In vivo assay of the potential gadolinium-induced toxicity for sensory hair cells using a zebrafish animal model.

Recently, intratympanic injection of gadolinium-based contrast agent (GdC) is growing in use to visualize the endolymphatic hydrops. Although GdC has been quite safely used over 20 years through intravenous injection, the biological influence of GdC on sensory hair cells needs to be thoroughly assessed for wider clinical application of it through intratympanic injection. In this in vivo experimental study, the summated number of sensory hair cells (SO1, SO2, O1 and OC1 neuromasts) showed a steep decrease in the group exposed to 10% and 20% GdC (35.7 ± 7.3, 15.09 ± 10.82, respectively, P < .01) compared with the control group (47.18 ± 2.30). An increase in apoptosis was also observed in the group exposed to 20% gadolinium (7.20 ± 5.56), as compared with the control group (0.08 ± 0.72) or the group exposed to 10% gadolinium (3.48 ± 3.32). A significant reduction in the viable cytoplasmic mitochondria was observed in embryos exposed to 20% GdC (369 ± 124 µm2 , P = .01) as compared with control embryos (447 ± 118 µm2 ) or embryos exposed to 10% GdC (420 ± 108 µm2 ). GdC administration did not impact peripheral neural structures. GdC caused a significant reduction in sensory hair cell counts in response to high concentrations along with increased apoptosis and mitochondrial damage. However, it may not be likely that GdC will lead to hair cell toxicity, as the estimated concentration in the inner ear after clinically tried intratympanic injection is far more diluted than the non-toxic concentration (0.625%) that was tested in this study.

Morphological and functional changes in a new animal model of Ménière's disease.

The purpose of this study was to clarify the underlying mechanism of vertiginous attacks in Ménière's disease (MD) while obtaining insight into water homeostasis in the inner ear using a new animal model. We conducted both histopathological and functional assessment of the vestibular system in the guinea-pig. In the first experiment, all animals were maintained 1 or 4 weeks after electrocauterization of the endolymphatic sac of the left ear and were given either saline or desmopressin (vasopressin type 2 receptor agonist). The temporal bones from both ears were harvested and the extent of endolymphatic hydrops was quantitatively assessed. In the second experiment, either 1 or 4 weeks after surgery, animals were assessed for balance disorders and nystagmus after the administration of saline or desmopressin. In the first experiment, the proportion of endolymphatic space in the cochlea and the saccule was significantly greater in ears that survived for 4 weeks after surgery and were given desmopressin compared with other groups. In the second experiment, all animals that underwent surgery and were given desmopressin showed spontaneous nystagmus and balance disorder, whereas all animals that had surgery but without desmopressin administration were asymptomatic. Our animal model induced severe endolymphatic hydrops in the cochlea and the saccule, and showed episodes of balance disorder along with spontaneous nystagmus. These findings suggest that administration of desmopressin can exacerbate endolymphatic hydrops because of acute V2 (vasopressin type 2 receptor)-mediated effects, and, when combined with endolymphathic sac dysfunction, can cause temporary vestibular abnormalities that are similar to the vertiginous attacks in patients with MD.

cAMP-Epac1 signaling is activated in DDAVP-induced endolymphatic hydrops of guinea pigs.

