Effect of asphyxia on the composition of cationic elements in the perilymph

To elucidate the role of free radicals in the cochlea, cationic elements in the perilymph of the scala tympani in normal and asphyxic guinea pigs were measured by an inductively coupled plasma atomic emission spectrometer. The concentrations of 7 cationic elements (i.e., calcium, copper, iron, magnesium, phosphorus, lead and zinc) were measured simultaneously in a sample of the perilymph taken before, during and after asphyxia. There was no significant change in these cationic elements during asphyxia for 3 min. In contrast to the stationary level during asphyxia, the iron concentration was significantly elevated at 5 min after re-ventilation following asphyxia and decreased rapidly toward the pre-asphyxic level thereafter, although no significant change was observed in other cationic elemebts. These results were comparable to the theory of non-protein-bound ioon accumulation produced by an O₂-dependent mechanism during re-perfusion.     

儿童阻塞性睡眠呼吸暂停低通气综合征与血清中铜锌铁钙镁关系的研究

目的 探讨儿童阻塞性睡眠呼吸暂停低通气综合征（OSAHS）与血清中部分微量元素水平变化的关系。方法 选取OSAHS患儿65例作为实验组,另选健康儿童43例作为对照组,测量其血清铜、锌、铁、钙、镁的含量,并用SPSS13.0软件包对测量数据进行统计学分析。结果 OSAHS组儿童血清铜高于对照组(P<0.01),血清锌、钙的含量低于对照组(P<0.01),而两组儿童镁和铁的含量无明显差异(P>0.05)。结论 OSAHS患儿血清中铜含量增加,钙、锌含量降低。     

Effect of gentamicin on the concentration of calcium, magnesium and zinc in perilymph of the guinea pig]

The calcium, magnesium and zinc ions in perilymph of the guinea pig were measured after hearing loss induced by gentamicin. The concentration of the calcium ions and magnesium ions increased, the zinc ions decreased in perilymph. The ototoxicity of gentamicin was related to the changes of these electrolytes in inner ear circumstance. The results showed that the changes of ions were the step of gentamicin ototoxicity.     

喉癌外周血淋巴细胞金属元素测定及其意义

测定了１５例喉鳞癌患者及１０例正常人外周血淋巴细胞中钙、镁、铜、锌、铁、锰含量，采用原子吸收光谱仪进行测定。结果表明：患病组铜含量增高（Ｐ＜００１），锌含量降低（Ｐ＜００５）。提示淋巴细胞金属元素含量异常可能为喉癌发病的原因之一。本方法操作简单，为探讨某些疾病发病机制的一项有效的检测手段。     

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 microM) to 7.6 microM and 8.8 microM 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 microM before asphyxia, and also significantly increased to 11.5 microM and 16.2 microM 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.     

Electrochemical profile for calcium ions in the stria vascularis: cellular model of calcium transport mechanism

The d.c. potential and the Ca2+ concentration in the stria vascularis of the chincilla were measured using double-barreled Ca2+-selective microelectrodes with fine tips. The impalement of the microelectrode through the stria vascularis indicated two or three regions showing low Ca2+ concentrations. The first region with a low Ca2+ concentration, presumably corresponding to the basal cell, showed a Ca2+ concentration of 2.5 +/- 1.9 microM. The low Ca2+ region, adjacent to the endolymph, showed 0.31 +/- 0.15 microM and presumably corresponds to the marginal cell. In contrast, the Ca2+ concentrations in the spiral ligament, intrastrial space and endolymph were 1.6 +/- 0.2 mM, 0.75 +/- 0.12 mM, and 17 +/- 7 microM, respectively. Based upon the electrochemical profile for Ca2+ ions, we hypothesize mechanisms of cellular Ca2+ transport where Ca2+ ions from perilymph are accumulated into endolymph via the action of Ca2+-ATPase located in apical membranes of both the basal and marginal cells.     

