Background: Cisplatin, or cis-diamminedichloroplatinum (CDDP), is an inorganic member of the platinum-based chemotherapeutic family used to treat different types of malignant tumors. CDDP has been utilized as an important drug for decades, however, it has dose-related serious or irreversible side effects that have a great impact on the patient's quality of life. Previous literature suggested numerous mechanisms for CDDP-induced hearing loss; the proposed mechanisms included oxidative stress to the outer hair cells and DNA damage. However, the exact mechanism of CDDP-induced hearing loss is not clear and its impact on the cochlear immune cells has not been elucidated. Interestingly, recent studies have revealed the existence of macrophages within the cochlea, vestibular system, and the cochlea-vestibular nerve, suggesting that macrophages may be essential for inner ear homeostasis and are a potential target for protection or treatment of hearing loss. In order to understand the role of cochlear macrophages in hearing loss development clearly, we developed multiplex immunohistochemistry (mIHC) and image cytometry for cochlear samples. We investigated the cell population within the cochlea and characterized the various types of cochlear resident macrophages in a CDDP-induced hearing loss mouse model.
Materials and methods: Mice four weeks of age were injected with 5mg/kg/day of CDDP intraperitoneally for six consecutive days. Hearing levels were investigated using auditory brainstem response (ABR) at day 0 prior to CDDP exposure and on days 7 and 14 following CDDP exposure. Mice cochleae were collected at day 0 prior to CDDP exposure and on days 8 and 15 following CDDP exposure and fixed in formalin and paraffin sections before mIHC, which can stain 6 different markers within the same section and identify different macrophage subtypes. Finally, the sections are digitally scanned by nanozoomer digital scanner, and computed image cytometry was applied to these digitally scanned images to precisely interpret the chromogenic signals for the identification and quantification of macrophages.
Results: CDDP exposed mice developed an ABR threshold shift at day 8 post-CDDP which began to recover at day 15 post-CDDP. This threshold shift was associated with a decrease in the number of macrophages of monocyte origin (F4/80⁺). Additionally, there was an increase in the expression of both pro-and anti-inflammatory macrophage marker ratios in the auditory nerve and spiral ganglia areas on day 8 which also started to resolve on day 15, suggesting a new subcategory of mixed macrophages in the inner ear. Furthermore, the Iba1⁺ macrophages ratio was increased at day 8 post-CDDP, suggesting microglial activation in the auditory nerve. These findings propose that CDDP exposure causes a state of temporary neuronal inflammation in the auditory nerve and spiral ganglia. This inflammation triggers macrophages polarization towards a new subcategory of macrophages that express markers of M1, M2, and microglia in a single macrophage.
Conclusion: This is the first study that provides an in-depth analysis of macrophage diversity within the inner ear prior to and following CDDP exposure using mIHC. Inner ear macrophages do not exhibit conventional macrophages category markers and are a novel subtype of macrophages. An increased Iba1 expression ratio following CDDP exposure suggests that the auditory nerve and spiral ganglia undergo neuronal inflammation. Thus, inner ear macrophages play a significant role in the mechanism of hearing loss following CDDP exposure.