Experimental ischaemia of the vertebral artery and its effect on brain stem auditory evoked responses in guinea pigs

The aim of the study was to examine the effect of artificial ischaemia of the vertebral artery region on brain stem evoked auditory potentials (BAEP) in guinea pigs. The results indicate that BAEP are influenced mainly by unilateral irritation of the vertebral artery. An increase in the audibility threshold was observed in the experimental guinea pigs in group C (an increase of 10-40 dB on the side of irritation of the vertebral artery and of 10 dB on the opposite side). Lengthening of the latency period of the P2 wave and of the conduction period between waves P1 and P5 was also observed.     

A Study of the Histologic and Enzyme Histochemical Changes in the Cochlea of Guinea Pigs After Non-Focused Ultrasound Irradiation

We evaluated the histologic and histochemical (succinate dehydrogenase, SDH) changes in cochleas of guinea pigs after non-focused ultrasound (NFU) irradiation. For this purpose, the cochleas of 50 guinea pigs (both ears in Groups 1–10) were irradiated at 2.5–8.0 MHz NFU for 6 h and the histologic/histochemical changes were determined. Our data show that after NFU irradiation for 6 h, no histological changes were observed in the cochleas by silver staining and scanning electron microscopic examination; however, the SDH activity of the basilar membranes and stria vascularises at the corresponding position decreased significantly. The SDH values of basilar membranes and stria vascularis were higher at 8 h than at 30 min after the NFU irradiation. SDH activity of the outer hair cells presented more declination than that of the inner hair cells. It was, therefore, concluded that a certain dosage of NFU irradiation at various frequencies could lead to metabolic changes in the basilar membrane and stria vascularis at different areas of cochlea. Moreover, these changes were found to be reversible or partially reversible. These changes also suggest that the cochlear hair cells located at different areas might be related to ultrasonic perception.     

一氧化氮对过氧化氢所致听力损失的保护作用

通过全耳蜗灌流法在体观察一氧化氮(N0)能否通过一氧化氮/环磷酸鸟苷(NO/cGMP)途径对抗过氧化氢这种氧自由基所致的听力损失。实验选用耳廓反射灵敏、无耳毒性药物使用史的健康杂色豚鼠(250-350 g)50只,雌雄不拘,随机分为5组,每组10只动物,分别行全耳蜗灌流人工外淋巴液;过氧化氢(H2O2);L-精氨酸(合成NO的底物);H2O2+L-精氨酸;H2O2+L-精氨酸+L-NNA(一氧化氮合成酶的抑制剂),均灌流2 h。通过圆窗龛电极,每隔30 min记录复合动作电位(compound action potential,CAP:由短声Click诱发)阈值,耳蜗微音器电位(cochlear microphonic,CM;由短纯音Tone Burst诱发)幅度,了解耳蜗功能的变化,并分离取出耳蜗基底膜并制备基底膜硬铺片,通过碘化毗啶(PI)和Hoecbst双染色方法,观察耳蜗组织各类细胞损伤情况。结果显示,灌流H2O2+L-精氨酸组的CAP阈移和CM下降幅度值明显低于单独灌流H2O2组,差异有显著性(氏P<0.05);前者形态学观察未见明显的细胞损伤,后者可见大量坏死红染的细胞。H2O1+L-精氨酸+L-NNA组CAP阈移和CM下降幅度与单独灌流H2O2组比较无统计学差异。实验结果提示NO可能通过NO/cGMP途径部分对抗过氧化氢所致的听力损失。     

Fibroblast growth factor-2 immunoreactivity is present in the central and peripheral auditory pathways of adult rats

Recent findings have pointed out the role of neurotrophic factors in the survival and maintenance of neurons of the auditory system. Basic fibroblast growth factor (bFGF, FGF-2) is a potent neurotrophic molecule whose actions can be seen in the central and peripheral nervous systems. In the present study, FGF-2 immunoreactivity was analyzed in the auditory pathways of the adult rat, employing a well-characterized polyclonal antibody against FGF-2. In the cochlea, FGF-2 immunoreactivity was observed in the inner and outer hair cells of the organ of Corti, spiral ganglion neurons, spiral limbus, and stria vascularis. Stereological methods employing optical fractionator revealed the presence of 84.5, 15, and 0.5% of spiral ganglion neurons possessing FGF-2 immunoreactivity of strong, moderate, and weak intensity, respectively. In the central auditory pathways, FGF-2 immunoreactivity was found in the cytoplasm of the neurons of the cochlear nuclei, trapezoid body nuclei, medial geniculate nucleus, and inferior colliculus. The two-color immunoperoxidase method showed FGF-2 immunoreactivity in the nuclei of astrocytes throughout the central auditory pathway. Computer-assisted microdensitometric image analysis revealed higher levels of specific mean gray values of FGF-2 immunoreactivity in the trapezoid body and ventral cochlear nucleus and also in the spiral ganglion and inner hair cells. Sections incubated with FGF-2 antibody preabsorbed with human recombinant FGF-2 showed no immunoreaction in the majority of the studied regions, exhibiting only a slight immunoreactive product in the hair cells of the organ of Corti. Furthermore, no changes in immunoreactivity were observed in sections incubated with FGF-2 antiserum preincubated with human recombinant acidic FGF (FGF-1). The findings suggest that FGF-2 may exert paracrine and autocrine actions on neurons of the central and peripheral auditory systems and may be of importance in the mechanism of hearing diseases.     

