离体豚鼠前庭神经节细胞的分离及胞内钙离子活动的观察

目的进一步了解前庭神经节细胞的形态和电生理特性.方法用体重250～300g的豚鼠,断头后取出颞骨,剔除面神经和听神经,取出前庭神经节.经酶消化后用机械法分离成单细胞,用钙离子敏感的荧光染料Fura-2染色,于数字荧光显微镜下观察.结果分离出的单个前庭神经节细胞形状为园形、椭园形和不规则型,直径从20μm到30μm不等,分为有微绒毛的Ⅰ型细胞和无绒毛的Ⅱ型细胞.在体外室温22～24℃下可存活5～6h.用150 mmol/L KCl灌流1分钟,灌流开始20秒后,前庭神经节细胞胞内钙离子荧光强度比率从静止时的0.47±0.04,增至1.18±0.19,40秒后达峰值1.43±0.18,然后缓慢下降.300秒后降至峰值一半,约10分钟后基本恢复至正常,重复刺激可得到类似的结果.在无钙外环境中,150 mmol/L KCl不能诱发胞内钙离子浓度增加.1μM ionomycin可诱发不可逆的胞内钙离子浓度增加.结论本研究使用酶消化后机械分离法分离出活性良好的单个神经节细胞,前庭神经节细胞膜存在电压依赖性钙离子通道.     

Extracellular adenosine 5'-ATP-induced calcium signaling in isolated vestibular ganglion cells of the guinea pig

Extracellular adenosine 5'-triphosphate (ATP)-induced intracellular calcium concentration ([Ca2+]i) changes in acutely isolated vestibular ganglion cells (VGCs) of the guinea pig were investigated using the Ca2+ -sensitive dye Fura-2. Extracellular ATP induced an increase in [Ca2+]i in VGCs in a dose-dependent manner. ATP induced an increase in [Ca2+]i even in the absence of extracellular Ca2+ (1 mM Ethylene Glycol-bis (beta-aminoethyl Ether) N,N,N',N'-Tetraacetic Acid (EGTA)), thus suggesting that ATP induces Ca2+ release from the intracellular stores. The P2-receptor antagonists suramin and reactive blue 2 inhibited the ATP-induced [Ca2+]i increase in a dose-dependent manner. The P1-receptor agonist adenosine did not induce any changes in [Ca2+]i. These results suggest that VGCs may possess a P2-purinergic receptor but not a P1-purinergic receptor. La3+, a receptor-mediated calcium channel blocker, inhibited the ATP-induced [Ca2+]i increase but, in contrast, nifedipine, a L-type calcium channel blocker, did not. These results suggest that ATP induces both a Ca2+ -release from the intracellular stores and a Ca2+ influx from the extracellular space through La3+ -sensitive and nifedipine-insensitive Ca2+ channels in VGCs. Our results also suggest that extracellular ATP may act as a neurotransmitter or neuromodulator of the vestibular peripheral system in the guinea pig.     

Fine structure of guinea pig vestibular kinocilium

The fine structure of the utricular kinocilium of the guinea pig was examined with transmission electron microscopy after treatment with tannic acid to enhance resolution of internal morphology. The utricular kinocilium was devoid of inner dynein arms and a central pair of microtubules, while a set of outer dynein arms and radial spokes was found. This supports the hypothesis that the vestibular kinocilium is non-motile. Internal electron-dense particles at the attachment sites of the stereo-kinociliar bonds were situated in the immediate periphery of the outer dynein arms, although no visible connection existed between these structures. Findings obtained in the present study seem to give insight on the mechanism of mechanosensory transduction in the vestibular sensory cells.     

Carbohydrates of the guinea pig vestibular supporting cells

Ultrastructural localization of glycoconjugates with special reference to the synthesizing process was studied in the guinea pig vestibular supporting cells using the tannic acid and ruthenium red staining technique. The extracellular glycoconjugate (glycocalyx) was well stained by the ruthenium red which may be synthesized by GERL complex. The supporting cell has a numerous number of granules which also contain tannic acid positive glycoconjugate. These granules were closely related to the Golgi complex as well as the apical cell surface. It may be indicated that the glycoconjugate of the supporting cells is glycosylated at the Golgi complex, stored in the granules, transported, secreted, and form otoconial membrane and the cupula.     

