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  • br Material and methods br Results

    2022-07-04


    Material and methods
    Results Consistent with our previous study, SR-SIM and confocal microscopy showed a large number of IBA1-expressing lactone in all parts of the human cochleae (Fig. 1). IBA1, the microglial and macrophage-specific calcium-binding protein, is involved in the membrane ruffling and phagocytosis of the activated microglia/macrophage. Macrophages having different shapes resided densely in adult human cochleae, along the auditory nerve and in all other cochlear tissues (also refer to Liu et al., 2018). Here, among the molecular markers tested with immunohistochemistry (TMEM119, CD163, P2Y12, CD68, CD11b, MHCII), CD68, CD11b and MHCII were stably expressed in macrophages and they seemed to co-localize in all the macrophages but in different subcellular compartments. TMEM119 and P2Y12 were not seen in our experiments, indicating that the cells labeled with IBA1, MHCII and with other fore-mentioned markers in human cochleae were not microglia. The presence of CD4+ and CD8+ cells and the interactions between these two types of lymphocytes and macrophages in the human cochleae were observed using immunohistochemistry and super-resolution structured-illumination microscopy (SR-SIM) (Fig. 2). In human cochleae, the CD4+ and CD8+ cells were seen around vessels of the modiolus and along the border of the Rosenthal's canal. The T cells were seen in the medial wall of the scala tympani (Fig. 3) that is part of the modiolus between the Rosenthal's canal and the scala tympani. This space contains vessels. The spaces bordering the apical and basal side of the Rosenthal canal contain bigger blood vessels. While a large number of macrophages were seen in the stria vascularis, among the neurons in the Rosenthal's canal and occasionally in organ of Corti, CD4+ and CD8+ cells were not found in these areas. A few isolated CD4+ and CD8+ cells were seen in the spiral ligament and in the peripheral region of the Rosenthal's canal. In the modiolus of the cochleae, macrophages, CD4+ and CD8+ cells were often in clusters. Interaction between these different cells was observed with super-resolution structured illumination microscopy, showing closely located lymphocyte cell bodies to macrophages which often sent processes to reach and touch the CD4+ and CD8+ cells (Fig. 2). The number of macrophages exceeds any other type of the labeled immune cells, in the whole cochlea. The CD4+ and CD8+ cells in human cochleae were mostly separately scattered, and the majority appeared round-shaped except a few that looked flat lying against the outer vessel wall and expressing CD8 (Fig. 2D). These rare cells seemed to have just migrated out of the modiolar blood vessels.
    Discussion Macrophages are ubiquitously located in the human cochlea including the stria vascularis, organ of Corti, nerve bundles and spiral ganglion (O'Malley et al., 2016; Liu et al., 2018). They could independently accomplish innate immune functions such as phagocytosis of potentially harmful substances including foreign bodies, pathogens, as well as apoptotic cells. In addition they also repair and maintain the intactness of the neurons and hair cells, therefore they are named “multitaskers”. The homeostasis maintenance are going-on activities, not necessarily relying on lymphocytes or other adaptive immune contrivances. This may explain why the number of macrophages exceeds that of T lymphocytes anywhere in the human cochleae although we only compare macrophages with CD4 and CD8 cells. Within the stria vascularis, organ of Corti and spiral ganglion where the densely packed cells have crucial functions for hearing perception, macrophages but not CD4+ or CD8+ cells could be seen in these compartments of the human cochleae. T helper cells and cytotoxic T cells were seen in the modiolus, mostly around the blood vessels, including the space between the Rosenthal's canal and the medial wall of the scala tympani. A few CD4+ and CD8+ cells were scattered in the peripheral region of the Rosenthal's canal and very few in the spiral ligament.