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[Background] The blood-labyrinth barrier (BLB) separates the inner ear from the circulation and is critical for maintaining ionic homeostasis and limiting the entry of deleterious agents. BLB integrity is disrupted by bacterial lipopolysaccharide (LPS), which elicits a strong inflammatory response in the inner ear leading to irreversible otic damage. Prolonged administration of systemic corticosteroids is the available treatment, but it shows both limited efficacy and major adverse effects. SPT-2101 is a novel in situ-forming gel formulation of dexamethasone allowing slow and sustained drug release after single intratympanic administration., [Methods] We used a rat model of LPS-induced injury to define the functional, cellular and molecular mechanisms associated to BLB dysfunction and the protection by SPT-2101. Hearing was assessed by auditory brainstem response (ABR) recording, BLB permeability by gadolinium dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and Evans blue extravasation. Gross cochlear histology and cellular alterations were studied by hematoxylin-eosin staining and immunofluorescence. RT-qPCR, PCR array and western blotting were used to assess transcriptional and protein changes., [Results] LPS-challenged rats showed BLB breakdown and altered permeability as shown by the progressive increase in cochlear gadolinium uptake and Evans blue incorporation. LPS administration increased the cochlear expression of the LPS toll-like receptors Tlr2 and co-receptor Cd14, pro-inflammatory cytokines and receptors such as Il1b and ll1r1, and also the oxidative stress and inflammasome mediators NRF2 and NLRP3. LPS also increased IBA1-positive macrophage infiltration in the lateral wall and spiral ganglion. A single intratympanic injection of SPT-2101 protected BLB integrity and prevented otic injury. Comparable effects were obtained by repeated administration of systemic dexamethasone, but not by a single dose. SPT-2101 administration normalized molecular inflammatory mediators and suppressed macrophage infiltration., [Conclusions] Our data indicate that single local administration of dexamethasone formulated as SPT-2101 protects BLB functional integrity during endotoxemia, providing a novel therapeutic opportunity to treat diseases related to BLB dysfunction.

Oxidative stress is a major triggering factor for noise-induced hearing loss. Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the rate-limiting step in the pentose-phosphate pathway, and is a key molecule in the cellular antioxidant response. G6PD overexpression in the transgenic mouse delays the onset of age-related hearing loss. However, its antioxidant potential against noise has not been explored yet. In this work, it has been shown that G6PD overexpression transiently protects against hearing damage caused by noise exposure, through activation of the antioxidant response mediated by NRF2-NQO1 and SOD

Cis-diammine-dichloroplatinum[II] (cisplatin) is a potent and widely used chemotherapeutic agent with significant efficacy against several forms of cancer in adults and children. Unfortunately, cisplatin has multiple adverse effects and causes irreversible hearing loss (HL) in a large proportion of patients, which severely impacts their quality of life.1 Here, we confirm that treatment with LPT99, a small molecule inhibitor of the apoptosome, preserves hearing levels in a rat model of cisplatin-induced HL and prevents apoptosis in cisplatin-exposed HEI-OC1 cells. These data are encouraging and support the potential of LPT99 for the prevention of the secondary effects of cisplatin for HL.

Stress-activated protein kinases (SAPK) are associated with sensorineural hearing loss (SNHL) of multiple etiologies. Their activity is tightly regulated by dual-specificity phosphatase 1 (DUSP1), whose loss of function leads to sustained SAPK activation. Dusp1 gene knockout in mice accelerates SNHL progression and triggers inflammation, redox imbalance and hair cell (HC) death. To better understand the link between inflammation and redox imbalance, we analyzed the cochlear transcriptome in Dusp1−/− mice. RNA sequencing analysis (GSE176114) indicated that Dusp1−/− cochleae can be defined by a distinct profile of key cellular expression programs, including genes of the inflammatory response and glutathione (GSH) metabolism. To dissociate the two components, we treated Dusp1−/− mice with N-acetylcysteine, and hearing was followed-up longitudinally by auditory brainstem response recordings. A combination of immunofluorescence, Western blotting, enzymatic activity, GSH levels measurements and RT-qPCR techniques were used. N-acetylcysteine treatment delayed the onset of SNHL and mitigated cochlear damage, with fewer TUNEL+ HC and lower numbers of spiral ganglion neurons with p-H2AX foci. N-acetylcysteine not only improved the redox balance in Dusp1−/− mice but also inhibited cytokine production and reduced macrophage recruitment. Our data point to a critical role for DUSP1 in controlling the cross-talk between oxidative stress and inflammation

