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{"project":"2_test","denotations":[{"id":"15320950-11152663-12070953","span":{"begin":228,"end":230},"obj":"11152663"},{"id":"15320950-11152663-12070954","span":{"begin":3389,"end":3391},"obj":"11152663"},{"id":"15320950-10079033-12070955","span":{"begin":4494,"end":4496},"obj":"10079033"},{"id":"15320950-10079033-12070956","span":{"begin":5570,"end":5572},"obj":"10079033"},{"id":"15320950-11717350-12070957","span":{"begin":6337,"end":6339},"obj":"11717350"},{"id":"15320950-11788352-12070958","span":{"begin":7059,"end":7061},"obj":"11788352"}],"text":"Methods\nThe endocochlear potential and the endolymphatic and perilymphatic K+ concentrations were measured in young adult mice (1–4 month of age) that either have (Slc26a4+/+) or lack (Slc26a4-/-) a functional gene for pendrin [14]. The mouse strain 129Sv/Ev (Taconic, Germantown, NY) was chosen as the source of Slc26a4+/+ controls, since Slc26a4-/- mice were propagated in the this strain and generated using a stem cell line derived from 129Sv/Ev. Slc26a4+/+ and Slc26a4-/- were agouti. They did not differ in coat color. Differences in pigmentation were verified using coisogenic age-matched Slc26a4+/+ and Slc26a4-/- that were bred in parallel.\nExpression of key proteins involved in the generation of the endocochlear potential and the transport of K+ was studied using confocal immunocytochemistry and quantitative RT-PCR. K+ secretion and the generation of the endocochlear potential were measured using electrophysiological techniques. All experiments were approved by the Institutional Animal Care and Use Committee of Kansas State University.\n\nConfocal immunocytochemistry\nAnimals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.) and transcardially perfused with a Cl--free phosphate-buffered Na-gluconate solution containing 4% paraformaldehyde. Temporal bones were removed and cochleae fixed by perilymphatic perfusion, decalcified in EDTA, processed through a sucrose gradient and infiltrated with polyethylene glycol. Mid-modiolar cryosections (12 μm, CM3050S, Leica, Nussloch, Germany) were blocked in PBS with 0.2% Triton-X (PBS-TX) and 10% bovine serum albumin. Slides were incubated overnight at 4°C with primary antibodies in PBS-TX with 1–3% BSA [rabbit anti-pendrin, 1:500 (h766–780); rat anti-ZO-1, 1:100 (Chemicon, Temecula, CA); goat anti-KCNQ1, 1:200 (C20, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-KCNE1, 1:200 (Alomone, Jerusalem, Israel); rabbit anti-KCNJ10, 1:300 (Alomone); rabbit anti-SLC12A2, 1:100 (Chemicon); and rabbit anti-connexin 26, 1:100 (Zymed, San Francisco, CA)]. Slides were washed in PBS-TX and incubated for 1 h at 25°C with appropriate secondary antibodies at a 1:1000 dilution in PBS-TX with 1–3% BSA [donkey anti-rabbit Alexa 488, chicken anti-rat Alexa 594, and chicken anti-goat Alexa 594 (Molecular Probes, Eugene, OR)]. Actin filaments were visualized by staining with Alexa 488 conjugated phalloidin (Molecular Probes). After incubation, slides were washed with PBS-TX, mounted with FluorSave (Calbiochem, La Jolla, CA), and viewed by confocal microscopy (LSM 5 Pascal or LSM 510 Meta, Carl Zeiss, Jena or Göttingen, Germany). Laser scanning brightfield images were collected to document structural preservation, for morphometric analysis and for analysis of pigmentation.\n\nQuantitative RT-PCR\nAnimals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.). Kidneys and brain were removed and pulverized in liquid N2. Temporal bones were removed and two preparations of inner ear tissues were obtained by microdissection: (1) stria vascularis without spiral ligament, and (2) spiral ganglia including the surrounding bone and the organ of Corti. The dissection medium was changed twice during the microdissection and isolated tissues were washed three times to minimize cross-contamination. The Slc26a4-/-genotype was verified by the observation of large otoconia in the utricular macula [14]. Total RNA was isolated and residual DNA contamination was removed by enzymatic digestion (RNeasy micro, Qiagen, Valencia, CA). Quality and quantity of 18S rRNA obtained from kidneys and brain were determined (Nano Assay, 2100 Bioanalyzer, Agilent, Palo Alto, CA). Further, the quality of RNA preparations obtained from stria vascularis and spiral ganglia was assessed (Pico Assay, 2100 Bioanalyzer). Real time RT-PCR was performed on RNA obtained from individual animals (OneStep RT-PCR, Qiagen; Smart Cycler, Cepheid, Sunnyvale, CA) in the presence of 0.2× SYBR green I (Molecular Probes). Transcripts of 18S rRNA and mRNA for the K+ channels KCNJ10, KCNQ1 and KCNQ4 were amplified using gene-specific primers (Table 1). RT was performed for 30 min at 50°C and 15 min at 95°C. PCR consisted of 50 cycles of 1 min at 60°C, 1 min at 72°C, 7s heating to hot-measurement temperature, 13s hot-measurement at 3–5°C below product melting temperature (Table 1) and 1 min at 94°C. Hot-measurements were performed to eliminate the detection of primer-dimers that had melting temperatures between 72 and 75°C [15]. PCR was followed by a melt (60 to 95°C). The generation of a single product of appropriate size was routinely checked by the presence of a single melt peak and by agarose gel-electrophoresis. Product identity was confirmed by sequencing.\nTable 1 Primers\nTemplate Primers Product Length Melting Temperature Hot Measurement Fidelity n\n18S gag gtt cga aga cga tca ga (sense) 316 bp 83.2 ± 02°C 78°C 1.89 ± 0.02 17\ntcg ctc cac caa cta aga ac (antisense)\nKCNJ10 tgg tgt ggt gtg gta tct gg (sense) 411 bp 83.2 ± 02°C 78°C 1.86 ± 0.02 15\ntga agc agt ttg cct gtc ac (antisense)\nKCNQ1 ttt gtt cat ccc cat ctc ag (sense) 239 bp 82.5 ± 02°C 79°C 1.85 ± 0.02 17\ngtt gct ggg tag gaa gag (antisense)\nKCNQ4 ccc gga aac cct tct gtg tc (sense) 245 bp 83.2 ± 02°C 80°C 1.87 ± 0.01 17\naaa gat gag cac cag gaa cc (antisense) Template molecules (T) were quantified according to T = P / (F ^Ct) where P is the number of product molecules, F is the fidelity of the reaction and Ct is the cycle at which the number of product molecules reaches a chosen threshold [15]. Fidelity (F) was obtained from the slope (S) of the log-linear phase of the growth curve via a best-fit fifth-order polynomial: F = 7.39 + 3.80 × S + 1.05 × S2 + 0.15 × S3 + 11.38 × 10-3 * S4 + 3.39 × 10-4 × S5. The number of product molecules at threshold (PCt) was determined by amplification of known amounts of 18S rRNA according to PCt = T × F ^Ct. Quantifications of 18S rRNA were used to compare tissue amounts. Genomic contamination of inner ear samples was assessed to be \u003c0.02% by omission of the RT step.\n\nIn vitro electrophysiology\nAnimals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.). Stria vascularis without spiral ligament was obtained by microdissection. Currents generated by the stria marginal epithelium were recorded [16]. A Pt-Ir wire microelectrode with a Pt-black tip was positioned 20–30 μm from the apical surface of the epithelium and vibrated at 200–400 Hz by piezo-electric bimorph elements (Applicable Electronics, Forest Dale, MA; ASET version 1.05, Science Wares, East Falmouth, MA). A Pt-black electrode (26-gauge) served as reference in the bath chamber. The signals from the phase-sensitive detectors were digitized (16 bit) at a rate of 0.5 Hz. The output was expressed as current density at the electrode.\n\nIn situ electrophysiology\nAnimals were anesthetized with inactin (thiobutabarbital sodium salt, 140 mg/kg ip). The endocochlear potential and the endolymphatic [K+] were measured with double-barreled microelectrodes [17]. Measurements were made in the basal turn by a round-window approach through the basilar membrane and in the apical turn after thinning the bone over the stria vascularis and picking a small hole (~30 μm). K+-selective electrodes were calibrated in solutions of constant cation (K+ and Na+) concentration of 150 mM. The minor selectivity of the K+ electrodes for Na+ produced a nonlinearity in the calibration curve, which was closely fitted by the Nicolski equation using nonlinear curve-fitting software (OriginLab, Northampton, MA): V = Vi + S × log ([K+] + A × [Na+]), where Vi is an offset term, S is slope, A is selectivity, and [Na+] is Na+ concentration. Calibrations were made immediately after withdrawal of the electrodes from the cochlea. Plasma K+ concentrations were obtained using a blood analyzer (Stat Profile M, Nova Biomedical, Waltham, MA).\nData are presented as mean ± sem; n denotes the number of experiments. Differences were considered significant when p \u003c 0.05."}

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PMC:516044 / 4235-12284 JSON TXT