Perilymph Of Guinea Pigs Research Articles

Impact of Systemic versus Intratympanic Dexamethasone Administration on the Perilymph Proteome.

Glucocorticoids are the first-line treatment for sensorineural hearing loss, but little is known about the mechanism of their protective effect or the impact of route of administration. The recent development of hollow microneedles enables safe and reliable sampling of perilymph for proteomic analysis. Using these microneedles, we investigate the effect of intratympanic (IT) versus intraperitoneal (IP) dexamethasone administration on guinea pig perilymph proteome. Guinea pigs were treated with IT dexamethasone (n = 6), IP dexamethasone (n = 8), or untreated for control (n = 8) 6 h prior to aspiration. The round window membrane (RWM) was accessed via a postauricular approach, and hollow microneedles were used to perforate the RWM and aspirate 1 μL of perilymph. Perilymph samples were analyzed by liquid chromatography-mass spectrometry-based label-free quantitative proteomics. Mass spectrometry raw data files have been deposited in an international public repository (MassIVE proteomics repository at https://massive.ucsd.edu/) under data set # MSV000086887. In the 22 samples of perilymph analyzed, 632 proteins were detected, including the inner ear protein cochlin, a perilymph marker. Of these, 14 proteins were modulated by IP, and three proteins were modulated by IT dexamethasone. In both IP and IT dexamethasone groups, VGF nerve growth factor inducible was significantly upregulated compared to control. The remaining adjusted proteins modulate neurons, inflammation, or protein synthesis. Proteome analysis facilitated by the use of hollow microneedles shows that route of dexamethasone administration impacts changes seen in perilymph proteome. Compared to IT administration, the IP route was associated with greater changes in protein expression, including proteins involved in neuroprotection, inflammatory pathway, and protein synthesis. Our findings show that microneedles can mediate safe and effective intracochlear sampling and hold promise for inner ear diagnostics.

In‐depth proteome of perilymph in guinea pig model

The vast majority of sensorineural hearing loss is caused by impairment of the inner ear cells. Proteomic analysis of perilymph may therefore improve our understanding of inner ear diseases and hearing loss. However, the investigation of the human perilymph proteome was limited due to technical difficulties in perilymph sampling. The guinea pig (Cavia porcellus) is frequently used as an experimental model in preclinical hearing research. In this study, we analyzed samples of perilymph collected from 12 guinea pigs to overcome limited experimental information regarding its proteome. We identified a total of 1413 proteins, establishing a greatly expanded proteome of the previously inferred guinea pig perilymph. This provides a comprehensive proteomic resource for the research community, which will facilitate future molecular-phenotypic studies using the guinea pig as an experimental model of relevance to human inner ear biology.

An LCMS-based untargeted metabolomics protocol for cochlear perilymph: highlighting metabolic effects of hydrogen gas on the inner ear of noise exposed Guinea pigs

IntroductionNoise-induced hearing loss (NIHL) is an increasing problem in society and accounts for a third of all cases of acquired hearing loss. NIHL is caused by formation of reactive oxygen species (ROS) in the cochlea causing oxidative stress. Hydrogen gas (H2) can alleviate the damage caused by oxidative stress and can be easily administered through inhalation.ObjectivesTo present a protocol for untargeted metabolomics of guinea pig perilymph and investigate the effect of H2 administration on the perilymph metabolome of noise exposed guinea pigs.MethodsThe left ear of guinea pigs were exposed to hazardous impulse noise only (Noise, n = 10), noise and H2 (Noise + H2, n = 10), only H2 (H2, n = 4), or untreated (Control, n = 2). Scala tympani perilymph was sampled from the cochlea of both ears. The polar component of the perilymph metabolome was analyzed using a HILIC-UHPLC-Q-TOF–MS-based untargeted metabolomics protocol. Multivariate data analysis (MVDA) was performed separately for the exposed- and unexposed ear.ResultsMVDA allowed separation of groups Noise and Noise + H2 in both the exposed and unexposed ear and yielded 15 metabolites with differentiating relative abundances. Seven were found in both exposed and unexposed ear data and included two osmoprotectants. Eight metabolites were unique to the unexposed ear and included a number of short-chain acylcarnitines.ConclusionsA HILIC-UHPLC-Q-TOF–MS-based protocol for untargeted metabolomics of perilymph is presented and shown to be fit-for-purpose. We found a clear difference in the perilymph metabolome of noise exposed guinea pigs with and without H2 treatment.

