Guinea Pig Cochlea Research Articles

A 3D Microscaffold Cochlear Electrode Array for Steroid Elution.

In cochlear implants, the electrode insertion trauma during surgery can cause damage residual hearing. Preserving the residual hearing is an important challenge and the localized administration of drugs, such as steroids, is one of the most promising ways, but remains a challenge. Here, a microscaffold cochlear electrode array (MiSCEA) consisting of a microfabricated flexible electrode array and a 3D microscaffold for steroid reservoir is reported. The MiSCEA without loaded drug is tested by measuring the electrically evoked auditory brainstem response of the cochlea in guinea pigs (n = 4). The scaffold is then coated with steroid (dexamethasone) encapsulated in polylactic-co-glycolic acid and the continuous release of the steroid into artificial perilymph during six weeks is monitored. The steroid-containing scaffolds are then implanted into guinea pigs (n = 4) and threshold shifts are analyzed for four weeks by measuring the acoustically evoked auditory brainstem response. The threshold shifts tend to be lower in the group implanted with the steroid-containing MiSCEAs. The feasibility of 3D MiSCEA opens up the development of potential next-generation cochlear electrode with improved steroid release dynamics into cochlea.

Early Alterations of Endothelial Nitric Oxide Synthase Expression Patterns in the Guinea Pig Cochlea After Noise Exposure

Constitutively expressed endothelial nitric oxide synthase (eNOS) is supposed to play a role in noise-induced nitric oxide (NO)-production. It is commonly known that intense noise exposure results in inducible NOS (iNOS) expression and increased NO-production, but knowledge about a contribution of the eNOS isoform is still lacking. Effects of noise exposure on eNOS immunolabeling were determined in male guinea pigs (n=24). For light microscopic analysis, 11 animals were exposed to 90 dB for 1 hr and 6 animals were used as controls. After exposure, eNOS immunostaining was performed on paraffin sections, and the staining intensities were quantified for 4 cochlear regions. For electron microscopic analysis, 2 animals were exposed for 2 hr to 90 dB and 5 animals were used as controls. The intensity of eNOS immunolabeling was found to be already comprehensively increased 1 hr after noise exposure to 90 dB. At the ultrastructural level, a clear increase in eNOS immunolabeling was found in microtubules-rich areas of cochlear cuticular structures. Hence, our findings indicate that the reticular lamina forming the endolymph-perilymph barrier at the apical side of the organ of Corti is involved in a fast intrinsic otoprotective mechanism of the cochlea.

Zinc-finger protein A20 protects hair cells from damage made by high-power microwave

Abstract Inner ear hair cells are important for maintaining hearing. Irreversible damage to hair cells is an important cause of sensorineural deafness. Electromagnetic radiation, especially high-power microwave, is an important threat to human health in modern society and war. However, it is not clear whether high-power microwave has an effect on cochlea hair cells. This study aimed to assess the effects of high-power microwave on cochlear hair cells in guinea pigs, and investigate the potential protection of these cells against high-power microwave-induced damage by recombinant adenovirus A20. Based on experimental results, a 65 W/cm2 irradiation density applied to guinea pigs in this study to establish a high-power microwave inner ear injury model. In addition, pAdEeay-1/A20 was injected via a round window into experimental guinea pig cochlea, whereas artificial perilymph was injected into the control group. Auditory function was assessed by testing the auditory brainstem response threshold, and damage to cochlear hair cells was investigated by cell counting and scanning electron microscopy observations of the basilar membrane. Inner ear injury was observed 6hours after 65 W/cm2 of irradiation and the auditory brainstem response threshold was significantly higher in the irradiation group (P < 0.05) compared with other groups. Propidium iodide staining and scanning electron microscopy results indicated that significant morphological changes occurred after radiation, especially to inner hair cells, which exhibited remarkable damage and the presence of several unknown spherical substances. Auditory brainstem response threshold was decreased in the pAdEeay-1/A20 group compared with the artificial perilymph group; moreover, damage to hair cells was milder in the pAdEeay-1/A20 group compared with the control group (P < 0.01). Thus, high-power microwave can cause damage to cochlear hair cells, as well as hearing loss with prolonged exposure and/or high dosage. In this regard, 65 W/cm2 of irradiation for 6hours is a reliable target dose for observation of damage. The zinc finger protein A20 can protect cochlear hair cells from high-power microwave-induced damage and prevent further hearing loss. This study was approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University, China on April 18, 2017.