电针对庆大霉素耳毒性的预防作用

目的和方法 :本实验用耳廓反射 (PR)、脑干听觉诱发电位 (BAEP)、组织化学方法 ,观察电针疗法对豚鼠庆大霉素 (GE)耳毒性的防治作用 ,并探讨其机制。结果 :电针疗法能降低GE引起的PR阈值和BAEP反应阈的上移幅度及BAEP波峰潜伏期和波峰间期的延长 ,能维持毛细胞溶酶体的完整 ,防止毛细胞的自溶性破坏。结论 :电针疗法有对抗GE耳毒性作用。维持耳蜗毛细胞内溶酶体完整 ,防止溶酶体内水解酶的逸出而发生细胞自溶 ,可能是电针疗法的作用机制之一。     

内耳减压病豚鼠听力和耳蜗组织的病理变化

研究探讨了内耳减压病豚鼠皮层听觉诱发电位阈值、耳蜗火棉胶切片、酶组织化学和透射电镜观察的变化。结果表明,豚鼠内耳减压病导致听力损失,耳蜗广泛的病理损害.毛细胞琥珀酸脱氢酶活性降低。提出了加压治疗内耳减压病时配合改善微循环、增加能量供应等见解。     

脑细胞生长肽对GM耳毒性的预防作用

目的：观察脑细胞生长肽（cerebral cell growth peptide,CCGP)对庆大霉素（gentamicin,GM)引起的豚鼠耳中毒的预防作用）。方法：用脑干听觉诱发电位（brainstem auditory evoked potential,BAEP)和组织化学方法分别检测动物听阀的变化和耳蜗毛细胞线粒体内琥珀酸脱氢酶（succinate dehydrogenase,SDH)及溶酶体内的酸性磷酸酶（acid phosphatase,ACP)活性变化,并在三组动物进行耳蜗受损毛细胞计数。结果：CCGP能降低GM引起的BAEP反应阈的上升幅度,保护耳蜗毛细胞SDH和维持溶酶体的完整性,减轻毛细胞的损伤。结论：CCGP能降低GM的中毒性;保护耳蜗毛细胞SDH,维持毛细胞的能量代谢,减少溶酶体损伤,降低溶酶体内水解酶逸出引起的毛细胞自溶性损伤,可能是CCGP预防GM耳毒性的机制之一。     

ROCK-dependent and ROCK-independent control of cochlear outer hair cell electromotility

Outer hair cell electromotility is crucial for the proper function of the cochlear amplifier, the active process that enhances sensitivity and frequency discrimination of the mammalian ear. Previous work (Kalinec, F., Zhang, M., Urrutia, R., and Kalinec, G. (2000) J. Biol. Chem. 275, 28000-28005) has suggested a role for Rho GTPases in the regulation of outer hair cell electromotility, although the signaling pathways mediated by these enzymes remain to be established. Here we have investigated the cellular and molecular mechanisms underlying the homeostatic regulation of the electromotile response of guinea pig outer hair cells. Our findings defined a ROCK-mediated signaling cascade that continuously modulates outer hair cell electromotility by selectively targeting the cytoskeleton. A distinct ROCK-independent pathway functions as a fast resetting mechanism for this system. Neither pathway affects the function of prestin, the unique molecular motor of outer hair cells. These results extend our understanding of a basic mechanism of both normal human hearing and deafness, revealing the key role of the cytoskeleton in the regulation of outer hair cell electromotility and suggesting ROCK as a molecular target for modulating the function of the cochlear amplifier.     

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.     

