We describe a model of heterotopic abdominal heart transplantation in rats, implying modifications of current strategies, which lead to a simplified surgical approach. Additionally, we describe a novel rejection model by in-ear injection of vital cardiac muscle cells, allowing further transplant immunological analyses in rats.
Heterotopic heart transplantation in rats has been a commonly used model for diverse immunological studies for more than 50 years. Several modifications have been reported since the first description in 1964. After 30 years of performing heterotopic heart transplantation in rats, we have developed a simplified surgical approach, which can be easily taught and performed without further surgical training or background.
After dissection of the ascending aorta and the pulmonary artery and ligation of superior and inferior caval and pulmonary veins, the donor heart is harvested and subsequently perfused with ice-cold saline solution supplemented with heparin. After clamping and incising the recipient abdominal vessels, the donor ascending aorta and pulmonary artery are anastomosed to the recipient abdominal aorta and inferior vena cava, respectively, using continuous running sutures.
Depending on different donor-recipient combinations, this model allows analyses of either acute or chronic rejection of allografts. The immunological significance of this model is further enhanced by a novel approach of in-ear injection of vital cardiac muscle cells and subsequent analysis of draining cervical lymphatic tissue.
Heterotopic heart transplantation is a frequently used experimental model for different investigations regarding transplantation tolerance, acute and chronic allograft rejection, ischemia-reperfusion injury, machine perfusion or cardiac remodelling. Among other advantages, the graft function can be monitored noninvasively by palpation and graft failure does not lead to a vital impairment of the recipient in contrast to other organs, such as kidneys or livers.
In 1964, Abbott et al. initially described heterotopic abdominal heart transplantation in rats1. Later, in 1966, the end-to-side technique for anastomoses was described by Tomita et al.2. The groundwork for the currently used model was reported by Ono and Lindsey in 19693. During the last decades, several modifications have been published to create different types of unloaded, partially loaded or loaded left ventricular heart grafts including combined heterotopic heart-lung transplantation4,5,6. For immunological analyses a non-volume loaded heart graft transplantation is most commonly performed. In this case, the blood flow retrogradely enters the donor ascending aorta and subsequently the coronary arteries. The venous drainage occurs along the coronary sinus into the right atrium and ventricle (Figure 1A-B). Therefore, the left ventricle is excluded from blood flow, apart from marginal amounts of blood from Thebesian veins. This also makes it a useful model for studying the pathophysiological mechanisms during left ventricular assist device therapy7.
Heterotopic heart transplantation has been performed in various species including mice, rabbits, pigs and has even been used as a uni- or biventricular assist device in humans8,9,10,11. The rat still represents a popular experimental animal for transplant models, especially since the graft survival times for different rat strain combinations have been well-defined in the past and a large number of immunological reagents are accessible12,13. Unlike mice, rats are larger making surgery and access to lymphatic tissue for immunological analyses more feasible12. Furthermore, the introduction of commercial cloning technologies in rats in recent years will most likely lead to a recurring interest in experimental rat models14.
In general, heterotopic heart grafts can be attached to the recipient vessels either by performing cervical or abdominal anastomosis. However, a few studies suggest that a femoral anastomosis facilitates improved monitoring due to better access for manual palpation or transfemoral echocardiography and thus allows a more precise detection of graft failure15,16.
It has been shown that there is no difference regarding operation time, complication rate, outcome and graft survival time between both anastomosis techniques17. Clearly, the availability of a sufficient number of draining lymph nodes must be mentioned as a benefit of cervical anastomosis; however, longer training periods are required. In contrast, the abdominal anastomosis is less complicated and equally valuable for immunological investigations, especially when combined with results from a novel method of in-ear injection of allogenic cardiac muscle cells and subsequent cervical lymphadenectomy. A combination of both models offers a broad spectrum of post-interventional immunological analyses.
The following protocol refers to operating in pairs of surgeons in order to reduce ischemia time. However, all experiments can be performed by a single person. The setup of instruments and materials for heart explantation and implantation is displayed in Figure 2A-B.
All animal experiences have been performed according to the guidelines of the local Ethics Animal Review Board of the regional authorities for consumer protection and food safety of Lower Saxony (LAVES, Oldenburg, Germany) with the approval IDs 12/0768 and 17/2472.
1. Heart explantation and perfusion
NOTE: As graft donors, female or male rats at an age of 7-22 weeks were used.
2. Heart implantation
NOTE: As recipients, 10-14 weeks old female or male rats were used. Donors and recipients were approximately weight matched.
