Buprenorphine HCl Pharmaceutical, Inc 42023-179-01
Dilating Forceps Fine science tools (FST) 18131-12
Dissecting Scissors 15 cm, Round Handle 8 mm diameter, Straight Slender Tapered Blade 7 mm, Lipshultz Pattern ASSI ASSI.SAS15RVL
Double Micro Clamps 5.5 x 1.5 mm Fine science tools (FST) 18040-22
Elizabethan collar Braintree Scientific EC-R1
Forceps 13.5 cm long, Flat Handle, 9 mm wide Straight Tips 0.1 mm diameter (x2) ASSI ASSI.JFL31
Halsey Micro Needle Holder Fine science tools (FST) 12500-12
Heparin Lock Flush Solution, USP, 100 units/mL BD PosiFlush 306424
Isoflurane Patterson Veterinary 14043-704-06
Jewelers Bipolar Forceps Non Stick 11 cm, straight pointed tip, 0.25 mm tip diameter ASSI ASSI.BPNS11223
Lone Star Elastic Stays CooperSurgical 3314-8G Used to retract the inguinal ligament for femoral vessels dissection
Lone Star Self-Retaining Retractors CooperSurgical 3301G
Micro-Mosquito Hemostats Fine science tools (FST) 13010-12 Used to retract the inguinal fat pad distally
Needle Holder, 15 cm Round Handle, 8 mm diameter, Superfine Curved Jaw 0.2 mm tip diameter, without lock ASSI ASSI.B1582
Nylon Suture Black Monolfilament 8-0, 6.5 mm 3/8c Ethilon 2808G Used to ligate collateral branches on the femoral vessels
Offset Bone Nippers Fine science tools (FST) 16101-10
S&T Vascular Clamps 5.5 x 1.5 mm Fine science tools (FST) 00398-02
Walton scissors Fine science tools (FST) 14077-09

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