TRPC3 ion channel subunit immunolocalization in the cochlea

Canonical transient receptor potential (TRPC) subunits assemble as tetramers to form ion channels with high calcium (Ca²⁺) permeability. Here, we investigated the possibility that TRPC3 ion channels are broadly expressed in the adult guinea pig and mouse cochleae. Using immunofluorescence, pronounced labeling occurred in the spiral ganglion (SG) neurons, inner hair cells (IHC), outer hair cells (OHC) and epithelial cells lining scala media. TRPC3 expression was homogeneous in the SG throughout the cochlea. In contrast, there was marked spatial variation in the immunolabeling in the cochlear hair cells with respect to location. This likely relates to the tonotopy of these cells. TRPC3 immunolabeling was more pronounced in the IHC than OHC. Both basal region IHC and OHC had higher TRPC3 expression levels than the corresponding cells from the apical region of the cochlea. These data suggest that TRPC3 ion channels contribute to Ca²⁺ homeostasis associated with the hair cells, with higher ion fluxes in more basal regions of the cochlea, and may also be a significant pathway for Ca²⁺ entry associated with auditory neurotransmission via the SG neurons. TRPC3 expression was also identified within the spiral limbus region, inner and outer sulcus, but without evidence for spatial variation in expression level. Expression in these gap junction-coupled epithelial cells lining scala media is indicative of a contribution of TRPC3 channels to cochlear electrochemical homeostasis.     

Developmental regulation of TRPC3 ion channel expression in the mouse cochlea

Canonical transient receptor potential type 3 (TRPC3) ion channels assemble from TRPC3 subunits and exhibit multiple activation mechanisms. TRPC3 has been proposed to contribute to Ca²⁺ entry supporting Ca²⁺ homeostasis in cochlear hair cells and to be activated by G protein-coupled receptor (GPCR) signaling in spiral ganglion neurons. The present study was designed to determine the spatiotemporal profile of TRPC3 expression during mouse cochlear ontogeny. TRPC3 immunofluorescence of cryosectioned cochleae was performed using E16–adult tissue. We found that prior to birth, TRPC3 expression was strongest in epithelial cells that form the cochlear partition. In the early postnatal period, to the onset of hearing (~P12), immunofluorescence was strongest in the hair cells, with increased expression in stria vascularis and Reissner’s membrane. Afferent neurite labeling in inner spiral plexus and outer spiral bundles developed transiently in the perinatal period, corresponding to the critical period of synaptic consolidation, while signal in the spiral ganglion soma increased from the perinatal period through to adulthood. Compared with the late embryonic/early postnatal levels, hair cell expression was relatively weaker from the third postnatal week, whereas spiral ganglion soma labeling was stronger. In the adult, TRPC3 expression was primarily in the soma of spiral ganglion neurons, the hair cells, and the inner and outer sulcus regions. This spatiotemporal profile of TRPC3 expression was consistent with this ion channel contributing to development of sensory, neural and epithelial cochlear tissues, as well as hair cell Ca²⁺ homeostasis and regulation of auditory neurotransmission via GPCR signaling.     

电码针对庆大霉素耳中毒的防治作用及其机制的研究

目的：观察电码针对豚鼠庆大霉素（GE）耳毒性的防治作用,方法：测定脑干听觉诱发电位（BAEP）和用组织化学方法测定耳蝇毛细胞及血管纹的琥珀酸脱氢酶（SDH）。结果：电针能降低CE引起的BAEP反应阈的上升幅度,缩小BAEP波峰潜伏期及波峰间期的延长;能保护毛细胞及耳蜗血管纹细胞线粒体呼吸酶的活性。结论：电针能降低GE5的耳毒性,保护毛细胞及耳蜗血管纹细胞线粒体酶的活性。保证这些细胞能量代谢,维持细胞所需要能量的各种功能的活动。减少细胞的损伤,可能有是电针防治GE耳毒性的机制之一。     

In vivo outer hair cell length changes expose the active process in the cochlea

Background

Mammalian hearing is refined by amplification of the sound-evoked vibration of the cochlear partition. This amplification is at least partly due to forces produced by protein motors residing in the cylindrical body of the outer hair cell. To transmit power to the cochlear partition, it is required that the outer hair cells dynamically change their length, in addition to generating force. These length changes, which have not previously been measured in vivo, must be correctly timed with the acoustic stimulus to produce amplification.

Methodology/Principal Findings

Using in vivo optical coherence tomography, we demonstrate that outer hair cells in living guinea pigs have length changes with unexpected timing and magnitudes that depend on the stimulus level in the sensitive cochlea.

Conclusions/Significance

The level-dependent length change is a necessary condition for directly validating that power is expended by the active process presumed to underlie normal hearing.     

