Lymphangiography and preliminary evaluation of N-butyl cyanoacrylate, Lipiodol, and ethanol mixtures; sodium tetradecyl sulphate with Lipiodol and air; and ethanol with Lipiodol in rats

Introduction

Lymphatic interventional radiology plays a crucial role, particularly in managing post-operative lymphatic leakage (1). In clinical practice, N-butyl 2-cyanoacrylate (NBCA) (Histoacryl; B. Braun, Melsungen, Germany) has been widely used as the only liquid embolic material for the lymphatic system. While NBCA is undoubtedly reliable, its primary drawback is the risk of catheter adhesion. Catheter insertion is not always required in certain procedures, such as pelvic lymphatic interventions and thoracic duct embolization via a puncture needle (2). However, catheterization is essential for other procedures, including thoracic duct embolization via transabdominal or transvenous approaches and catheterization into the lymphatic duct in the lower limbs for inguinal lymphorrhea (3). Although the risk of catheter adhesion, as seen in vascular interventions, should also be considered in lymphatic interventions, knowledge regarding effective alternative embolic materials remains limited. In vascular settings, NBCA combined with iodized oil (Lipiodol; Guerbet, Villepinte, France) and ethanol—referred to as NLE—was designed as a non-adhesive agent (4). Additionally, sclerosant agents such as sodium tetradecyl sulphate (STS) (Fibro-Vein 1%; S.T.D. Pharmaceutical Products, Hereford, UK), when combined with Lipiodol and air, or ethanol and Lipiodol (EL), have shown a lower risk of catheter adhesion. However, their efficacy in lymphatic interventions has not yet been explored.

To evaluate such alternatives to NBCA, a suitable animal model is essential. While lymphatic interventions have been conducted in mice (5), rats (6,7), rabbits (8,9), dogs (10) and swine (11), a universal animal model for lymphatic interventions has not yet been developed (8). While larger animals tend to offer easier access to the lymphatic system, they are also more costly and harder to handle than smaller animals. Rats are less expensive to house and maintain compared to rabbits, and they do not require microscopy during procedures, unlike mice, due to their size. Additionally, the lymphatic anatomies of rats, such as cisterna chyli, thoracic duct and confluence at the left venous angle, are very similar to those of humans (12,13). Therefore, the aim of this study was to develop a rat model for lymphatic intervention using two different lymphatic access methods, as well as to preliminarily assess the feasibility of using NLE, STS, and EL as alternative embolic agents to NBCA. We present this article in accordance with the ARRIVE reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-25-25/rc).

Methods

A total of 18 male rats, 12 Lewis and 6 Sprague-Dawley [body weight: 347 g (interquartile range: 323–428 g); Charles River Laboratories, Raleigh, North Carolina, USA], were included in this study. An acclimatisation period of at least 1 week was observed after arrival at the institution. Two different lymphatic approaches were performed: (I) percutaneous cisterna chyli/retroperitoneal lymphatic duct puncture, and (II) an exposed lymph node approach. The first approach was performed in 6 Lewis rats, while the second approach was conducted in 6 Lewis and 6 Sprague-Dawley rats. Under isoflurane anesthesia (1–2% with 1–2 mL/min O₂ supply), the rats were placed in the supine position on a standard fluoroscopy table for the procedure, with a heating pad to maintain a constant body temperature. A microscope was not used for either approach. Once lymphatic access was successfully established, embolization was performed using two NLE ratios (2:2:1 and 1:5:1), STS foam and EL to evaluate their effectiveness. The living rats were humanely euthanized using an intravenous Euthanasia Solution (VetOne) following the procedures. The study was approved by the institutional animal care and use committee of Oregon Health and Science University (No. IP00000417). Experiments were performed in compliance with the principles and guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. A protocol was prepared before the study without registration.

Percutaneous transabdominal puncture of the cisterna chyli/retroperitoneal lymphatic duct

A direct puncture to cisterna chyli or retroperitoneal lymphatic duct was performed at the 2nd–3rd lumbar level using a 25-G needle (3/4 in.) under fluoroscopic guidance (OEC 9800; GE HealthCare, Milwaukee, Wisconsin, USA). Using forceps, the needle tip was advanced to the lumbar vertebrae and then retracted slightly while 50% diluted iodinated contrast media (Omnipaque 350; GE HealthCare) was injected. The injection was repeated while adjusting the needle tip location until the thoracic duct was visualized fluoroscopically.

Exposed lymph node puncture

Hair was clipped from the abdominal to the popliteal region for exposed lymph node puncture. First, 0.05 mL isosulfan blue (Lymphazurin 1%) was subcutaneously injected into the left or/and right rear foot pad using a 34-G needle. Next, the blue-stained popliteal lymph nodes were exposed through a small incision, and additional 0.05 mL dye was injected intranodally using the same needle. Finally, a 5.0-cm midline incision was made to expose the stained iliolumbar lymph node, and direct intranodal lymphangiography was performed using a 27-G needle (1/2–1 1/4 in.) using Lipiodol.