Aging of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative damage in macromolecules, which contributes to cellular malfunction, compromises cell viability, and, ultimately, leads to functional decline. Cellular detoxification relies in part on the production of NADPH, which is an important cofactor for major cellular antioxidant systems. NADPH is produced principally by the housekeeping enzyme glucose‐6‐phosphate dehydrogenase (G6PD), which catalyzes the rate‐limiting step in the pentose phosphate pathway. We show here that G6PD transgenic mice (G6PD‐Tg), which show enhanced constitutive G6PD activity and NADPH production along life, have lower auditory thresholds than wild‐type mice during aging, together with preserved inner hair cell (IHC) and outer hair cell (OHC), OHC innervation, and a conserved number of synapses per IHC. Gene expression of antioxidant enzymes was higher in 3‐month‐old G6PD‐Tg mice than in wild‐type counterparts, whereas the levels of pro‐apoptotic proteins were lower. Consequently, nitration of proteins, mitochondrial damage, and TUNEL+ apoptotic cells were all lower in 9‐month‐old G6PD‐Tg than in wild‐type counterparts. Unexpectedly, G6PD overexpression triggered low‐grade inflammation that was effectively resolved in young mice, as shown by the absence of cochlear cellular damage and macrophage infiltration. Our results lead us to propose that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the production of ROS and cellular detoxification power along aging and thus prevents hearing loss progression.

Mitogen-activated protein kinases (MAPK) p38 and c-Jun N-terminal kinases (JNKs) are activated during the cellular response to stress signals. Their activity is regulated by the MAPK-phosphatase 1 (DUSP1), a key component of the anti-inflammatory response. Stress kinases are well-described elements of the response to otic injury and the otoprotective potential of JNK inhibitors is being tested in clinical trials. In contrast, there are no studies exploring the role of DUSP1 in hearing and hearing loss. Here we show that Dusp1 expression is age-regulated in the mouse cochlea. Dusp1 gene knock-out caused premature progressive hearing loss, as confirmed by auditory evoked responses in Dusp1-/- mice. Hearing loss correlated with cell death in hair cells, degeneration of spiral neurons and increased macrophage infiltration. Dusp1-/- mouse cochleae showed imbalanced redox status and deregulated expression of cytokines. These data suggest that DUSP1 is essential for cochlear homeostasis in the response to stress during ageing.

World Health Organization estimates that around 466 million people worldwide have disabling hearing loss (HL), and 34 million of these are children. The most common is sensorineural hearing loss (SNHL), a heterogeneous disorder which is produced mainly by the irreversible loss of sensory cells or neurons in the cochlea. Insulin like growth factor type 1 (IGF-1) is a neurotrophic factor key for the regulation of postnatal cochlear growth and differentiation. Human IGF-1 deficiency is a rare disease (ORPHA73272) associated with growth retardation, microcephaly and SNHL. The mouse model lacking the Igf1 gene reproduces the syndrome and presents dwarfism and SNHL. IGF-1 deficiency causes important cellular alterations in the mouse cochlea, such as the early apoptosis of auditory neurons and the deficit in myelination. Analysis of downstream signalling in the Igf1-/- cochlea has shown the activation of p38 MAPK pathway, involved in response to stress, whereas ERK1/2 and AKT pathways, which regulate proliferation and survival, are impaired. A transcriptomic study carried out in the Igf1-/- showed the altered expression of the cell cycle modulator Foxm1 and of the myocyte enhancer factor-2 (Mef2), a key factor for cellular differentiation, during inner ear development and early postnatal ages.IGF-1 haploinsufficiency has been also associated with growth retardation and HL in human genetic disorders such as Laron syndrome. In contrast, the Igf1+/- mouse does not show congenital HL, but adult mice hearing thresholds progressively increase with ageing and mice show increased susceptibility to noise insult. Adult Igf1+/- cochleae show unbalanced redox and inflammation biomarkers, as well as altered IGF-1 downstream signalling, which have been proposed as molecular mechanism underlying susceptibility. Mouse models of IGF-1 deficiency constitute a valuable tool to study the molecular bases of deafness and to identify potential targets to develop new HL therapies.

