Inhibition of phosphodiesterase 3, 4, and 5 induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4T MRI.

CONCLUSION: The data indicate important roles for phosphodiesterase (PDE) 3, 4, 5, and related cAMP and cGMP pools in the regulation of inner ear fluid homeostasis. Thus, dysfunction of these enzymes might contribute to pathologies of the inner ear. OBJECTIVE: The mechanisms underlying endolymphatic hydrops, a hallmark of inner ear dysfunction, are not known in detail; however, altered balance in cAMP and cGMP signaling systems appears to be involved. Key components of these systems are PDEs, enzymes that modulate the amplitude, duration, termination, and specificity of cAMP and cGMP signaling. METHOD: To evaluate the role of PDE3, 4, and 5 and associated cAMP and cGMP pools in inner ear function, the effect of cilostamide (PDE3 inhibitor), rolipram (PDE4 inhibitor), and sildenafil (PDE5 inhibitor), administrated via mini-osmotic pumps, on mouse inner ear fluid homeostasis was evaluated using 9.4T in vivo MRI in combination with intraperitoneally administered Gadolinium contrast. Also, using human saccule as a model, the expression of PDEs and related signaling molecules and targets was studied using immunohistochemistry. RESULTS: PDE3, PDE4, as well as PDE5 inhibitors resulted in the development of endolymphatic hydrops. Furthermore, PDE3B, PDE4D, and some related signaling components were shown to be expressed in the human saccule.

Molecular changes associated with the endolymphatic hydrops model.

HYPOTHESIS: Hearing loss and cochlear degeneration in the guinea pig model of endolymphatic hydrops (ELH) results, in part, from toxic levels of excitatory amino acids (EAAs) such as glutamate, which in turn leads to changes in the expression of genes linked to intracellular glutamate homeostasis and apoptosis, leading to neuronal cell death. BACKGROUND: EAAs have been shown to play a role in normal auditory signal transmission in mammalian cochlea, but have also been implicated in neurotoxicity when levels are elevated. Changes in the expression of specific genes involved in the glutamatergic and apoptotic pathway would serve as evidence for excitotoxicity linked to elevated levels of glutamate. METHODS: Guinea pigs underwent surgical obliteration of the endolymphatic duct, and then a timed harvest of the treated (right) and control (left) cochlea and subsequent quantification of gene expression via real-time quantitative polymerase chain reaction. RESULTS: Quantitative polymerase chain reaction data show significant upregulation of glutamate aspartate transporter and neuronal nitric oxide synthase mRNA levels 3 weeks postsurgery and Caspase 3 mRNA levels 1 week postsurgery. No significant changes were detected in glutamine synthetase expression levels. CONCLUSION: Upregulation of genes involved in glutamate homeostasis and the apoptotic pathway in animals treated with endolymphatic duct obstruction (usually associated with secondary ELH) support the hypothesis that EAAs may play a role in the pathophysiology of ELH-related cochlear injury. Inhibitors to these pathways can be useful for the study of new avenues to delay or prevent ELH-related hearing loss.

Effects of a nitric oxide synthase Type II inhibitor on compound action potential thresholds in experimental endolymphatic hydrops.

HYPOTHESIS: Nitric oxide (NO) is likely to be synthesized by nitric oxide synthase Type II (NOS II) action and may partake in the origin of changes of compound action potential (CAP) threshold observed in guinea pigs with induced endolymphatic hydrops. This study aimed to assess the action of a NOS II inhibitor on CAP thresholds in these experimental samples. BACKGROUND: In guinea pigs with experimental endolymphatic hydrops, there are lesions on the cochlea and progressive increase of CAP threshold. NOS II was found in the cochlea of this animal model, and it was inferred that NO can contribute by such alterations. METHODS: The animals were divided into two groups, in which eight received an intake of a NOS II inhibitor, aminoguanidine, and another eight served as a control group. During 16 weeks, CAP thresholds at 1,000, 2,000, 4,000 and 6,000 on electrocochleography were compared between the groups. RESULTS: The group that had an intake of aminoguanidine showed a lower increase on CAP thresholds at 2,000 (p < 0.05) and 6,000 Hz (p < 0.05) at the 12th postoperative week, and at 1,000 (p < 0.05), 2,000 (p < 0.001), 4,000 (p < 0.001), > and 6,000 Hz (p < 0.001) at the 16th week. CONCLUSION: We conclude that NOS II inhibitor reduced the elevation of CAP thresholds in experimentally induced endolymphatic hydrops.