Expression pattern of Connexin 26 and Connexin 30 in mature cochlea of the monkey

Connexin26 (Cx26) and Cx30 are the predominant connexin subtypes found in the cochlea. They play an essential role in the cochlear functions. However, most studies use mice and the data on the cochlear expression profiles of the two Cxs in higher animals (e.g., humans) are scarce. Studies using the cochleae from non-human primate other than mice may provide information needed to narrow this gap. Here we studied cellular distributions of Cx26 and Cx30 in the adult monkey and guinea pig cochleae by immunofluorescent labeling and confocal microscopy observations. We detected Cx26 and Cx30 expressions in the type I, II& V fibrocytes in the spiral ligament, fibrocytes of the spiral limbus, in the supporting cells of organ of Corti, inner and outer sulcus cells, and in the basal cells of the stria vascularis. Both Cx26 and Cx30 were not detected in hair cells, in mesenchymal cells under the basilar membrane and cells lining the scala vestibule. Cells of the Reissner's membrane and spiral ganglion neurons are also negative. These findings demonstrate that cochlear expressions of Cx26 and Cx30 in the adult mouse, guinea pig and non-human primate have a common cellular pattern.

Computational Simulation Expands Understanding of Electrotransfer-Based Gene Augmentation for Enhancement of Neural Interfaces

The neural interface is a critical factor in governing efficient and safe charge transfer between a stimulating electrode and biological tissue. The interface plays a crucial role in the efficacy of electric stimulation in chronic implants and both electromechanical properties and biological properties shape this. In the case of cochlear implants, it has long been recognized that neurotrophins can stimulate growth of the target auditory nerve fibers into a favorable apposition with the electrode array, and recently such arrays have been re-purposed to enable electrotransfer (electroporation)-based neurotrophin gene augmentation to improve the “bionic ear.” For both this acute bionic array-directed electroporation and for chronic conventional cochlear implant arrays, the electric fields generated in target tissue during pulse delivery are central to efficacy, but are challenging to map. We present a computational model for predicting electric fields generated by array-driven DNA electrotransfer in the cochlea. The anatomically realistic model geometry was reconstructed from magnetic resonance images of the guinea pig cochlea and an eight-channel electrode array embedded within this geometry. The model incorporates a description of both Faradaic and non-Faradaic mechanisms occurring at the electrode-electrolyte interface with frequency dependency optimized to match experimental impedance spectrometry measurements. Our simulations predict that a tandem electrode configuration with four ganged cathodes and four ganged anodes produces three to fourfold larger area in target tissue where the electric field is within the range for successful gene transfer compared to an alternate paired anode-cathode electrode configuration. These findings matched in vivo transfection efficacy of a green fluorescent protein (GFP) reporter following array-driven electrotransfer of the reporter-encoding plasmid DNA. This confirms utility of the developed model as a tool to optimize the safety and efficacy of electrotransfer protocols for delivery of neurotrophin growth factors, with the ultimate aim of using gene augmentation approaches to improve the characteristics of the electrode-neural interfaces in chronically implanted neurostimulation devices.

Age‑associated variation in the expression and function of TMEM16A calcium‑activated chloride channels in the cochlear stria vascularis of guinea pigs.