慢性扁桃体炎与扁桃体肥大患者扁桃体中铁和锌含量的研究

目的　通过测定扁桃体组织中铁和锌元素的含量来研究这两种元素在慢性扁桃体炎和扁桃体肥大发病机制中扮演的角色。方法　选取40例在我院行扁桃体切除术的患者,根据病史、临床特征和病理学表现分为扁桃体肥大组和慢性扁桃体炎组。用微波消解-石墨炉原子吸收法测定其扁桃体组织中铁和锌的含量。结果　铁和锌的含量在扁桃体肥大组和慢性扁桃体炎组中均有显著性差异（t 铁=3.89,t 锌=3.63,P 均＜0.001）。扁桃体肥大组中铁和锌的含量均高于慢性扁桃体炎组。结论　扁桃体组织中铁和锌含量的降低会导致扁桃体炎反复发作。     

Quantification of solute entry into cochlear perilymph through the round window membrane.

The administration of drugs to the inner ear via the round window membrane is becoming more widely used for both clinical and experimental purposes. The actual drug levels achieved in different regions of the inner ear by this method have not been established. The present study has made use of simulations of solute movements in the cochlear fluids to describe the distribution of a marker solute in the guinea pig cochlear fluid spaces. Simulation parameters were derived from experimental measurements using a marker ion, trimethylphenylammonium (TMPA). The distribution of this ion in the cochlea was monitored without volume disturbance using TMPA-selective microelectrodes sealed into the first and second turns of scala tympani (ST). TMPA was applied to perilymph by irrigation of the intact round window membrane with 2 mM solution. At the end of a 90 min application period, TMPA in the first turn, 1.4 mm from the base of ST, reached an average concentration of 330 microM (standard deviation (S.D.) 147 microM, n = 8). TMPA in the second turn, 7.5 mm from the base of ST reached a concentration of 15 microM (S.D. 33 microM, n = 5). The measured time courses of TMPA concentration change were interpreted using the Washington University Cochlear Fluids Simulator (V 1.4), a public-domain program available on the internet at http ://oto.wustl.edu/cochlea/. Simulations with parameters producing concentration time courses comparable to those measured were: (1) round window permeability: 1.9 x 10(-80 cm/s; (2) ST clearance half-time: 60 min; (3) longitudinal perilymph flow rate: 4.4 nl/min, directed from base to apex. Solute concentrations in apical regions of the cochlea were found to be determined primarily by the rate at which the solute diffuses, balanced by the rate of clearance of the solute from perilymph. Longitudinal perilymph flow was not an important factor in solute distribution unless the bony otic capsule was perforated, which rapidly caused substantial changes to solute distribution. This study demonstrates the basic processes by which substances are distributed in the cochlea and provides a foundation to understand how other applied substances will be distributed in the ear.     

Calcium transport mechanism in the endolymph of the chinchilla

The Ca2+ transport mechanism between endolymph and perilymph was evaluated by the effects of vanadate and amiloride on the endocochlear potential (EP) and the Ca2+ concentration in endolymph using Ca2+-selective microelectrodes. Under normal conditions, the EP was 81.8 +/- 0.9 mV, and the Ca2+ concentrations in endolymph and perilymph were 16.6 +/- 1.3 microM and 1.85 +/- 0.11 mM (N = 12), respectively. Therefore, the uphill electrochemical potential gradient for Ca2+ from perilymph to endolymph, 20.2 +/- 2.0 mV, indicates the existence of an active uptake of Ca2+ into endolymph. Vanadate, the inhibitor of Ca2+-ATPase, topically applied to the round window membrane caused biphasic changes of the EP and the endolymph Ca2+ concentration; the former in a transient increase followed by a consistent decrease and the latter in a slow decrease followed by a slow increase. Amiloride induced a slight EP depression and a concomitantly slight elevation of the Ca2+ concentration in endolymph. The electrochemical potential gradient for Ca2+ between endolymph and perilymph vanished with the use of vanadate but was not affected by amiloride. These results suggest that Ca2+-ATPase, sensitive to vanadate, maintained the bulk of active Ca2+ transport in the cochlea and that the participation of Na+-Ca2+ exchange is negligible.     

