Response of cochlear blood flow to prostaglandin E1 applied topically to the round window.

CONCLUSIONS: The increase in cochlear blood flow (CBF) after administration of prostaglandin E1 (PGE1) to the round window depends on increased blood flow through the anterior inferior cerebellar artery (AICA). OBJECTIVES: To evaluate the response of CBF to PGE1 applied topically to the round window, and to investigate the origin of blood flow changes after this topical application. MATERIAL AND METHODS: The response of CBF to topically applied PGE1 was measured by placing the tip of a laser Doppler probe on the bony wall of the basal turn of the cochlea after the middle ear mucosa over the cochlea had been removed in guinea pigs and rats. In rats, the CBF response to PGE1 administration was investigated after occlusion of the AICA or stapedial artery. RESULTS: CBF increased following PGE1 administration in both guinea pigs and rats. In rats, CBF increased from 100% to 132%+/-10% (mean+/-SD) after the topical application of 0.5 microl of a 0.014% PGE1 solution. CBF decreased after occlusion of the AICA or stapedial artery but did not increase after PGE1 administration during occlusion of the AICA. The CBF response to PGE1 administration was similar before and after occlusion of the stapedial artery.

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.

The immunohistochemical analysis of pendrin in the mouse inner ear.

Pendred's syndrome (PS) is an autosomal recessive disorder characterized by deafness and goiter, which are caused by mutations in the Pendred's syndrome gene (PDS). PDS encodes a membrane protein named pendrin that is considered to act as an anion transporter. An expression pattern of the PDS ortholog (Pds) mRNA in the auditory and vestibular systems has been reported in mice, and the localization of pendrin has been reported recently. We generated antipeptide antibodies against human pendrin, and performed immunohistochemical analysis of mouse inner ears. We detected pendrin in the endolymphatic duct and sac, and the utricle, saccule, and external sulcus. In the endolymphatic duct and sac, the expression of pendrin was apparent at the apical membrane. In addition, we detected pendrin in the spiral ligament, Claudius cells, Deiter's cells, and the spiral ganglion of the cochlea. Our results are key to defining the role of pendrin in inner ear development and elucidating the pathogenic mechanisms underlying deafness in PS.

Effects of trolox, locally applied on round windows, on cisplatin-induced ototoxicity in guinea pigs.

OBJECTIVE: Cisplatin (CDDP), an antitumor agent widely used in the treatment of pediatric solid tumors, has dose-limiting side effects such as ototoxicity and nephrotoxicity. Recently, evidence has been accumulated to demonstrate that these side effects are closely related to oxidative stress. In the present study, we attempted to suppress CDDP-induced ototoxicity in guinea pigs by administering trolox, a water-soluble analogue of alpha-tocopherol which is a natural lipid-soluble antioxidant, locally on round windows. METHODS: Hartley albino guinea pigs (250-300 g) were treated with CDDP (0.3 mg/ml) in the presence or absence of a combined treatment of trolox (5 mM). Both drugs were administered locally on round windows. RESULTS: The combined treatment of trolox distinctly improved the ototoxic side effects induced by CDDP. These were: elevation of auditory brain stem response threshold at 4, 8 and 16 kHz and substantial losses of outer hair cells with the base-to-apex gradient. CONCLUSION: Trolox, locally applied on round windows, showed a suppression on CDDP-ototoxicity. The results obtained in the present study suggest that a local application of trolox in the tympanic cavity can be a promising candidate to prevent the CDDP-ototoxicity in the future.

Imaging of a congenital perilymphatic fistula.

A 7-year-old boy with a history of purulent meningitis and watery rhinorrhea was studied using computed tomography (CT) and magnetic resonance imaging (MRI). He had a common cavity in the left inner ear. With high-resolution heavily T2-weighted MRI, leakage of the inner ear fluid into the middle ear at the oval window area through a congenital perilymphatic fistula could be visualized. Surgery to close the fistula showed a perforation in the stapes footplate.

Round window membrane in young and aged C57BL/6 mice.

Although there have been many studies on the round window membrane (RWM), little information has been reported about changes in the membrane associated with aging. We have undertaken morphological studies of RWMs using young (7-8 weeks old) and aged (27-29 months old) C57BL/6 mice. The RWM was thinner in mice from the aged group compared with that of the young group. The cell density in the epithelial and inner layers was also reduced in the aged group. In the middle layer of the RWM in aged mice, transmission electron microscopy revealed many degenerated short and thick elastic fibers. Confocal laser microscopy using fluorescein isothiocyanate(FITC)-wheat germ agglutinin (WGA) staining was used to identify WGA-positive fibers in the middle layer of the RWM, which changed in a similar manner to the fibers in aging skin.

In vitro activation of extracellular signal-regulated kinase1/2 in the inner ear of guinea pigs.

Growth factors such as vascular endothelial growth factor (VEGF) exert their proliferative properties partly through activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK1/2). Although both VEGF and inactive ERK could be detected in the inner ear of guinea pigs, under normal conditions activated ERK (phospho-ERK) was found only sparely. Cochleae of adult guinea pigs were removed, incubated with VEGF in a carbogen-gased organ-bath for 5, 15, 30 and 60 min (n=6 in each group), fixed with PFA 4%, embedded in paraffin and sectioned, followed by immunohistochemical staining to inactive and active ERK. Whereas inactive ERK was found in all cochleae, in sensory and supporting cells of the apex activated ERK was strongly detected after 5-min VEGF-incubation. After 15 min all Corti-organs showed clear staining corresponding to activated ERK, which decreased again after 30 min. Faint staining in endothelial cells of the spring-coil-vessels and in the spiral ganglion cells was found after 30 min and was increased after 60 min, while the staining in the Corti-organs vanished. Addition of the MEK-inhibitor PD 98059 to the organ-bath led to diminished phospho-ERK1/2 immunostaining. These findings provide evidence for a VEGF-dependent phosphorylation of ERK1/2 in the cochlea. Activated ERK1/2 is thought to support axonal outgrowth, enhancement of cell survival and to regulate the turnover of the NO/cGMP-pathway.

Mechanism of leflunomide-induced proliferation of mitochondria in mammalian cells.

Leflunomide (LFM) is an inhibitor of mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) that catalyzes the conversion of dihydroorotate to orotate coupled with the generation of reactive oxygen species (ROS) from mitochondria. We demonstrate here that LFM causes an unrestrained proliferation of mitochondria both in human osteosarcoma cell line 143B cells and rat liver derived RL-34 cells. Increases in the total mass of mitochondria per cell in LFM-treated cells were evidenced by the application of Green FM or 10-n-nonyl acridine orange to flow cytometry, an enhanced replication of mtDNA and electron microscopy. Externally added uridine improved the disturbance in cell cycle progression in LFM-treated cells, but failed to suppress such unrestrained mitochondrial proliferation. On the contrary, lapacol and 5-fluoroorotate, inhibitors of DHODH besides LFM, suppressed the biogenesis of mitochondria during the cell cycle progression. LFM, but not lapacol or 5-fluoroorotate, caused increases of the intracellular level of acetylated alpha-tubulin. These data suggest that the inhibition of DHODH may not be at least primarily related to the LFM-induced abnormal proliferation of mitochondria, and support our recent published observation that changes in the physicochemical properties of microtubules may be in someway concerned with the biogenesis of mitochondria.