Systemic lipopolysaccharide induces cochlear inflammation and exacerbates the synergistic ototoxicity of kanamycin and furosemide

Abstract

Aminoglycoside antibiotics are highly effective agents against gram-negative bacterial infections, but they cause adverse effects on hearing and balance dysfunction as a result of toxicity to hair cells of the cochlea and vestibular organs. While ototoxicity has been comprehensively studied, the contributions of the immune system, which controls the host response to infection, have not been studied in antibiotic ototoxicity. Recently, it has been shown that an inflammatory response is induced by hair cell injury. In this study, we found that lipopolysaccharide (LPS), an important component of bacterial endotoxin, when given in combination with kanamycin and furosemide, augmented the inflammatory response to hair cell injury and exacerbated hearing loss and hair cell injury. LPS injected into the peritoneum of experimental mice induced a brisk cochlear inflammatory response with recruitment of mononuclear phagocytes into the spiral ligament, even in the absence of ototoxic agents. While LPS alone did not affect hearing, animals that received LPS prior to ototoxic agents had worse hearing loss compared to those that did not receive LPS pretreatment. The poorer hearing outcome in LPS-treated mice did not correlate to changes in endocochlear potential. However, LPS-treated mice demonstrated an increased number of CCR2(+) inflammatory monocytes in the inner ear when compared with mice treated with ototoxic agents alone. We conclude that LPS and its associated inflammatory response are harmful to the inner ear when coupled with ototoxic medications and that the immune system may contribute to the final hearing outcome in subjects treated with ototoxic agents.

FIG. 1

Treatment protocol. Mice were treated according to the following protocol: On 2 consecutive days, 0.5 mg/kg LPS was injected IP (saline for control mice), and 24 h after the second LPS injection, mice were injected with kanamycin (1,000 mg/kg) and 40 min later, with furosemide (180 mg/kg). Auditory brainstem responses (ABRs) and endocochlear potentials (EPs) were tested on day 5 after kanamycin-furosemide administration. After physiologic testing, the mice were perfused through the heart with fixative, and cochleas were harvested for histologic analysis.

FIG. 2

ABR thresholds after LPS pretreatment and ototoxic medications. Hearing thresholds are shown at day 5 after ototoxic treatment in four experimental groups, saline controls (saline), LPS alone (no ototoxic agents), kanamycin-furosemide (no LPS pretreatment), and LPS pretreatment with kanamycin-furosemide. Mice that received LPS alone (LPS) had no significant change in hearing thresholds compared with saline controls. Those treated with kanamycin-furosemide (KF) showed a moderate hearing loss (30–40-dB threshold shift). LPS-kanamycin-furosemide mice (LPS-KF) had the highest hearing thresholds (40–60-dB threshold shift). Statistical analysis: asterisks demonstrate frequencies at which significant differences were present between the three groups (LPS vs KF, LPS vs LPS-KF, KF vs LPS-KF). Mice tested at days 14 and 28 showed similar threshold shifts as shown at day 5.

FIG. 3

Endocochlear potential 5 days after treatment. Endocochlear potential (EP) was measured in the basal turn of the cochlea in control, kanamycin-furosemide, and LPS-kanamycin-furosemide-treated mice 5 days after exposure to ototoxic agents. The control mice showed an average endocochlear potential of 109 mV (bar) with a range of 101–112 mV (individual data plotted in open circles). The kanamycin-furosemide-treated mice demonstrated a reduction in the average EP (average 94 mV). Mice treated with LPS-kanamycin-furosemide also showed decline in EP (average 86 mV). The difference between KF and LPS-KF was not statistically significant (Mann-Whitney U test). Controls demonstrated a statistically significant difference when compared with LPS-KF (asterisk).

FIG. 4

Morphometry of the stria vascularis. Strial thickness was markedly increased 5 days after antibiotic-diuretic treatment (B) when compared to controls (A). The strial thickness was measured in the upper basal turn in the midmodiolar plane in all cochlear sections, and the three experimental groups were compared with saline controls (C). Strial thickness was significantly different in control mice when compared to KF or LPS-KF mice. There was no significant difference between KF and LPS-KF mice. The width of the SV marked by white line in A is 30 μm, in B is 80 μm. Scale bar 30 μm.

FIG. 5

Fluorescent-labeled monocytes and macrophages in the cochlea. Representative sections of the cochlea in saline controls (A, B, C), LPS (D, E, F), kanamycin-furosemide (G, H, I), and LPS-kanamycin-furosemide (J, K, L)-treated ears in double heterozygous knock-in mice (CX3CR1^+/GFP CCR2^+/RFP). CX3CR1⁺ cells are shown in green (A, D, G, J), CCR2⁺ cells in magenta (B, E, H, K), and merged images (C, F, I, L). Arrowheads indicate the lower spiral ligament, in the location of type IV fibrocytes, where inflammatory cells typically appear. Most inflammatory cells in the cochlea after kanamycin-furosemide treatment expressed CX3CR1 and not CCR2, while LPS-pretreated mice demonstrated CCR2⁺ monocytes in abundance (K). Scale bar in C 50 μm, applies to all figures.

FIG. 6

CCR2⁺ and CX3CR1₊ cell counts. LPS treatment resulted in a significant increase in both CX3CR1₊ and CCR2₊ cells in the cochlea. In kanamycin-furosemide-treated animals, few specimens demonstrated CCR2 expression LPS was the most potent stimulus for migration of inflammatory cells into the cochlea. Statistical analysis demonstrates significantly more CCR2₊ cells in LPS controls compared to saline controls and significantly more CCR2₊ cells in LPS-pretreated compared to saline-pretreated mice receiving ototoxic agents. There was no significant change in CCR2 expression caused by ototoxicity alone. Both LPS and ototoxic agents independently caused increases in the CX3CR1₊ monocyte/macrophage population in the inner ear.

FIG. 7

Representative sections of the mouse cochlea after kanamycin-furosemide and LPS-kanamycin-furosemide. Plastic-embedded mouse cochleas were sectioned and analyzed for histologic changes after treatment. Representative sections of the mouse basal turn (A), middle turn (C), and apical turn (E) after kanamycin-furosemide. In this specimen, the basal turn showed preserved hair cells while the apical hair cells were absent. The organ of Corti was otherwise preserved, and the inner hair cells were present. B, D, F representative sections after LPS-kanamycin-furosemide treatment. All turns showed complete loss of outer hair cells. In other specimens in this series, we observed sporadic retention of outer hair cells mostly in the basal half. Scale bar in A 50 μm (applies to A, B), C 25 μm (applies to C, D, E, and F). Arrows indicate area of outer hair cells.

FIG. 8

Cytocochleograms. Five days after ototoxic injury, cochleas were harvested and processed for light microscopy. Some inner hair cells in the basal turn were missing after kanamycin-furosemide treatment (KF). LPS pretreatment with kanamycin-furosemide (LPS-KF) did not alter the amount or pattern of inner hair cell damage. LPS pretreatment resulted in a significant reduction of outer hair cells when combined with kanamycin-furosemide. Statistical analysis demonstrated no significant difference in inner hair cell survival. Outer hair cell survival was lower in LPS-kanamycin-furosemide-treated animals compared with kanamycin-furosemide alone when comparing average OHC loss over the entire cochlear duct (KF n = 12, LPS-KF n = 12, error bars represent standard error of the means).