The effect of furosemide on glucose oxidation of the cochlea and other tissues

Summary We tested the hypothesis that furosemide interferes with energy generation in the cochlea, and determined its effect on CO₂ formation from glucose and glyceroaldehyde-3-phosphate dehydrogenase (GAPDH) activity by examining biochemical and histochemical changes in the cochlea, the kidney, and the liver. We found that furosemide suppressed CO₂ formation in vitro at relatively low concentrations in all tissues examined. GAPDH was inhibited as soon as 2 min after its administration (80 mg/kg, i.v.). Recovery of this enzyme activity was most rapid in the liver. We concluded that furosemide does interfere with energy generation in the cochlea, kidney, and liver as a result of its inhibition of GAPDH.     

Drug delivery to the cochlea using PLGA nanoparticles

OBJECTIVES: This study aimed to investigate the efficacy of encapsulating therapeutic molecules in poly lactic/glycolic acid (PLGA) nanoparticles for drug delivery to the cochlea. STUDY DESIGN: An experimental study. METHODS: We examined the distribution of rhodamine, a fluorescent dye, in the cochlea, liver, and kidney of guinea pigs. Intravenous injection of rhodamine or rhodamine-encapsulated PLGA nanoparticles was used to target the fluorescent dye systemically to the liver, kidney, and cochlea, and these molecules were applied locally to the round window membrane (RWM) of the cochlea. The localization of rhodamine fluorescence in each region was quantitatively analyzed. RESULTS: After systemic application of rhodamine nanoparticles, fluorescence was identified in the liver, kidney, and cochlea. The systemic application of nanoparticles had a significant effect on targeted and sustained delivery of rhodamine to the liver but not the kidney or cochlea. Rhodamine nanoparticles placed on the RWM were identified in the scala tympani as nanoparticles, indicating that the PLGA nanoparticles can permeate through the RWM. Furthermore, the local application of rhodamine nanoparticles to the RWM was more effective in targeted delivery to the cochlea than systemic application. CONCLUSIONS: These findings indicate that PLGA nanoparticles can be an useful drug carrier to the cochlea via local application.     

Noxious effects upon cochlear metabolism

The influence of various toxic substances and of drugs with ototoxic side effects upon energy generation, energy utilization, and membrane processes of the cochlea were studied. None of the drugs tested interfered with energy generation to as great an extent as did anoxia or cyanide and 2,4-dinitrophenol. Ouabain produced a pronounced interference with energy utilization of the stria vascularis. The “loop” diuretics ethacrynic acid and furosemide produced a reduction of energy utilization of a lesser degree than did ouabain. The “loop” diuretics do not seem to exert their toxic action upon strial Na+K+-ATPase, but may act by interfering with strial adenylate cyclase. Aminoglycoside antibiotics and diuretic and nondiuretic mercurials seem to exert their primary noxious action upon cochlear function by interfering with membrane processes of the structures bounding the cochlear duct.     

Alternating current induced otoacoustic emissions in the guinea pig.

Injection of alternating current (AC) into the scala media of the guinea pig cochlea induced otoacoustic emissions (OAEs) at the frequency of the AC fundamental, together with harmonic and intermodulation distortion products. Although the waveform of the injected ACs was distorted, probably due to nonlinear polarization of the metal electrodes, and was composed of the fundamental plus distortion products of every order, only a few of the lowest order distortion products were selectively emitted with the fundamental. AC injection at a basal site extended the high frequency limit of OAEs. Electrical stimulation of the crossed olivocochlear bundle inhibited the sideband emissions with little change in the fundamental. OAE was reduced reversibly by temporary impairment of the cochlea due to exposure to fatiguing sound, by intravenous application of furosemide and by temporary anoxia. Irreversible reduction resulted from intracochlear perfusion with excess K+ solution, acoustic trauma and cardiac arrest. These facts imply that AC-induced OAE is not an artifact generated electrically; rather, such emissions originate in the cochlea and normal metabolic activity in the cochlea is essential. A proposed mechanism of generation includes two components: 1) electromechanical transduction from AC to mechanical vibration in the cochlea and 2) a distortion-producing process; the contribution of each component to the receptor mechanism is discussed.     