3. Postoperative care
4. Enzymatic digestion of the heart and subcutaneous injection of heart cells in the ear
In the past, different immunological issues have been addressed on the basis of the model, which was validated in the work group by more than 500 transplants with a survival rate of more than 95%13,18,19,20,21,22,23,24. Total operating times (including graft explantation and implantation) usually did not exceed 60 minutes, whereas combined cold and warm ischemia times were around 30 minutes. The strain combinations applied were mainly based on Lewis (Lew) background. Syngenic transplants survived up to 100 days without signs of graft failure, but significant weight and size reduction upon graft explantation. Most recently, we performed heterotopic heart transplantation in two different donor-recipient combinations simulating a fast and a prolonged rejection model: Lew.1a → Lew wt leading to fast rejection (mean survival time of 7.4 days) and Lew.1u-7B→ Lew.1a leading to a more prolonged rejection (mean survival time of 42.5 days) (Figure 6). Macroscopically, the rejected grafts showed a thrombosis accompanied by a livid discoloration and swelling, whereas non-rejected grafts show distinct atrophy, most likely as a consequence of an unloaded left ventricle. Furthermore, we drew up cryostat sections of transplanted hearts in order to detect cell infiltration using an alkaline phosphatase-antialkaline phosphatase (APAAP) staining method. Single frames with a 50x magnification were merged to one composite image, giving an overview of the complete graft and the distribution of infiltrating cells. Histological analysis showed an increased infiltration of (e.g., CD4+, TCR+, or NKR-P1A/B+) immune cells in the allogenic grafts, whereas syngenic grafts were largely free of cell infiltration (Figure 7A-C).
Cervical lymphadenectomy and re-stimulation assays of draining lymph node cells after in ear injection of cardiac muscle cells in the above mentioned strain combinations revealed distinct strain-specific immune responses towards allogenic cardiac tissue and allowed further immunological analyses, such as cytokine profiling (Figure 8A-C).
Figure 1: Schematic presentation of the end-to-side anastomoses and the resulting blood flow through the heart. After anastomosing the donor ascending aorta end-to-side to the recipient abdominal aorta and analogously the pulmonary artery to the recipient inferior vena cava (A), blood flow enters the coronary arteries via the ascending aorta. The venous drainage occurs via the sinus coronarius into the right atrium and ventricle and through the pulmonary artery into the recipient inferior vena cava (B). Please click here to view a larger version of this figure.
Figure 2: Required surgical instruments and materials. (A) Explantation: 1: elastic limb band, 2: retractors, 3: 5-0 ligature, 4: probe pointed scissors, 5-6: micro needle holders, 7: scissors, 8: surgical forceps, 9: micro forceps, 10: eye lubricant, 11: compresses, 12: cotton swabs, 13: perfusion base, 14: saline solution on ice. (B) Implantation: 1: elastic limb band, 2: retractors, 5-6: micro needle holders, 7: scissors, 8: forceps, 9: micro forceps, 10: eye lubricant, 11: compresses, 12: cotton swabs, 15: micro scissors, 16: micro forceps, 17: Potts scissors, 18: needle holder, 19: arched cannula, 20: Cooley vascular clamp, 21: 8-0 monofilament sutures, 22: hemostatic gauze, 23: 3-0 polyfilament sutures, 24: Petri dish, 25: Carprofen (5 mg/mL), 26: local anesthetic (lidocaine 0.2%), 27: saline solution. Please click here to view a larger version of this figure.
Figure 3: Heart explantation. After heparinization (A), thoracotomy is performed and ascending aorta and pulmonary artery are severed as distal as possible (B). With one single ligature pulmonary and both caval veins are occluded (C) and the heart is removed from the thoracic cavity (D). (E) shows the heart before and (F) after perfusion with 30 mL saline solution containing heparin. Please click here to view a larger version of this figure.
Figure 4: Heart implantation. After exposition of abdominal vessels and placement of a Cooley vascular clamp (A) the vessels are cannulated (B) and a longitudinal incision is performed using Potts scissors (C-D). The donor ascending aorta is fixated with one knot each at the cranial and caudal corner of the incision of the recipient abdominal aorta (E) and anastomosis is performed by continuously running sutures (E-F). Note that the graft is placed on the right side of the vessels for the first half of the anastomosis (E) and onto the left side of the vessels for the second half of the anastomosis (F) and the subsequent venous anastomosis. The first half of the venous anastomosis is performed by an intraluminal suture (G). After finishing the second half of the venous anastomosis, the graft is ready for reperfusion (H). Please click here to view a larger version of this figure.
