The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.
It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details – details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.
In an era when it is possible to perform kidney, lung, liver and pancreas transplants in mice, the cornerstone of basic organ transplant and immunology research since 19731-4 remains the heterotopic heart transplant model in the mouse. In the intervening years several papers have been published detailing improvements/refinements5,6 to this procedure.
As a model of solid organ primarily vascularized transplantation this procedure is second to none. Once mastered this procedure lends itself to research into allogeneic rejection responses7, the development of chronic vasculopathies8 and the mechanisms of ischemia reperfusion injury9.
The keys to successfully learning this procedure are just like any other surgery, patience on the part of the instructor and the trainee and attention to detail. At the beginning of the process the new surgeon will find that they will spend many hours on each transplant. As experience is gained, surgical times, and therefore ischemia, will drastically reduce. Paying attention to the details of every step will sooner or later lead to success.
While the instructor can do their best to pass on, and to anticipate, all of the possible pit falls that may be encountered during these surgeries, the “creative” trainee will likely find some of their own!
The basics of the procedure are as follows. The donor ascending aortic arch is end-to-side anastomosed to the recipient abdominal aorta and the donor pulmonary artery is end-to-side anastomosed to the recipient abdominal inferior vena cava (IVC). Blood flows from the recipient aorta in retrograde fashion through the donor aorta to the coronary arteries. Once the blood has flowed through the coronary system it drains into the right atrium via the coronary sinus, is pumped into the right ventricle and then via the pulmonary artery into the recipient IVC. In this way the coronary system is supplied arterial blood and sinus rhythm returns to the graft within 1-2 min of reperfusion. Since the left chambers of the heart are essentially pressure under-loaded the left ventricular free wall will atrophy over time.
All animals were housed under pathogen-free conditions at the University of Colorado Barbara Davis Center Animal Facility under IACUC approval and cared for according to NIH guidelines.
Depth of anesthesia is judged by toe pinch initially and by observance of respiration rate once the procedure has begun.
1. Donor Heart Harvest
2. Heart Implant Technique
3. Graft assessment
IMPORTANT NOTES:
All instruments are sterilized, sterile gloves are worn throughout the procedure and a sterile field is maintained. The donor and recipient surgeries are performed with the use of an operating microscope. Ensure that the anastomoses are “clean”. That is, that the back walls are not caught when placing stitches. This will cause a significant constriction to flow that will more than likely result in a failed graft and in extreme cases to hind-limb paralysis. It is also vitally important that full thickness passes including the vascular adventitia and the intima of the suture needle are achieved. Evertion of the edges also ensures that there is intima-to-intima contact, which aids in sealing and healing of the anastomoses. Another vitally important factor is ensuring that the tension of the anastomotic suture lines is also optimal. Too loose and there will be irreversible leaking, too tight and stricture to flow will result. If on the arterial side this will result in poor perfusion of the graft, if on the venous side a congested heart will result.
The utilization of this surgical technique opens the way for either simple graft survival/rejection studies, or quite complex experimental protocols. In the study briefly described in the figure below, we sought to define the involvement, if any, of Fas and/or perforin as mechanisms of CD4 T cell mediated cardiac rejection. This was made possible by the extraordinary array of mouse strains that are available today. Results demonstrate that the direct rejection of cardiac allografts by CD4 effector T cells requires the alternative contribution of graft Fas expression and T cell perforin expression. To our knowledge, this is the first demonstration that cytolytic activity by CD4 T cells can play an obligate role for primary acute allograft rejection in vivo.