OBJECTIVE: To explore whether Cyclic Adenosine Monophosphate (cAMP)-Epac1 signaling is activated in 1-Desamino-8-D-arginine-Vasopressin-induced Endolymphatic Hydrops (DDAVP-induced EH) and to provide new insight for further in-depth study of DDAVP-induced EH. METHODS: Eighteen healthy, red-eyed guinea pigs (36 ears) weighing 200-350 g were randomly divided into three groups: the control group, which received intraperitoneal injection of sterile saline (same volume as that in the other two groups) for 7 consecutive days; the DDAVP-7d group, which received intraperitoneal injection of 10 mg/mL/kg DDAVP for 7 consecutive days; and the DDAVP-14d group, which received intraperitoneal injection of 10 µg/mL/kg DDAVP for 14 consecutive days. After successful modeling, all animals were sacrificed, and cochlea tissues were collected to detect the mRNA and protein expression of the exchange protein directly activated by cAMP-1 and 2 (Epac1, Epac2), and Repressor Activator Protein-1 (Rap1) by Reverse Transcription (RT)-PCR and western blotting, respectively. RESULTS: Compared to the control group, the relative mRNA expression of Epac1, Epac2, Rap1A, and Rap1B in the cochlea tissue of the DDAVP-7d group was significantly higher (p <  0.05), while no significant difference in Rap1 GTPase activating protein (Rap1gap) mRNA expression was found between the two groups. The relative mRNA expression of Epac1, Rap1A, Rap1B, and Rap1gap in the cochlea tissue of the DDAVP-14d group was significantly higher than that of the control group (p <  0.05), while no significant difference in Epac2 mRNA expression was found between the DDAVP-14d and control groups. Comparison between the DDAVP-14d and DDAVP-7d groups showed that the DDAVP-14d group had significantly lower Epac2 and Rap1A (p <  0.05) and higher Rap1gap (p < 0.05) mRNA expression in the cochlea tissue than that of the DDAVP-7d group, while no significant differences in Epac1 and Rap1B mRNA expression were found between the two groups. Western blotting showed that Epac1 protein expression in the cochlea tissue was the highest in the DDAVP-14d group, followed by that in the DDAVP-7d group, and was the lowest in the control group, showing significant differences between groups (p <  0.05); Rap1 protein expression in the cochlea tissue was the highest in the DDAVP-7d group, followed by the DDAVP-14d group, and was the lowest in the control group, showing significant differences between groups (p <  0.05); no significant differences in Epac2 protein expression in the cochlea tissue were found among the three groups. CONCLUSION: DDAVP upregulated Epac1 protein expression in the guinea pig cochlea, leading to activation of the inner ear cAMP-Epac1 signaling pathway. This may be an important mechanism by which DDAVP regulates endolymphatic metabolism to induce EH and affect inner ear function. OXFORD CENTRE FOR EVIDENCE-BASED MEDICINE 2011 LEVELS OF EVIDENCE: Level 5.

Live Imaging of the Dehydration Effect of Isosorbide on the Normal and Hydropic Guinea Pig Cochleae Using Optical Coherence Tomography.

OBJECTIVE: To investigate the isosorbide-induced dehydration effect on the endolymphatic space by intratympanic administration of isosorbide. BACKGROUND: Isosorbide, an osmotic diuretic, is used orally as a typical conservative therapy for Menière's disease (MD) in Japan. The dehydration effect occurs 6 hours after isosorbide ingestion. Intratympanic administration of isosorbide resolves endolymphatic hydrops faster than oral ingestion. In addition, the dehydration effect has never been shown directly. Therefore, we investigated the dehydration effect of intratympanic administration of isosorbide on endolymphatic hydrops using optical coherence tomography. METHODS: We used eight Hartley guinea pigs, divided into normal and hydrops groups. In the hydrops group, the animals underwent endolymphatic sac obliteration to create endolymphatic hydrops. We obtained midmodiolar section images of the cochleae using optical coherence tomography. Then, 50 to 70% isosorbide was sequentially administered intratympanically for 5 minutes, and the apical turn of the cochlea was observed. The relative midmodiolar cross-sectional area of the scala media was calculated for quantitative assessment of the endolymphatic space. RESULTS: In the normal group, 50% isosorbide had a slight but significant dehydration effect on the scala media; at 55 to 70%, Reissner's membrane became flat. In the hydrops group, 50% isosorbide slightly reduced endolymphatic hydrops; 65% flattened Reissner's membrane, and 70% slightly concaved it toward the basilar membrane. CONCLUSION: The results suggest that we could select the concentration of isosorbide according to the stage or severity of MD and relief from acute attack. Intratympanic administration of isosorbide may be a promising treatment for patients with MD.

Effect of electroacupuncture on arginine vasopressin-induced endolymphatic hydrops.

