Our data suggest that PLX5622, a specific inhibitor of CSF1R, at a dose of 1200 mg/kg for 7 days eliminated almost all cochlear mononuclear phagocytes. Following cochlear implantation, a cellular infiltration, including macrophages, with fibrotic tissue deposition occurs adjacent to the electrode array in the basal scala tympani and was associated with increased electrode impedance. When cochlear implantation was performed in mice with ongoing PLX 5622 macrophage depletion, cellular infiltration (including macrophage infiltration) was inhibited but the volume of fibrotic response was not. Electrical impedance following cochlear implantation trended higher in the PLX5622-treated group. Moreover, PLX5622 treatment was associated with the degeneration of SGNs in the base of the cochlea independent of cochlear implantation.
With short-term (7 days) administration of PLX5622 at a dose of 1200 mg/kg for 7 days, most CX3CR1-positive cells can be depleted from the cochlea. This dosage is also sufficient to deplete the brain microglia population . It has been shown previously that CSF1R inhibition by PLX5622 is not microglia-specific; it can affect other mononuclear phagocyte populations (monocyte, macrophage, and dendritic cells) as well . Although there is preliminary evidence of a cochlear microglia population, their relative abundance compared to other mononuclear phagocytes like macrophages and dendritic cells has not been established . CX3CR1 is expressed in all types of mononuclear phagocytes [12, 22, 53]. Here we observed that most, but not all, cochlear CX3CR1 + cells were depleted with short (or even long-term) treatment with PLX5622, suggesting differing susceptibility among mononuclear phagocytes. The relative susceptibility of individual types of mononuclear phagocytes to CSF1R inhibition by PLX5622 is yet to be determined as are dose-specific effects. Our data also support the previously published literature showing that sustained treatment with PLX5622 in CX3CR1GFP/+ mice results in a significant elimination of resident macrophages (∼ 94%) without causing elevation of the ABR threshold. This study also suggests that CSF1R is expressed on the cochlear CX3CR1 + cells explaining the depletion of CX3CR1 + cells with CSF1R inhibitor PLX5622.
PLX5622 not only depleted resident CX3CR1 + cells before cochlear implantation, but it also caused sustained depletion of the infiltrating CX3CR1 + cells after placement of the electrode array. In this study, we have cautiously used the definition of ‘tissue-resident macrophages’ as a group of macrophages present in non-traumatized, uninflamed cochlear tissue from young mice. In our case, ‘cochlear tissue-resident macrophages’ represented by CX3CR1 + macrophages in a young mouse cochlea that has not been implanted. We determined whether PLX5622 can deplete ‘cochlear tissue-resident macrophages’ by treating unimplanted, CX3CR1+/GFP reporter mice with PLX5622 (PLX) and comparing them with age-matched, unimplanted CX3CR1+/GFP reporter mice.
On the other hand, we have defined ‘infiltrating/inflammatory macrophages’ as macrophages that infiltrate following a traumatic/inflammatory event (in our case, cochlear implantation). Infiltrating/inflammatory macrophages are thought to be derived from circulating monocytes. To determine whether cochlear implantation can deplete infiltrating macrophages, we first depleted ‘cochlear tissue-resident macrophages’ with 7 days of treatment of PLX5622. Then, we implanted the cochlea where ‘cochlear tissue-resident macrophages’ were depleted. After implantation in these ‘cochlear tissue-resident macrophage’ free cochlea, we continued to treat these mice with PLX5622. This treatment effectively tests whether PLX5622 can deplete the infiltration of CX3CR1 + macrophages following cochlear implantation.
To the best of our knowledge, this is the first study to explore the role of CSF1R inhibition in a cochlear implant model. Studies on brain implants demonstrated similar effects on the brain microglial population . They have shown that although PLX5622 treatment depletes microglia from the rat brain, astrocytes encapsulate the neuro-implant suggesting that microglia are redundant for this FBR in the brain. The reduction in cellular density in the scala tympani of PLX-treated mice following CI might be a direct effect of the elimination of resident and infiltrating macrophage population. Also, in the spiral ganglia, we observed degeneration of SGNs that can contribute to the decline in cellular density within the spiral ganglia. Moreover, macrophages also secrete growth and angiogenic factors .