Ageing of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative-derived damage in macromolecules, which contribute to cellular malfunction, compromise cell viability and, finally, causes functional decline. The cellular detoxification power partially relies in the production of the cofactor for the activity of key antioxidant enzymes NADPH. This is mainly produced by the glucose-6-phosphate dehydrogenase (G6PD), an enzyme that catalyzes the rate-limiting step in the pentose phosphate pathway. We show here that the transgenic mouse hG6PD-Tg, which shows enhanced NADPH production along life, maintains lower auditory thresholds than wild type mice during ageing. G6PD overexpression preserves irreplaceable cochlear cell populations, thus hG6PD-Tg mice exhibit significantly higher number of inner and outer hair cells (OHC) and more widespread OHC innervation than wild type mice. Transcripts for antioxidant enzymes were significantly increased in 3-month old hG6PD-Tg, particularly remarkable those of glutathione reductase and thioredoxin reductase 1, whose activity depends on NADPH availability. Accordingly, tyrosine modification by nitration in proteins was reduced in 9-month old G6PD-Tg compared with wild type mice. As well as lesser TUNEL positive apoptotic cells were detected in whole mount preparations of the organ of Corti of hG6PD-Tg mice. Western blotting analysis of cochlear extracts further showed increased survival and reduced pro-apoptotic signaling in the transgenic versus the wild type mice. In conclusion, we propose that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the generation of ROS and the cell detoxification power along ageing and, therefore to prevent hearing loss progression.

Mitogen-activated protein kinases (MAPK) such as p38 and the c-Jun N-terminal kinases (JNKs) are activated during the cellular response to stress signals. Their activity is regulated by the MAPK-phosphatase 1 (DUSP1), a key component of the anti-inflammatory response. Stress kinases are well-described elements of the response to otic injury and the otoprotective potential of JNK inhibitors is being tested in clinical trials. By contrast, there are no studies exploring the role of DUSP1 in hearing and hearing loss. Here we show that Dusp1 expression is age-regulated in the mouse cochlea. Dusp1 gene knock-out caused premature progressive hearing loss, as confirmed by auditory evoked responses in Dusp1-/- mice. Hearing loss correlated with cell death in hair cells, degeneration of spiral neurons and increased macrophage infiltration. Dusp1-/- mouse cochleae showed imbalanced redox status and dysregulated expression of cytokines. These data suggest that DUSP1 is essential for cochlear homeostasis in the response to stress during ageing.

