Summary

大鼠异位心脏腹部/在卷装配置单肺移植

Published: May 29, 2015
doi:

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 heart2,3. 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 anastomosis6,9. 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

所有的动物饲养和照顾按照国家和护理和使用实验动物的制度准则。伦理批准该协议被授予由不列颠哥伦比亚省的动物护理委员会的大学。男,SD大鼠的体重300 – 450克的用于本协议。 1.供体手术有有约100ml心麻痹(RT)的一个圆筒形烧瓶通过三路活塞相连长静脉(IV)导管管材。用支架提升烧瓶约80厘米工作表面上方,靠重力使心脏停搏交付。 为了可视化结构充…

Representative Results

上述HAHLT技术是高度技术性,需要密切注意细节。 表1列出了一些与成功与不成功程序关联的关键因素,并且可以用作用于排除故障的技术困难的指南。 经过收件人主动脉松开,移植冠状动脉应被视为填补氧合血。因此,心肌应该成为粉红色和灌注良好。在技​​术上是成功的实验,心脏就会开始移植再灌注后不久击败。接枝应留在无负载状态(与收件人IVC夹紧?…

Discussion

成功与此处所描述的技术将被断定几个因素。其中的关键是确保捐助国和受援国动物的稳定性,采用细致的手术技术,是安全的和最小的失血,确保完成心脏停搏均匀移植冷却,最大限度地减少总缺血时间,并适当去宣扬移植。如上文承认,该技术的技术复杂性是其主要缺点。

我们拥有先进的先前HAHLT技术在几个方面。在供体和受体的操作中描述的变型提供执行以受控和有效…

Disclosures

The authors have nothing to disclose.

Acknowledgements

马克J.卡恩斯接收来自UBC临床医生调查程序(通过医生和加拿大皇家外科学院授予),以及UBC 4YF博士生奖学金的支持。

约翰·博伊德H.是国家疗养院协会和迈克尔·史密斯基金会健康研究学者。通过资金CIHR。作者要感谢M.阿拉德博士和Richard Wamboldt他们与安装和维护设备的灌注援助。

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.

References

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  2. Ma, Y., Wang, G. Comparison of 2 heterotopic heart transplant techniques in rats: cervical and abdominal heart. Exp Clin Transplant. 9 (2), 128-133 (2011).
  3. Wiedemann, D., Boesch, F., Schneeberger, S., Kocher, A., Laufer, G., Semsroth, S. Graft function after heterotopic rat heart transplant with an isolated reperfused working heart: a methodic consideration. Exp Clin Transplant. 10 (2), 154-157 (2012).
  4. Chen, Z. H., Xia, S. S. The technique of heterotopic heart-lung transplantation in the rat. J Tongji Med Univ. 6 (2), 67-70 (1986).
  5. Ibrahim, M., Navaratnarajah, M., et al. Heterotopic abdominal heart transplantation in rats for functional studies of ventricular unloading. J Surg Res. 179 (1), e31-e39 (2013).
  6. Asfour, B., Hare, J. M., et al. A simple new model of physiologically working heterotopic rat heart transplantation provides hemodynamic performance equivalent to that of an orthotopic heart. J Heart Lung Transplant. 18 (10), 927-936 (1999).
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  9. Didié, M., Biermann, D., et al. Preservation of left ventricular function and morphology in volume-loaded versus volume-unloaded heterotopic heart transplants. A.Am J Physiol Heart Circ Physiol. 305 (4), H533-H541 (2013).
  10. Galiñanes, M., Zhai, X., Hearse, D. J. The effect of load on atrophy, myosin isoform shifts and contractile function: studies in a novel rat heart transplant preparation. J Mol Cell Cardiol. 27 (1), 407-417 (1995).
  11. Yokoyama, H., Ohmi, M., Murata, S., Nakame, T., Tabayashi, K., Mohri, H. Proposal of a working left heart model with a heterotopic transplantation technique in rats. J Heart Lung Transplant. 14 (4), 706-712 (1995).
  12. Maruyama, T., Swartz, M. T., McBride, L. R., Pennington, D. G. Working heart model of heterotopic heart-lung transplantation in rats. J Thorac Cardiovasc Surg. 107 (1), 210-215 (1994).
  13. Pacher, P., Nagayama, T., Mukhopadhyay, P., Bátkai, S., Kass, D. A. Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats. Nat Protoc. 3 (9), 1422-1434 (2008).
  14. Jespersen, B., Knupp, L., Northcott, C. A. Femoral arterial and venous catheterization for blood sampling, drug administration and conscious blood pressure and heart rate measurements. J Vis Exp. (59), 3496-3410 (2012).
  15. Habertheuer, A., Kocher, A., et al. Innovative, simplified orthotopic lung transplantation in rats). J Surg Res. 185 (1), 419-425 (2013).

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Cite This Article
Kearns, M. J., Wang, Y., Boyd, J. H. Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration. J. Vis. Exp. (99), e52418, doi:10.3791/52418 (2015).

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