OBJECTIVE: To investigate the influence of electroacupuncture (EA) on experimentally induced endolymphatic hydrops (EH) in guinea pigs, and elucidate the association between the dehydrating effect of EA and changes in stria vascularis ultrastructure and expression of vasopressin type 2 receptor (V2R), cyclic adenosine monophosphate (cAMP), and aquaporin 2 (AQP2) in the endolymphatic sac (ES). METHODS: The EH model was established by intraperitoneal injection of arginine vasopressin (AVP). As a treatment, EA was delivered to Baihui (GV 20) and Tinggong (SI 19) acupoints, once daily for 10 consecutive days. For histomorphological studies, degree of cochlear hydrops was evaluated by hematoxylin-eosin staining, and the ratio of scala media (SM) area to SM + scala vestibuli area was calculated. In mechanical studies, ultrastructural changes in stria vascularis tissue were examined by transmission electron microscopy. In addition, cAMP levels and mRNA expression levels of V2R and AQP2 in the ES were compared among groups. RESULTS: EA treatment significantly reduced cochlear hydrops compared with hydropic guinea pigs (P = 0.015). Furthermore, EA attenuated ultrastructural changes in the stria vascularis tissue following EH, significantly upregulated the expression of V2R (P = 0.016), and attenuated AVP-induced upregulation of both cAMP (P = 0.038) and AQP2 expression (P = 0.017) in the ES. CONCLUSION: Collectively, the results of the present study suggest that the dehydrating effect of EA is associated with improvement of stria vascularis ultrastructure and V2R-cAMP-AQP2 signaling pathway regulation in the ES.

Endolymphatic hydrops induced by different mechanisms responds differentially to spironolactone: a rationale for understanding the diversity of treatment responses in hydropic inner ear disease.

Background: The exact pathophysiological mechanism(s) underlying endolymphatic hydrops (EH) remain elusive. We have previously shown that chronic administration of vasopressin and inhibitors of the cAMP/cGMP degrading enzymes (PDE3, PDE4, PDE5) results in the development of EH to mice. Aims/objectives: Evaluate the ability of spironolactone, an aldosterone antagonist, to prevent EH, when induced by different pathways. Material and methods: Mice were treated for 4 weeks with vasopressin, the PDE3 inhibitor cilostamide and the PDE4 inhibitor rolipram in the presence or absence of spironolactone. EH was assessed using high resolution 9.4T MRI. The expression of proteins in human saccule sensory epithelium was studied with immunohistochemistry. Results: Spironolactone prevents EH induced by vasopressin and rolipram, but not hydrops induced by cilostamide. The aldosterone target ENaC and the mineralocorticoid receptor were expressed in the human saccule sensory epithelium. Conclusions: The effect of spironolactone on EH appears to be pathway-dependent and may provide explanations why certain drugs may be effective in some patients with hydropic ear disease while not in others. Significance: Extrapolating this finding to the clinic supports that a personalized medicine approach is probably necessary in the treatment of diseases involving EH, as different pathways may be needed to be targeted for treatment.

A new animal model for Ménière's disease.

CONCLUSION: A new murine model for the study of Ménière's disease has been developed by treatment with both lipopolysaccharide (LPS) and aldosterone. Induction of vestibular dysfunction in the hydropic animal model may entail additional stress such as reduced inner ear blood flow, and sudden acute changes in endolymph volume and/or pressure. OBJECTIVE: The purpose of this study was to develop a more suitable animal model, showing closer resemblance to the pathophysiological process in Ménière's disease. MATERIALS AND METHODS: Adult CBA/J mice were treated by intratympanic injection of LPS, intraperitoneal injection of aldosterone, or injection of both LPS and aldosterone. Morphological analyses were performed in the cochlea and endolymphatic sac. RESULTS: All experimental animals showed mild to moderate endolymphatic hydrops. Those treated with both LPS and aldosterone showed reversible vestibular dysfunction after the intratympanic injection of epinephrine.

Sudden hearing loss after cialis (tadalafil) use: A unique case of cochlear hydrops.

We discuss a unique case of sudden sensorineural hearing loss after Cialis (tadalafil) use, a phosphodiesterase 5 (PDE5) inhibitor, and the implication of ipsilateral cochlear hydrops seen on magnetic resonance imaging (MRI). We report a case of a 53-year-old male with unilateral low-frequency sudden sensorineural hearing loss (SSNHL) after ingestion of tadalafil. The SSNHL occurred 1 day after ingestion and was associated with aural fullness and tinnitus. There were no symptoms of vertigo. He received oral prednisone immediately after the onset of hearing loss without improvement. Delayed intravenous contrast-enhanced three-dimensional Fluid-attenuated inversion recovery MRI revealed ipsilateral dilation of the cochlear duct without any hydronic change in the vestibular system. Acetazolamide therapy was initiated, and his symptoms improved. A posttreatment audiogram revealed an increase in threshold of 15 dB. To the best of our knowledge, this is the first case of cochlear hydrops visualized on imaging after a PDE5 inhibitor induced SSNHL. Tadalafil and other PDE5 inhibitors have a known association with SSNHL. Despite several proposed mechanisms, there is inconclusive evidence of a causal relationship. Our presented case suggests that cochlear hydrops may be one possible mechanism of PDE5 inhibitor-associated SSNHL. MRI should be considered in the evaluation of such patients who do not respond to oral steroids as initial treatment. Laryngoscope, 2615-2618, 2018.