PMCA2 mutation causes structural changes in the auditory system in deafwaddler mice

Homozygous deafwaddler mice (dfw/dfw) have a mutation in the gene encoding plasma membrane Ca²⁺ATPase isoform 2 (Pmca2). They walk with a hesitant and wobbly gait, display head bobbing and are deaf. Light microscopy and transmission electron microscopy were used to evaluate the nature and relationship of morphological changes in the cochlea, spiral ganglion cells and spherical cells of the cochlear nucleus in homozygous and heterozygous mice of different ages and controls. Ultrastructural findings showed that in 7 week old homozygous (dfw) mice, inner hair cells and their afferent terminals were present although outer hair cells appeared apoptotic. Stereocilia were absent from the second and third rows of outer hair cells. Ganglion cells were also present although abnormal in appearance. In older homozygous mutants there was a loss of hair cells and spiral ganglion cells. Remaining ganglion cells in this group contained very few cytoplasmic organelles apart from a few hypertrophied mitochondria. In the anteroventral cochlear nucleus, spherical cell soma size was smaller in all homozygous (dfw) mutants than in heterozygous mice and controls. The ultrastructural appearance of the end bulbs of Held in homozygous mutants was abnormal compared with controls, and in the younger group were seen to be swollen, with less distinct synaptic densities and containing large numbers of small synaptic vesicles arranged in clumps. In the older group these synapses were distorted and contained hypertrophied mitochondria and no synaptic densities could be seen, suggesting that these synapses may be non-functional. This study has shown that in homozygous (dfw) mice structural abnormalities occurred not only in cochlear hair cells but also in the spiral ganglion neurones and spherical cells in the cochlear nucleus. It seems likely that these changes are the result of the Pmca2 mutation and the subsequent accumulation of toxic levels of calcium that may lead to alterations in their functional integrity.     

Radioprotective Effect of Aminothiol PrC-210 on Irradiated Inner Ear of Guinea Pig

Radiotherapy of individuals suffering with head & neck or brain tumors subserve the risk of sensorineural hearing loss. Here, we evaluated the protective effect of Aminothiol PrC-210 (3-(methyl-amino)-2-((methylamino)methyl)propane-1-thiol) on the irradiated inner ear of guinea pigs. An intra-peritoneal or intra-tympanic dose of PrC-210 was administered prior to receiving a dose of gamma radiation (3000 cGy) to each ear. Auditory Brainstem Responses (ABRs) were recorded one week and two weeks after the radiation and compared with the sham animal group. ABR thresholds of guinea pigs that received an intra-peritoneal dose of PrC-210 were significantly better compared to the non-treated, control animals at one week post-radiation. Morphologic analysis of the inner ear revealed significant inflammation and degeneration of the spiral ganglion in the irradiated animals not treated with PrC-210. In contrast, when treated with PrC-210 the radiation effect and injury to the spiral ganglion was significantly alleviated. PrC-210 had no apparent cytotoxic effect in vivo and did not affect the morphology or count of cochlear hair cells. These findings suggest that aminothiol PrC-210 attenuated radiation-induced cochlea damage for at least one week and protected hearing.     

Effect of c-myc on the ultrastructural structure of cochleae in guinea pigs with noise induced hearing loss

Noise over-stimulation may induce hair cells loss and hearing deficit. The c-myc oncogene is a major regulator for cell proliferation, growth, and apoptosis. However, the role of this gene in the mammalian cochlea is still unclear. The study was designed to firstly investigate its function under noise condition, from the aspect of cochlear ultrastructural changes. We had established the adenoviral vector of c-myc gene and delivered the adenovirus suspension into the scala tympani of guinea pigs 4 days before noise exposure. The empty adenoviral vectors were injected as control. Then, all subjects were exposed to 4-kHz octave-band noise at 110 dB SPL for 8 h/day, 3 days consecutively. Auditory thresholds were assessed by auditory brainstem response, prior to and 7 days following noise exposure. On the seventh days after noise exposure, the cochlear sensory epithelia surface was observed microscopically and the cochleae were taken to study the ultrastructural changes. The results indicated that auditory threshold shift after noise exposure was higher in the ears treated with Ad.EGFP than that treated with Ad.c-myc-EGFP. Stereocilia loss and the disarrangement of outer hair cells were observed, with greater changes found in the Ad.EGFP group. Also, the ultrastructure changes were severe in the Ad.EGFP group, but not obvious in the Ad.c-myc-EGFP group. Therefore, c-myc gene might play an unexpected role in hearing functional and morphological protection from acoustic trauma.     

