Electrochemical properties of the non-excitable tissue stria vascularis of the mammalian cochlea are sensitive to sounds.

Excitable cochlear hair cells convert the mechanical energy of sounds into the electrical signals necessary for neurotransmission. The key process is cellular depolarization via K+ entry from K+ -enriched endolymph through hair cells' mechanosensitive channels. Positive 80 mV potential in endolymph accelerates the K+ entry, thereby sensitizing hearing. This potential represents positive extracellular potential within the epithelial-like stria vascularis; the latter potential stems from K+ equilibrium potential (EK ) across the strial membrane. Extra- and intracellular [K+ ] determining EK are likely maintained by continuous unidirectional circulation of K+ through a putative K+ transport pathway containing hair cells and stria. Whether and how the non-excitable tissue stria vascularis responds to acoustic stimuli remains unclear. Therefore, we analysed a cochlear portion for the best frequency, 1 kHz, by theoretical and experimental approaches. We have previously developed a computational model that integrates ion channels and transporters in the stria and hair cells into a circuit and described a circulation current composed of K+ . Here, in this model, mimicking of hair cells' K+ flow induced by a 1 kHz sound modulated the circulation current and affected the strial ion transport mechanisms; the latter effect resulted in monotonically decreasing potential and increasing [K+ ] in the extracellular strial compartment. Similar results were obtained when the stria in acoustically stimulated animals was examined using microelectrodes detecting the potential and [K+ ]. Measured potential dynamics mirrored the EK change. Collectively, because stria vascularis is electrically coupled to hair cells by the circulation current in vivo too, the strial electrochemical properties respond to sounds. KEY POINTS: A highly positive potential of +80 mV in K+ -enriched endolymph in the mammalian cochlea accelerates sound-induced K+ entry into excitable sensory hair cells, a process that triggers hearing. This unique endolymphatic potential represents an EK -based battery for a non-excitable epithelial-like tissue, the stria vascularis. To examine whether and how the stria vascularis responds to sounds, we used our computational model, in which strial channels and transporters are serially connected to those hair cells in a closed-loop circuit, and found that mimicking hair cell excitation by acoustic stimuli resulted in increased extracellular [K+ ] and decreased the battery's potential within the stria. This observation was overall verified by electrophysiological experiments using live guinea pigs. The sensitivity of electrochemical properties of the stria to sounds indicates that this tissue is electrically coupled to hair cells by a radial ionic flow called a circulation current.

Rapid optical tomographic vibrometry using a swept multi-gigahertz comb.

We propose a rapid tomographic vibrometer technique using an optical comb to measure internal vibrations, transient phenomena, and tomographic distributions in biological tissue and microelectromechanical system devices at high frequencies. This method allows phase-sensitive tomographic measurement in the depth direction at a multi-MHz scan rate using a frequency-modulated broadband electrooptic multi-GHz supercontinuum comb. The frequency spacing was swept instantaneously in time and axisymmetrically about the center wavelength via a dual-drive Mach-Zehnder modulator driven by a variable radio frequency signal. This unique sweeping method permits direct measurement of fringe-free interferometric amplitude and phase with arbitrarily changeable measurement range and scan rate. Therefore, a compressive measurement can be made in only the depth region where the vibration exists, reducing the number of measurement points. In a proof-of-principle experiment, the interferometric amplitude and phase were investigated for in-phase and quadrature phase-shifted interferograms obtained by a polarization demodulator. Tomographic transient displacement measurements were performed using a 0.12 mm thick glass film and piezo-electric transducer oscillating at 10-100 kHz with scan rates in the range 1-20 MHz. The depth resolution and precision of the vibrometer were estimated to be approximately 25 µm and 1.0 nm, respectively.

Analysis of Pharmacokinetics in the Cochlea of the Inner Ear.

Hearing loss affects >5% of the global population and therefore, has a great social and clinical impact. Sensorineural hearing loss, which can be caused by different factors, such as acoustic trauma, aging, and administration of certain classes of drugs, stems primarily from a dysfunction of the cochlea in the inner ear. Few therapeutic strategies against sensorineural hearing loss are available. To develop effective treatments for this disease, it is crucial to precisely determine the behavior of ototoxic and therapeutic agents in the microenvironment of the cochlea in live animals. Since the 1980s, a number of studies have addressed this issue by different methodologies. However, there is much less information on pharmacokinetics in the cochlea than that in other organs; the delay in ontological pharmacology is likely due to technical difficulties with accessing the cochlea, a tiny organ that is encased with a bony wall and has a fine and complicated internal structure. In this review, we not only summarize the observations and insights obtained in classic and recent studies on pharmacokinetics in the cochlea but also describe relevant analytical techniques, with their strengths, limitations, and prospects.

