A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.
The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.
The surgical technique of heterotopic abdominal heart transplantation in mice represents a standard model for research in transplantation immunology1,2,3. However, it is very challenging to perform and this implicates a restriction to the widespread use of this model4,5.
In traditional mouse heart transplantation (THTx), the donor aorta and the recipient abdominal aorta are anastomosed while the pulmonary artery is anastomosed to the recipient inferior vena cava6,7,8.
In this modified mouse heart transplantation technique, the donor aorta is anastomosed to the recipient abdominal aorta and the donor IIVC is anastomosed to the recipient inferior vena cava(3,4,6) (Figure 2 and Figure 3).
All animal experiments were conducted following the guidelines from the directive 2010/63/EU of the European Parliament on protection of animals used for scientific purposes (Ethic committee approved, #G1071/09).
NOTE: Preliminary preparation, anesthesia, post-operative care and monitoring work are the same as performed in traditional surgical methods1,2,4. BALB/c mice served as heart donors and C57BL/6J as transplant recipients. Mice were aged 8-12 weeks, weighed~30 g at transplantation and were housed under standard conditions.
1. Preparatory steps
2. Donor operation procedure
3. Recipient operation procedure
NOTE: The initial operation steps are similar to those previously shown for the donor mouse, including anesthesia and disinfection.
Here, a modified technique of heterotopic abdominal heart transplantation in mice that has been previously developed in our laboratory and has proven useful for the last 16 years is presented. Previously, it was reported that in overall 40 cases of vena cava to vena cava (V-V group) compared to 40 cases of the traditional pulmonary artery to vena cava (P-V group) anastomosis procedure4 (Table 1) the vessel anastomosis took 20.8±1.3 min in the V-V group, which was significantly shorter than in the P-V group (27.5±1.3 min, p<0.01). The warm ischemia time, total recipient operation time and postoperative heart repeating time, which were 25.5±1.2 min, 42.0±1.5min, and 1.1±0.2 min, respectively, were also significantly shortened than observed in the traditional P-V group (all p<0.05) (Figure 1). Even though there are no differences for longtime survival rates in this model10,11, the modified technique facilitated abdominal heart transplantation in mice resulting both in a reduced warm ischemia and grafted heart rebeating time.
With respect to these previously published data and our experience with this model within the last 16 years, we recommend that the key operation steps should take a limited amount of time to ensure a success rate of >90%4. Therefore, optimal results are achieved if the harvesting of the donor heart takes no longer than 60 min, the cold ischemia time should be limited to 40 min at maximum, and the construction of the IIVC anastomosis should not take longer than 15 min as this is directly associated with a reduced warm ischemia time.
Figure 1. Comparison of procedure times between the two operation techniques (n =40 each, mean + SE) Please click here to view a larger version of this figure.
Figure 2. Donor heart preparation using the traditional (a, upper left) and the modified (b, lower left) mouse heart transplantation model. The photographs in panels a and b depict the aorta (A), the pulmonary artery (PA), the right atrium (RA), the left atrium (LA) and the intrathoracic inferior vena cava (IIVC) of the donor heart. Notice the difference in vessel length of the PA compared to the IIVC. Panel c shows the recipient situs prepared for the heterotopic abdominal HTX. Please click here to view a larger version of this figure.
Figure 3. Vascular anastomosis of the modified model: aorta (A), intrathoracic inferior vena cava (IIVC), abdominal inferior vena cava (aivc), abdominal aorta (aa). Please click here to view a larger version of this figure.
Figure 4. Demonstration of vessel length for the pulmonary artery (PA) and the intrathoracic inferior vena cava (IIVC). Please click here to view a larger version of this figure.
Figure 5. Physiological trumpet shape at the end of the intrathoracic inferior vena cava. Aorta (A), intrathoracic inferior vena cava (IIVC), right atrium (RA), left atrium (LA). The left red double arrow on the left highlights the diameter and the red circle on the right the structure of trumpet shape. Please click here to view a larger version of this figure.
Vena cava to vena cava (new method) | Pulmonary A to vena cava(traditional method) | |
Donor procurement (min) | 12.8 + 0.3 | 11.4 + 0.4 |
Recipient operation (min) | 42.0 + 1.5 | 48.6 + 1.4** |
Vessel anastomosis (min) | 20.8 + 1.3 | 27.5 + 1.3** |
Cold ischemia (min) | 32.8 + 0.6 | 34.2 + 0.7 |
Warm ischemia (min) | 25.5 + 1.2 | 32.6 + 1.3** |
Rebeat postoperation (min) | 1.1 + 0.2 | 2.1 + 0.4* |
Success rate | 92.50% | 90.00% |
*P < 0.05 | ||
**P < 0.01. |
Table 1. Comparison of Time Distributions and Initial Outcomes in Two Operations (n – 40, mean + SE)
Reprinted from Wu, K., Zhang, J., Fu, J., Wu, S., Philipp, T., Uwe, H., Kribben, A. and Witzke, O. Novel technique for blood circuit reconstruction in mouse heart transplantation model. Microsurgery. 26, 594-598 (2006).
The surgical technique of heterotopic abdominal heart transplantation in mice is very challenging and this implicates a restriction to the widespread use of this model.
