Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.
Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.
Currently, there is a large discrepancy between the number of donated liver organs and the number of patients waiting for a donated liver. The shortage of liver organs is a global problem. To expand the donor pool, split liver transplantation (LTx) and living donor LTx were developed to use a partial liver as a graft1.
To further investigate the mechanism behind partial liver transplantation (PLTx), relevant animal models have been established2,3,4,5. In rat PLTx, the liver lobes are resected in vivo and ex vivo to mimic the conditions of the living donor LTx and split LTx, respectively, in human. A paper published in the Journal of Visualized Experiments presented a detailed protocol involving a 50% rat PLTx using an ex vivo hepatectomy5. However, a rat PLTx with an in vivo hepatectomy has not yet been reported in the visualized literature.
In addition to the difference between in vivo and ex vivo hepatectomies, the technique of performing a hepatectomy itself also plays an important role in determining the outcome of PLTx. Currently, in many surgical studies using the rat PTLx model, the liver lobes were resected after placing a simple ligation at the pedicle of the liver lobe2,3,6,7,8,9. However, placing a simple ligation before resection is not suitable for all liver lobes, as different liver lobes have different shapes and sizes. A simple ligation at the base of the median lobe carries a high risk of causing a constriction of the vena cava, which might eventually affect the outflow of the partial liver graft10,11. Therefore, an update hepatectomy technique based on knowledge of the rat hepatic anatomy is required in the field of rat PTLx.
In the protocol described in this study, an updated vessel-oriented 70% hepatectomy was incorporated into the procedure of the rat PLTx. The portal veins and hepatic arteries of the left lateral lobe (LLL) and median lobe (ML) were dissected and divided individually before removal of the liver parenchyma. Then, the hepatic veins of the LLL and ML were ligated by piercing sutures. By using individual ligations and multiple piercing sutures rather than placing a simple ligation, the remnant stump of the ML was able to spread over the vena cava. Hence, the constriction of the infrahepatic vena cava caused by a surgical ligation was avoided. Additionally, occlusion of the blood supply of the LLL and ML by individual ligations before removal of the liver parenchyma decreased the rate of bleeding in the remnant liver stump, thereby minimizing the influence of blood loss on the experiments11.
For microsurgeons, it is a significant challenge to reconstruct the proper hepatic artery (PHA) of a liver graft because of the extremely small diameter of this vessel. Although the question of whether re-arterialization in LTx is truly necessary is still under debate12,13,14, numerous microsurgical techniques for reconstructing the hepatic artery have been proposed14. Here, we introduce a novel technique for re-arterialization of the liver graft, which anastomoses the common hepatic artery (CHA) to the enlarged PHA in an end-to-side manner. By using this end-to-side technique, two hepatic arteries are connected with an anastomosis of a larger diameter. The larger the diameter of the anastomosis is, the easier hand suturing is to perform and the greater the improvement in the patency of the anastomosis becomes.
All of the procedures followed the guidelines of rodent surgery approved by the Wenzhou Medical University Animal Policy and Welfare Committee 15.
1. Animals
2. Operative Environment
3. Basic Microsurgical Maneuvers
4. Preparation of the Cuff and Biliary Stent
5. Anesthesia and Fixation of the Rats
6. Donor Operation
7. Back-Table Operation (Graft Preparation)
8. Recipient Operation
9. Postoperative Treatments (Analgesia and Antibiotics)
In total, 31 cases of syngeneic arterialized rat PLTx were completed using this protocol. All of the recipients survived until the end of the observation time. The body weight of the recipients began to recover after postoperative day (POD) 4. The slope of the body weight was close to that of a normal Lewis rat after POD 6 (Figure 8), which indirectly implied the recovery of the recipient. Histologically, a slight proliferation of the bile duct was observed in the recipients, and the liver lobular architecture of the recipients was intact. No necrosis or obvious sinusoidal dilatation was observed (Figure 9). The serum level of alanine aminotransferase (ALT) was within the normal range at POD 30 (Figure 10). No cases of yellow urine caused by jaundice were observed in these recipients, which was indicated by checking the cage bedding daily, indicating the patency of the biliary stent. The normal serum level of bilirubin on POD 30 further confirmed the successful reconstruction of the bile duct (Figure 11). The patency of the hepatic artery was checked by transecting the PHA on the proximal side of the anastomosis during sacrifice. Bleeding from the PHA was observed in all of the recipients, implying the patency of the anastomosis. All of these results suggested that the liver grafts functioned well.
