The ototoxic aminoglycoside antibiotics are essential to treat severe bacterial infections, particularly in neonatal intensive care units. Using a bacterial lipopolysaccharide (LPS) experimental model of sepsis, we tested whether LPS-mediated inflammation potentiates cochlear uptake of aminoglycosides and permanent hearing loss in mice. Using confocal microscopy and enzyme-linked immunosorbent assays, we found that low-dose LPS (endotoxemia) greatly increased cochlear concentrations of aminoglycosides and resulted in vasodilation of cochlear capillaries without inducing paracellular flux across the blood-labyrinth barrier (BLB), or elevating serum concentrations of the drug. Additionally, endotoxemia increased expression of both serum and cochlear inflammatory markers. These LPS-induced changes, classically mediated by Toll-like Receptor 4 (TLR4), were attenuated in TLR4-hyporesponsive mice. Multiday dosing with aminoglycosides during chronic endotoxemia induced greater hearing threshold shifts and sensory cell loss compared to mice without endotoxemia. Thus, endotoxemia-mediated inflammation enhanced aminoglycoside trafficking across the BLB, and potentiated aminoglycoside-induced ototoxicity. These data indicate that patients with severe infections are at greater risk of aminoglycoside-induced hearing loss than previously recognized.

Rationale Hearing loss in neonatal intensive care unit (NICU) graduates range from 2–15% compared to 0.3% in full-term births, and the etiology of this discrepancy remains unknown. The majority of NICU admissions receive potentially ototoxic aminoglycoside therapy, such as gentamicin, for presumed sepsis. Endotoxemia and inflammation are associated with increased cochlear uptake of aminoglycosides and potentiated ototoxicity in mice. We tested the hypothesis that sepsis or systemic inflammatory response syndrome (SIRS) and intravenous gentamicin exposure increases the risk of hearing loss in NICU admissions. Methods The Institutional Review Board at Oregon Health & Science University (OHSU) approved this study design. Two hundred and eight infants met initial criteria, and written, informed consent were obtained from parents or guardians of 103 subjects ultimately enrolled in this study. Prospective data from 91 of the enrolled subjects at OHSU Doernbecher Children’s Hospital Neonatal Care Center were processed. Distortion product otoacoustic emissions (DPOAEs; f2 frequency range: 2,063 to 10,031 Hz) were obtained prior to discharge to assess auditory performance. To pass the DPOAE screen, normal responses in >6 of 10 frequencies in both ears were required; otherwise the subject was considered a “referral” for a diagnostic hearing evaluation after discharge. Cumulative dosing data and diagnosis of neonatal sepsis or SIRS were obtained from OHSU’s electronic health record system, and the data processed to obtain risk ratios. Results Using these DPOAE screening criteria, 36 (39.5%) subjects would be referred. Seventy-four (81%) subjects had intravenous gentamicin exposure. Twenty (22%) had ≥4 days of gentamicin, and 71 (78%) had <4 days. The risk ratio (RR) of referral with ≥4 days of gentamicin was 1.92 (p=0.01). Eighteen subjects had sepsis or met neonatal SIRS criteria, 9 of whom had ≥5 days of gentamicin and a DPOAE referral risk ratio of 2.12 (p=0.02) compared to all other subjects. Combining subjects with either vancomycin or furosemide overlap with gentamicin treatment yielded an almost significant risk ratio (RR = 1.77, p=0.05) compared to the rest of the cohort. Conclusions We report an increased risk of referral with DPOAE screening for those receiving ≥4 days of intravenous gentamicin administration that may contribute to the greater prevalence of hearing loss in NICU graduates. We propose an expanded prospective study to gather a larger cohort of subjects, identifying those with sepsis or neonatal SIRS, to increase the statistical power of this study design. Subsequent studies also need to obtain follow-up diagnostic audiological data to verify whether the outcomes of DPOAE screening, in addition to the standard AABR screen, is a reliable predictor of permanent hearing loss following gentamicin exposure in the NICU.

Normal microvessel structure and function in the cochlea is essential for maintaining the ionic and metabolic homeostasis required for hearing function. Abnormal cochlear microcirculation has long been considered an etiologic factor in hearing disorders. A better understanding of cochlear blood flow (CoBF) will enable more effective amelioration of hearing disorders that result from aberrant blood flow. However, establishing the direct relationship between CoBF and other cellular events in the lateral wall and response to physio-pathological stress remains a challenge due to the lack of feasible interrogation methods and difficulty in accessing the inner ear. Here we report on new methods for studying the CoBF in a mouse model using a thin or open vessel-window in combination with fluorescence intra-vital microscopy (IVM). An open vessel-window enables investigation of vascular cell biology and blood flow permeability, including pericyte (PC) contractility, bone marrow cell migration, and endothelial barrier leakage, in wild type and fluorescent protein-labeled transgenic mouse models with high spatial and temporal resolution. Alternatively, the thin vessel-window method minimizes disruption of the homeostatic balance in the lateral wall and enables study CoBF under relatively intact physiological conditions. A thin vessel-window method can also be used for time-based studies of physiological and pathological processes. Although the small size of the mouse cochlea makes surgery difficult, the methods are sufficiently developed for studying the structural and functional changes in CoBF under normal and pathological conditions.