One of the disadvantages of the conventional technique is the limiting length of the donor's pulmonary artery (PA). It is usually of about 2 mm of length, whereas the length of the IIVC of the donor heart used in our model is generally about 1 cm (Figure 2). That means that in the modified model, the IIVC anastomosis offers a clearer sight of the operation situs enabling improved surgery of anastomoses and preventing the undesired development of too strict sutures or even damaging lesions of the vessels (Figure 4). Both the construction of the PA as well as the IIVC anastomosis are challenging even for experienced operators. The PA is very delicate and thin-walled and the IIVC is even thinner and potentially more fragile in mice. Therefore, operators need to be aware of this restriction and should apply caution when suturing the IIVC. However, at this point it is important to emphasize that although the IIVC vessel wall is very thin, it has the great advantage that the vessel length is not associated with tension to the blood vessel connection, thereby making the application of a precise suture easier and less prone to a damage. Since the cautious and safe application of the vessel sutures are very critical for a successful operation outcome, a magnification of the situs of 10 to 20-fold is recommended.
In addition, the ending of the thoracic segment of the IIVC forms a typical trumpet shaped structure (Figure 5). Its larger opening diameter represents one of the important and beneficially reasons why the IIVC can be chosen. Its usage facilitates the application of a sufficient anastomosis. This reduces both the difficulty of the operation and the operation time.
A possible event that may compromise the outcome of the transplant procedure represents a thrombosis of the vessel anastomosis, often fostered by a stenosis. Although in our model the length of the vessel does increase on purpose, this was not associated with the formation of thrombi. The trumpet shaped anastomosis of the IIVC may also exert a positive effect while greatly reducing the occurence of an anastomotic stenosis. Therefore, for this procedure a posttransplant heparinization is not necessary.
A previous analysis of the IVC-aivc anastomosis method revealed several advantages and improvements when compared to the conventional technique10. With respect to these results and from the retrospective point of view of our longstanding experience4,10,12,13, this technique results both in a reduced warm ischemia and rebeating time of the grafted heart. Even though there are no differences for longtime survival rates for this model10, the modified technique presented here is facilitating the anostomotic vessel reconstruction thus reducing the difficulty of abdominal heart transplantation in mice. The training and application of this model may therefore improve the accessibility and widespread application of abdominal heart transplantation in mice for immunological as well as cardiac research purposes.
The authors have nothing to disclose.
We thank Dr. Yun Xu for her help as voice actor, Dr. med. Jianhua Peng for her help in video editing and Dr. Annika Kuckhahn for her comments and support. This work was supported in part by the German Research Foundation (DFG) to promote international collaborations (HO2581/4-1 to AH), and the National Science Foundation of China (NSFC; #81760291 to FJ).
30G-needles | Braun | 456300 | |
acepromazine | CP Pharma | Tranquisol P | |
BALB/c AnNCrl mice | Charles River. Germany | no catalog number | |
Bepanthen eye ointment | Haus-Apotheke | PZN 01578675 | |
Bonn Micro Forceps | FST | 11083-07 | |
Box for insulation and oxygen supply device | RUSKINN | INVIV | |
C57BL/6J mice | Charles River. Germany | no catalog number | |
Carprofen | Zoetis | Rimadyl 50 mg/ml | |
CATHETER-FEP 26G | TERUMO | Surflo-W | |
Clip Applicator Forceps Style | FST | 18057-14 | |
Curved forceps | WPI | 14114-G | |
custodiol/HTK | Dr. Franz Köhler Chemie | no catalog numer | |
Cutasept skin disinfection | VWR | BODL980365 | |
electrosurgical pen | Bovie | CHANGE-A-TIP | |
gauze pads, cotton swabs | Lohmann-Rauscher | 13353 | |
Heating mat | THERMO MAT PRO 30W | HTP-30 | |
Hemostatic sponge | CuraSpon | J1276A | |
heparine-solution | Haus-Apotheke | PZN 03029820 | |
Ice box | PETZ | No Catalog Number available | |
Inhalation anesthesia device | GROPPLER | BKGM 0616 | |
insulation and oxygen supply device | RUSKINN | INVIV | |
isoflurane | CP Pharma | Isofluran CP 1 ml/ml | |
ketamine | Zoetis | no catalog numer | |
metamizole | WDT | no catalog numer | |
Micro scissors | FST | 15000-00,15000-10 | |
Micro Serrefine ( Clamp ) Angled / 16 mm | FST | 18055-06 | |
Microscope | Leica | LEICAMZ6 | |
Microscope light | SCHOTT | KL2500LED | |
Saline solution (NaCl 0.9%) | Haus-Apotheke | PZN 06178437 | |
Scissors | Peha Instruments | 991083/4 | |
small Petri dish | Sarstedt | 833900 | |
Straight forceps | WPI | 14113-G | |
surgical tape | BSN | 4120 | |
Suture Tying Forceps – 10 cm | FST | 18025-10 | |
Sutures(10-0) | Medtronic | N2540 | |
Sutures(4-0) | ETHILON | V4940H | |
Sutures(7-0) | ETHILON | 1647H | |
Syringe (0.3 mL) | BD | 324826 | |
Syringe (1 mL) | BD | 320801 | |
xylazine | Bayer | Rompun |