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![FIG URE 1 Both kanamycin/furosemide (KF) and noise exposure result in profound hearing loss and hair cell death. (a, c) All three genotypes have normal auditory brainstem response (ABR) thresholds prior to noise exposure or saline injection (black line). ABR thresholds are permanently elevated without any evident recovery after cochlear damage induced by either aminoglycoside injection (a) or noise over exposure (c). (") indicate that mice did not respond to any stimulus frequency. Data expressed as mean 6 SD. n 5 4-9 for each genotype for either injury model. Statistical significance computed using two-way analyses of variance (ANOVA) followed by Tukey's multiple comparisons test yielded highly significant (p < .0001) effect of days and frequency. Difference among genotypes prior noise exposure or saline injection and at several times post injury is not significant. (b, d) Representative confocal images of cochlear whole mounts immunolabeled for hair cells (Myosin VIIa) from CX 3 CR1 1/1 mice injected with KF (b) or exposed to loud noise (d) demonstrate extensive loss of both inner and outer hair cells in all the frequency regions of the cochlea. dB SPL 5 decibel sound pressure level; dpi 5 days post injection; DPNE 5 days post noise exposure; IHC 5 inner hair cell; kHz 5 kilohertz; OHC 5 outer hair cell. Scale 5 55 lm [Color figure can be viewed at wileyonlinelibrary. com]](profile/Tejbeer-Kaur-2/publication/321684118/figure/fig3/AS:669631610109975@1536664025426/FIG-URE-1-Both-kanamycin-furosemide-KF-and-noise-exposure-result-in-profound-hearing.png)
FIG URE 1 Both kanamycin/furosemide (KF) and noise exposure result in profound hearing loss and hair cell death. (a, c) All three genotypes have normal auditory brainstem response (ABR) thresholds prior to noise exposure or saline injection (black line). ABR thresholds are permanently elevated without any evident recovery after cochlear damage induced by either aminoglycoside injection (a) or noise over exposure (c). (") indicate that mice did not respond to any stimulus frequency. Data expressed as mean 6 SD. n 5 4-9 for each genotype for either injury model. Statistical significance computed using two-way analyses of variance (ANOVA) followed by Tukey's multiple comparisons test yielded highly significant (p < .0001) effect of days and frequency. Difference among genotypes prior noise exposure or saline injection and at several times post injury is not significant. (b, d) Representative confocal images of cochlear whole mounts immunolabeled for hair cells (Myosin VIIa) from CX 3 CR1 1/1 mice injected with KF (b) or exposed to loud noise (d) demonstrate extensive loss of both inner and outer hair cells in all the frequency regions of the cochlea. dB SPL 5 decibel sound pressure level; dpi 5 days post injection; DPNE 5 days post noise exposure; IHC 5 inner hair cell; kHz 5 kilohertz; OHC 5 outer hair cell. Scale 5 55 lm [Color figure can be viewed at wileyonlinelibrary. com]
Source publication
Cochlear hair cells are vulnerable to a variety of insults like acoustic trauma and ototoxic drugs. Such injury can also lead to degeneration of spiral ganglion neurons (SGNs), but this occurs over a period of months to years. Neuronal survival is necessary for the proper function of cochlear prosthetics, therefore it is of great interest to unders...
