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Multiplex immunohistochemistry reveals cochlear macrophage heterogeneity and local auditory nerve inflammation in cisplatin-induced hearing loss

Inner ear macrophages play a vital role in cochlear homeostasis. Recent studies have demonstrated the existence of macrophages at different sites of the cochlea, with increased cochlear infiltration as an inflammatory response mechanism to injury. However, current methods, such as conventional immun...

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Autores principales: Bedeir, Mai Mohamed, Ninoyu, Yuzuru, Nakamura, Takashi, Tsujikawa, Takahiro, Hirano, Shigeru
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9633043/
https://www.ncbi.nlm.nih.gov/pubmed/36341090
http://dx.doi.org/10.3389/fneur.2022.1015014
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author Bedeir, Mai Mohamed
Ninoyu, Yuzuru
Nakamura, Takashi
Tsujikawa, Takahiro
Hirano, Shigeru
author_facet Bedeir, Mai Mohamed
Ninoyu, Yuzuru
Nakamura, Takashi
Tsujikawa, Takahiro
Hirano, Shigeru
author_sort Bedeir, Mai Mohamed
collection PubMed
description Inner ear macrophages play a vital role in cochlear homeostasis. Recent studies have demonstrated the existence of macrophages at different sites of the cochlea, with increased cochlear infiltration as an inflammatory response mechanism to injury. However, current methods, such as conventional immunohistochemistry and flow cytometry, provide limited information about the diversity of cochlear macrophages. Recently, multiplex immunohistochemistry (mIHC) successfully identified the heterogeneity of immune cells in cancer tissue and thereby improved our understanding of the disease prognosis. In this study, we modified the mIHC technique for cochlear tissue and utilized it to investigate cochlear macrophage behavior and heterogeneity before and after exposure to ototoxic drugs such as cisplatin. Four-week-old C57BL/6N female mice were intraperitoneally injected with cisplatin at 5 mg/kg/day consecutively for 6 days. Their hearing levels were assessed before and after the injection. Their cochleae were harvested before (day 0) and on days 8 and 15 after the cisplatin injection. Paraffin-embedded sections were sequentially immunostained using macrophage surface markers to identify the different categories of macrophages. Each immunostaining cycle included incubation with primary antibody, incubation with secondary antibody, chromogenic staining, and image scanning. Thereafter, all antibodies were stripped out, and antigen retrieval was performed to prepare the tissue for the next cycle. The results revealed that activated cochlear macrophages were not entirely differentiated into M1 or M2 categories but into multi-marker M1/M2 mixed macrophages. Furthermore, the ratio of these mixed (M1/M2) macrophages to Iba1(+) macrophages increased in the auditory nerve after cisplatin exposure, suggesting local auditory nerve inflammation. The increase in the population of activated macrophages in the auditory nerve region was concomitant with the temporary shift of hearing threshold on day 8 post-cisplatin injection. The findings of this study indicate the effectiveness of mIHC in identifying cochlear macrophage heterogeneity both in the resting state and after cisplatin exposure. Therefore, mIHC could be a powerful tool in cochlear immunology research. Our findings may provide new insights into the co-relation between the cochlear macrophage and cisplatin exposure.
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spelling pubmed-96330432022-11-04 Multiplex immunohistochemistry reveals cochlear macrophage heterogeneity and local auditory nerve inflammation in cisplatin-induced hearing loss Bedeir, Mai Mohamed Ninoyu, Yuzuru Nakamura, Takashi Tsujikawa, Takahiro Hirano, Shigeru Front Neurol Neurology Inner ear macrophages play a vital role in cochlear homeostasis. Recent studies have demonstrated the existence of macrophages at different sites of the cochlea, with increased cochlear infiltration as an inflammatory response mechanism to injury. However, current methods, such as conventional immunohistochemistry and flow cytometry, provide limited information about the diversity of cochlear macrophages. Recently, multiplex immunohistochemistry (mIHC) successfully identified the heterogeneity of immune cells in cancer tissue and thereby improved our understanding of the disease prognosis. In this study, we modified the mIHC technique for cochlear tissue and utilized it to investigate cochlear macrophage behavior and heterogeneity before and after exposure to ototoxic drugs such as cisplatin. Four-week-old C57BL/6N female mice were intraperitoneally injected with cisplatin at 5 mg/kg/day consecutively for 6 days. Their hearing levels were assessed before and after the injection. Their cochleae were harvested before (day 0) and on days 8 and 15 after the cisplatin injection. Paraffin-embedded sections were sequentially immunostained using macrophage surface markers to identify the different categories of macrophages. Each immunostaining cycle included incubation with primary antibody, incubation with secondary antibody, chromogenic staining, and image scanning. Thereafter, all antibodies were stripped out, and antigen retrieval was performed to prepare the tissue for the next cycle. The results revealed that activated cochlear macrophages were not entirely differentiated into M1 or M2 categories but into multi-marker M1/M2 mixed macrophages. Furthermore, the ratio of these mixed (M1/M2) macrophages to Iba1(+) macrophages increased in the auditory nerve after cisplatin exposure, suggesting local auditory nerve inflammation. The increase in the population of activated macrophages in the auditory nerve region was concomitant with the temporary shift of hearing threshold on day 8 post-cisplatin injection. The findings of this study indicate the effectiveness of mIHC in identifying cochlear macrophage heterogeneity both in the resting state and after cisplatin exposure. Therefore, mIHC could be a powerful tool in cochlear immunology research. Our findings may provide new insights into the co-relation between the cochlear macrophage and cisplatin exposure. Frontiers Media S.A. 2022-10-20 /pmc/articles/PMC9633043/ /pubmed/36341090 http://dx.doi.org/10.3389/fneur.2022.1015014 Text en Copyright © 2022 Bedeir, Ninoyu, Nakamura, Tsujikawa and Hirano. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Neurology
Bedeir, Mai Mohamed
Ninoyu, Yuzuru
Nakamura, Takashi
Tsujikawa, Takahiro
Hirano, Shigeru
Multiplex immunohistochemistry reveals cochlear macrophage heterogeneity and local auditory nerve inflammation in cisplatin-induced hearing loss
title Multiplex immunohistochemistry reveals cochlear macrophage heterogeneity and local auditory nerve inflammation in cisplatin-induced hearing loss
title_full Multiplex immunohistochemistry reveals cochlear macrophage heterogeneity and local auditory nerve inflammation in cisplatin-induced hearing loss
title_fullStr Multiplex immunohistochemistry reveals cochlear macrophage heterogeneity and local auditory nerve inflammation in cisplatin-induced hearing loss
title_full_unstemmed Multiplex immunohistochemistry reveals cochlear macrophage heterogeneity and local auditory nerve inflammation in cisplatin-induced hearing loss
title_short Multiplex immunohistochemistry reveals cochlear macrophage heterogeneity and local auditory nerve inflammation in cisplatin-induced hearing loss
title_sort multiplex immunohistochemistry reveals cochlear macrophage heterogeneity and local auditory nerve inflammation in cisplatin-induced hearing loss
topic Neurology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9633043/
https://www.ncbi.nlm.nih.gov/pubmed/36341090
http://dx.doi.org/10.3389/fneur.2022.1015014
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