쥐 이소성 복부 심장 / 볼륨로드 구성에서 단일 폐 이식

Summary

We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft.

Abstract

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft involves anastomosis of donor inferior vena cava (IVC) to recipient IVC, and donor ascending aorta (Ao) to recipient abdominal Ao. The right upper and middle lung lobes are preserved and function as conduits for blood flow from right heart to left heart.

There are several advantages to using this technique, and it lends itself to a broad range of applications. Because the graft is transplanted in a configuration that allows for dyamic volume-loading, cardiac function may be directly assessed in vivo. The use of pressure-volume conductance catheters permits characterization of load-dependent and load-independent hemodynamic parameters. The graft may be converted to a loaded configuration by applying a clamp to the recipient’s infra-hepatic IVC. We describe modified surgical techniques for both donor and recipient operations, and an ideal myocardial protection strategy. Depending on the experimental aim, this model may be adapted for use in both acute and chronic studies of graft function, immunologic status, and variable ventricular loading conditions. The conducting airways to the transplanted lung are preserved, and allow for acute lung re-ventilation. This facilitates analysis of the effects of the mixed venous and arterial blood providing coronary perfusion to the graft.

A limitation of this model is its technical complexity. There is a significant learning curve for new operators, who should ideally be mentored in the technique. A surgical training background is advantageous for those wishing to apply this model. Despite its complexity, we aim to present the model in a clear and easily applicable format. Because of the physiologic similarity of this model to orthotopic transplantation, and its broad range of study applications, the effort invested in learning the technique is likely to be worthwhile.

Introduction

The first rodent model of heterotopic abdominal heart transplantation (HAHT) was described by Abbott and colleagues in 1964¹. This technique, and subsequent modifications have been widely applied to characterize transplant graft function and immunologic status. The majority of HAHT techniques described involve a non-volume loaded heart^2,3. Models of HAHT involving volume-loaded ventricles have been described, but they are frequently limited in one or more respects.

Heterotopic abdominal heart-lung transplantation (HAHLT) with a volume-loaded left ventricle (LV) has been described previously. Chen and colleagues⁴, and subsequently Ibrahim and colleagues⁵ described HAHLT with a single aorto-aortic (donor ascending to recipient abdominal aorta) anastomosis. The only volume load presented to the ventricle in this circulation is the coronary venous return. Asfour and colleagues described a HAHT technique in which the lung circuit was eliminated by anastomosing donor pulmonary artery (PA) to donor left atrium (LA)⁶. In this circulation, venous inflow to right ventricle (RV) occurs via a donor SVC to recipient IVC anastomosis, and the subsequent LV load is ejected into the aorto-aortic anastomosis. Cardiac function was partially assessed in vivo, and also in vitro using a Langendorff rig. Figueiredo and colleagues described a HAHLT model similar to our own⁷, but in mice. Venous inflow to the RV occurs via donor SVC to recipient IVC anastomosis. Blood subsequently passes through the single lung circulation and LV load is ejected into the aorto-aortic anastomosis. Cardiac function in their study was assessed by magnetic resonance imaging (MRI). Wen and colleagues described a unique HAHT technique in which the LV is loaded by means of a recipient aorta to donor LA anastomosis⁸. The LV, therefore, fills at systemic pressures. Cardiac function, and whether LV stroke volume is ejected antegradely in their model was not assessed.

Many of the techniques referenced above involve non-physiologic LV loading conditions, including the techniques whose partial LV load is represented only by coronary venous return. On the other hand, many techniques do approach physiologic LV loading. The majority of these techniques, as with the technique of Asfour and colleagues, omit the pulmonary circulation and utilize a donor PA to donor LA anastomosis^6,9. The circulation described by Galinanes and colleagues¹⁰ employs a direct recipient cava to donor LA anastomosis, omitting the pulmonary circulation and the right heart. Yokoyama and colleagues achieve the same effect by ligating the donor PA and creating an interatrial communication in the donor heart (omitting donor lung and right heart circulations)¹¹. The circulation of Maruyama and colleagues¹² involves an anastomosis between donor left PA and recipient Ao, which permits LV filling via the pulmonary circulation as a conduit, but effectively excludes the right heart.

