De Ming Zhang

Lipopolysaccharide-induced expression of inducible nitric oxide synthase in the guinea pig organ of Corti

The purpose of the investigation was to ascertain whether inoculation of bacterial lipopolysaccharide (LPS) into the cochlea of the guinea pig could elicit formation of inducible nitric oxide synthase (iNOS). Immunohistochemical study revealed that immunoreactivity to iNOS was seen below outer hair cells representing nerve fibers and synaptic nerve endings. iNOS-staining could also be observed in phalangeal dendrites of Deiters cells pointing to the cuticular membrane, Hensens cells and on stria vascularis 48 h after inoculation with LPS. Immunohistochemical investigation with a specific anti-nitrotyrosine antibody also revealed intense immunoreactivity identical to that of iNOS, suggesting formation of peroxynitrite in the organ of Corti by the reaction of NO with O(2)(-). On the basis of these findings, it can be concluded that NO together with O(2)(-), which form the more reactive peroxynitrite, are the most important pathogenic agents in LPS-induced damage of cochlea in the guinea pig.

Pharmacological Models for Inner Ear Therapy with Emphasis on Nitric Oxide

Nitric oxide (NO)-mediated neurotoxicity may be an appropriate pathophysiological model with which to explain a variety of inner ear diseases characterized by acute or progressive hearing loss, tinnitus and vertigo. The localization of NO synthase (NOS) isoforms was examined in the inner ear of the pigmented guinea pig after intratympanic injection of 1 mg lipopolysaccharide (LPS) or 5 mg gentamicin (GM) using an immunohistochemical method, revealing the expression of NOS II in the inner ear. Production of NO in the isolated organ of Corti and utricle or in the isolated vestibular and cochlear hair cells after stimulation with L-arginine, glutamate, GM and LPS was investigated using the fluorescence indicator 4,5-diaminofluorescein diacetate. The fluorescence intensity of the sensory cells was augmented by stimulation with L-arginine, glutamate, GM and LPS. A significant increase in NO production was also noted in the LPS-treated animals. These findings imply that NO from constitutive NOS may mediate ototoxicity in the early phase, whereas NO from NOS II may contribute to the late phase of tissue damage in the inner ear. Based on this hypothesis, reduction of glutamatergic excitotoxicity and inhibition of NOS, scavenging superoxide and scavenging peroxynitrite are thought to attenuate NO-mediated otoneurotoxicity.Nitric oxide (NO)-mediated neurotoxicity may be an appropriate pathophysiological model with which to explain a variety of inner ear diseases characterized by acute or progressive hearing loss, tinnitus and vertigo. The localization of NO synthase (NOS) isoforms was examined in the inner ear of the pigmented guinea pig after intratympanic injection of 1 mg lipopolysaccharide (LPS) or 5 mg gentamicin (GM) using an immunohistochemical method, revealing the expression of NOS II in the inner ear. Production of NO in the isolated organ of Corti and utricle or in the isolated vestibular and cochlear hair cells after stimulation with L-arginine, glutamate, GM and LPS was investigated using the fluorescence indicator 4,5-diaminofluorescein diacetate. The fluorescence intensity of the sensory cells was augmented by stimulation with L-arginine, glutamate, GM and LPS. A significant increase in NO production was also noted in the LPS-treated animals. These findings imply that NO from constitutive NOS may mediate ototoxicity in the early phase, whereas NO from NOS II may contribute to the late phase of tissue damage in the inner ear. Based on this hypothesis, reduction of glutamatergic excitotoxicity and inhibition of NOS, scavenging superoxide and scavenging peroxynitrite are thought to attenuate NO-mediated otoneurotoxicity.

Formation and Fate of Giant Otoconia of the Guinea Pig following Streptomycin Intoxication

Formation and fate of abnormal (giant) otoconia of the guinea pig following streptomycin intoxication were investigated using scanning electron microscopy. The giant otoconia formed as multifaceted morphology in their early developmental period. They grew up the the transitional type and finally to the cylindrical type. It has been suggested that the giant otoconia found following streptomycin intoxication may be formed mainly by dissolution of normal otoconia due to the loss of environmental calcium, followed by recrystallization as giant crystals. These phenomena seemed to be closely related to the otoconial dynamics which may regulate calcium ion homeostasis of the endolymph.

Electron Probe X-ray Microanalysis of Otoconia in Guinea Pig Inner Ear: A comparison Between Young and Old Animals

Using the electron probe X-ray microanalysis technique, the elemental composition of otoconia was analysed in both young and old normal pigmented guinea pigs. Calcium concentration in both the utricular and the saccular otoconia was lower in the old animals, which indicates that the loss of calcium from otoconia and/or decrease in calcium ion uptake may occur in the old animals. The present study has established that calcium and other elements (P, S, Cl and K) in the otoconia of the young animals are related via a linear function, indicating that P, S, Cl and K are present in the mineral phase of the otoconia. The associations of Ca-P, Ca-K and Ca-S are not maintained in the old animals. It has been suggested that the aging phenomenon may influence the ionic metabolism in the vestibular end organs resulting in the altered mineral composition of otoconia.