Ectopic expression of ROR1 prevents cochlear hair cell loss in guinea pigs with noise‐induced hearing loss

Noise‐induced hearing loss (NIHL) is one of the most frequent disabilities in industrialized countries. Evidence shows that hair cell loss in the auditory end organ is responsible for the majority of various ear pathological conditions. The functional roles of the receptor tyrosine kinase ROR1 have been underscored in various tumours. In this study, we evaluated the ability of ROR1 to influence cochlear hair cell loss of guinea pigs with NIHL. The NIHL model was developed in guinea pigs, with subsequent measurement of the auditory brainstem response (ABR). Gain‐of‐function experiments were employed to explore the role of ROR1 in NIHL. The interaction between ROR1 and Wnt5a and their functions in the cochlear hair cell loss were further analysed in response to alteration of ROR1 and Wnt5a. Guinea pigs with NIHL demonstrated elevated ABR threshold and down‐regulated ROR1, Wnt5a and NF‐κB p65. The up‐regulation of ROR1 was shown to decrease the cochlear hair cell loss and the expression of pro‐apoptotic gene (Bax, p53) in guinea pig cochlea, but promoted the expression of anti‐apoptotic gene (Bcl‐2) and the fluorescence intensity of cleaved‐caspase‐3. ROR1 interacted with Wnt5a to activate the NF‐κB signalling pathway through inducing phosphorylation and translocation of p65. Furthermore, Wnt5a overexpression decreased the cochlear hair cell loss. Collectively, this study suggested the protection of overexpression of ROR1 against cochlear hair cell loss in guinea pigs with NIHL via the Wnt5a‐dependent NF‐κB signalling pathway.     

Caspase-3在老年豚鼠耳蜗螺旋神经节细胞的表达

目的检测caspase-3在老年豚鼠耳蜗的表达。方法实验分两组:实验组和对照组,实验组豚鼠年龄为33至35个月之间,对照组豚鼠年龄为2至3个月。用免疫组织化学方法检测caspase-3在两组豚鼠耳蜗的表达。结果Caspase-3在实验组耳蜗的表达呈阳性,阳性区域主要存在于耳蜗螺旋神经节细胞。在对照组耳蜗的表达呈阴性。结论Caspase-3在老年豚鼠耳蜗螺旋神经节细胞中呈阳性表达,提示caspase-3在豚鼠耳蜗老化过程中起重要作用。     

卡那霉素耳慢性中毒对豚鼠耳蜗毛细胞中Bcl-2表达的影响

目的:探讨卡那霉素耳慢性中毒对豚鼠耳蜗毛细胞中Bcl-2表达的影响。方法:取20只豚鼠随机分为2组,实验组连续14d肌肉注射硫酸卡那霉素,200mg/(kg.d),对照组连续14d等量肌肉注射生理盐水,停药14d处死动物后制作耳蜗标本,处死前检测其ABR的变化,免疫组化及原位杂交法测定Bcl-2的表达。结果:豚鼠卡那霉素耳慢性中毒后,ABR阈值较对照组明显上升,Bcl-2阳性表达减低,与对照组比较差异有显著性(P<0.05)。结论:卡那霉素耳慢性损害可能与抑制Bcl-2的表达有关。     

Addition of Exogenous NAD+ Prevents Mefloquine-Induced Neuroaxonal and Hair Cell Degeneration through Reduction of Caspase-3-Mediated Apoptosis in Cochlear Organotypic Cultures

Background

Mefloquine is widely used for the treatment of malaria. However, this drug is known to induce neurological side effects including depression, anxiety, balance disorder, and sensorineural hearing loss. Yet, there is currently no treatment for these side effects.

Principal Findings

In this study, we show that the coenzyme NAD⁺, known to play a critical role in maintaining the appropriate cellular redox environment, protects cochlear axons and sensory hair cells from mefloquine-induced degeneration in cultured rat cochleae. Mefloquine alone destroyed hair cells and nerve fiber axons in rat cochlear organotypics cultures in a dose-dependent manner, while treatment with NAD⁺ protected axons and hair cells from mefloquine-induced degeneration. Furthermore, cochlear organs treated with mefloquine showed increased oxidative stress marker levels, including superoxide and protein carbonyl, and increased apoptosis marker levels, including TUNEL-positive nuclei and caspases-3. Treatment with NAD⁺ reduced the levels of these oxidative stress and apoptosis markers.

Conclusions/Significance

Taken together, our findings suggest that that mefloquine disrupts the cellular redox environment and induces oxidative stress in cochlear hair cells and nerve fibers leading to caspases-3-mediated apoptosis of these structures. Exogenous NAD⁺ suppresses mefloquine-induced oxidative stress and prevents the degeneration of cochlear axons and sensory hair cells caused by mefloquine treatment.