Changing cation levels (Mg2+, Ca2+, Na+) alters the release of glutamate, GABA and other substances from the guinea pig cochlea.

We examined the effects of changes in cation levels (increased Mg2+ concentration combined with low Ca2+ concentration, and two low concentrations of Na+) on the perilymph levels of gamma-aminobutyric acid (GABA), glutamate (Glu), aspartate (Asp) and other substances. Artificial perilymph solutions containing normal (5 mM) and high (50 mM) levels of K+ were perfused through the perilymphatic compartment of the guinea pig cochlea to examine basal release (5 mM K+) and depolarization-induced release (50 mM K+). Each of the two K+ concentrations were contained in four different solutions: [I] normal artificial perilymph (NARP; NaCl, 137 mM; CaCl2, 2 mM; MgCl2, 1 mM;); [II] high Mg2+ (20 mM)/low Ca2+ (0.1 mM) (HMgLCa); [III] low Na+ (117 mM; LNa), and [IV] very low Na+ (NaCl, 0 mM; VLNa). The effluent was collected and assayed for eighteen primary amines by HPLC. Compared with NARP, the HMgLCa group had an increase in the high K(+)-induced release of Asp and Glu with no change in GABA. VLNa increased the normal K+ levels of Asp, Glu and GABA up to those observed with high K+ in NARP. VLNa increased the high K+ levels of Asp and Glu over fivefold compared with the high K+ levels in NARP, but decreased GABA. We ascribe the results to an interference with either a Na(+)-dependent uptake processes or a Na+/Ca2+ exchange carrier.     

The quinoxalinediones DNOX, CNOX and two related congeners suppress hair cell-to-auditory nerve transmission

We tested 6,7-dinitroquinoxaline-2,3-dione (DNQX); 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX); 6,7-dichloro-3-hydroxy-2-quinoxalinecarboxylic acid (DHQC); and 3-hydroxy-2-quinoxalinecarboxylic acid (3HQC), new kainate and quisqualate receptor antagonists, upon cochlear potentials in guinea pig. Perilymph spaces of guinea pig cochleae were perfused with artificial perilymph solutions containing up to 1000 microM concentrations of DHQC and 3HQC, and 500 microM concentrations of DNQX and CNQX, at a rate of 2.5 microliters/min for 10 min. Cochlear potentials evoked by 10 kHz tone bursts of varying intensity were recorded from the basal turn scala vestibuli. Cochlear perfusion of the four drugs resulted in a dose-related suppression of the compound action potential of the auditory nerve (CAP; N1-P1), a prolongation of N1 latency at suprathreshold levels, an elevated CAP threshold, and a decreased N1 latency at CAP threshold. None of the drugs had significant effects on cochlear microphonics (CM) or the summating potential (SP). EC50 values (concentrations causing a 50% reduction in CAP amplitude at 68 dB SPL) were 8 microM for DNQX, 30 microM for DHQC, 35 microM for CNQX, and 1 mM for 3HQC. Results support the hypothesis that kainate and quisqualate receptors are involved in neurotransmission between the hair cell and afferent nerve.     

Isosorbide Concentration in Perilymph of the Guinea Pig After Oral Administration Versus That After Round Window Perfusion

Objectives

The aims of this study were to investigate the feasibility of isosorbide delivery into perilymph through the round window membrane (RWM), and to compare the intracochlear isosorbide concentration in perilymph after oral administration (PO) versus that after round window perfusion (RWP).

Methods

Sixteen male guinea pigs (32 ears) were used. Isosorbide, an osmotic diuretic, was administered via RWP or PO. First, to investigate the optimal perfusion time, perilymph sampling of scala tympani from the RWM was performed after RWP for 15, 30, or 60 minutes. Second, to compare the drug concentration after RWP versus that after PO, perilymph was aspirated at 3 and 6 hours after administration. Intracochlear concentration of isosorbide was analyzed by high-performance liquid chromatography coupled to refractive index detection.

