The Use of Lucifer Yellow to Investigate the Intercellular Coupling of Mouse Spiral Ligament Fibrocytes

Abstract

Gap junctions expressed in the spiral ligament are implicated in K+ cycling and metabolite homeostasis. Within the spiral ligament, the fibrocyte types (I-V) have differ in their expression of connexin isoforms. The functional intercellular linking between the fibrocyte types has not been studied extensively. In this project, we utilise the gap junction-permeant fluorescent tracer dye, Lucifer Yellow to visualise the gap junction connectivity of fibrocytes in the spiral ligament. Mice (C57/BL at Postnatal age 21 days) were used in a combination of in vitro and in vivo experiments. To determine the presence of the spiral ligament gap junction network, the scrape loading technique of dye uptake was used. The in vitro scrape loading experiments had the best results with 30 minutes exposure to 0.05% LY in the in vitro spiral ligament (n=5). The scrape-load-induced fluorescence indicated that the LY diffused greater along the spiral ligament rather than across. The diffusion of LY throughout the spiral ligament was sensitive to the gap junction blocker, carbenoxolone (200 μM, n=1). Cells were identified using DAPI (1.5 μg/ml) and WGA Alexa Fluor® 594 (5μg/L), which also showed that LY had significant nuclear and cytoplasmic localisation (n=2). The in vivo experiments were optimised at a higher concentration of 0.25% LY (n=3). With more site-specific scrape load injury to the regions expressing fibrocyte types, I-IV, it was shown that the type II region had the strongest uptake and diffusion along the spiral ligament (n=3). This was followed by the type IV and I fibrocyte regions, that both showed similar uptake and lack of diffusion patterns (n=3), which indicates reduced intercellular communication. The type III fibrocyte area did not illustrate any uptake or diffusion of LY (n=3). Overall, these results confirm the presence of gap junctions expressed in the spiral ligament that serves as part of an intercellular communication network among the fibrocytes, where by the largest amount of intercellular communication was shown in the type II fibrocyte region due to their widespread diffusion of Lucifer Yellow.