Ensemble spontaneous activity in the guinea-pig cochlear nerve

Spectral analysis of electrical noise recorded from the round window (RW) of the cochlea is referred to as the ensemble spontaneous activity (ESA) of the cochlear nerve. The ESA is considered to represent the summed spontaneous activity of single fibers of the auditory nerve and changes in the spectral characteristics of the ESA have been observed in humans with tinnitus. Experiments were undertaken to determine the relationship of the ESA to auditory neurotransmission. The ESA consisted of energy centered at approximately 900 Hz, similar to the spectral peak of single auditory neuron discharges. The amplitude of the ESA was correlated with good auditory sensitivity in the 12-30 kHz region of the cochlea. Constant pure tones of 12-22 kHz suppressed the ESA reducing its amplitude in a frequency and intensity dependent manner implying that the ESA recorded at the RW is generated or dominated by neurons in the basal region of the cochlea. The ESA was significantly suppressed by round window perfusion of the P2X receptor agonist adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) (10 mM) the glutamate receptor antagonist 6-7-dinitroquinoxaline-2,3-dione (DNQX) (1 mM), and the sodium channel antagonist tetrodotoxin (TTX) (20 microM). Following intravenous furosemide injection (40 mg/kg) reduction and recovery of the ESA correlated with similar changes in the endocochlear potential (EP). Following DNQX and ATPgammaS an additional spectral peak at 200 Hz was often observed. This peak has been postulated to be a correlate of tinnitus in humans but had not previously been observed in a guinea-pig model of tinnitus. These data confirm the spectral characteristics of the ESA in guinea-pigs and show it is dependent on the sensitivity of the auditory nerve and intact auditory neurotransmission. In addition these experiments support the view that the ESA represents summed spontaneous neural activity in the cochlea and provide a platform for studies of the influence of ototoxic compounds on the spontaneous neural outflow of the cochlea as a model of tinnitus.     

Deafness and renal failure due to potassium bromate poisoning.

Several substances are known to have a profound effect on both the kidney and the inner ear. The aminoglycoside antibiotics are toxic to both of these organs. Ethacrinic acid and furosemide affect transport mechanisms in the loop of Henle as well as the inner ear. In addition, studies have suggested antigenic similarities between the stria vascularis of the cochlea and the renal tubules. This report of potassium bromate poisoning and its resultant deafness and renal failure further emphasizes the curious relationship between these two organ systems.     

Chronic Reduction of Endocochlear Potential Reduces Auditory Nerve Activity: Further Confirmation of an Animal Model of Metabolic Presbyacusis

Gerbils aged in quiet show a decline of the endocochlear potential (EP) and elevated auditory nerve compound action potential (CAP) thresholds. However, establishing a direct relationship between an age-related reduction in the EP and changes in the activities of primary auditory neurons is difficult owing to the complexity of age-related histological changes in the cochlea. To address this issue, we developed a young gerbil model of “metabolic” presbyacusis that uses an osmotic pump to deliver furosemide into the round window niche for 7 days, resulting in a chronically reduced EP. In this model, the only major histopathologic changes were restricted to the hook region of the cochlea and consisted of loss of strial intermediate cells and massive edema in the lateral wall. The morphological and physiological evidence suggests that the cochlea can adapt to furosemide application over time. The morphology of spiral ganglion cells and hair cells appeared normal throughout the cochlea. CAP responses and EP values in this model are similar to those of quiet-aged ears. The spontaneous activity of single auditory fibers (n = 188) was assessed in 15 young gerbils treated with furosemide for 7 days. The percentage of recorded low-spontaneous rate (SR) fibers at characteristic frequencies (CFs) ≥ 6 kHz was significantly lower in furosemide-treated than in control ears. Recovery function tests of CAP responses after prior stimulation also showed a decline in activity of the low-SR population with CFs ≥ 6 kHz in the treated cochleas. A similar loss in the activity of low-SR fiber has been previously shown in quiet-aged gerbils. These results suggest that dysfunction of the cochlear lateral wall and subsequent chronic reduction in the EP can directly affect the activity patterns of primary auditory neurons in a manner similar to that seen in aged gerbils.     

Changes in endolymph chloride concentration following furosemide injection

Endocochlear potential (EP) and chloride concentration in endolymph were monitored with microelectrodes in the basal turn of the cochlea of the chinchilla. After intravenous injection of furosemide (25-100 mg/kg), the EP dropped precipitously and rapidly reached its minimum value, however, the chloride activity in endolymph decreased more gradually. Possible mechanisms for this phenomenon include a reduced electrostatic attraction of chloride ions to the scala media due to a decreased EP and a reduction of passive influx of chloride into endolymph, resulting from a reduction of active inward potassium transport by furosemide.     