Contexts in source publication
Context 1
... in ABR thresholds were insignificant among the three genotypes at all days and frequencies tested after ototoxic-or noise-injury (p > .05). Based on Myosin 7a immunolabeling (from 4 to 6 temporal bones at each time point from CX 3 CR1 1/1 mice), both inner and outer hair cells (OHC) were intact in saline treated ( Figure 1b) or unexposed control mice (Figure 1d). A single KF treatment resulted in nearly-complete ablation of OHC along the entire length of the cochlea by 14 days post injection (dpi). ...Context 2
... in ABR thresholds were insignificant among the three genotypes at all days and frequencies tested after ototoxic-or noise-injury (p > .05). Based on Myosin 7a immunolabeling (from 4 to 6 temporal bones at each time point from CX 3 CR1 1/1 mice), both inner and outer hair cells (OHC) were intact in saline treated ( Figure 1b) or unexposed control mice (Figure 1d). A single KF treatment resulted in nearly-complete ablation of OHC along the entire length of the cochlea by 14 days post injection (dpi). ...Context 3
... single KF treatment resulted in nearly-complete ablation of OHC along the entire length of the cochlea by 14 days post injection (dpi). Inner hair cells (IHC) were completely missing in the base region of these cochleae and a partial loss of IHCs was detected in the middle and apical region of the cochlea at 60 days post KF injection (Figure 1b). Hair cells were intact at 1 day following 2 hr exposure to 8-16 kHz noise (120 dB) but, by 7 days post-noise exposure (DPNE), a nearly-complete loss of ...Similar publications
The loss of sensory hair cells in the cochlea is the major cause of sensorineural hearing loss, and inflammatory processes and immune factors in response to cochlear damage have been shown to induce hair cell apoptosis. The expression and function of Nfatc4 in the cochlea remains unclear. In this study, we investigated the expression of Nfatc4 in t...
Citations
... Inside the cochlea, the sensory epithelium includes inner and outer hair cells, along with various supporting cell types. Although macrophages are seldom observed within the intact cochlear sensory epithelium, they are present in adjacent tissues [57,58]. Hair cell death initiates diverse cellular removal processes by engaging both supporting cells and macrophages. ...
... This hair cell loss triggers the infiltration of immune cells, including macrophages and neutrophils, into the cochlea [57,[61][62][63][64]. Macrophages play a multi-pronged role in auditory pathology, particularly cochlear synaptopathy, and are important for maintaining auditory function. One significant aspect is their involvement in fractalkine signaling mediated by the ligand CX3CL1 and its receptor CX3CR1 [58,60,65]. This signaling pathway is pivotal for neuronal survival and communication with SGNs. ...
Inner ear macrophages, also known as cochlear macrophages, are immune cells that play a crucial role in maintaining the homeostasis and hearing function of the inner ear. They are responsible for responses to cochlear insults, such as noise exposure, ototoxic drugs, and surgical injuries. These cells have been shown to be present in the spiral ganglion, spiral ligament, and stria vascularis of the inner ear. As our understanding of inner ear macrophages continues to evolve, it is becoming evident that these cells are not just inert populations in the auditory system but are active participants in the complicated mechanics of inner ear homeostasis. Nevertheless, a comprehensive understanding of the roles and functions of macrophages within the auditory system is lacking. This review explores the presence, origin, and multifaceted roles of inner ear macrophages, elucidating their significance in maintaining auditory function, while also highlighting their potential inimical role in inner ear inflammation. The information collated herein has important implications for the development of therapeutic strategies aimed at preserving or restoring auditory function.
... The increase in the number of macrophages in the spiral ganglion is accompanied by macrophage activation. These events have been shown in the ganglion after aminoglycoside-induced deafening in rats (Rahman et al., 2023) and mice (Kaur et al., 2018) and after noise-induced trauma in mice (Hirose et al., 2005;Kaur et al., 2018). Macrophages are early responders to damage or infection and can be involved in limiting tissue damage. ...
... The increase in the number of macrophages in the spiral ganglion is accompanied by macrophage activation. These events have been shown in the ganglion after aminoglycoside-induced deafening in rats (Rahman et al., 2023) and mice (Kaur et al., 2018) and after noise-induced trauma in mice (Hirose et al., 2005;Kaur et al., 2018). Macrophages are early responders to damage or infection and can be involved in limiting tissue damage. ...
... Macrophages are early responders to damage or infection and can be involved in limiting tissue damage. Consistent with this, some studies have implied that macrophages can reduce neurodegeneration following cochlear trauma (Kaur et al., 2015(Kaur et al., , 2018. In contrast, we have previously shown that anti-inflammatory drugs reduce the extent of SGN death after deafening (Rahman et al., 2023), suggesting instead that macrophages contribute to SGN death. ...