Rapid analysis of neomycin in cochlear perilymph of guinea pigs using disposable SPE cartridges and high performance liquid chromatography-tandem mass spectrometry

Irreversible hearing loss induced by aminoglycoside in human through local or systemic administration route negatively impacts quality of life. The aim of this work was to develop and validate an analytical method suitable for the detection and quantification of neomycin in cochlear perilymph of guinea pig after local application. The SupelMIP SPE column was used for the pre-treatment of matrix. Chromatographic separation was conducted by a reversed phase ODS column (100 × 2.1 mm, 3 μm) at 40 °C in gradient mode with 0.2‰ (v/v) HFBA in water and 0.2‰ (v/v) HFBA in acetonitrile as mobile phase, at a flow rate of 0.30 mL/min, with retention time of 3.50 and 3.62 min for internal standard tobramycin and analyte neomycin, respectively. The MS was performed with positive ionization mode, with data acquisition in Multi Reaction Monitor (MRM) mode. This method was proved to be specific, accurate (97.1–115% of nominal values) and precise (CV% < 15%). Calibration curves for matrix matched standard of neomycin ranged from 1.25 to 200 μg/mL, with LOD and LLOQ of 0.625 and 1.25 μg/mL in blank matrix. The matrix effect was corrected to (−0.1) - 1.33 by adding internal standard. The relative SPE recovery values were ≥98.9% in low, medium and high QC samples. Neomycin in matrix proved to be stable under room temperature - and −20 °C, or under three freeze-thawing cycles, or under processing as well. Finally, the proposed method was successfully applied to a toxicokinetics study of neomycin in perilymph after round window membrane (RWM) administration, which was in accordance with threshold shift of auditory brainstem response (ABR) test related to hearing loss.

Pharmacokinetics of sodium thiosulfate in Guinea pig perilymph following middle ear application

HypothesisTo determine the pharmacokinetics of sodium thiosulfate in the inner ear perilymph following middle ear application in Guinea pigs. BackgroundCisplatin chemotherapy is often associated with a dose-dependent high frequency sensorineural hearing loss. Sodium thiosulfate has been shown to reduce cisplatin-induced ototoxicity when given intravenously, but this may limit the tumoricidal effects of the chemotherapy. Recent animal studies looking at middle ear application of sodium thiosulfate have shown prevention of outer hair cell and hearing loss, but the perilymph pharmacokinetics have not yet been established. MethodsTwenty Guinea pig ears were split into two groups and administered sodium thiosulfate to the middle ear at either a concentration of 250 mg/mL or 50 mg/mL for 30 min. Perilymph samples were then obtained serially through the round window over 6 h. Sodium thiosulfate concentrations were obtained using high-pressure liquid chromatography. ResultsThe 250 mg/mL group had a maximum perilymph concentration of 7.27 mg/mL (±0.83) that decreased to 0.94 mg/mL (±0.03) over 6 h. The 50 mg/mL group had an initial concentration of 1.63 mg/mL (±0.17) and was undetectable after 1 h. The half-life of sodium thiosulfate within perilymph was 0.74 h. Conclusionsand Relevance: The results of this study show that sodium thiosulfate is capable of diffusing through round window and into the inner ear perilymph. Peak levels decline over several hours after exposure. This has a potential application as a localized therapy in the prevention of cisplatin induced ototoxicity.

Perilymph pharmacokinetics of marker applied through a cochlear implant in guinea pigs.