Betaine-homocysteine S-methyltransferases (BHMTs) are methionine cycle enzymes that remethylate homocysteine; hence, their malfunction leads to hyperhomocysteinemia. Epidemiologic and experimental studies have revealed a correlation between hyperhomocysteinemia and hearing loss. Here, we have studied the expression of methionine cycle genes in the mouse cochlea and the impact of knocking out the Bhmt gene in the auditory receptor. We evaluated age-related changes in mouse hearing by recording auditory brainstem responses before and following exposure to noise. Also, we measured cochlear cytoarchitecture, gene expression by RNA-arrays and quantitative RT-PCR, and metabolite levels in liver and plasma by HPLC. Our results indicate that there is an age-dependent strain-specific expression of methionine cycle genes in the mouse cochlea and a further regulation during the response to noise damage. Loss of Bhmt did not cause an evident impact in the hearing acuity of young mice, but it produced higher threshold shifts and poorer recovery following noise challenge. Hearing loss was associated with increased cochlear injury, outer hair cell loss, altered expression of cochlear methionine cycle genes, and hyperhomocysteinemia. Our results suggest that BHMT plays a central role in the homeostasis of cochlear methionine metabolism and that Bhmt2 up-regulation could carry out a compensatory role in cochlear protection against noise injury in the absence of BHMT.—Partearroyo, T., Murillo-Cuesta, S., Vallecillo, N., Bermúdez-Muñoz, J. M., Rodríguez-de la Rosa, L., Mandruzzato, G., Celaya, A. M., Zeisel, S. H., Pajares, M. A., Varela-Moreiras, G., Varela-Nieto, I. Betaine-homocysteine S-methyltransferase deficiency causes increased susceptibility to noise-induced hearing loss associated to plasma hyperhomocysteinemia.

Aminoglycoside antibiotics are used widely in medicine despite their ototoxic side-effects. Oxidative stress and inflammation are key mechanisms determining the extent and severity of the damage. Here we evaluate the protective effect of a treatment with resveratrol plus N-acetylcysteine on the ototoxic actions of kanamycin and furosemide in the rat. Resveratrol (10 mg/kg) and N-acetylcysteine (400 mg/kg) were administered together to Wistar rats on 5 consecutive days. The second day, a concentrated solution of kanamycin and furosemide was placed on the round window to induce ototoxicity. Hearing was assessed by recording auditory brainstem responses before and 5, 16 and 23 days after the beginning of the treatment. Cochlear samples were taken at day 5 (end of the treatment) and at day 23, and targeted PCR arrays or RT-qPCR were performed to analyze oxidative balance and inflammation related genes, respectively. In addition, the cytoarchitecture and the presence of apoptosis, oxidative stress and inflammation markers were evaluated in cochlear sections. Results indicate that administration of resveratrol plus N-acetylcysteine reduced the threshold shifts induced by ototoxic drugs at high frequencies (≈10 dB), although this protective effect fades after the cessation of the treatment. Gene expression analysis showed that the treatment modulated the expression of genes involved in the cellular oxidative (Gpx1, Sod1, Ccs and Noxa1) and inflammatory (Il1b, Il4, Mpo and Ncf) responses to injury. Thus, co-administration of resveratrol and NAC, routinely used individually in patients, could reduce the ototoxic secondary effects of aminoglycosides.

Age-related hearing loss, presbycusis, is the most frequently occurring sensory abnormality associated with aging. A steady growth of life expectancy will lead to a rapid increase in the number of older people with expressed presbycusis. Its etiology is multifactorial, comprising effects of noise exposure, ototoxic drugs, inflammation, and last but not least, genetic factors. Traditionally presbycusis was described as a complex of pathological changes appearing in the inner ear in the process of aging. At present, we understand presbycusis to be a complex of changes occurring in the whole auditory system. Long-term hearing deprivation can impact cognitive performance by decreasing the quality of communication, leading to a social isolation and depression [WHO, February 2017]. Age-related pathological vulnerability is present in the sensory hair cells and in their ribbon synapses that contact auditory nerve fibers, but also in auditory neurons. The activation of stress MAPK precedes cellular loss in different scenarios of cochlear insult and its pharmacological inhibition has proved to be otoprotective in animal models. The MAPK dual phosphatases are natural regulators of their activity but their role in hearing loss has not been studied. Here we show that Mkp1-/- mice suffered premature and progressive ARHL, showing higher ABR thresholds and wave 1 latencies than WT at all the ages studied. Histological analysis and immunohistochemistry indicated loss of hair cells, degeneration of afferent spiral neurons, loss of spiral ligament fibrocytes and increased macrophage infiltration. Comparative gene expression studies (RNA-Seq and RTqPCR) showed deregulation of GSH synthesis and cycle enzymes, altered balance of pro- and anti-inflammatory cytokines and of apoptosis mediators. In summary, we show here that MKP1 deficiency causes an exacerbated inflammatory response and accelerates progressive hearing loss.

