Carolyn O. Dirain

Ciprofloxacin Decreases Collagen in Mouse Tympanic Membrane Fibroblasts

Objectives To determine how collagen production by tympanic membrane fibroblasts is affected by ciprofloxacin at levels found in eardrops. Study Design Prospective, controlled, and blinded cell culture study. Setting Academic tertiary medical center. Subjects Cell culture of mouse fibroblasts. Methods A primary fibroblast culture was established from mouse tympanic membranes. Fibroblasts were cultured until they were 75% confluent, then treated with dilute hydrochloric acid (control) or ciprofloxacin (0.01% or 0.3%) for 24 or 72 hours for Western blotting and for 24 or 48 hours for cytotoxicity assay. Cells were observed with phase-contrast microscope. Western blotting was performed for collagen type 1 α1 (collagen 1A1) and α-tubulin. Results Fibroblasts treated with 0.01% and 0.3% ciprofloxacin for 24 hours had lower levels of collagen 1A1 (P = .0005 and P < .0001, respectively) and α-tubulin (both P < .0001) than control fibroblasts. Collagen 1A1 and α-tubulin levels were lower in fibroblasts treated with 0.3% than with 0.01% ciprofloxacin (P = .02 and P = .014). After 72 hours, 0.3% ciprofloxacin completely eliminated collagen 1A1 and α-tubulin (P < .001). Cells treated with 0.01% ciprofloxacin for 72 hours also had lower collagen 1A1 (P < .0001) and α-tubulin (P = .005) as compared with the control. Seventy-two-hour incubation in 0.01% or 0.3% ciprofloxacin resulted in lower levels of collagen 1A1 (P = .009 and P < .0001, respectively) and α-tubulin (P = .007 and P < .0001, respectively) than 24-hour incubation. Cytotoxicity assay and phase-contrast microscopy mirrored these findings. Conclusions Treatment of tympanic membrane fibroblasts with 0.3% ciprofloxacin, as found in eardrops, reduces fibroblast viability and collagen and α-tubulin protein levels. These findings could explain tympanic membrane healing problems associated with quinolone eardrops.

Mitochondria-Targeted Antioxidant Mitoquinone Reduces Cisplatin-Induced Ototoxicity in Guinea Pigs

Objective To determine if mitoquinone (MitoQ) attenuates cisplatin-induced hearing loss in guinea pigs. Study Design Prospective and controlled animal study. Setting Academic, tertiary medical center. Subjects and Methods Guinea pigs were injected subcutaneously with either 5 mg/kg MitoQ (n = 9) or normal saline (control, n = 9) for 7 days and 1 hour before receiving a single dose of 10 mg/kg cisplatin. Auditory brainstem response thresholds were measured before MitoQ or saline administration and 3 to 4 days after cisplatin administration. Results Auditory brainstem response threshold shifts after cisplatin treatment were smaller by 28 to 47 dB in guinea pigs injected with MitoQ compared with those in the control group at all tested frequencies (4, 8, 16, and 24 kHz, P = .0002 to .04). Scanning electron microscopy of cochlear hair cells showed less outer hair cell loss and damage in the MitoQ group. Conclusion MitoQ reduced cisplatin-induced hearing loss in guinea pigs. MitoQ appears worthy of further investigation as a means of preventing cisplatin ototoxicity in humans.

Assessment of mitochondrial membrane potential in HEI-OC1 and LLC-PK1 cells treated with gentamicin and mitoquinone.