Patients undergoing cochlear implantation could benefit from a simultaneous application of drugs into the ear, helping preserve residual low-frequency hearing and afferent nerve fiber populations. One way to apply drugs is to incorporate a cannula into the implant, through which drug solution is driven. For such an approach, perilymph concentrations achieved and the distribution in the ear over time have not previously been documented. We used FITC-labeled dextran as a marker, delivering it into perilymph of guinea pigs at 10 or 100 nL/min though a cannula incorporated into a cochlear implant with the outlet in the mid basal turn. After injections of varying duration (2 hours, 1 day or 7 days) perilymph was collected from the cochlear apex using a sequential sampling technique, allowing dextran levels and gradients along scala tympani to be quantified. Data were interpreted quantitatively using computer simulations of the experiments. For injections of 2 hours duration, dextran levels were critically influenced by the presence or absence of fluid leakage at the cochleostomy site. When the cochleostomy was fluid-tight, substantially higher perilymph levels were achieved at the injection site, with concentration declining along scala tympani towards the apex. Contrary to expectations, large dextran gradients along scala tympani persisted after 24 hours of sustained injection and were still present in some animals after 7 days injection. Functional changes associated with implantation and dextran delivery, and the histological state of the implant and cannula were also documented. The persistent longitudinal gradients of dextan along the ear were not readily explained by computer simulations of the experiments based on prior pharmacokinetic data. One explanation is that inner ear pharmacokinetics are altered in the period after cochlear implantation, possibly by a permeabilization of the blood-labyrinth barrier as part of the immune response to the implant.

Protein cross linker enzymes and food applications

Irreversible hearing loss induced by aminoglycoside in human through local or systemic administration route negatively impacts quality of life. The aim of this work was to develop and validate an analytical method suitable for the detection and quantification of neomycin in cochlear perilymph of guinea pig after local application. The SupelMIP SPE column was used for the pre-treatment of matrix. Chromatographic separation was conducted by a reversed phase ODS column (100 × 2.1 mm, 3 μm) at 40 °C in gradient mode with 0.2‰ (v/v) HFBA in water and 0.2‰ (v/v) HFBA in acetonitrile as mobile phase, at a flow rate of 0.30 mL/min, with retention time of 3.50 and 3.62 min for internal standard tobramycin and analyte neomycin, respectively. The MS was performed with positive ionization mode, with data acquisition in Multi Reaction Monitor (MRM) mode. This method was proved to be specific, accurate (97.1–115% of nominal values) and precise (CV% < 15%). Calibration curves for matrix matched standard of neomycin ranged from 1.25 to 200 μg/mL, with LOD and LLOQ of 0.625 and 1.25 μg/mL in blank matrix. The matrix effect was corrected to (−0.1) - 1.33 by adding internal standard. The relative SPE recovery values were ≥98.9% in low, medium and high QC samples. Neomycin in matrix proved to be stable under room temperature - and −20 °C, or under three freeze-thawing cycles, or under processing as well. Finally, the proposed method was successfully applied to a toxicokinetics study of neomycin in perilymph after round window membrane (RWM) administration, which was in accordance with threshold shift of auditory brainstem response (ABR) test related to hearing loss.

In Vivo Electrochemical Monitoring of the Change of Cochlear Perilymph Ascorbate during Salicylate-Induced Tinnitus

As one of the most important neurochemicals in biological systems, ascorbate plays vital roles in many physiological and pathological processes. In order to understand the roles of ascorbate in the pathological process of tinnitus, this study demonstrates an in vivo method for real time monitoring of the changes of ascorbate level in the cochlear perilymph of guinea pigs during the acute period of tinnitus induced by local microinfusion of salicylate with carbon fiber microelectrodes (CFMEs) modified with multiwalled carbon nanotubes (MWNTs). To accomplish in vivo electrochemical monitoring of ascorbate in the microenvironment of the cochlear perilymph, the MWNT-modified CFME is used as working electrode, a microsized Ag/AgCl is used as reference electrode, and Pt wire is used as counter electrode. Three electrodes are combined together around a capillary to form integrated capillary-electrodes. The integrated capillary-electrode is carefully implanted into the cochlear perilymph of guinea pigs and used both for externally microinfusing of salicylate into the cochlear perilymph and for real time monitoring of the change of ascorbate levels. The in vivo voltammetric method based on the integrated capillary-electrodes possesses a high selectivity and a good linearity for ascorbate determination in the cochlear perilymph of guinea pigs. With such a method, the basal level of cochlear perilymph ascorbate is determined to be 45.0 ± 5.1 μM (n = 6). The microinfusion of 10 mM salicylate (1 μL/min, 5 min) into the cochlear decreases the ascorbate level to 28 ± 10% of the basal level (n = 6) with a statistical significance (P < 0.05), implying that the decrease in ascorbate level in the cochlear may be associated with salicylate-induced tinnitus. This study essentially offers a new method for in vivo monitoring of the cochlear perilymph ascorbate following the salicylate-induced tinnitus and can thus be useful for investigation on chemical essences involved in tinnitus.

