Hyperoxia in portal vein causes enhanced vasoconstriction in arterial vascular bed

Abstract

Long-term perfusion of liver grafts outside of the body may enable repair of poor-quality livers that are currently declined for transplantation, mitigating the global shortage of donor livers. In current ex vivo liver perfusion protocols, hyperoxic blood (arterial blood) is commonly delivered in the portal vein (PV). We perfused porcine livers for one week and investigated the effect of and mechanisms behind hyperoxia in the PV on hepatic arterial resistance. Applying PV hyperoxia in porcine livers (n = 5, arterial PV group), we observed an increased need for vasodilator Nitroprussiat (285 ± 162 ml/week) to maintain the reference hepatic artery flow of 0.25 l/min during ex vivo perfusion. With physiologic oxygenation (venous blood) in the PV the need for vasodilator could be reduced to 41 ± 34 ml/week (p = 0.011; n = 5, venous PV group). This phenomenon has not been reported previously, owing to the fact that such experiments are not feasible practically in vivo. We investigated the mechanism of the variation in HA resistance in response to blood oxygen saturation with a focus on the release of vasoactive substances, such as Endothelin 1 (ET-1) and nitric oxide (NO), at the protein and mRNA levels. However, no difference was found between groups for ET-1 and NO release. We propose direct oxygen sensing of endothelial cells and/or increased NO break down rate with hyperoxia as possible explanations for enhanced HA resistance.

Figure 1

Schematic illustration of the perfusion loops for arterial PV (a) and venous PV groups (b). (a) In arterial PV group, arterial blood was provided in the PV with total PV flow rate of 1 l/min. For this purpose blood flow was split after the oxygenator into the PV and HA lines with the same oxygen content. (b) In venous PV group, venous blood was provided in the PV with total PV flow rate of 1 l/min. For this purpose, oxygen deprived blood from the liver output was mixed with oxygenated blood after oxygenator. Flow rates in the arterial and deoxygenated lines were automatically adjusted to maintain an oxygen saturation at the liver output/outlet of ~ 65%. (c) Representative illustration of the perfusion machine. HA hepatic artery, PV portal vein, VC vena cava.

Figure 2

Oxygen delivery, vasculature resistance and patterns of vasoactive substance activation in experimental groups. Arterial PV group blue circles, n = 5 experiments, venous PV group red triangles, n = 5 experiments. (a,b) Representative experiment data of the HA and PV resistance from Group 0 without Nitroprussiat application. The increased HA resistance resulted in a decreased flow between perfusion day 1 and 3. From perfusion day 4, HA resistance decreased and was accompanied with increased PV resistance and absence of bile flow. (c) Oxygen delivery rate in the PV was higher with arterial PV compared to venous PV. (d,e) Oxygen saturation in the vena cava was higher in arterial PV group compared with venous PV group with no difference in oxygen uptake rate between groups during perfusion. (f) Resistance in arterial PV and venous PV groups controlled with Nitropurssiat. (g–l) Vasoactive substance release at mRNA and protein level without significant difference among experimental groups; (g) Endothelin-1 at mRNA level, (h) Endothelin-1 at protein level in tissue, (i) endothelial NO synthase (eNOS) and (j) inducible NO synthase (iNOS) at mRNA level, (k) NO level in perfusate, (l) heme oxygenase 1 at mRNA level. Superoxide dismutase (SOD) at mRNA (m) and protein (n) levels. P value * < 0.05, ** < 0.01, *** < 0.001. ns not significant.

Figure 3

Injury markers and liver function in experimental groups. Arterial PV group blue circles, n = 5 experiments, venous PV group red triangles, n = 5 experiments. (a–c) Injury marker release in perfusate shown for AST (a), cytochrome c (b) and 8-OHdG (c). (d,e) Representative staining showing integrity on H&E staining (g) and preserved glycogen stores in PAS staining (h) after one week of perfusion. (f) Representative immunohistochemistry staining for Caspase 3 showing absence of relevant apoptosis on perfusion day 7. (d) Macrophages were not activated in both groups as shown with TLR4 at mRNA level. (e) Endothelial cell activation at mRNA level expressed with ICAM-1. (i) similarly to ICAM-1, von Willebrand Factor immunohistochemistry staining showed absence of relevant endothelial cell activation. (j) Bile flow was constantly present in both experimental groups for one week. (k,l) Livers cleared lactate (k) and maintained albumin level (l) in perfusate. P value * < 0.05, ** < 0.01, *** < 0.001. ns not significant.