A Mouse Model of Donor Heart Lymphatic Ablation via Electrocautery for Transplantation Research

Summary

We developed a novel mouse model of donor cardiac lymphatic ablation in heart transplantation. This model demonstrates that the disruption of donor lymphatics attenuates acute rejection, reduces myocardial injury, and prolongs graft survival, providing new insights into cardiac lymphatic function in transplantation immunity.

Abstract

Although cardiac lymphatic vessels have garnered increasing attention in recent years, the relationship between the donor cardiac lymphatic system and acute rejection in heart transplantation remains to be elucidated.

Cardiac electrophysiological activity was assessed via electrocardiography (ECG) in normal mice before and after cardiac lymphatic ablation. Major lymphatic vessels of donor hearts were visualized by Evans Blue injection and subsequently ablated via electrocautery. A heart transplantation model featuring donor cardiac lymphatic dysfunction was established using hearts subjected to lymphatic ablation. Cardiac graft function was assessed via echocardiography, western blotting, and ELISA. The impact of donor lymphatic ablation on acute rejection was assessed by hematoxylin-eosin (HE) staining and monitoring of graft survival time.

ECG results indicated no significant changes in cardiac electrophysiology before and after lymphatic ablation, confirming that major cardiac vessels remained undamaged. Evans Blue was injected into the cardiac apex to label the lymphatic vessels. Major lymphatics were then ablated using electrocautery. Following the completion of the ablation procedure, the mice were heparinized, and their hearts were subsequently harvested. The aorta and pulmonary artery were transected, and the superior/inferior vena cava, along with the pulmonary veins, were ligated. The prepared (ablated) donor hearts were then transplanted. Levels of cTnI and ejection fraction demonstrated that lymphatic ablation did not exacerbate cardiac injury. HE staining revealed that ablation of donor cardiac lymphatics alleviated acute rejection, reduced myocardial injury, and prolonged graft survival.

We successfully established a murine heart transplantation model with donor cardiac lymphatic dysfunction and demonstrated that ablation of donor cardiac lymphatics can mitigate acute rejection, attenuate myocardial damage, and extend graft survival. These findings provide new insights and a foundation for understanding the role of the cardiac lymphatic system in heart transplantation.

Introduction

Cardiac allograft rejection remains one of the most common and serious complications following heart transplantation, primarily classified into acute cellular rejection and antibody-mediated rejection. Although modern immunosuppressive therapies have significantly improved graft survival rates, rejection continues to be a major determinant of transplant outcomes. According to literature, approximately 12% of patients experience at least one episode of moderate or severe acute rejection within the first-year post-transplantation¹.

The cardiac lymphatic system has garnered increasing attention for its potential role in transplant rejection. Beyond its well-established functions in fluid balance and immune surveillance, the lymphatic system plays an important role in modulating immune responses. Lymphatic vessels serve as the primary conduits for immune cells to enter and exit tissues^2,3. In the heart, the lymphatic network provides the fundamental infrastructure for the transportation and recirculation of immune cells, including lymphocytes. Through this pathway, lymphocytes migrate from the bloodstream into the interstitial space, where they perform immune surveillance. Subsequently, they are drained to the lymph nodes and ultimately return to the systemic circulation^2,4. Moreover, studies indicate that T cells and their subsets play a central role in acute rejection responses, and the activation and migration of these cells may be influenced by the lymphatic system⁵.

Although lymphatic vessels are known to exist in the heart and form networks analogous to blood vessels, their significance has not received comparable attention over the years. Studies have demonstrated that the absence of functional lymphatic vessels leads to marked cardiac hypertrophy and edema, indicating a crucial role of lymphatics in maintaining cardiac tissue homeostasis⁶. Furthermore, impaired or absent lymphatic drainage may reduce the efficiency of inflammatory cell clearance, thereby exacerbating local inflammatory responses and influencing the process of cardiac remodeling⁷.

In cardiac diseases, various interventions targeting lymphatic vessels exist. In mouse models, cardiac lymphatic deficiency can be induced by generating Vegfc+/- mice⁸. Clinically, interventional mediastinal lymphatic embolization⁹ and minimally invasive pericardial window surgery¹⁰ can be employed to treat chylous pericardial effusion. For patients with Fontan circulation physiology, lymphatic decompression may be achieved surgically¹¹, via percutaneous thoracic duct decompression¹², or by establishing lymphovenous anastomosis¹³. Occlusion techniques include thoracic duct embolization¹⁴, selective lymphatic duct embolization¹⁵, and thoracic duct ligation¹⁶. However, direct interventions targeting the cardiac lymphatic vessels themselves remain limited.

