Intratesticular and peritesticular methylene blue injection in visualizing lymphatic vessels and its impact on testicular injury in rat testes

Abstract

Objective: Methylene blue is a vital dye used for lymphatic mapping in laparoscopic varicocelectomy. However, some clinical studies have reported testicular calcifications following its use, raising concerns about its potential impact on testicular health. We aimed to experimentally investigate the pathological changes associated with the application of different concentrations of methylene blue through both intratesticular and peritesticular routes.

Materials and methods: This study examined methylene blue-induced testicular damage in 45 male Wistar albino rats. Rats were divided into seven groups and received intratesticular or peritesticular injections: control (G1), saline (G2), needle-only (G3), methylene blue 100% intratesticular (G4), methylene blue 50% intratesticular (G5), methylene blue 100% peritesticular (G6), and methylene blue 50% peritesticular (G7). Testicular tissues were histologically analyzed for structural changes, including edema, inflammation, calcification, hyalinization and spermatogenesis alterations, using hematoxylin-eosin staining and Johnsen scoring.

Results: Histopathological analysis showed significant testicular changes. Hyalinization was absent in G1-G3 but present in G4 (28.6%), G5 (35.7%), and G6 (35.7%) (p = 0.001). Calcification peaked in G4 (50%) and G5 (42.9%) (p = 0.004). Seminiferous tubule necrosis (ST necrosis) was highest in G4 (28.6%) and G5 (21.4%) (p = 0.027). Tubular atrophy was most severe in G5 (42.9%) (p = 0.011). Inflammation and edema were prominent in G4 (42.9%), G5 (35.7%), and G6 (100%) (p < 0.001). Two-way ANOVA indicated significant differences between testicular sides (p < 0.001). Peritesticular injection caused milder changes.

Conclusion: Intratesticular methylene blue injection, especially at high concentrations, caused significant testicular damage, including calcification, tubular atrophy, ST necrosis, and hyalinization.

Keywords: Methylene blue; Testis; Varicocelectomy; Vital dye.