Characterisation of the static offset in the travelling wave in the cochlear basal turn.

In mammals, audition is triggered by travelling waves that are evoked by acoustic stimuli in the cochlear partition, a structure containing sensory hair cells and a basilar membrane. When the cochlea is stimulated by a pure tone of low frequency, a static offset occurs in the vibration in the apical turn. In the high-frequency region at the cochlear base, multi-tone stimuli induce a quadratic distortion product in the vibrations that suggests the presence of an offset. However, vibrations below 100 Hz, including a static offset, have not been directly measured there. We therefore constructed an interferometer for detecting motion at low frequencies including 0 Hz. We applied the interferometer to record vibrations from the cochlear base of guinea pigs in response to pure tones. When the animals were exposed to sound at an intensity of 70 dB or higher, we recorded a static offset of the sinusoidally vibrating cochlear partition by more than 1 nm towards the scala vestibuli. The offset's magnitude grew monotonically as the stimuli intensified. When stimulus frequency was varied, the response peaked around the best frequency, the frequency that maximised the vibration amplitude at threshold sound pressure. These characteristics are consistent with those found in the low-frequency region and are therefore likely common across the cochlea. The offset diminished markedly when the somatic motility of mechanosensitive outer hair cells, the force-generating machinery that amplifies the sinusoidal vibrations, was pharmacologically blocked. Therefore, the partition offset appears to be linked to the electromotile contraction of outer hair cells.

In vivo tomographic visualization of intracochlear vibration using a supercontinuum multifrequency-swept optical coherence microscope.

This study combined a previously developed optical system with two additional key elements: a supercontinuum light source characterized by high output power and an analytical technique that effectively extracts interference signals required for improving the detection limit of vibration amplitude. Our system visualized 3D tomographic images and nanometer scale vibrations in the cochlear sensory epithelium of a live guinea pig. The transverse- and axial-depth resolution was 3.6 and 2.7 µm, respectively. After exposure to acoustic stimuli of 21-25 kHz at a sound pressure level of 70-85 dB, spatial amplitude and phase distributions were quantified on a targeted surface, whose area was 522 × 522 µm2.

Establishment of a simpler method for measuring HDL-microRNAs.

BACKGROUND: MicroRNAs are present not only in exosomes but also in high-density lipoprotein (HDL) and have the potential as biomarkers for various diseases. Various purification methods have been developed to quantify HDL-miRNAs; however, they are unsuitable for clinical applications. Therefore, we aimed to establish a simpler analytical method to quantify HDL-miRNAs for clinical applications. METHODS: We purified HDL fraction from pooled plasma using a three-step protocol consisting of ultracentrifugation, phosphotungstic acid/MgCl2 precipitation and desalting/buffer exchange followed by the quantification of HDL-miRNAs by quantitative real-time PCR. In order to establish a method to quantify HDL-miRNAs by quantitative real-time PCR, we prepared standard curves for miR-223 and miR-92. The HDL-miRNAs of 10 volunteers were assessed. RESULTS: Exosomes and LDL were not detected in the purified HDL fraction. Furthermore, we confirmed that only HDL was purified and that the HDL recovery rate of our method was at least approximately 50%. The threshold cycle values of miR-223, miR-92, miR-146a and miR-150 in the same subject were 32.11 ± 0.58, 32.50 ± 0.35, 34.30 ± 0.70 and 34.91 ± 0.77, respectively ( n = 10). The coefficient of variation values for these miRNAs were 1.08-2.21%. In addition, the standard curve for the quantitative analysis of miRNAs showed high linearity (30-30,000 copies/ µL) with a correlation coefficient of >0.99. The concentrations of HDL-miR-223 and HDL-miR-92 in the plasma of 10 subjects were 1.98 ± 0.32 and 0.90 ± 0.14 copies/mL (×104). CONCLUSIONS: We established a simple method for quantifying HDL-miRNAs and improved the sample processing capacity compared with conventional methods.

Maternal fructose intake disturbs ovarian estradiol synthesis in rats.