Influence of carbon dioxide laser irradiation on the morphology and function of guinea pig cochlea

Both experimental and clinical studies have demonstrated that carbon dioxide laser is suitable for stapedotomy. The aim of this study was to investigate morphological, electrophysiological and functional changes in the inner ear after irradiation with CO(2) laser set with different energy parameters. A cochleostomy in the basal cochlear turn of guinea pig cochleae was performed with CO(2) laser of 1, 2 and 3 w, respectively. The cochleae were removed three weeks after laser irradiation. The auditory evoked brainstem response (ABR) was measured before and after laser application and immediately before removal of the cochlea. Immunohistochemical methods were used to examine inducible nitric oxide synthase (iNOS/NOSII) and heat-shock protein 70 (Hsp70) concentrations in the cochlea after laser application. The organ of Corti was studied by scanning electron microscopy. Worse hearing loss was observed in animals receiving higher-power CO(2) laser. These findings correlated with more intense injury of the cochlear ultrastructure and with positive expression of iNOS and Hsp70 in spiral ganglion cells, nerve fibres, supporting cells of the organ of Corti and cells of the spiral ligament. The CO(2) laser as a noncontact procedure is shown to be effective and safe if the total amount of energy is kept within the limits applied in this study. Nitric oxide and stress proteins play important roles in the traumatic mechanism of the inner ear, which are related to hearing loss and injury of the ultrastructure of the inner ear.     

The cochlea magnesium content is negatively correlated with hearing loss induced by impulse noise

Objective

Magnesium is proved to attenuate acoustic trauma, and reactive oxygen species (ROS) formation is a critical role that involves hearing loss induced by impulse noise. We aimed to investigate the relationship between the cochlea magnesium content, ROS formation and hearing loss induced by impulse noise.

Methods

Ninety pigmented guinea pigs were exposed to impulse noise. Auditory thresholds were assessed by sound-evoked auditory brainstem response (ABR) 24 h before and 72 h after exposure to impulse noise. 4-Hydroxynonenal(HNE) used as a marker of ROS was determined immunohistochemically. The cochlea magnesium content was examined with the method of energy dispersive x-ray analysis, and the cochlea was also detected with scanning electron microscope. The relationship between the cochlea magnesium content, ROS formation and hearing loss was analyzed.

Results

There was loss of outer hair cell cilia accompanying with significant auditory threshold shift after impulse noise exposure. ROS was positive in the organ of Corti of all animals. The cochlea magnesium content was negatively correlated with ROS formation and hearing loss.

Conclusions

Inhibiting ROS formation is one of the mechanisms for magnesium to reduce acoustic trauma, and difference in cochlea magnesium contents is one of the factors that induce varying degrees of cochlear damage among each individual after acoustic trauma.     

Cochlear damage resulting from kanamycin and furosemide.

Permanent cochlear damage has been shown to occur in guinea pigs following the combined administration of kanamycin and furosemide. At the doses used, only a transient effect was measured with furosemide alone and no effect was detectable with kanamycin alone. This interaction results when a single subcutaneous dose of 400 mg/kg of kanamycin is followed in 2 hours by a single intravenous dose of furosemide. The dosage range for furosemide was 50 mg/kg for a just-detectable effect to 100 mg/kg for a very severe effect. Damage to the cochlea was ascertained by measures of the a.c. cochlear potential as well as surface preparation histology.     

Smad4 基因在野生型和基因缺陷小鼠耳蜗内的定位和分布

目的检测Smad4基因任成年小鼠耳蜗中是否有表达及表达部位的分布情况，以进一步研究Smad4基因在内耳发育以及听功能调控上的作用。方法动物由军事医学科学院发育和疾病遗传学研究室杨晓研究员提供．动物获取是在C57BL／6J和Blackswiss两种遗传背景的小鼠应用Cre—LoxP系统进行条件基因打靶，于小鼠胚胎早期在软骨细胞内将Smad4基因剔除，并生成同窝三种基因型小鼠-野生型Smad4＋/＋，杂合子Smad4＋／-，纯合子Smad4-／-用于本研究。对同窝三种不同基因型小鼠的耳蜗冰冻切片应用免疫组织化学方法染色观察．阴性对照为野生型小鼠耳蜗的冰冻切片并用PBS代替一抗．阳性对照为小鼠肾脏组织冰冻切片。结果Smad4在三种基因型小鼠耳蜗均有广泛表达，表达部位主要集中于血管纹、螺旋韧带、基底膜、盖膜、毛细胞、支持细胞、螺旋神经节细胞等处，其中血管纹和基底膜表达最为明显．Smad4在野生型和杂合型小鼠的耳蜗内表达情况基本一致，但纯合子小鼠耳蜗内表达与前两学相比为较低的水平．但未完全消失。结论Smad4在正常小鼠的耳蜗广泛存在，该基因是耳蜗中广泛表达的蛋白中的一种。作为Bmp4信号通路下游信号因子，它可能在骨性耳蜗的形成、内耳感觉细胞的分化成熟过程中起着重要的作用。     