Figure 5: In-ear injection of allogenic cardiac muscle cells. After fixing the recipient's ear on a finger using double-sided tape (A) allogenic vital cardiac muscle cells are injected subcutaneously close to visual capillary vessels (B). After an observation period draining cervical lymph nodes (*) are extracted (C). Please click here to view a larger version of this figure.
Figure 6: Heart survival in different syngenic and allogenic donor-recipient combinations. Kaplan-Meier analysis shows the survival of syngenic (n=10 fast rejection model; n=5 prolonged rejection model) and allogenic grafts (n=11 fast rejection model; n=14 prolonged rejection model). In the prolonged rejection model six of 14 recipients reached the end of the observation period (60 days) without graft failure, leading to a prolonged graft survival in this group. Please click here to view a larger version of this figure.
Figure 7: Histological analysis of syngenic and allogenic heart grafts. (A-B) show the infiltration of CD4+ cells using the APAAP staining method in a syngenic graft (A) and an allogenic graft upon rejection (B). (C) presents the increase of cell infiltration in allogenic grafts when compared to syngenic grafts (serving as reference group) for different immune cell subsets. The classification applied to quantify cell infiltration was modified from Hirschburger et al.25: 0 = infiltration comparable to syngenic grafts; 0.5 = slight increase of stained cells in isolated tissue sections; 1 = increase of singular stained cells over the whole tissue section; 1.5 = increase of stained cell clusters uniformly distributed over the whole tissue section; 2 = strong; 2.5 = very strong; 3 = strongest increase of stained cell clusters throughout the whole tissue section. The histological sections of the grafts were analyzed using a 50x magnification. Five grafts per group (syngenic and allogenic, respectively) were included in the analysis. Please click here to view a larger version of this figure.
Figure 8: Analysis of draining lymph node cells after in-ear injection of allogenic cardiac muscle cells. Specific re-stimulation (with 2 x 105 splenocytes of the respective donor strain) of 2 x 105 lymphocytes harvested from either draining cervical or mesenteric lymph nodes (LN) of fast rejecting Lew wt and prolonged rejecting Lew.1a recipients showed significantly reduced proliferation of draining lymph node cells in Lew.1a recipients (A), whereas proliferative capacity in general was still observable after unspecific stimulation with CD3/CD28 antibody (B). Surprisingly, cytokine profiling revealed an increase of inflammatory cytokines in the lymph nodes of prolonged rejecting recipients (C). Results are presented as mean ± SEM of at least 4 animals per group. Significance is indicated with * for p-values ≤ 0.05 and **** for p-values ≤ 0.0001. (This figure has been modified from Beetz et al.24). Please click here to view a larger version of this figure.
The previously described method of heterotopic cardiac transplantation in rats is mainly based on the description of Ono and Lindsey in 19693. Since then, several modifications have been introduced in various species leading to a wide diversity of this model. Combining several of these modifications and introducing our own experience resulting from over 30 years of performing heterotopic heart transplants in the laboratory, we created a feasible surgical approach, which does not require long training periods or surgical background. In the following, we will discuss general limitations of this model and underline critical steps of the protocol. Furthermore, we will emphasize the benefits of combining heterotopic heart transplantation with a novel method of in-ear injection of cardiac muscle cells.
Anesthesia and general complications
Although it has been reported that isoflurane anesthesia is superior to injection anesthesia regarding early survival after heterotopic heart transplantation, intraoperative respiratory depression still represents one of the most common complications and thus requires a careful narcosis management26. Instead of serial graft explantation and implantation, respectively, we advise beginning the preparation of the recipient and the abdominal vessels immediately after thoracotomy of the donor animal, particularly because operation times of less than one hour are associated with a better outcome regarding graft and recipient survival26,27. Besides already mentioned complications such as hypothermia due to long operating times and missing heating mats, and intestinal necrosis or obstruction by unphysiological placement of the intestine, a paresis of hinder limbs represents a further complication, which can be prevented by atraumatic vascular clamping and thorough flushing of the anastomoses to avoid peripheral embolism28,29.