AIMS: Recent increases in fructose consumption have raised concerns regarding the potential adverse intergenerational effects, as maternal fructose intake may induce physiological dysfunction in offspring. However, no reports are available regarding the effect of excess maternal fructose on reproductive tissues such as the ovary. Notably, the maternal intrauterine environment has been demonstrated to affect ovarian development in the subsequent generation. Given the fructose is transferred to the fetus, excess fructose consumption may affect offspring ovarian development. As ovarian development and its function is maintained by 17ß-estradiol, we therefore investigated whether excess maternal fructose intake influences offspring ovarian estradiol synthesis. Rats received a 20% fructose solution during gestation and lactation. After weaning, offspring ovaries were isolated. KEY FINDINGS: Offspring from fructose-fed dams showed reduced StAR and P450(17α) mRNA levels, along with decreased protein expression levels. Conversely, attenuated P450arom protein level was found in the absence of mRNA expression alteration. Consistent with these phenomena, decreased circulating levels of estradiol were observed. Furthermore, estrogen receptor α (ERα) protein levels were also down-regulated. In accordance, the mRNA for progesterone receptor, a transcriptional target of ERα, was decreased. These results suggest that maternal fructose might alter ovarian physiology in the subsequent generation.

Hearing Loss Controlled by Optogenetic Stimulation of Nonexcitable Nonglial Cells in the Cochlea of the Inner Ear.

Light-gated ion channels and transporters have been applied to a broad array of excitable cells including neurons, cardiac myocytes, skeletal muscle cells and pancreatic ß-cells in an organism to clarify their physiological and pathological roles. Nonetheless, among nonexcitable cells, only glial cells have been studied in vivo by this approach. Here, by optogenetic stimulation of a different nonexcitable cell type in the cochlea of the inner ear, we induce and control hearing loss. To our knowledge, deafness animal models using optogenetics have not yet been established. Analysis of transgenic mice expressing channelrhodopsin-2 (ChR2) induced by an oligodendrocyte-specific promoter identified this channel in nonglial cells-melanocytes-of an epithelial-like tissue in the cochlea. The membrane potential of these cells underlies a highly positive potential in a K+-rich extracellular solution, endolymph; this electrical property is essential for hearing. Illumination of the cochlea to activate ChR2 and depolarize the melanocytes significantly impaired hearing within a few minutes, accompanied by a reduction in the endolymphatic potential. After cessation of the illumination, the hearing thresholds and potential returned to baseline during several minutes. These responses were replicable multiple times. ChR2 was also expressed in cochlear glial cells surrounding the neuronal components, but slight neural activation caused by the optical stimulation was unlikely to be involved in the hearing impairment. The acute-onset, reversible and repeatable phenotype, which is inaccessible to conventional gene-targeting and pharmacological approaches, seems to at least partially resemble the symptom in a population of patients with sensorineural hearing loss. Taken together, this mouse line may not only broaden applications of optogenetics but also contribute to the progress of translational research on deafness.

Multifrequency-swept optical coherence microscopy for highspeed full-field tomographic vibrometry in biological tissues.

Because conventional laser Doppler vibrometry or Doppler optical coherence tomography require mechanical scanning probes that cannot simultaneously measure the wide-range dynamics of bio-tissues, a multifrequency-swept optical coherence microscopy with wide-field heterodyne detection technique was developed. A 1024 × 1024 × 2000 voxel volume was acquired with an axial resolution of ~1.8 µm and an acquisition speed of 2 s. Vibration measurements at 10 kHz were performed over a wide field of view. Wide-field tomographic vibration measurements of a mouse tympanic membrane are demonstrated to illustrate the applicability of this method to live animals.

A microsensing system for the in vivo real-time detection of local drug kinetics.

Real-time recording of the kinetics of systemically administered drugs in in vivo microenvironments may accelerate the development of effective medical therapies. However, conventional methods require considerable analyte quantities, have low sampling rates and do not address how drug kinetics correlate with target function over time. Here, we describe the development and application of a drug-sensing system consisting of a glass microelectrode and a microsensor composed of boron-doped diamond with a tip of around 40 µm in diameter. We show that, in the guinea pig cochlea, the system can measure-simultaneously and in real time-changes in the concentration of bumetanide (a diuretic that is ototoxic but applicable to epilepsy treatment) and the endocochlear potential underlying hearing. In the rat brain, we tracked the kinetics of the drug and the local field potentials representing neuronal activity. We also show that the actions of the antiepileptic drug lamotrigine and the anticancer reagent doxorubicin can be monitored in vivo. Our microsensing system offers the potential to detect pharmacological and physiological responses that might otherwise remain undetected.

