The Experimental Study Of Future Remnant Liver Regeneration Induced By Selective PVL/PPVL Combined With In Situ Splitting

BackgroundPostoperative liver failure due to insufficient remnant liver after hepatectomy is a fatal complication. In the 1980s, Professor Makuuchi from Japan first applied selective portal vein embolization in patients requiring extended hepatectomy with the aim of avoiding postoperative liver failure, which induced hypertrophy of the nonoccluded liver segments and atrophy of the occluded liver segments by embolizing the branch of the portal vein. Originally planned extended hepatectomy was implemented when the remnant liver volume could meet the physical needs of patients. This procedure made lots of patients requiring major liver resection in whom the estimated functional residual liver volume was insufficient obtained the opportunity of surgery. Since then, portal vein embolization (PVE) or portal vein ligation (PVL) has been accepted by more and more hepatobiliary surgeons and widely used in the world.However, some shortcomings and disadvantages of this procedure have been paid attention with the increasing application of preoperative PVE or PVL. Recent studies have shown that PVE/PVL could increase the future remnant liver volume 8% to 46% between 2 and 8 weeks. That is to say, sufficient hypertrophy of the remnant liver is not always achieved using PVE or PVL. In addition, the mean period between PVE and liver surgery was 36.9 days according to literature. It provided the necessary conditions in time for tumor growth, invasion and metastasis. In addition, numerous clinical practice and animal experiments reports showed PVE/PVL enhanced tumor progression in the occluded lobe.In 2012, Schnitzbauer et al introduced a new strategy of accelerating remnant liver regeneration, which was a right PVL combined with in situ splitting(PVL+ISS) along the falciform ligament in initial surgical exploration to induce hypertrophy of the left lateral lobe in patients. This new strategy induced a 74% median volume increase in the left lateral lobe in a mean of 9 days, then a second radical hepatectomy was carried out. de Santibanes and Clavien named this technique "Associating Liver Partition and Portal vein ligation for Staged hepatectomy", or ALPPS. This approach significantly accelerated remnant liver regeneration in a short time and obviously shorten waiting time of the second surgery.ALPPS attracted worldwide attention and discussion in hepatobiliary surgery. However, the technical complexity of ALPPS and a high incidence of morbidity and mortality restricted its application. There are many related issues that need further clarification. Moreover, the exact mechanisms of the spectacular regenerative response in PVL+ISS are not clear. So we think it is necessary to establish an animal model of PVL+ISS and disclose the mechanisms of the spectacular liver regeneration in terms of hemodynamic, histopathology and molecular biology through compared with PVL. This will also lay a foundation for further research of ALPPS related issues.Due to in situ splitting could promote remnant liver regeneration obviously, we assume it may induce remnant liver regeneration through partial portal vein ligation or simultaneous hepatic artery ligation of occluded liver lobe, and combined with in situ splitting based on this model. Then we compare the hepatic regeneration rate of remnant liver lobe and degree of partial occluded lobe necrosis in different operation groups and evaluate its potential clinical value in order to provide theory and new ideas for solving some related problems after PVE/PVLPart Ⅰ Establishing a "portal vein ligation combined with in situ splitting" model in ratsOBJECTIVE:Establishing a mature and stable rat model of portal vein ligation combined with in situ splitting in order to lay the experimental foundation for further researching ALPPS related issues.METHODS:Adult male SD rats, weight 250-280g. A midline laparotomy was done after successful anesthesia of ether inhalation. Abdominal retractor will be used for expanding the incision and exposing the visual field of operation clearly. Firstly, peripheral hepatic ligament and liver lobes were freed under the operating microscope. Then according to preoperative plan, the corresponding portal veins of the right lobe, the caudal lobe, and the left lateral and left median lobes were ligated or suture ligated after careful dissection. The right median lobe was preserved. The ischemic line can emerge immediately at the right side of the falciform ligament when left branch of portal vein was ligated, then the in situ splitting was performed along the border between the left median lobe and the right median lobe. Compression or suture ligature was used for hematischesis to hemorrhage in the transection surface. Then the abdomen was closed when no active bleeding or errhysis was found. We divided the whole learning process into two stages