In cases where near physiologic loading conditions were met, we advance the technique of HAHLT in 2 major respects. First, to our knowledge, the exact configuration we report has not been described in rats. It is possibly the most versatile circulation for investigators wishing to study the physiology, structure, and immunology of the transplanted heart-lung graft. Second, we describe how the function of the transplant graft can be directly characterized in vivo. For this application, pressure-volume conductance catheters can be introduced directly into the LV apex of the transplant graft, which allows for complete cardiac functional characterization.

The technique described here can be applied to both acute and chronic studies of transplant graft function, while the functional assessment may be performed either in vivo or in vitro. We present a model in which the loading conditions can be near physiologic, however the degree of ventricular loading may be manipulated both acutely and chronically by diverting venous return towards or away from the graft. Afterload conditions can also be manipulated. Because the lung and its airway are retained in this transplant configuration, investigators can re-ventilate the donor lung acutely. Uniquely, lung re-ventilation changes the composition of blood perfusing the transplant coronary arteries. Under non-ventilated conditions, blood ejected from the donor aorta is deoxygenated, and mixes with oxygenated blood in the recipient aorta. Under acutely ventilated conditions, ejected blood becomes oxygenated. Thus, transplant graft function can be compared under ventilated and non-ventilated conditions, and also under variably loaded conditions.

The protocol below describes important modifications to previously described HAHLT donor and recipient operations. It also describes an optimal technique for protecting the transplant graft throughout the period of ischemia (time between donor explant and recipient implant). Advantages of this technique include physiologic conditions potentially approaching that of an orthotopically transplanted graft, and a wide range of investigative applications. An important limitation is its technical complexity. With adequate mentoring and practice, the advantages of this technique will likely outweigh the challenges in adopting it.

Protocol

모든 동물은 보관과 전국 및 관리 및 실험 동물의 사용을위한 제도적 지침에 따라 마음에 든다고했다. 이 프로토콜에 대한 윤리 승인은 브리티시 컬럼비아의 동물 관리위원회의 대학에 의해 수여되었다. 300 사이 계량 남성, 흰쥐 – 450 g을이 프로토콜에 사용 하였다. 1. 기부자 운영 3 방향 스톱 콕 장거리 정맥 내 (IV) 카테터 튜브에 연결된 원통형 플라스크에 심정지 (RT…

Representative Results

위에서 설명한 HAHLT 기술은 고도의 기술과 세부 사항입니다. 표에 세심한주의를 필요로 한 하이라이트 실패 절차 대 성공과 관련된 중요한 요소의 일부, 그리고 기술적 인 문제를 해결하기위한 가이드로 사용할 수 있습니다. 받는 사람 대동맥이 클램핑 해제 한 후, 이식 관상 동맥은 산소가 혈액으로 채우기 위해 볼 수 있어야합니다. 따라서, 심근는 …

Discussion

여기에 설명 된 기술과 성공은 여러 가지 요인에 입각한다. 그 중 주요 균일 이식 냉각 완전한 심정지 체포, 총 허혈 시간을 최소화을 보장, 안전하고 최소한의 혈액 손실과 관련된 세심한 수술 기법을 채택, 모두 기증자와받는 사람 동물의 안정성을 보장하고, 적절하게 탈 방송 이식을 할 것이다. 위의 인정으로, 기술의 기술적 인 복잡성은 최고 제한 사항입니다.

우리는 여?…

Materials

Material/Equipment	Company	Catalog Number	Comments
Celsior Cardioplegic Soution	Genzyme		The solution is kept on ice throughout the procedure. We prepare our own solution, with slight modifications.
Rodent Ventilator	Harvard Apparatus	Model 683
Vital Sign Monitor	Nonin	Model 9847V	Displays SpO2 and heart rate.
IV Cannulae	Jelco	3063	24-26G x 3/4" cannulae.
IV Tubing	CareFusion	MP9259-C	Short-length connector tubing (18cm).
Surgical Clips	Teleflex Medical	001204	Horizon titanium ligating clips.
Sutures	Ethicon, Sharpoint	LA54G, AK-0107	3-0 silk reel, and 9-0 prolene suture (single-armed, DR5 needle).
Surgical Instruments	Not Applicable	Not Applicable	The instruments used are generic, and can be purchased from any surgical supply company.