Stereociliary glycocalyx and interconnections in the guinea pig vestibular organs

Stereociliary glycocalyx and interconnections in the guinea pig vestibular organs were investigated at the electron microscopic level by using the ruthenium red staining technique. This staining visualized the glycocalyx and ciliary interconnections as well as the contact zone between the sensory hair bundle and the otolithic membrane or the cupula. Glycocalyx was found over the entire length of the stereocilia and the kinocilium of the sensory cells as well as over the entire apical surface of the supporting cells, in the form of a fuzzy filamentous layer. The ciliary structures were found interconnected by different types of connections, such as between the kinocilium and the neighboring stereocilia, and connections between stereocilia. These connections all emerged from the fuzzy layer of the glycocalyx and were also stained identically by ruthenium red, and were therefore considered to be morphologically similar. In addition, the tips of the sensory hairs were firmly attached to the otolithic membrane or the cupula. These findings suggested that the glycocalyx in the cell lining serves as an anchoring structure for the mechanical coupling not only between the ciliary structures, but also between the sensory hair bundle and the otolithic membrane or cupula.     

Obliteration of vestibular and cochlear aqueducts in the guinea pig

The study involved 42 healthy adult guinea pigs placed into two major groups. In group A, only histologic studies were performed after obliteration of the right vestibular aqueduct, the cochlear aqueduct, or both the vestibular and cochlear aqueducts. In group B, hearing changes were monitored by repeated determination of averaged temporal response thresholds after the same obliterative operations as in group A. At the conclusion of the experiments, histologic studies of temporal bone were performed and hearing changes were correlated with the severity of disease found in the inner ear. Histologic examination of 32 temporal bones from animals in which the vestibular aqueduct or both the vestibular and cochlear aqueducts were obliterated consistently demonstrated endolymphatic hydrops. The endolymphatic hydrops was usually progressive, and its severity was proportional to the length of time from the operation. Serial determination of averaged temporal response thresholds in 18 guinea pigs after these operations demonstrated a progressive deterioration in thresholds. The hearing loss was greater at the lower frequencies. The audiometric changes correlated positively with the severity of endolymphatic hydrops. No significant audiometric or temporal bone histologic changes were found after obliteration of the cochlear aqueduct.     

Nitric oxide in guinea pig vestibular sensory cells following gentamicin exposure in vitro.

Gentamicin-induced production of nitric oxide (NO) in the vestibular end organs of the guinea pig was investigated using the new fluorescence indicator 4,5-diaminofluorescein diacetate for direct detection of NO. Utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with gentamicin. This increase in fluorescence was inhibited by the presence of the non-specific inhibitor for nitric oxide synthase, L-N(G)-nitroarginine methylester, and by the non-specific N-methyl-D-aspartic acid antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate. These findings indicate that NO may play an important role in the ototoxicity of aminoglycoside.     

Central projections from singular parts of the vestibular labyrinth in the guinea pig.

Primary afferent projections from singular parts of the vestibular labyrinth were studied in the guinea pig. The posterior ampullary nerve, the common trunk of the anterior and lateral ampullary nerves, as well as fibers innervating the macula sacculi or the macula utriculi were traced with crystals of horseradish peroxidase (HRP) lyophilisate. Posterior, as well as anterior and lateral ampullary fibers were found to project extensively to the superior vestibular nucleus, but also reached the other main vestibular nuclei. Saccular fibers projected mainly to the lateral parts of the lateral vestibular nucleus and to the adjoining descending and superior vestibular nuclei as well as to group y. Modest projections could be followed to the medial vestibular nucleus. Furthermore, a distinct saccular projection to the cochlear nuclei was evident. Utricular projections reached the four main vestibular nuclei with a denser accumulation of fibers within ventral parts of the lateral, descending and superior vestibular nuclei.     

Distribution of tyrosine hydroxylase-like immunofluorescence in guinea pig vestibular ganglia and sensory areas.

The distribution of tyrosine hydroxylase (TH)-like immunofluorescence was analyzed in the guinea pig vestibular ganglia and end organs using a monoclonal antibody to TH. TH was chosen as a marker for the sympathetic fibers because TH regulates the first step of catecholamine synthesis by converting tyrosine to dopa. In the vestibular ganglia, there were TH-positive nerve fibers having distinct varicosities surrounding the vestibular ganglion cells. In the sensory areas, there was a sympathetic plexus in the subepithelial tissue of the saccule, the utricle, and the crista ampullaris. We speculated that the sympathetic innervation has a direct influence on the vestibular ganglion cells and diffuse influence on the capillary permeability.     