Given the critical role of cardiac lymphatic vessels, we established a novel murine heart transplantation model by ablating major lymphatic vessels in donor hearts via electrocautery. This model was utilized to investigate the impact of donor graft lymphatic dysfunction on acute rejection post-transplantation.

Protocol

Male C57BL/6J and BALB/c mice (male，6-8 weeks old, 25-28 g body weight) were maintained under specific pathogen-free conditions. All experimental procedures were conducted in accordance with protocols approved by the Animal Ethics Committee of Fuwai Hospital (Approval No.: 0108-7-800-ZX(X)-019).

Visualization and ablation of cardiac lymphatic vessels in mice
To simulate cardiac lymphatic dysfunction, major cardiac lymphatic vessels were ablated via electrocautery.

All mice were anesthetized via intraperitoneal injection of 1.25% tribromoethanol at a dosage of 0.2 mL per 10 g body weight and mechanically ventilated throughout the procedure. Following anesthesia with tribromoethanol, BALB/c mice underwent endotracheal intubation using a 22-gauge catheter and were connected to electrocardiogram (ECG) leads (Figure 1A, Supplementary Figure S1). In the ventilator parameters, the respiratory ratio was set at 1:1 or 1:2, with a tidal volume of 0.3 mL and a respiratory rate of 100 breaths/min. After connecting the catheter to the ventilator, the condition of the mouse was closely monitored; intubation was considered successful if the mouse's respiratory rate matched that of the ventilator, while inconsistent rates or abdominal distension indicated failure requiring reintubation. A thoracotomy was then performed in the left 3-4 intercostal space with an incision length of approximately 1 cm, and a retractor was used to expose the entire heart (Figure 1B). Subsequently, 5 µL of Evans blue dye was injected intramuscularly into the cardiac apex to visualize the lymphatic vessels, with a 1-minute waiting period after injection to allow complete opacification (Figure 1C, E; Supplementary Figure S2A-B), noting that Evans blue should be injected very slowly. After visualization, the location of the major cardiac lymphatic vessels was confirmed by comparing with previously reported anatomical data^17,18, and ablation was performed using an electrocautery pen set at 10 W in continuous mode, with a coagulation time of approximately 3-7 seconds (Figure 1D,F). Electrocardiography (ECG) was monitored before and after ablation to ensure the absence of vascular injury (Figure 1G,H). Finally, following major lymphatic ablation, the mice were heparinized in preparation for subsequent donor heart harvest. A schematic representation of the surgical procedure is provided in Figure 2.

Abdominal heterotopic heart transplantation
Heterotopic heart transplantation in the abdominal cavity of mice was performed as previously described¹⁹. A brief protocol is summarized below:

Donor procedure
A laparotomy was performed along the linea alba in BALB/c mice, and 1 mL of 50 U/mL heparin saline at 4 °C was injected into the inferior vena cava, while the abdominal aorta was cut to induce systemic heparinization. Following this, the heart was exposed, and 8-0 sutures were used to ligate the left and right superior vena cava, which were then transected at the distal end of the sutures (Figure 3A,B). Subsequently, the aorta was isolated from the surrounding adipose tissue and cut near the aortic arch (Figure 3C), and a similar procedure was performed to isolate and transect the pulmonary artery (Figure 3D). The inferior vena cava in the thoracic segment was ligated with 8-0 sutures and transected at the distal end (Figure 3E), after which the pulmonary veins were ligated and transected using 7-0 silk braided sutures (Figure 3F). Finally, the excised donor heart was preserved in physiological saline at 4 °C for subsequent transplantation, with all hearts preserved for the same duration of approximately 30 min to ensure experimental consistency.

Recipient procedure
Anesthetize the C57BL/6J recipient mice in preparation for abdominal heart transplantation. A laparotomy was performed along the linea alba to expose the abdominal aorta and inferior vena cava of the recipient mouse (Figure 4A). After ligating the dorsal branch vessels of the inferior vena cava with 10-0 nylon sutures, vascular clamps were used to occlude blood flow through the abdominal aorta and inferior vena cava (Figure 4B). Subsequently, a window was created in the abdominal aorta and inferior vena cava of the recipient mouse, and the blood was flushed from the vessels with heparin saline. Using 11-0 nylon sutures, end-to-side anastomosis was performed to connect the pulmonary artery of the donor heart to the inferior vena cava of the recipient and the aorta of the donor heart to the abdominal aorta of the recipient (Figure 4C-E). Finally, the vascular clamps were released to restore blood flow to the recipient mouse (Figure 4F).