The present study was designed to investigate the expression and function of transmembrane protein 16 (TMEM16A), a calcium-activated chloride channel (CaCC), in the stria vascularis (SV) of the cochlea of guinea pigs at different ages, and to understand the role of CaCCs in the pathogenesis of presbycusis (age-related hearing loss), the most common type of sensorineural hearing loss that occurs with natural aging. Guinea pigs were divided into the following groups: 2 weeks (young group), 3 months (youth group), 1 year (adult group), D-galactose intervention (D-gal group; aging model induced by subcutaneous injection of D-galactose) and T16Ainh-A01 (intraperitoneal injection of 50 µg/kg/day TMEM16A inhibitor T16Ainh-A01 for 2 weeks). Differences in the hearing of guinea pigs between the various age groups were analyzed using auditory brainstem response (ABR), and immunofluorescence staining was performed to detect TMEM16A expression in the SV and determine the distribution. Reverse transcription-quantitative PCR and western blot analyses were conducted to detect the mRNA and protein levels of TMEM16A in SV in the different age groups. Morris water maze behavior analysis demonstrated that spatial learning ability and memory were damaged in the D-gal group. Superoxide dismutase activity and malondialdehyde content assays indicated that there was oxidative stress damage in the D-gal group. The ABR thresholds gradually increased with age, and the increase in the T16Ainh-A01 group was pronounced. Immunofluorescence analysis in the cochlear SV of guinea pigs in different groups revealed that expression of TMEM16A increased with increasing age (2 weeks to 1 year); fluorescence intensity was reduced in the D-gal model of aging. As the guinea pigs continued to mature, the protein and mRNA contents of TMEM16A in the cochlea SV increased gradually, but were decreased in the D-gal group. The findings indicated that CaCCs in the cochlear SV of guinea pigs were associated with the development of hearing in guinea pigs, and that downregulation of TMEM16A may be associated with age-associated hearing loss.

Intracochlear PLGA based implants for dexamethasone release: Challenges and solutions

The effective treatment of diseases of the inner ear is currently an unmet medical need. Local controlled drug delivery to the cochlea is challenging due to the hidden location, small volume and high sensitivity of this organ. A local intracochlear delivery of drugs would avoid the problems of intratympanic (extracochlear) drug application, but is more invasive. The requirements for such a delivery system include a small size and appropriate flexibility. The delivery device must be rigid enough for surgical handling but also flexible to avoid traumatizing cochlear structures. We developed biodegradable dexamethasone loaded PLGA extrudates for the controlled intracochlear release. In order to achieve the desired flexibility, Polyethylene glycol (PEG) was used as a plasticizer. In addition to the drug release, the extrudates were characterized in vitro by differential scanning calorimetry (DSC) and texture analysis. Simulation of the pharmacokinetics of the inner ear support the expectation that a constant perilymph drug level is obtained after few hours and retained over several weeks. Ex vivo implantation of the extrudates into a guinea pig cochlea indicate that PEG containing extrudates have the desired balance between mechanical strength and flexibility for direct implantation into the cochlea. The location of the implant was visualized by computer tomography. In summary, we postulate that intracochlear administration of drug releasing biodegradable implants is a new and promising approach to achieve local drug delivery to the cochlea for an extended time.

Different rates of endocytic activity and vesicle transport from the apical and synaptic poles of the outer hair cell.

BackgroundIntense endocytic activity at the apex of outer hair cells (OHCs)—the electromechanical cells of the cochlea—has been demonstrated using the vital plasma-membrane marker FM1-43 and confocal laser-scanning microscopy. Vesicular traffic toward the cell nucleus to distinct locations of the endoplasmic reticulum has also been shown.ObjectiveThe current study characterizes the dynamics of endocytic activity, as well as apicobasal and basoapical trafficking, using a local perfusion technique that we recently developed and published to visualize bidirectional trafficking in isolated bipolar cells.Materials and methodsThe fluorescent plasma-membrane markers FM1-43 (10 µM) and FM4-64 (10 µM), together with a fluid-phase marker, Lucifer yellow (50 µM), were used to label endocytosed vesicles in isolated OHCs of the guinea pig cochlea. Targets of endocytosed vesicles were examined with a fluorescent marker of subsurface cisternae, DiOC6 (0.87 µM). Single- and two-photon confocal laser-scanning microscopy was used to visualize labeled vesicles.ResultsThe plasma-membrane markers presented more intense vesicle internalization at the synaptic pole than at the apical pole of the OHC. Intracellular basoapical vesicle trafficking was faster than apicobasal trafficking. Vesicles endocytosed at the synaptic pole were transcytosed to the endoplasmic reticulum system. An intracellular Lucifer yellow signal was not detected.ConclusionThe larger endocytic fluorescent signals in the synaptic pole and the faster basoapical trafficking imply that membrane internalization and vesicle trafficking are more efficient at the synaptic pole than at the apical pole of the OHC.