Objective To determine the effects of concurrent treatment with gentamicin and the mitochondria-targeted antioxidant mitoquinone (MitoQ; which may prevent gentamicin ototoxicity) on change in the mitochondrial membrane potential (Δψm), a precursor of apoptosis. Study Design Prospective and controlled. Setting Academic research laboratory. Subjects and Methods LLC-PK1 (Lilly Laboratories Culture–Pig Kidney Type 1) and HEI-OC1 (House Ear Institute Organ of Corti 1) cells—renal and auditory cell lines, respectively—were used in this study. Δψm was assessed by flow cytometry through the MitoProbe JC-1 Kit for Flow Cytometry in untreated LLC-PK1 and HEI-OC1 cells and cells exposed to low- (100µM) or high- (2000µM) dose gentamicin for 24 hours, with and without 0.5µM each of MitoQ or idebenone (IDB; an untargeted ubiquinone). Results Δψm was not different in untreated LLC-PK1 cells and cells coincubated with low-dose gentamicin and MitoQ or IDB (P > .05). In HEI-OC1 cells, coincubation with low-dose gentamicin and MitoQ decreased Δψm (P = .002). Coincubation of LLC-PK1 cells with high-dose gentamicin and DMSO, MitoQ, or IDB depolarized Δψm (P < .0001), with MitoQ depolarizing the Δψm to a greater extent than that of IDB (P = .03). In contrast, HEI-OC1 cells demonstrated a hyperpolarized Δψm when coincubated with high-dose gentamicin and DMSO, MitoQ, or IDB (P < .001). Conclusion The combination of gentamicin and MitoQ holds the potential to disrupt Δψm. This suggests a heightened need to monitor for toxicity in patients receiving both agents.

Collagen and α-Tubulin of Mouse Tympanic Membrane Fibroblasts Treated with Quinolones and Aminoglycosides

Objective To assess collagen and α-tubulin levels of mouse tympanic membrane fibroblasts treated with quinolone and aminoglycoside antibiotics at concentrations found in eardrops. Study Design Prospective controlled cell culture study. Setting Academic tertiary medical center. Subjects Mouse tympanic membrane fibroblasts. Methods In experiment 1, fibroblasts were treated with the following for 24 or 48 hours: phosphate-buffered saline (negative control), dilute hydrochloric acid (positive control), 0.5% gatifloxacin, or commercially available 0.3% ciprofloxacin, 0.3% ciprofloxacin + 0.1% dexamethasone, 0.3% ofloxacin, 0.5% moxifloxacin, 0.3% gentamicin, or 3.5 mg/mL of neomycin + polymyxin B sulfate + hydrocortisone. In experiment 2, cells were treated with the pure form of gatifloxacin, gentamicin, ofloxacin, or ciprofloxacin. Cells were observed with phase-contrast microscope until harvested. Proteins were extracted for Western blotting with antibodies against collagen α1 type I (collagen 1A1) and α-tubulin, and for densitometry to quantify levels. Results Collagen and tubulin levels in fibroblasts treated with ofloxacin, moxifloxacin, gatifloxacin, or gentamicin for 24 hours were not different from the saline control. Fibroblasts treated with neomycin + polymyxin B + hydrocortisone, ciprofloxacin + dexamethasone, or ciprofloxacin for 24 hours had lower collagen 1A1 and α-tubulin levels (all P < .001) than the negative control. After 48 hours, fibroblasts treated with neomycin + polymyxin B sulfate + hydrocortisone, ciprofloxacin + dexamethasone, ciprofloxacin, or moxifloxacin had lower collagen 1A1 (P ≤ .007) and α-tubulin (P < .0001; except ciprofloxacin, P = .033) as compared with control. In experiment 2, only cells treated with ciprofloxacin had lower collagen 1A1 and α-tubulin levels and cell viability (all P < .0001) than control. Cytotoxicity assay and phase-contrast images mirrored the protein findings. Conclusion The adverse impact of topical antibiotic exposure on tympanic membrane collagen and tubulin protein levels is drug specific. This may be important for selection of ototopical therapy.