Vibrational stimulation induces osteoblast differentiation and the upregulation of osteogenic gene expression in vitro.

Vibrational stimulation is an accepted non-invasive method used to improve bone remodeling. However, the underlying mechanisms of this phenomenon remain unclear. In this study, we developed a new vibration-loading system to apply vibrational stimulation to cells based on a previously reported in vivo study. We hypothesized that osteoblasts respond to vibrational strain by expressing osteogenic marker genes, such as alkaline-phosphatase (ALP), Runx2, and Osterix. To test our hypothesis, we developed a vibration-loading system to apply a precise vibrational force to an osteoblast culture on a silicone membrane. The system regulated frequency and acceleration of the vibration, and strain on the silicone membrane culture surface was measured using the strain gauge method. After vibrational stimulation, cellular gene expression was analyzed using real-time polymerase chain reaction. We obtained clear strain signals from the culture surface at vibrational ranges of 1.0-10 m/s2 acceleration and frequencies of 30, 60, and 90 Hz, respectively. The strain increased in a linear fashion, depending on the acceleration magnitude. Vibrational stimulation also significantly upregulated expression of the osteogenic marker genes Runx2, Osterix, type I collagen, and ALP. In conclusion, we developed a new vibration-loading system that can precisely regulate frequency and acceleration, and we established the presence of dynamic cellular strain on a culture surface. Our findings suggest that vibrational stimulation may directly induce osteoblast differentiation.

The unique electrical properties in an extracellular fluid of the mammalian cochlea; their functional roles, homeostatic processes, and pathological significance.

The cochlea of the mammalian inner ear contains an endolymph that exhibits an endocochlear potential (EP) of +80 mV with a [K(+)] of 150 mM. This unusual extracellular solution is maintained by the cochlear lateral wall, a double-layered epithelial-like tissue. Acoustic stimuli allow endolymphatic K(+) to enter sensory hair cells and excite them. The positive EP accelerates this K(+) influx, thereby sensitizing hearing. K(+) exits from hair cells and circulates back to the lateral wall, which unidirectionally transports K(+) to the endolymph. In vivo electrophysiological assays demonstrated that the EP stems primarily from two K(+) diffusion potentials yielded by [K(+)] gradients between intracellular and extracellular compartments in the lateral wall. Such gradients seem to be controlled by ion channels and transporters expressed in particular membrane domains of the two layers. Analyses of human deafness genes and genetically modified mice suggested the contribution of these channels and transporters to EP and hearing. A computational model, which reconstitutes unidirectional K(+) transport by incorporating channels and transporters in the lateral wall and connects this transport to hair cell transcellular K(+) fluxes, simulates the circulation current flowing between the endolymph and the perilymph. In this model, modulation of the circulation current profile accounts for the processes leading to EP loss under pathological conditions. This article not only summarizes the unique physiological and molecular mechanisms underlying homeostasis of the EP and their pathological relevance but also describes the interplay between EP and circulation current.

The novel bisphosphonate disodium dihydrogen-4-[(methylthio) phenylthio] methanebisphosphonate increases bone mass in post-ovariectomy rats.

The novel bisphosphonate (BP) disodium dihydrogen-4-[(methylthio) phenylthio] methanebisphosphonate (MPMBP) is a non-nitrogen-containing BP with an antioxidant side chain that possesses anti-inflammatory properties. We investigated the systemic effects of this compound on bone loss induced by ovariectomy (OVX) in adult rats. Micro-computed tomography revealed that MPMBP increased bone mass and density in both the metaphysis and diaphysis, and improved the structural properties important for mechanical strength of osteoporotic bone. Sequential bone labeling with tetracycline and calcein indicated that MPMBP decreased longitudinal growth of the primary spongiosa (PS), but stimulated cortical bone formation in the diaphysis. MPMBP increased type I collagen accumulation in the PS, and decreased the number and size of adipocytes in the bone marrow, suggesting inhibition of increased bone marrow adipogenesis induced by OVX. Furthermore, MPMBP reduced the number of bone resorbing cathepsin K-positive osteoclasts induced by OVX. These results suggest that MPMBP could improve bone loss induced by estrogen deficiency. Both stimulation of bone formation and inhibition of bone resorption might play a role in the increase in bone mass and bone density after MPMBP treatment.