Clin Exp Otorhinolaryngol.  2021 May;14(2):179-184. 10.21053/ceo.2020.00395.

Three-Dimensional Distribution of Cochlear Macrophages in the Lateral Wall of Cleared Cochlea

• Bae SH
• Kwak SH
• Yoo JE
• Kim KM
• Hyun YM
• Choi JY
• Jung J

• KMID: 2515430
• DOI: http://doi.org/10.21053/ceo.2020.00395

Abstract

Objectives
. Resident macrophages are well known to be present in the cochlea, but the exact patterns thereof in spiral ligaments have not been discussed in previous studies. We sought to document the distribution of macrophages in intact cochleae using three-dimensional imaging.
Methods
. Cochleae were obtained from C-X3-C motif chemokine receptor 1^+/GFP mice, and organ clearing was performed. Three-dimensional images of cleared intact cochleae were reconstructed using two-photon microscopy. The locations of individual macrophages were investigated using 100-μm stacked images to reduce bias. Cochlear inflammation was then induced by lipopolysaccharide (LPS) inoculation into the middle ear through the tympanic membrane. Four days after inoculation, three-dimensional images were obtained.
Results
. Macrophages were scarce in areas adjacent to the stria vascularis, particularly the area just beneath it even though many have suspected macrophages to be abundant in this area. This finding remained consistent upon LPS-induced cochlear inflammation, despite a significant increase in the number of macrophages, compared to non-treated cochlea.
Conclusion
. Resident macrophages in spiral ligaments are scarce in areas adjacent to the stria vascularis.

Keyword

Macrophages; Cochlea; Spiral Ligament of Cochlea; Stria Vascularis

Figure

• 

  Fig. 1. Image of a cleared intact cochlea from a CX3CR1+/GFP mouse using two-photon microscopy. Green cells denote macrophages, blue areas denote the bony capsule of the cochlea, and white colored structures denote vessels. The internal structures of the cochlea are clearly distinguishable without any staining due to the endogenous fluorescence induced by the two-photon laser excitation. (A) Three-dimensional (3D) image of part of the cochlear middle turn. The dotted line marks the microstructures of the cochlea. The X-axis is parallel to the cochlear turn, the Y-axis is parallel to the spiral limbus, and the Z-axis is parallel to the modiolus. (B) XY-plane image obtained from the 3D image. Three layers marked by dotted square brackets. The stria vascularis (SV) shows brighter endogenous fluorescence than that of the spiral ligament. (C) YZ-plane image obtained from the 3D image. Roman numerals in the gradated area denote the area of each fibrocyte type [8]. OC, organ of corti.

• 

  Fig. 2. Image along the YZ-plane (100 µm extended) of the spiral ligament of the cochlea from a CX3CR1+/GFP mouse. Green cells denote resident macrophages. The dotted line represents the spiral ligament, and the dotted area marks the area with a lack of macrophages. (A) Middle turn of an untreated cochlea. (B) Basal turn of an untreated cochlea. (C) Middle turn of an inflamed cochlea. (D) Basal turn of an inflamed cochlea. Compared to the untreated cochlea, the inflamed cochlea shows a higher density of green cells. Newly localized macrophage/monocytes are seen in the basilar membrane and in the spiral limbus of the inflamed cochlea. Notably, the areas adjacent to the stria vascularis and beneath it show very few macrophages.

• 

  Fig. 3. Statistical analysis of macrophage counts in each sample revealed a significant increase in the number of macrophages after lipopolysaccharide (LPS) injection. At 4 and 10 days after LPS injection, significant increases in macrophages were noted in the type III fibrocyte area, compared to phosphate-buffered saline (PBS)-injected cochlea. PBS, sacrificed at 4 days after PBS injection (n=8); LPS 1, sacrificed at 1 day after LPS injection (n=4); LPS 4, sacrificed at 4 days after LPS injection (n=8); LPS 7, sacrificed at 7 days after LPS injection (n=4); LPS 10, sacrificed at 10 days after LPS injection (n=4). *Statistical significance, P<0.05.

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  Fig. 4. Schematic image of the macrophage distribution pattern in untreated spiral ligaments. Macrophages are colored black, and the dotted area indicates areas lacking macrophages.

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