The average duration of the anhepatic phase was 23.03 ± 2.3 min. The cuff anastomosis of the PV took 4.68 ± 0.77 min, while the cuff anastomosis of the IHVC only took 2.05 ± 0.71 min. Compared with the cuff anastomosis of the PV, the cuff anastomosis of the IHVC saved the step of placing stay sutures and thereby further shortened the anastomosis time (Table 1).
Two cases of bleeding in the remnant stump of the ML during the 70% hepatectomy were observed among the 31 cases of vessel-oriented hepatectomy. In a previous experiment, bleeding in the remnant stump of the ML was observed in 8 out of 18 cases of parenchyma-preserving vessel-oriented hepatectomy (Figure 12). This result indicated a lower rate of bleeding in the remnant liver stump of the ML after using vessel-oriented 70% hepatectomy.
Figure 1. The cuffs and biliary stent.
(A,C) The size of cuffs. (B) The fillister on the cuff. (D) The body part and handle part of the cuff. (E) (a) IHVC cuff; ( b) PV cuff. (F) Biliary stent. Scale bar = 5 mm. Please click here to view a larger version of this figure.
Figure 2. To occlude the blood supply of LLL and ML.
(A)The common trunk of the PV, hepatic artery and bile duct of the LLL and LML.
(B)The discoloration of the LLL and LML after transecting the common trunk.
(C)The portal vein of the RML.
(D)All PVs, hepatic arteries and bile ducts of the LLL and the ML were transected.
Scale bar = 5 mm. Abbreviations: LLL left lateral lobe, ML median lobe, LML left median lobe, RML right median lobe, RSL right superior lobe, RIL right inferior lobe, SCL superior caudate lobe. Please click here to view a larger version of this figure.
Figure 3. To resect the left median lobe.
(A) Resect the liver mass of the LML immediately above the forceps.
(B) Place three piercing sutures on the incision. ()
Scale bar = 5 mm. Abbreviations: RML right median lobe, RSL right superior lobe, RIL right inferior lobe, SCL superior caudate lobe. Please click here to view a larger version of this figure.
Figure 4. To resect the right median lobe (RML).
(A)The base of the RML.
(B)Place mosquito hemostasis forceps around the base of RML.
(C)Resect the liver parenchyma of RML immediately above the forceps.
(D)Leave a plain and thin layer of the remnant liver tissue on the SHVC.
Scale bar = 5 mm. Abbreviations: ML median lobe, RSL right superior lobe. Please click here to view a larger version of this figure.
Figure 5. To install the cuff.
(A) Pull the PV through the lumen of a cuff.
(B) Fix the cuff handle and PV together by a vessel clamp.
(C) Evert the PV vascular wall to cover the outside surface of the cuff.
(D) Secure the vascular wall and the cuff in position.
Scale bar = 5 mm. Please click here to view a larger version of this figure.
Figure 6. To reconstruct the SHVC.
(A&B) Pull two stay sutures to the right and left, respectively.
(C) Anastomose the posterior wall of the SHVC by a running suture in the vascular lumen.
(D) Anastomose the anterior wall of the SHVC out of the vascular lumen.
Scale bar = 5 mm. Please click here to view a larger version of this figure.
Figure 7. To reconstruct the IHVC.
(A) A cylindrical vascular lumen of the IHVC in the recipient.
(B) Secure the cuff anastomosis of the IHVC.
(C) Start the reperfusion.
(D) Trim off the surrounding liver parenchyma above the circumferential silk suture.
Scale bar = 5 mm. Abbreviations: PV portal vein, IHVC inferior infrahepatic vena cava, RIL right inferior lobe. Please click here to view a larger version of this figure.
Figure 8. Postoperative body weight recovery.
