Ortotópico Hind Limb Transplantation no mouse

Summary

Este novo modelo de transplante de membro posterior ortotópico em ratos, aplicando uma técnica manguito não sutura para a anastomose super-microvascular, fornece uma ferramenta poderosa para in vivo pesquisa imunológica mecanicista relacionadas com alotransplante composta vascularizado (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

Todos os experimentos foram conduzidos de acordo com o Guia para o Cuidado e Uso de Animais de Laboratório do Instituto Nacional de Saúde (NIH) e foram aprovados pela Comissão de Johns Hopkins University animal Cuidado e Uso (JHUACUC). Os procedimentos específicos foram realizados sob a MO13M108 protocolo ACUC aprovado. Operação 1. Donor Administrar analgesia no ponto de tempo adequado para cada formulação farmacológica antes da cirurgia. De acordo com a cuidar dos animais aprovados e uso de …

Representative Results

Executando alotransplante compósito vascularizada num modelo de ratinho utilizando uma técnica não-sutura braçadeira permite atingir uma excelente e de longa sobrevivência do enxerto prazo e animal, tal como mostrado na Figura 1. Além disso, representa um método confiável para a obtenção de resultados reprodutíveis de rejeição de aloenxertos gradual no compósito vascularizado alotransplante como documentado por as imagens mostradas …

Discussion

Vascularizado Composite Alotransplante, como membro superior e transplante de rosto para reconstrução de defeitos de tecidos devastadores, tem evoluído como uma opção de tratamento válida para pacientes não alterada para procedimentos reconstrutivos convencionais. Os avanços técnicos no campo da microcirurgia reconstrutiva, bem como uma vasta experiência com potente terapêuticas imunossupressoras e imunomoduladoras em transplante de órgãos sólidos, agora permite a sobrevivência do enxerto a longo prazo ne…

Materials

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	N/A	10MM, 30°, 33G
Lactated Ringers Solution	Fisher Scientific	NC9968051
Buprenorphine	N/A	N/A	DEA Number required; Obtained from hosptial pharmacy.
Enrofloxacin; Baytril	Bayer Health Care	186599
Heparin	N/A	N/A	Obtained from hosptial pharmacy