Effect of Streptomycin on the Otoconial Layer of the Guinea Pig

The effects of streptomycin on the otoconial layer of the adult guinea pig were investigated using scanning electron microscopy. Administration of streptomycin induced a reduction of otoconia with formation of giant otoconia. These phenomena reached their maximum 4 weeks after the cessation of streptomycin intoxication in the utricle and between 2 and 6 weeks after in the saccule. Otoconia began to recover in the utricle 4 weeks and in the saccule 6 weeks after the cessation of streptomycin intoxication with an increasing number of small otoconia. Eight or 10 weeks after the cessation of streptomycin intoxication, the otoconial layer has completely recovered in both the utricle and saccule.

Localization of Nitric Oxide Synthase Isoforms (I, II and III) in the Endolymphatic Sac of the Guinea Pig

The localization of nitric oxide (NO) synthase (NOS) isoforms was examined in the endolymphatic sac (ES) of the pigmented guinea pig by indirect immunohistochemistry. The cytoplasm of the epithelial cells in the ES showed both NOS-I and -III immunoreactivity, whereas their nuclei appeared negative. NOS-III staining was also observed in the endothelial lining of the blood vessels. These findings support the hypothesis that NO in the epithelial cells may play an important role for the active intracellular transport of the endolymph and ion. NO may also be crucially involved in the regulation of ES blood flow. Immunostaining for NOS II revealed no reactivity in general, while in lipopolysaccharide-inoculated animals, intense reactivity was found in the cytoplasm of the ES epithelial cells as well as macrophages in the lumen. Thus it has been indicated that NO also may play an important role in the immunodefensive mechanisms in the ES.

Localization of soluble guanylate cyclase activity in the guinea pig inner ear

The aim of this study was to characterize the nitric oxide (NO) receptor soluble guanylate cyclase (sGC), to determine the cells targeted by NO and to elucidate the function of the NO/cGMP pathway in the inner ear. sGC activity in the inner ear was localized by immunohistochemical detection of NO-stimulated cGMP. Soluble guanylate cyclase activity in the cochlea was detected in the nerve endings underneath the outer and inner hair cells, supporting cells, stria vascularis and vessels. In the vestibular organs, sGC activity was detected in the cytoplasm of sensory cells, nerve fibres, dark cells and transitional cells and vessels. These findings suggest that the NO/cGMP pathway may be involved in regulatory processes in neurotransmission, blood flow and inner ear fluid homeostasis.The aim of this study was to characterize the nitric oxide (NO) receptor soluble guanylate cyclase (sGC), to determine the cells targeted by NO and to elucidate the function of the NO/cGMP pathway in the inner ear. sGC activity in the inner ear was localized by immunohistochemical detection of NO-stimulated cGMP. Soluble guanylate cyclase activity in the cochlea was detected in the nerve endings underneath the outer and inner hair cells, supporting cells, stria vascularis and vessels. In the vestibular organs, sGC activity was detected in the cytoplasm of sensory cells, nerve fibres, dark cells and transitional cells and vessels. These findings suggest that the NO/cGMP pathway may be involved in regulatory processes in neurotransmission, blood flow and inner ear fluid homeostasis.

Uptake of Tetracycline in Otoconia of the Guinea Pig

Calcium ion turnover in the otoconia of adult guinea pigs was investigated by observing the uptake of tetracycline. Oral administration of tetracycline resulted in the deposition of tetracycline (fluorescence) on the outer surface of otoconia, indicating the occurrence of dynamic exchange and/or uptake of calcium ions in the otoconia. Prolonged administration of tetracycline induced with fluorescence deposition in the central portion as well as on the surface of the otoconia. These findings suggest the occurrence of neogenesis, regeneration and/or growth of otoconia even in adult animals.

Polychromatic labeling of otoconia for the investigation of calcium turnover

The calcium ion turnover into otoconia of adult guinea pigs was investigated by the use of different fluorochromes, i.e. tetracycline (TC), calcein (Cal) and alizarin complexone (AC). The administration of all fluorochromes induced yellow (TC), green (Cal) and red (AC) fluorescence on the outer surface of otoconia, respectively. Sequential polychromatic labeling with AC followed by TC induced only red fluorescence after 1 week administration of TC, combined fluorescence after 2 weeks administration of TC and only yellow fluorescence after more than 3 weeks of administration of TC. The otoconia labeled with both AC and TC showed red fluorescence on the side faces of otoconia and yellow fluorescence on the terminal faces. These results may indicate the existence of a dynamic exchange of calcium ions in the otoconia and this exchange is mainly restricted to the terminal faces. The sequential polychromatic labeling may thus be of great use for the further investigation of the calcium dynamics of otoconia.

Incorporation of Tetracycline into Otoconia of the Guinea-pig following Streptomycin Intoxication

The effects of streptomycin on the calcium ion turnover into otoconia of adult guinea-pigs investigated by the use of tetracycline. The oral administration of tetracycline induced the deposition of tetracycline (fluorescence) on the outer surface of otoconia indicating the existence of dynamic exchange and/or uptake of calcium ions in the otoconia. The significant finding is that streptomycin specifically interfered with calcium uptake into the otoconia which indicated that the decrease in calcium uptake caused by streptomycin may be closely related to the loss of otoconia as well as to a decrease in the calcium contents of otoconia. The decrease in calcium incorporation into otoconia caused by streptomycin was recovered within 6 weeks after the last injection of streptomycin. The number of otoconia with fluorescence in the central portion as well as their outer surface was increased. It is therefore suggested that the recovery of calcium uptake as well as new otoconial regeneration may play an important role for the recovery from loss of otoconia.