Ligature of pulmonary and caval veins and bleeding complications
In order to reduce warm ischemia time, we use one single ligature for both caval and all four pulmonary veins. As a possible complication resulting from a too proximal/ventral placement of the ligature, the disruption of venous backflow by occlusion of the sinus coronarius has to be mentioned. In case of severe bleeding after removal of the Cooley clamp and visible beating of the graft, the ligature has to be checked for insufficiency immediately. We observed this type of complication especially if the dissection of the pulmonary veins was performed too close to the ligature while removing the graft from the thoracic cavity. Otherwise, severe bleeding is mainly caused by insufficiency of the vascular anastomoses. Additionally, a coronary artery running along the ascending aorta which is often severed during explantation is described to cause lethal bleeding upon reperfusion27.
Ischemia time and perfusion
Regardless of the transplantation model, it is always indispensable to reduce ischemia time, especially as the heart is considered as a vulnerable organ regarding ischemia damage. By performing surgery with two surgeons, we are able to achieve a minimum warm and cold ischemic time and therefore forgo the usage of cardioplegic solutions in order to reduce ischemia-reperfusion damage30. Generally, the perfusion of the graft plays a key role and is essential to cool the graft and remove blood cells, which could result in thrombosis or embolism. Whereas low perfusion pressures lead to insufficient perfusion and thus to an incomplete removal of blood cells, high perfusion pressures can cause endothelial damage31,32. We advise perfusion of the pulmonal artery as well as the ascending aorta until the coronary arteries are visibly flushed clear.
Incision of recipient vessels
A critical step in the protocol constitutes the incision of the recipient vessels without causing damage to the vessels posterior wall: Schmid et al. described the benefit of aortotomy or venotomy performing a small transverse incision followed by longitudinal enlargement in cranial and caudal direction, which further leads to a decreased stenosis rate of aortal anastomosis33. In our model, the recipient vessels are punctured using a small cannula. Afterwards, the puncture site is enlarged by using Potts scissors to create a longitudinal incision. For a better feasibility, we recommend bending the tip of the cannula to an angle of 30-45° leading to a decreased risk of damaging the vessels posterior wall. We did not observe clinically relevant vascular stenoses in any of our recipients. Similar benefits of opening recipient vessels by puncturing the abdominal aorta and the inferior vena cava with a cannula have been described by Shan et al.34.
Mastering the model of heterotopic heart transplantation
During the last decade, the model has been performed by researchers in the department with little or no surgical background. As stated above, we operate in pairs of surgeons, whereas the more experienced researcher is in charge of guaranteeing the success of the transplantation and at the same of gradually improving the skill set of the unexperienced researcher. After a short training period of approximately ten graft explantations and implantations in dead animals, the unexperienced researcher is in charge of performing the heart explantation and assisting the graft implantation in approximately ten live animals. Subsequently, the unexperienced researcher actively performs the vascular anastomoses, so that after approximately ten further transplantations, the former unexperienced researcher is usually capable of performing all critical steps of the model.
Applying this training concept, past publications from our department using heterotopic heart transplantation in rats showed no differences regarding morbidity, mortality or graft function despite several different teams of surgeons13,18,19,20,21,22,23,24.
Of note, performing the vascular anastomoses represents the most critical step in this protocol and solid organ transplant models in general. We, therefore, recommend prolonged training periods using dead animals until anastomoses are performed accurate and fast, especially if an experienced researcher is not available for guidance in live animals.
General advantages and disadvantages of the model
Whereas spontaneous tolerance induction is often described as a phenomenon in liver transplantation and also observed in kidney transplantation, the heart is considered as a rather immunogenic organ and thus enables reliable rejection in transplant models35,36. Contrary to these findings, we could also notice long-term survival and absence of rejection after heterotopic heart transplantation in certain donor-recipient combinations despite complete major histocompatibility complex disparity.
An often-mentioned criticism of heterotopic heart transplantation is the subjectivity of graft monitoring by manual palpation. Therefore, the model has been extended to femoral anastomosis techniques in order to facilitate the access for palpation and introduce further monitoring techniques such as transfemoral echocardiography15,16. On the other hand, Mottram et al. demonstrated that the monitoring of the graft via palpation correlates well with electrocardiographic measurements37. Thus, manual palpation in heterotopic heart transplants seems sufficient for monitoring graft function in an acute rejection model.
As a consequence of heterotopic placement and left ventricular unloading, the heart does not function under anatomical or physiological conditions assuming that this does not impact immunological analyses. Contrary to this assumption, it had been shown that cardiac remodeling resulting from left ventricular unloading during left ventricular assist device therapy leads to a decreased cytokine release38,39. On the other hand, Tang-Quan et al. described the unloaded setting as a more appropriate approach for immunological analysis, since long-term ischemic damage of the graft resulting from perfusion with partially deoxygenated blood in the left ventricular loaded model was observed40.
