The distribution of perivascular-resident cells in blood-labyrinth barrier observed with two-photon fluorescence microscope and Imaris deconvolution

Abstract

Objective:To determine the cell types composing blood-labyrinth barrier in stira vascularis of cochlear lateral wall, analyze the distribution of these composing cells in blood-labyrinth barrier, and to investigate the relationship between perivascular-resident macrophages （PVMs）, endothelial cells and pericytes in blood-labyrinth barrier. Method:Cochlear lateral wall tissues were harvested from adult GFP-transgenic mice（C57BL/6）. Then the isolated whole stria vascularis tissue was scanned at 0.5 um intervals on the Z axis by two-photon confocal microscope and a 3D-structure of stria vascularis was reconstructed to observe the distribution of capillaries in blood-labyrinth barrier. Cochlear stria vascularis isolated from Balb/c mice was stained by mulit-immunofluorescence and then 3D real time deconvolution of stria vascularis was performed by Imaris software to investigate the distribution of PVMs and pericytes, and their contacting with basement membrane of capillaries was also observed. The ultrastructure of endothelial cells, pericytes and PVMs in blood-labyrinth barrier was observed using transmission electron microscope. Result:The vessels in stria vascularis are parallel to modiolus, distinct from that in spiral ligament which are perpendicular to modiolus. Numerous pericyts in stria vascularis are ensheathed by a vascular basement membrane shared with endothelial cells and closely attaching to the lateral wall of endothelial cells, while PVMs are located outside basement membrane of capillaries. Unlike pericytes that surround one capillary, PVMs branch to connect with more than one capillary. Conclusion:Serial layers on the Z axis scanned by two-photon confocal microscope and a 3D-structure reconstructed by Imaris 3D deconvolution helps to display the micro structure of capillaries in cochlear lateral wall clearly, which could be applied to analyze the 3D structure and function of blood-labyrinth barrier. PVMs in stria vascularis contact with more than one vessel through cytoplasmic processes, suggesting that PVMs may play a more significant role than pericytes in the integrity of blood-labyrinth barrier.