Idiopathic hair cell loss in the guinea pig

Summary An incidence of specific, marked sensory cell loss affecting 12–88% of the outer hair cells in the third cochlear turn was found in a group of 130 healthy, coloured, intact guinea pigs divided into three age groups (6 weeks, 7 months, and 3 years). The pathological findings were compared with a known physiological norm. At 7 months, the disorder occurred in 8% of the animals, whereas at 3 years 30% were involved. The etiology of the above finding is at present still obscure.     

The histone deacetylase inhibitor sodium butyrate protects against cisplatin-induced hearing loss in guinea pigs

OBJECTIVE: There is a need for otoprotective agents that can be administered systemically without compromising cancer treatment. Histone deacetylase inhibitors are anticancer agents that act by upregulating the expression of cell-cycle control genes. They are also neuroprotective, leading us to hypothesize that they might be otoprotective. The goal of this study was to determine if the antitumor agent sodium butyrate (a histone deacetylase inhibitor) protects against cisplatin ototoxicity when administered systemically. STUDY DESIGN: This was an animal study. METHODS:: Cisplatin was administered to guinea pigs who received either 12 days of sodium butyrate (7 d before and 5 d after cisplatin) or equivolume saline injections. Hearing was tested with distortion product otoacoustic emission (DPOAE) analysis before the start of the study and 2 weeks after cisplatin treatment. RESULTS: Guinea pigs given a single intraperitoneal injection of 14 mg/kg cisplatin experience a mean hearing loss of 8 dB across the frequencies of 3.5, 5, 7, 10, 14, and 20 kHz. Intraperitoneal injection of 1.2 mg/kg sodium butyrate per day for 7 days before and 5 days after cisplatin almost completely eliminates this threshold shift (P=.0011). CONCLUSIONS: The histone deacetylase inhibitor sodium butyrate gives almost complete protection in a single-dose model of cisplatin ototoxicity in guinea pigs. Because histone deacetylase inhibitors are anticancer agents with very few side effects, they may be candidates for clinical use during cisplatin chemotherapy.     

Voltage-dependent channels in dissociated outer hair cells of the guinea pig

Voltage-dependent channels in outer hair cells (OHCs) dissociated from the guinea pig cochlea were investigated by the use of a whole-cell patch-clamp technique. Two types of K⁺ current were recorded from OHCs. One was a slowly inactivating K⁺ current that was activated at a potential more positive than ?30 mV. Another is a K⁺ current that was already activated at resting membrane potential. After suppressing both K⁺ currents, depolarizing voltage steps elicited a slowly inactivating inward current that was dependent on external Ca²⁺ and was indicative of an L-type Ca²⁺ channel in OHCs. Aminoglycoside antibiotics known to be ototoxic selectively inhibited the Ca²⁺ current.     

豚鼠耳蜗外毛细胞的变性机制

目的:探讨外毛细胞维持形态及变性过程发生的可能机制。方法:倒置相差显微镜下,对于急性酶分离所得的外毛细胞进行6 h的连续性观察。结果:半数以上活性良好的单离外毛细胞可保持活性平均约6 h,变性过程中均出现自表皮板至基底部的纵行褶线。含钙和不含钙的细胞维持活性时间相同。静纤毛缺失可以同样维持较长时间的活性。结论:钙离子浓度和静纤毛损伤不是外毛细胞变性的决定性因素,而环细胞表面的某种弹性机制可能是维持形态保持活性的根本原因。     

The continuing search for outer hair cell afferents in the guinea pig spiral ganglion.

Antidromic stimulation of the stump of the VIIIth nerve was combined with microelectrode recording in the spiral ganglion of the guinea pig cochlea in an attempt to identify a sub-population of neurons with long-latency antidromic action potentials that might correspond to the thin unmyelinated afferent neurons emanating from the outer hair cells. The techniques used were similar but not identical to those employed in an earlier study by Brown (1994). By far the largest population of cells contacted had short antidromic latencies (0.58+/-0.12 ms, 76 units) and also responded to acoustic stimulation. These were assumed to be type I afferents emanating from inner hair cells. Eight cells had antidromic latencies larger than 1 ms, all but one of which had a zero spontaneous rate. All eight of these longer-latency cells were unresponsive to acoustic stimulation despite the fact that short-latency neurons in the same cochleas showed robust responses to sound before and after they were contacted. Four of these longer-latency cells had their antidromic thresholds accurately measured and two had significantly higher thresholds to electrical stimulation (0.1 ms duration) than type I cells in the same animal while two had similar electrical thresholds. Attempts to trace the eight long-latency neurons to the outer hair cells using intracellular injection of horseradish peroxidase were unsuccessful. On the basis of present evidence, we cannot conclude definitively that the long-latency neurons found in the spiral ganglion belong to the outer hair cell afferent population.     

