Showing papers by "Hiroshi Hibino published in 2017"

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

Abstract: 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. A system consisting of a glass microelectrode and a boron-doped diamond microsensor can simultaneously track, in rat brains and in the guinea pig cochlea, the local real-time kinetics of injected drugs and the resulting electrophysiological activity.

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

Abstract: 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.

Computer modeling defines the system driving a constant current crucial for homeostasis in the mammalian cochlea by integrating unique ion transports.

Abstract: The cochlear lateral wall—an epithelial-like tissue comprising inner and outer layers—maintains +80 mV in endolymph. This endocochlear potential supports hearing and represents the sum of all membrane potentials across apical and basolateral surfaces of both layers. The apical surfaces are governed by K+ equilibrium potentials. Underlying extracellular and intracellular [K+] is likely controlled by the “circulation current,” which crosses the two layers and unidirectionally flows throughout the cochlea. This idea was conceptually reinforced by our computational model integrating ion channels and transporters; however, contribution of the outer layer’s basolateral surface remains unclear. Recent experiments showed that this basolateral surface transports K+ using Na+, K+-ATPases and an unusual characteristic of greater permeability to Na+ than to other ions. To determine whether and how these machineries are involved in the circulation current, we used an in silico approach. In our updated model, the outer layer’s basolateral surface was provided with only Na+, K+-ATPases, Na+ conductance, and leak conductance. Under normal conditions, the circulation current was assumed to consist of K+ and be driven predominantly by Na+, K+-ATPases. The model replicated the experimentally measured electrochemical properties in all compartments of the lateral wall, and endocochlear potential, under normal conditions and during blocking of Na+, K+-ATPases. Therefore, the circulation current across the outer layer’s basolateral surface depends primarily on the three ion transport mechanisms. During the blockage, the reduced circulation current partially consisted of transiently evoked Na+ flow via the two conductances. This work defines the comprehensive system driving the circulation current. In in vivo mammalian cochlea, ionic current constantly and unidirectionally flows—this unique “circulation current”, which contributes to high sensitivity of sensory cells transducing atomic scale acoustic vibrations to electrical signals, likely depends upon ion transports across a multiple-layered epithelial tissue. To determine how the circulation current is established, a team conducted by Hiroshi Hibino at Niigata University in Japan used a theoretical approach, because ionic currents are unmeasurable in vivo. A conceptual computational model they previously developed lacked involvement of an epithelial tissue membrane recently found to show unusual ion transport profile; integration and coupling of this element to other membrane transport machineries resulted in reproducing experimental measurements. This work defines the comprehensive system driving the circulation current, which remains uncertain for nearly 20 years, and helps us to understand the mechanism for hearing.

An approach to the research on ion and water properties in the interphase between the plasma membrane and bulk extracellular solution

Abstract: The article An approach to the research on ion and water properties in the interphase between the plasma membrane and bulk extracellular solution, written by Hiroshi Hibino, Madoka Takai, Hidenori Noguchi, Seishiro Sawamura, Yasufumi Takahashi, Hideki Sakai and Hitoshi Shiku, was originally published Online First without open access.

Time-controllable Nkcc1 knockdown replicates reversible hearing loss in postnatal mice

Abstract: Identification of the causal effects of specific proteins on recurrent and partially reversible hearing loss has been difficult because of the lack of an animal model that provides reversible gene knockdown. We have developed the transgenic mouse line Actin-tTS::Nkcc1 tetO/tetO for manipulatable expression of the cochlear K+ circulation protein, NKCC1. Nkcc1 transcription was blocked by the binding of a tetracycline-dependent transcriptional silencer to the tetracycline operator sequences inserted upstream of the Nkcc1 translation initiation site. Administration of the tetracycline derivative doxycycline reversibly regulated Nkcc1 knockdown. Progeny from pregnant/lactating mothers fed doxycycline-free chow from embryonic day 0 showed strong suppression of Nkcc1 expression (~90% downregulation) and Nkcc1 null phenotypes at postnatal day 35 (P35). P35 transgenic mice from mothers fed doxycycline-free chow starting at P0 (delivery) showed weaker suppression of Nkcc1 expression (~70% downregulation) and less hearing loss with mild cochlear structural changes. Treatment of these mice at P35 with doxycycline for 2 weeks reactivated Nkcc1 transcription to control levels and improved hearing level at high frequency; i.e., these doxycycline-treated mice exhibited partially reversible hearing loss. Thus, development of the Actin-tTS::Nkcc1 tetO/tetO transgenic mouse line provides a mouse model for the study of variable hearing loss through reversible knockdown of Nkcc1.

Hearing loss controlled by optogenetic stimulation of nonexcitable nonglial cells in the cochlea of the inner ear

Abstract: 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 β-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.

Fibrocytes in the cochlea of the mammalian inner ear: their molecular architecture, physiological properties, and pathological relevance

Abstract: Fibroblasts are a cell type that dominates connective tissues in a broad array of organs and plays key roles in formation of the extracellular matrix and wound healing. The cochlea of the mammalian inner ear harbors loose connective tissues such as the spiral ligament and spiral limbus, and their cellular components are called “fibrocytes.” The fibrocytes in the ligament are functionally differentiated and specialized for ion transport that is essential for proper actions of the cochlea. Molecular biological and histological assays have shown that these cells express specific types of ion channels and transporters. Results of in vivo electrophysiological experiments have integrated activities of individual channels and transporters into the ionic flow that circulates throughout the organ and maintains the electrochemical properties in various tissues and extracellular fluids. Moreover, analyses of deafness genes in humans as well as transgenic experiments on mice recently revealed the relevance of fibrocyte dysfunction to hearing disorders. In this review article, we not only describe molecular architecture and physiological and pathological significance of cochlear fibrocytes but also provide insights into next-generation therapies targeting these cells.