Effects of Common Ear Drops on Tympanic Membrane Healing in Rats

Objective Ciprofloxacin + dexamethasone ear drops have been associated with higher rates of tympanic membrane perforations than ofloxacin. This was thought to be an effect of the steroid; however, ciprofloxacin (sans steroid) has been found to be more toxic to tympanic membrane fibroblasts than ofloxacin in vitro. This study aimed to compare the effect of these agents on tympanic membrane healing in vivo. Study Design Controlled animal study. Setting Academic research laboratory. Methods Perforations were created in 54 rats with a carbon dioxide laser. Rats were randomized to 6 groups (9/group), with 1 ear receiving ofloxacin, ciprofloxacin, dexamethasone, ofloxacin + dexamethasone, ciprofloxacin + dexamethasone, or neomycin, and the contralateral ear receiving saline twice daily for 10 days. Healing was assessed over 40 days. Results Ofloxacin did not delay healing relative to saline. All other treatments delayed healing relative to ofloxacin at day 10 (P < .0001). Dexamethasone and ofloxacin + dexamethasone delayed healing up to day 14 (P < .0001). Neomycin and ciprofloxacin + dexamethasone further delayed healing up to day 28 (P = .009) and day 35 (P = .043), respectively. All eardrums healed by day 10 with ofloxacin, day 20 with ciprofloxacin, day 28 for dexamethasone, and day 35 for ofloxacin + dexamethasone. At day 40, 2 of 9 ciprofloxacin + dexamethasone–treated eardrums had not healed. All saline-treated eardrums in the ofloxacin, ciprofloxacin, dexamethasone, and ofloxacin + dexamethasone groups were healed by day 14, but this was delayed to day 20 in the ciprofloxacin + dexamethasone group (P = .007). Conclusions Ototopical quinolones delay rat tympanic membrane healing in a drug-specific manner, with ciprofloxacin having a greater impact than ofloxacin. This effect is potentiated by steroids.

Microflora of Retained Intracochlear Electrodes from Infected Cochlear Implants

Objectives Cochlear implant infections may be refractory to medical management and require device removal with subsequent reimplantation. During device removal, the intracochlear electrode array is commonly left in place to prevent obliteration of the cochlear lumen. If the electrode is colonized with pathogens, this risks contaminating the replacement implant. In this study, we compare the microorganisms detected on infected cochlear implants against those on the retained electrode using culture and microbial gene-sequencing techniques. Study Design Prospective single-cohort study. Setting Tertiary medical center. Subjects and Methods Six patients with refractory cochlear implant infections had the receiver-stimulator and extracochlear electrode removed to facilitate treatment of the infection. The intracochlear electrode was removed at (delayed) reimplantation. Implant specimens were analyzed by microbial culture and 16S DNA gene sequencing. Results Staphylococcus aureus was the organism most commonly identified. None of the 6 patients’ intracochlear electrodes yielded microbes by culture. Two intracochlear electrodes revealed bacterial species, and 1 revealed fungal species by gene sequencing. There was no correlation between the microbes on the infected extracochlear implants and the retained intracochlear electrodes. All subjects underwent reimplantation after resolution of their infections. One of 6 subjects developed a second infection after reimplantation, with S aureus in the primary and secondary infections. Conclusions The intracochlear electrodes of infected cochlear implants carry a low microbial burden. Preserving intracochlear electrodes upon removal of infected cochlear implants appears to carry a low risk of contaminating a replacement cochlear implant.

The Adenoid Microbiome in Recurrent Acute Otitis Media and Obstructive Sleep Apnea

OBJECTIVE To compare the microbial flora of adenoids from patients with recurrent acute otitis media (AOM) and patients with obstructive sleep apnea (OSA). MATERIALS AND METHODS This study was prospective and controlled. Adenoids were obtained from children undergoing adenoidectomy for recurrent AOM (n=7) or OSA (n=13). Specimens were processed for total deoxyribonucleic acid (DNA) isolation. 16s DNA 454-pyrosequencing was performed on AOM (n=5) and OSA (n=5) specimens. All specimens were analyzed by real-time polymerase chain reaction for the quantification of the oral commensal bacteria, Streptococcus salivarius. RESULTS All adenoid specimens had evidence of microbes. Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus were among the dominant species in all samples. S. salivarius, Prevotella sp. and Terrahaemophilus aromaticivorans were more common on adenoids from OSA patients (p<0.05). Bradyrhizobium sp. was more common on adenoids from patients with recurrent AOM (p<0.05). The microbial profiles associated with recurrent AOM were different from, but overlapped with OSA. S. salivarius quantified by real-time PCR was not different between the two groups. CONCLUSION Microbes are present on all adenoid specimens, though the microbial profile differs between recurrent AOM and OSA. The clinical significance of these differences remains to be determined.

