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Anat Cell Biol 2024; 57(4): 598-604

Published online December 31, 2024

https://doi.org/10.5115/acb.24.101

Copyright © Korean Association of ANATOMISTS.

The lymphatic drainage of the goat heart

Chuan-Xiang Ma , Wei-Ren Pan , Zhi-An Liu , Yao Li , Fan-Qiang Zeng

Department of Human Anatomy, College of Biomedical Sciences, Xuzhou Medical University, Xuzhou, China

Correspondence to:Wei-Ren Pan
Department of Human Anatomy, College of Biomedical Sciences, Xuzhou Medical University, Xuzhou 221004, China
E-mail: weirenpan@126.com

Received: April 16, 2024; Revised: May 15, 2024; Accepted: May 26, 2024

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

The detailed knowledge of the morphological structure, drainage pathways and patterns, the first tier lymph node of the cardiac lymphatic and its relationship with the circulatory system has not yet been completed. Although, the cardiac lymphatics had been described with renewed interest in past years, which was attributed to the transparent nature of lymphatic vessels that are difficult to be observed. In this study, cardiac lymphatics of the goat heart were perfused by a direct microinjecting technique with a radiopaque mixture. This demonstrated the subepicardial and subendocardial lymph capillary networks communicating with transmyocardial lymph vessels and then entering to subepicardial collecting lymph vessels that were directed toward the atrio-ventricular sulcus where they form a confluence from which the main cardiac lymph channels. We also found that: 1) the quantity and caliber of collecting lymph vessels varied in each goat heart; 2) drainage patterns of lymph vessels in the goat heart were different in individuals; 3) the first tier lymph node that each major lymph vessel drained to was different; and 4) multiple lymphatic-venous anastomosis sites have been confirmed to exist in the subepicardium of the left and right ventricles of each goat heart, which may be the morphological structure to accelerate the return of intercellular fluid to the venous system during excessive exercise of the heart. Therefore, the information may provide reference for further study in physiological and pathological conditions of the human heart.

Keywords: Goats, Heart, Lymphatic vessel, Lymph nodes, Lymphovenous anastomosis

As a part of the cardiac vasculature, the heart lymphatic system plays an important role in collecting and returning the excess interstitial fluid to the circulation system under both the physiological and pathological processes [1-4]. The cardiac lymphatics was first described by Rudbeck [5] in 1653. Since then, many studies had been carried out by using the mercury perfusion, the dye or ink injections, or application of hydrogen peroxide and so on [6-8]. On the basis of previous studies, the outline of the cardiac lymphatic system had been drawn. But the detailed knowledge of the morphological structure, drainage pathways and patterns, the first tier lymph node of the cardiac lymphatic and its relationship with the circulatory system has not yet been completed although the cardiac lymphatics had been described with renewed interest in past years [1, 3, 4, 9]. Therefore, it would be helpful to review the detailed lymphatic system of the heart if using the direct microinjection technique with a radiopaque mixture [10] and immunofluorescence (IF) staining examination.

The information from this study may provide reference for further study in physiological and pathological conditions of the human heart.

A total of 26 fresh goat hearts attached with the aorta, pulmonary artery, superior and inferior vena cava were obtained from a local abattoir. Twenty-four of them were used for the direct lymphatic injection, two for histological and immunohistochemistry (IHC)/IF studies. After sufficient flushing with normal saline, at room temperature, a small amount of 6% hydrogen peroxide (Zhonglian Chemical Co., Ltd.) was injected into the subendocardium, subepicardium and myocardium at multiple points from the apex to the base of the heart (the technique of the direct lymphatic injection has been used and described previously) [10-14]. Under a surgical microscope (Leica Microsystems Ltd.), the distended lymph vessels were found in these areas. A fine glass needle (Zhejiang KDL Medical Devices Co.) or 30-gauge needle (PrecisionGlide Needle; Becton Dickinson & Co.), attached to an extension tube (B. Braun) and 1 ml syringe was holding on a micromanipular (MN-153, Narishige Scientific Instrument Laboratory), was inserted into each inflated lymphatic vessel and then passed through a pair of lymphatic valves to prevent leakage. Radio-opaque mixture (Barium Sulphate 15 g; Shanghai Silian Indusry Co., Ltd., Milk powder 5 g; Heinz Inc.; Concentrated poster color - dark green 3 g; Liaoyuan Arts & Stationery Ltd., Water 20 ml, mixed them thoroughly in a mortar) was slowly and gently pulsed into the lymphatic vessel via 1 ml syringe. Lymphatic vessels were traced, measured, photographed and radiographed (Digital X-ray Diagnostic System - Multix Select DR; Siemens Healthineers Corp.) to reveal their distribution. The above process was repeated in 24 goat hearts. In two hearts, the aorta was inserted by a 14# urinary catheter (14#; Jiangxi Fenglin Medical Equipment Co., Ltd.) and tied by a string. A red poster color mixture (6 g of concentrated poster color - red; Liaoyuan Arts & Stationery Ltd., 6 g of gelatin; Xuzhou Fengrui Natural Biotechnology Co., Ltd., mixed in 200 ml of 90°C normal saline, cooled down to 40°C for use) was filled prior to the lymphatic perfusion (Fig. 1A).