Spiral ganglion neurons (SGNs) transmit auditory information from cochlear hair cells to the brain. SGNs are thus not only important for normal hearing, but also for effective functioning of cochlear implants, which stimulate SGNs when hair cells are missing. SGNs slowly degenerate following aminoglycoside-induced hair cell loss, a process thought to involve an immune response. However, the specific immune response pathways involved remain unknown. We used RNAseq to gain a deeper understanding immune-related and other transcriptomic changes that occur in the rat spiral ganglion after kanamycin-induced deafening. Among the immune and inflammatory genes that were selectively upregulated in deafened spiral ganglia, the complement cascade genes were prominent. We then assessed SGN survival, as well as immune cell numbers and activation, in the spiral ganglia of rats with a CRISPR-Cas9-mediated knockout of complement component 3 (C3). Similar to previous findings in our lab and other deafened rodent models, we observed an increase in macrophage number and increased expression of CD68, a marker of phagocytic activity and cell activation, in macrophages in the deafened ganglia. Moreover, we found an increase in MHCII expression on spiral ganglion macrophages and an increase in lymphocyte number in the deafened ganglia, suggestive of an adaptive immune response. However, C3 knockout did not affect SGN survival or increase in macrophage number/activation, implying that complement activation does not play a role in SGN death after deafening. Together, these data suggest that both innate and adaptive immune responses are activated in the deafened spiral ganglion, with the adaptive response directly contributing to cochlear neurodegeneration.
... The increase in the number of macrophages in the spiral ganglion is accompanied by macrophage activation. These events have been shown in the ganglion after aminoglycoside-induced deafening in rats (Rahman et al., 2023) and mice (Kaur et al., 2018) and after noise-induced trauma in mice (Hirose et al., 2005;Kaur et al., 2018). Macrophages are early responders to damage or infection and are typically associated with protection. ...
... The increase in the number of macrophages in the spiral ganglion is accompanied by macrophage activation. These events have been shown in the ganglion after aminoglycoside-induced deafening in rats (Rahman et al., 2023) and mice (Kaur et al., 2018) and after noise-induced trauma in mice (Hirose et al., 2005;Kaur et al., 2018). Macrophages are early responders to damage or infection and are typically associated with protection. ...
... Macrophages are early responders to damage or infection and are typically associated with protection. Consistent with this, some studies have shown macrophages playing a protective role in response to cochlear damage (Kaur et al., 2015;Kaur et al., 2018). In contrast, we have previously shown that anti-inflammatory drugs reduce the extent of SGN death (Rahman et al., 2023), suggesting a detrimental role for macrophages. ...
Spiral ganglion neurons (SGNs) transmit auditory information from cochlear hair cells to the brain. SGNs are thus not only important for normal hearing, but also for effective functioning of cochlear implants, which stimulate SGNs when hair cells are missing. SGNs slowly degenerate following aminoglycoside-induced hair cell loss, a process thought to involve an immune response. However, the specific immune response pathways involved remain unknown. We used RNAseq to gain a deeper understanding immune-related and other transcriptomic changes that occur in the rat spiral ganglion after kanamycin-induced deafening. Among the immune and inflammatory genes that were selectively upregulated in deafened spiral ganglia, the complement cascade genes were prominent. We then assessed SGN survival, as well as immune cell infiltration and activation, in the spiral ganglia of rats with a CRISPR-Cas9-mediated knockout of complement component 3 (C3). Similar to previous findings in our lab and other deafened rodent models, we observed infiltration of macrophages and increased expression of CD68, a marker of phagocytic activity and cell activation, in the deafened ganglia. Moreover, we found that the immune response also includes MHCII+ macrophages and CD45+ and lymphocytes, indicative of an adaptive response. However, C3 knockout did affect SGN survival or macrophage infiltration/activation, indicating that complement activation does not play a role in SGN death after deafening. Together, these data suggest that both innate and adaptive immune responses are activated in the deafened spiral ganglion, with the adaptive response directly contributing to cochlear neurodegeneration.
