ABSTRACT
NOTCH pathway proteins, including the transcriptional factor HES1, play crucial roles in the development of the inner ear by means of the lateral inhibition mechanism, in which supporting cells have their phenotype preserved while they are prevented from becoming hair cells. Genetic manipulation of this pathway has been demonstrated to increase hair cell number. The present study aimed to investigate gene expression effects in hair cells and supporting cells after Hes1-shRNA lentivirus transduction in organotypic cultures of the organ of Corti from postnatal-day-3 mice. Forty-eight hours after in vitro knockdown, Hes1 gene expression was reduced at both mRNA and protein levels. Myo7a (hair cell marker) and Sox2 (progenitor cell marker) mRNA levels also significantly increased. The modulation of gene expression in the organ of Corti upon Hes1 knockdown is consistent with cell phenotypes related to lateral inhibition mechanism interference in the inner ear. The lentivirus-based expression of Hes1-shRNA is a valuable strategy for genetic interference in the organ of Corti and for future evaluation of its efficacy in protocols aiming at the regeneration of hair cells in vivo.
Subject(s)
Animals , Rats , Cochlea , Basic Helix-Loop-Helix Transcription Factors/genetics , Organ of Corti , Cell Differentiation , Receptors, Notch , Transcription Factor HES-1/genetics , Hair Cells, AuditoryABSTRACT
In mammals, damage to sensory receptor cells (hair cells) of the inner ear results in permanent sensorineural hearing loss. Here, we investigated whether postnatal mouse inner ear progenitor/stem cells (mIESCs) are viable after transplantation into the basal turns of neomycin-injured guinea pig cochleas. We also examined the effects of mIESC transplantation on auditory functions. Eight adult female Cavia porcellus guinea pigs (250-350g) were deafened by intratympanic neomycin delivery. After 7 days, the animals were randomly divided in two groups. The study group (n=4) received transplantation of LacZ-positive mIESCs in culture medium into the scala tympani. The control group (n=4) received culture medium only. At 2 weeks after transplantation, functional analyses were performed by auditory brainstem response measurement, and the animals were sacrificed. The presence of mIESCs was evaluated by immunohistochemistry of sections of the cochlea from the study group. Non-parametric tests were used for statistical analysis of the data. Intratympanic neomycin delivery damaged hair cells and increased auditory thresholds prior to cell transplantation. There were no significant differences between auditory brainstem thresholds before and after transplantation in individual guinea pigs. Some mIESCs were observed in all scalae of the basal turns of the injured cochleas, and a proportion of these cells expressed the hair cell marker myosin VIIa. Some transplanted mIESCs engrafted in the cochlear basilar membrane. Our study demonstrates that transplanted cells survived and engrafted in the organ of Corti after cochleostomy.
Subject(s)
Animals , Female , Organ of Corti/surgery , Stem Cells , Stem Cell Transplantation/methods , Hair Cells, Auditory, Inner/transplantation , Hearing Loss, Sensorineural/surgery , Auditory Threshold , Immunohistochemistry , Protein Synthesis Inhibitors , Neomycin , Cell Survival , Cells, Cultured , Reproducibility of Results , Evoked Potentials, Auditory, Brain Stem , Treatment Outcome , Guinea Pigs , Mice, Inbred BALB CABSTRACT
Os autores estudam o padräo de normalidade para a audiometria do tronco cerebral e sua variaçäo em relaçäo à faixa etária. Foram selecionados 40 indivíduos de 9 a 59 anos de idade sem história pregressa de patologia otológica e com exame físico e audiometria tonal normal. Além do estudo médio das latências, conclui-se que o limiar ao B.E.R.A. aumenta com a idade em todas as curvas, apesar da audiometria tonal normal, e que as curvas I, III e V säo as mais consistentes e com valor clínico, e que a amplitude tem pouco valor prático