Supplementary Material for: Low-Molecular-Weight Hyaluronic Acid Contributes to Noise-Induced Cochlear Inflammation

Introduction: Our previous work indicated that the activation of the Toll-like receptor (TLR) 4 signaling pathway contributed to noise-induced cochlear inflammation. Previous studies have reported that low-molecular-weight hyaluronic acid (LMW-HA) accumulates during aseptic trauma and promotes inflammation by activating the Toll-like receptor (TLR) 4 signaling pathway. We hypothesized that LMW-HA or enzymes synthesizing or degrading HA might be involved in noise-induced cochlear inflammation. Methods: The present study included two arms. The first arm was the noise exposure study, in which TLR4, proinflammatory cytokines, HA, hyaluronic acid synthases (HASs) and hyaluronidases (HYALs) in the cochlea as well as auditory brainstem response (ABR) thresholds were measured before and after noise exposure. The second arm was analysis of HA delivery-induced reactions, in which control solution, high-molecular-weight hyaluronic acid (HMW-HA) or LMW-HA was delivered into the cochlea by cochleostomy or intratympanic injection. Then, the ABR threshold and cochlear inflammation were measured. Results: After noise exposure, the expression of TLR4, proinflammatory cytokines, HAS1 and HAS3 in the cochlea significantly increased over the 3rd to 7th day post-noise exposure (PE3, PE7). The expression of HYAL2 and HYAL3 dramatically decreased immediately after noise exposure, gradually increased thereafter to levels significantly greater than the preexposure level on PE3, and then rapidly returned to the preexposure level on PE7. The expression of HA, HAS2 and HYAL1 in the cochlea remained unchanged after exposure. After cochleostomy or intratympanic injection, both the hearing threshold shifts and the expression of TLR4, TNF-α and IL-1β in the cochleae of the LMW-HA group were obviously greater than those of the control group and HMW-HA group. The expression of proinflammatory cytokines in the LMW-HA and control groups on the 7th day (D7) after cochleostomy tended to increase compared to that on the 3rd day (D3), whereas levels in the HMW-HA group tended to decrease on D7 compared to D3. Conclusion: HAS1, HAS3, HYAL2 and HYAL3 in the cochlea are involved in acoustic trauma-induced cochlear inflammation through the potential proinflammatory function of LMW-HA.