Takamasa Yoshida

The mechanism underlying maintenance of the endocochlear potential by the K+ transport system in fibrocytes of the inner ear

•  The endocochlear potential (EP) of +80 mV in cochlear endolymph is essential for audition and controlled by K+ transport across the lateral cochlear wall composed of two epithelial barrier layers, the syncytium containing the fibrocytes and the marginal cells. •  The EP depends upon the diffusion potential elicited by a large K+ gradient across the apical surface of the syncytium. •  We examined by electrophysiological approaches an involvement of Na+,K+‐ATPase, which occurs at the syncytiums basolateral surface comprising the fibrocytes’ membranes and would mediate K+ transport across the lateral wall, in maintenance of the EP. •  We show that the Na+,K+‐ATPase sustains the syncytiums high [K+] that is crucial for the K+ gradient across the apical surface of the syncytium. •  The results help us better understand the mechanism underlying the establishment of the EP as well as the pathophysiological process for deafness induced by dysfunction of the ion transport apparatus.

Management of chylous fistula after neck dissection using negative‐pressure wound therapy: A preliminary report

Chylous fistula is a distressing complication resulting from thoracic duct injury during neck dissections. We have successfully managed chylous fistula using negative‐pressure wound therapy (NPWT) in a case where all conservative treatments failed. A 60‐year‐old man with tongue cancer underwent subtotal glossectomy and bilateral neck dissections. On postoperative day 4, a chylous fistula with large drainage developed in the right neck. Conservative treatments were not effective, therefore, NPWT was started from postoperative day 9. The drainage volume then began to decrease, and the chylous fistula was closed 6 days after starting NPWT. In our case, the effects of wound shrinkage and fluid removal by NPWT were considered to contribute to early closure. Although preliminary, NPWT can be an important treatment choice for the management of a chylous fistula after neck dissections.

A minimally invasive method to prevent postlaryngectomy major pharyngocutaneous fistula using infrahyoid myofascial flap

INTRODUCTION To prevent postoperative pharyngocutaneous fistula (PCF) after total (pharyngo)laryngectomy, simultaneous coverage of pharyngeal anastomosis with vascularised flaps such as pectoralis major muscle, anterolateral thigh or radial forearm, has been reported to be effective. As an alternative to the invasive methods using distant flaps, we used the infrahyoid myofascial flap (IHMFF), which was harvested from the same operation field of (pharyngo)laryngectomy, for covering the site of pharyngeal anastomosis. Herein, we describe the safety and effectiveness of our minimally invasive method for preventing PCF. METHODS Eleven patients who were at a high risk of developing PCF due to previous chemoradiotherapy underwent simultaneous coverage of pharyngeal anastomosis with IHMFF after total (pharyngo)laryngectomy. The incidence of PCF and the rate of major fistula requiring surgical closure were determined, and the results were compared with the control group (23 patients without IHMFF cover after laryngectomy). RESULTS PCF developed in 2 of the 11 patients (18.2%). The fistulae of these two patients were closed conservatively and did not require additional surgery. PCF developed in 6 of 23 patients (26.1%) in patients without IHMFF cover. All the six patients with fistula required additional closure surgery. The incidence of PCF did not differ in patients with or without IHMFF cover (Fishers exact probability test; p=0.939, NS). However, the rate of major PCF requiring surgical closure was significantly lower in patients with IHMFF cover (Fishers exact probability test; p=0.036<0.05). CONCLUSIONS For (pharyngo)laryngectomy patients, IHMFF cover is a minimally invasive method that can prevent major PCF.

Molecular architecture of the stria vascularis membrane transport system, which is essential for physiological functions of the mammalian cochlea

Stria vascularis of the mammalian cochlea transports K+ to establish the electrochemical property in the endolymph crucial for hearing. This epithelial tissue also transports various small molecules. To clarify the profile of proteins participating in the transport system in the stria vascularis, membrane components purified from the stria of adult rats were analysed by liquid chromatography tandem mass spectrometry. Of the 3236 proteins detected in the analysis, 1807 were membrane proteins. Ingenuity Knowledge Base and literature data identified 513 proteins as being expressed on the ‘plasma membrane’, these included 25 ion channels and 79 transporters. Sixteen of the former and 62 of the latter had not yet been identified in the stria. Unexpectedly, many Cl− and Ca2+ transport systems were found, suggesting that the dynamics of these ions play multiple roles. Several transporters for organic substances were also detected. Network analysis demonstrated that a few kinases, including protein kinase A, and Ca2+ were key regulators for the strial transports. In the library of channels and transporters, 19 new candidates for uncloned deafness‐related genes were identified. These resources provide a platform for understanding the molecular mechanisms underlying the epithelial transport essential for cochlear function and the pathophysiological processes involved in hearing disorders.