Vestibular Function Change in a Vasopressin-Induced Hydrops Model.

HYPOTHESIS: A vasopressin-induced endoymphatic hydrops model can represent an acute vertiginous attack in Menière's disease (MD). BACKGROUND: Previous animal models are not appropriate to evaluate the efficacy of new treatments for hydrops because they cannot represent an acute attack of MD. Recently, a new dynamic model was introduced for acute hydrops exacerbation using the vasopressin type 2 receptor agonist, desmopressin (1-deamino-8-D-Arginine vasopressin, VP); however, resulting changes in vestibular function have not been investigated. METHODS: A total of 37 guinea pigs were used. Two to 4 weeks after surgical ablation of endolymphatic sacs in 33 guinea pigs, acute exacerbation of hydrops was induced by a single VP injection in 18 animals (group A). Next, two VP injections at 1 hour interval were administered to investigate the effect of multiple VP doses on vestibular function in the other 15 animals (group B). In the remaining four animals, VP was injected without surgery for the control group (control). Bidirectional sinusoidal harmonic acceleration (SHA) tests of vestibular function were performed. "Type I response" was defined as when the maximum slow-phase velocity (SPV) during left rotation (toward the operated ear) was lower than that during right rotation (toward the normal ear). In contrast, "Type II response" was defined as when maximum SPV at the left rotation was higher than that at the right rotation. Vestibular symmetry scores were analyzed at baseline and after each of two VP injections given 1 hour apart. RESULTS: Vestibular symmetry scores increased at 1 hour after VP injection in all 18 animals in group A (p < 0.001). Two hours after VP injection, symmetry score decreased to the initial score. Two different types of vestibular response were observed after VP. However, the symmetry scores between type I and II responses were not significantly different (p = 0.173). In all 15 animals of Group B, vestibular asymmetry was sustained over 3 hours when two VP injections were given 1 hour apart. In three of Group B, the type of vestibular response changed from type II response to type I response after the 2nd VP injection; however, no animal demonstrated a shift from type I to type II response. CONCLUSION: VP can transiently induce an acute exacerbation of hydrops and asymmetric vestibular dysfunction in guinea pigs. This model could help in studying new treatments for acute hydrops and in explaining the mechanism of bidirectional nystagmus in MD.

Effects of vasopressin on gene expression in rat inner ear.

Vasopressin regulates water excretion from the kidney by increasing water permeability of the collecting duct as a hormone secreted from the posterior pituitary. A clinical study reported that plasma levels of arginine vasopressin were significantly higher in patients suffering from Meniere's disease. It was histologically confirmed that chronic administration of vasopressin induced endolymphatic hydrops in guinea pigs. However, the mechanism of endolymphatic hydrops induced by vasopressin is still unclear. We use cDNA microarray to study the effects of vasopressin on gene expression profiles in rat inner ear to elucidate the possible mechanism of the induced hydrolabyrinth. Wistar rats were intraperitoneally injected with 50 microg/kg arginine vasopressin once a day for one week. Hydrolabyrinth in rat inner ear induced by administration of vasopressin was detected by HE stain. The bullae were dissected out for total RNA extraction. cDNAs were synthesized by reverse transcription and labeled with Cyanine3 (Cy3) or Cyanine5 (Cy5). The BiostarR-40s cDNA microarray was hybridized with the above cDNAs and the changes of mRNA expression intensity were showed by data analysis. Furthermore, the changes of aquaporins expression level were measured by reverse transcription polymerase chain (RT-PCR). Endolymphatic hydrops were present in rats intraperitoneally injected with vasopressin. 226 known differentially expressed genes were screened out in rat inner ear induced by vasopressin injection. Of the 226 genes, 18 transcripts were increased by 5-fold or more, and 7 transcripts were decreased to 0.2-fold or less. Ten differentially expressed genes were identified that associate with cell signal transduction, 14 differentially expressed genes were identified that relate to ion transport, 7 differentially expressed genes were involved in vesicle-mediated transport, and 2 differentially expressed genes were aquaporin 2 (AQP2) and aquaporin 7 (AQP7). The expression level of AQP2 was significantly higher and AQP7 was significantly lower. These results suggest that there are obvious differences in gene expression profiles in inner ear between vasopressin injected rats and normal control rats. Vasopressin may disturb fluid homeostasis in inner ear by way of signal transduction, ion transport, vesicle-mediated transport and aquaporins. It is likely that up-regulated expression of AQP2 mRNA and down-regulated expression of AQP7 mRNA in the rat inner ear caused by vasopressin induce an increased production and a decreased absorption of endolymph, resulting in endolymphatic hydrops.