Effect of the pulsed Er:YSGG and Ho:YAG laser on the organ of Corti of the guinea pig cochlear--a scanning electron microscopy study

BACKGROUND: Recent experimental studies have demonstrated that, apart from the continuous wave lasers, several pulsed laser systems are also suitable for stapedotomy. The aim of this study was to clarify whether irradiation of the basal convolution of the guinea pig cochlea could cause morphological inner-ear changes using the Er:YSGG and Ho:YAG laser with laser parameters required for stapedotomy. METHODS: After opening the bulla, the basal convolution of the guinea pig cochlea, whose thickness is similar to that of the human stapes footplate, was irradiated with the Er:YSGG and Ho:YAG laser. The laser parameters used were those necessary for an adequate perforation of a human stapes footplate (500-600 microns). The cochleae were removed 90 minutes, 1 day, 2 weeks, or 4 weeks after laser application. The organ of Corti was examined by scanning electron microscopy in all convolutions. RESULTS: Application of Er:YSGG laser parameters effective for stapedotomy (5 pulses, energy: 85 J/pulse, energy density: 36 J/cm2, total energy: 0.425 J) had no adverse effects on the organ of Corti in the guinea pig cochlea. On the other hand, effective Ho:YAG laser parameters (10 pulses, energy: 210 J/pulse, energy density: 90 J/cm2, total energy: 2.1 J) caused damage to the outer hair cells with fusion of stereocilia and formation of giant cilia leading to partial or total cell loss. The inner hair cells and supporting cells were usually normal. CONCLUSION: Our results clearly demonstrate that the Er:YSGG laser has high application safety. It could prove to be a viable alternative to the thermically acting CO2 laser for stapedotomy. The Ho:YAG laser is not well tolerated in animals and has low application safety. Its use in stapedotomy would be unreliable and dangerous for the inner ear.     

Effect of albumin-bound furosemide on the endocochlear potential of the chinchilla. Alleviation of furosemide-induced ototoxicity

The effects of albumin-unbound furosemide and albumin-bound furosemide on the cochlear function were compared by the continuous observation of the endocochlear potential (EP) in the chinchilla using the microelectrode method. The EP depression following the intravenous injection of 50 mg/kg of furosemide was 108.5 +/- 2.7 mV, while the addition of 1.0 and 1.3 g/kg of albumin induced the EP depression to be 35.0 +/- 4.8 and 8.1 +/- 1.9 mV, respectively, and both prolonged the time to attain the minimum EP. However, there was no difference in the recovery time of the EP between the two groups. The results indicate that access to the site of furosemide action in the cochlea is dependent on the unbound fraction of furosemide and that the albumin-bound furosemide alleviates the EP depression induced by furosemide alone with the augmentation of diuresis.     

Reduction of acute cisplatin ototoxicity and nephrotoxicity in rats by oral administration of allopurinol and ebselen

Cisplatin ototoxicity has been associated with the generation of toxic levels of reactive oxygen species (ROS) which can lead to injury or loss of outer hair cells in the organ of Corti, damage to the stria vascularis, and loss of spiral ganglion cells, resulting in permanent hearing loss. In an attempt to reduce the formation of ROS and to bolster the innate oxidative stress defenses of the cochlea, we tested individual and combined formulations of allopurinol, a xanthine oxidase inhibitor, and ebselen, a glutathione peroxidase mimic. We used an acute cisplatin toxicity rat model (16 mg/kg i.p.) to analyze allopurinol and ebselen alone and in combination for their ability to reduce cisplatin associated hearing loss and nephrotoxicity. The results from our studies indicate that a combined formulation of ebselen and allopurinol affords significant protection to the cochlea and kidney from cisplatin toxicity. In the cochlea, protection is dependent on the preservation of outer hair cell number, while in the kidney, protection is associated with the preservation of proximal tubular epithelia. Further evaluation of the chemoprotective effects of ebselen and allopurinol on cisplatin side effects in the presence of tumor appears warranted.     