Figure 1. Subepicardium lymphatics of the goat heart. (A) The relationship between subepicardium lymphatics (green vessels) and coronary blood vessels (red vessels) of the goat heart. (B) Each group of subepicardium lymphatics on the radiograph was painted with different colors. (C) The caliber of the subepicardium lymphatic was measured by a micrometer (M) under the surgical microscopy.

Next, small tissues (1.5×2.5 mm) of the bilateral side of the atrial and ventricular walls were harvested from 2 fresh goat hearts and fixed immediately in 4% polyformaldehyde. After 24 hours, paraffin blocks were made, histological sections (5 µm) were cut and mounted on glass slices, hematoxylin and eosin stain were performed in a routine procedure. Slices were scanned by an automatic digital scanner (Pannoramic MIDI; 3Dhistech) for image analysis.

Thereafter, 5 µm sections were taken from the same paraffin blocks and mounted on glass slices for IHC examination. Slices were passed through xylol, alcohol solutions and distilled water. After heat-mediated antigen restoration with citrate buffer (pH 8) was performed, samples were blocked with 1% bovine serum albumin (BSA ST023-50g; Beyotime Biotechnology) for 30 minutes, and incubated with primary antibody solutions (LYVE-1 [PA1-16635] 1:400 dilution; Thermo Fisher Scientific) overnight at 4°C. Samples were then washed 3 times with phosphate-buffered saline (PBS) before being incubated with secondary antibodies (Goat Anti-Rabbit IgG H&L 5220-0336, 1:500 dilution; SeraCare KPL) at room temperature in the dark for 50 minutes. Subsequently, samples were counterstained with diaminobenzidine (C0424649335, Nanjing Chemical Reagent Co., Ltd.) and hematoxylin at room temperature after being washed 3 times with PBS. Finally, slices were mounted with antifade mounting medium (P0128; Beyotime Biotechnology) and were scanned by an automatic digital scanner for image analysis.

Radiographs were transferred to a computer (Dell Vostro 200; Dell Computer Inc.) for image processing using Photoshop software (Adobe Photoshop CS5 V12; Adobe Systems Software Co., Ltd.). Each group of lymphatic vessels on the radiograph was painted with different colors (Fig. 1B). All data including the caliber of vessels measured by a micrometer under the surgical microscopy (Figs. 1C, 2) were transferred to the computer for analyzing with Microsoft Excel software 2016 (Microsoft).

Figure 2. The quantity and caliber of the lymph vessel in the subepicardium of the goat heart. (A) RVSLV of the goat heart, (B) LVSLV of the goat heart, (C) LASLV of the goat heart, and (D) AILV and PILV of the goat heart. RVSLV, right ventricular subepicardium lymph vessel; LVSLV, left ventricular subepicardium lymph vessel; LASLV, left atrial subepicardium lymph vessel; AILV, anterior interventricular lymph vessel; PILV, posterior interventricular lymph vessel.

Abundant lymphatic capillaries, precolleting and collecting lymph vessels were found in the subendocardium, myocardium and subepicardium of the goat heart (Fig. 3). They drained to the precollecting lymph vessel and collecting lymph vessel, the latter travelled parallel with corresponding blood vessels and formed right coronary lymph vessel (RCLV), left coronary lymph vessel (LCLV), posterior coronary lymph vessel (PCLV) respectively and then entered into relative lymph nodes (Figs. 4, 5). The quantity and caliber of collecting lymph vessels varied in each case, listed in Fig. 2. Drainage patterns of vessels were different in individuals.

Figure 3. Distribution of the lymphatics in the goat heart. (A) Lymphatics in the subepicardium. (B) Lymphatic capillaries in the subepicardium. (C) Lymphatic capillaries in the subendocardium. Red arrowheads indicate sites that branches of lymphatic capillaries enter the myocardium. (D) The lymphatic vessel in the myocardium. Red arrows indicate the direction of the lymph flow. AA, arch aorta; T, trachea; BABLN, brachiocephalic arterial bifurcation lymph node; PTLN, para tracheal lymph node; RTBLN, right tracheal bronchial lymph node; BA, brachiocephalic artery.