Results

Isosorbide passed through the RWM into perilymph after RWP. After RWP for 15, 30, and 60 minutes, mean isosorbide concentrations in perilymph were 116.27±44.65, 245.48±112.84, and 279.78±186.32 mM, respectively. The intracochlear concentration after RWP for 30 minutes was higher than that after RWP for 15 minutes (P=0.043). At 3 and 6 hours after PO, isosorbide concentrations in perilymph were 28.88±4.69 and 12.67±2.28 mM, respectively. In contrast, the corresponding concentrations after RWP were 117.91±17.70 and 75.03±14.82 mM at 3 and 6 hours, respectively. Isosorbide concentrations in perilymph following RWP were significantly higher than those following PO at both 3 and 6 hours (P=0.025 and P=0.034, respectively).

Conclusion

Isosorbide can rapidly pass through the RWM after RWP in guinea pigs, and 30 minutes of perfusion is considered to be appropriate. In addition, over a 6-hour period, RWP can deliver higher concentrations of isosorbide into perilymph than those achieved with PO.     

Intracellular free calcium concentrations in single taste receptor cells in the guinea pig.

Single, viable taste receptor cells were isolated from the tongue of the guinea pig by enzymatic digestion and mechanical dissociation. The cells could be classified into flask, spindle and intermediate shapes. The intracellular free calcium ion concentrations [(Ca2+)i] of these cells were determined using the Ca2+ sensitive dye fura-2 and digital imaging microscopy. All types of cells produced an irreversible increase in (Ca2+)i upon addition of Ca2+ ionophore ionomycin (1 microM) and denatonium (10 microM). There was no evidence of any increase in (Ca2+)i in the taste receptor cells in nominally Ca2+ free solution, and when stimulated by denatonium (10 microM). When 3 mM CaCl2 was added, the (Ca2+)i remarkably increased. This would suggest that the (Ca2+)i increase in the presence of denatonium mainly depended on calcium influx from the extracellular space. There was no increase in case of high potassium (50 mM and 150 mM) or saccharose (1 mM and 5 mM) stimulation. The hypothesis that the increase in (Ca2+)i controls biochemical mechanisms related to the bitter taste transduction process is worthy of further study.     

Systemic Lipopolysaccharide Compromises the Blood-Labyrinth Barrier and Increases Entry of Serum Fluorescein into the Perilymph

The blood vessels that supply the inner ear form a barrier between the blood and the inner ear fluids to control the exchange of solutes, protein, and water. This barrier, called the blood-labyrinth barrier (BLB) is analogous to the blood-brain barrier (BBB), which plays a critical role in limiting the entry of inflammatory and infectious agents into the central nervous system. We have developed an in vivo method to assess the functional integrity of the BLB by injecting sodium fluorescein into the systemic circulation of mice and measuring the amount of fluorescein that enters perilymph in live animals. In these experiments, perilymph was collected from control and experimental mice in sequential samples taken from the posterior semicircular canal approximately 30 min after systemic fluorescein administration. Perilymph fluorescein concentrations in control mice were compared with perilymph fluorescein concentrations after lipopolysaccharide (LPS) treatment (1 mg/kg IP daily for 2 days). The concentration of perilymphatic fluorescein, normalized to serum fluorescein, was significantly higher in LPS-treated mice compared to controls. In order to assess the contributions of perilymph and endolymph in our inner ear fluid samples, sodium ion concentration of the inner ear fluid was measured using ion-selective electrodes. The sampled fluid from the posterior semicircular canal demonstrated an average sodium concentration of 145 mM, consistent with perilymph. These experiments establish a novel technique to assess the functional integrity of the BLB using quantitative methods and to provide a comparison of the BLB to the BBB.     