Hearing loss is the most prevalent sensorial impairment of the elderly according to WHO. Age-related hearing loss (ARHL) is mainly caused by the death of irreplaceable cellular populations in the cochlea. ARHL is commonly associated with cognitive deficit, social isolation and depression. The onset and progression of the pathology rely on genetic factors that are not well characterized, and are often aggravated by environmental factors such as noise exposure or ototoxic agents.

[Background]: Aminoglycoside antibiotics are widely used in medicine but they show ototoxic side-effects. The generation of reactive oxygen species is a central element in ototoxicity, leading to oxidative stress, inflammation and ultimately activation of caspase-dependent apoptosis. Several antioxidants have been used independently in clinical trials against ototoxicity, with positive but limited effects. The combination of antioxidant drugs with complementary mechanisms of action is a novel approach that could provide stronger ROS scavenging potentiate the otoprotection and prevent the oxidation of the drugs themselves. [Objective]: Toevaluate the protective effect of a treatment with resveratrol plus N-acetylcysteine (NAC) on the ototoxic actions of kanamycin and furosemide in the rat. [Methods]: Resveratrol (10 mg/kg) and NAC (400 mg/kg) were administered together intraperitoneally to male 2 monthold Wistar rats on 5 consecutive days. The second day, a concentrated solution of kanamycin and furosemide was placed unilaterally on the round window by bullostomy to induce ototoxicity. Auditory brainstem responses were registered before and 5, 16 and 23 days after the beginning of the treatment. Cochlear samples were taken at day 5 and 23 to analyze oxidative balance and inflammation related genes by targeted PCR arrays or RT-qPCR, respectively. The cytoarchitecture and the presence of apoptosis, oxidative stress and inflammation markers were evaluated in cochlear sections. [Results and Conclusions]: Co-administration of resveratrol plus NAC reduced the threshold shifts induced by ototoxic drugs, although this protective effect fades after the cessation of the treatment. The treatment modulated the expression of genes involved in the cellular oxidative (Gpx1, Sod1, Ccs and Noxa1) and inflammatory (Il1b, Il4, Mpo and Ncf) responses to injury.

Age-related hearing loss (ARHL) is the most prevalent sensorial impairment of the elderly (WHO, February 2017). Its onset and progression rely on not well-characterized genetic factors, often aggravated by noxious factors such as noise and ototoxic agents. Stress kinases, p38 and JNK, activation precedes cellular loss and its pharmacological inhibition has proved to be otoprotective in animal models. The MAP kinase phosphatases (MKP) are natural regulators of the activity of stress kinases and central elements in the cellular response triggered by these enzymes but their role in hearing loss has not been studied. ABR and DPOAE hearing thresholds were measured, data analysis showed that Mkp1-/- mice suffered premature and progressive hearing loss. Higher ABR latencies of wave I indicated delayed transmission. Functional decline along life correlated with morphological and cellular cochlear alterations. Histological analysis and immunohistochemistry revealed loss of sensory cells in the organ of Corti, degeneration of afferent spiral neurons, loss of the spiral ligament fibrocytes and increased macrophages infiltration. Gene expression data (RNA-Seq and RT-qPCR) confirmed the altered transcriptome profile of null mice, concretely deregulation of GSH biosynthesis and antioxidant enzymes, unbalance of pro- and anti- inflammatory cytokines. In summary, we show here that MKP1 deficiency causes an exacerbated inflammatory response and accelerates progressive hearing loss.