Contents of amino acids and their conversions in the guinea pig's perilymph

Contents of amino acids and their conversions in the guinea pig's perilymph

Transport of aspirin and sodium salicylate to perilymph of guinea pig

Transport of aspirin and sodium salicylate to perilymph of guinea pig

Entry of Substances Into Perilymph Through the Bone of the Otic Capsule After Intratympanic Applications in Guinea Pigs

Drugs applied to the middle ear enter perilymph through the bony otic capsule. Drugs applied intratympanically in humans are thought to enter the cochlea primarily through the round window membrane (RWM). Local drug treatments of the ear are commonly evaluated in rodent models. The otic capsule is much thinner at the cochlear apex in rodents than in humans. We therefore investigated whether drugs applied to the middle ear could enter perilymph through the otic capsule as well as through the RWM. The distribution of gentamicin and the marker trimethylphenylammonium (TMPA) along the guinea pig cochlea was assessed with sequential apical perilymph sampling after 2 delivery paradigms that included 1) completely filling the tympanic bulla with solution and 2) applying the solution to the RWM only. In addition, TMPA entry into perilymph of the third turn was measured with ion-selective electrodes after the bulla was filled with TMPA solution. In application protocols that allowed drug to contact the otic capsule (by completely filling the bulla), markedly higher drug concentrations were found in the apical, low-frequency regions of the cochlea compared with drug applications to the RWM only. Gentamicin and TMPA can enter perilymph of guinea pigs through the RWM and simultaneously through the bony otic capsule. Drug distribution along the cochlea after intratympanic applications will therefore be dramatically different in rodents and humans. Results obtained from intratympanic drug treatments of animals, in which the bulla is filled with solution and contacts the bony capsule of the cochlea, do not provide a good model for the situation in humans.

Dexamethasone Pharmacokinetics in Guinea Pig Inner Ear Perilymph

Aim: To study the dexamethasone pharmacokinetics in the inner ear perilymph of guinea pigs using high-pressure liquid chromatography. Methods: Sixty-five guinea pigs were divided into three groups. In the first group, the drug application protocol used an intra-abdominal dose of 0.5% dexamethasone 4 mg·kg^–1. In the second group, an intratympanic application dose of 0.5% dexamethasone 150 µl was used. The third group was the control group. The concentrations of dexamethasone in inner ear perilymph were determined by high-pressure liquid chromatography. Results: The perilymph concentration-time curves of dexamethasone conformed to a one-compartment open model after an intra-abdominal application. The C_maxwas 0.927 ± 0.008 mg·l^–1, the T_max1.47 ± 0.04 h, the T_1/2K 2.92 ± 0.056 h, the AUC 5.533 ± 0.05 mg·h·l^–1, the T_1/2Ka 0.47 ± 0.024 h. After an intratympanic application, the perilymph concentration-time curves of dexamethasone also conformed to a one-compartment open model. The C_maxwas 0.201 ± 0.006 mg·l^–1, the T_max 0.117 ± 0.06 h, the AUC 0.868 ± 0.004 mg·h·l^–1, the T_1/2K 2.918 ± 0.089 h, the T_1/2Ka 0.161 ± 0.009 h. Compared to the intra-abdominal application, the intratympanic application resulted in similar levels of inner ear perilymph drug concentrations in 30 min. Conclusion: Dexamethasone can penetrate the blood-labyrinthine barrier after intra-abdominal application. Dexamethasone can enter into perilymph after intratympanic application. Under the condition of the study, the intratympanic application resulted in a similar level of inner ear perilymph drug concentrations compared to the intra-abdominal application in 30 min.