High potassium concentrations protect inner and outer hair cells in the newborn rat culture from ischemia-induced damage

Several studies indicate that an increase in the extracellular potassium (K+) concentration is a factor exerting a damaging effect on cochlear hair cells (HCs). The present study was designed to examine the effects of high extracellular K+ concentrations on the HCs under normoxic and ischemic conditions. Organotypic cultures of the organ of Corti of newborn rats were exposed to normoxia and ischemia at K+ concentrations of 5-70 mM in artificial perilymph for 3-4h. The number of IHCs and OHCs in the apical, medial and basal parts of the cochlea were counted 24h later. The work resulted in two main findings: (1) extracellular K+ concentrations of 30-70 mM had no effect on the HCs under normoxic conditions; (2) under ischemic conditions, a clear HC loss, mainly in the medial and basal cochlear parts, was observed at 5 mM K+ as previously reported. In contrast, a high extracellular K+ concentration strongly attenuated the HC loss. This effect nearly completely disappeared by the addition of both eosin, an inhibitor of the plasma membrane calcium ATPase (PMCA), and linopirdine, an inhibitor of the KCNQ4 channel, indicating that a normal activity of the PMCA and the KCNQ4 channels are key factors for HC survival under ischemia and depolarizing conditions.     

The effects of histamine and its antagonists on the cochlear microphonic and the compound action potential of the guinea pig

OBJECT: we studied the effects of histamine, the H1 receptor antagonist pyrilamine, and the H2 receptor antagonist cimetidine on the cochlear potential of guinea pigs (cochlear microphonic, CM; compound action potential, CAP). METHODS: histamine was applied into the cochlear perilymph at three different dosages (10 microM, 50 microM or 10 mM). Pyrilamine and cimetidine were applied at 50 microM each. RESULTS: histamine increased the CAP at 10 and 50 microM without any significant effects on the CM. The effects of histamine at 50 microM were suppressed by the 50-microM of pyrilamine and cimetidine. At 10 mM of histamine, CAP and CM amplitudes were significantly decreased. CONCLUSION: in low concentrations, histamine may act as an extracellular signal on inner hair cells (IHCs) or it may stimulate the afferent nerve by binding to their H1 and H2 receptors. A possible explanation for the inhibitory effects of histamine at 10-mM dosage was apparently found in that the effects of the high concentration may be supraphysiological; and furthermore, there is a difference in the mechanism by which histamine exerts its effects mediated by the histamine receptors on the cochlea.     

Caffeine and ryanodine demonstrate a role for the ryanodine receptor in the organ of Corti

The hypothesis that the release of Ca(2+) from ryanodine receptor activated Ca(2+) stores in vivo can affect the function of the cochlea was tested by examining the effects of caffeine (1-10 mM) and ryanodine (1-333 microM), two drugs that release Ca(2+) from these intracellular stores. The drugs were infused into the perilymph compartment of the guinea pig cochlea while sound (10 kHz) evoked cochlear potentials and distortion product otoacoustic emissions (DPOAEs; 2f(1)-f(2)=8 kHz, f(2)=12 kHz) were monitored. Caffeine significantly suppressed the compound action potential of the auditory nerve (CAP) at low intensity (56 dB SPL; 3.3 and 10 mM) and high intensity (92 dB SPL; 10 mM), increased N1 latency at high and low intensity (3 and 10 mM) and suppressed low intensity summating potential (SP; 10 mM) without an effect on high intensity SP. Ryanodine significantly suppressed the CAP at low intensity (100 and 333 microM) and at high intensity (333 microM), increased N1 latency at low intensity (33, 100 and 333 microM) and at high intensity (333 microM) and suppressed low intensity SP (100 and 333 microM) and increased high intensity SP (333 microM). The cochlear microphonic (CM) evoked by 10 kHz tone bursts was not affected by caffeine at high or low intensity, and ryanodine had no effect on it at low intensity but decreased it at high intensity (10, 33, 100 and 333 microM). In contrast, caffeine (10 mM) and ryanodine (33 and 100 microM) significantly increased CM evoked by l kHz tone bursts and recorded from the round window. Caffeine (10 mM) and ryanodine (100 microM) reversibly suppressed the cubic DPOAEs evoked by low intensity primaries. Overall, low intensity evoked responses were more sensitive and were suppressed to a greater extent by both drugs. This is consistent with the hypothesis that release of Ca(2+) from ryanodine receptor Ca(2+) stores, possibly in outer hair cells and supporting cells, affects the function of the cochlear amplifier.     

