Suture Techniques for Vascular Reconstruction in Mouse Orthotopic Liver Transplantation

Summary

Vascular reconstructions in recipients, including the suprahepatic inferior vena cava (SHIVC), portal vein, and infrahepatic inferior vena cava (IHIVC), are the key procedures for successful mouse orthotopic liver transplantation. We herein provide suture techniques and tips for SHIVC and IHIVC reconstruction in the mouse liver transplant model.

Abstract

Translational research using animal models is essential for addressing the clinical issues in organ transplantations. Although mouse orthotopic liver transplantation (LT) is a useful tool for investigating the mechanisms of liver regeneration, ischemia-reperfusion injury, and immune responses following LT, technical challenges in this procedure restrict its feasibility. The key aspects for successful mouse orthotopic LT are vascular reconstruction in recipients, including the suprahepatic inferior vena cava (SHIVC), portal vein (PV), and infrahepatic inferior vena cava (IHIVC). In addition to the technical difficulties in reconstructing these vessels, the time limitation of the anhepatic phase to within 20 min makes this procedure more challenging. Although a cuff technique can be used for PV reconstruction, a safe and quick suture technique is required to reconstruct the SHIVC during the anhepatic phase. For IHIVC reconstruction, while both cuff and suture techniques are applicable, the suture technique leads to a shorter operation time in donor surgery and back-table preparation and can maintain a larger vascular lumen than the cuff technique. We herein provide guidelines for SHIVC and IHIVC reconstruction using a suture technique to facilitate mouse orthotopic LT.

Introduction

With advances in surgical techniques, immunosuppressive drugs, and intensive care medicine, organ transplantation has been established as a standard therapy for end-stage organ diseases. Liver transplantation (LT) is currently the only curative treatment for end-stage liver disease and certain hepatic malignancies. Translational research using animal models is essential to address clinical issues in LT. As various genetically modified animals are available in mouse models, mouse orthotopic LT is an important tool to explore mechanistic insights into liver regeneration, ischemia-reperfusion injury, and immune responses following LT¹. However, as the technical challenges in this procedure restrict its feasibility, research groups working on this model are limited^{2,3,4,5,6,7,8,9,10}.

Mouse orthotopic LT was first described by Qian et al. in 1991². The surgical procedure, developed by applying orthotopic LT techniques in the rats, comprises three steps: donor surgery, back-table preparation of the liver graft, and recipient operation⁶. The technically challenging parts of mouse LT are biliary and vascular reconstruction in recipients, including the suprahepatic inferior vena cava (SHIVC), portal vein (PV), and infrahepatic inferior vena cava (IHIVC). In addition to the surgical techniques, the anhepatic phase from clamping to releasing the PV flow should be within 20 min for successful mouse orthotopic LT ^2,6,11. Furthermore, in our experience, the total reperfusion time from PV clamping to IHIVC release should be within 30 min to achieve long-term survival in mouse LT. This is consistent with the previous report for rat LT describing the preferable time of unstable systemic hemodynamic state as within approximately 30 min¹². Thus, technical difficulties and time limitations in mouse LT make this procedure more challenging.

Although the cuff technique can be used for PV reconstruction, a suture technique is required to reconstruct the SHIVC during the anhepatic phase. Therefore, a safe and rapid suture technique is essential for SHIVC anastomosis. While both cuff and suture techniques are applicable for IHIVC reconstruction, most previous studies have employed a cuff technique^5,6,7,11,13. However, the use of a cuff technique in IHIVC reconstruction leads to a longer operation time in donor and back-table operations. In addition, inserting a cuff may be harder than suturing for beginners because of the short margin of recipient inferior vena cava (IVC)¹³.

We herein provide guidelines for successful SHIVC and IHIVC reconstruction using a suture technique in mouse orthotopic LT.