Vasopressin induces endolymphatic hydrops in mouse inner ear, as evaluated with repeated 9.4 T MRI.

From histopathological specimens, endolymphatic hydrops has been demonstrated in association with inner ear disorders. Recent studies have observed findings suggestive of hydrops using MRI in humans. Previous studies suggest that vasopressin may play a critical role in endolymph homeostasis and may be involved in the development of Ménière's disease. In this study we evaluate the effect of vasopressin administration in vivo in longitudinal studies using two mouse strains. High resolution MRI at 9.4 T in combination with intraperitoneally delivered Gadolinium contrast, was performed before and after chronic subcutaneous administration of vasopressin via mini-osmotic pumps in the same mouse. A development of endolymphatic hydrops over time could be demonstrated in C57BL6 mice (5 mice, 2 and 4 weeks of administration) as well as in CBA/J mice (4 mice, 2 weeks of administration; 6 mice, 3 and 4 weeks of administration). In most C57BL6 mice hydrops developed first after more than 2 weeks while CBA/J mice had an earlier response. These results may suggest an in vivo model for studying endolymphatic hydrops and corroborates the future use of MRI as a tool in the diagnosis and treatment of inner ear diseases, such as Ménière's disease. MRI may also be developed as a critical tool in evaluating inner ear homeostasis in genetically modified mice, to augment the understanding of human disease.

Endolymphatic hydrops induced by chronic administration of vasopressin.

Recently, many lines of evidence have supported the possibilities that vasopressin (VP) is closely linked to the formation of endolymphatic hydrops in Meniere's disease. In the present study, it was examined whether or not the chronic administration of VP might induce endolymphatic hydrops. For this purpose, histological studies and VP radioimmunoassay were independently performed in 20 and 40 guinea pigs, respectively. The degree of hydrops was quantitatively assessed by the increase ratio (IR) of the scala media area in the mid-modiolar sections of the cochlea. The IR was defined by the following equation: 100x(A-B)/B (A: the cross-sectional area of the bulging scala media; B: the no-bulging scala media, enclosed by an idealized straight Reissner's membrane). VP was administered at the rates of 200 microU/kg/min, 400 microU/kg/min and 1000 microU/kg/min for 1 week via the osmotic mini-pump. The IR of the total of the apical, second, third and basal turns (means+/-S.D.s) were 4.4+/-0.7, 10.4+/-1.8, 17.4+/-7.9 (n=10 ears, each) in respective doses of VP. Comparing with that of the control animals (5.2+/-1.7, n=10 ears), the area increased significantly in the VP dosage of 400 and 1000 microU/kg/min (Bonferroni's method, P<0.05). Plasma VP concentrations produced by the VP administration in these dosages were 2.2+/-0.4, 3.5+/-0.8 and 14.0+/-3.9 (n=10, each) pg/ml. Although 3.5 pg/ml is the upper limit of plasma VP concentration in normal human subjects, 14.0 pg/ml was almost the same concentration as those observed in the acute phase of Meniere's disease (Takeda et al., 1995). Therefore, the formation of endolymphatic hydrops in cases of Meniere's disease might be caused by high concentrations of plasma VP.

Signs of endolymphatic hydrops after perilymphatic perfusion of the guinea pig cochlea with cholera toxin; a pharmacological model of acute endolymphatic hydrops.