Although abdominal placement of the grafts offers surgical benefits in terms of practicability, it is difficult to harvest sufficient numbers of draining retroperitoneal lymph nodes upon graft rejection impairing further analyses. For this reason, we introduced a novel method of in-ear injection of allogenic cardiac muscle cells. Originating from parasitological research, this concept has not been applied for immunological analyses in transplantation, despite its feasibility41,42.The advantage of this model is the possibility of identifying and harvesting a significant number of draining lymph nodes, which offers the possibility of performing complex immunological analyses. Of note, both models could be combined in one recipient offering further insights into the mechanisms of rejection and tolerance in cell and organ transplantation in rats.
Our models of rat heart and cardiac muscle cell transplantation represent a practicable and well-studied approach and can be performed without further surgical training or background. Facing the fact that new cloning technologies for rats have been introduced and developed recently, these models offer vast opportunities for transplant immunological researchers.
The authors have nothing to disclose.
We want to thank Britta Trautewig, Corinna Löbbert and Ingrid Meder for their commitment.
Anesthesia device (including isoflurane vaporizer) | Summit Anesthesia Solutions | No Catalog Number available | |
Cannula (27 G) | BD Microlance | 302200 | |
Carprofen | Pfizer | Rimadyl 50 mg/mL | |
Cellstar Tubes (15 mL) | GreinerBioOne | 188271 | |
Cell strainer (40 µm) | BD Falcon | 2271680 | |
Collagenase Type CLSII | Biochrome | C2-22 | |
Compresses 5×5 cm | Fuhrmann | 31501 | |
Compresses 7.5×7.5 cm | Fuhrmann | 31505 | |
Cotton swabs | Heinz Herenz Medizinalbedarf | 1032128 | |
Dexpathenol (5 %) | Bayer | "Bepanthen" | |
DPBS BioWhittaker | Lonza | 17-512F | |
Forceps | B. Braun | Aesculap BD557R | |
Forceps | B. Braun | Aesculap BD313R | |
Forceps | B. Braun | Aesculap BD35 | |
Heating mat | Gaymar Industries | "T/Pump" | |
Hemostatic gauze | Ethicon | Tabotamp | |
Heparin-Natrium 25 000 I.E. | Ratiopharm | No Catalog Number available | |
Isofluran CP | CP-Pharma | No Catalog Number available | |
Large-pored sieve (stainless steel) | Forschungswerkstätten Hannover Medical School | No Catalog Number available | |
Lidocaine | Astra Zeneca | 2 % Xylocain | |
Metamizol-Natrium | Ratiopharma | Novaminsulfon 500 mg/mL | |
Micro forceps | B. Braun | Aesculap BD3361 | |
Micro needle holder | Codman, Johnson & Johnson Medical | Codmann 80-2003 | |
Micro needle holder | B. Braun | Aesculap BD336R | |
Micro needle holder | B. Braun | Aesculap FD241R | |
Micro scissors | B. Braun | Aesculap FD101R | |
Micro scissors | B. Braun | Aesculap FM471R | |
Needle holder | B. Braun | Aesculap BM221R | |
Penicillin/Streptomycin/Glutamine (100x) | PAA | P11-010 | |
Peripheral venous catheter (18 G) | B. Braun | 4268334B | |
Peripheral venous catheter (22 G) | B. Braun | 4268091B | |
Probe pointed scissors | B. Braun | Aesculap BC030R | |
Retractors | Forschungswerkstätten Hannover Medical School | No Catalog Number available | |
RPMI culture medium | Lonza | BE12-702F | |
Saline solution (NaCl 0.9 %) | Baxter | No Catalog Number available | |
Scissors | B. Braun | Aesculap BC414 | |
Surgical microscope | Carl-Zeiss | OPMI-MDM | |
Sutures (anastomoses) | Catgut | Mariderm 8-0 monofil | |
Sutures (ligature) | Resorba | Silk 5-0 polyfil | |
Sutures (skin, fascia) | Ethicon | Mersilene 3-0 | |
Syringe (1 mL) | B. Braun | 9166017V | |
Syringe (10 mL) | B. Braun | 4606108V | |
Syringe (20 mL) | B. Braun | 4606205V | |
Vascular clamp | B. Braun | Aesculap FB708R |