according to the accumulation of experience, the microscopic operation proficiency and the successful rate of operation during the learning process of rat model. There were 15 rats in the first stage and 10 rats in the second stage. We evaluated the stability of the model through comparing the operation time, successful rate of operation, complication incidence rate and postoperative survival rate in two stages of learning process. All survival rats and 5 normal rats (weight 250-280g) were sacrificed on the 10th day after operation, then all right median lobes obtained were measured by laboratory micro scale to evaluate the liver regeneration of right median lobe. All Statistical analysis of experimental data were performed using SPSS statistical software (version 18.0). A significant difference was assumed when P was less than 0.05.RESULTS:1. The time of PVL was no significant difference between the two stages. The time of ISS and the whole operation in stage Ⅱ was significantly shorter than stage Ⅰ (p<0.05).6 rats died during surgery in stage I and no rats died in stage Ⅱ successful rate of operation was 60%VS100%(p<0.05).2.9 of the 15 rats in stage Ⅰ and 1 of the 10 rats in stage Ⅱ had intraoperative or postoperative complications. The complication incidence in stage II was lower than in stage Ⅰ (p<0.05). The survival rate was 100% in stage Ⅱ and 66.67% in stage Ⅰ. The difference was significant (p<0.05).3. All survival rats were sacrificed on the 10th day after operation. The mean weight of right median lobes increased from preoperative 2.02±0.23g to postoperative 5.58±0.46g, the right median lobes regenerated significantly.CONCLUSIONS:The operation time and the incidence of complications were significantly reduced through two stages of exploration and learning. The successful rate of surgery and postoperative survival rate achieved 100% in stage Ⅱ. Postoperative hepatic hypertrophy of right median lobes induced by the model increased significantly. These indicated that we had successfully established a mature and stable rat model of portal vein ligation combined with in situ splitting and could continue the next related experimental study using this model.Part Ⅱ The study on assessment of postoperative HRR and mechanisms of accelerated liver regeneration after PVL+ISS OBJECTIVE:Evaluating postoperative liver regeneration rate of portal vein ligation combined with in situ splitting. We will reveal the mechanisms of accelerating liver regeneration in terms of hemodynamic, histopathology and molecular biology through comparing with selective portal vein ligation.METHODS:The animals were the same as part Ⅰ. All animals were randomly assigned to one of three experimental groups according to different treatment. Group 1:sham operation (SHAM). The liver was freed from its ligaments after a midline laparotomy. The hepatic artery, portal vein and bile duct were dissected without ligation, and then the abdomen was closed. Group2:Selective portal vein ligation (PVL). The corresponding portal veins of the caudal lobe, left lateral and left median lobes, and the right lobe were ligated after careful dissection. The right median lobe was preserved. Group3:Selective portal vein ligation combined with in situ splitting (PVL+ISS). After PVL as group2, then the in situ splitting was performed along the border between the left median lobe (LML) and the right median lobe (RML). Laser speckle contrast imaging (LSCI) was used to detect the microcirculation blood perfusion of the right median and left median lobes during surgery. The animals of each group were sacrificed at 24,48, and 72 h and 7 days after the operation (n= 6 for each time point). The samples (serum and liver tissue) were collected. The hepatic regeneration rate (HRR) of the right median lobe was calculated after liver lobes were measured by laboratory micro scale. The collected serum was analyzed for ALT, AST, ALB and TBIL. Histopathologic changes of right median and left median lobes were observed under optical microscopy and immunohistochemistry was used to detect the number of Ki-67-positive hepatocytes of right median lobe. Cytokines such as TNF-α, IL-6, HGF and HSP70 in regenerating liver lobes or serum were investigated by RT-PCR and ELISA. All Statistical analysis of experimental data was the same as part Ⅰ.RESULTS:1. The hepatic regeneration rate (HRR) for both PVL and PVL+ISS were obviously higher than the SHAM group at all time points. Compared to the PVL group, PVL+ISS induced a greater regeneration response (p<0.05) at 72h and at 7 days after operation. There were no significant differences between the two groups at 24h and 48h.2. The results of Laser speckle contrast imaging (LSCI) showed the right median lobes had an increased microcirculation blood perfusion and the microcirculation blood perfusion of the left median lobes obviously decreased after selective PVL. The microcirculation blood perfusion of the left median lobes further decreased (p<0.05) after ISS and the right median lobes had no significant difference.3. The biochemical parameters of the serum:Both PVL and PVL+ISS caused an obvious increase in ALT and AST levels compared with the SHAM group at 24 and 48h. The ALT and AST levels in the PVL+ISS group were significantly different compared with the PVL group at 24h(p<0.05), There were no significant differences at 72 h and 7 days among these three groups. Significant differences in the serum albumin concentrations were observed in the PVL and PVL+ISS groups compared with the SHAM group at all time points. Serum albumin concentrations in the PVL+ISS group decreased considerably compared with the PVL group until the 72-h time point. TBIL levels in all groups were not significantly different at any time point.4. Detection of histopathology:Hematoxylin-eosin staining of the left median lobes after PVL and PVL+ISS revealed necrosis scores that were significantly larger after PVL+ISS than after PVL at 24 h after surgery. Immunohistochemical results showed the number of Ki-67-positive hepatocytes in the regenerating liver lobe was greater in the PVL+ISS group than in the PVL group at 48 h and 72 h (p<0.05). However, there was no significant difference between the two groups at 24 h and on day 7.5. Detection of cytokines in the regenerating liver lobes:All cytokines were highly upregulated in the regenerating lobes 24 h after PVL or PVL+ISS compared to sham-operated animals. In addition, we observed a significant increase in TNF-a and IL-6 mRNA in the PVL+ISS group compared with the PVL group. There was no significant difference for the mRNA levels of HGF and HSP70 between the PVL and PVL+ISS groups. Hepatic TNF-a, IL-6 and HGF protein levels with ELISA at 24 h post-surgery showed that hepatic TNF-a, IL-6 and HGF levels in the PVL+ISS group had a significant difference compared with the PVL group (p<0.05). Both groups were significantly different from the SHAM group (p<0.05). Serum TNF-a, IL-6 and HGF in PVL and PVL+ISS group increased compared with SHAM group (p<0.05). Serum TNF-a, IL-6 level in PVL+ISS group were higher than PVL group (p<0.05).CONCLUSIONS:1. Both PVL and PVL+ISS could induce hepatic regeneration of remnant liver effectively compared to the SHAM group. However, PVL+ISS induced a greater regeneration response, which also confirmed the reliability of the rat model of PVL+ISS.2. The possible mechanisms of accelerated liver regeneration included the reduced microcirculation blood perfusion of the left median lobe and up-regulation of cytokines (especially TNF-α and IL-6) which are closely related to hepatocyte proliferation in the regenerating lobes or serum after ISS.Part Ⅲ The impact and clinical significance of selective partial PVL or simultaneous hepatic artery ligation combined with in situ splitting on liver regeneration and injuryOBJECTIVE:To compare the impact of selective partial portal vein ligation (PPVL) or simultaneous hepatic artery ligation (HAL) combined with in situ splitting (ISS) on liver regeneration and injury and evaluate its potentially clinical significance.METHODS:We firstly preliminary evaluated the impact of different degree PPVL for left portal branch(left lateral and left median lobes) or simultaneous HAL combined with ISS on HRR of remnant liver and injury of occluded lobe in rats. The blood flow of left branch of PV was restricted through the portal branch was ligated with a needle by silk then the needle removed. We set up 4 experimental groups according to different operational treatment.(1)PPVL:The portal branches of caudal lobe and the right lobe were ligated, the left portal branch was partial ligated. (2)PPVL+ISS: (1)+ISS. (3)PPVAL:(1)+HAL of left lateral and left median lobes. (4)PPVAL+ISS: (3)+ISS. Each group was divided into 4 subgroups according to different degree of PPVL (diameter 0.6,0.5,0.4,0.25mm needle) and 4 rats in each subgroup. The rats were sacrificed at 72h after surgery for evaluating HRR of right median lobe and necrosis of left median lobe.We selected 2 experimental groups (PPVL using 0.4mm needle) for further research according to above preliminary experimental results and PVL group in part I was chose as control.1. PPVL+ISS.2. PPVAL+ISS.3. PVL. The animals were sacrificed at 24,48,72 h and 7 days after the operation (n= 6 for each time point). The samples were collected. The ultrasound flowmeter and LSCI were used to detect the blood flow of left portal branch and the microcirculation blood perfusion of the right median and left median lobes before and after PPVL or HAL during surgery. The methods of detecting HRR, HE staining, serum biochemical parameters, Immunohistochemistry for Ki-67 and RT-PCR for TNF-α、IL-6、HGF、HSP70 mRNA were similar to part Ⅱ. All Statistical analysis of experimental data was the same as part Ⅰ.RESULTS:The preliminary results of HRR and necrosis of liver lobes through different degree PPVL or simultaneous HAL combined with ISS in rats:1. Comparison within groups:The HRR of each experimental group enhanced with the degree of PPVL increasing except 0.4 or 0.25mm PPVL in PPVAL and PPVAL+ISS groups. The necrosis area of LML was enlarged with the increasing degree of PPVL (p<0.05).2. Comparison between groups:0.6 and 0.5mm PPVL, the HRR of PPVAL and PPVAL+ISS were obviously higher than PPVL and PPVL+ISS(p<0.05), no significant differences were found between PPVAL and PPVAL+ISS or PPVL and PPVL+ISS. There was no necrosis of LML when 0.6mm PPVL in each group and only mild necrosis when 0.5mm PPVL in PPVAL and PPVAL+ISS groups. 