Authors
M. DE RUDDER (1), A. DILI (2), B. PIRLOT (3), C. BOUZIN (4), I. LECLERCQ (3) / [1] Université catholique de Louvain, Brussels, Belgium, Laboratory of Hepato-Gastroenterology, [2] CHU UCL Namur, Site de Godinne, Yvoir, Belgium, HPB surgery, [3] UCLouvain, Université catholique de Louvain, Brussels, Belgium, Laboratory of Hepato-gastroenterology, Institut de Recherche Expérimentale et Clinique, [4] UCLouvain, Université catholique de Louvain, Brussels, Belgium, Imaging plateform, Institut de Recherche Expérimentale et Clinique
Introduction
After hepatectomy, hepatocytes proliferate first and before the proliferation of sinusoidal endothelial cell (SEC) causing a transient imbalance between cell populations and a transient perturbation of the lobular architecture with proliferating hepatocytes forming avascular, hypoxic, clusters. The larger the liver resection, the highest the portal hyperperfusion and the more hepatocytes proliferate. Hence, the larger the liver resection, the larger the avascular, and thus non-functional, hepatocyte islands. Hypoxia is, thus, considered at the origin of liver dysfunction, also called a "small-for-size syndrome" (SFSS). The new surgical technique called “Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy” (ALPPS) allows for a rapid remnant hypertrophy with high resection rates compared to the more conventional techniques. Recent work from our lab on rats showed that ALPPS rescued survival compared to a standard SFSS-setting hepatectomy. Also, ALPPS was associated with hypoxia in the remnant. Confirmatory, induction of hypoxia after a SFSS hepatectomy rescued survival. Hypoxia also activated HIF pathways and triggered a pro-angiogenic response. We hypothesize that hypoxia-induced angiogenesis helps to maintain the lobular structure and thus liver function during liver regeneration.
Aim
The aim of the present study is to analyze the remodeling of the liver sinusoidal network during SFSS-hepatectomy with of without exposure to hypoxia.
Methods
Directly after a SFSS-setting hepatectomy (where 80% of the liver is removed) (referred to as time T), mice were housed in a gas tight chamber and exposed to a controlled hypoxic environment (11% FiO2) (PHx80 HC) or to ambient air (20,9% FiO2) (PHx80). We sacrificed mice at T+24H, T+48H, T+72H and T+7days. Survival was assessed twice a day. Endothelial cell proliferation was assessed by double immunofluorescence to detect the proliferative marker Ki67 and CD31/PECAM-1 to endothelial cells: proliferative endothelial cells were Ki67+/CD31+. CD31- cells were divided by their size as such: <45µm² = non-parenchymal cells (except endothelial cells); >45µm² = hepatocytes. Feret's diameter of liver sinusoids and sinusoidal area were assessed manually and semi-automatically respectively on x20 images of CD31 immunostained liver sections. Mitosis were counted on x20 fields on H&E staining.
Results
In Animals with SFSS hepatectomy, mortality was high with only 30% of survival at T+7days with most of the deaths occurring in the first 3 days. Exposure of the animal to the hypoxic environment directly after the surgery dramatically improved their survival as 95% of the mice survived at T+7days. Liver cells' proliferation was assessed by immunofluorescence. Proliferative hepatocytes were hardly present in the regenerating livers at T+24H while hypoxia chamber slightly but not significantly increased their numbers at T+48H (0.36±0.38 in PHx80 vs 4.55±5.77 in PHx80 HC, p value= 0.08). Mitosis counts confirmed these results, as hardly any mitosis were found at T+24H. At T+48H, mitosis count increased in both groups. As so, liver weight was not different between PHx80 and PHx80 HC during the course of liver regeneration. On the other hand, endothelial cell proliferation was greatly enhanced with hypoxia: differences between PHx80 and PHx80+HC were already seen at T+24H (0.52±0.39 vs 5.56±2.87, p = 0.0018) and even larger at T+48H (0.71±0.88 vs 8.81±4.07, p value< 0.0001). The vascular bed (as the mean sinusoidal diameter) was increased at T+24H in PHx80 HC compared to PHx80 (6.19±0.47 vs 4.94±0.21, p value= 0.001). Also, the sinusoidal area was larger in PHx80 HC compared to PHx80 at T+48H (54055±10850 vs 19864±9201, p value= 0.0007).
Conclusions
Our data demonstrate that hypoxia rescues survival after a SFSS-setting hepatectomy. Hypoxia had no effect on hepatocyte proliferation but accelerated LSEC proliferation, increased the diameter of liver sinusoids and the overall vascular sinusoidal areas. Our results are compatible with the hypothesis that hypoxia triggers and angiogenic response as to ensure proper blood supply to regenerating hepatocytes, hence supporting liver function.