There are indications that endolymph homeostasis is controlled by intracellular cAMP levels in cells surrounding the scala media. Cholera toxin is a potent stimulator of adenylate cyclase, i.e. it increases cAMP levels. We hypothesized that perilymphatic perfusion of cholera toxin might increase endolymph volume by stimulating adenylate cyclase activity, providing us with a pharmacological model of acute endolymphatic hydrops (EH). Guinea pig cochleas were perfused with artificial perilymph (15 min), with or without cholera toxin (10 microg/ml). The endocochlear potential (EP) was measured during and after perfusion. The summating potential (SP), evoked by 2, 4 and 8 kHz tone bursts, was measured via an apically placed electrode 0, 1, 2, 3 and 4 h after perfusion. Thereafter, the cochleas were fixed to enable measurement of the length of Reissner's membrane, reflecting EH. After perfusion the EP increased significantly over time in the cholera toxin group as compared to the controls. Also, the SP increased gradually at all frequencies in the cholera toxin group. Comparison within animals showed that the increase in SP became significant after 2 h at 4 kHz, after 3 h at 2 kHz and after 4 h at 8 kHz. In the control group the SP did not change significantly. The compound action potential (CAP) amplitude decreased monotonically over time at all frequencies in both the cholera toxin group and the control group, but it decreased faster in the cholera toxin group. Also, the cochlear microphonics amplitude decreased over time at all frequencies in both groups, but the decrease was significant only in the cholera toxin group after 3 h at 2 and 4 kHz. Quantification of the length of Reissner's membrane showed a small but insignificant enlargement in the cholera toxin treated animals compared to controls. These results are in accord with our view that EH is accompanied by an increase in SP and a decrease in CAP. Our results partially confirm previous results of Feldman and Brusilow (Proc. Natl. Acad. Sci. USA (1973) 73, 1761-1764). New aspects in relation to that study are the significantly increased EP and SP. In the classical EH model, based on obstruction of the absorptive function of the endolymphatic sac, increased SPs are accompanied by decreased EPs. In this cholera toxin model of EH, it is unlikely that the endolymphatic sac is involved. Apparently, EH can be based on mechanisms located in the cochlea itself as opposed to mechanisms located in the endolymphatic sac.

Time sequence of degeneration pattern in the guinea pig cochlea during cisplatin administration. A quantitative histological study.

We investigated the key tissues that are implicated in cisplatin ototoxicity within the time window during which degeneration starts. Guinea pigs were treated with cisplatin at a dose of 2 mg/kg/day for either 4, 6, or 8 consecutive days. Histological changes in the organ of Corti, the stria vascularis and the spiral ganglion were quantified at the light microscopical level. Outer hair cell (OHC) loss started between 4 and 6 days of cisplatin administration, but is only significantly different from the non-treated group after 8 days of treatment. Midmodiolar OHC counts were comparable to the cytocochleogram data. The cross-sectional area of the stria vascularis did not differ from the non-treated group, nor did an endolymphatic hydrops develop during the course of treatment. Spiral ganglion cell (SGC) densities did not decrease. After 6 days, however, detachment of the myelin sheath of the type-I SGCs was seen in the lower basal turn, whereas after 8 days it was also present in the more apically located turns. Myelin sheath detachment is the result of perikaryal shrinkage and swelling of the myelin sheath. The present study confirms that cisplatin at a daily dose of 2 mg/kg has a detrimental effect on the OHCs as well as on the type-I SGCs. These intracochlear effects occur simultaneously; OHC loss and SGC shrinkage start between the fourth and sixth day of cisplatin administration and appear to develop in parallel. At this dose, no histological effect on the stria vascularis could be observed, although previous electrophysiological experiments demonstrated a clear effect on the endocochlear potential

The effects of V2 antagonist (OPC-31260) on endolymphatic hydrops.

In the present study, two experiments were performed to investigate the influence of OPC-31260 on experimentally induced endolymphatic hydrops in guinea pigs and the regulation of aquaporin-2 (AQP2) mRNA expression in the rat inner ear. In morphological studies, the increases in the ratios of the length of Reissner's membrane (IR-L) and the cross-sectional area of the scala media (IR-S) were quantitatively assessed among normal guinea pigs (normal ears) and three groups with hydropic ears: hydropic ears with no infusion (non-infusion hydropic ears), hydropic ears with an infusion of physiological saline into the scala tympani (saline-infused hydropic ears) and hydropic ears with infusion of 0.3% OPC-31260 into the scala tympani (OPC-infused hydropic ears). IR-Ls in the experimental groups were markedly larger than in the normal ear group, but there was no significant difference among the groups of non-infusion hydropic ears, saline-infused hydropic ears and OPC-infused hydropic ears. The IR-Ss of non-infusion hydropic ears and saline-infused hydropic ears (48.8-49.3%) were statistically different from that of normal ears (6.5%) (Dunnet multiple comparison test, P<0.01). However, IR-S of the OPC-infused hydropic ears (-14.8%) was significantly smaller than those of non-infusion hydropic ears and saline-infused hydropic ears (one-way ANOVA, P<0.01). In the quantitative polymerase chain reaction study, a comparison of the ratio of AQP2 and beta-actin mRNA (MAQP2/Mbeta-actin) was made between water-injected and OPC-31260-injected rats. An intravenous injection of OPC-31260 resulted in a significant decrease in MAQP2/Mbeta-actin both in the cochlea and in the endolymphatic sac (t-test, P<0.001). These results indicate that water homeostasis in the inner ear is regulated via the vasopressin-AQP2 system, and that the vasopressin type-2 antagonist OPC-31260 is a promising drug in the treatment of Meniere's disease.

