Macrophages in the Human Cochlea: Saviors or Predators-A Study Using Super-Resolution Immunohistochemistry

Abstract

The human inner ear, which is segregated by a blood/labyrinth barrier, contains resident macrophages [CD163, ionized calcium-binding adaptor molecule 1 (IBA1)-, and CD68-positive cells] within the connective tissue, neurons, and supporting cells. In the lateral wall of the cochlea, these cells frequently lie close to blood vessels as perivascular macrophages. Macrophages are also shown to be recruited from blood-borne monocytes to damaged and dying hair cells induced by noise, ototoxic drugs, aging, and diphtheria toxin-induced hair cell degeneration. Precise monitoring may be crucial to avoid self-targeting. Macrophage biology has recently shown that populations of resident tissue macrophages may be fundamentally different from circulating macrophages. We removed uniquely preserved human cochleae during surgery for treating petroclival meningioma compressing the brain stem, after ethical consent. Molecular and cellular characterization using immunofluorescence with antibodies against IBA1, TUJ1, CX3CL1, and type IV collagen, and super-resolution structured illumination microscopy (SR-SIM) were made together with transmission electron microscopy. The super-resolution microscopy disclosed remarkable phenotypic variants of IBA1 cells closely associated with the spiral ganglion cells. Monitoring cells adhered to neurons with "synapse-like" specializations and protrusions. Active macrophages migrated occasionally nearby damaged hair cells. Results suggest that the human auditory nerve is under the surveillance and possible neurotrophic stimulation of a well-developed resident macrophage system. It may be alleviated by the non-myelinated nerve soma partly explaining why, in contrary to most mammals, the human's auditory nerve is conserved following deafferentiation. It makes cochlear implantation possible, for the advantage of the profoundly deaf. The IBA1 cells may serve additional purposes such as immune modulation, waste disposal, and nerve regeneration. Their role in future stem cell-based therapy needs further exploration.

Keywords: cochlea; human; immunohistochemistry; ionized calcium-binding adaptor molecule 1; macrophages; structured illumination microscopy.

Figure 1

Human organ of corti (OC) contains migrating macrophages. (A–D) Horizontal serial sections (1 μm) of the apical turn of the OC dissected from a middle-aged female with normal hearing in both ears during surgery for petroclival meningioma (40). (B,F) Dendritic macrophage (Ma) with lysosomes can be seen in the corti tunnel near a degenerating inner hair cell (IHC). (E) Many IHCs (supernumerary) are present beneath the reticular lamina. (F) Framed area in panel (B) is shown in higher magnification in panel (F). (G) Framed area in panel (A) is viewed at higher magnification in panel (G) and displays an IHC undergoing degeneration. TCF, tunnel crossing fiber; OPC, outer pillar cell; IPS, inner pillar cell; ISC, inner sulcus cell; IPhC, inner phalangeal cell (1% osmium tetroxide and toluidine blue staining). The broken arrow displays top-down sectioning. (H) Immunofluorescence of an ionized calcium-binding adaptor molecule 1 (IBA1)-IR cell associated with an outer hair cell in the OC. The reticular lamina expresses fractalkine. (I) Hypothetical representation of macrophage disposal of dying hair cells in the human OC. Hair cells retract beneath the reticular lamina and are removed by activated macrophages.

Figure 2

Human stria vascularis contains a myriad of macrophages closely related to blood vessels. (A) Immunofluorescence of ionized calcium-binding adaptor molecule 1 (IBA1)-IR cells in the lateral wall of the apical turn of the human cochlea. Most cells are located in the stria vascularis (broken line), but there are also cells in the spiral ligament. (B–E) There are many perivascular cells. There is one intra-capillary IBA1 cell (B). Col. IV, collagen IV; Cap, capillary; E, endolymph; MC, marginal cells; BC, basal cell; Nu, nucleus.

Figure 3

Ionized calcium-binding adaptor molecule 1 (IBA1) cells in the stria vascularis are not intermediate cells (ICs). Confocal microscopy (A,B) and SR-SIM [(C,D) maximal intensity projections] images of IBA1-positive cells in the lateral wall of the human cochlea. Their long pseudopodia reach the wall of the strial capillaries and MCs. A terminal bulb can be seen at the capillary wall [arrow in panel (D)]. The IBA1 cells contain no melanin granules [arrows in panel (B)], suggesting they are not ICs. MC, marginal cells; BCs, basal cells; E, endolymph; Cap, capillary of the StV. Inset in panel (A) shows perivascular macrophages (IBA1, green) in the wall of the scala tympani.