Perilymph sampling from the cochlear apex: A reliable method to obtain higher purity perilymph samples from scala tympani

Measurements of drug levels in the fluids of the inner ear are required to establish kinetic parameters and to determine the influence of specific local delivery protocols. For most substances, this requires cochlear fluids samples to be obtained for analysis. When auditory function is of primary interest, the drug level in the perilymph of scala tympani (ST) is most relevant, since drug in this scala has ready access to the auditory sensory cells. In many prior studies, ST perilymph samples have been obtained from the basal turn, either by aspiration through the round window membrane (RWM) or through an opening in the bony wall. A number of studies have demonstrated that such samples are likely to be contaminated with cerebrospinal fluid (CSF). CSF enters the basal turn of ST through the cochlear aqueduct when the bony capsule is perforated or when fluid is aspirated. The degree of sample contamination has, however, not been widely appreciated. Recent studies have shown that perilymph samples taken through the round window membrane are highly contaminated with CSF, with samples greater than 2 μL in volume containing more CSF than perilymph. In spite of this knowledge, many groups continue to sample from the base of the cochlea, as it is a well-established method. We have developed an alternative, technically simple method to increase the proportion of ST perilymph in a fluid sample. The sample is taken from the apex of the cochlea, a site that is distant from the cochlear aqueduct. A previous problem with sampling through a perforation in the bone was that the native perilymph rapidly leaked out driven by CSF pressure and was lost to the middle ear space. We therefore developed a procedure to collect all the fluid that emerged from the perforated apex after perforation. We evaluated the method using a marker ion trimethylphenylammonium (TMPA). TMPA was applied to the perilymph of guinea pigs either by RW irrigation or by microinjection into the apical turn. The TMPA concentration of the fluid sample was compared with that measured in perilymph prior to taking the sample using a TMPA-selective microelectrode sealed into ST. Data were interpreted with a finite element model of the cochlear fluids that was used to simulate each aspect of the experiment. The correction of sample concentration back to the perilymph concentration prior to sampling can be performed based on the known ST volume (4.7 μL in the guinea pig) and the sample volume. A more precise correction requires some knowledge of the profile of drug distribution along the cochlear prior to sampling. This method of sampling from the apex is technically simple and provides a larger sample volume with a greater proportion of perilymph compared to sampling through the RW.

Hydroxyl radical formation in the perilymph of asphyxic guinea pig

To elucidate the role of hydroxyl radical ( OH) species in the generation mechanism of the cochlear pathology induced by transient asphyxia and subsequent re-ventilation, the concentrations of 2,3-hydroxybenzoic acid (DHBA) and 2,5-DHBA, major products arising from the attack of OH upon salicylate, were measured in the perilymph of the guinea pig by the high performance liquid chromatography–electrochemical/UV method. The mean value of 2,3-DHBA concentration in the perilymph significantly increased from the pre-asphyxic level (6.4 μM) to 7.6 μM and 8.8 μM during asphyxia of 3 min duration and at 5 min after the onset of re-ventilation, respectively. The 2,5-DHBA concentration was 7.9 μM before asphyxia, and also significantly increased to 11.5 μM and 16.2 μM during and after asphyxia, respectively. These results strongly indicated that OH was generated in the perilymph of the asphyxic and re-ventilated guinea pig cochlea, and the significance of this increased OH in generating anoxia and re-perfusion injury is discussed with respect to iron and oxygen-derived free radicals.

Morphological and physiological effects of long duration infusion of strychnine into the organ of Corti.

Acute strychnine administration has long been used as a method to eliminate the effects of efferent activity. It has been shown that long after termination of chronic strychnine infusion into the cochlea, the ear becomes more susceptible to acoustic trauma suggesting that chronic strychnine infusion results in long lasting or permanent disruption of efferent function. Much research has been directed towards the functional significance of the olivocochlear system. However, there is little information concerning the effect of long duration inactivation of the medial olivocochlear system in an awake behaving animal. This study was designed to determine the structural and functional consequences of inactivation of the efferents by chronic infusion of strychnine into the cochlear perilymph of guinea pigs for two weeks via an osmotic pump. Physiological evaluations showed that the strychnine infusion eliminated the efferent induced reduction of the cochlear whole-nerve action potential three weeks after cessation of strychnine infusion. Contralateral efferent function remained unaltered. Histological evaluation at the light and electron microscopic levels revealed disoriented efferent synapses under the outer hair cells.