... Specifically, macrophages in the lateral wall and SGNs were activated, as revealed by the increased number of amoeboid cells with a pro-inflammatory and phagocytic phenotype. These findings agree with macrophages activation observed in different experimental models of cochlear damage, such as aminoglycoside ototoxicity, age-related hearing loss and noise exposure [38,[102][103][104][105][106]. Our western blot analyses also revelated high CD68 protein levels. ...
Background
Redox imbalance and inflammation have been proposed as the principal mechanisms of damage in the auditory system, resulting in functional alterations and hearing loss. Microglia and astrocytes play a crucial role in mediating oxidative/inflammatory injury in the central nervous system; however, the role of glial cells in the auditory damage is still elusive.
Objectives
Here we investigated glial-mediated responses to toxic injury in peripheral and central structures of the auditory pathway, i.e., the cochlea and the auditory cortex (ACx), in rats exposed to styrene, a volatile compound with well-known oto/neurotoxic properties.
Methods
Male adult Wistar rats were treated with styrene (400 mg/kg daily for 3 weeks, 5/days a week). Electrophysiological, morphological, immunofluorescence and molecular analyses were performed in both the cochlea and the ACx to evaluate the mechanisms underlying styrene-induced oto/neurotoxicity in the auditory system.
Results
We showed that the oto/neurotoxic insult induced by styrene increases oxidative stress in both cochlea and ACx. This was associated with macrophages and glial cell activation, increased expression of inflammatory markers (i.e., pro-inflammatory cytokines and chemokine receptors) and alterations in connexin (Cxs) and pannexin (Panx) expression, likely responsible for dysregulation of the microglia/astrocyte network. Specifically, we found downregulation of Cx26 and Cx30 in the cochlea, and high level of Cx43 and Panx1 in the ACx.
Conclusions
Collectively, our results provide novel evidence on the role of immune and glial cell activation in the oxidative/inflammatory damage induced by styrene in the auditory system at both peripheral and central levels, also involving alterations of gap junction networks. Our data suggest that targeting glial cells and connexin/pannexin expression might be useful to attenuate oxidative/inflammatory damage in the auditory system.
... Immune cells in the cochlea respond to noise-induced damage [56]. Rai et al. used flow cytometry, immunostaining, and existing scRNAseq data [57] to demonstrate that both innate and adaptive immune system cell types exist in the cochlea, especially B, T, NK, and bone marrow cells (macrophages and neutrophils) are the major immune cells [58]. ...
Hearing loss and deafness, as a worldwide disability disease, have been troubling human beings. However, the auditory organ of the inner ear is highly heterogeneous and has a very limited number of cells, which are largely uncharacterized in depth. Recently, with the development and utilization of single-cell RNA sequencing (scRNA-seq), researchers have been able to unveil the complex and sophisticated biological mechanisms of various types of cells in the auditory organ at the single-cell level and address the challenges of cellular heterogeneity that are not resolved through by conventional bulk RNA sequencing (bulk RNA-seq). Herein, we reviewed the application of scRNA-seq technology in auditory research, with the aim of providing a reference for the development of auditory organs, the pathogenesis of hearing loss, and regenerative therapy. Prospects about spatial transcriptomic scRNA-seq, single-cell based genome, and Live-seq technology will also be discussed.
... We employed a local injection of kanamycin through the unilateral round window membrane to make a deaf model in mice. Compared with the systemic injection, the local injection of kanamycin can rapidly induce hearing loss and inflammation which includes recruitment of macrophages 23 . Furthermore, this model can minimize the adverse effects of kanamycin including the renal toxicity, and reduce the mortality rate 24 . ...
... Regarding the underlying mechanisms, our results suggest a role of neuron-macrophage interaction in the setting of macrophage activation and neurodegeneration in the spiral ganglion after kanamycin administration, as previously reported 29 . Kaur et al., have identified critical roles of Fractalkine (CX 3 CL1)/CX 3 CR1 signaling between neuron and macrophage for the recruitment of macrophage and protection of SGNs from degeneration 23,30 . The discrepancy between their neuroprotective function and our neurotoxic function of macrophage after hair cell damage may suggest the existence of several signaling pathways which determine the protective/toxic fates of macrophage. ...
