Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Summary

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their occurrence.

Abstract

Vascularized composite allotransplantation (VCA) is a relatively new field in the reconstructive surgery. Clinical achievements in human VCA include hand and face transplants and, more recently, abdominal wall, uterus, and urogenital transplants. Functional outcomes have exceeded initial expectations, and most recipients enjoy an improved quality of life. However, as clinical experience accumulates, chronic rejection and complications from the immunosuppression must be addressed. In many cases where grafts have failed, the causative pathology has been ischemic vasculopathy. The biological mechanisms of the acute and chronic rejection associated with VCA, especially ischemic vasculopathy, are important areas of research. However, due to the very small number of VCA patients, the evaluation of proposed mechanisms is better addressed in an experimental model. Multiple groups have used animal models to address some of the relevant unsolved questions in VCA rejection and vasculopathy. Several model designs involving a variety of species are described in the literature. Here we present a reproducible model of VCA heterotopic hindlimb osteomyocutaneous flap in the rat that can be utilized for translational VCA research. This model allows for the serial evaluation of the graft, including biopsies and different imaging modalities, while maintaining a low level of morbidity.

Introduction

Reconstructive surgery for the catastrophic tissue loss from amputation, blast injuries, malignancies, and congenital defects are limited by the availability of tissue from the patient and the additional morbidity caused at the donor site. In some cases, such as burn victims or quadrilateral amputees, viable tissue for reconstruction is not available from the patient. In 1964, the first modern hand transplant was performed in Ecuador. While this was a technical success, immunosuppression available at the time was insufficient to prevent rejection, and the graft was lost in less than 3 weeks¹. In 1998 and 1999, the first hand transplants in the ....

Protocol

All animal surgeries were performed in accordance with protocols approved by the University of Louisville's Institutional Animal Care and Use Committee (IACUC-approved protocol 18198) and the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals¹⁰. Four-month-old male Brown-Norway (RT1.Aⁿ) and 4-month-old male Lewis (RT1.A^l) rats were used as VCA donor and recipients, respectively.

1. Donor Allograft Harvest

1. Sedate the donor animal using vaporized isoflurane applied through a chamber.
2. Shave the graft donor area (hindlimb), as well....

Results

The rat VCA heterotopic hindlimb osteomyocutaneous flap model allows for long-term allograft survival under immunosuppression. The model is reliable, reproducible, and simple to perform. The flap is well hidden in the groin area and constitutes minimal morbidity and discomfort to the animal. The skin presentation is a clinical manifestation of the allograft’s survival and rejection (Figure 1). The flap design allows for gross clinical monitoring and creates an opportunity for various imaging.......

Discussion

In developing this model of VCA, several key issues were considered. First, it was important to include intact bone (tibia and fibula), bone marrow, and skin in the graft. While clinical hand transplants from adult donors do not transfer significant amounts of actively hematopoietic marrow, studies of the role of the bone marrow niche are better mirrored using an intact, vascularized bone rather than a cut long bone, which results in fibrosis of the exposed marrow. Moreover, the closed bone osteomyocutaneous flap design .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acepromazine	Henry Schein	5700850
Adventitia Scissors	ASSI	SAS15R8
Approximator Clamp (Double)	ASSI	ABB2V, ABB22V
Approximator Clamp (single)	FST	00398-02
Clamp Applying Forceps	ASSI	CAF4
Dissecting Scissors	ASSI	SDS18R8
Flushing blunt needle 27 G	SAI
Heparin Sodium	Sagent	25021-400-30
Isoflurane	Patterson Veterinary	14043-704-06
Jewelers Bipolar	ASSI	103000BPS03
Jewelers forceps #3	FST	11231-30
Ketamine HCl 100 mg/mL	Zoetis	043-304	DEA License required
Lactated Ringer Solution	Hospira	0409-7953-03
Lactated Ringer Solution + 5% Dextrose	Hospira	0409-7953-09
Meloxicam	Henry Schein	11695-6925-2
Micro forceps	ASSI	JFAL3
Micro needle holder	ASSI	B138
Prograf (Tacrolimus) 5 mg/mL	Astellas	0469-3016-01
Suture, 10-0 Prolene	Ethicon	W2790	or 10-0 Ethilon (2830)
Suture, 4-0 Coated Vicryl	Ethicon	J714D
Vessel Dilator Forceps	ASSI	D5AZ
Xylazine	VetOne	13985-612-50