Transportation of Salicylate to Perilymph of Guinea Pig

Transportation of Salicylate to Perilymph of Guinea Pig

外リンパへの陰イオン透過性 ペニシリンＧ濃度定量

The concentrations of benzylpenicillin (penicillin G; PG) in sequential samples from the scala tympani (ST), scala vestibule (SV), cerebrospinal fluid (CSF) and serum of guinea pigs after the intravenous injection of 200mg/kg PG were measured by highperformance liquid chromatography. We also investigated PG concentrations in the perilymph of guinea pigs pretreated with the competitive organic anion transport inhibitor, probenecid (PR).We found a pronounced difference in the clearance curve of PG between ST and SV. Two hundred mg/kg of PR significantly increased the half-life time of PG in the serum, and also increased the PG concentration in the CSF. Pretreatment with the same dosage of PR inhibited the accumulation of PG in the ST and abolished the peaking of PG. This curve was very similar to that in the SV. PG concentrations in the SV were unaffected by pretreatment with PR. These data are analogous to the results of the furosemide experiments reported by Hara et al (1987).Our data suggest a difference in the anion transport mechanism between ST and SV.

フロセミドに対する血液‐内耳関門の透過性に関する実験的研究

To clarify the characteristics and the control of the blood-cochlea barrier, we measured EP (the endocochlear potential) and the furosemide concentration in the perilymph of guinea pigs. It was recognised that: 1) glycerol increased the toxicity of furosemide against the EP in adults, 2) the toxicity of furosemide against EP was stronger in neonates than in adults, 3) glycerol did not increase furosemide toxicity against EP in neonates, 4) acute hypertension also increased the ototoxicity of furosemide, 5) glycerol increased furosemide concentration in the perilymph.These findings suggest that glycerol facilitates the permeability of blood-cochlea barrier to furosemide. Therefore, it is assumed that glycerol and acute hypertension can facilitate the access of some drugs used to treat inner ear diseases.

Effect of hypertonic solutions on oxygen tension in the cochlear perilymph

The effect of glycerol on oxygen tension was studied in the cochlear perilymph of guinea pigs with a polarographic technique. Following a transient fall immediately after the intravenous administration of glycerol, the oxygen tension increased to reach a peak 10 minutes later, and then decreased gradually. These changes suggest alterations of the cochlear blood flow due to the local vasodilating effect of glycerol. The increase of oxygen tension and blood flow in the cochlea is considered to be one of the possible contributory factors in the improvement of hearing in patients with Meniere's disease after glycerol administration.

Effect of pentobarbital anesthesia on amikacin concentrations in plasma and perilymph and evaluation of multiple sampling in perilymph of guinea pigs.

The purpose of this study was to determine whether a multiple-sampling procedure could be used in guinea pigs to study the kinetics of amikacin in perilymph. Amikacin was infused intravenously for 6 h into conscious anesthetized guinea pigs, and the concentrations of the drug in plasma and perilymph were measured. From each anesthetized guinea pig, five to six perilymph samples were collected from one ear, and one sample was collected from the other ear at 6 h. The concentrations of amikacin in perilymph were dose proportional and increased slowly during the 6-h infusion. However, after 6 h of intravenous infusion, the concentrations of amikacin in perilymph of the multiply sampled ears were significantly higher than those of the singly sampled ears, indicating that the multiple-sampling procedure should not be used as is to study the kinetics of amikacin in perilymph. Amikacin concentrations in perilymph were linearly related to amikacin concentrations in plasma in pentobarbital-anesthetized animals, as had previously been observed for conscious guinea pigs. However, the slope of the regression line was only 0.09 for anesthetized animals compared with 0.24 for conscious animals. Drug concentrations in plasma were found to be threefold higher in anesthetized animals, whereas drug levels in perilymph were the same in both groups at similar dosing rates. These results indicate that the amikacin concentration in perilymph is not solely dependent upon its concentration in plasma and that other factor(s) can affect the entry of amikacin into the inner ear.