Partial Heterotopic Hindlimb Transplantation Model in Rats

Summary

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

Abstract

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.

Introduction

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 defects^4,5. 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....

Protocol

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 with 1.5-3% isoflurane inhalation through a breathing cone.
2. Apply eye lubricant before surgery in survival procedures. Shave the surgical site....

Results

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

Discussion

Orthotopic limb transplantation models in rodents have been described in the literature^16,17,18; 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.......

Materials

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