Stretch-activated ion channels in guinea pig outer hair cells.

Two types of stretch-activated (SA) ion channels have been found in the lateral wall of isolated outer hair cells (OHC) from the guinea pig cochlea. One type had a reversal potential of -12 mV and was non-selective to cations, passing Ca2+ as well as monovalent ions. The channel had a conductance of 38-50 pS and the amplitude of the current through the open SA channel was independent of suction. The probability of the channel being open increased with applied suction and was voltage dependent with the maximum probability occurring at pipette potentials of -40 to -60 mV. The second type of SA channel had a conductance of approximately 150 pS and a reversal potential of approximately -50 mV. The ionic selectivity of this channel has not yet been determined, but it is probably K+ selective. OHCs have been shown to undergo a slow change in length in response to acoustic stimulation directed at the lateral wall of the OHC. The SA channels reported here could affect the motile response by altering the membrane potential or by allowing the entry of free Ca2+ which could lead to a change in OHC length through the interaction of actin and myosin. SA channels could also play an important role in regulating the osmotic pressure of OHC thereby influencing its electro-mechanical response.     

豚鼠耳蜗单离外毛细胞外向钾电流的特性

目的：探讨豚鼠耳蜗单离外毛细胞（ＯＨＣ）钾电流（Ｉｋ）的正常值及其特性。方法：应用膜片钳全细胞记录技术及多种辅助方法,测试在不同细胞内、外液和电压刺激条件下的Ｉｋ、钾尾电流（Ｉｋｔａｉｌ）和反转电位。结果：Ｉｋ具有明显的电压依赖性和时间依赖性,在２０ｍｓ内达峰值,平均激活电压约为－３２．７ｍＶ,从激活电压至０电压时Ｉｋ增长最快,在４０ｍＶ时接近饱和。正常条件下Ｉｋ无明显“ｒｕｎ－ｄｏｗｎ现象”。细     

Effect of capsaicin on potassium conductance and electromotility of the guinea pig outer hair cell

Capsaicin, the classic activator of TRPV-1 channels in primary sensory neurons, evokes nociception. Interestingly, auditory reception is also modulated by this chemical, possibly by direct actions on outer hair cells (OHCs). Surprisingly, we find two novel actions of capsaicin unrelated to TRPV-1 channels, which likely contribute to its auditory effects in vivo. First, capsaicin is a potent blocker of OHC K conductances (I(K) and I(K,n)). Second, capsaicin substantially alters OHC nonlinear capacitance, the signature of electromotility - a basis of cochlear amplification. These new findings of capsaicin have ramifications for our understanding of the pharmacological properties of OHC I(K), I(K,n) and electromotility and for interpretation of capsaicin pharmacological actions.     

Prestin-dependent and prestin-independent motility of guinea pig outer hair cells

The motile response of isolated guinea pig outer hair cells (OHCs) was investigated using a combination of whole-cell patch clamp recording and continuous video image analysis. OHC's length, width, and area were measured from video images and the cell volume estimated from these values. Morphological data was then correlated with electrophysiological recordings of whole-cell current, membrane potential and voltage-dependent non-linear capacitance. Electromotility was evoked either by manipulating the membrane potential under voltage-clamp conditions or by exposing OHCs to high K+ solutions. Other motile responses were investigated in voltage-clamp experiments at constant holding potential, or exposing OHCs to solutions that did not affect the membrane potential. We found that electrical stimulation evoked voltage-dependent changes in OHC's length, width and area but not in cell volume regardless of the time course of stimulation. Moreover, changes in cell area were always associated with both voltage-dependent motility and non-linear capacitance, suggesting prestin dependency. In contrast, voltage-independent motile responses at constant membrane potential, which are presumed to be prestin-independent, were associated with changes in cell length, width and volume without significant changes in area. Area measurements, then, become a tool to investigate the simultaneous occurrence of both prestin-dependent and prestin-independent OHC motilities, and for evaluating the individual contribution of each mechanism to the total cell movement.     

