Contribution of macrophages to intracochlear tissue remodeling responses following cochlear implantation and neural survival

Abstract

Introduction: Cochlear implants (CIs) restore hearing to deafened patients. The foreign body response (FBR) following cochlear implantation (post-CI) comprises an infiltration of macrophages, other immune and non-immune cells, and fibrosis into the scala tympani; a space that is normally devoid of cells. This FBR is associated with negative effects on CI outcomes including increased electrode impedances and loss of residual acoustic hearing. This study investigates the extent to which macrophage depletion by an orally administered CSF-1R specific kinase (c-FMS) inhibitor, PLX-5622, modulates the tissue response to CI and neural health.

Materials and methods: 10-12-week-old CX3CR1+/GFP Thy1+/YFP mice on C57Bl6 background with normal hearing were fed chow containing 1200 mg/kg PLX5622 or control chow for the duration of the study. 7-days after starting the diet, 3-channel cochlear implants were implanted ear via the round window. Serial impedance and neural response telemetry (NRT) measurements were acquired throughout the study. Electric stimulation began 7 days post-CI until 28- days post-CI for 5 hrs/day, 5 days/week, with programming guided by NRT and behavioral responses. Cochleae harvested at 10-, 28- or 56-days post-CI were cryosectioned and labeled with antibody against α-smooth muscle actin (α-SMA) to identify myofibroblasts and quantify the fibrotic response. Using IMARIS image analysis software, the outlines of scala tympani, Rosenthal canal, modiolus and lateral wall for each turn were traced manually to measure region volume. Density of nuclei, CX3CR1+ macrophages, Thy1+ spiral ganglion neuron (SGN) numbers and ratio of volume of α-SMA+ space/volume of scala tympani were calculated.

Results: Cochlear implantation in control diet subjects caused infiltration of cells, including macrophages, into the cochlea: this response was initially diffuse throughout the cochlea and later localized to the scala tympani of the basal turn by 56-days post-CI. Fibrosis was evident in the scala tympani adjacent to the electrode array. Mice fed PLX5622 chow showed reduced macrophage infiltration throughout the implanted cochleae across all timepoints. However, scala tympani fibrosis was not reduced relative to control diet subjects. Further, mice treated with PLX5622 showed increased electrode impedances compared to controls. Finally, treatment with PLX5622 decreased SGN survival in implanted and contralateral cochleae.

Discussion: The data suggest that macrophages play an important role in modulating the intracochlear tissue response following CI and neural survival.

Keywords: Cochlear implant; biomaterials; fibrosis; foreign body response; inflammation.

Figure 1

Depletion of Cochlear CX3CR1+ cells by oral administration of CSFR1 inhibitor, PLX5622. A. Study design for oral administration of CSFR1 inhibitor, PLX5622. Two groups of 10–12-week-old CX3CR1^+/GFP Thy1^+/YFP mice with C57B6 background were fed chow mixed with PLX5622 (PLX) or control chow (No PLX). At day 7, all mice were euthanized, labeled with Hoechst 3342 for histopathologic examination and confocal imaging. Confocal microscopy images showing CX3CR1-positive cells in (B) No-PLX and (C) PLX groups. D. Graphical representation of effect of 7 days of PLX-5622 treatment on cochlear resident CX3CR1-positive cells.

Figure 2

Cochlear implantation following elimination of cochlear macrophages. A. Study design for cochlear implantation in PLX5662 treated mice. Cochlear implantation was performed on 2 groups of CX3CR1^+/GFP Thy1^+/YFP mice: one group was fed on chow with 1200ppm of PLX-5622 (PLX) and the other group with control chow (No PLX) for 7 days. CI surgeries were performed on the left ear of both groups of mice after that (day 0). Following recovery from surgery, mice were continued with respective chow (PLX or No PLX) until they were sacrificed at 10-, 28- and 56-days post implantation. Starting on post-operative day 7, mice within stimulation cages, connected to the CI processor were stimulated for 5 h per day, 5 days a week. B-M: Fluorescent microscopic images of representative mid-modiolar, 30μm thick sections of the basal turn of the cochleae from respective groups. The following labels were used (Hoechst, blue), macrophages (CX3CR1-GFP, green) and neurons, (Thy1-YFP, yellow). In the presence of functional cochlear implant, macrophage (CX3CR1+ cells) infiltration into the cochlea appears to be sustained until the end of the study period (56 days post-CI). This was associated with infiltration of other cells labeled with nuclear labeling Hoechst.