Histological effects of co-administration of an ACTH((4-9)) analogue, ORG 2766, on cisplatin ototoxicity in the albino guinea pig.

Cisplatin is one of the most potent antineoplastic drugs presently known, but its therapeutic efficacy is seriously limited by several side effects such as ototoxicity. Several compounds that are known for their nephroprotective effects also seem to reduce the incidence and severity of cisplatin-induced ototoxicity. Hamers et al. (1994) and De Groot et al. (1997) investigated the possibly protective effect of concomitant administration of the ACTH((4-9)) analogue ORG 2766 upon cisplatin ototoxicity in guinea pigs. Animals were treated with cisplatin at a daily dose of 2.0 mg/kg for 8 consecutive days and ORG 2766 at a daily dose of 75 mcg/kg for 9 days. Concomitant administration of cisplatin plus ORG 2766 resulted in a bimodal distribution of the electrophysiological data (compound action potential and cochlear microphonics amplitudes) and the histological data (outer hair cell (OHC) counts). It was surmised that this dichotomy might occur at a certain cisplatin dose. We investigated whether this protective effect of ORG 2766 could be enhanced by reducing the daily dose of cisplatin while maintaining the same dose of ORG 2766. Thirty-six animals were treated with daily i.p. injections of cisplatin at a dose of 1.0 mg/kg (n=18) or 1.5 mg/kg (n=18) for 8 consecutive days. When comparing the mean OHC counts of the different experimental groups, treatment with cisplatin at a daily dose of 1.5 mg/kg for 8 consecutive days resulted in a considerable loss of OHCs, which was significantly reduced after co-administration of ORG 2766. Co-treatment with ORG 2766 did not result in a change in the volume of the scala media. The present results are in agreement with the electrophysiological results published earlier (Stengs et al., 1998b).

Induction of endolymphatic hydrops in the guinea pig by perisaccular deposition of sepharose beads carrying and not carrying immune complexes.

We tried to induce endolymphatic hydrops in guinea pig cochleas by unilateral, perisaccular deposition of sepharose beads carrying immune complexes. Controls consisted of the deposition of sepharose beads without immune complexes and the contralateral, untreated ear. The effects of the treatment were studied by light microscopy and electrophysiological recordings of the gross cochlear potentials 1, 2, and 6 weeks after treatment. Each condition included six animals. Analysis of variance of the morphometric data concerning the ears treated with deposition of the beads showed a statistically significant difference (P = 0.04) between the degree of hydrops found for the beads with immune complexes and for those without. The difference between the treated ears and the contralateral untreated ears was significant (P = 0.01) for the beads with immune complexes and not significant (P = 0.8) for those without immune complexes while there was no significant effect of post-treatment time interval. Analysis of variance of the electrophysiological data, collected in response to tone bursts at the apex of the cochlea, showed no significant differences between the results for the beads with and without immune complexes. Therefore these results were pooled. One week after treatment the pooled results for the compound action potential showed a small decrease in amplitude, just significant at 2 kHz, but not at 4 and 8 kHz. This decrease disappeared completely after 6 weeks. The pooled results for the negative summating potential (SP) showed a significant increase in magnitude at all frequencies decreasing with post-treatment interval. The cochlear microphonics did not demonstrate any change in amplitude after treatment. The results indicate that deposition of sepharose beads with immune complexes induces endolymphatic hydrops. Also, deposition of the sepharose beads itself may have induced hydrops together with enhancement of the SP. SP enhancement may be related to the development of endolymphatic hydrops rather than to the presence of hydrops as such. This may be based on pressure build-up while hydrops develops.