Assessment of NLE, STS and EL

The effectiveness of embolization was assessed using two NLE ratios: 2:2:1 (NLE221) and 1:5:1 (NLE151) for NBCA, Lipiodol and ethanol, according to previous reports (14,15). STS was prepared using a 3:2:3 ratio of air, STS and Lipiodol, which was modified to 3:2:1 in Sabri et al.’s report to allow better visualization under fluoroscopy (16). EL was prepared at a 2:1 ratio of ethanol to Lipiodol (EL21) in accordance with the report by Gao et al. (17). The embolic effect was evaluated by measuring the travel distance of the mixture along the lymphatic duct.

Statistical analysis

Statistical methods were not employed, as this was a preliminary feasibility study.

Results

Lymphangiography using the percutaneous transabdominal puncture of the cisterna chyli/retroperitoneal lymphatic duct, which was the first approach, was successfully performed in 4 out of 6 rats. The diameter of the thoracic duct was approximately equal to that of the access needle (0.5 mm) (Figure 1A), and the intestinal lymph trunk was visualized (Figure 1B). Embolization could not be performed with this access due to insufficient stability of the needle. In the second approach access, the popliteal and iliolumbar lymph node were visualized in all 12 and 11 rats, respectively (Figure 2A,2B). The size of the latter lymph node was approximately 5 mm. Lymphangiography using Lipiodol was performed in one rat (Figure 2C), with the Lipiodol ascending toward the confluence at the left venous angle. Direct embolization was achieved in 10 rats. Four embolic materials—two NLE ratios (NLE221 and NLE151), STS foam, and EL—were tested in 3, 2, 3, and 2 rats, respectively. All four materials were visually cleared under fluoroscopy (Figure 2D-2G). Lymphatic flow ceased in all cases, as demonstrated by the observation that the embolic materials did not advance upward for 5 minutes. The average travel distance the embolic material traveled along lymphatic channels was 1.2 cm for NLE221 (Figure 2D), 3.5 cm for NLE151 (Figure 2E), 4.3 cm for STS (Figure 2F) and 4.0 cm for EL (Figure 2G).

Figure 1 Images of the percutaneous transabdominal puncture of the cisterna chyli using a 25-G needle. (A) Visualization of the thoracic duct. (B) The needle tip is within the cisterna chyli (arrow), which is dilated during the contrast media injection. The arrowhead indicates intestinal lymph trunk. CAU, caudal; CRA, cranial; L, left; R, right.

Figure 2 Images of exposed lymph node puncture. (A) A left popliteal lymph node is stained blue (arrow). (B) A blue-stained iliolumbar lymph node is visualized (arrow). The urinary bladder is marked with an asterisk (*). (C) In a case of lymphangiography using Lipiodol, it advances upward through the thoracic duct (arrow) and finally reaches the confluence at the left venous angle. (D) In a case where the ratio of N-butyl 2-cyanoacrylate combined with Lipiodol and ethanol (NLE) is 2:2:1, a 0.8-mL mixture was injected via the iliolumbar lymph node (arrowhead) and traveled 2.5 vertebral segments (arrow), equivalent to 2.5 cm. (E) In a case of NLE151, a 1.2-mL mixture was injected via the iliolumbar lymph node (arrowhead) and traveled 4.5 cm (arrow). (F) In a case using sodium tetradecyl sulphate, a 2.0-mL mixture was injected via the iliolumbar lymph node (arrowhead) and traveled 5.0 cm (arrow). (G) In a case of ethanol and Lipiodol, a 0.6 mL mixture was injected via the iliolumbar lymph node (arrowhead) and traveled 3.0 cm (arrow). CAU, caudal; CRA, cranial; L, left; R, right.

Discussion

Two different lymphatic approaches were successfully achieved; however, the direct intranodal approach was more technically feasible for subsequent embolization. Additionally, all materials were effective for embolization.

NBCA is commonly used as the liquid embolic material in the lymphatic system. While NBCA is reliable, there remains concerns with catheter adhesion (18) and non-target embolization to the pulmonary artery (18,19), and the left internal jugular vein (18). However, alternative agents to NBCA in lymphatic interventions have not been thoroughly investigated. NLE has primarily been promoted to reduce catheter adhesion in vascular interventions (4). Its efficacy in the lymphatic system has not been previously reported. Although STS (18,20,21) and ethanol (22,23) have been applied in the lymphatic interventions, reports remain limited. Conversely, Onyx is not recommended due to the porosity of its cast (24). Thus, there is a crucial need to explore optimal embolic agents for lymphatic interventions; however, the lack of a suitable animal model has been a major limitation.