Protocol

All animal experiments were approved by the Kyoto University Animal Experimentation Committee and performed in accordance with the Kyoto University Animal Protection Guidelines. Mouse surgeries were performed under general anesthesia using isoflurane inhalation. The vaporizer was set to 3% isoflurane with 1 L/min oxygen to induce anesthesia. After laparotomy, isoflurane was reduced to 1% to maintain anesthesia. The microsurgical tools used in this protocol, including those used in our previously published vascular reconstruction protocol¹³, are listed in the Table of Materials. A Microscope was required to perform mouse orthotopic LT¹³. A Heating pad was used to maintain the mouse body temperature¹³.

1. Animals

1. Use a mouse weighing 25-30 g.
   NOTE: Although mice weighing 20-35 g can be used, approximately 25 g mice are suitable for both donors and recipients. As age and liver steatosis affect LT outcomes, appropriate mice should be selected for each purpose of the experiments.

2. Dissection of the IHIVC in the donor liver

1. Isolate the IHIVC from the surrounding tissue. Seal the lumbar veins with an electrocautery if needed.
2. Remove the fat from IHIVC to expose the vessel wall.
3. Dissect the IHIVC above the right renal artery and vein (Figure 1).
   NOTE: If the suture technique is used for the IHIVC reconstruction, dissection of the right renal vein in the donor site is unnecessary. Donor surgery usually takes less than 30 min when applying the suture technique for IHIVC reconstruction.

3. Dissection of the SHIVC in the donor liver

1. After perfusion of the storage solution from the PV, cut the anterior and posterior walls of the SHIVC (Figure 1).
   NOTE: Isolation of the SHIVC posterior wall from the diaphragm crus is crucial to obtain a sufficient margin for reconstruction.

4. Back -table preparation of the liver graft

1. Attach a cuff to the PV, as previously described¹³.
2. Attach a clamp clip to the IHIVC.
3. Turn the liver upside down and expose the stump of SHIVC.
4. Place a stay suture with 10-0 nylon on both edges of the SHIVC and hold them with bulldog clamps.
   NOTE: Back-table preparation takes approximately 10 min without a cuff attachment to IHIVC.

5. Reconstruction of the SHIVC in the recipient

1. Encircle the SHIVC with 4-0 silk to retract the liver downward.
2. Reduce the concentration of isoflurane to 0.3-0.5% before the anhepatic phase.
3. Clamp the IHIVC above the right renal vein using a microserrefine.
4. Clamp the PV using a Pean forceps¹³.
5. Clamp the SHIVC with the diaphragm using a bulldog clamp while retracting the liver downward.
6. Cut the anterior wall of the SHIVC, and then cut the posterior wall (Figure 1).
   NOTE: Isolation of the SHIVC posterior wall from the diaphragm crus in the recipient and donors is crucial to obtain a sufficient margin for the reconstruction.
7. After dissecting PV and IHIVC closely to the recipient's liver, remove the liver from the recipient (Figure 1).
8. Put the prepared liver graft into the abdominal cavity of the recipient and place the liver appropriately.
9. Place and ligate a stay suture on the right end of the SHIVC and hold it with a bulldog clamp to pull the suture toward the ventral side of the recipient (Figure 2A).
   NOTE: The right side of the SHIVC lies deep in the abdominal cavity. Pull out the right side of the SHIVC so that the posterior wall is placed in front.
10. Place and ligate a stay suture on the left end of the SHIVC.
11. Start continuous suturing with an intraluminal method on the posterior wall of the SHIVC from the left side (Figure 2A).
12. When reaching the right end of the posterior wall of the SHIVC, turn the operating table 90° counterclockwise.
    NOTE: Rotate the operating table 90° clockwise for left-handed surgeons. Alternatively, surgeons can suture the anterior wall from the front towards the back without rotation.
13. Continue the suture with an over-and-over suture on the anterior wall of the SHIVC from the right side (Figure 2A).
    NOTE: Approximately six to eight stitches for the posterior wall and eight to ten stitches for the anterior wall are required. The suture on the left end of the anterior wall is important to prevent bleeding.
14. After flushing the air in the SHIVC, ligate the continuous suture with the stay suture on the left side of the SHIVC.
15. Rotate the operating table 90° clockwise to restore the recipient position to the original position.
16. After the reconstruction and reperfusion of the PV, unclamp the SHIVC.
    NOTE: It takes 10-15 min for the SHIVC reconstruction. As the PV clamping time can affect LT outcomes, the anhepatic time (from PV clamping to PV unclamping) should be fixed for up to 20 min based on the surgeon's skill. After PV reperfusion, isoflurane is reset to 1% to maintain anesthesia.