Drug Diffusion Along an Intact Mammalian Cochlea.

Intratympanic drug administration depends on the ability of drugs to pass through the round window membrane (RW) at the base of the cochlea and diffuse from this location to the apex. While the RW permeability for many different drugs can be promoted, passive diffusion along the narrowing spiral of the cochlea is limited. Earlier measurements of the distribution of marker ions, corticosteroids, and antibiotics demonstrated that the concentration of substances applied to the RW was two to three orders of magnitude higher in the base compared to the apex. The measurements, however, involved perforating the cochlear bony wall and, in some cases, sampling perilymph. These manipulations can change the flow rate of perilymph and lead to intake of perilymph through the cochlear aqueduct, thereby disguising concentration gradients of the delivered substances. In this study, the suppressive effect of salicylate on cochlear amplification via block of the outer hair cell (OHC) somatic motility was utilized to assess salicylate diffusion along an intact guinea pig cochlea in vivo. Salicylate solution was applied to the RW and threshold elevation of auditory nerve responses was measured at different times and frequencies after application. Resultant concentrations of salicylate along the cochlea were calculated by fitting the experimental data using a mathematical model of the diffusion and clearing of salicylate in a tube of variable diameter combined with a model describing salicylate action on cochlear amplification. Concentrations reach a steady-state at different times for different cochlear locations and it takes longer to reach the steady-state at more apical locations. Even at the steady-state, the predicted concentration at the apex is negligible. Model predictions for the geometry of the longer human cochlea show even higher differences in the steady-state concentrations of the drugs between cochlear base and apex. Our findings confirm conclusions that achieving therapeutic drug concentrations throughout the entire cochlear duct is hardly possible when the drugs are applied to the RW and are distributed via passive diffusion. Assisted methods of drug delivery are needed to reach a more uniform distribution of drugs along the cochlea.

Expression and localization of diacylglycerol kinase ζ in guinea pig cochlea and its functional implication under noise-exposure stress conditions.

Cochlear hair cells are essential for the mechanotransduction of hearing. Sensorineural hearing loss can be irreversible because hair cells have a minimal ability to repair or regenerate themselves once damaged. In order to develop therapeutic interventions to prevent hair cell loss, it is necessary to understand the signaling pathway operating in cochlear hair cells and its alteration upon damage. Diacylglycerol kinase (DGK) regulates intracellular signal transduction through phosphorylation of lipidic second messenger diacylglycerol. We have previously reported characteristic expression and localization patterns of DGKs in various organs under pathophysiological conditions. Nevertheless, little is known about morphological and functional aspects of this enzyme family in the cochlea. First RT-PCR analysis reveals predominant mRNA expression of DGKα, DGKε and DGKζ. Immunohistochemical analysis shows that DGKζ localizes to the nuclei of inner hair cells (IHCs), outer hair cells (OHCs), supporting cells and spiral ganglion neurons in guinea pig cochlea under normal conditions. It is well known that loud noise exposure induces cochlear damage, thereby resulting in hair cell loss. In particular, OHCs are highly vulnerable to noise exposure than IHCs. We found that after 1week of noise exposure DGKζ translocates from the nucleus to the cytoplasm in damage-sensitive OHCs and gradually disappears thereafter. In sharp contrast, DGKζ remains to the nucleus in damage-resistant IHCs. These results suggest that DGKζ cytoplasmic translocation is well correlated with cellular damage under noise-exposure stress conditions and is involved in delayed cell death in cochlear outer hair cells.