0.4mm PPVL, the HRR of PPVAL+ISS was significantly different from another 3 groups (p<0.05). No significant difference was found between PPVAL and PPVL+ISS and both groups were higher than PPVL group. The necrosis scores of LML in PPVAL and PPVAL+ISS were higher than PPVL and PPVL+ISS (p<0.05), PPVAL+ISS was higher than PPVAL and no difference was found between PPVL and PPVL+ISS.0.25mm PPVL,2 rats died after surgery in PPVAL+ISS. The HRR of PPVL+ISS and PPVAL+ISS enhanced obviously compared with PPVL and PPVAL (p<0.05). There were no significant differences between PPVL+ISS and PPVAL+ISS or PPVL and PPVAL. Large areas of macroscopic necrosis could be found in PPVAL and PPVAL+ISS, the necrosis score of LML in PPVL+ISS was higher than PPVL group (p<0.05).The various experimental results of PPVL+ISS and PPVAL+ISS using 0.4mm needle compared with PVL:1. The blood flow of left branch of PV was 6.35±0.50ml/min before PPVL and reduced to 1.85±0.32ml/min after PPVL using 0.4mm needle. The blood flow was 1.88±0.33ml/min after the concomitant hepatic artery was ligated, no significant difference was found before and after concomitant hepatic artery was ligated.2. The results of LSCI showed the microcirculation blood perfusion of RML increased and LML decreased after PPVL(p<0.05). The microcirculation blood perfusion of LML further decreased obviously and the RML had no significant change after concomitant hepatic artery was ligated.3. The HRR of each group had no significant differences at 24h after surgery. The HRR of PPVAL+ISS were higher than PPVL+ISS and PVL at 48h、72h and 7d (p<0.05). There were no significant differences between PPVL+ISS and PVL at all time points.4. The expression of Ki-67 in the regenerating liver lobe had no significant differences at 24h and 7d after operation. The expression of Ki-67 in PPVAL+ISS were stronger than in PPVL+ISS and PVL (p<0.05). There was significant difference between PPVL+ISS and PVL at 72h but not at 48h.5. The total necrosis scores of LML in PPVAL+ISS were significantly larger than PVL and PPVL+ISS and the necrosis scores of LML in PPVL+ISS were smaller than another 2 groups at 24h after surgery (p<0.05).6. PPVAL+ISS caused an obvious increase in ALT and AST levels compared with PPVL+ISS and PVL at 24h and 48h, the ALT level in PPVL+ISS was lower than PVL (p<0.05). AST level in PPVL+ISS was lower than PVL at 48h. Serum ALB concentrations in PPVAL+ISS decreased compared with PVL and PPVL+ISS at 24h and 48h (p<0.05). ALB level in PVL was lower than PPVL+ISS at 48h. TBIL levels in all groups were not significantly different except obviously increased in PPVAL+ISS at 24h and 48h.7. TNF-α and IL-6 mRNA in PPVAL+ISS increased significantly compared with PPVL+ISS and both groups were upregulated compared to PVL group (p<0.05). HGF mRNA in PPVAL+ISS increased compared to another 2 groups (p<0.05). HSP70 mRNA levels among these 3 groups were no significant different.CONCLUSIONS:1. Associating HAL or ISS with selective PPVL were both beneficial to the liver regeneration. The HRR will be enhanced with increasing degree of PPVL. However, the ISS promote liver regeneration only under the condition that PPVL achieved a certain intensity.2. Associating HAL aggravated injury of the partial occluded lobes. So how to control the degree of PPVL accurately was crucial to avoid severe hepatic necrosis or liver abscess after operation.3. The HRR of PPVL+ISS using 0.4mm needle was equivalent to that of PVL and the injury of partial occluded lobes were lighter than that of PVL. Both the HRR and hepatic necrosis area of PPVAL+ISS using 0.4mm needle increased significantly compared to that of PVL. However, no large areas of macroscopic necrosis appeared in the partial occluded lobes. Therefore, it is safe and feasible.4. Both PPVL+ISS and PPVAL+ISS were secure and effective and feasible to induce hypertrophy of the remnant liver as long as the degree of PPVL was controlled accurately. PPVL+ISS have some clinical value for patients who have a light poor liver function or whose hepatic artery become stenosis or occlusion due to invasion of lesions. While PPVAL+ISS may make atrophy or necrosis of tumor in the occluded lobe due to complete loss of hepatic artery blood supply during process of liver regeneration, which will provide a new idea for solving rapid tumor progression in the occluded lobe after PVE/PVL...