Figure 4. The relationship between subepicardial lymphatics and major cardiovascular vessels of the goat heart after lymphatics were injected by a radio-opaque mixture and major cardiovascular vessels were filled by a red poster color mixture. (A) Anterior view. (B) Posterior view. (C) Right side view. (D) Left side view. AA, arch aorta.

Figure 5. The lymphatic drainage of the goat heart. (A) A radiograph of the goat heart after subepicardial lymphatics were injected by a radio-opaque mixture. (B) A photograph of the goat heart after subepicardial lymphatics were injected by a radio-opaque mixture. BABLN, brachiocephalic arterial bifurcation lymph node; RTBLN, right tracheal bronchial lymph node; LTBLN, left tracheal bronchial lymph node; PCLV, posterior coronary lymph vessel; RCLV, right coronary lymph vessel; LCLV, left coronary lymph vessel; CLV, circumflex lymph vessel; RVSLV, right ventricular subepicardium lymph vessel; LVSLV, left ventricular subepicardium lymph vessel; AILV, anterior interventricular lymph vessel; PILV, posterior interventricular lymph vessel; LASLV, left atrial subepicardium lymph vessel; PTLN, para tracheal lymph node; T, trachea; AA, arch aorta; BA, brachiocephalic artery.

In the subepicardium

Presenting mesh-like fashion, lymph capillaries were relatively small and sparse beneath the atrial epicardium, large and dense under the ventricular epicardium of the goat heart. They drained to precollecting lymph vessels and then merged to collecting lymph vessels presenting tree-root-like fashion (Figs. 3A, B, 4) that travelled towards the cardiac base. Two to three lymphatic-venous anastomosis sites were found in the subepicardium of left and right ventricles of each specimen (Fig. 6). Occasionally, the lymphatic ampulla was also seen (Fig. 6).

Figure 6. Subepicardial lymphatics of the goat heart. (A) Lymphatics in the subepicardium. A lymphatic ampulla is seen LA. Black arrows indicate preclecting lymph vessels; red arrows indicate the direction of the lymph flow. (B) The lymphatic-venous anastomosis site is seen in the subepicardium (indicating by a blue arrow). LC, lymphatic capillaries; LA, lymphatic ampulla; V, venule.

Five major groups of the collecting lymph vessel were found in the subepicardium of the goat heart.

Right ventricular subepicardium lymph vessel

An average of 6 collecting lymph vessels (ranging from 5 to 9) were found in the right ventricular subepicardium layer of each heart (Fig. 2). They travelled beneath the epicardium towards the cardiac base, meandering their way and merging with each other. Vessels travelled within/nearby the right coronary sulcus and formed 1 or 2 RCLV, and then continued their journey to drain into the brachiocephalic arterial bifurcation lymph node (in 1 case, Fig. 3A) along the surface of the ascending aorta and brachiocephalic artery, right tracheal bronchial lymph node (RTBLN; in 9 cases, 7 of them by traveling under the pulmonary artery and the arch aorta and the remaining 2 through the gap between the pulmonary artery and the arch aorta), left tracheal bronchial lymph node (LTBLN; in 2 cases through the gap between the pulmonary artery and the arch aorta) or LCLV directly (in 12 cases) (Figs. 35). Lymph vessels next to the anterior and posterior interventricular grooves drained into anterior interventricular lymph vessel (AILV) and posterior interventricular lymph vessel (PILV) (Figs. 4, 5, 7).

Figure 7. Posterior view of subepicardial lymphatics of the goat heart. (A) PILV drains to CLV. (B) PILV drains to LTBLN via PCLV. (C) PILV drains to PTBLN (behind the adipose tissue) via PCLV. PILV, posterior interventricular lymph vessel; CLV, circumflex lymph vessel; LTBLN, left tracheal bronchial lymph node; PCLV, posterior coronary lymph vessel; PTBLN, posterior tracheal bifurcation lymph node.

Left ventricular subepicardium lymph vessel

An average of 7 collecting lymph vessels (ranging from 4 to 9) were found in the left ventricular subepicardium layer of each heart (Fig. 2). Vessels arising from the left anterior surface of the left ventricle of the heart travelled beneath the epicardium towards the cardiac base, meandering their way and merging with each other to form 1 or 2 LCLV that ran under the arch aorta and the pulmonary artery and drained into RTBLN (in 15 cases), LTBLN (in 6 cases) or the para tracheal lymph node (in 6 cases) (Figs. 5, 6). Vessels arising from the dorsal surfaces of the left ventricle of the heart merged into LCLV via circumflex lymph vessel (CLV) (Figs. 35). Lymph vessels next to the anterior and posterior interventricular grooves drained into AILV and PILV (Figs. 4, 5, 7).

