The Experimental Study Of Future Remnant Liver Regeneration Induced By SPVL Combined With LPT/P-LPT In Rats

[Background] Up to now, liver resection remains to be first considered by the hepatobiliary surgeon as the treatment of patients with hepatic carcinoma. Along with the development and improvement of the related surgical concepts and surgical techniques, more patients have the opportunity to receive a liver resection to cure the disease. However, various complications related with liver resection have always been accompanied with liver surgery. Just as a professor said in reviewing the development of the liver tumor surgery:"Such phenomenal achievement did not come easily, but with the’blood, sweat, and tears’of patients and their families and of courageous surgeons"。the liver failure caused by insufficient volume of remaining liver is a deadly complication after hepatectomy. It’s gratifying that the liver’s ability to regenerate itself could provide a unique way to solve this problem. Therefore, to find the effective way to increase the residual liver volume after hepatectomy has become the focus of attention and efforts of liver surgeons and related fields.Portal vein embolization (PVE) and Portal vein ligation (PVL) have been widely accepted and applied in the clinic. However, the shortcomings and disadvantages were focused and discussed by people. It’s reported that waiting time for hyperplasia of PVE/PVL was much longer while a part of patients faced the tumor progress during the waiting time.Liver surgeons never stop the pace for exploration and continue to seek more effective ways to improve the liver regeneration. In 2012 ALPPS was reported as a new method which was able to induce the rapid proliferation of reserved hepatic lobe.This method means to ligature the portal vein of the intended resection side of the liver, and then combined with the liver parenchyma transection, meanwhile retained all the arteries, bile duct and hepatic venous not changing. So after the sufficient proliferation of induced reserved hepatic lobe, a second operation was performed to remove the liver of the tumor side.The arising of ALPPS quickly attracted the attention of the hepatobiliary surgery and became the focus of research and discussion. More medical centers in the world have tried and carried out this technology. Therefore the issues of the mechanisms of the regeneration and the risk of surgery need further study. When they conducted heated discussion in the clinical meeting, they also turned light on the basic research.In our experiment, we first established the animal model of associating liver partition and portal vein ligation in rats and tried to understand the mechanism of liver regeneration from those aspects including hemodynamics, liver function, pathological and molecular biology. Then we supposed whether different degree of transection of liver parenchyma would induce different degree of liver regeneration, or whether a certain extent of the transection of liver parenchyma would guarantee the abundant regeneration which would decrease the time of operation. We even assumed whether it could reduce trauma and further reduce the risk of surgery. When it was extended to clinical application, it was difficult to separate for complete liver parenchyma transection and it will prolong the operation time for some patients. If it is enough to do only a portion of the liver parenchyma transection, these patients will benefit. Based on this assumption, our experiment did the related research on rats in order to provide ideas and evidence for clinical application.Part I Establishing the model of selective portal vein ligation combined with liver parenchyma transaction in ratsOBJECTIVE:In order to make experimental basis for further study on ALPPS, we have established a repeatable and stable rat model for associating liver partition and portal vein ligation for staged liver resection.METHODS:Choose healthy male SD rats weighing 250-280g. The rats were anesthetized by isoflurane inhalation which was sustained by small animal anesthesia machine. With the help of a surgical microscope, we first transected the ligamentum teres hepatis as well as the liver lobe, and opened the corresponding portal sheath, suturing or ligaturing right lobe of the rat portal vein branch, caudate lobe branch, left branch (left lateral lobe of the left middle of the portal vein branch), and reserved the right middle lobe rat portal vein branch while retaining all the bile duct and artery branch. The lobe will become ischemia after the ligation of the right portal vein and caudate lobe branch. The left lateral lobe and left middle lobe will appear ischemia and dark after the ligation of the left portal vein