The body weight of recipients began to recover after postoperative day 4. The slope of the body weight was close to that of a normal Lewis rat after postoperative day 6, which indirectly implied the recovery of the recipient.
Figure 9. Histology of liver graft after 30 days.
In histology, a slight proliferation of the bile duct was observed in recipients, and the liver lobular architecture of recipients was intact. No necrosis or obvious sinusoidal dilatation was observed (400X). Scale bar = 20 um Please click here to view a larger version of this figure.
Figure 10. Serum level of ALT.
The serum level of ALT was within the normal range on POD 30. (**, P<0.05).
Figure 11. Serum Level of bilirubin.
The serum level of bilirubin on POD 30 was comparable to that of a normal Lewis rat ( P>0.05).
Figure 12. Bleeding rates of different methods of hepatectomy.
A lower rate of bleeding in the remnant liver stump of the ML was observed in the method using the vessel-oriented 70% hepatectomy.
Procedures | Removal of liver | SHVC anastomosis | PV anastomosis | IHVC anastomosis | Anhepatic time |
Time (minutes) | 2.62±0.46 | 15.73±2.05 | 4.68±0.77 | 2.05±0.71 | 23.03±2.30 |
Data were expressed in Mean±SD |
Table 1. Time of individual surgical procedures during anhepatic phase
Table 2. Difference between two visualized protocols of the rat PLTx. Please click here to view a larger version of this table.
Supplementary Materials
Rat PLTx is a sophisticated microsurgical procedure with a training program that is high in cost and long in duration20. The complexity of the rat PLTx protocol has prevented researchers from using this animal model. Compared with full-size rat LTx, rat PLTx presents the microsurgeon not only with the challenge of a transplantation procedure but also with the challenge of liver resection in a small animal. Therefore, a visualized microsurgical protocol describing the whole procedure in detail is essential for establishing this complex model. A previously visualized article reported by Nagai et al. presented a protocol of 50% partial orthotopic LTx with hepatic arterial reconstruction in rats5 (Table 2). This protocol used an ex vivo hepatectomy, which is clinically similar to the procedure of split LTx.
Compared with split LTx, living donor LTx resects the partial liver graft from the living donor rather than splitting the liver in the back-table operation. The in vivo liver resection may lead to more surgical manipulations, longer portal hypertension, and even more warm ischemia injuries to the partial liver graft. Hence, several rat PLTx models utilizing in vivo hepatectomy have been established in relevant studies6,8,21,22. Many protocols involve resection of the liver lobes after simple ligation at the pedicle or base2,3,6,8,21,23. However, different liver lobes are of different shapes. The ML of the rat has a wide base that semi-surrounds the vena cava10,24. Placing a simple ligation at the base of the ML easily constricts the inferior vena. To avoid the constriction of the vena cava, Madrahimov et al. developed a parenchyma-preserving vessel-oriented hepatectomy to remove the ML in 2 steps after four piercing sutures in the parenchyma were made, leaving the remnant stump of the ML flat10. However, the PVs of the liver lobes ligated by the piercing sutures were hidden in the parenchyma. Bleeding in the remnant stump caused by missing or injuring the small branches of the PV could not be completely avoided 11. In our protocol, the corresponding PVs and hepatic arteries were further dissected first. Then, the blood supply of the resected liver lobes was individually occluded by ligations before the resection. Therefore, the rate of bleeding in the remnant stump was lowered using our protocol (Figure 10). The technique of placing four piercing sutures in the stump of the ML was also employed in our protocol to ligate the hepatic veins but not the PV and hepatic arteries. With these ligations using multiple piercing sutures and a step-wise resection, the flat shape of the remnant liver stump was maintained, which avoided the constriction of the vena cava.
For the IHVC reconstruction, a slight modification was made to the cuff anastomosis described by Kamada et al.25. The inferior vena cava of the recipient was transected in the parenchyma of the right inferior lobe, rather than on the IHVC vessel, which left a circumferential liver parenchyma around the vena cava. The liver parenchyma around the vena cava helped maintain a cylindrical shape of the vascular end of the IHVC of the recipient rather than a flat shape. Hence, the lumen of the IHVC was naturally opened without the help of stay sutures and forceps. This modification helped save the step of placing stay sutures and facilitated the insertion of the IHVC cuff into the vascular lumen of the recipient. Moreover, this modification prolonged the vascular length of the IHVC, thereby reducing the tension between the two ends during the cuff anastomosis.
