Thirty-two male New Zealand rabbits (2.5 ± 0.2 kg, 10 wk) were obtained from the animal center of Lanzhou University, Gansu, China, and individually housed under a 12-h light-dark cycle at least one week prior to the study. Animals were fasted for 12 h and deprived of water 4 h prior to the operation. The experiment was carried out in accordance with the ARRIVE guidelines for animal studies[13], and was approved by the Ethics Committee of the Lanzhou University Second Hospital. At the end of the study, all the animals were sacrificed using diethyl ether.

Animals were randomly allocated into four groups (n = 8 each): an anhepatic phase group (APH); a renal vein ligation group (RVL); a renal vein and hepatic pedicle ligation group (RVHP); and a sham operation (SOP) group. All four procedures were performed on a given day, in a random sequence, to eliminate any effect of training by the experimenters.

The rabbits were anesthetized with chloral hydrate (0.2 g/kg) by an i.v. injection in the ear, and catheters were inserted through the external jugular vein to collect blood samples and the right femoral artery for blood pressure monitoring. A large median laparotomy was performed after disinfecting the area with 0.5% iodine. For animals in the APH group, the hepatic pedicle, supra-hepatic vena cava, and infra-hepatic vena cava were dissected and ligated[14-16]. Animals in the RVHP group received dissection and ligation of the hepatic pedicle, supra-hepatic vena cava and renal veins, leaving the inferior vena cava open. Only the renal veins were ligated in the RVL group. The hepatic pedicles, and supra- and infra-hepatic vena cavae of rabbits were all dissected but not ligated in the SOP group. Data were collected at five time points during the procedures (0 min, 10 min, 20 min, 30 min, and 45 min) (Figure 1).

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Figure 1 Time-line flow chart. APH: Anhepatic phase group; RVHP: Renal veins, supra-hepatic vena cava, and hepatic pedicle ligation group; RVL: Renal vein ligation group; SOP: Sham operation group.

Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial blood pressure (MABP) were monitored continuously with a multichannel physiologic recorder (RM 6000; Datex-Ohmeda, Madison, WI, United State) through an arterial cannula in the right femoral artery[17,18].

Blood samples collected from the jugular vein were centrifuged, and the serum was stored at -80 °C. Serum concentrations of BK and ANGII were determined using enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

Groups were compared using two-way analyses of variance followed by a Bonferroni-Dunn or Tukey’s post-hoc test. Associations between hemodynamic parameters and serum vasoactive substance concentrations were analyzed by Spearman correlation analysis. Data are shown as mean ± SD, and P < 0.05 was considered statistically significant. All of the analyses were performed using SPSS software, version 18 (SPSS Inc., Chicago, IL, United States). The statistical methods of this study were reviewed by Hu XB from Lanzhou University School of Public Health.

The surgical operations were successfully carried out in all animals. The mean survival time was 50.7 ± 4.6 min, and only one animal in the RVHP group died prior to completion of the study. The baseline hemodynamic parameters and vasoactive substance concentrations did not differ among the groups (Table 1).

The changes in hemodynamic parameters (SBP, DBP, and MABP) are shown in Figure 2. There were no changes in any blood pressure measures in the SOP group throughout the procedure. However, all hemodynamic parameters were significantly decreased in the APH, RVL, and RVHP groups compared with baseline (0 min) (P < 0.05 for all). Moreover, SBP, DBP, and MABP values were significantly different from the SOP group at 10 min, 20 min, 30 min, and 45 min (P < 0.05 for all). Although values in the RVL group were higher than those in the APH and RVHP groups, the differences were not significant.

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Figure 2 Hemodynamic parameters. A: Systolic; B: Diastolic; C: Mean arterial blood pressures at various time points. ^aP < 0.05 vs SOP. APH: Anhepatic phase group; RVHP: Renal veins, supra-hepatic vena cava, and hepatic pedicle ligation group; RVL: Renal vein ligation group; SOP: Sham operation group.

Serum BK levels did not change over the course of the procedure in the SOP group (Figure 3). In contrast, serum BK levels at 10 min, 20 min, 30 min, and 45 min were significantly higher in the APH, RVL, and RVHP groups compared to baseline value (P < 0.05 for all). Furthermore, these BK levels were significantly increased compared to SOP controls at 20 min, 30 min, and 45 min (P < 0.05 for all).

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Figure 3 Serum bradykinin levels. ^aP < 0.05 vs 0 min and SOP. APH: Anhepatic phase group; RVHP: Renal veins, supra-hepatic vena cava, and hepatic pedicle ligation group; RVL: Renal vein ligation group; SOP: Sham operation group.

Serum ANGII levels did not change over the course of the procedure in the SOP group (Figure 4). In contrast, serum ANGII levels at 10 min, 20 min, 30 min, and 45 min were significantly lower in the APH, RVL, and RVHP groups compared to baseline value (P < 0.05 for all). Furthermore, these ANGII levels were significantly decreased compared to SOP controls at all postoperative time points (P < 0.05 for all).

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Figure 4 Angiotensin II levels. ^aP < 0.05, ^bP < 0.01 vs 0 min and SOP. APH: Anhepatic phase group; RVHP: Renal veins, supra-hepatic vena cava, and hepatic pedicle ligation group; RVL: Renal vein ligation group; SOP: Sham operation group.

Spearman correlation analyses revealed that BK levels were negatively correlated with SBP, DBP, and MABP in all experimental groups (P < 0.05 for all) (Table 2). In contrast, ANGII levels in the APH, RVL, and RVHP groups were positively correlated with all three hemodynamic measures (P < 0.05 for all).

Although orthotopic liver transplantation (OLT) has become the optimal treatment for end-stage liver diseases, it is still associated with substantial morbidity and mortality. Hemodynamic disorders are of primary concern, as they can determine the success of OLT.

Many factors can contribute to hemodynamic instability in OLT. Blood loss and ischemia/reperfusion syndrome are the most common factors, though major fluid shifts, acidosis, and electrolyte and coagulation abnormalities also contribute. Despite efforts to address these factors, hemodynamics has not been improved and complications remain high.

Although the reduction of blood volume typically increases peripheral vascular resistance, this is not observed during the anhepatic phase of OLT. Therefore, additional elements, such as vasoactive substances, may be involved in the pathogenesis of hemodynamic disorders in these patients. This study shows that renal vein congestion significant impacts hemodynamics, possibly through mechanism involving changes in serum levels of bradykinin (BK) and angiotensin II (ANGII).

Hemodynamic disorders in the anhepatic phase of OLT are not solely due to a decrease in vein blood volume returning to the heart, but may involve renal vein congestion. Serum BK and ANGII levels may be indicators of postoperative complications in OLT.

The kallikrein-kinin and renin-angiotensin-aldosterone systems play a vital role in regulating body blood pressure, and BK and ANGII are the key regulating factors in the two systems. BK is a potent vasodilator, and ANGII is a vasoconstrictor, which have an antagonistic effect on each other. When angiotensin-converting enzyme transforms ANGI into ANGII, it simultaneously inactivates BK.

This is an experimental study in 32 rabbits regarding the effect of renal vein congestion on hemodynamics and vasoactive substances in the anhepatic phase of OLT. The authors found significant hemodynamic differences in the OLT models compared with the sham group due to alterations of BK and ANGII. It provides a new avenue for the prevention of postoperative complications in OLT.