Left atrial subepicardium lymph vessel

One to three collecting lymph vessels were found in the left artrial subepicardium layer of each heart (Fig. 2). They ran obliquely and upwardly towards the root of the aorta, and then merged into LCLV (Figs. 3A, 5A).

Right atrial subepicardium lymph vessel

Some lymph capillaries found around the sinoatrial node area, formed 1 or 2 very small and short precollecting lymph vessels and drained into the nearby right ventricular subepicardium lymph vessel (RVSLV).

Anterior interventricular lymph vessel

One collecting lymph vessel along with the anterior interventricular blood vessel was found in the anterior interventricular groove of each heart. They ran upwardly and merged into LCLV near the left coronal sulcus (Figs. 4, 5). Along the way, AILV received several RVSLV and left ventricular subepicardium lymph vessel (LVSLV) nearby.

Posterior interventricular lymph vessel

One collecting lymph vessel along with the posterior interventricular blood vessel was found in the posterior interventricular groove of each heart. They ran upwardly and drained into LTBLN (in 9 cases), posterior tracheal bifurcation lymph node (PTBLN; in 7 cases) via PCLV, or joined with CLV (in 8 cases) (Figs. 4, 5, 7). Along the way, PILV received several RVSLV and LVSLV nearby.

In the subendocardium

A mesh of avalvular lymphatic capillaries with different calibers existed beneath the endocardium in the goat heart (Fig. 3C) and drained into the precollecting and collecting vessels in the myocardium layer via the trench between the trabeculae carneae (Figs. 3D, 8).

Figure 8. Lymphatics in the myocardium of the goat heart. (A) L is accompanied by a V. The lymphatic valve is seen (green arrow). Hematoxylin and eosin stain, scale bar: 20 µm. (B, C) L are accompanied by Ar and V. Both Ar and V contained blood cells. L, lymphatic vessel; V, vein; Ar, arteries.

In the myocardium

Arising from the subendocardium, lymph vessels travelled intricately in the myocardium accompanied with blood vessels (Figs. 3D, 8). They merged to collecting lymph vessels in the subepicardium.

Based previous studies (Fig. 9) [1-4, 6-8], it was generally believed that there were subepicardial and subendocardial lymph capillary networks communicating with transmyocardial lymph vessels and then entering subepicardial collecting lymph vessels that directed toward the atrio-ventricular sulcus where they form a confluence from which the main cardiac lymph channels. In this study, these lymph vessels have not only been confirmed in the goat heart, but have also found some additional information:

Figure 9. The lymphatic distribution of the heart. Adapted from [Anatomie, physiologie, pathologie des vaisseaux lymphatiques, considerés chez l’homme et chez les vertébrés]. Adrien Delahaye; 1874. French. [6].

1. The quantity and caliber of collecting lymph vessels varied in each goat heart.

2. Drainage patterns of lymph vessels of the goat heart were different in individuals.

3. The first tier lymph node that each major lymph vessel drained to was different.

4. Multiple lymphatic-venous anastomosis sites have been confirmed to exist in the subepicardium of the left and right ventricles of each goat heart, which may be the morphological structure to accelerate the return of intercellular fluid to the venous system during excessive exercise of the heart.

Therefore, the above information will provide reference for further study in physiological and pathological conditions of the human heart.

Tissue engineering and 3D printing of the heart are still making progress [15, 16]. Doctors and scientists tried to use this technique to repair damaged or ineffective heart valves and myocardium, with the possibility to replace the entire heart. When conducting cardiac tissue engineering and 3D printing, the cardiac lymphatic vessels should also be taken into consideration to avoid cardiac lymphedema that may affect its function.

In conclusion, the detailed lymphatic anatomy of the goat heart has been presented. The result may provide basic knowledge of animal experiment for further scientific study and clinical applications.

Conceptualization: WRP, CXM. Data acquisition: CXM, YL, WRP. Data analysis or interpretation: WRP, CXM, ZAL, YL. Drafting of the manuscript: WRP. Critical revision of the manuscript: WRP, CXM, ZAL. Approval of the final version of the manuscript: all authors.

No potential conflict of interest relevant to this article was reported.

National Natural Science Foundation of China (No: 31671253); Xuzhou Medical University President special fund (No: 53051116); Xuzhou Medical University Foreign Experts Special Fund, Department of International Cooperation and Exchange, Xuzhou Medical University (No: 537101).

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