Experimental design
Experimental group: Cardiac lymphatic vessels in BALB/c mice were visualized using Evans blue dye and ablated with an electrocautery pen. The hearts from BALB/c mice with ablated lymphatic vessels were transplanted into C57BL/6J mice.

Control group: Cardiac lymphatic vessels in BALB/c mice were visualized using Evans Blue dye, but no ablation was performed. The hearts from BALB/c mice were transplanted into C57BL/6J mice.

In this study, the sample size was standardized across all experimental groups and time points, with n=5 mice per group for assays conducted at 24 hours post-cardiac transplantation, n=5 per group for assays at 7 days post-transplantation, and n=5 per group for the observation of cardiac graft survival time.

The harvested donor hearts were preserved in 4 °C ice-cold saline for 30 min prior to cardiac transplantation. Both experimental and control group donor hearts were maintained in saline for an identical duration.

The beating duration of cardiac grafts was assessed daily via palpation in both groups (n=5 per group).

Histological assessment and parenchymal rejection (PR) scoring
Myocardial tissue samples underwent fixation in 10% formalin for 48 h, followed by dehydration through a graded ethanol series (70%, 80%, 95%, 100%), xylene clearing, paraffin embedding, and microtome sectioning (4 µm), with resultant sections stained using hematoxylin and eosin (H&E). Graft rejection was evaluated using the PR score for heart transplantation, and the degree of graft injury was assessed in a blinded manner²⁰.

Echocardiographic examination
Cardiac function was assessed using the ultrasound system (40 MHz 550 probe transducer). Mice were placed in a supine position on a 360-degree rotatable animal platform. After removing abdominal hair, cardiac function was evaluated under 1.0% isoflurane anesthesia. The ejection fraction (EF) of the cardiac graft was measured via M-mode echocardiography.

Detection of myocardial injury markers
The extent of myocardial tissue damage was evaluated by measuring the expression of cardiomyocyte-specific enzymes in serum. Cardiac troponin I (cTnI) levels were analyzed according to the manufacturer's instructions.

Western blotting
At 24 h post-cardiac transplantation, myocardial tissue from the main lymphatic vessel regions of the cardiac grafts in both the control and experimental groups was collected. Following the previously described protocol²¹, the expression of lymphatic vessel-associated markers was analyzed. The cardiac tissues were thoroughly lysed in RIPA lysis buffer containing protease inhibitors (phenyl methane sulfonyl fluoride). Prior to heat denaturation, the protein concentration was determined using a BCA kit and adjusted to a uniform concentration. The total protein from each group was separated via SDS-PAGE and subsequently transferred onto a PVDF membrane. The PVDF membrane was blocked with 5% skimmed milk (room temperature for 1-2 h) and then incubated overnight at 4 °C with the respective primary antibodies. The following day, the membrane was treated with a secondary antibody (anti-rabbit, 1:1000) at room temperature for 30 min. The membrane strips were incubated with ECL solution (1-2 mL) and exposed to capture the images.

The antibodies used were as follows: LYVE1 Polyclonal antibody (1:500), Podoplanin Antibody (1:500), and Anti-VEGF Receptor 3 (1:50).

Statistical analysis
All results are expressed as mean ± standard error of mean (SEM). When comparing two groups, statistical analyses were performed using Student's t-test or the Wilcoxon rank-sum test, depending on data distribution and characteristics. Statistical significance was defined as p < 0.05.

Results

Assessment of cardiac graft function
Mouse models of cardiac transplantation were established in both control and experimental groups according to the described protocol. The functional status of the grafts was evaluated at 24 h post-transplantation. The expression level of serum cardiac troponin I (cTnI) was higher in the experimental group than in the control group, but the difference was not statistically significant (Figure 5A). Concurrently, echocardiography reveale...

Discussion

The cardiac lymphatic system constitutes a vital component in maintaining tissue fluid homeostasis and supporting immune function within the heart. By facilitating interstitial fluid reabsorption, lipid transport, and immune cell trafficking, it ensures cardiac performance under both physiological and pathological conditions. In recent years, growing evidence has highlighted the critical involvement of the cardiac lymphatic system in multiple cardiovascular diseases, such as myocardial infarction, heart failure, and card...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Electrocoagulation pen	Weitai	V70
Microscopic forceps	Jinzhong	WA3030
Microscopic forceps	Jinzhong	WA2040
Microscopic needle holder	Jinzhong	YZE010
Microscopic needle holder	Jinzhong	YZE020
Microscopic scissors	Jinzhong	YBC010
Microscopic scissors	Jinzhong	YBC020
Needle holder	Jinzhong	J32010
Scissor	Jinzhong	Y00010
Small animal ventilator	Zhuodi	DW-300