p27Kip1 down-regulation as achieved by two clinically feasible means did not induce proliferation of supporting cells in the rat neonatal cochlea in vivo

Publication date: Available online 6 December 2018

Source: Hearing Research

Author(s): Sebastián A. Silva, Juan C. Maass

Abstract

In mammals, the cochlear sensory epithelium becomes quiescent early during development. After the first postnatal week, there is no cell replacement or proliferation, and severe damage leads to permanent deafness. Supporting cells’ trans-differentiation has been suggested as a way to regenerate cochlear hair cells after damage. However, they are also needed for proper functionality. Cdkn1b (p27^Kip1) participates in the cochlear terminal mitosis state achieved during development. Its expression is maintained in adult supporting cells and its postnatal deletion has induced cochlear proliferation in vitro and in vivo. Therefore, its manipulation has been proposed as a feasible way to induce proliferation of supporting cells after birth. Nevertheless, the literature is scarce regarding feasible methods to directly decrease p27^Kip1 in the clinical domain. The effects of p27^Kip1 knockdown using viral vectors are not completely elucidated and no pharmacological approaches to decrease p27^Kip1 in the cochlea have been tested in vivo before. This study explores the ability of p27^Kip1 messenger knockdown and pharmacological transcriptional inhibition to induce proliferation of supporting cells in the P0 neonatal rat cochlea in vivo. Respectively, lentiviral vectors transducing shRNA against p27^Kip1 were administered into the scala media or Alsterpaullone 2-Cyanoethyl into the round window niche. Cell markers and gene expression were assessed through immunostaining and qRT-PCR. Despite both methods significantly decreasing p27^Kip1 expression in vivo, signs of toxicity in the organ of Corti were not found; however, relevant proliferation was not found either. Finally, cochlear damage was added to increase the response in vitro, achieving only a mild to moderate proliferation induction. We conclude that our approaches were not able to stimulate the recall of supporting cell proliferation despite significantly decreased p27^Kip1 levels in vivo. Considering the evaluation of the cochlea at a very responsive stage, we propose that the level of isolated modification of p27^Kip1 expression in living mammals achievable through these approaches is insufficient to induce proliferation of supporting cells. Future proliferation induction experiments in the cochlea should study other methods and genes.

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