Experimental endolymphatic hydrops under action of a type II nitric oxide synthase inhibitor: otoacoustic emissions evaluation and electrocochleography.

UNLABELLED: In experimental endolymphatic hydrops distortion-products otoacoustic emission (dpoae) amplitudes decrease and there is elevation on electrocochleographic thresholds. Some authors found type ii nitric oxide synthase (nos ii) expression in hydropic cochleas and they suggest nitric oxide (no) may be involved in endolymphatic hydrops pathogenesis. The aim of this study was to evaluate the action of a nos ii inhibitor on dpoae and electrocochleography in experimental endolymphatic hydrops. MATERIAL AND METHODS: endolymphatic hydrops was induced in 16 guinea pigs by obliterating the endolymphatic duct and sac in the right ear. They were divided in two groups: eigth guinea pigs under the action of aminoguanidine, a nos ii inhibitor and eigth control guinea pigs. We compared dpoae amplitudes at geometric means of frequencies 1062, 2187, 4375 and 7000 hz, compound action potential threshold at 1000, 2000, 4000 and 6000 hz and summating potential to action potential (sp/ap) ratio between the groups during the postoperative observation period of 16 weeks. RESULTS: there were no significant changes in the dpoae amplitudes and in the sp/ap ratio. The group that received aminoguanidine had a lower degree of threshold increase at 2000 (p<0.05) And 6000 hz (p<0.05) In 12th postoperative week and at 1000 (p<0.05), 2000 (P<0.001), 4000 (P<0.001) And 6000 hz (p<0.001) At 16th postoperative week. CONCLUSIONS: nos ii inhibitor decreased the electrocochleography threshold elevation on experimental endolymphatic hydrops.

Hidropsia endolinfática experimental sob ação de inibidor da óxido nítrico sintase tipo II: avaliação com emissões otoacústicas e eletrococleografia / Experimental endolymphatic hydrops under action of a type II nitric oxide synthase inhibitor: otoacoustic emissions evaluation and electrocochleography

No modelo experimental de hidropsia endolinfática há redução na amplitude das emissões otoacústicas produtos de distorção (EOAPD) e elevação nos limiares eletrofisiológicos na eletrococleografia. Estudos mostraram que há expressão da óxido nítrico sintase tipo II (ONS II) na cóclea com hidropsia, sugerindo a participação do óxido nítrico (ON) na patogênese desta doença. O objetivo deste trabalho foi avaliar a ação de um inibidor da ONS II nas EOAPD e eletrococleografia em cobaias com hidropisia endolinfática experimental. MATERIAL E MÉTODOS: Foram estudadas 16 cobaias nas quais se induziu hidropsia endolinfática experimental por obliteração do ducto e saco endolinfático na orelha direita durante 16 semanas, divididas em dois grupos: oito cobaias recebendo um inibidor da ONS II, a aminoguanidina, por via oral e um grupo de oito cobaias como controle. Comparamos as amplitudes das EOAPD nas médias geométricas de freqüências de 1062, 2187, 4375 e 7000Hz, os limiares eletrofisiológicos nas freqüências de 1000, 2000, 4000 e 6000Hz e a relação entre os potenciais de somação e de ação (PS/PA) entre os grupos. RESULTADOS: Não houve diferença significante nas EOAPD e na relação PS/PA entre os grupos. O grupo que recebeu a aminoguanidina apresentou menor elevação nos limiares eletrofisiológicos nas freqüências de 2000 (p<0,05) e 6000 Hz (p<0,05) na 12ª semana e nas freqüências de 1000 (p<0,05), 2000 (p<0,001), 4000 (p<0,001) e 6000Hz (p<0,001) na 16ª semana. CONCLUSÕES: O inibidor da ONS II reduziu a elevação dos limiares eletrofisiológicos na eletrococleografia na hidropsia endolinfática experimental.

Activation of caspase-3 is associated with oxidative stress in the hydropic guinea pig cochlea.