Effect of salicylate on potassium currents in inner hair cells isolated from guinea-pig cochlea

Although salicylate is one of the most widely used nonsteroidal anti-inflammatory drugs, it causes moderate hearing loss and tinnitus at high-dose levels. In the present study, salicylate effects on the K currents in inner hair cells were examined. Salicylate reversibly reduced the outward K currents (I(K,f)), but did not affect the inward current (I(K,n)). Salicylate blocked the outward K currents in a concentration-dependent manner according to Hill equation with a half-blocking concentration of 1.66mM, and the Hill coefficient of 1.86.

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

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.Sensory neuroscience: let ion run through a cochlear labyrinthIn 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.

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.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.

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 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.

Diagnosis and following up of Ménière’s disease using multifrequency tympanometry—Cutoff values and temporal changes in measurements

OBJECTIVE This study aimed to verify cutoff values for G width (the width of bimodal peaks for the waveform obtained when measuring conductance at 2000Hz) in Japanese individuals diagnosed with Ménières disease (MD) using multifrequency tympanometry (MFT) and to determine the relationship between the G width and ability to hear low-pitched sounds using measurements over time. METHODS The study included 51 patients with clinically diagnosed MD, who had not undergone endolymphatic sac surgery, but had no other known ear disease (57 ears in patients aged 22-80 years were affected, and 45 ears in patients aged 18-83 years were unaffected; mean age: 53.3±16.9 years). We also enlisted 80 healthy controls with no prior history of ear disease (160 ears, aged 22-76 years, mean age: 40.8±15.7 years). MFT was used to measure the bimodal peak width of the waveform obtained when measuring conductance at resonance frequency of 2000Hz. For patients who had G width measured several times over multiple outpatient visits, we used initial test data to analyze cutoff values. In nine cases with four or more measurements over time, we evaluated a possible correlation between G width and the sum of the hearing threshold for three low-pitched frequencies (125Hz, 250Hz, and 500Hz). We used Students t-test to determine significance. RESULTS The both ears in the MD patients had a G width wider than the distribution in the control group. There was a significant difference between G width in the control group and in affected ears with MD (p=0.00026) and there was also a significant difference between G width in the control group and in unaffected ears of MD patients (p=0.0056). The cutoff value set with a specificity of 95% was 200daPa, with a sensitivity of 35.1% and specificity of 95.6%. The cutoff value set with a sensitivity of 50% was 140daPa, with sensitivity of 50.9% and specificity of 78.8%. There was no significant difference between resonance frequency of ears in the control group and ears with MD (p=0.41). In nine cases with four or more measurements over time, a case showed a statistically significant positive correlation between the G width and hearing ability threshold for low-pitched sounds (125Hz, 250Hz, and 500Hz) (p=0.03), while an another case showed a tendency toward a positive correlation, which was not statistically significant (p=0.08). Further, there were cases that did not show significant differences in the present study, but might have shown a negative correlation if the number of measurements had been increased. CONCLUSION Measurement of G width using MFT may have accuracy as the traditional endolymphatic hydrops test. MFT is non-invasive, causes little discomfort for patients, requires little time to perform, and can be performed by paramedics. MFT was shown to be useful in screening for MD and it is effective in diagnosing MD to measure the change over time of G width using MFT.

Theoretical and experimental analysis of ototoxic mechanism in the spiral ligament fibrocytes by multi-level simulation with ion transports in the cochlea

生 体 機 能 の 多 階 層 的 理 解 と 創 薬 研 究 へ の 応 用 2 要約:空気の振動である音は,内耳蝸牛に存在する 音の感覚細胞である有毛細胞を振動させる.この時, 有毛細胞の毛に存在するイオンチャネルが開口し, 常時+80 mVを示す特殊な内リンパ液からイオンが流 入する.この「内リンパ液高電位」は,有毛細胞の興 奮に不可欠であり,蝸牛側壁の血管条が成立させる. Na,K,2Cl共輸送体(NKCC)や Na,K-ATPaseの 阻害薬は,内リンパ液高電位を低下させることで,薬剤 性難聴を惹起することが報告されているが,その電位 低下のメカニズムは明らかにされていなかった.我々 はこれまでに,血管条に発現する NKCCと Na,KATPaseが制御する内リンパ液高電位成立機構を電気 生理学的手法により示し,さらに蝸牛内の多階層イオ ン輸送モデル「Nin-Hibino-Kurachi(NHK)model」の 構築とコンピュータシミュレーションによって,阻害 薬を経動脈的に投与した時に起こる内リンパ液高電 位低下のメカニズムを説明した.血管条に隣接し,そ の一部と一体化しているらせん靭帯を構成する線維 細胞にも,NKCCと Na,K-ATPaseが発現している ことが知られているが,薬剤性難聴時のこれらの関与 は不明である.近年我々は,らせん靭帯ではおもに Na,K-ATPaseが,K輸送と K濃度バランスに寄与 すること,そしてNKCCはほとんど機能していないこ とを明らかにした.これらに基づいて,NHKモデル を改訂した.今後シミュレーションを行うことにより, 輸送体阻害薬の経動脈投与の実験結果を正確に再現す ることが期待される. 1. はじめに