The first approach—percutaneous transabdominal puncture of the cisterna chyli/retroperitoneal lymphatic duct—mirrors the human procedure; however, its application in rats has not been previously reported. In the second approach, Kreel described lymphangiography from the iliolumbar lymph node via the popliteal lymph node (6), although procedural details, such as whether it was percutaneous or involved a small incision, and methods for detecting the popliteal lymph node were not described. Sugawara et al. (5) and Matsumoto et al. (8) demonstrated lymphangiography in mice and rabbits, respectively, by visualizing the popliteal lymph node after subcutaneous dye injection into the rear foot. Based on these reports, we developed the second approach. Lymphangiography was successfully achieved with both approaches. However, maintaining the tip of the needle inside the cisterna chyli/retroperitoneal lymphatic duct was more challenging than inside the iliolumbar lymph node due to its small size.

Lymphatic flow ceased in all cases following the injection of NLE, STS, and EL. This is the first report comparing the efficacy of these agents in the lymphatic system. While all materials have the potential to embolize lymphatic vessels, NLE221 exhibits the strongest embolic power, while the other agents showed comparable effectiveness in terms of travel distance. These findings offer novel insights into the relative embolic properties of these agents in the lymphatic system. This may help guide the selection of embolic materials in future preclinical lymphatic interventions. NLE forms a paste- or noodle-like polymerized structure, which reduces the likelihood of catheter adhesion (25). Various NLE ratios have been reported to impact embolic efficacy. The stability of the mixture and distal embolization depend on the concentration of NBCA and ethanol. High ethanol and low NBCA concentration decrease both adhesion ability and mixture stability (26). In clinical procedures, Fujitsuna et al. used NLE141 for type 2 endoleak (25), and Koizumi et al. used NLE221 for congenital anastomoses between bronchial and pulmonary arteries (14). The choice of ratio depended on the procedures. Furthermore, Hama et al. reported embolization of wide-neck aneurysms models in swine using NLE221, NLE361, NLE271 and NLE151 (15). Mixtures with less 30% NBCA concentration require balloon assistance to prevent distal migration. Thus, we tested different two ratios above and below a 30% NBCA concentration. The NBCA concentration in the NLE should be adjusted according to the distance between the injection site and the leakage point. However, attention must be paid to two important factors: (I) the risk of protein coagulation necrosis caused by high-concentration ethanol, and (II) the need for a Lipiodol concentration over 40% for clear visualization under fluoroscopy (26). Regarding STS, Crawford et al. (18) used a combination of STS and Lipiodol at a ratio of 2:1 for thoracic duct embolization. They expressed concerns about the risk of air embolism when using STS foam. Conversely, Srinivasa et al. used STS foam with Lipiodol and air (21), and both reported its effectiveness. STS foam commonly prepared at a ratio of 3:2:1 for air, STS and Lipiodol (16). In this study, to achieve clear fluoroscopic visualization, the Lipiodol concentration was increased to approximately 40%, as noted in the Nakai report (26). The optimal ethanol-to-Lipiodol ratios has not been established. Gao et al. evaluated several ratios—EL13, 12, 11, 21, 31 and 41—in a rabbit hepatoma model (17). Among them, EL12, 11, 21, 31, and EL41 were equally effective, and EL21 demonstrated a better long-term therapeutic effect, as indicated by higher apoptosis indices. EL21 may induce sufficient endothelial injury similar to vascular embolization.

This study has several limitations. As a pilot study, there were limited numbers of rats, and only male rats were used. Additionally, the rat stains were not uniform. Furthermore, we did not assess histological changes to the lymphatic ducts after NLE, STS and EL embolization.

Conclusions

We describe two different lymphatic access techniques for lymphatic interventions in rats. While both techniques were successful, the direct intranodal approach using the popliteal and iliolumbar lymph nodes was technically more feasible for subsequent embolization. Further research using this model may optimize the development of ideal agents for lymphatic embolization.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-25-25/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-25/dss

Peer Review File: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-25/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-25-25/coif). K.Y. receives consulting fees from Kaneka Medical America and GE HealthCare for consulting agreement to provide advice from a professional perspective. K.F. reports grants from Guerbet, LLC, W.L. Gore, and AstraZeneca (paid to institution); receives consulting fees from Cook Medical, Baylis Medical, and Inquis Medical; receives personal payments from Neuwave Medical for Training/Education; reports the patent “Oregon Health and Science University; US 2021/0346180 A1; EP4228560A1”; participates in a Data Safety Monitoring Board or Advisory Board of Eisai and Genentech; serves as a board examiner of American Board of Radiology; serves on Consensus Guidelines Committee of American College of Radiology, Guidelines and Statements Division of Society of Interventional Radiology, and Multidisciplinary Consensus Panel of American Society of Digestive Disease Interventions; holds stock of Auxetics, Inc. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was approved by the institutional animal care and use committee of Oregon Health and Science University (No. IP00000417). Experiments were performed in compliance with the principles and guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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