Orthotopic Hind Limb Transplantation in the Mouse

Summary

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

Abstract

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research.

Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

Introduction

The late nineties heralded the pioneering days of reconstructive transplantation with the first successful hand transplant performed in France in 1998. Since then, the use of VCAs for reconstruction of devastating tissue defects has been successfully employed in a wide spectrum of patients. To date, the world counts 76 recipients of 112 upper extremities as well as 31 faces ^1-3. In addition, several other types of VCAs such as abdominal wall ⁴, larynx ⁵, trachea ⁶, vascularized joints ⁷, and even penis ⁸ have been performed. Furthermore, the live birth of a baby was recently reported after uterus transplantation ⁹. This growing world experience is indicative for how reconstructive transplantation has become a valid therapeutic option for patients suffering of significant functional tissue defects not amendable to conventional reconstructive and restorative surgery and treatment.

While the idea of replacing "like with like" sparked clinical enthusiasm, initial skepticism still prevails with regards to side effects of conventional high-dose immunosuppression required to maintain allografts and their function ^10,11. However, as shown by seminal work of Lee et al., these composite grafts are less likely to reject than its individual components, and furthermore, some of the tissue components such as the vascularized bone compartment have fueled optimism as they might exert unique immunological effects onto the balance of immune acceptance and rejection ¹².

Our group pioneered several microsurgical animal models for solid organ transplantation, as well as vascularized composite allotransplantation ^13-19. Here we describe a novel surgical procedure using a non-suture cuff technique to perform super micro-vascular anastomosis in an orthotopic mouse hind limb transplantation model. This transplant model provides a useful tool for investigating immune acceptance and rejection mechanisms, as well as the role of individual tissue components, such as the vascularized bone marrow compartment, towards tolerance induction in the immunologically versatile setting of the mouse species. Additionally, the orthotopic placement of the limb opens the possibilities for nerve regeneration and functional outcome studies, which are critically important to the setting of VCA.

Protocol

All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institute of Health (NIH) and were approved by the Johns Hopkins University Animal Care and Use Committee (JHUACUC). The specific procedures were performed under the approved ACUC protocol MO13M108. 

1. Donor Operation

1. Administer analgesia at the appropriate time point for each pharmacological formulation prior to surgery. As per the approved animal care and use protocol use 0.1 mg/kg BW of buprenorphine subcutaneously 1 hr prior to skin incision.
2. Sedate the donor with isoflurane applied through a chamber attached to an isoflurane vaporizer at 4%; maintain sedation and anesthesia at 2% through a nose cone. Perform toe pinch withdrawal reflection to monitor the depth of anesthesia prior to the initiation of the procedure.
3. Wear masks, disposable isolation gowns and gloves.
4. Shave the surgical area, in particular the hind limb and groin, and prep with 10% Povidone - Iodine.
5. Use a sterile field drape, autoclaved instruments and a high magnification microscope (40X).
6. Make groin skin incision using scissors proximally to the mid-thigh area and circumferentially connect the incision to demarcate the hind limb from the rest of the mouse body.
7. Identify and dissect the femoral artery, vein and nerve. Separate all three structures using forceps and micro-scissors.
8. Once the vascular pedicle is dissected divide the vessels at the level of the inguinal ligament using micro scissors.
9. Next, continue to divide the individual ventral (gracilis and medial thigh muscles) and dorsal muscle groups ²⁰ proximally at the level of the mid-thigh to separate the graft from the donor animal using scissors.
10. Transect the femur and cut at the mid of the femoral shaft using scissors.
11. Euthanize animal by isoflurane overdose followed by cervical dislocation. Confirm cessation of heart beat and respiration.
12. Flush the limb with 2 ml heparinized (30 I.E.) cold (4 °C) saline by using a 33 G flushing needle mounted on a syringe (see Materials Table).
13. Place one polyimide cuff on the femoral vein and artery, respectively.
14. Wrap graft into wet cotton gauze, place in petri dish and store at 4 °C until inset.

