Bone Marrow Stromal Cells Accelerate Hearing Recovery via Regeneration or Maintenance of Cochlear Fibrocytes in Mouse Spiral Ligaments.

Mammalian cochleae have limited capacity for regeneration, which is one of the major difficulties in the treatment of sensorineural hearing loss. In the current study, we examined the potential of bone marrow-derived stromal cells (BMSCs) for functional restoration of mouse cochleae through regeneration or maintenance of cochlear fibrocytes in the spiral ligament (SL). We used a mouse model of degeneration of cochlear fibrocytes in the SL using local application of 3-nitropropionic acid (3-NP), in which disruption of the gap junction network in the SL resulted in the reduction of the endocochlear potential (EP). Mouse BMSCs were infused into the posterior semicircular canal 7 days after 3-NP application. Transplanted BMSCs were frequently observed in the cochlear fluid space 4 weeks after transplantation, although a few transplants had migrated into the cochlear tissues including the SL. BMSC-treated cochleae exhibited higher cell densities in the SL and greater EP levels than the control ones. Immunohistochemistry further demonstrated the restoration of functional proteins in the SL. Significant recovery in thresholds of auditory brainstem responses following BMSC transplantation was found only at 40 kHz in a mild degeneration model. Our cumulative findings indicated that BMSCs accelerated regeneration or maintenance of fibrocytes in damaged SLs, leading to partial functional restoration of the mouse cochleae. Anat Rec, 303:478-486, 2020. © 2019 American Association for Anatomy.

Downregulation of inwardly rectifying potassium channel 5.1 expression in C57BL/6J cochlear lateral wall.

Age-related hearing loss (AHL) is one of the most common sensory disorders among elderly persons. The inwardly rectifying potassium channel 5.1 (Kir5.1) plays a vital role in regulating cochlear K(+) circulation which is necessary for normal hearing. The distribution of Kir5.1 in C57BL/6J mice cochleae, and the relationship between the expression of Kir5.1 and the etiology of AHL were investigated. Forty C57BL/6J mice were randomly divided into four groups at 4, 12, 24 and 52 weeks of age respectively. The location of Kir5.1 was detected by immunofluorescence technique. The mRNA and protein expression of Kir5.1 was evaluated in mice cochleae using real-time polymerase-chain reactions (RT-PCR) and Western blotting respectively. Kir5.1 was detected in the type II and IV fibrocytes of the spiral ligament in the cochlear lateral wall of C57BL/6J mice. The expression levels of Kir5.1 mRNA and protein in the cochleae of aging C57BL/6J mice were down-regulated. It was suggested that the age-related decreased expression of Kir5.1 in the lateral wall of C57BL/6J mice was associated with hearing loss. Our results indicated that Kir5.1 may play an important role in the pathogenesis of AHL.

Age-related morphological changes in the basement membrane in the stria vascularis of C57BL/6 mice.

Basement membrane anionic sites (BMAS) are involved in the selective transport of electrically charged macromolecules in cochlear capillaries. Using cationic polyethyleneimine (PEI), we examined age-related changes in BMAS in the cochleae of C57BL/6 mice. The mice were grouped according to age as follows: 3 days, 4 weeks, 8 weeks, 6 months, and 12 months. In the right bony labyrinths, widths of the stria vascularis were measured in paraffin-embedded sections using light microscopy. The left bony labyrinths were immersed in a 0.5 % cationic PEI solution and embedded in epoxy resin. Ultrathin sections of the left cochlea were examined using transmission electron microscopy. A significant difference in stria vascularis width was observed between the 4-week-old and 12-month-old mice. The PEI distribution in the capillary and epithelial basement membranes (BMs) of the cochlea was observed. In all animals, PEI particles were evenly distributed in the capillary BM of the spiral ligament and in the subepithelial BM of Reissner's membrane. In the stria vascularis, PEI particles were evenly distributed in the capillary BM in 3-day-old mice. In 4- and 8-week-old mice, PEI particle sizes were markedly lower than those observed in 3-day-old mice. In 6- and 12-month-old mice, PEI particles were hardly detected in the strial capillary BM. In the strial capillary BM in these mice, the laminae rarae externa and interna disappeared, but the lamina densa became larger. We speculated that age-related changes of strial capillary BMAS may affect electrically charged macromolecule transport systems in the stria vascularis of C57BL/6 mice.

Acoustic overstimulation activates 5'-AMP-activated protein kinase through a temporary decrease in ATP level in the cochlear spiral ligament prior to permanent hearing loss in mice.

Inner ear disorders are known to be elicited by mitochondrial dysfunction, which decreases the ATP level in the inner ear. 5'-AMP-activated protein kinase (AMPK) is a serine/threonine kinase activated by metabolic stress and by an increase in the AMP/ATP ratio. To elucidate the involvement of AMPK-derived signals in noise-induced hearing loss, we investigated whether in vivo acoustic overstimulation would activate AMPK in the cochlea of mice. Std-ddY mice were exposed to 8kHz octave band noise at a 90-, 110- or 120-dB sound pressure level (SPL) for 2h. Exposure to the noise at 110 or 120dB SPL produced outer hair cell death in the organ of Corti and permanent hearing loss. Exposure to the noise at 120-dB SPL elevated the level of the phospho-AMPK α-subunit (p-AMPKα), without affecting the protein level of this subunit, immediately and at 12-h post-exposure in the lateral wall structures including the spiral ligament and stria vascularis. In the hair cells and spiral ganglion cells, no marked change in the level of p-AMPKα was observed at any time post-exposure. The level of phospho-c-Jun N-terminal kinase (p-JNK) was increased in the lateral wall structures at 2- to 4-h post-exposure at 120dB SPL. Noise exposure significantly, but temporarily, decreased the ATP level in the spiral ligament, in an SPL-dependent manner at 110dB and above. Likewise, elevation of p-AMPKα and p-JNK levels was also observed in the lateral wall structures post-exposure to noise at an SPL of 110dB and above. Taken together, our data suggest that AMPK and JNK were activated by ATP depletion in the cochlear spiral ligament prior to permanent hearing loss induced by in vivo acoustic overstimulation.

Late-phase recovery in the cochlear lateral wall following severe degeneration by acute energy failure.

We previously reported a model of acute cochlear energy failure using a mitochondrial toxin, 3-nitropropionic acid (3-NP), to study mechanisms of inner ear disorders such as inner ear ischemia. In this model, the main cause of hearing loss is apoptosis of fibrocytes in the cochlear lateral wall. Here, we analyzed the time course of structural and hearing level changes in the cochlea from the acute phase to the chronic phase up to 2 months after surgery. Hearing levels as determined by auditory brainstem response (ABR) thresholds exceeded the maximum acoustic output (>87 dBSPL) of the system at all frequencies 1 day after 3-NP treatment. Histology showed nearly complete loss of fibrocytes 2 weeks after 3-NP treatment. However, after 2 months, ABR showed significant recovery at low frequency (8 kHz) in four of five rats treated with 3-NP. ABR thresholds at 20 kHz occasionally showed some recovery. At 40 kHz, recovery of ABR thresholds was not observed. Histology of 3-NP-treated rats revealed partial recovery of the lateral wall and the regenerated fibrocytes in the spiral ligament expressed Na/K-ATPase in the cochlear basal turn 2 months after 3-NP treatment. These results indicate that ABR recovery is caused by regeneration of the cochlear lateral wall. Our findings demonstrate the recoverable capacity of the cochlear lateral wall that leads to functional recovery after severe damage.