while the right middle lobe will become congestion. Besides, a significant ischemia zone appeared between the left and right middle lobe which was located on the right side of Falciform ligament about 2 millimeters. On this basis, the liver parenchyma was transected along with the ischemic zone by suturing or ligation with microsurgical technique and then the left middle lobe was separated from the right middle lobe while the section was stanch bleeding properly. The abdominal cavity was washed by warm saline after checked no abnormal and the abdomen was closed by silk continuous suturing to end the surgery. The whole model was divided into two stages according to the learning and skilling level. There were 20 rats in the first stage while 10 rats in the second stage. The learning processes and repeatability and stability of the model were studied by comparing the operation time, the rate of success, complication rate as well as the survival rate between these two stages. In the first 9 days after the operation, all surviving rats and 6 normal control rats were killed and the corresponding lobe were removed and weighed by the electronic balance to assess the regeneration on state of the reserved hepatic lobe. Statistical analysis was performed using SPSS standard version 18.0. P<0.05 was considered statistically significant.RESULTS:1. The comparison between the operative time and surgical success rate:the time of portal vein ligation, liver parenchyma transection and the whole operative time of the second stage was significantly shorter than those of the first stage (p<0.05). There were 7 rats died in the first stage during the surgery and the surgical success rate was 65%. The operation of the second stage was much more skillful while the success rate was 100% which was significantly improved comparing with the first stage (p<0.05)2. The comparison of surgical complications and postoperative survival rate:during the first stage, of all the 20 rats,7 rats died in the operation, and there were 5 rats died in the remaining 13 rats,3 rats in the first 3 days after operation and 2 in the 7 days after operation. The postoperative survival rate was 61.54%. After the skill training and experience of the first stage,10 rats were all survival after the operation and the survival rate was 100% which was significantly higher than that of the first stage (p<0.05).3. All rats were sacrificed in the ninth day after the operation. The weight of the right middle lobe was 5.72 ± 0.86g which was much higher than that of the control group.CONCLUSION:1.This experiment has established a repeatable and stable rat model for associating selective portal vein ligation with liver parenchyma transaction after familiar with the vascular anatomy of liver lobe. This has provided a basis and techniques for further research.2.This animal model requires higher microsurgical techniques and the learning phase before the experiment is necessary and advantageous. The procedure and skill of liver parenchyma transaction was difficult and essential.3.When the surgery is skillful, the survival rate of the rats could reach 100%. In our model, the regeneration effect of right middle lobe of rats is very obvious.Part II The role of SPVL+LPT for postoperative HRR and mechanisms of accelerated liver regenerationOBJECTIVE:Postoperative HRR were valuated for selective portal vein ligation combined with liver panrenchyma transaction(SPVL+LPT) compared With SPVL or LPT respectively. And the possible mechanisms of accelerating regeneration of FLR in terms of hemodynamic, histopathology and molecular biology were revealed.METHODS:The experimental groups were as follws. Groupl:sham operation (SHAM). Just finish the procedure before SPVL,including free of liver ligaments and branch of portal vein. Group2:liver panrenchyma transection (LPT). After temporary occlusion by vascular clips of the left portal vein, a significant ischemia zone appeared between the left and right middle lobe, the liver parenchyma was transected along with the ischemic zone. Group3:Selective portal vein ligation (SPVL). The portal vein branchs of the caudal lobe, left lateral and left median lobes, and the right lobe were ligated. Group4:Selective portal vein ligation combined with liver panrenchyma transection (SPVL+LPT). After SPVL as group3, then the liver panrenchyma transection was performed as group 2. Ultrasonic detector(TS 420) was used to detect portal vein blood flow volume. Laser speckle contrast imaging (LSCI) was used to detect the microcirculation blood perfusion of RML and LML. The rats were sacrificed atl2h,24h,48h, and 72 h and 7 days after operation and the liver tissue and blood were collected. The hepatic regeneration rate (HRR) of