Quisqualate excites spiral ganglion neurons of the guinea pig

The effects of quisqualate on primary afferent auditory neurons were examined by comparing the unit activity of guinea pig spiral ganglion neurons before, during, and after the infusion of artificial perilymph containing 1 mM quisqualate into the basal turn scala tympani. In 10 of 13 preparations quisqualate infusion increased unit activity above baseline rates established prior to infusion while control infusions of quisqualate-free artificial perilymph had no appreciable influence on the unit activity of four preparations. Postexcitatory depression typically followed peak evoked excitation, but no purely inhibitory responses were observed. Postexcitatory depression developed into a temporary cessation of spike production in three cases, suggesting depolarization blockade had developed; however, all of these preparations gradually regained some degree of spontaneous unit activity following the termination of quisqualate infusion. Quisqualate-induced excitation may be attributable to the activation of receptors for the afferent neurotransmitter released by hair cells. This interpretation is consistent with our working hypothesis that the primary afferent neurotransmitter is L-glutamate or a structural analog of this excitatory amino acid.     

Glutamate-induced production of nitric oxide in guinea pig vestibular sensory cells

Glutamate-induced production of nitric oxide (NO) in the vestibular organ of the guinea pig was investigated using the new fluorescence indicator, DAF-2DA, for direct detection of NO. Utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with glutamate, NMDA and AMPA. This is the first direct evidence of NO production in the vestibular end organs. NO may play an important role in the glutamate-induced ototoxicity and also be involved in disease of the inner ear.     

Cisplatin ototoxicity in the guinea pig: vestibular and cochlear damage

The aim of the present study was to investigate both vestibular and cochlear cisplatin toxicity. Twelve albino guinea pigs were divided into an experimental (n=8) and a control saline group (n=4) and were treated with cisplatin at a daily dose of 2.5 mg/kg for 6 consecutive days. Vestibular dysfunction was evaluated by computing the gain of the vestibular ocular reflex (VOR) evoked by stimulation in the horizontal (HVOR) and vertical (VVOR) planes. Changes in cochlear function were characterised as compound action potential threshold shifts. After the functional testing, tympanic bullae were removed and processed for morphological examination of the sensorineural epithelium. The onset of vestibular functional impairment was observed on the third day, although the VOR gain decrease was not significant. The impairment of the vestibular function progressed until the sixth day becoming statistically significant particularly at VVOR mid frequencies of stimulation. At these frequencies both macula and crista ampullaris functions are involved. Concomitantly a progressive auditory threshold shift was observed at all stimulus frequencies. The decline of the auditory function was statistically significant from the third day of treatment and it was more evident at high frequencies. Morphological observations showed a massive loss of outer hair cells and a degeneration of the organ of Corti in the basal/middle turns and only a slight loss of hair cells of the cristae ampullares and maculae. In conclusion, functional and morphological data provide evidence that the toxic effect of cisplatin is more pronounced in the organ of Corti than in the vestibular epithelium.     

Hydrostatic fluid pressure in the vestibular organ of the guinea pig

Since inner ear hair cells are mechano-electric transducers the control of hydrostatic pressure in the inner ear is crucial. Most studies analyzing dynamics and regulation of inner ear hydrostatic pressure performed pressure measurements in the cochlea. The present study is the first one reporting about absolute hydrostatic pressure values in the labyrinth. Hydrostatic pressure of the endolymphatic system was recorded in all three semicircular canals. Mean pressure values were 4.06?cmH₂O?±?0.61 in the posterior, 3.36?cmH₂O?±?0.94 in the anterior and 3.85?cmH₂O?±?1.38 in the lateral semicircular canal. Overall hydrostatic pressure in the vestibular organ was 3.76?cmH₂O?±?0.36. Endolymphatic hydrostatic pressure in all three semicircular canals is the same (p?=?0.310). With regard to known endolymphatic pressure values in the cochlea from past studies vestibular pressure values are comparable to cochlear values. Until now it is not known whether the reuniens duct and the Bast’s valve which are the narrowest passages in the endolymphatic system are open or closed. Present data show that most likely the endolymphatic system is a functionally open entity.     

