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The first successful cervical heterotopic heart transplantation in mice was reported by Chen et al[1] in 1991. But there have been only a few studies using this method owing to its difficulty in operation, especially microvascular suture. Matsuura et al[2-3] successfully applied the cuff technique to cervical heterotopic heart transplantation in mice. Although this method reduces the operative mortality and greatly shortens the operative time, there still have been some technical problems.
In the present study, we make further improvements to mouse models of cervical heterotopic heart transplantation using the tail-cuff technique. Results show that the improved tail-cuff technique can shorten the ischemic time of the grafts and increase the successful rate of surgery and is an ideal method for establishing mouse models of cervical heterotopic heart transplantation. 

The cervical heterotopic heart transplantation model reported by Chen et al[1] in mice have some advantages, such as relative small injuries, the lower incidence rate of infection, the evidence for acute rejection using direct palpation of heartbeat, and higher survival rate. However, the anastomosis of small vessels is of technical difficulty because of small size, which widely limits the use of the mouse model. Then, cuff technique was successfully used in cervical heart transplantation model in mice, which greatly reduces the difficulty of the operation and shortens the operative time. There still have been some technical problems. The difficult part of cuff technique is to evert the vessels to wrap around the cuffs, which frequently spends lots of time. To solve this problem, Matsuura et al[2] considered that it was necessary for three retention sutures at the free edge of vessel. However, the cuff remained easy to move because of having no point of support. It prolonged ischemia time of the graft and was difficult to perform by one surgeon. Recently, Wang et al[6] prepared the cuff on PA of the graft. But often, it is impossible to make the cuff when the PA trunk is too short. In our previous experiments, a cuff with a mandril was used to evert the vessels[3]. However, insertion of the mandril will easily lead to damage to vascular intima and finally result in thrombus.
In this study, we perfectly solved the problem of lacking of support point at the base of cuff. The tail of arterial cuff or venous cuff was immobilized with the CA or the right external jugular vein by microhemostat clamps. Then, we can unhurriedly evert the vessels to wrap around the cuffs with the superfine-forceps. Compared with the cuff made from segmental epidural catheter used in our previous model, the tube wall of intravenous catheter made of 24 G or 22 G intravenous catheter is thinner, and the inside diameter is more larger. So, blood flow entering the cardiac graft is sufficient, and the reflux of venous blood is unobstructed. In addition, the tail-cuff techniques would not require three retention sutures, which simplifies and improves the surgical procedures. The whole process could be performed by one surgeon with little training for microvascular surgery. The total ischemic time of grafts was shortened. Especially, the average time for vascular everting and reconnection of both vessels was obviously shortened, and the surgical success rate was 100%. In addition, the histopathological changes showed an identical mild inflammatory response in isografts and the significant rejection in allografts, which are consistent with previous results reported by Wu et al[7], which proves that the model is stable.
Several reports previously published described complications such as thrombus formation related to the application of the cuff technique in rabbits and rats[8]. However, there were no complications related to thrombus formation in this study. The present results are consistent with many other researchers who reported no thrombus formation or stenosis in heart transplantation operations performed using the cuff  technique[2, 5-6]. Results from this study demonstrated that this improved technique using the tail-cuff for mouse heart transplantation was success and practicable.
Additionally, it is necessary to pay attention to the following factors to ensure the success rate of operation. One is to make a notch at the middle of cuff body to prevent slippage of donor heart, especially after regaining of donor heart. Another is to avoid twisting of the vessels, which is difficult to find because vascular wall is very thin. So it is helpful to remember the position of the retention suture before everting the vessels. Thirdly, we suggest that it should be not necessary to resect the right sternocleidomastoid muscle, which often results in errhysis. It is more important that reservation of sternocleidomastoid muscle can avoid twisting of the cardiac grafts, because the pulmonary artery and aorta of donor heart are just on the two sides of sternocleidomastoid muscle following implantation of donor hearts.
In summary, this improved tail-cuff technique we described facilitates vascular anastomosis in cervical heart transplantation in mice, shortens ischemia time of the graft, and finally leads to a higher success rate of surgery. This technique has its superiority and can serve as an ideal method for establishing cervical heterotopic heart transplantation model in mice.

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