2. Recipient Operation

1. Removal of the Hind Limb
   1. Administer analgesia at the appropriate time point for each pharmacological formulation prior to surgery. As per the approved animal care and use protocol use 0.1 mg/kg BW of Buprenorphine SC 1 hr prior to skin incision.
   2. Sedate the donor with isoflurane applied through a chamber attached to an isoflurane vaporizer at 4%; maintain sedation and anesthesia at 2% through a nose cone. Perform toe pinch withdrawal reflection to monitor the depth of anesthesia prior to the initiation of the procedure.
   3. Use veterinary ointment on the eyes of the mouse to prevent dryness while under anesthesia.
   4. Shave the surgical area, in particular the hind limb and groin and prep with 10% Povidone - Iodine.
   5. Make groin skin incision using scissors proximally to the mid-thigh area and circumferentially connect the incision to demarcate the hind limb from the rest of the mouse body.
   6. Identify and dissect the femoral artery, vein and nerve and separate all three structures using forceps and micro-scissors.
   7. Once the vascular pedicle is dissected, clamp the femoral vessels at the level of the inguinal ligament.
   8. Cut the vessels distal at the level of the superficial epigastric artery.
   9. Next, continue to divide the individual ventral (gracilis and medial thigh muscles) and dorsal muscle groups ²⁰ proximally at the level of the mid-thigh to separate the native hind limb from the recipient animals using scissors.
   10. Transect the femur in the middle of the femoral shaft using scissors.
   11. Cauterize previously transected thigh muscles to prevent bleeding of the dissection site and thus recipient blood loss.
2. Implantation
   1. Minimize fluid loss by irrigating the operative field with warm saline (37 °C) and injecting 0.3 ml warm saline before and after the operation.
   2. Place the graft in a way that reflects the accurate anatomical position of the native hind limb by aligning the femur bone of the recipient and the graft and connect them using a 20 G spinal needle as an intramedullary rod.
   3. Coapt the ventral and dorsal muscle groups using absorbable suture material (6-0 Polysorb).
   4. Connect the femoral vessels using the non-suture cuff technique; in detail, pull the recipient side of the vessel over the cuffs previously mounted on the vessel ends of the graft. Use a 10-0 Nylon suture and perform a circumferential tie to fix the recipient vessel onto the cuff.
   5. Next release the clamps. At this stage visually verify cuff rotation and optimal positioning to prevent mis-rotation and kinking of the vessels.
   6. Perform meticulous hemostasis using electro cautery with a particular focus on the muscle recipient donor interface and the bone ends.
   7. Close the skin using non-absorbable Nylon sutures (6-0 Ethilon).
   8. Establish normothermic conditions by allowing the animal to recover in its cage under a heating lamp. Continue regular monitoring for at least 4 hr prior to returning it to the housing facility.
   9. Provide post-operative analgesia with Buprenorphine at a dose of 0.1mg/kg SC every 6-8 hr for 3 days.

Results

Performing vascularized composite allotransplantation in a mouse model using a non-suture cuff technique allows to achieve excellent and long term graft and animal survival as shown in Figure 1. Furthermore, it represents a reliable method of obtaining reproducible outcomes of gradual allograft rejection in vascularized composite allotransplantation as documented by the images shown in Figure 2. H&E histology of tissues obtained from animals undergoin...

Discussion

Vascularized Composite Allotransplantation, such as upper extremity and face transplantation for reconstruction of devastating tissue defects, has evolved as a valid treatment option for patients not amendable to conventional reconstructive procedures. Technical advances in the field of reconstructive microsurgery as well as a vast experience with potent immunosuppressive and immune modulatory therapies in solid organ transplantation, now enables long-term allograft survival in this unique patient population ^3,21

Materials

Name	Company	Catalog Number	Comments
Suture, 6-0 Nylon	MWI	31849
Suture, 6-0 Polysorb	MWI	72667
Suture, 10-0 Nylon	Aero Surgical	TK-107038
Polyimide Tubing, Size 25	Vention Medical	141-0023
Polyimide Tubing, Size 27	Vention Medical	141-0015
Microvascular Clamps (Single)	Synovis	00396
Microvascular Clamps (Double)	Synovis	00414
Micro-Scissors	Synovis	SAS-18
Micro-Forceps	Synovis	FRS-15 RM-8
Micro-Dilators	Synovis	FRS-15 RM-8d.1
Micro-Needledriver	Synovis	C-14
Micro-Clamp Applicator	Synovis	CAF-4
Micro-Flushing Needle	Hamilton		10MM, 30°, 33G
Lactated Ringers Solution	Fisher Scientific	NC9968051
Buprenorphine			DEA Number required; Obtained from hosptial pharmacy.
Enrofloxacin; Baytril	Bayer Health Care	186599
Heparin			Obtained from hosptial pharmacy