Thus, the elimination of macrophages could indirectly reduce cellularity by decreasing cell proliferation and angiogenesis. Although macrophages are widely viewed as master regulators of the FBR to biomaterials, other innate and adaptive immune cells including T and B lymphocytes and mast cells contribute to these tissue responses . In the cochlea, the FBR to the implanted electrode array occurs in a unique environment in the scala tympani that is otherwise devoid of cells. While the role of these other immune cells has not been studied intensively in the cochlea, it has been documented that T and B lymphocytes infiltrate the cochlea following cochlear implantation . A wide range of cytokines can be secreted by activated macrophages; these include Interleukins (e.g., IL-1, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-18), TNF-α, and TGF-β . Many of these cytokines act as chemotactic factors for other immune cells. It is possible that depletion of resident and infiltrating macrophages by PLX5622 impacts recruitment of other immune cells and thus overall cellular infiltration of the scala tympani following CI.
One significant finding of these experiments is that the fibrotic response, as measured by anti-α-SMA immunolabeling, was not significantly reduced by PLX5622 treatment. These results mirror other studies that explored the role of CSF1R inhibition with PLX5622 on the FBR to neuro-implants in the brain. Sharon et al. showed that PLX5622 depletes microglia in rat brains , however, it does not inhibit the astrocyte response encapsulating the neural implant . These results with cochlear and neural implants are in sharp contrast with findings in non-neural tissue following the implantation of biomaterials. Doloff et al. demonstrated that following the implantation of biomaterials in non-neural tissue, the elimination of macrophages with CSF1R inhibitor effectively suppressed the fibrotic response. There are several plausible explanations for this difference between cochlear implantation and implantation in the peritoneal cavity performed by Doloff et al. including: 1. the likelihood that the pathophysiology of the FBR in neural tissue differs from that in non-neural tissue. Moreover, the peritoneal cavity provides a unique immunological niche that harbors specialized leukocyte populations supported by fat-associated lymphoid clusters (FALCs). On the other hand, among the neuronal tissues, the cochlea has a specialized immune environment where immune cells and non-immune, resident cells of cochlea, play immune functions . Therefore, the comparison between implantation in the cochlea and peritoneal cavity appears to be a comparison between two specialized immunological niches. 2. The implanted biomaterials were different in cases of implantation in non-neural (peritoneal) tissue and elicited different, material-specific types of FBR. 3. The pharmacological agents that were used to deplete macrophages are different from what has been used in the neural tissue and had a different impact on fibrotic response. 4. Finally, cochlear implants were electrically stimulated, whereas the tissues implanted in Doloff et al. were not electrically stimulated. We have recognized an important limitation of our study: we used only one relatively specific marker (α-SMA) for quantification of the fibrotic response. However, the sensitivity of α-SMA as a marker for cochlear fibrotic response is not known. In our study, the depletion of CX3CR1 + cells with PLX5622 resulted in no change in α-SMA + fibrotic response. However, there are other markers of fibrotic response. While both α-SMA and collagen type 1A have been used as markers for post-CI fibrosis by Bas et al., the relative sensitivity of α-SMA as a marker of post-CI fibrotic response has never been examined . This area needs to be further investigated. Using Col-EGFP/α-SMA-RFP dual reporter mice, Sun et al. showed that only a minority of collagen-producing cells co-express α-SMA in the fibrotic lung and kidney suggesting that α-SMA may not be a sensitive marker of fibrotic response in those organs. Therefore, our study does not necessarily exclude changes in other molecular markers of fibrosis following cochlear implantation.
Following CI, there is a gradual rise in electrode impedances consistent with an evolving tissue response. PLX 5622 treatment leads to a more rapid rise in electrical impedance compared to No PLX. As PLX5622 treatment reduces cellular infiltration into the cochlea, it appears that reducing cellular infiltration alone is not sufficient to prevent the rise in electrical impedance associated with the FBR. Further, the extent of fibrosis, as measured by anti-α-SMA immunolabeling, is not affected by PLX5622 treatment suggesting that the fibrotic tissue might be the factor maintaining the high electrical impedance in PLX5622 treated implanted cochlea. Moreover, electrode impedances in mice treated with PLX5622 rose more rapidly than the impedances in control mice raising the possibility that there are functional differences in the nature of the fibrotic response in the absence of macrophages. Post-implantation cochlear fibrosis is often accompanied by neo-ossification in humans and mice. The current study methods employed decalcification for histological preparation, prohibiting assessments of cochlear neo-ossification after implantation. This aspect is important for future studies, as CSF1R inhibition is associated with alterations in osteoclast activity that could impact post-CI neo-ossification and differentially affect electrode impedance compared to the less dense, non-mineralized fibrotic tissue .
Degeneration of SGNs in the base of the cochlea with PLX5622 treatment is noteworthy. The role of cochlear macrophages in the protection of SGNs depends on the model of cochlear insult. In a mouse model of selective hair cell destruction, fractalkine-mediated infiltration of CX3CR1 + mononuclear cells protect SGNs from degeneration . Conversely, anti-inflammatory therapy with ibuprofen or dexamethasone has been shown to suppress the infiltration of macrophages in the spiral ganglion following aminoglycoside-induced hair cell loss in a rat model; this suppression of macrophage infiltration is associated with SGN protection .
