The simple surgery to safely extend the anhepatic time to 30 minutes for rat liver transplantation

Background Orthotopic rat liver transplantation (OLT) is widely used, it remains complicated although many renovations have been made. Methods OLT was performed for tolerance induction, including whole graft OLTs from close male Sprague Dawley (SD) rat to male SD (whole graft group, n = 21) and 30 minute anhepatic time group (AHT group, n = 11), OLT was performed from 50% male Lewis allograft to male Brown Norway (BN) rats to induce tolerance (half graft group, n = 28), Cyclosporine A was injected once daily for 14 days.


Introduction
Rat OLT is well-accepted model on immunology, ischemia-reperfusion injury and regeneration since Lee and his colleagues developed [1,2], nowadays it is complicated requiring microsurgical techniques, especially for new microsurgeons although many valuable modi cations have come into use [3][4][5]. It is documented that the short anhepatic time plays a critical role of the success of LT and improves survival greatly, and that cardiac arrest in the recipient frequently occurred when AHT was more than 26 minutes [6,7]. Higher mortality rate during AHT necessitates researchers to extend AHT. Here we rst introduced the simplest method to extend AHT to 30 minutes while facilitating this complicated procedure.

Methods And Materials
For our project, OLT was performed to study liver regeneration and immunological tolerance through stem cells (detail out of scope).Following the procedure of literature, we observed that clamping the diaphragm would lead to abnormal breathing and rat death (supplemental video 1), and that the change of clamping the diaphragm would bene t this microsurgery and rat survival, it is generalized in this procedure. Whole OLT were performed from close male SD to male SD with different AHT, partial OLT performed with male Lewis to male BN as the acute rejection model (Table 1).

Surgical Procedure
Under iso urane inhalation anesthesia with a few modi cations, a transverse incision was made to enter the abdominal cavity of rats. In the donor procedure, the liver was ushed through the aorta with lactated Ringer's solution, and then re ushed through the portal vein (PV). The graft was immersed in lactated Ringer's solution. Cuffs were prepared for PV and infra-hepatic vena cava (IVC). For 50% graft, the caudate lobes, the left lateral one and the left portion of the median lobe were removed at the back table.
Cold storage time was less than 3 hours in all cases. In the recipient part, after the abdominal cavity was opened like the donor, all ligaments of the liver were cut. The proper liver artery was ligated proximally, and the accessory liver artery was ligated and cut, a blunt separation behind the liver was made to create a tunnel. The left sub-diaphragmatic vein was ligated closed to the diaphragm. The recipient PV and IVC were cross-clamped with microvascular clamps, and iso urane was immediately decreased to 0.3 volume %. A mosquito forceps was placed through the tunnel on the part of diaphragm ring (left side) to occlude SHVC and stabilized (Fig. 1), SHVC was anastomosed with 8 − 0 polypropylene running suture (Fig. 2), when this anastomosis was completed, the forceps was replaced with a vascular bulldog on the real SHVC while the diaphragm ring was declamped (Fig. 3), the anhepatic time was generally less than 20 minutes. PV was reconnected with the cuff, followed by blood ow restoration once the clamp on the PV was released. The IVC reconnection was made as was for PV. For 30 minute AHT group, the clamp on the PV was waited and released until 30 minutes. Proximal ligation of the gastroduodenal artery was made with a suture and an opening was made on the anterior wall of the common hepatic artery into which the stent in the donor hepatic artery was inserted and secured with 8 − 0 silk suture and xed from both ends (Fig. 4, supplemental video 2), bile duct continuity was made when a tube in the donor bile duct was inserted into the recipient bile duct. The abdomen was closed with two layers and the animals were kept in a cage under an infrared light. Tape water and 10% glucose solution were both supplied for rst 24 hours, and later regular food and tape water were offered. Antibiotic was injected subcutaneously once a day, 4 days in total. For half graft group, cyclosporine A was subcutaneously injected once daily for 14 days and ceased afterwards.

Results
For the whole graft, 30-day survival rate was 85.5% (18/21), the reasons of death were gas embolism due to missed suturing during the anastomosis in 2 cases, blood loss in 1 case. For AHT group, 30-day survival rate 72.7% (8/11), the causes of death were respiratory failure in one case, unknown reasons were in 2 cases. For half graft group, 30-day survival rate was 64.3% (18/28) (Fig. 5), the reasons of death were diarrhea in 4 cases, BN rats ate themselves in 6 cases. There were no differences for survival for 3 groups(p = 0.289).Histological examination revealed almost normal structures of liver without brosis, ductopenia, thickened wall in the liver arterioles and venules in half graft group (H&E staining not shown).

Discussion
Since Kamada introduced the cuff method for OLT, it has greatly bene ted OLT in basic and clinical medicine 1,2,6 . AHT as a surgical skill has been prioritized to minimize as soon as possibly by clinicians and microsurgeons who perform liver transplantation. Clinically it ranged from 37 to 321 minutes, it was reported that over 100-minute AHT was associated with a higher incidence of graft dysfunction [6,8]. Experimentally, the AHT ceiling of rat OLT is 26 minutes in the literature [1,6]. For our report, it is safely prolonged to 30 minutes, or even 35 minutes whereas survival rates were not signi cantly different, in the time-e cient manner, the different anhepatic time has little impact on recipients and survival.
Continuous suture and cuff method comprise the reconnection of SHVC [1.2.9-14], suturing should be completed rapidly while SHVC is blocked and it necessitates clamping the diaphragmatic ring a little more without ventilator assisting. The diaphragm is to maintain respiratory movement, once clamped, that dramatically affects respiratory and causes rats to move due to compromised respiration (supplemental video 1), some surgeons might add anesthesia, consequently the rats will die, especially under plain and simple mask anesthesia inhalation (ether etc), this is the reason that cardiac arrest occurred and higher mortality came forth during AHT [7]. Cuff method e ciently shortens SHVC anastomosis, but it is not universally applicable due to short SHVC in length. Magnetic ring is a cuff method in nature and precludes future MRI examination [13,14].
Pharmaceutics extension AHT came forth in some centers, prostaglandin and its analogue were used to extend AHT and improve survival [15,16]. Liu et al reported that clamping the supra-celiac aorta one minute can effectively improve rat OLT by increasing the tolerable time of AHT [6], this maneuver does not surpass the AHT ceiling. Our maneuver is surgically easy and effectively extends AHT to 30 minutes.
A few references were reviewed to reveal that the diaphragm was clamped much more [17,18], we followed that procedure which led to irregular breathing (supplemental video 1). In the literature almost no attention has been paid to how to clamp SHVC or the diaphragm on the reconnection of SHVC, our report is the rst description of clamping the diaphragm. As to tolerance induction in our report, it is ongoing research of our project and beyond the scope here.
In summary, OLT can be safely performed with 30 minute AHT extended through the change of clamping the diaphragm; this procedure facilitates its application in the research.