Age-related hearing loss (ARHL), a sensorineural hearing loss of multifactorial origin, increases its prevalence in aging societies. Besides hearing aids and cochlear implants, there is no FDA approved efficient pharmacotherapy to either cure or prevent ARHL. We hypothesized that selegiline, an antiparkinsonian drug, could be a promising candidate for the treatment due to its complex neuroprotective, antioxidant, antiapoptotic, and dopaminergic neurotransmission enhancing effects. We monitored by repeated Auditory Brainstem Response (ABR) measurements the effect of chronic per os selegiline administration on the hearing function in BALB/c and DBA/2J mice, which strains exhibit moderate and rapid progressive high frequency hearing loss, respectively. The treatments were started at 1 month of age and lasted until almost a year and 5 months of age, respectively. In BALB/c mice, 4 mg/kg selegiline significantly mitigated the progression of ARHL at higher frequencies. Used in a wide dose range (0.15–45 mg/kg), selegiline had no effect in DBA/2J mice. Our results suggest that selegiline can partially preserve the hearing in certain forms of ARHL by alleviating its development. It might also be otoprotective in other mammals or humans.

The administration of immune checkpoint inhibitors (ICIs) often leads to immune-related adverse events. However, their effect on auditory function is largely unexplored. Thorough preclinical studies have not been published yet, only sporadic cases and pharmacovigilance reports suggest their significance. Here we investigated the effect of anti-PD-1 antibody treatment (4 weeks, intraperitoneally, 200 μg/mouse, 3 times/week) on hearing function and cochlear morphology in C57BL/6J mice. ICI treatment did not influence the hearing thresholds in click or tone burst stimuli at 4–32 kHz frequencies measured by auditory brainstem response. The number and morphology of spiral ganglion neurons were unaltered in all cochlear turns. The apical-middle turns (<32 kHz) showed preservation of the inner and outer hair cells (OHCs), whilst ICI treatment mitigated the age-related loss of OHCs in the basal turn (>32 kHz). The number of Iba1-positive macrophages has also increased moderately in this high frequency region. We conclude that a 4-week long ICI treatment does not affect functional and morphological integrity of the inner ear in the most relevant hearing range (4–32 kHz; apical-middle turns), but a noticeable preservation of OHCs and an increase in macrophage activity appeared in the >32 kHz basal part of the cochlea.

Osteoclasts are multinucleated cells of hematopoietic origin which are critically involved in physiological and pathological bone resorption. They develop from myeloid progenitors through characteristic gene expression changes and intercellular fusion. This process is directed by M-CSF and RANKL which are also able to trigger osteoclast development from bone marrow cells in vitro. Osteoclasts are conventionally visualized by histochemical staining followed by manual counting, which hinders kinetic studies and automated quantification. Here we describe two fluorescence-based assays for the real-time analysis of myeloid cell to osteoclast development (FRAMCO) in primary mouse bone marrow cell cultures. Both assays rely on red-to-green fluorescence conversion of the membrane-targeted tdTomato/membrane-targeted eGFP (mTmG) transgene by Cre recombinase driven by the osteoclast-specific cathepsin K promoter (Ctsk-Cre). In the first assay (FRAMCO1.1), osteoclast-specific gene expression triggers red-to-green color conversion of cells carrying both the Ctsk-Cre and mTmG transgenes. In the second assay (FRAMCO1.2), red-to-green fluorescence conversion is triggered by fusion of neighboring co-cultured bone marrow cells separately carrying either the Ctsk-Cre or the mTmG transgenes. The two assays were tested using a high-content confocal fluorescence imaging system, followed by automated quantification. The FRAMCO1.1 assay showed robust red-to-green fluorescence conversion of more than 50% of the culture (including mononuclear cells) within 3 days under osteoclastogenic conditions. The FRAMCO1.2 assay showed a less robust but still readily measurable red-to-green color conversion in multinuclear cells within 5 days of differentiation. The assays required both the Ctsk-Cre and the mTmG transgenes and gave no signals in parallel macrophage cultures. The proper functioning of the two assays was also confirmed at the DNA, mRNA and bulk protein level. The assay systems were validated using lisophosphatidylcholine, a previously reported inhibitor of preosteoclast fusion. Taken together, our assays allow high-throughput automated real-time analysis of two critical aspects of osteoclast development, facilitating the screening for novel drug candidates for the pharmacological control of osteoclast-mediated bone resorption.

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