Exploring the Enhanced Liver Regeneration Patterns Following ALPPS Versus Selective Portal Vein Ligation in an Experimental Model

Abstract

Background: Associating liver partition and portal vein ligation (PVL) for staged hepatectomy (ALPPS) and selective PV embolization (PVE) are important clinical strategies in liver surgery. Even though it has been demonstrated that ALPPS induces a more rapid and expressed hypertrophy than PVL/PVE, this phenomenon is still not well understood.

Aim: In the present study, we aimed to characterize enhanced regeneration patterns in a rat model.

Methods: Male Wistar rats were used (n = 84; 220-250 g). Selective PVL and ALPPS were achieved using microsurgical techniques (RML-regenerating/LML-non-regenerating). Parameters of liver regeneration, microcirculation, hepatocyte morphology, hepatocellular injury, and activation status of certain protein kinases involved in liver regeneration were investigated.

Results: Right median lobe (RMLs) in the ALPPS group exhibited a more significant and rapid hypertrophy compared to PVL (regeneration ratio, 1.669 ± 0.155 vs. 1.980 ± 0.189, p = 0.009, PVL vs. ALPPS). ALPPS led to a more prominent hepatocellular injury. Hypertrophy was associated with increased microcirculation of the RML and a prominent increase of hepatocellular size (300.43 ± 31.92 μm² vs. 374.48 ± 58.34 μm², PVL vs. ALPPS) and morphology. There was an early pAkt/Akt activation after surgery which was significantly higher in ALPPS (5 ± 2 vs. 9.7 ± 3 RQ-fold-change, p = 0.0087, PVL vs. ALPPS).

Conclusions: Our results suggest that the enhanced regeneration in ALPPS is associated with characteristic changes in liver microcirculation, cell division, hepatocyte morphology, and activation of pAkt/Akt.

Keywords: ALPPS; animal model; hypertrophy; liver; regeneration.

FIGURE 1

Study flowchart and experimental design. (A) Protocol for sampling and sacrifice. Animals were randomized into three experimental groups: PVL (n = 36), ALPPS (n = 36), and sham (n = 12) operation. Black bars represent the groups according to the different observation periods (n = 6/group/timepoint). (B–F) Schematic diagrams (B, E) and photographs (C, D, F, G) of the operative procedure. Portal branches of the caudal lobe (CL), left lateral (LLL), left median (LML), and the right lobe (RL) were ligated. The immediate changes in the circulation of the liver created a visible demarcation line (dashed line) between the portally perfused RML and portally not perfused LML. Parenchymal transection was performed along the falciform ligament. (G) Photo represents the macroscopic changes of the liver lobes on the postoperative 3rd day after parenchymal transection.

FIGURE 2

Changes in regeneration rate and cell‐cycle entry. (A) Regeneration rate at the indicated timepoints. Regeneration rate was expressed as a ratio of relative liver weight of RML (g/100 g b.w.) at sacrifice and relative liver weight of RML (g/100 g b.w.) in the control group at 12, 24, 48, 72, and 168 h PVL versus ALPPS. (B) Weight gain of RML in relation to the total body weight (BW) at 12, 24, 48, 72, and 168 h PVL versus ALPPS. (C) Alteration of Ki‐67 index at 24, 48, 72, and 168 h PVL versus ALPPS. (D) Body weight changes at 4, 12, 24, 48, 72, and 168 h PVL versus ALPPS. Values are mean ± standard deviation; n = 6/group/timepoint). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 PVL versus ALPPS. (E, F) Representative immunohistochemistry images showing Ki‐67 immunostaining at 48 h. Arrows show Ki‐67 positive cells. Original magnification ×400.

FIGURE 3

Alterations of liver microcirculation. (A–C) Microcirculatory flow, velocity, and oxygen saturation of RML and LML at 4, 12, 24, 48, 72, and 168 h PVL versus ALPPS. Values are mean ± standard deviation; n = 6/group/time point. Continuous lines represent the regenerating RML, while dashed lines show the ligated LML. **p < 0.001, ****p < 0.0001 PVL‐LML versus PVL‐RML, ⁺⁺⁺⁺ p < 0.0001 PVL‐LML versus ALPPS‐RML, ^## p < 0.01, ^#### p < 0.0001 PVL‐LML versus ALPPS‐RML, ^xxxx p < 0.0001 ALPPS‐LML versus ALPPS‐RML, ^& p < 0.05 PVL‐RML versus ALPPS‐RML.

FIGURE 4

Changes in hepatocyte cell size and morphology. (A, B) Alterations in serum transaminase levels during the observation period at 4, 12, 24, 48, 72, and 168 h PVL versus ALPPS. Values are mean ± standard deviation; n = 6/group/time point. (C–E) Percentages of cells by cell size at 48 h (C), 72 h (D), and 168 h (E). ⁺ p > 0.05 PVL versus Sham, *p < 0.01, **p < 0.001 PVL versus ALPPS, ^# p < 0.01, ^## p < 0.001 ALPPS versus Sham. (F–H) Mean cell size in μm² at 48 h (F), 72 h (G), and 168 h (H), ****p < 0.0001 PVL versus ALPPS. Values are mean ± standard deviation; n = 6/group/time point. (I, J) Representative image of beta catenin immunofluorescence staining in PVL (I) and ALPPS (J) at POD3. Original magnification ×400.

FIGURE 5

Serum TNF‐alpha and IL‐6 levels. (A, B) Alterations in serum cytokine levels during the observation period at 4, 24, 72, and 168 h PVL versus ALPPS. Values are mean ± standard deviation; n = 6/group/time point. *p < 0.01, **p < 0.001, ****p < 0.0001 PVL versus ALPPS.

FIGURE 6

Tissue protein levels pAKT/Akt, pERK/ERK, pJNK/JNK. (A–C) Relative protein expression of pAKT/Akt, pERK/ERK, pJNK/JNK in liver tissue at 4, 12, 24 h PVL versus ALPPS, ***p < 0.001. Values are mean ± standard deviation; n = 6/group/time point. The vertical dotted lines in the Western blot images denote that the blots have been spliced to remove the triplicate lanes. Western blot analysis shown is representative of independent experiments. Complete raw data has been provided in the online.