The aim of this study was to investigate the involvement of oxidative stress and apoptosis in an animal model of Meniere's disease. Endolymphatic hydrops (ELH) is generally accepted as the decisive histological characteristic of Meniere's disease. Closure of the endolymphatic duct (Kimura's method) was used to induce endolymphatic hydrops in guinea pigs. Sham-operated animals served as controls. After 4 weeks the animals operated showed a significant elevation of the hearing thresholds as measured by audiometric brainstem responses (ABR) pre- and postoperatively. Immediately after the second ABR measurement, the animals were sacrificed for further immunohistological examinations of the inner ear with specific antibodies to active caspase-3 (cas-3) as a marker for apoptosis and antibodies to 8-isoprostane (8-iso) and nitrotyrosine (NT) as indicators of oxidative stress. Compared with the sham-operated controls, hydropic cochleae showed strong immunostaining for both oxidative stress markers in spiral ganglion cells, in the blood-vessels and fibrocytes of the lateral wall, as well as in supporting cells of the organ of Corti. Activation of cas-3 in spiral ganglion cells and the lateral wall was found exclusively in hydropic cochleae. Our findings suggest that oxidative stress is involved in the development of endolymphatic hydrops and may lead to cellular damage which induces apoptosis by activation of cas-3. Apoptotic cell death might contribute to the sensorineural hearing loss found in later stages of Meniere's disease.

Changes in Ca2+-ATPase in a guinea pig endolymphatic hydrops model.

OBJECTIVE: To investigate the localization of Ca(2+)-ATPase (Ca(2+) pump) in the cochlear and its change after endolymphatic hydrops, and to study the relationship between compound action potential (CAP) threshold and the Ca(2+)-ATPase activety. METHODS: The left endolymphatic sac was ablated to induce endolymphatic hydrops in fourteen healthy guinea pigs with normal action potential thresholds measured after a sliver ball electrode placed on the round window. Ca(2+)-ATPase activity was studied cytochemically using a lead citrate reaction in control and hydropic ears. The reaction product was lead phosphate particles as an expression of Ca(2+)-ATPase activity, observed with an eletron microscope. RESULTS: Ca(2+)-ATPase activity is mainly found on the endolymphatic surface of Reis sner's membrane, the stereocilia and cuticular plate of inner and outer hair cells, and along the infolded plasma membrane of strial marginal cells. CAP thresholds of filtered click are increased and Ca(2+)-ATPase activity significantly decreased after endolymphatic hydrops in the mentioned locations. CONCLUSIONS: CAP thresholds are increased and Ca(2+)-ATPase activity are significantly decreased in the cochlea after endolymphatic hydrops. These results suggest that there is a negative correlation between them.

Expression of inducible nitric oxide synthase (iNOS/NOS II) in the hydropic cochlea of guinea pigs.

Immunohistochemical investigations of the guinea pig cochlea, using a specific antibody to the inducible isoform of NO synthase (iNOS/NOS II), have been performed 3 weeks after closure of the right endolymphatic duct (n=7). Endolymphatic hydrops, the morphological substrate of Meniere's disease, became evident by distension of the Reissner's membrane. iNOS expression could be noted in endothelium, spiral ganglion cells, in nerve fibers, in supporting cells of the organ of Corti and cells of the spiral ligament. Temporal bones of non-operated controls (n=6) as well as of sham-operated animals (n=3) did not show structures positive to iNOS. These findings imply that iNOS-generated NO could be involved in the pathophysiology of cochlear dysfunction in Meniere's disease.

Expression of inducible nitric oxide-synthase in the vestibular system of hydropic guinea pigs.

Immunohistochemical investigations of the guinea pig vestibular system, using a specific antibody to the inducible isoform of NO-synthase (iNOS/NOS II), have been performed 3 weeks after surgical closure of the right endolymphatic duct (n = 7). Endolymphatic hydrops (ELH) of the right temporal bone became evident by excavation of the Reissner's membrane in all seven animals. Those animals revealed iNOS-expression in ganglion cells, in the wall of blood vessels and in nerve fibers of the right vestibular system, while the corresponding left temporal bones and temporal bones of non-operated controls (n = 6) as well as of sham-operated animals (n = 3) did not show any iNOS-positive structures. iNOS-generated NO could be involved in the pathophysiology of vestibular dysfunction in Meniere's disease.

[Changes of Ca(2+)-ATPase in the cochlea of guinea pig during labyrinthine hydrop].

OBJECTIVE: To investigate the localization of Ca(2+)-ATPase (Ca2+ pump) in the cochlea and its change in labyrinthine hydrops. METHODS: The left endolymphatic sac was ablated to induce endolymphatic hydrops in fourteen healthy guinea pigs after the sliver ball electrode was placed on the round window. The Ca(2+)-ATPase was studied by the lead citrate reaction in the control and hydropic ears. The reaction product was lead phosphate particles as an expression of Ca(2+)-ATPase activity under the electron-microscope. RESULTS: The Ca(2+)-ATPase activity was found mainly on the endolymphatic surface of the Reissner's membrane, the stereocilia and cuticular plate of inner and outer hair cells, as well as along the infolding plasma membrane of the strial intermediate cells. The Ca(2+)-ATPase activity was significantly decreased during endolymphatic hydrops in the above-mentioned locations. CONCLUSION: The response thresholds of filtered click were increased and the Ca(2+)-ATPase significantly decreased in the cochlea during labyrinthine hydrops. These results suggest that correlation exists between the CAP threshold and the activity of Ca(2+)-ATPase in the model of labyrinthine hydrops.

