Orthotopic Rat Forelimb Transplantation

Summary

Here, we present a protocol to demonstrate the microsurgical feasibility of rat forelimb transplantation. This model may serve as an important translational platform for VCA research.

Abstract

Vascularized composite allotransplantation (VCA) involves the transplantation of multiple tissue types -- including skin, muscle, bone, and nerves -- offering a promising reconstructive option for patients with severe traumatic injuries or disfigurements. Despite its transformative potential, VCA has encountered significant challenges such as graft rejection, chronic immunosuppression complications, and neuromuscular recovery's intricacies.

We utilize a rat forelimb model as a cost-effective and anatomically relevant platform to address these challenges. The rat forelimb closely mirrors human limb anatomy, enhancing our findings' translational impact. Previous studies have validated this model for reliably and reproducibly measuring functional recovery, thereby establishing it as a key tool for assessing the rejection trajectory of forelimb grafts.

Moreover, the model offers a valuable opportunity to explore innovative therapeutic approaches and serve as a good translational platform for novel preservation techniques. Through further investigation of this model, we aim to deepen our understanding of the mechanisms behind graft rejection and neuromuscular recovery. Ultimately, this work strives to pave the way for improving clinical outcomes of VCA, addressing both current limitations and future challenges in transplant medicine.

Introduction

Vascularized Composite Allotransplantation (VCA) is a life-changing procedure to reconstruct severe tissue defects. VCA refers to the transplantation of a composite tissue consisting of skin, muscle, bones, connective tissue, blood vessels, and nerves. Further refinement and exploration of VCA techniques would profoundly benefit patients suffering traumatic injuries or disfigurements¹. However, its broader application is challenged by ongoing hurdles such as graft rejection, complications from long-term immunosuppression, neuromuscular recovery, and peri-transplant logistics.

The rat forelimb model has emerged as a p....

Protocol

Surgical procedures were carried out under the protocols approved by the Johns Hopkins University Animal Care and Use Committee (RA18M74) according to guidelines established by the National Institutes of Health and the American Association for the Accreditation of Laboratory Animal Care.

1. Donor procedure (Figure 1)

1. Conduct the induction of anesthesia via inhalation of isoflurane at a concentration of 5% and maintain at 1% throughout the procedure. Start the procedure after confirming the accurate anesthesia level via toe pinch.
2. Make a circumferential inc....

Results

Graft survival can be monitored and determined through macroscopic observation. Representative pictures of both short-term (POD7) and long-term follow-up (POD120) are shown in Figure 3. The graft could recover without apparent signs of necrosis. Survival data are shown in Figure 4. Both skin and muscle samples are harvested at the designated endpoint. Representative histological images are shown in Figure 5 and .......

Discussion

Orthotopic forelimb transplantation in rats is a commonly used model in experimental reconstructive transplantation as it not only contains vascularized composite tissues but also allows for the assessment of functional recovery. A conventional suture technique for vascular anastomosis is highly complex and demands advanced microsurgical skills that require years of microsurgical training. To greatly reduce the time and costs, a vascular non-suture cuff anastomosis technique was applied in this modified rat forelimb mode.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10-0 Non-Sterile Micro Suture	AROSurgical	TK-107038
4-0 Sharpoint Nylon Black	esutures	A1667N
4-0 Sharpoint Polysyn Taper	esutures	G214N
5 mL syringes	MDC	2045
6-0 silk	Fisher Scientific	NC9742105
ABB-22 V Double Micro Vessel	S&T	00480
B-1 V Single Micro Vessel Clamp	S&T	00462
BD Quincke Spinal needle	esutures	405073
Castroviejo Micro Needle Holders	F.S.T	12061-02
Chatilon DFE II force meter	Ametek	DFE II series
Cotton tiped	JH Supply store	100252
Fine Scissors - Tungsten Carbide	F.S.T	14569-09
Forceps	S&T	FRS-15 RM-8
Gauze Sponges	Fisher Scientific	22-362178
Halsted-Mosquito Hemostats	F.S.T	13009-12
Heparin	JHH Pharmacy	NDC 63323-540-05
I.V. Catheter Radiopaque 24 G	esutures	SMTH5053
Jewelers Bipolar Coagulating Forceps, Stainless Steel	ASSI	103000BPS03
Round Handled Vannas Spring Scissors	F.S.T	15400-12
Special-Forceps	S&T	FRAS-15 RM-8
Vessel Dilators - Balanced Instruments	F.S.T	18602-15