The current investigation was designed to determine the functional status of intercellular junctions in the organ of Corti following acoustic trauma. We revealed a site-specific disruption of the cell-cell junctions in the outer zone of the reticular lamina of the cochlear sensory epithelium. We also documented the magnitude of the disruption and the spatial correlation between the junction disruption and sensory cell damage. Moreover, we demonstrated that the increase in permeability occurs in both the reticular lamina and the basilar membrane. This is the first study that uses macromolecular probes to investigate the functional integrity of intercellular junctions during cochlear pathogenesis following acoustic overstimulation. This study provides important information for the understanding of molecular changes in cell adhesion in response to acoustic injury.
In the current investigation, we loaded dextran-FITC probes into the perilymph space through either the scala tympani or the scala vestibuli. Compared to the application of chemical agents to the endolymph space, this approach has the merits of decreased inner ear disturbance and a greater homogenous distribution of the loaded probes within the cochlea. Moreover, it allows us to assess the barrier function of the basilar membrane. To ensure that the large molecular probes used in the current study were able to reach the endolymph space through the vestibular membrane, we examined the dextran-FITC staining in normal cochleae where the vestibular membrane is functionally intact. We found an intracellular accumulation of dextran-FITC fluorescence in sensory cells when the probes were administrated to the perilymph space. This observation is consistent with a previous finding that showed that the vestibular membrane is permeable to thorium dioxide [38
], a large molecular tracer that is of comparable size to the dextran-FITC molecules used in the current study. Together, these results indicate that the vestibular membrane is permeable to macromolecular tracers, possibly through micropinocytosis [38
The organ of Corti consists of diverse sensory and supporting cells, each forming contacts with neighboring cells. Here, we provide evidence that functional changes in intercellular junctions are site-specific following acoustic trauma. The outer zone shows a massive accumulation of tracer molecules and also displays a greater level of damage, allowing the entry of 500
kDa dextran-FITC molecules in some noise-exposed ears. This finding suggests that the outer zone is the vulnerable site for the functional disruption of cell-cell junctions in the cochlear sensory epithelium. Unlike the outer zone, accumulation of dextran-FITC fluorescence in the middle zone does not occur in the intercellular junctions, but rather in the Corti’s lymph spaces beneath the level of cell-cell junctions. This finding raises the question as to how the macromolecules reach the pericellular spaces. As demonstrated in the current study and in previous observations [15
], acoustic trauma causes the degeneration of sensory cells. The demise of these cells can lead to structural defects in the reticular lamina, causing leaks. The dextran-FITC molecules can also diffuse from the disrupted junctions between the Hensen cells in the outer zone. Although the current study does not provide further evidence to assess the contribution of these routes of probe passage, our results suggest that the cell-cell junctions in the middle zone are less susceptible to acoustic trauma than those in the outer zone.
We estimated the magnitude of intercellular junction interruption using graded sizes of dextran-molecules and found that the cutoff size to pass through the damaged junction spaces varied between individual cochleae with the maximal leak between 40
kDa and 2,000
kDa. Based on Stoke’s radius analysis, dextrans with these molecule weights have the dimensions of approximately 14.7 to 27
nm. Because the actual sizes of the dextran macromolecules can be affected by multiple factors, including the homogeneity level of the molecules, the size of added domains (FITC), and the concentration of the working solution, the current analysis of molecular sizes can only provide a gross estimate of the magnitude of junction dysfunction.
The organ of Corti has two boundaries: the reticular lamina and the basilar membrane. While the noise-induced damage to the reticular lamina has been documented previously [18
], changes in the functional integrity of the basilar membrane are largely unstudied. The basilar membrane consists of the basement membrane, filaments, homogeneous ground substance, and mesothelial cells. In the physiological condition, this structure is permeable to certain macromolecular particles [38
] and has a greater permeability to small molecule agents than does the reticular lamina [39
]. Here, we demonstrate that the basilar membrane is impermeable to dextran-FITC probes (40
kDa) and that acoustic overstimulation causes the permeabilization of the membrane. Because no intracellular accumulation of dextran-FITC fluorescence was observed within mesothelial cells, we suspect that the probes entered the organ of Corti through the mesothelial cell junctions, which are desmosome-like junctions [41
]. The biological impact of leaks in the basilar membrane on sensory cell viability is not known. Considering the similarity of the ionic compositions of Corti’s lymph and the perilymph [42
], we suspect that the impact of leaks in the basilar membrane is not as detrimental as the leaks in the reticular lamina that cause a mixture of endolymph and Corti’s lymph, a known factor for the induction of sensory cell degeneration [22
Exposure to intense noise causes sensory cell damage in the organ of Corti. Ample evidence indicates that cell damage is initiated immediately following noise exposure and continues to develop hours, days, or even weeks afterwards [44
]. Acute cell death features apoptotic phenotypes and can occur rapidly post-exposure [27
]. Given the rapidity of acute sensory cell death, cell junction interruption, which is a direct consequence of mechanical stress, has been considered to be a triggering event for acute cell death [27
]. The current finding that acute cell death is evident 30 minutes following noise exposure is consistent with the previous finding of rapid sensory cell death. We also found general agreement regarding the site of junction disruption and the site of sensory cell lesions. However, there is not a one-to-one spatial correlation between the two events. This observation suggests that the disruption of cell-cell junctions is a contributing, but not the sole, factor for the initiation of acute sensory cell death. This hypothesis is supported by a previous finding that acute apoptosis is associated with both intrinsic and extrinsic cell death pathways [50
]. Future studies aimed at investigating the role of adhesion function in this multifactorial causation will shed light on the process of sensory cell degeneration induced by acoustic overstimulation.