Voltage readout from a piezoelectric intracochlear acoustic transducer implanted in a living guinea pig

The ability to measure the voltage readout from a sensor implanted inside the living cochlea enables continuous monitoring of intracochlear acoustic pressure locally, which could improve cochlear implants. We developed a piezoelectric intracochlear acoustic transducer (PIAT) designed to sense the acoustic pressure while fully implanted inside a living guinea pig cochlea. The PIAT, fabricated using micro-electro-mechanical systems (MEMS) techniques, consisted of an array of four piezoelectric cantilevers with varying lengths to enhance sensitivity across a wide frequency bandwidth. Prior to implantation, benchtop tests were conducted to characterize the device performance in air and in water. When implanted in the cochlea of an anesthetized guinea pig, the in vivo voltage response from the PIAT was measured in response to 80–95 dB sound pressure level 1–14 kHz sinusoidal acoustic excitation at the entrance of the guinea pig’s ear canal. All sensed signals were above the noise floor and unaffected by crosstalk from the cochlear microphonic or external electrical interference. These results demonstrate that external acoustic stimulus can be sensed via the piezoelectric voltage response of the implanted MEMS transducer inside the living cochlea, providing key steps towards developing intracochlear acoustic sensors to replace external or subcutaneous microphones for auditory prosthetics.

Nano, micro, and macro-scale effects on cochlear tuning

Reconciling the highly tuned and nonlinear basilar membrane (BM) response at the base with the nearly low-pass and weakly nonlinear response at the apex has presented a longstanding challenge to cochlear mechanics modelers. Recent experiments have shown that the BM centric view of cochlear mechanics is incomplete and have highlighted the importance of modeling and measuring the dynamics of the organ of Corti (OoC). Here, we describe a new computational model of the guinea pig cochlea that can correctly simulate the response at all frequencies. The model shows that the electromotile force from the outer hair cells modulate the differential motion between the reticular lamina and the BM. Model calculations at the apex show that the geometric taper of the scalae duct as well as the cytoarchitecture of the OoC breaks the scaling symmetry observed at the base. Further, the model predicts that the neural tuning at the base is primarily governed by the macroscopic dynamics of the cochlear partition, while the micro-scale fluid dynamics and the nano-scale channel dynamics dominate the neural tuning at the apex. Overall, the model provides a physiological explanation for the differences between high and low frequency hearing observed in psychophysical experiments. [Work supported by NIH-R01-04084.]

Pharmacokinetics and tissue distribution of neurotrophin 3 after intracochlear delivery

Neurotrophin therapy has potential to reverse some forms of hearing loss. However, cochlear pharmacokinetic studies are challenging due to small fluid volumes. Here a radioactive tracer was used to determine neurotrophin-3 retention, distribution and clearance after intracochlear administration. 125I-neurotrophin-3 was injected into guinea pig cochleae using a sealed injection technique comparing dosing volumes, rates and concentrations up to 750 μg/mL. Retention was measured by whole-cochlear gamma counts at five time points while distribution and clearance were assessed by autoradiography. Smaller injection volumes and higher concentrations correlated with higher retention of neurotrophin-3. Distribution of neurotrophin-3 was widespread throughout the cochlear tissue, decreasing in concentration from base to apex. Tissue distribution was non-uniform, with greatest density in cells lining the scala tympani and lower density in neural target tissue. The time constant for clearance of neurotrophin-3 from cochlear tissues was 38 h but neurotrophin-3 remained detectable for at least 2 weeks. Neurotrophin-3 was evident in the semi-circular canals with minor spread to the contralateral cochlea. This study is the first comprehensive evaluation of the disposition profile for a protein therapy in the cochlea. The findings and methods in this study will provide valuable guidance for the development of protein therapies for hearing loss.

Imaging Bioluminescent Exogenous Stem Cells in the Intact Guinea Pig Cochlea.