豚鼠耳蜗外毛细胞的简化分离

目的:为获得能够满足电生理需要的外毛细胞,探讨简单可靠的分离外毛细胞的方法。方法:暴露听泡后去除耳蜗骨壳,将螺旋韧带连同基底膜和蜗轴一起放入酶液消化,减少显微镜下分离基底膜的操作程序,同时避免去除螺旋韧带时部分基底膜片段的丢失。结果:获得较多的保持良好活性的单离外毛细胞,耳大约80%的细胞能保持活性平均约6h。结论:去除骨壳后的残余耳蜗整体消化并适当提高消化液浓度的分离法,操作简单,可以获得能够满足电生理实验需要的较多的单离的耳蜗外毛细胞,有利于内耳研究工作的开展。     

Acetylcholine-induced calcium oscillation in isolated outer hair cells in guinea pig

Objective This study is to explore the relationship between acetylcholine(ACh)-induced calcium release from intracellular Ca2 stores and function of outer hair cell(OHC) motors, in an attempt to elucidate the mechanism of OHC electromotility at resting state. Methods OHCs were isolated from adult guinea pig (200-300 g) cochlea and loaded with Fluo-3/AM. The cells were treated with ACh/dHBSS, ACh/HBSS, dHBSS only or HBSS only. Intracellular [Ca2 ]i variations in cells under the four treatments were observed using an Ar-Kr laser scan confocal microscope. Results [Ca2 ]i oscillations were recorded in five OHCs treated with ACh/dHBSS but not in other cells. This is the first time that Ach-excited [Ca2 ]i oscillations are reported in guinea pig OHCs independent of extracellular calcium. Conclusions ACh-excited [Ca2 ]i oscillations in OHCs originates from intracellular calcium release and may play a crucial role in maintaining active mechanical motility of the OHC at resting and modulating OHC electromotility.     

Cystine protects cochlear outer hair cells against glutamate toxicity

We previously reported that long-term exposure to glutamate (Glu) induced death of cochlear outer hair cells (OHCs). However, the mechanisms of OHC death induced by Glu were unclear. In the central nervous system, Glu is known to interfere with a cystine-Glu antiporter, leading to a decrease in cystine uptake and reducing the intracellular glutathione level. We therefore investigated the effect of cystine supplementation on degeneration of OHCs caused by long-term exposure to Glu. Supplementation of cystine significantly decreased the number of dying OHCs. These findings suggest that a cystine-Glu interaction may be involved in the mechanism of OHC degeneration caused by Glu.     

Simultaneous monitoring of slow cell motility and calcium signals of the guinea pig outer hair cells

'Slow' motility (shape changes over seconds to minutes) of the mammalian cochlear outer hair cell (OHC) could play a protection role from intense sound pressure and is associated with elevation of the cytosolic free Ca(2+) concentration ([Ca(2+)](i)). In the present work, a new approach was elaborated using fluorescent imaging for continuous monitoring of both [Ca(2+)](i) changes and slow motility of OHCs employing the Ca(2+) fluorescent indicator Fura-2. Whole OHC fluorescence and that of cell segments were analyzed to discriminate between fluorescence changes caused by [Ca(2+)](i) rise and those related to change of the cell shape. The reliability of the method was examined by simultaneous monitoring of [Ca(2+)](i) and OHC length changes induced by change of buffer osmolarity or by increase of KCl concentration. The method revealed that the time course of [Ca(2+)](i) increase and rate of cell shortening often do not coincide. It was also observed that [Ca(2+)](i) increased in 70 mM KCl more slowly than the rate of KCl delivery to OHCs. The comparison of the time courses of [Ca(2+)](i) elevation, induced by increase of K(+)/Na(+) ratio and by substitution of Na(+) with N-methyl-D-glucamine(+), indicated that the relatively slow kinetics of [Ca(2+)](i) increase in the OHC is partially attributed to regulation of Ca(2+) homeostasis by the Na(+)/Ca(2+) exchanger.     

高渗透压对豚鼠耳蜗离体外毛细胞膜电位的影响

为了解高渗透压对豚鼠耳蜗离体活外毛细胞膜电位及细胞长度的影响。我们运用粘附式细胞仪和膜电位敏感针ＤｉＢＡＣ，对豚鼠耳蜗离体外细胞在高渗环境中的膜电位相对值及细胞长度的变化进行了动态测定。     

CGRP- and cholinergic-containing fibers project to guinea pig outer hair cells

MU33, an antibody to calcitonin gene-related peptide (CGRP), was used to investigate the magnitude of CGRP-containing nerve fiber endings, compared to acetylcholinesterase (AChE)-containing nerve fiber endings on outer hair cells (OHCs). Results showed that CGRP-containing nerve fiber immunoreactivity mimicked the AChE fiber OHC staining pattern across the cochlea suggesting that CGRP can function as both a medial efferent (contacting primarily OHCs) and as a lateral efferent (contacting primarily inner hair cell afferents) neurotransmitter.     