[Effect of furosemide on the carbonic anhydrase activity in vestibule].

In order to explore the mechanism that the furosemide dehydrates and improves the vestibular function in the endolymphatic hydrops. The effects of furosemide on the carbonic anhydrase activity in vestibule were studied by using histocytochemistry and image analysis. The results demonstrated that the furosemide can obviously inhibit the carbonic anhydrase activity. There was no significant difference between the normal ear and the ear with endolymphatic hydrops in the location of the carbonic anhydrase in the vestibule. It suggested that one of the dehydrant mechanisms is inhibition of carbonic anhydrase activity.

Na,K-ATPase in the cochlear lateral wall of human temporal bones with endolymphatic hydrops.

Meniere's disease has traditionally been thought to arise from a disruption in longitudinal endolymphatic flow. This view has been brought into question by recent experimental studies that have focused attention on derangements of cochlear fluid and electrolyte homeostatic mechanisms in Meniere's disease, including abnormalities in Na,K-ATPase enzymes found in the cochlear lateral wall. The current study examined the immunohistochemical labeling pattern of the major ion-transporting enzyme of the stria vascularis, Na,K-ATPase, in archival sections of hydropic and nonhydropic human temporal bones for increased density of label that could indicate overproduction of fluid. The results showed good labeling of the stria vascularis in the celloidin sections. The hydropic ears tended to have darker label, but the difference was not statistically significant. The findings are consistent with normal functioning of the stria vascularis in cases of Meniere's disease.

Activities of carbonic anhydrase in the cochleae of guinea pigs with early experimental endolymphatic hydrops.

Normal auditory function depends on maintenance of the unique ion composition in the endolymph. Carbonic anhydrase in the inner ear has been suggested to play an important role in maintaining the ion concentration and regulating fluids of the inner ear. Cochlear dysfunction may indicate changes in the biochemical components and osmotic pressure of the inner ear fluids as well as inadequate generation of intracellular metabolic energy. Dysfunction of the inner ear was investigated in the early stages of endolymphatic hydrops. Normal adult albino guinea pigs were operated on to obliterate the endolymphatic ducts and sacs of the right ears to induce endolymphatic hydrops. The auditory function of experimental guinea pigs was assessed according to the difference between the preoperative and postoperative hearing thresholds of the auditory brainstem response (ABR). Vibratome sections of the hydropic cochlea (right) and control cochlea (left) were stained histochemically for the activities of carbonic anhydrase in this study. Decreased activity of this enzyme was not shown consistently in the stria vascularis and organ of Corti of the hydropic cochlea in the early stage of endolymphatic hydrops whereas auditory dysfunction in the hydropic ear was noted from the ABR threshold for the experimental animals with 2 months survival. The results of the present study suggest that further investigation concerning the role of carbonic anhydrase in the cochlear auditory function is necessary.

Reduced Na(+)-K(+)-ATPase activities in the cochleae of guinea pigs with experimental endolymphatic hydrops.

Normal auditory function depends on the maintenance of the unique ion composition in the endolymph. The Na(+)-K(+)-ATPase in the lateral wall of the cochlear duct has been suggested to play an important role in maintaining the endolymphatic ion concentration and in generating a positive endocochlear potential. Cochlear dysfunction may indicate changes in the biochemical components and osmotic pressure of the inner ear fluids as well as inadequate generation of intracellular metabolic energy. Dysfunction of the stria vascularis and spiral prominence was investigated in the early stages of endolymphatic hydrops. Vibratome sections of hydropic and normal cochleae were stained histochemically for Na(+)-K(+)-ATPase activity in this study. Decreased activity of this enzyme was shown in the stria vascularis and spiral prominence of the hydropic cochlea in the early stage of endolymphatic hydrops. The results coincide with those of studies of electrophysiologic changes in cochlear function in hydropic animals by others. The results of the present study provide further information concerning a possible deficit in ion transport activity and decreased enzymatic activity in the cochlea.