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

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.