Figure 3

Quantification of CX3CR1+ macrophage density in cochlea following cochlear implantation. Cochlear implantation was performed in CX3CR1^+/GFP Thy1^+/YFP mice, fed on chow with 1200ppm of PLX-5622 (PLX) or control chow (No PLX) for 7 days. Following surgery, mice were continued with respective chow (PLX or No PLX). Starting on post-operative day 7, mice within stimulation cages, connected to the CI processor were stimulated for 5 h per day, 5 days a week. Mice were euthanized at 10-, 28- and 56-days post-CI and imaged as in Figure 3. Image analysis was performed in IMARIS image analysis software. In 30μm thick midmodiolar sections, CX3CR1+ macrophage cells were counted on maximum intensity z-projections of 3D confocal image stacks. The outline of Rosenthal’s canal (RC) and lateral wall at base, middle and apex of cochlea, scala tympani of the base of the cochlea and modiolus were traced and volume of each area was measured. CX3CR1+ macrophages were counted using automated counting system in IMARIS image analysis software aided by custom made macros. The density of macrophages with visible, Hoechst+ nuclei was calculated per 10⁵ μm³ in each area mentioned. Values derived from every region of cochlea for an individual animal were averaged together from 3 sections; “n” is the total number of mice used in the study. Error bars indicate SEM.

Figure 4

Quantification of cellular density into scala tympani of the base of cochlea following cochlear implantation. Cochlear implantation was performed in CX3CR1^+/GFP Thy1^+/YFP mice, fed on chow with 1200ppm of PLX-5622 (PLX) or control chow (No PLX) for 7 days. Treatment with respective diets and electrical stimulation was continued until the desired endpoints (10, 28 or 56 days). Nuclei were labeled with Hoechst 3342 in 30μm thick mid-modiolar sections. Image analysis was performed in IMARIS image analysis software. Hoechst+ cells were counted on maximum intensity z-projections of 3D confocal image stacks in scala tympani of the base of the cochlea. Volume of scala tympani and nuclear counts were made using automated counting system in IMARIS image analysis software aided by custom made macros. Nuclear density (Hoechst+ cells) was calculated per 10⁵ μm³. An average of 3 sections per animal was taken with ‘n’ being the number of animals. Error bars indicate SEM.

Figure 5

Quantification of SMA+ tissue within scala tympani of the base of the cochlea following cochlear implantation. Following 7-day feeding on chow with 1200ppm of PLX-5622 (PLX) or control chow (No PLX), cochlear implantation was performed in CX3CR1^+/GFP Thy1^+/YFP mice, Respective diets were resumed following recovery from surgery. Electrical stimulation was continued until 28-day post-CI. Mice were euthanized at the desired endpoints (10, 28 or 56 days). Following euthanasia, 30μm midmodiolar sections were labeled with anti-alpha SMA antibodies. The volume of the scala tympani and SMA+ tissue volumes were measured using IMARIS image analysis software. Fibrosis was measured by dividing the volume of SMA+ tissue by volume of scala tympani, expressed in % volume. Error bars indicate SEM.

Figure 6

Electrode impedance following cochlear implantation plotted over time. Lines represent mean impedance values across all functional electrodes (without open circuit) at the representative timepoints for the No PLX (green line) and PLX groups (pink line). Error bars indicate SEM.

Figure 7

Quantification of spiral ganglion neuron density following cochlear implantation. 7-day feeding on chow with 1200ppm of PLX-5622 (PLX) or control chow (No PLX) was followed by cochlear implantation in CX3CR1^+/GFP Thy1^+/YFP mice. After recovery from surgical anesthesia, respective diets were resumed. Electrical stimulation was done as described before. Cochlea harvested at desired endpoints (10, 28 or 56 days) were sectioned at 30μm thickness. After measurement of spiral ganglia volume and quantification of spiral ganglion neurons, SGN density was calculated by dividing the SGN count by the volume and expressed as per 10⁵ μm³. Density in 3 sections per animal was averaged. Number of the animals is considered the ‘n’ for this experiment. Error bars indicate SEM.