With the advantage of fast vascular anastomosis, the two-cuff technique is frequently used in the rat orthotopic LTx for anastomosing the PV and IHVC. Miyata et al. even proposed a protocol involving a three-cuff technique to anastomose the PV, IHVC and SHVC. However, the cuff technique also has its disadvantages. First, a cuff with a fixed size cannot perfectly fit the size of the corresponding vessel. In addition, the cuff technique disturbs blood flow, resulting in a higher incidence of thrombosis and foreign body reaction to the cuff26. The SHVC, a main vessel in the rat, shows high blood flow. Any constriction or thrombosis in this vessel may cause severe postoperative complications. Therefore, in our PLTx protocol, we choose to use the hand suture technique to reconstruct the SHVC, which allows for the adjustment of the anastomosis as close as possible to the physical setting. However, high-quality SHVC anastomosis completed by the hand suture technique requires high microsurgical skill. For beginners, Carrel’s triangulation technique is recommended for anastomosing vessels 27. By using this technique, three stay sutures are placed at regular intervals around the circumference of the SHVC. The 3D shape of the SHVC is maintained by retracting these three stay sutures along three directions, which can prevent the SHVC from stenosis during anastomosis.
Re-arterialization of the liver graft is a controversial topic. In our opinion, re-arterialization is not the key step for ensuring a high survival rate of the recipient, but it might be the key step for ensuring a high-quality liver graft. In the traditional protocol of the rat LTx involving the two-cuff technique, the hepatic artery was not reconstructed, as the lack of perfusion of the hepatic artery did not affect the outcome of the rat LTx12. Moreover, a previous study on mouse LTx was conducted to investigate the effect of re-arterialization on long-term graft survival, histological alterations, ischemic liver damage and early immunologic activation pathways13. The researchers concluded that the re-arterialization of liver grafts did not have a major effect on the survival rate or the degree of immunologic activation. However, a recent study reported by Huang et al. suggests that re-arterialization is important for the recovery of liver parenchyma subjected to hepatectomy, especially with the obstruction of outflow28. Non-arterialization of the partial liver graft aggravated liver damage and delayed the recovery from focal necrosis. Therefore, re-arterialization of the partial liver graft is recommended. As illustrated here, an end-to-side anastomosis technique was used to reconstruct the hepatic artery, which was described previously by one of our authors 14. The advantage of this technique is the choice to anastomose two thick arteries, the CHA and GDA, rather than the thin PHA, which reduces the difficulty of reconstructing the hepatic artery and maintains a high rate of patency of anastomosis.
The classical rat LTx protocol proposed by Kamada et al. suggests controlling the anhepatic phase such that it is less than 26 min, as no animal survived when the anhepatic phase lasted longer than 26 min25. Our experience is consistent with this suggestion, although we previously observed several survival cases with an anhepatic phase time longer than 26 min. In our opinion, a stable anhepatic phase takes priority over a shorter anhepatic phase in surgical research. In our group, the anhepatic phase is precisely controlled to within a specific time. The anhepatic phase is not stopped until the preset anhepatic time is reached, although the anastomosis of the SHVC and PV could be completed much earlier. However, in a training project, a shorter anhepatic phase is encouraged.
Although many improvements have been made to shorten the training phase and eliminate unnecessary operations, rat LTx and PLTx are still complex microsurgical operations20. Therefore, in addition to a visualized protocol, a step-wise training protocol based on the Plan-Do-Check-Action cycle is important for achieving a high survival rate and high operation quality. Our preliminary experience in using this training protocol showed that 40-73 operations were required to master the rat orthotopic LTx. An additional 9-13 operations were required to master the rat re-arterialized PLTx. The training protocol proposed by Czigány et al. claims that 50-60 LTxs are sufficient to master the orthotopic LTx and the PLTx29.