Oxidative stress pathways in the potentiation of noise-induced hearing loss by acrylonitrile

We hypothesize that the disruption of antioxidant defenses is a key mechanism whereby chemical contaminants can potentiate noise-induced hearing loss (NIHL). This hypothesis was tested using acrylonitrile (ACN), a widely used industrial chemical whose metabolism is associated with glutathione (GSH) depletion and cyanide (CN) generation. CN, in turn, can inhibit Cu/Zn superoxide dismutase (SOD). We have shown previously that ACN potentiates NIHL, even with noise exposure approaching permissible occupational levels. However, the relative involvement of GSH depletion and/or CN production in this potentiation is still unknown. In this study, we altered these metabolic pathways pharmacologically in order to further delineate the role of specific antioxidants in the protection of the cochlea. We investigated the effects of sodium thiosulfate (STS), a CN inhibitor, 4-methylpyrazole (4MP), a drug that blocks CN generation by competing with CYP2E1, and l-N-acetylcysteine (l-NAC), a pro-GSH drug, in order to distinguish between GSH depletion and CN production as the mechanism responsible for potentiation of NIHL by ACN. Long-Evans rats were exposed to an octave-band noise (97 dB SPL, 4h/day, 5 days) and ACN (50 mg/kg). Separate pre-treatments with STS (150 mg/kg), 4MP (100 mg/kg) and l-NAC (4 x 400 mg/kg) all dramatically reduced blood CN levels, but only l-NAC significantly protected GSH levels in both the liver and the cochlea. Concurrently, only l-NAC treatment decreased the auditory loss and hair cell loss resulting from ACN + noise, suggesting that GSH is involved in the protection of the cochlea against reactive oxygen species generated by moderate noise levels. On the other hand, CN does not seem to be involved in this potentiation.     

An experimental study using sodium salicylate to reduce cochlear changes induced by furosemide

Furosemide is a loop diuretic which has been found to be ototoxic in humans and experimental animals. The ototoxic effects seem to be directed primarily towards the stria vascularis, since its shrinkage and extracellular edema have been observed in correlation with electrophysiologic changes. The present study was designed to examine the interaction of sodium salicylate and furosemide on the cochlear microstructures. Chinchillas weighing 400-600 g were used in all tests performed. The endocochlear potential (EP) was monitored continuously through a microelectrode inserted through the basilar membrane. A control group of animals was injected with 0.5 ml saline intravenously (IV) 30 min before 25 mg/kg furosemide was given. The experimental group of animals was injected with 50 mg/kg sodium salicylate IV 30 min before 25 mg/kg furosemide. The control animals were found to have a mean decrease in EP of 61.1 +/- 7.0 mV. In contrast, the experimental group had very little alteration of the EP following furosemide injection (18.7 +/- 3.9 mV). These findings suggest that sodium salicylate markedly reduces the ototoxic effect of furosemide. This effect may be mediated by an alteration of local or systemic prostaglandin metabolism, or may be due to inhibition of organic acid uptake in the cochlea.     

Effect of Carbon Dioxide on Cochlear Blood Flow in Guinea Pigs

The influence of carbon dioxide (CO2) on cochlear blood flow (CBF), blood pressure (SBP) and skin blood flow (SBF) was studied in anaesthetized guinea pigs. A transient acute respiratory acidosis was produced by inhalation of CO2 and oxygen (O2) gas mixtures. The blood flows were measured by laser Doppler flowmetry (LDF). High CO2 increased CBF and SBP, and decreased SBF in a dose-dependent manner. The responses of CBF, SBP and SBF to high CO2 were reversible. Our results indicate that high CO2 (and low pH) dilates the smooth muscle of the blood vessels, resulting in an increase in CBF. CO2 also activates the sympathetic nervous system in the whole body, producing an increase in SBP. The distribution of alpha-adrenergic fibres/receptors is abundant in skin and scarce in the cochlea. The constrictive effect on blood vessels is much greater in the skin than in the cochlea, thus our results showed a decrease in SBF during stimulation with higher CO2.