Pathological conditions in cochlea, such as ototoxicity, acoustic trauma, and age-related cochlear degeneration, induce cell death in the organ of Corti and degeneration of the spiral ganglion neurons (SGNs). Although macrophages play an essential role after cochlear injury, its role in the SGNs is limitedly understood. We analyzed the status of macrophage activation and neuronal damage in the spiral ganglion after kanamycin-induced unilateral hearing loss in mice. The number of ionized calcium-binding adapter molecule 1 (Iba1)-positive macrophages increased 3 days after unilateral kanamycin injection. Macrophages showed larger cell bodies, suggesting activation status. Interestingly, the number of activating transcription factor 3 (ATF3)-positive-neurons, an indicator of early neuronal damage, also increased at the same timing. In the later stages, the number of macrophages decreased, and the cell bodies became smaller, although the number of neuronal deaths increased. To understand their role in neuronal damage, macrophages were depleted via intraperitoneal injection of clodronate liposome 24 h after kanamycin injection. Macrophage depletion decreased the number of ATF3-positive neurons at day 3 and neuronal death at day 28 in the spiral ganglion following kanamycin injection. Our results suggest that suppression of inflammation by clodronate at early timing can protect spiral ganglion damage following cochlear insult.
... Currently, we do not understand the mechanisms by which cochlear resident and repopulated macrophages influence the recovery of ABR thresholds after noise trauma, but the data imply that macrophages may directly impact the functional properties of IHCs, synapses, and/or Type I SGNs that contribute to generation of ABRs without significantly impacting the function of OHCs. Previously, we did not observe restoration or exacerbation of cochlear function after profound sensorineural hearing loss where sensory hair cells and neurons are damaged and/or lost (Kaur et al., 2015(Kaur et al., , 2018. These data imply that macrophages may perform functional protective role in a context-dependent manner that is related to the degree of tissue damage and hearing loss. ...
Resident cochlear macrophages rapidly migrate into the inner hair cell synaptic region and directly contact the damaged synaptic connections after noise-induced synaptopathy. Eventually, such damaged synapses are spontaneously repaired, but the precise role of macrophages in synaptic degeneration and repair remains unknown. To address this, cochlear macrophages were eliminated using colony stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622. Sustained treatment with PLX5622 in CX 3 CR1 GFP/+ mice of both sexes led to robust elimination of resident macrophages (∼94%) without significant adverse effects on peripheral leukocytes, cochlear function, and structure. At 1-day after noise exposure of 93 or 90 dB SPL for 2 hours the degree of hearing loss and synapse loss were comparable in presence and absence of macrophages. At 30 days after exposure, damaged synapses appeared repaired in the presence of macrophages. However, in the absence of macrophages such synaptic repair was significantly reduced. Remarkably, upon cessation of PLX5622 treatment, macrophages repopulated the cochlea, leading to enhanced synaptic repair. Elevated auditory brainstem response (ABR) thresholds and reduced ABR peak 1 amplitudes showed limited recovery in the absence of macrophages, but recovered similarly with resident and repopulated macrophages. Cochlear neuron loss was augmented in the absence of macrophages, but showed preservation with resident and repopulated macrophages after noise exposure. While the central auditory effects of PLX5622 treatment and microglia depletion remains to be investigated, these data demonstrate that macrophages do not affect synaptic degeneration but are necessary and sufficient to restore cochlear synapses and function after noise-induced synaptopathy.
SIGNIFICANCE STATEMENT
The synaptic connections between cochlear inner hair cells and spiral ganglion neurons can be lost due to noise over exposure or biological aging. This loss may represent the most common causes of sensorineural hearing loss also known as hidden hearing loss. Synaptic loss results in degradation of auditory information leading to difficulty in listening in noisy environments and other auditory perceptual disorders. We demonstrate that resident macrophages of the cochlea are necessary and sufficient to restore synapses and function following synaptopathic noise exposure. Our work reveals a novel role for innate-immune cells such as macrophages in synaptic repair that could be harnessed to regenerate lost ribbon synapses in noise- or age-linked cochlear synaptopathy, hidden hearing loss and associated perceptual anomalies.