Dose-dependent effect of 8-day cisplatin administration upon the morphology of the albino guinea pig cochlea.

Numerous studies investigating cisplatin ototoxicity in animals have been performed, but it is difficult to derive a clear dose-effect relation from these studies. The degree of cisplatin-induced ototoxicity depends on a multitude of factors. Many parameters, such as dose, mode of administration, dosage schedule and concomitant administration of protective additives, vary among the published studies. Therefore, we performed a basic dose-effect study on cisplatin ototoxicity in the guinea pig. Albino guinea pigs were treated with cisplatin at daily doses of either 0.7, 1.0, 1.25, 1.5 or 2.0 mg/kg for 8 consecutive days. Electrocochleography was performed on day 10 after which the cochleas were removed and processed for histological examination. The electrophysiological results showed a marked transition from almost no ototoxic effect to a large effect between a daily dose of 1.25 and 1.5 mg/kg (Stengs et al., 1998). Outer hair cell (OHC) counts corresponded well with the electrophysiological results. At daily doses of 0.7, 1.0 and 1.25 mg/kg no statistically significant OHC loss was observed, whereas OHC loss averaged 60% and 65% in the basal turns at daily doses of 1. 5 and 2.0 mg/kg, respectively. Morphological changes in the stria vascularis were present only in cochleas from animals treated with cisplatin doses of 1.0, 1.25 and 1.5 mg/kg/day. Cochleas from animals treated with a daily cisplatin dose of 2.0 mg/kg for 8 consecutive days showed an endolymphatic hydrops. The present study shows that cisplatin, administered at a daily dose of 1.5 mg/kg for 8 consecutive days, provides a degree of OHC loss that is well suited to study the effects of putative protective agents and possible hair cell recovery.

Cisplatin-induced ototoxicity: morphological evidence of spontaneous outer hair cell recovery in albino guinea pigs?

Cisplatin is frequently used in the treatment of various forms of malignancies. Its therapeutic efficacy, however, is limited by the occurrence of sensorineural hearing loss. Little is known about the course of hearing loss over longer time intervals after cessation of cisplatin administration. Infrequently, recovery of hearing has been described in animals and humans. Stengs et al. (1997) treated guinea pigs with cisplatin at a daily dose of 1.5 mg/kg for 8 consecutive days and subsequently studied cochlear function after survival times varying from 1 day to 16 weeks. Spontaneous improvement of the hair cell-related potentials (cochlear microphonics and summating potentials) was observed starting 2 weeks after cessation of treatment. In the present study we examined light microscopically the cochleas used in the study of Stengs et al. (1997). One day after cessation of cisplatin administration outer hair cell (OHC) loss in the basal cochlear turn averaged 66%. In the 1-week survival group, OHC counts were similar to those of the 1-day survival group. In the 4-week survival group, however, a relatively small loss of OHCs was found in the basal cochlear turn; OHC loss averaged only 15%. A similar loss was found after 8 weeks. In the 16-week survival group, OHC loss in the basal turn increased to 48%, but this was not statistically significant. Our histological observations are in line with the electrophysiological data from the same animals. Our findings suggest that OHCs recover from cisplatin-induced damage 1-4 weeks after treatment. However, the results do not allow a conclusion as to whether the observed recovery is due to the formation of new OHCs or to (self-)repair of damaged OHCs.

Electrocochleographic evaluation of the guinea pig model of endolymphatic hydrops.

Electrocochleography (ECochG) was used to evaluate cochlear function in guinea pigs with experimentally induced endolymphatic hydrops (ELH) before and after osmotic dehydration with either glycerol or urea. We surgically induced ELH in the right ears of 9 guinea pigs, while the right ears of 6 guinea pigs received a sham operation. The left ears of the 15 animals constituted the normal group. Eight weeks after surgery, summating potential (SP) and action potential (AP) amplitudes were measured prior to and following the administration of glycerol or urea. The SPs and SP/AP ratios were reduced in all groups, with no significant differences among groups or between dehydrating agents. Some of the hydropic ears, however, did show an increased AP threshold and a recruitment effect. In measurements from 6 additional animals, serum osmolarity increased more with urea than with glycerol. The guinea pig model remains valuable for investigation of ELH, even though it differs in significant respects from ELH in humans.