# Validation of Indocyanine Green-Methylene Blue Dye in the Lymphedema Rat Tail Model

## TL;DR

Researchers tested a new imaging dye in a rat model of lymphedema to evaluate its effectiveness compared to an existing dye.

## Contribution

A newly synthesized ICG-MB dye was validated as a dual-mode imaging reagent in a rat tail lymphedema model.

## Key findings

- The ICG-MB reagent showed stable performance in lymphatic flow imaging.
- The rat tail model successfully induced lymphedema with measurable volume increases.
- Histological analysis confirmed lymphatic pathology consistent with lymphedema.

## Abstract

Background/Objectives: Lymphedema is characterized by edema; in severe cases, skin changes and ulceration significantly impair patients’ quality of life. Although several experimental rodent models for lymphedema have been established, a reproducible and practical model remains essential for evaluating new therapeutic and imaging agents. This study aimed to establish a lymphedema animal model and to evaluate the efficacy of a newly synthesized dual-mode imaging reagent as a potential alternative to indocyanine green (ICG). Methods: Eleven Sprague-Dawley rats were classified into two groups. Full-thickness skin excision was performed on the tails of nine rats to induce lymphedema; two rats served as controls. Five rats received ICG injections for 1 week postoperatively, while the remaining six rats were administered tail injections of chemically synthesized indocyanine green-methylene blue (ICG-MB) reagent. Lymphatic flow was photographed using a SPY camera. After euthanasia, tail segments were analyzed by microcomputed tomography (micro-CT) to measure volume and by hematoxylin–eosin staining for histological evaluation. Results: On postoperative day 7, lymphatic flow was confirmed in the ICG-MB group using the SPY Elite® fluorescence imaging system. On micro-CT scans, the preoperative rat tail volume was 3992.72 ± 144.80 mm3. Rat tail volume was 5216.71 ± 1131.88 and 4614.76 ± 468.29 mm3, respectively, at 1 and 2 weeks after lymphedema was induced. Histology revealed lymphocyte infiltration, inflammatory reaction, and thickened subcutaneous adipose tissue, with no significant difference between groups. Conclusions: The rat tail lymphedema model proved valuable for studying lymphedema pathology and diagnostic agents. The ICG-MB reagents demonstrate stable performance and favorable biocompatibility.