Noise induced hearing loss and vestibular dysfunction in the guinea pig

Abstract

This study analysed the acoustic and vestibular functional and morphological modifications in guinea pigs after acoustic trauma. Animals were exposed to noise (6 kHz, at 120 dB SPL for 60 minutes) and then auditory brainstem responses (ABR) and vestibuloocular reflex (VOR) were measured at 6 hours, 1 day, 3, 7, and 21 days after noise. Western blotting and immunostaining for 4-hydroxy-2-noneal (4-HNE) and vascular endothelial growth factor (VEGF) were performed in the cochlear and vestibular regions at 1 and 7 days after noise exposure. A significant decrease of VOR gain was observed on day 1 and the recovery was completed at day 21. ABR threshold values reached a level of 80 dB at day 1 after trauma reaching a value of about 50 dB SPL on day 21. 4-HNE expression, a marker of lipid peroxidation was strongly increased in the cochlea. In the vestibule, 4-HNE immunoreactivity was faint. However, VEGF was up-regulated both in the cochlea and vestibule. In conclusion, the expression of VEGF in both cochlear and vestibular structures suggests a reparative role with potentially therapeutic implications.

Sumario

Este estudio analiza las modificaciones vestibulares funcionales y morfológicas en los cobayos después de un trauma acústico. Los animales fueron expuestos a ruido (6kHz, a 120dB SPL durante 60 minutos) y se midieron las respuestas auditivas de tallo cerebral (ABR) y el reflejo vestíbulo-ocular (VOR); a las 6 horas, al siguiente día y los días 3, 7 y 21 después del ruido. Se realizaron pruebas de Western Blot e inmuno-reacciones para 4-hydroxi-2-noneal (4-HNE) y para factor de crecimiento vascular endotelial (VEGF) en las regiones coclear y vestibu-lar en los días 1 y 7 después de la exposición a ruido. Los resultados mostraron un descenso significativo de la ganancia del VOR en el día 1 y la recuperación fue completa en el día 21. Los umbrales de ABR llegaron a un nivel de 80dB en el día 1 después del trauma, alcanzando un valor de 50dB SPL en el día 21. El marcador 4-HNE, indicativo de peroxidación de los lípidos, estaba bastante aumentado en la cóclea. En la región vestibular, la reacción 4-HNE fue débil. Sin embargo, el VEGF se reguló por incremento tanto en la cóclea como en el vestíbulo. En conclusión, la expresión del VEGF en las estructuras tanto cocleares como vestibulares sugiere un papel reparador con potenciales implicaciones terapéuticas.     

Impulse noise induced damage in the vestibular end organs of the guinea pig. A light microscopic study

Guinea pigs were exposed to the impulse noise from 90-300 rifle shots (peak 158 dB SPL, maximal energy content at 1.1 kHz). This exposure induced severe cochlear damage. The vestibular end organs also showed damage of varying degree. The ampullary cristae were most severely damaged, but changes were also seen in the utricular and saccular maculae. The changes appear to be primarily mechanical and to result from the effect of the acoustic pressure wave on the vestibular labyrinth.     

Direct evidence of nitric oxide production in guinea pig vestibular sensory cells

Production of nitric oxide (NO) in the vestibular organ of the guinea pig was investigated using the new fluorescence indicator, DAF-2DA, for direct detection of NO. The utricular maculae and isolated vestibular sensory cells were examined to locate NO production sites. The fluorescence intensity of the sensory cells was augmented by stimulation with L-arginine, and significantly increased after inoculation with LPS. This is the first direct evidence of NO production in the vestibular end organs. NO may play an important role for the vestibular physiology and also be involved in disease of the inner ear.     

Neuronal responses to vestibular stimulation in the guinea pig hypothalamic paraventricular nucleus

We investigated the effects of caloric stimulation on neuronal activity in the hypothalamic paraventricular nucleus (PVN) in anesthetized guinea pigs. Hot water stimulation of the contralateral labyrinth produced excitation in 29.4% of the PVN neurons tested, while cold water produced excitation in 22.2% of the neurons. Hot water resulted in inhibition of 22.4% of the neurons and cold water inhibition of 24.7% of the neurons. Intracranial vestibular nerve section greatly reduced responsiveness of the PVN neurons to caloric stimulation, indicating that the majority of the responses observed were vestibular in origin. The response pattern of the individual PVN neurons was similar following hot and cold water stimulation and after stimulation of the contralateral and ipsilateral labyrinths. These results suggest that the PVN neurons receive vestibular afferents bilaterally according to the intensity of vestibular stimulation, with the information received probably integrated in the hypothalamus to participate in vestibulo-autonomic reflexes.