Figure 1. Perforin and Fas Represent Obligate and Parallel Pathways of CD4 T cell-Mediated Cardiac Rejection B6, B6 PFPKO (perforin knock-out), and B6gld (Fas-ligand deficient) CD4 T cells were utilized to reconstitute B6rag-/- recipients of C3H wild type or Fas-deficient C3Hlpr cardiac allografts. Removal of Fas alone (♦, p=NS vs. control Wt C3H + B6 CD4 T cells) from the donor hearts or removal or perforin alone from the CD4 T cells (■, p=NS vs. control Wt C3H + B6 CD4 T cells) did not abrogate rejection. Interestingly, the removal of FasL from effector CD4 T cells did delay rejection significantly (●, p < 0.02 vs. Wt C3H + B6 CD4 T cells, p < 0.01 vs. Wt C3H + B6 PFPKO CD4 T cells, and p< 0.01 vs. C3Hlpr + B6 CD4 T cells). However, most allografts were still rejected (4 of 5). Significantly, the simultaneous removal of both donor Fas and CD4 T cell perforin completely abrogated rejection (○, p < 0.002 vs. control Wt C3H + B6 CD4 T cells, Wt C3H + B6 PFPKO CD4 T cells, and C3Hlpr + B6 CD4 T cells). This abrogation was significantly more robust than the individual removal of CD4 T cell FasL (○, p < 0.003 vs. control Wt C3H + B6gld CD4 T cells). From Grazia et al10. Reprinted with permission.
This surgical technique is not easy to master, but once mastered is a powerful research tool. The researcher/surgeon is rewarded by consistency of technique and by attention to detail. Patience during the learning phase is key. As published by Niimi3, with the aid of a video based learning tool it takes an average of 11 attempts to achieve the first successful procedure and 78 attempts to achieve a 90% success rate. Videos have become an important teaching tool in surgery11,12.
Troubleshooting
Bleeding from the anastomoses may occur and this is likely due to either lack of correct tension in the sutures, or too few sutures. While a clotting inducing agent such as Gelfoam can be useful for reducing leaks, we recommend that the surgeon should rely on good technique. Congested non-beating heart is most commonly due to anastomoses that are too tight, particularly on the venous side. A non-beating, non-perfused graft is commonly caused by an air bubble that has traveled into one of the coronary arteries. It is important to maintain a damp-to-wet field to avoid the entry of bubbles into the vessels.
Limitations of the Technique
This technique is not suitable if a researcher wants to investigate effects on a fully functioning heart. That would require an orthotopic transplant technique, which to date has proved impossible to perform.
Significance with Respect to Existing Methods
If one wishes to study the effects on a fully vascularized, solid organ transplant in the mouse, then the heart model is probably the simplest to master. Mouse models of lung, kidney and liver transplantation do exist, but are much harder to learn and perfect.
Critical steps Within the Protocol
It is vitally important that full thickness passes including the vascular adventitia and the intima of the suture needle are achieved. Evertion of the edges also ensures that there is intima-to-intima contact, which aids in sealing and healing of the anastomoses. Another vitally important factor is ensuring that the tension of the anastomotic suture lines is also optimal. Too loose and there will be irreversible leaking, too tight and stricture to flow will result. If on the arterial side this will result in poor perfusion of the graft, if on the venous side a congested heart will result.
Above all, repetition, consistency of procedure and continual attention to detail will yield great results and fundable and publishable data.
The authors have nothing to disclose.
The authors wish to thank Dr. Biagio Pietra for his previous work in our lab.
Instrument | Roboz # | Fine Science Tools # | Arosurgical # |
Straight micro-dissecting forcep #5 | RS-5015 | 11295-51 | |
Curved micro-dissecting forcep #7 | RS-5047 | 11297-00 | |
Curved serrated forcep | RS-5137 | 11052-10 | |
Vannas micro-dissecting scissors, short | RS-5610 | 09.140.08 | |
Micro-dissecting scissors, straight, sharp, long | 11.602.11 | ||
Micro spring handle needle holder | 11.549.15 | ||
Straight mosquito forcep | 91308-12 | ||
Micro-dissecting scissors, straight, blunt | RS-5962 | 14078-10 | |
Micro-dissecting scissors, curved, blunt | RS-5981 | 14079-10 | |
Micro retractor | RS-6540 | ||
Instrument tray, 10” x 6 ½” x ¾” | RT-1350S | ||
Silk suture, 5/0, 22.5m spool | 18020-50 | ||
Suture | |||
10/0 nylon | T4A10Q07 | ||
5/0 silk | E19A05N | ||
Gloves | Drapes | ||
Biogel from Medex Supply | Precept, #64-9012-9 | ||
Syringes | Cotton applicators | ||
B-D 1cc insulin, #329424 | Fisher-brand, #23-400-100 | ||
Povidone-Iodine swabs | |||
PDI, #B40600 | |||
4/0 Cotton ties | |||
Domestic cotton autoclaved with instruments |