... Эти повреждения также могут привести к дегенерации спиральных ганглиозных нейронов, но это происходит в течение нескольких месяцев или лет. Как лечение аминогликозидами, так и чрезмерная акустическая стимуляция приводили к потере волосковых клеток [3]. Хотя потеря слуха больше не считается неизбежным вредом для пациентов с лекарственно-устойчивым туберкулезом (ЛУТ), ототоксичные ЛП продолжают использоваться для лечения ряда инфекционных и онкологических заболеваний по всему миру. ...
... As is known, macrophages can be classified as M1 and M2 subtypes, M1 macrophages produce pro-inf lammatory cytokines, whereas M2 macrophages produce anti-inf lammatory cytokines (37). Pro-inf lammatory mediators, such as IL-1β and IL-6, were demonstrated to play roles in the basilar membrane (38,39). However, no difference in mRNA levels of pro-inf lammatory cytokines Il6 and Il1b was observed in both Fam73a or Fam73b KO mice compared with the WT controls ( Fig. 5A−B). ...
Mitochondrial dynamics is essential for maintaining the physiological function of the mitochondrial network, and the disorders of it leads to a variety of diseases. Our previous study identified mitochondrial dynamics controlled anti-tumor immune responses and anxiety symptoms. However, how mitochondrial dynamics affects auditory function in the inner ear remains unclear. Here, we show that deficiency of FAM73a or FAM73b, two mitochondrial outer membrane proteins that mediate mitochondrial fusion, lead to outer hair cells damage and progressive hearing loss in FVB/N mice. Abnormal mitochondrial fusion causes elevated oxidative stress and apoptosis of hair cells in early stage. Thereafter, the activation of macrophages and CD4+ T cell is found in the mutant mice with the increased expression of the inflammatory cytokines IL-12 and IFN-γ compared to control mice. Strikingly, dramatically decreased number of macrophages by LCCA treatment alleviates the hearing loss of mutant mice. Collectively, our finding highlights that FAM73a or FAM73b deficiency affects hair cells survival by disturbing mitochondrial function, and the subsequent immune response in the cochleae worsens the damage of hair cells.
... HCs form synapses with the efferent axons responsible for modulating their response to external stimuli [58][59][60][61] . Macrophages are required for promoting/maintaining synapses between HCs and neurons in several models of cochlear HCs lesions [62][63][64] . To assess the role of IL-10 and/or IL-4 signaling in HC synapses formation/maintenance during HC regeneration, we generated il10ra (IL-10 signaling) and stat6 (downstream effector of IL-4 signaling 65 ) double heterozygous mutants. ...
Macrophages are essential for tissue repair and regeneration. Yet, the molecular programs, as well as the timing of their activation during and after tissue injury are poorly defined. Using a high spatio-temporal resolution single cell analysis of macrophages coupled with live imaging after sensory hair cell death in zebrafish, we find that the same population of macrophages transitions through a sequence of three major anti-inflammatory activation states. Macrophages first show a signature of glucocorticoid activation, then IL-10 signaling and finally the induction of oxidative phosphorylation by IL-4/Polyamine signaling. Importantly, loss-of-function of glucocorticoid and IL-10 signaling shows that each step of the sequence is independently activated. Lastly, we show that IL-10 and IL-4 signaling act synergistically to promote synaptogenesis between hair cells and efferent neurons during regeneration. Our results show that macrophages, in addition to a switch from M1 to M2, sequentially and independently transition though three anti-inflammatory pathways in vivo during tissue injury in a regenerating organ. Macrophages are important for tissue regeneration after injury. Here, using a single cell sequencing approach in a zebrafish injury model, the authors show that macrophages transition through a three stage process during tissue regeneration after hair cell damage.