In our Plan-Do-Check-Action training protocol, quality control at each training step and in each individual manipulation must be emphasized, especially in such a complex model. A high survival rate is not tantamount to a high-quality procedure. In the representative results, several criteria for evaluating the quality of the microsurgery were listed, including histology, liver enzyme, body weight recovery, jaundice (serum bilirubin and yellow urine), duration of the anhepatic phase, and patency of the hepatic artery. High quality is achieved only when all of the criteria are met.
The authors have nothing to disclose.
This research was supported by National Natural Science Foundation of China (Grant No. 81501382) and Zhejiang Provincial Natural Science Foundation of China (Grant No. LQ13H100003 and LQ16H100002).
Surgical microscope | Möller-Wedel, Germany | 654359 | |
Light source | OSRAM (China) Lighting Co., Ltd | 64627 | |
Isoflurane Vaporizer | MIDMARK Corporation | VIP3000 | |
Isoflurane | Baxter Healthcare Corporation | 40032609 | For Inhalation anesthesia |
Normal Saline | Zhejiang Tianrui Pharmaceutical Co., Ltd. | 716092103 | |
Povidone Iodine Solution | HANGZHOU MINSHENG PHARMACEUTICAL | H33021567 | For disinfection |
Hemostatic forceps | Shanghai Medical Instruments(Group) Ltd | J31020 | |
Surgical scissors | Shanghai Medical Instruments(Group) Ltd | JC2116 | |
Needle-holder | Shanghai Medical Instruments(Group) Ltd | J32030 | |
Dressing Forceps | Shanghai Medical Instruments(Group) Ltd | J42020 | |
Mosquito hemostatic forceps | Shanghai Medical Instruments(Group) Ltd | W40340 | For liver resection; For retracting the stay sutures. |
Micro-scissors | Shanghai Medical Instruments(Group) Ltd | WA1040 | |
Micro needle-holder | Shanghai Medical Instruments(Group) Ltd | WA2060 | |
Curved micro typing forceps | Shanghai Medical Instruments(Group) Ltd | WA3061 | |
straight micro typing forceps | Shanghai Medical Instruments(Group) Ltd | WA3060 | |
Micro hemostatic forceps | Shanghai Medical Instruments(Group) Ltd | WA3020 | |
Bulldog clamp | Shanghai Medical Instruments(Group) Ltd | XEC350 | |
Vessel clamps | Shanghai Medical Instruments(Group) Ltd | W40160 | |
Micro Serrefine-curved | Fine Science Tools, Inc | 18055-05 | |
Dacryosyrinx | Shi Zhuo medical instruments Co., Ltd. | 5# curve | Use as the curved needle for forcing out the air bubbles in the vascular lumen |
Sterilized gauze swabs | Fuqing Health & Integral Medical | 20230R | |
5mL syringe | Zhejiang Yusheng Medical Instrument Co.,Ltd | 811932416 | |
18G needles | Shanghai Kindly Enterprise Development Group Co., Ltd | 01-014 | For fixing the upper abdomen wall |
Intima II 22G intraveous catheter | Becton Dickinson Medical Devices (Shanghai) Co Ltd. | 383207y | For making biliary stent; For infusing perfusate |
7-0 polypropylene sutures | Ningbo Medical Needle Co., Ltd | 151026 | For SHVC anastomsis |
6-0 silk suture | Shanghai Pudong Jinhuan Medical Products Co., Ltd | 2650182 | For ligation |
Culture dish (100mm) | Corning Incorporated | 430165 | used in back table for storage ice. The back table dish was placed on it. |
Culture dish (30 mm) | Corning Incorporated | 3296 | As a vessel for organ preservation |
Angiocath 12G intravenous catheter | Becton Dickinson Medical Devices (Shanghai) Co Ltd. | 382277 | For making a IHVC cuff |
Angiocath 14G intravenous catheter | Becton Dickinson Medical Devices (Shanghai) Co Ltd. | 382269 | For making a PV cuff |
Buprenorphine (hydrochloride) | Tianjin Institute of Pharmaceutical Research Pharmaceutical Co., Ltd | H12020275 | For analgesic |
Cefuroxime sodium for injection | Esseti FarmaceuticiS.r.l | H20160013 | As an antibiotics |