ABSTRACTStem‐cell‐based therapy may be used to replace damaged or lost neurons in the cochlear nerve of patients suffering from severe‐to‐profound sensorineural hearing loss. In order to achieve functional recovery in future clinical trials, knowledge about survival of grafted cells and their differentiation into functional neurons is a prerequisite. This calls for non‐invasive in vivo visualization of cells and long‐term monitoring of their survival and fate after cochlear transplantation. We have investigated if molecular optical imaging enables visualization of exogenous cells in the intact cochlea of guinea pig cadaver heads. Transduced (stem) cells, stably co‐expressing fluorescent (copGFP) and bioluminescent (Luc2) reporter molecules, were injected into the internal auditory meatus or directly into the cochlea through the round window. After injection of the cells into the internal auditory meatus, a bright bioluminescent signal was observed in the cavum conchae of the auricle, indicating that light generated by Luc2 is passing through the tympanic membrane and the external auditory meatus. Similar results were obtained after injection of the cells through the round window membrane, either directly into the scala tympani or in Rosenthal's canal within the modiolus of the basal cochlear turn. Imaging of the auditory bulla demonstrated that the bioluminescent signal passes through the tympanic membrane and crevices in the bony wall of the bulla. After opening the auditory bulla, the bioluminescent signal was emanating from the round window. This is the first study demonstrating that bioluminescence imaging enables visualization of luciferase‐expressing cells injected into the intact guinea pig cochlea. Anat Rec, 303:427–440, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Use of scanning electron microscopy in the cochlea of guinea pigs

IntroductionThe use of electron microscopy in the study of the inner ear has allowed us to observe minute details of the hair cells, especially in ototoxicity studies; however, the preparation of this material is a difficult and delicate task. In an attempt to simplify the handling of these materials, two agents, toluidine blue and ethylenediamine tetra-acetic acid were tested, in addition to the elimination of osmium tetroxide during the preparation of albino guinea pig cochleae. We also tested the applicability of these methodologies in an ototoxicity protocol. ObjectiveTo verify the quality of the images obtained with and without the use of ethylenediamine tetra-acetic acid, toluidine blue and osmium tetroxide in the preparation of cochleae of albino guinea pigs for the scanning electron microscopy. MethodsThree groups of cochleae were used. In Group 1, 10 cochleae were prepared with the usual methodology, dissecting the optical capsule without decalcification and using osmium tetroxide as a post-fixative agent. In Group 2, we prepared 10 cochleae decalcified with ethylenediamine tetra-acetic acid, injecting toluidine blue in the endolymphatic space to facilitate the identification of the organ of Corti. In Group 3, we used 4 cochleae of guinea pigs that received 3 doses of cisplatin (7.5mg/kg, D1–D5–D6), two prepared according to the methodology used in Group 1 and two with that used in Group 2. Scanning electron microscopy images were obtained from the organ of Corti region of the basal turn of each cochlea. ResultsThe organ of Corti was more easily identified with the use of toluidine blue. The dissection of the cochlea was more accurate in the decalcified cochleae. The quality of the images and the preservation of the organ of Corti obtained with the two methodologies were similar. ConclusionThe proposed modifications resulted in images of similar quality as those observed using the traditional methodology.

Photoacoustic effect invokes auditory response in infrared neuron stimulation

Infrared neuron stimulation is regarded as an innovative approach for stimulating cochleae in animals while the exact mechanism still remains unknown. In this paper, we studied compound action potentials of guinea pig cochleae with chronic or acute deafness. We recorded optical compound action potentials and analyzed stretched cochlear preparations by fluorescence microscopy. Photoacoustic signals were measured by hydrophone and microphone, respectively. In our experiment, we observed a switch response effect in vitro and in vivo experiments. Therefore, we proposed photoacoustic effect could invoke auditory response in infrared neuron stimulation.

Development of in vivo drug sensing system with needle-type diamond microelectrode

Continuous and real-time measurement of local concentrations of systemically administered drugs in vivo must be crucial for pharmacological studies. Nevertheless, conventional methods require considerable samples quantity and have poor sampling rates. Additionally, they cannot determine how drug kinetics correlates with target function over time. Here, we describe a system with two different sensors. One is a needle-type microsensor composed of boron-doped diamond with a tip of ~40 μm in diameter, and the other is a glass microelectrode. We first tested bumetanide. This diuretic can induce deafness. In the guinea-pig cochlea injected intravenously with bumetanide, the changes of the drug concentration and the extracellular potential underlying hearing were simultaneously measured in real time. We further examined an antiepileptic drug lamotrigine in the rat brain, and tracked its kinetics and at the same time the local field potentials representing neuronal activity. The action of the anticancer reagent doxorubicin was also monitored in the cochlea. This microsensing system may be applied to analyze pharmacokinetics and pharmacodynamics of various drugs at local sites in vivo, and contribute to promoting the pharmacological researches.

