Transplantation/Immunology
Innovative, simplified orthotopic lung transplantation in rats

https://doi.org/10.1016/j.jss.2013.05.006Get rights and content

Abstract

Background

Various techniques of orthotopic single lung transplantation in rats have been reported; however, their widespread use has been limited owing to the complexity of the procedure. We report a novel microsurgical lung transplantation model in rats with a high survival rate that can be performed by one surgeon alone.

Methods

A total of 90 left lung allografts were transplanted from Fischer to Wistar Kyoto rats. We developed a triple axis precision system to place and stabilize the vascular clips intrathoracically to clamp the bronchovascular structures, thereby avoiding interference with the heart and contralateral lung movement. A single-suture bronchial anastomosis technique and proximal cuffing approach for vascular anastomosis was used, rendering surgical assistance unnecessary.

Results

In our recent series, both short-term (12 h) and long-term (21 d) survival was 100%. The lungs showed excellent perfusion and ventilation immediately on transplantation. Blood gas samples drawn from the left pulmonary vein and the histologic sections revealed excellent graft function. The donor operation lasted 20 ± 2 min, donor left lung dissection required 20 ± 2 min, and implantation required 90 ± 5 min.

Conclusions

The present innovative method of left orthotopic single lung transplantation can be performed by one experienced surgeon alone, with excellent results and a high degree of reproducibility.

Introduction

Lung transplantation represents the only curative treatment option for patients with end-stage pulmonary disease. Refinements in the surgical techniques during the past 30 years and novel immunosuppressive regimens have resulted in marked improvements in the overall outcome and patient survival after lung transplantation [1]. Despite these improvements, long-term graft survival has been significantly limited by bronchiolitis obliterans syndrome (BOS) [1], [2]. Ischemia–reperfusion injury is an additional, rather acute, problem resulting in the risk of acute graft failure in clinical lung transplantation [2], [3], [4], [5]. Our understanding of the molecular processes involved in ischemia–reperfusion injury is that inflammatory and mechanical insults to the endothelium of the microvasculature trigger an injury with a deleterious tissue response [6], [7]. The mechanisms underlying BOS development, however, are not clearly understood. When considering the incidence of BOS (i.e., developing in 50% of recipients within 5 years and 75% within 10 years after lung transplantation), it becomes clear that novel therapeutic strategies are urgently desired for BOS treatment [2].

Novel approaches using molecular tools in counteracting ischemia–reperfusion injury and BOS necessitate standardized lung transplant models in animals. Although large animal models are currently available [8], rodent transplant models have the advantage of significantly lower costs and the availability of several plasmids and vectors for application in this species [9], [10]. Asimacopoulos et al. [11] first reported on orthotopic rat lung transplantation in 1971. Mizuta et al. [12] reported the cuff technique for rat lung transplantation, which facilitates anastomoses of the pulmonary artery and vein, in 1989. Furthermore, they described a suture technique for bronchial anastomosis [12]. Reis et al. [13] described a nonsuture external cuff technique that did not require microscopic suturing of the vessels or bronchus. More recently, Goto et al. [14] reported a technique of interposing parts of the donor rat's descending aorta to complete the anastomoses of the corresponding bronchovascular structures. However, the major obstacles associated with these models have been that they were not physiological, such as was the case for the model reported by Goto et al. [14], had high dropout rates and high complication rates, or required extensive microsurgical training and the presence of two surgeons to perform the procedure. Complications, such as arterial bleeding, venous bleeding, anastomotic leakage, bronchial stenosis, bronchial leakage, hematothorax, and pneumothorax, have been the major limiting factors in animal survival and operative outcome.

Thus, we established a simplified and reproducible orthotopic single lung transplant procedure in rats with excellent short- and long-term survival rates that can be performed by a single surgeon. We report on a precision, triple axis, vascular clip stabilizer (Fig. 1A), which markedly facilitated the procedure and allowed reproducible anastomoses. We have provided blood gas analysis results of blood drawn directly from the left pulmonary vein 60 min after reperfusion and the examination results of histologic sections of the left lung allografts 60 min and 24 h after transplantation to prove regular histologic features, physiological integration, and proper post-transplant function.

Section snippets

Development of the triple axis stabilizer

An aluminum plate served as the base for a 15-cm-long steel cylinder (Fig. 1A) that was probed with an L-shaped 2-mm steel wire. The cylinder was tapped on the side to allow for vertical movement in the cylinder and fixation of the wire with a screw (Fig. 1A). A commercial mosquito clamp was mounted on top and fixed with an articulated joint (Fig. 1B). Intraoperatively, an aneurysm clip (Aesculap FE720 Miniclip, Aesculap, Central Valley, PA), clamping the cuffed vessels and the recipient

Results

A total of 90 transplantations were performed. On average, the donor operation lasted 20 ± 2 min, and the left lung dissection of the donor bronchovascular structures lasted 20 ± 2 min. Implantation and revascularization required 90 ± 5 min, on average. Including the time needed to perform all anastomoses (10 ± 1 min for the bronchial anastomoses and 5 ± 1 min for each vascular anastomosis), we achieved ischemia times as short as 40 min.

Our technical success rate was 95%. Perioperative

Discussion

We have reported on a novel model of orthotopic single lung transplantation in rats. It is a straightforward procedure that addresses many of the limitations described with previous methods. In contrast to other investigators using the cuff technique, we are the first group to use a proximal cuff technique by placing an 18-gauge polyethylene intravenous catheter on the recipient pulmonary artery and a 16-gauge cuff on the pulmonary vein, instead of cuffing the donor vasculature [12], [13], [15]

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