Figure 4

Ionized calcium-binding adaptor molecule 1 (IBA1) macrophages are physically related to spiral ganglion cells. (A) Confocal microscopy of immunohistochemistry for collagen IV and IBA1 in the spiral ganglion of the basal turn of the human cochlea. (B) SR-SIM of an IBA1-immunoreactive cell that projects into the cytoplasm of an axon process. Framed area is shown in higher magnification in panel (C). Inset shows 3D video reconstruction shown in Video S1 in Supplementary Material. (D) Unprocessed SR-SIM showing TUJ1-positive type I spiral ganglion cell with associated IBA1 cell process (*) shown in panel (E). Macrophage cell nuclei typically expressed IBA1 protein. (E) SR-SIM (maximal intensity projection) of the synapse-like IBA1 process facing the surface of the type I ganglion cell. A thin cell process (arrow) can be seen projecting into the neuron. (F, G) Transmission electron microscopy of unidentified cell processes directly facing the type I ganglion cell. There are cell membrane specializations and signs of possible cell fusion.

Figure 5

Ionized calcium-binding adaptor molecule 1 (IBA1) macrophage interaction in the brain and ear. (A,C) Confocal microscopy of IBA1 and TUJ1 immunoreactivity in the guinea pig bran. IBA1 cells are seen both in the white and gray matter. (B) SR-SIM (maximum intensity projection) show IBA1 cells closely related to the surface of the TUJ1-positive neurons. Framed area is magnified in panel (D). (D) Higher magnification showing guinea pig brain neurons. IBA1-positive cells are seen in close association with the dendritic processes (arrow). The IBA1 cell nucleus express IBA1 protein (*). (E) SR-SIM of the human spiral ganglion showing IBA1-immunoreactive cell (*) physically related to the collagen IV basal lamina (BL) surrounding the satellite glial cell (SGC). (F) Transmission electron microscopy of human spiral ganglion shows cells believed to represent macrophages (*). They show slender processes (arrows) and contain typical electron-dense bodies (left inset). Cytoplasmic processes project against the outer surface of the SGCs (arrow in right inset). Type I, type I spiral ganglion cell soma; Ax, axon.

Figure 6

(A) Surveilling macrophages with projecting “antennae” exist around the human type I cells. (A) SR-SIM (maximum intensity projection) of an ionized calcium-binding adaptor molecule 1 (IBA1)-positive macrophage at the bone trabecula in Rosenthal canal of the first turn of the cochlea. Inset shows relationship to a TUJ1-positive neuron. (B) Higher magnification shows “horn”-like projections (arrow). Cytoplasmic vesicles as well as its nucleus contain IBA1 protein.

Figure 7

Ionized calcium-binding adaptor molecule 1 (IBA1) cells accumulate at habenula perforate and fractalkine is expressed in the spiral ganglion. (A) SR-SIM of IBA1 and myelin basic protein (MBP) expression at the habenula perforata of the human cochlea. A macrophage is shown around the unmyelinated fibers. It has a thin projection against the habenular opening (inset). (B) The tympanic covering layer also contains IBA1-positive cells firmly attached to the inferior surface of the basilar membrane (BM). (C) Confocal microscopy of numerous IBA1-positive cells around unmyelinated portion of the acoustic nerve bundle (arrow). There is no strong fractalkine expression in the organ of corti. (D) Inset in panel (D) shows a cell positive for the P2 Y12 receptor. SR-SIM (maximal intensity projection) at corresponding location showing IBA1 cells with thin projection into the habenular opening. (E) Confocal microscopy of IBA1 and fractalkine expression in the human spiral ganglion (basal turn). (F) SR-SIM (maximum intensity projection) of fractalkine expression with IBA1-positive macrophages surrounding the type I ganglion cell.

Figure 8

Ionized calcium-binding adaptor molecule 1 (IBA1) cells perforate basal lamina (BL) of Schwann cells. (A) SR-SIM (maximum intensity projection) of IBA1 macrophages (M) in a cross-sectioned nerve bundle in the spiral lamina (second turn, inset). Framed areas are shown in higher magnification in panels (B,C). Macrophages (M, green) send projections to adjacent axons (Ax). (B) An IBA1 cell process penetrates the BL of a Schwann cell (arrow). Inset shows 3D video reconstruction in Video S2 in Supplementary Material. Serial optical sections (B) show IBA1 cell process entering the peri-neuronal space (insets, arrows). (C) IBA1 cells send many projections to surrounding axons in the spiral lamina (arrows). Sch, Schwann cell.

Figure 9

Central auditory nerve axons are connected to ionized calcium-binding adaptor molecule 1 (IBA1)-positive processes. SR-SIM (single optical section) shows IBA1-positive macrophages among central axons in the cochlea (inset, framed area). The IBA1 protein is also expressed in the nucleus (inset right). These macrophages project thin branches that contact adjacent axons (short arrows, magnified in left and middle insets). Nu, nucleus.

Figure 10

Drawing showing possible macrophage interaction in the human cochlea. Macrophages [ionized calcium-binding adaptor molecule 1 (IBA1) cells] can be observed in the human cochlea, both in the spiral ganglion and more seldom in the organ of corti. Macrophages may interact to form a protective link between hair cells and neurons via a fractalkine/CX3CR1 signaling system as demonstrated experimentally by Kaur et al. (23) (illustration by Karin Lodin).