RML was evaluated. Liver function including ALT, AST, ALB and TBIL were tested. Histopathologic changes of LML were observed and Ki-67-positive index of RML were evaluated. The mRNA and protein lever of TNF-a, IL-6, HGF in RML or serum were tested by RT-PCR and ELISA respectively. Statistical analysis was performed using SPSS 18.0. P<0.05 was considered statistically significant.RESULTS:1. The HRR since 24h for both SPVL and SPVL+LPT were obviously higher than SHAM and LPT group. Comparing with the SPVL, SPVL+LPT induced more greater regeneration (p<0.05) at 72h and 7 days. There were no significant differences between the two groups atl2h、24h and 48h.2. IHC results showed the Ki-67-positive index in RML was higher in the SPVL and SPVL+LPT than LPT at 24h 48 h and 72 h (p<0.05). And SPVL+LPT was more higher than SPVL at 48h and72h.3.The intraoperative portal blood flow volume changes:The portal blood flow LPT and SHAM were both no significant change compared with the preoperative (p> 0.05), The portal blood flow of SPVL and SPVL+LPT were below the LPT and SHAM group (p<0.05). And SPVL has no difference with SPVL+LPT group (p> 0.05).The PBFVo of per g liver tissue of RML was obviously higher in post-SPVL than pre-SPVL.4. The results of Laser speckle contrast imaging (LSCI):The microcirculation blood perfusion of RML increased and that of the LML obviously decreased after SPVL(p<0.05). After SPVL+LPT, The microcirculation blood perfusion of LML further decreased (p<0.05) and that of RML was same as before.5. HE staining showed that necrosis scores of LML in SPVL+LPT were higher than SPVL at 24h post-operation.6. The serum test of liver function:The ALT and AST in LPT group were higher than SHAM at 12h、24h. Both SPVL and SPVL+LPT showed an obvious increase in ALT and AST compared to the SHAM and LPT group at 12h、24h and 48h. The ALT and AST levels in the SPVL+LPT group were higher than SPVL group at 12h, 24h(p<0.05). ALB lever were lower in the SPVL and SPVL+LPT compared to the SHAM and LPT group after 24h. ALB lever were lower in SPVL+LPT group than SPVL at 24h、48h. TBIL levels in 4 groups had no significantly different at all point.7. Results of PCR and ELISA:All cytokines were highly upregulated in the RML at 24 h in LPT、SPVL and SPVL+LPT than SHAM group (p<0.05). Moreover, a significant increase of TNF-a and IL-6 mRNA in the SPVL+LPT group compared to the SPVL group (p<0.05). There was no significant difference for the mRNA levels of HGF between the SPVL and SPVL+LPT groups. ELISA showed the comparing results of protein lever were same as mRNA both in liver tissue and serum.CONCLUSIONS:1. LPT can not induce hepatic regeneration effectively. Both SPVL and SPVL+LPT could induce hepatic regeneration of future remnant liver effectively compared to the SHAM and LPT group. However, SPVL+LPT induced more greater than SPVL.2. Changes of portal vein blood flow and changes in liver tissue perfusion were basis for liver regeneration, on this basis, liver parenchyma transaction can be more strongly promote liver regeneration.3. The possible mechanisms of liver regeneration were changes of microcirculation blood perfusion in the ML and up-regulation of TNF-a and IL-6 in the RML or serum after SPVL+LPT.Part III The impact and potential clinical meanings of SPVL combined with different degree Partial-LPT on liver regenerationOBJECTIVE:by conducting Selective portal vein ligation (SPVL) combing with partial liver parenchyma transection(P-LPT) on rats, to compare that with SPVL (that is, SPVL+0%LPT) and SPVL+LPT (that is, SPVL+100%LPT). To compare and evaluate the influence of different degrees of liver parenchyma transaction on FLR liver regeneration and the potential clinical values.METHODS:1. The animals were the same as before.The cross section of rat liver parenchyma of RML was observed and measured. By preliminary experimental observation, the influence on reserved liver regeneration was observed and evaluated. Different experiment groups were set according to the degree of liver parenchyma:(1) Selective portal vein ligation+25% LPT group (SPVL+25%LPT):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, and the liver parenchyma transaction was 25%. (2)Selective portal vein ligation+50% LPT group (SPVL+50%LPT):on the basis of listed selective portal vein ligation, combining 50% liver parenchyma dividing. (3)Selective portal vein ligation+75% LPT group (SPVL+75%LPT):on the basis of listed selective portal vein ligation, combining 75% liver parenchyma dividing. There were 6 rats in each experiment group. At 7d after operation, rats were killed and hepatic regeneration rate of the right middle lobe of rats from each group was evaluated. Based on the listed results,10 rats were added to 