6. Reconstruction of the IHIVC in the recipient

1. Place and ligate stay sutures with 10-0 nylon on both ends of the IHIVC.
2. Hold the stay sutures with bulldog clamps to pull toward both sides, so that the IHIVC stump becomes wide and straight.
3. Start the continuous suturing with an intraluminal method on the left side of the posterior wall (Figure 2B).
4. After reaching the right side, continue suturing over and over on the anterior wall from the right side of the recipient (Figure 2B).
   NOTE: Rotation of the operating table is not required for IHIVC reconstruction. Approximately six to eight stitches for each posterior and anterior wall are sufficient.
5. Flush the air in the IHIVC.
6. Unclamp the IHIVC clips from the graft side, and then the recipient side.
   NOTE: Ligation of the continuous suture with the stay suture on the left side of the IHIVC is unnecessary. It takes 8-10 min for IHIVC reconstruction. The total reperfusion time from PV clamping to IHIVC release should be within 30 min.

Results

A mouse recipient recovered from anesthesia within 30 min when successful vascular reconstruction was performed using a normal liver graft without an extended cold ischemic time. A success rate of >90% can be considered skilled for this procedure. We examined the learning curves in one surgeon for post-LT recovery rates (recovery from anesthesia), anhepatic time (from clamping to releasing the PV flow), and total reperfusion time (from clamping the PV to releasing the IHIVC flow). Over 60 cases were required to obtain...

Discussion

This article documented the detailed techniques for the reconstruction of the SHIVC and IHIVC with a suture technique in mouse orthotopic LT. Vascular reconstruction, especially SHIVC anastomosis, which requires a suturing technique during the anhepatic phase, is a crucial skill for successful mouse LT. In addition to the safe and secure suturing technique, quick suturing is necessary to keep the anhepatic phase within 20 min for reproducible outcomes in mouse orthotopic LT^2,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10-0 Micro suture	AROSurgical Instruments Corporation	T4A10N07
16 G intravenous catheter	TERUMO	SR-FF2032	IHIVC cuff
20 G intravenous catheter	TERUMO	SR-FF1651	PV cuff
8-0 braid silk	Natsume Seisakusho	CR9-80B2	8-0 silk
Belzer UW Cold Storage Solution	Astellas		organ preservation fluid
Bulldog clamp	B BRAUN	FB329R	Bulldog clamp
Bulldog clamps	Natsume Seisakusho	C-42-S-2	For SHIVC clamping
C57BL/6 mice	Oriental Bio Service
Forceps	Fine Science Tools	11231-30
Isoflurane inhalation solution	Viatris		Anesthesic
Micro Blunted Tips 0.1 mm x 0.06 mm	Fine Science Tools	11253-20	Straight microforceps
Micro scissors	Muranaka Medical Instruments	451-609-13
Micro serrefine	Fine Science Tools	18055-03	For IHIVC clamping
Micro serrefine	Fine Science Tools	18055-04	For PV clamping
Micro Serrefine Clamp Applicator with Lock	Fine Science Tools	18056-14	Vessel clip applicator
Micro Serrefines	Fine Science Tools	18055-4	Vessel clip
Micro suture foeceps	Aesculap	FD281R
Micro-serrefine clamp applicator	Fine Science Tools	18056-14
Needle holder	Aesculap	FD231R
No.11 Spare Blades	FEATHER Safety Razor	11	Blades
Ophthalmic scissor, round handle	B BRAUN	FD103R	Microscissor
Plastic rectangular-shaped container	Daiso		10 cm long, 15 cm wide and 6 cm high
SuperGrip Tips	Fine Science Tools	00649-11	Curved microforceps
SZX7	Olympus	SZX7	Microscope
Vannas scissors	World Precision Instruments	501232
Vascular clip	BEAR Medic Corporation	TKF-1-30	For IHIVC clamping on the liver graft