Hypothesis: Both toll-like receptor 4 (TLR4) and downstream neutrophil activity are required for endotoxemia-enhanced blood–labyrinth barrier (BLB) trafficking. Background: Aminoglycoside and cisplatin are valuable clinical therapies; however, these drugs often cause life-long hearing loss. Endotoxemia enhances the ototoxicity of aminoglycosides and cisplatin in a TLR4 dependent mechanism for which downstream proinflammatory signaling orchestrates effector immune cells including neutrophils. Neutrophil-mediated vascular injury (NMVI) can enhance molecular trafficking across endothelial barriers and may contribute to endotoxemia-enhanced drug-induced ototoxicity. Methods: Lipopolysaccharide (LPS) hypo-responsive TLR4-KO mice and congenitally neutropenic granulocyte colony-stimulating factor (GCSF) GCSF-KO mice were studied to investigate the relative contributions of TLR4 signaling and downstream neutrophil activity to endotoxemia-enhanced BLB trafficking. C57Bl/6 wild-type mice were used as a positive control. Mice were treated with LPS and 24 hours later cochleae were analyzed for gene transcription of innate inflammatory cytokine/chemokine signaling molecules, neutrophil recruitment, and vascular trafficking of the paracellular tracer biocytin-TMR. Results: Cochlear transcription of innate proinflammatory cytokines/chemokines was increased in endotoxemic C57Bl/6 and GCSF-KO, but not in TLR4-KO mice. More neutrophils were recruited to endotoxemic C57Bl/6 cochleae compared with both TLR4 and GCSF-KO cochleae. Endotoxemia enhanced BLB trafficking of biocytin-TMR in endotoxemic C57Bl/6 cochleae and this was attenuated in both TLR4 and GCSF-KO mice. Conclusion: Together these results suggest that TLR4-mediated innate immunity cytokine/chemokine signaling alone is not sufficient for endotoxemia-enhanced trafficking of biocytin-TMR and that downstream neutrophil activity is required to enhance BLB trafficking. Clinically, targeting neutrophilic inflammation could protect hearing during aminoglycoside, cisplatin, or other ototoxic drug therapies.

BackgroundCisplatin neuro-, oto-, and nephrotoxicity are major problems in children with malignant tumors, including medulloblastoma, negatively impacting educational achievement, socioemotional development, and overall quality of life. The blood-labyrinth barrier is somewhat permeable to cisplatin, and sensory hair cells and cochlear supporting cells are highly sensitive to this toxic drug. Several chemoprotective agents such as N-acetylcysteine (NAC) were utilized experimentally to avoid these potentially serious and life-long side effects, although no clinical phase I trial was performed before. The purpose of this study was to establish the maximum tolerated dose (MTD) and pharmacokinetics of both intravenous (IV) and intra-arterial (IA) NAC in adults with chronic kidney disease to be used in further trials on oto- and nephroprotection in pediatric patients receiving platinum therapy.MethodsDue to ethical considerations in pediatric tumor patients, we used a clinical population of adults with non-neoplastic disease. Subjects with stage three or worse renal failure who had any endovascular procedure were enrolled in a prospective, non-randomized, single center trial to determine the MTD for NAC. We initially aimed to evaluate three patients each at 150, 300, 600, 900, and 1200 mg/kg NAC. The MTD was defined as one dose level below the dose producing grade 3 or 4 toxicity. Serum NAC levels were assessed before, 5 and 15 min post NAC. Twenty-eight subjects (15 men; mean age 72.2 ± 6.8 years) received NAC IV (N = 13) or IA (N = 15).ResultsThe first participant to experience grade 4 toxicity was at the 600 mg/kg IV dose, at which time the protocol was modified to add an additional dose level of 450 mg/kg NAC. Subsequently, no severe NAC-related toxicity arose and 450 mg/kg NAC was found to be the MTD in both IV and IA groups. Blood levels of NAC showed a linear dose response (p < 0.01). Five min after either IV or IA NAC MTD dose administration, serum NAC levels reached the 2–3 mM concentration which seemed to be nephroprotective in previous preclinical studies.ConclusionsIn adults with kidney impairment, NAC can be safely given both IV and IA at a dose of 450 mg/kg. Additional studies are needed to confirm oto- and nephroprotective properties in the setting of cisplatin treatment. Clinical Trial Registration URL: https://eudract.ema.europa.eu. Unique identifier: 2011-000887-92