Suppression Of Inflammation Delays Functional Regeneration Of Lateral Line Hair Cells In Zebrafish Larvae

Human cochlear hair cells,once destroyed due to infection,genetic factors,hypoxia,autoimmune disorder,ototoxic drugs or noise exposure,do not regenerate,which results in permanent deafness.In contrast,the hair cells of lower animals,such as birds,reptiles,amphibians and fish,have a remarkable ability to regenerate.Previous studies mainly focus on mechanisms of hair cell regeneration,such as Wnt and Notch signals.It is still unknown whether the inflammation has any effects on hair cell regeneration and its recovery of function as in the course of hair cell damage there always exists inflammatory responses.We used a transgenic zebrafish line Tg(corola-eGFP;lyz-Dsred)to observe the migration of the innate immune neutrophils and macrophages after lateral line hair cells were damaged by acute copper exposure.Zebrafish larvae,before being immerged in copper sulfate solution,were treated with BRS-28,an anti-inflammatory agent,to suppress the production of proinflammatory factors and chemokines such as NO?IL-1??TNF-??cox-2 and iNOS.And then,with confocal microscope,we observed the morphology and numbers of regenerated hair cells.Since the rheotaxis can reflect the overall function of hair cells after regeneration,a U-shaped tank was used to detect this behavior in AB/WT larvae,and a behavioral analysis software was applied for quantitative evaluation of the rheotaxis.The function of regenerated hair cells in a single neuromast were observed by in vivo real time calcium live imaging.The calcium activity of the regenerated hair cells can be observed in transgenic zebrafish line Huc:h2b-gcamp6f after the neuromasts were stimulated.When the production of proinflammatory factors was suppressed,the number of neutrophils and macrophages migrating to damaged hair cells were significantly reduced.We found the regeneration of lateral line hair cells was delayed and the recovery of the rheotaxis of the larvae was also delayed.Calcium imaging suggested that the function of neuromast in the inflammatory inhibition group was weaker than that in the non-inflammatory inhibition group at the early stage of regeneration,and returned to normal at the late stage.Our results,meanwhile,suggested that suppressing inflammation delays hair cell regeneration and functional recovery when hair cells are damaged.Based on the above results,we believe that BRS-28 suppress the production of proinflammatory factors,thereby reducing the number of neutrophils and macrophages migrating to the injured neuromast and slowing down the process of inflammation resolution.This may attribute to the blockage of the activation of JNK signaling pathway of microphages,which influences the initiation of compensatory proliferation of support cells and ultimately leads to a delay in hair cell regeneration.However,inflammatory inhibition does not affect the function when hair cells regeneration was completed.The results suggest that inflammation play positive and permissive roles in regeneration of hair cells.We expect our results can reveal a part of mechanisms of hair cells regeneration in vertebrates and the regulation of inflammation on regeneration.This study provides a suitable method for objective evaluation of hair cell function in vivo for the first time,and explores the function of regenerated hair cells for the first time by calcium imaging technology,and finally provides a new idea for how to make hair cell regeneration and functional recovery in adult mammals.