Macrophage infiltration can be associated with the protection or degeneration of SGNs depending on context and chemokine receptor expression. CX3CR1 receptor deletion (CX3CR1KO) induces a distinct phenotype when compared to the depletion of CX3CR1 expressing cells, as we demonstrate here with PLX5622. One potential explanation is that macrophages play diverse roles in SGN protection in deafening models: the fractalkine pathway is involved in SGN protection, whereas macrophages are involved in additional mechanisms that contribute to SGN degeneration. Therefore, selective inhibition of fractalkine is neurotoxic whereas non-selective inhibition of inflammation provides neuroprotection following deafening. In a noise-induced cochlear synaptopathy model, macrophages promote synapse regeneration .
In the present study, macrophage infiltration into the spiral ganglia following cochlear implantation itself does not appear to cause SGN degeneration. We observed SGN degeneration in the base of the cochlea following PLX5622 treatment independent of cochlear implant surgery. This observation suggests a general protective role of macrophages for SGNs. Bas et al. have shown that 7 days after cochlear implantation in a murine model, arginase 1 (Arg1) positive, M2 macrophages infiltrate into the cochlea, primarily into the spiral ganglion .
M2 macrophages are thought to engulf and digest dead cells, debris, and extracellular matrix components that promote activation of tissue-damaging M1 macrophages . M2 macrophages are also believed to secrete immunoregulatory cytokines (e.g., IL-10) and activate immunoregulatory T lymphocytes (Treg) . A potential explanation for SGN degeneration following the depletion of macrophages with PLX5622 is that it depletes the neuroprotective, anti-inflammatory M2 macrophages within the spiral ganglion.
However, there is an important confounder that makes the interpretation of our data more complex. Our experiments were done on mice with a C57BL/6J/B6 background, whereas other deafening and synaptopathy experiments were performed on CBA/J mice and rats. C57BL/6J/B6 mice exhibit early onset hearing loss and SGN loss that is not observed in CBA/J mice or rats . Importantly, the C57BL/6J/B6 background mimics hearing loss patterns seen in many human CI candidates with the post-lingual onset of high-frequency hearing loss . One plausible explanation for our results is that macrophages play a protective role for SGNs in C57BL/6J/B6 mice and in the absence of macrophages, early onset SGN degeneration is accelerated. Experiments inhibiting CSF1R with PLX5622 in mice with CBA/J backgrounds can provide additional insights into this issue.
We would like to mention a potential limitation of the method of SGN quantification that we used. We observed variation in Thy1-driven YFP expression among the SGN population. Therefore, the use of Thy1-reporter expression as a marker for SGN might present issues with reliability. Moreover, the sensitivity of Thy1-reporter as a marker for SGN is not currently known. These present findings are relevant to current efforts to develop pharmacologic-based therapies to mitigate the effects of CI insertion trauma, as the effect of dexamethasone eluting electrode arrays (NCT04750642, NCT04450290) on macrophage suppression and subsequent SGN preservation or degeneration is not yet clear. In a human study, post-CI inflammatory foreign body response has been shown to be associated with degeneration of SGNs . Data from guinea pig model of cochlear implantation suggested that a healthy SGN population is required for optimum neural response to electrical stimulation [36, 40]. Elucidating the impact of macrophages on post-CI SGN health is relevant for the development of more targeted strategies for selectively mitigating maladaptive aspects of the inflammatory response. As corticosteroids are nonspecific immunosuppressive agents, they might exert unwanted side effects and a more specific immunosuppressive agent might be more beneficial in this context.
In summary, our study suggests that macrophages (mononuclear phagocytes) play an important role in the intracochlear tissue remodeling that occurs following CI and in SGN health. Depletion of macrophages with PLX5622 reduces cellular infiltration into the scala media, but not fibrosis, following cochlear implantation. Moreover, macrophages appear to modulate the dynamics of fibrosis contributing to increases in electrode impedances. Depleting a specific subset of mononuclear phagocytes (e.g., dendritic cells), lymphocytes, or non-immune cells will provide valuable information about their role in the post-CI FBR and inform translational efforts to mitigate this response. The current study describes the unique role of macrophages in cellular infiltration, fibrosis, and SGN health following implantation. Further work is needed to understand the interplay of other immunologic cells following cochlear implantation that, along with macrophages, contribute to post-CI cochlear inflammation and FBR.