Development of <i>in vivo</i> drug sensing system with needle-type diamond microelectrode.

Continuous and real-time measurement of local concentrations of systemically administered drugs in vivo must be crucial for pharmacological studies. Nevertheless, conventional methods require considerable samples quantity and have poor sampling rates. Additionally, they cannot determine how drug kinetics correlates with target function over time. Here, we describe a system with two different sensors. One is a needle-type microsensor composed of boron-doped diamond with a tip of ~40 μm in diameter, and the other is a glass microelectrode. We first tested bumetanide. This diuretic can induce deafness. In the guinea-pig cochlea injected intravenously with bumetanide, the changes of the drug concentration and the extracellular potential underlying hearing were simultaneously measured in real time. We further examined an antiepileptic drug lamotrigine in the rat brain, and tracked its kinetics and at the same time the local field potentials representing neuronal activity. The action of the anticancer reagent doxorubicin was also monitored in the cochlea. This microsensing system may be applied to analyze pharmacokinetics and pharmacodynamics of various drugs at local sites in vivo, and contribute to promoting the pharmacological researches.

Primary Cultivation and Identification of Vascular Smooth Muscle Cells from the Spiral Modiolar Artery of Guinea Pigs.

BackgroundThis article reports a method to obtain vascular smooth muscle cells (SMCs) from the spiral modiolar artery (SMA) of guinea pigs and provides materials for related experimental studies.Material/MethodsSMA was separated from the cochlea of guinea pigs, digested with trypsin (1.25 g/L) and allowed to adhere in a 35-mm culture dish. The morphology of the sample was investigated, and the sample was identified by immunofluorescence analysis, flow cytometry, Western blot, and RT-PCR. Cell viability was calculated using trypan blue and flow cytometry. Whole-cell patch clamp was used to record the membrane input resistance (Rinput), reciprocal membrane input conductance (Ginput), membrane input capacitance (Cinput), and resting membrane potential (RP) of the SMCs.ResultsMicroscopy results showed that the cells had typical peak–valley growth pattern. The cell growth curve was similar to an S curve, and flow cytometry results showed that the cell apoptosis rate was less than 10%. Moreover, flow cytometry, immunofluorescent staining, Western blot and RT-PCR detected the specific and intensely positive expression of cell type-specific markers α-SM-actin, SM22α, calponin and desmin. Furthermore, following properties of the P3 and P6 cells were obtained: Rinput, 2611±356 and 2477±338 MΩ; Ginput, 0.454±0.071 and 0.273±0.037 ns; Cinput, 17.029±0.917 and 18.042±1.051 pF, and RP −20.602±1.503 and −22.192±1.905 mV.ConclusionsVarious highly purified SMCs were obtained from the SMA of guinea pigs. We provide an ideal experimental material for the study of the pathogenesis of diseases related to the circulation disturbances in the inner ear in vitro. The results can be used to evaluate the effects of drugs on vascular smooth muscle.

Unusual mechanical processing of sounds at the apex of the Guinea pig cochlea

One of the tenets of mammalian auditory physiology is that the frequency selectivity at the cochlear base decreases as a function of stimulus level. Changes in frequency selectivity have been shown to be accompanied by changes in response phases as a function of stimulus level. The existence of such nonlinear properties has been revealed by the analysis of either direct or indirect recordings of mechanical vibrations of the cochlea. Direct measurements of cochlear mechanical vibrations, however, have been carried out with success primarily in cochlear regions that are tuned to frequencies >7 kHz, but not in regions sensitive to lower frequencies. In this paper we continue to analyze recently published data from measurements of sound-induced vibrations at four locations near the apex of the intact guinea pig cochlea, in a region encompassing approximately 25% of its total length. Analysis of the responses at all locations reveal level-dependent phase properties that are rather different from those usually reported at the base of the cochlea of laboratory animals such as the chinchilla. Cochlear group delays, for example, increase or remain constant with increasing stimulus. Similarly, frequency selectivity at all the regions increases as a function of stimulus level.