Cytotoxicity of Ciprofloxacin and Steroids in Mouse Tympanic Membrane Fibroblasts

Objective Ciprofloxacin, commonly given as eardrops, has been shown to adversely affect tympanic membrane fibroblasts. Dexamethasone potentiates this effect. A newly available eardrop contains ciprofloxacin and fluocinolone, a more potent steroid. We evaluated the cytotoxic effects of this preparation on mouse tympanic membrane fibroblasts. Study Design Prospective, in vitro. Setting Academic laboratory. Subjects and Methods In experiment 1, fibroblasts were exposed to 1:10 dilutions of commercially available 0.3% ofloxacin, 0.3% ciprofloxacin, 0.3% ciprofloxacin + 0.1% dexamethasone, 0.3% ciprofloxacin + 0.025% fluocinolone, or dilute hydrochloric acid (control), twice within 24 hours. In experiment 2, cells were also treated with the dilutions of the pure form of dexamethasone 0.1% or fluocinolone 0.025%, alone and in combination with ofloxacin or ciprofloxacin. Cells were exposed to the solutions for 2 hours each time and were placed back in growth media after the treatments. Cells were observed with phase-contrast microscope until the cytotoxicity assay was performed. Results Survival of fibroblasts treated with ofloxacin was not different from the control. Fibroblasts treated with ciprofloxacin, ciprofloxacin + dexamethasone, or ciprofloxacin + fluocinolone had much lower survival (all P < .0001). Cells treated with ciprofloxacin + fluocinolone had lower survival than ciprofloxacin (P < .0001) and ciprofloxacin + dexamethasone (P = .0001). Steroids alone also decreased fibroblast survival compared to control (P < .0001). The combination of dexamethasone or fluocinolone with ciprofloxacin, but not ofloxacin, further decreased fibroblast survival (P < .0001). Phase-contrast images mirrored the cytotoxicity findings. Conclusion Tympanic membrane fibroblast cytotoxicity of ciprofloxacin is potentiated by corticosteroids. This effect may be deleterious when treating a healing perforation but beneficial when treating granulation tissue on the tympanic membrane.

Predictors of returns to the emergency department after head and neck surgery

Thirty‐day hospital readmissions have become a measure of quality of care. Many readmissions enter through the emergency department. The purposes of this study were to determine the rate, risk factors, and costs of 30‐day returns to the emergency department (30dEDRs) after head and neck surgery.

Evaluation of Mitoquinone for Protecting Against Amikacin-Induced Ototoxicity in Guinea Pigs

HYPOTHESIS Mitoquinone (MitoQ) attenuates amikacin ototoxicity in guinea pigs. BACKGROUND MitoQ, a mitochondria-targeted derivative of the antioxidant ubiquinone, has improved bioavailability and demonstrated safety in humans. Thus, MitoQ is a promising therapeutic approach for protecting against amikacin-induced ototoxicity. METHODS Both oral and subcutaneous administrations of MitoQ were tested. Amikacin-treated guinea pigs (n = 12-18 per group) received water alone (control) or MitoQ 30 mg/l-supplemented drinking water; or injected subcutaneously with 3 to 5 mg/kg MitoQ or saline (control). Auditory brainstem responses and distortion product otoacoustic emissions were measured before MitoQ or control solution administration and after amikacin injections. Cochlear hair cell damage was assessed using scanning electron microscopy and Western blotting. RESULTS With oral administration, animals that received 30 mg/l MitoQ had better hearing than controls at only 24 kHz at 3-week (p = 0.017) and 6-week (p = 0.027) post-amikacin. With subcutaneous administration, MitoQ-injected guinea pigs had better hearing than controls at only 24 kHz, 2-week post-amikacin (p = 0.013). Distortion product otoacoustic emission (DPOAE) amplitudes were decreased after amikacin injections, but were not different between treatments (p > 0.05). Electron microscopy showed minor difference in outer hair cell loss between treatments. Western blotting demonstrated limited attenuation of oxidative stress in the cochlea of MitoQ-supplemented guinea pigs. CONCLUSIONS Oral or subcutaneous MitoQ provided limited protection against amikacin-induced hearing loss and cochlear damage in guinea pigs. Other strategies for attenuating aminoglycoside-induced ototoxicity should be explored.