Cell proliferation was attenuated by selectively inhibiting the MAP kinase cascade and the Rb–Raf-1 interaction during the early development of the inner ear.

<p>Otocysts were explanted at HH16–18 and kept in culture medium with DMSO (A1–A4, E1–E4), RRD-251 10 µM (B1–B4), RRD-251 20 µM (C1–C4), RRD-251 40 µM (D1–D4), U0126 30 µM (F1–F4), U0126 50 µM (G1–G4), or a combination of RRD-251 20 µM and U0126 30 µM (H1–H4) for 24 h and then exposed to 10 mM EdU for 30 min. Cell proliferation is indicated by EdU (green) staining and the nuclei are counterstained with DAPI (blue). Based on the morphological changes that occur in culture that mimic the normal development of the inner ear and the expression of Tuj1 (red) that serves as a marker of neural processes, the cultured otocysts were divided into the otic vesicle area (OV, Tuj1-negative) and the acoustic-vestibular ganglia area (AVG, Tuj1-positive), which would develop into sensory epithelium and the spiral ganglion, respectively. The OV and AVG areas were measured with the ImageJ software, and the data are presented as means ± SEM relative to control values (I, J). High magnification pictures were taken from the center of the otocysts (A4–H4), and the EdU-positive cells in each 63×high power field (HPF) were counted under the different conditions (K). * = <i>P</i><0.05 compared to control, # = <i>P</i><0.05 compared to RRD-251 10 µM, $ = <i>P</i><0.05 compared to RRD-251 20 µM, and & = <i>P</i><0.05 compared to U0126 30 µM (I–K). The changes in the ratios between the OV area and AVG area after treatment with U0126 and RRD-251 are shown in (L). * = <i>P</i><0.05 and # = <i>P</i><0.01 compared with controls (L). At least six otic vesicles per condition from three different experiments were evaluated, and statistical significance was estimated using one-way ANOVA. Scale bar = 50 µm.</p

Sensory epithelium treated with U0126 or RRD-251.

<p>(A–C) Neuroblasts and progenitor cells of the sensory epithelium had distinct responses to the inhibition of the two independent pathways of pRb phosphorylation. (D1–D2) The explanted otic vesicles were cultured with DMSO, RRD-251 40 µM, or U0126 50 µM for 24 hours and then lysed to detect the level of phosphorylated and unphosphorylated ERK, pRb, and activated Caspase-3. β-actin was used as the loading control. Green arrows indicate the EdU-positive cells, red arrows indicate the Tuj1-positive cells, and the white arrows indicate the Tuj1 and EdU double-positive cells. At least three different experiments were evaluated, and the statistical significance was estimated using one-way ANOVA. * = <i>P</i><0.05 and # = <i>P</i><0.01 compared to controls. Scale bar = 50 µm.</p

Primers used in the study.

<p>Primers used in the study.</p

The expression of G1/S specific <i>cyclins</i>, <i>Rb1</i> and <i>Raf-1</i> during the development of otocyst.

<p>The expression of G1/S specific cyclins (A1-3), <i>Raf-1</i> and <i>Rb1</i> (B1-2) mRNA was analyzed by qRT-PCR at different embryonic development stages. Gene expression was normalized to the levels at stage HH18. (C1–C3) The protein lysates at different stages of inner ear development were analyzed by western blots to determine the levels of Raf-1 and pRb. β-actin was used as the loading control. At least three different experiments were evaluated, and statistical significance was estimated using one-way ANOVA. * = <i>P</i><0.05 compared to HH 18, # = <i>P</i><0.05 compared to HH 20.</p

RRD-251 promoted apoptosis during the early development of otic vesicles.

<p>After treatment with DMSO (A1–A4), RRD-251 20 µM (B1–B4), or RRD-251 40 µM (C1–C4), the balance between proliferation, apoptosis, and quiescence during otic development was shifted. In the DMSO control group, nearly half of the cells incorporated EdU (48.78%), only 7.32% of the cells were Caspase-3–positive, and 43.9% of the cells in the otic vesicles were double negative (A4). After treatment with RRD-251, the portion of EdU-positive cells was reduced to 29.85% and 20.84% for doses of 20 µM (B4) and 40 µM (C4), respectively, and the portion of cleaved Caspase-3–positive cells was increased to 7.46% and 47.22%, respectively. Furthermore, compared with controls, the portion of double-negative cells was increased at the lower dose of RRD-251 (B4) to 62.69% and was reduced with the higher dose (C4) to 31.94%. Green arrows indicate the EdU-positive cells, red arrows indicate the cleaved Caspase-3–positive cells, and the white arrows indicate the double-negative cells. The differences between the three separate cell populations within each group were estimated using the χ² test (<i>P</i><0.001). Scale bar = 10 µm.</p

Expression of genes involved in cell cycle progression after the attenuation of pRb phosphorylation by inhibiting the two different pathways.

<p>The estimated gene expression was calculated as 2^−ΔΔCt and statistical significance was estimated using one-way ANOVA. * = <i>P</i><0.05, ** = <i>P</i><0.01, and *** = <i>P</i><0.001 compared to controls.</p

Image_1_Multiple Sevoflurane Exposures During the Neonatal Period Cause Hearing Impairment and Loss of Hair Cell Ribbon Synapses in Adult Mice.pdf

ObjectivesThis study aims to investigate the effects of multiple sevoflurane exposures in neonatal mice on hearing function in the later life and explores the underlying mechanisms and protective strategies.Materials and MethodsNeonatal Kunming mice were exposed to sevoflurane for 3 days. Auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) tests, immunofluorescence, patch-clamp recording, and quantitative real-time PCR were performed to observe hearing function, hair cells, ribbon synapses, nerve fibers, spiral ganglion neurons, and oxidative stress.ResultsCompared to control group, multiple sevoflurane exposures during the neonatal time significantly elevated ABR thresholds at 8 kHz (35.42 ± 1.57 vs. 41.76 ± 1.97 dB, P = 0.0256), 16 kHz (23.33 ± 1.28 vs. 33.53 ± 2.523 dB, P = 0.0012), 24 kHz (30.00 ± 2.04 vs. 46.76 ± 3.93 dB, P = 0.0024), and 32 kHz (41.25 ± 2.31 vs. 54.41 ± 2.94 dB, P = 0.0028) on P30, caused ribbon synapse loss on P15 (13.10 ± 0.43 vs. 10.78 ± 0.52, P = 0.0039) and P30 (11.24 ± 0.56 vs. 8.50 ± 0.84, P = 0.0141), and degenerated spiral ganglion neuron (SGN) nerve fibers on P30 (110.40 ± 16.23 vs. 55.04 ± 8.13, P = 0.0073). In addition, the Vhalf of calcium current become more negative (−21.99 ± 0.70 vs. −27.17 ± 0.60 mV, P ConclusionThe findings suggest that multiple sevoflurane exposures during neonatal time may cause hearing impairment in adult mice. The study also demonstrated that elevated oxidative stress led to ribbon synapses impairment and SGN nerve fibers degeneration, and the interventions of antioxidants alleviated the sevoflurane-induced hearing impairment.</p