The Simple Maneuver to Safely Extend the Anhepatic Time to 30 Minutes for Rat Liver Transplantation

Background: Orthotopic rat liver transplantation (OLT) is widely used; it remains to be challenging although many renovations have been made. Methods: For tolerance induction, we performed OLT including whole graft OLT from close Sprague Dawley (SD) rat to SD (whole graft group, control group, n=21) and 30 minute anhepatic time (AHT) group (AHT group, n=11). For good outcomes of AHT group and generalization of this maneuver, partial OLT was performed from 50% Lewis allograft to Brown Norway (BN) rats to induce tolerance (half graft group, n=28), Cyclosporine A was injected once daily for 14 days. Results: For whole graft group, 30-day survival rate was 85.5% (18/21), the reasons of death were gas embolism due to the missed suturing in 2 cases, blood loss in 1 case. For AHT group and tolerance group, 30-day survival rate was 72.7% (8/11), 64.3% (18/28), the causes of death were diverse. There were no differences in survival in 3 groups (p=0.289). Conclusion: The anhepatic time can be extended simply through the change of clamping the diaphragm, which facilitates its application in the research.

Results: For whole graft group, 30-day survival rate was 85.5% (18/21), the reasons of death were gas embolism due to the missed suturing in 2 cases, blood loss in 1 case. For AHT group and tolerance group, 30-day survival rate was 72.7% (8/11), 64.3% (18/28), the causes of death were diverse. There were no differences in survival in 3 groups (p=0.289).
Conclusion: The anhepatic time can be extended simply through the change of clamping the diaphragm, which facilitates its application in the research.

Background
Rat OLT is well-accepted model especially in the research of tolerance induction since Lee and his colleagues developed [1,2], it is complicated in microsurgical techniques, especially for new microsurgeons although many valuable modi cations have come into forth [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 introduce the simplest method to extend AHT to 30 minutes while facilitating this complicated procedure.

Methods
For our project, rat OLT was performed to study liver regeneration and immunological tolerance through stem cells (detailed protocol out of scope here).Following the procedure in the literature, we observed that clamping the diaphragm led to abnormal breathing and rat death (supplemental video 1), and that the change of clamping the diaphragm bene ted this microsurgical procedure and improved rat survival.
Whole OLT was performed from close SD to SD with different AHT, and partial OLT was performed with Lewis to BN as the acute rejection model whereas this technique was generalized and applied in our project (Table 1). Surgical procedure Iso urane inhalation anesthesia was applied with a few modi cations. In the donor procedure, a transverse incision was made to enter the abdominal cavity of the rat. 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, 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 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 (Figure 1), SHVC was anastomosed with 8-0 polypropylene running suture (Figure 2), when this anastomosis was completed, the forceps was replaced with a vascular bulldog on the real SHVC while the diaphragm ring was de-clamped (Figure 3), the anhepatic time was generally less than 20 minutes. PV was reconnected with the cuff; blood ow was restored once the clamp on the PV was released. IVC reconnection was made as was for PV. For AHT group, the clamp on the PV was released until 30 minutes. The gastroduodenal artery was proximally ligated and an opening was made on the common hepatic artery into which the stent in the donor hepatic artery was inserted and secured ( Figure 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. 10% glucose solution and puri ed water were both supplied for rst 3 days, and later regular food and tap water were offered. Sodium ceftizoxime (100mg/kg) 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. Sacri ce was made on deep anesthesia with iso urane for euthanasia.

Statistical analysis
The cumulative survival rates of different groups was evaluated with the Kaplan-Meier Curve, the analysis was made with SPSS 22.0 software (IBM Corp, Armonk, NY, USA), and P<0.05 was considered signi cant

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 30-min 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 research [1,2,6]. Short 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 extended to 30 minutes (even 35 minutes) whereas survival rates were not signi cantly different, in the time-e cient manner (30 min), the different anhepatic time has little impact on recipients survival.
Continuous suture and cuff method comprise the reconstruction of SHVC [1.2.9-14], suturing should be completed rapidly while SHVC is blocked and it necessitates the diaphragmatic ring being clamped a little more without ventilator aid. The diaphragm is to maintain respiratory movement, once clamped and retracted downward (caudalad), it dramatically affects respiratory and causes the rat to move due to compromised respiration (supplemental video 1), some surgeons might add anesthesia, resultantly the rat will die, especially under plain and simple mask anesthesia inhalation (ether etc), this is the reason that cardiac arrest and higher mortality occurred 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 the protocol is out of scope here.

Conclusion
OLT can be safely performed with 30 minute AHT extended simply through the change of clamping the diaphragm; this procedure facilitates its application in the research. The mosquito forceps and the bulldog for occlusion of SHVC.

Figure 2
Clamping the diaphragmatic ring with the mosquito forceps.

Figure 3
The diaphragmatic ring declamped with the bulldog. Intraoperative view. The hepatic artery was reconnected Figure 5 Survivals for different groups.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.