Irina Omelchenko

The influence of NF-κB signal-transduction pathways on the murine inner ear by acoustic overstimulation

Nuclear factor‐kappa B (NF‐κB) comprises a family of inducible transcription factors that serve as important regulators of the host immune and inflammatory responses. The NF‐κB signals are activated via the canonical and/or noncanonical pathways in response to diverse stimuli. The excessive action of NF‐κB signal‐transduction pathways frequently causes self‐injurious phenomena such as allergic diseases, vascular disorders, and ischemia–reperfusion neuronal damage. In the inner ear, the role of NF‐κB has not been clarified because the activated NF‐κB signals potentially induce both cytoprotective and cytotoxic target genes after ototoxic stimulation. In the present study, we investigated the response of NF‐κB in both the canonical and noncanonical pathways to acoustic overstimulation (117 dB/SPL/2 hr) and followed the change of inflammatory factors (inducible nitric oxide synthase [iNOS], intracellular adhesion molecule‐1 [ICAM‐1], and vascular cell adhesion molecule‐1 [VCAM‐1]) in the cochlear lateral wall (CLW) and the rest of cochlea (RoC). By means of immunohistochemistry combined with confocal microscopy and reverse transcriptase–polymerase chain reaction techniques, we found the response of NF‐κB family members (NF‐κB1, 2, RelA, and RelB) at the transcription level. After the NF‐κB signaling, the inflammatory factors were significantly increased in the CLW and the RoC. Additionally, at the protein level, the prominent expression of adhesion molecules (ICAM‐1 and VCAM‐1) was observed in the tissue around the capillaries in the stria vascularis. These results show that acoustic overstimulation causes the NF‐κB signaling to overexpress the inflammatory factors in the inner ear, and the up‐regulation of the adhesion molecules (ICAM‐1 and VCAM‐1) and iNOS potentially influence the hemodynamics and the cellular integrity in the stria vascularis.

Bone Marrow Cell Recruitment Mediated by Inducible Nitric Oxide Synthase/Stromal Cell-Derived Factor-1α Signaling Repairs the Acoustically Damaged Cochlear Blood-Labyrinth Barrier

Using a mouse model with noise-induced cochlear blood-labyrinth-barrier (CBLB) injury, we examined the effects of inducible nitric oxide synthase (iNOS) on the recruitment of bone marrow-derived cells (BMDCs) to the CBLB after acoustic injury. Lethally irradiated C57BL/6J and B6.129P2-Nos2(tm1Lau)/J mice were transplanted with GFP(+)-BMDCs from C57Bl/6-Tg (UBC GFP) mice. Four weeks after transplantation, we assessed the population of GFP(+)-BMDCs in the CBLB. Only small numbers of GFP(+)-BMDCs were found to infiltrate the area of the CBLB in the control recipient mice. However, robust GFP(+)-BMDC migration occurred in the area of the CBLB within the injured cochlea during the first week following acoustic trauma, and further BMDC accumulation was seen by 2 weeks posttrauma. After 4 weeks, the BMDCs were integrated into vessels. Local iNOS from perivascular resident macrophages was found to be important for BMDC infiltration, since mice deficient in iNOS (Inos(-/-)) and mice with iNOS that had been inhibited by 1400W displayed reduced BMDC infiltration. Stromal cell-derived factor-1α (SDF-1α) and its chemokine receptor 4 (CXCR4) were required for the iNOS-triggered recruitment. BMDC recruitment was significantly reduced by the inhibition of SDF-1α activity. Inhibition of the iNOS/SDF-1α signaling pathway reduced vascular repair as observed by reduced vascular density. Our study revealed an intrinsic signaling pathway of iNOS that mediates SDF-1α to promote GFP(+)-BMDC infiltration/targeting in cochlear vascular repair.

Nitric oxide and mitochondrial status in noise-induced hearing loss

This study investigated the distribution of nitric oxide (NO) within isolated outer hair cells (OHCs) from the cochlea, its relationship to mitochondria and its modulation of mitochondrial function. Using two fluorescent dyes—4,5-diaminofluorescein diacetate (DAF-2DA), which detects NO, and tetramethyl rhodamine methyl ester (TMRM+), a mitochondrial membrane potential dye—it was found that a relatively greater amount of the DAF fluorescence in OHCs co-localized with mitochondria in comparison to DAF fluorescence in the cytosole. This study also observed reduced mitochondrial membrane potential of OHCs and increased DAF fluorescence following exposure of the cells to noise (120 dB SPL for 4 h) and to an exogenous NO donor, NOC-7 (>350 nm). Antibody label for nitrotyrosine was also increased, indicating NO-related formation of peroxynitrite in both mitochrondria and the cytosol. The results suggest that NO may play an important physiological role in regulating OHC energy status and act as a potential agent in OHC pathology.

Neurotoxic Potential of Three Structural Analogs of β-N-oxalyl-α,β-Diaminopropanoic Acid (β-ODAP)

Lathyrism is a non-progressive motor neuron disease produced by consumption of the excitatory amino acid, 3-N-oxalyl-L-2,3-diaminopropanoic acid (β-ODAP). To learn more about the mechanisms underlying Lathyrism three structural analogs of β-ODAP were synthesized. Carboxymethyl-α,β-diaminopropanoic acid (CMDAP) evoked inward currents which were antagonized by APV (30 μM), but not by CNQX (10 μM). N-acetyl-α,β-diaminopropanoic acid (ADAP) evoked no detectable ionic currents but potentiated N-methyl-D-aspartate (NMDA)-activated currents. The potentiation of NMDA currents by ADAP was blocked by 7-chlorokynurenic acid. Carboxymethylcysteine (CMC) did not activate any detectable ionic currents. None of the three β-ODAP analogs produced visible symptoms of toxicity in day old chicks when administered for 2–3 consecutive days. Ligand binding studies demonstrated that all the three compounds were effective to in displacing [3H]glutamate. The maximum inhibition was 92% for CMDAP, 61% for ADAP, 65% for CMC and 99% for β-ODAP. These data indicate that analogs of β-ODAP may interact with glutamate receptors without producing neurotoxicity.

Expression and function of channelrhodopsin 2 in mouse outer hair cells

Outer hair cell (OHC) is widely accepted as the origin of cochlear amplification, a mechanism that accounts for the extreme sensitivity of the mammalian hearing. The key process of cochlear amplification is the reverse transduction, where the OHC changes its length under electrical stimulation. In this study, we developed a method to modulate electro-mechanical transduction with an optogenetic approach based on channelrhodopsin 2 (ChR2), a direct lightactivated non-selective cation channel (NSCC). We specifically expressed ChR2 in mouse cochlea OHCs through in uterus injection of adenovirus vector with ChR2 in fusion with the fluorescent marker tdTomato. We also transfected ChR2(H134R), a point mutant of ChR2, with plasmid to an auditory cell line (HEI-OC1). With whole cell recording, we found that blue light (470 nm) elicited a current with a reversal potential around zero in both mouse OHCs and HEI-OC1 cells and generated depolarization in both cell types.