The effect on perilymph of the alteration of serum glucose or calcium concentration.

In the experimentally-induced hyper- or hypoglycemic state, perilymph glucose concentration paralleled the blood concentration, although a delay of about one hour was observed between the time of maximum concentration of glucose in perilymph and its concentration in blood. In hypercalcemia, perilymph calcium concentration steadily increased over a three-hour period, although blood calcium concentration fluctuated during this time. After an initial increase in the CSF calcium concentration, there were insignificant changes during the second and third hours. In animals with thyrocalcitonin-induced hypocalcemia, although blood calcium concentration steadily decreased, its concentration in perilymph and CSF remained practically constant. The present findings suggest that the chemical composition of perilymph can be altered by changing the blood concentration of glucose or calcium. The marked difference of behavior noted between glucose and calcium in these experiments would indicate the existence of different mechanisms for maintaining the homeostatic state for these two substances.     

Effect of glycerol on inner ear fluid electrolytes and osmolalities in guinea pigs

Endolymph of the scala media (SM) and perilymph of the scala vestibuli (SV) and scala tympani (ST) were collected from the basal turn of anesthetized guinea pigs before and after intravenous administration of glycerol (3 g/kg). Sound-evoked responses were recorded during the test periods. Blood, CSF, and perilymph of the ST were also collected continuously after the injection. The osmolalities and chloride concentrations of the collected samples were determined. In another experiment, the continuous changes of potassium and chloride concentrations in endolymph and perilymph of the ST before and after the injection were measured by ion-selective electrodes. The osmolalities in CSF and perilymph lagged behind the increase in serum osmolality. The osmolalities in endolymph and perilymph increased gradually after the injection, reached maximum values after 90 minutes, and then decreased. The changes in chloride and potassium concentrations in endolymph and perilymph had similar tendencies. But the increases in chloride concentrations in perilymph of the SV and ST were much less than that in endolymph. We propose that most of the osmolality increase in perilymph is due to glycerol or other osmotically active substances and that the osmolality increase in endolymph is due to water shift.     

Permeability to chloride ions of the cochlear partition in normal guinea pigs

The endocochlear potential and Cl- activities in the endolymph and perilymph of guinea pigs were simultaneously measured with a pair of double-barreled Cl--sensitive liquid-membrane electrodes. Under normal conditions the mean Cl- activity was 96.6 mEg/l in the endolymph and 93.5 mEq/l in the perilymph. The mean Cl- concentration determined by potentiometric titration was 132.3 mM in the endolymph and 121.8 mM in the perilymph. The permeability to Cl- of the cochlear partition was determined after active transport was abolished by permanent anoxia. The modified permeability coefficient and conductance for Cl- of the cochlear partition were computed from the rate of decrease in the Cl- concentration of the endolymph and its electrochemical potential difference between the endolymph and perilymph. The mean modified Cl- conductance and permeability coefficient of the cochlear partition were (10.7 +/- 4.25) X 10(-3) omega -1 . cm-3 and (22.58 +/- 8.95) X 10(-6) s-1 respectively, when averaged from 10 to 30 min after onset of anoxia. Our results indicate that the cochlear partition is relatively permeable to Cl-, when compared to its permeability to K+. Possible coupling between Cl- and water movement across the cochlear partition is discussed.