豚鼠前庭感觉上皮损伤中细胞凋亡的观察

细胞凋亡对细胞增殖、器官发生和功能维持起着重要作用。一定剂量的庆大霉素连续注射，造成豚鼠前庭器官损伤。采用半薄切片，透射电镜（TEM）和TUNEL（TdT－modidedbiotin-dUTPNick-endlabeling，末端脱氧核苷酸转移酶介导的生物素标记）原位杂交技术，特异标记DNA片段3′－OH末端，原位显示凋亡细胞。在半薄切片和TEM观察中发现两种类型的细胞损伤方式：①毛细胞肿胀，胞浆空泡化，细胞体从顶端表面挤出；②毛细胞在上皮内变性，显示出细胞凋亡的形态特征，包括细胞核凝缩，核膜消失，成碎块状，并由支持细胞吞噬。原位杂交显示：细胞凋亡标记阳性细胞主要分布在上皮表层，较高水平标记主要发生于给药后第3到7天。提示细胞凋亡是内耳前庭感觉细胞损伤的一种重要方式，凋亡的主动发生可能是一种潜在的介入方式来减少氨基甙类抗生素对毛细胞造成的急性损伤，并与感觉上皮损伤后的修复过程有关。     

Filter function of the guinea pig cochlea after degeneration of outer hair cells

After kanamycin induced degeneration of outer hair cells from guinea pigs the tuning properties of primary auditory nerve fibres are compared with those of normal untreated guinea pigs. The existence of fibres with no alteration of the tuning properties leads to the conclusion that there is no neural interaction between inner and outer hair cells needed to enhance the frequency selectivity.     

Filter function of the guinea pig cochlea after degeneration of outer hair cells

Summary After kanamycin induced degeneration of outer hair cells from guinea pigs the tuning properties of primary auditory nerve fibres are compared with those of normal untreated guinea pigs. The existence of fibres with no alteration of the tuning properties leads to the conclusion that there is no neural interaction between inner and outer hair cells needed to enhance the frequency selectivity.This study was supported by the Deutsche Forschungsgemeinschaft (Bo 421/1-3). Kanamycin was kindly provided by Chemie Grünenthal.     

Effect of neuroregulators on the intracellular calcium level in the outer hair cell isolated from the guinea pig.

The cytosolic calcium concentration [( Ca2+]i) of the isolated outer hair cell of the guinea pig was measured using fluorescence imaging microscopy and the effects of efferent neuroregulators such as acetylcholine, ATP, GABA, substance P, enkephalin, calcitonin gene-related peptide, serotonin, dopamine, norepinephrine, and glutamate were investigated. Among the drugs tested only ATP induced an elevation of the [Ca2+]i of the outer hair cell. In the resting condition, [Ca2+]i averaged 104.5 +/- 31.1 nM (n = 27), while 100 microM ATP significantly increased [Ca2+]i to 146.3 +/- 43.5 nM (n = 19). Superfusion with Ca2(+)-free solution (pCa = 7.5) abolished the increase in [Ca2+]i induced by ATP, suggesting that ATP causes an entry of external Ca2+. The relevance of [Ca2+]i to the inhibitory actions of efferent neuroregulators is discussed.     

Hyposmotic stimulation-induced nitric oxide production in outer hair cells of the guinea pig cochlea

Nitric oxide (NO) production during hyposmotic stimulation in outer hair cells (OHCs) of the guinea pig cochlea was investigated using the NO sensitive dye DAF-2. Simultaneous measurement of the cell length and NO production showed rapid hyposmotic-induced cell swelling to precede NO production in OHCs. Hyposmotic stimulation failed to induce NO production in the Ca(2+)-free solution. L-N(G)-nitroarginine methyl ester (L-NAME), a non-specific NO synthase inhibitor and gadolinium, a stretch-activated channel blocker inhibited the hyposmotic stimulation-induced NO production whereas suramin, a P2 receptor antagonist did not. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor inhibited the hyposmotic stimulation-induced increase in the intracellular Ca(2+) concentrations ([Ca(2+)](i)) while L-NAME enhanced it. 1H-[1,2,4]oxadiazole[4,3a]quinoxalin-1-one, an inhibitor of guanylate cyclase and KT5823, an inhibitor of cGMP-dependent protein kinase (PKG) mimicked effects of L-NAME on the Ca(2+) response. Transient receptor potential vanilloid 4 (TRPV4), an osmo- and mechanosensitive channel was expressed in the OHCs by means of immunohistochemistry. 4alpha-phorbol 12,13-didecanoate, a TRPV4 synthetic activator, induced NO production in OHCs. These results suggest that hyposmotic stimulation can induce NO production by the [Ca(2+)](i) increase, which is presumably mediated by the activation of TRPV4 in OHCs. NO conversely inhibits the Ca(2+) response via the NO-cGMP-PKG pathway by a feedback mechanism.