50% LPT group.2.combining the preliminary result of the preliminary experiment, SPVL SPVL+25%LPT、SPVL+75%LPT and SPVL+100%LPT group were selected for the further experiment study. The operating methods of all groups were as before. After operation, the rats were killed and the samples were taken at 5 time points (12h,24h, 48h,72h,7d), six rats at each time point for every group. During the operation, laser speckle imaging (LSCI) was used to detect the microcirculation blood flow change of left and right middle liver lobe after different degree of liver parenchyma dividing. The hepatic regeneration rate of each group at different time points was calculated. The serum ALT, AST, ALB, TBIL were detected. The immunohistochemical method was adopted to detect the index of Ki-67 in the RML. RT-PCR and ELISA were adopted to detect cytokines TNF-a, IL-6 in the RML or serum at 24h. The statistical method was the same as the part Ⅱ.RESULTS:1.the difference of hepatic regeneration rate (HRR) between SPVL+25%LPT group and SPVL group had no statistical significance and the difference of hepatic regeneration rate (HRR) between SPVL+75%LPT group and SPVL+LPT (that is, SPVL+100%LPT) group had no statistical significance. While the hepatic regeneration rates of SPVL+50%LPT group had no statistical significance with SPVL+25%LPT group and SPVL group. We considered that the sample amount was too small, so we added 10 more rats, but the result was the same with that before. Considering that the fracture surface of rat liver is too small and unable to be grouped in a further degree precisely, so 50%LPT group was not included into the further study.2.The comparative result of SPVL、SPVL+25%LPT group, SPVL+75%LPT group and SPVL+100%LPT group.2.1 At 12h,24h,48h after operation, HRR of all groups had no difference (p>0.05). At 72h and 7d after operation, HRR of SPVL+75%LPT group and SPVL+100%LPT group were all higher than that of SPVL+25%LPT group and SPVL group (p<0.05). HRR of SPVL+75%LPT group and SPVL+100%LPT group had no statistical difference at different time points (p>0.05).2.2 Ki-67 expression in the hyperplastic liver lobe of all groups at 12h,24h,7d after operation had no evident difference. At 48h,72h after operation, the expression levels of SPVL+75%LPT group and SPVL+100%LPT group were evidently higher than that of SPVL+25%LPT group and SPVL group (p<0.05). Ki-67 of SPVL+75%LPT group and SPVL+100%LPT group had no statistical difference at all time points (p>0.05). 2.3After the left branch of portal vein ligatured and the blood flow smooth, and different degree of LPT, laser speckle was used to detect the microcirculation change in the right middle lobe, and there was no evident difference (p>0.05). The microcirculation blood flow of left middle lobe of SPVL+25%LPT group was close to that of SPVL group, while SPVL+75%LPT group and SPVL+100%LPT were both lowe r than that of SPVL+25%LPT group (p<0.05).2.4 HE staining of the left median lobes after SPVL and SPVL+25%LPT revealed necrosis scores that were significantly smaller than in SPVL+75%LPT and SPVL+100% LPT at 24h after surgery(p<0.05).2.5 Serum liver function tests:At 24h, after operation, ALT and AST levels of SPVL+75%LPT group and SPVL+100%LPT group were evidently higher than that of SPVL+25%LPT group and PVL group (p<0.05), while at 12h 24h, ALT AST levels of SPVL+75%LPT group were lower than that SPVL+100%LPT group (p<0.05). At 24h, 48h, ALB level of SPVL+75%LPT group and SPVL+100%LPT group was lower than that of SPVL+25%LPT group and PVL group (p<0.05). The difference of TBIL level among four groups had no statistical significance.2.6 TNF-α, IL-6 mRNA level and protein in the RML of SPVL+75%LPT group and SPVL+100%LPT were evidently higher than that of SPVL+25%LPT group and SPVL group (p<0.05), while TNF-α of SPVL+75%LPT group were lower than SPVL+100%LPT group. In the serum TNF-α, IL-6 protein expression level of SPVL+75%LPT group and SPVL+100%LPT were higher than that of SPVL+25%LPT group and SPVL group (p<0.05), and the TNF-α, IL-6 protein of SPVL+75%LPT group were lower than SPVL+100%LPT group (p<0.05).CONCLUSIONS:1. The effect of SPVL+25%LPT stimulating hepatic regeneration is similar to SPVL while the effect of SPVL+75%LPT stimulating hepatic regeneration is evidently higher than SPVL+25%LPT.2. SPVL+75%LPT can reach the liver regeneration effect that is close toSPVL+100%LPT. Moreover, SPVL+75%LPT group has the effect of relieving liver function damage and reduce the inflammatory level. When it is difficult to conduct complete liver parenchyma transection, selective portal vein ligation combining partial liver parenchyma transaction(SPVL+P-LPT) can be used to reduce the operation length, bleeding amount, inflammation and trauma.