| INTRODUCTIONMany inner ear disorders are associated with the damage of hair cells. The mechanisms underlying various harmful factors maybe the same or alike. A number of investigations have been carried out with the intention of understanding the injury mechanisms and devising means to prevent inner ear damage. By using cochlear organotypic culture techniques on rat Corti's Organ, we investigated if a nitric oxide synthase (NOS) inhibitor [ L - N - nitroarginind methylester (L -NAME) ] combined with a calpain inhibitor [ leupeptin ] provides synergetic protection for cochlear hair cell damaged by gentamicin (GM ) .MATERIALS AND METHODS1. Animal grouping:20 Wistar rats (40 ears) on post - natal days 3-5 were randomly divided into five groups,4 rats in each group.(1) group; normal control group, cultivated only; (2)group:be given GM(2mg/ml)only; (3)group:be given GM(2mg/ml) + leupeptin(1 mM); (4)group:be given GM(2mg/ml) + L-NAME(100p,M) ; (5) group: be given GM ( 2mg/ml) + L - NAME (100 uM ) + leupeptin (1mM);2. Major reagent and instrument:inner ear organotypic culture medium, gentamicin, ( GM ) ( Sigma GM -1264 ), L - NAME ( Sigma - 5751) , leupeptin ( Amresco ZJ580) inner ear organoiypic culture instrumentmicrography system3. Method:Wistar rats on post - natal days 3-5 were killed , cochleas were dissected with the aid of watchmaker forceps and a stereomicroscope. Organs of Corti were freed from surrounding tissues and placed on a 30mm culture dish with culture supplement and treatment. After 48 hours, specimens were fixed for 4 hours in 4%. paraformal - dehyde in 0. 1M phosphate buffer(PH7. 4). Spacimens were rinsed in 0. 1M PBS, incubated in 0. 25% Triton X - 100 for 5 min and immersed in TRITC - labled PHALLOIDIN in PBS for 30 min. Specimens were examined under a fluorescence microscope with appropriate filters for TRITC, the data were analyzed by AN OVA.RESULTSHair cell incubated in normal culture supplement for 48 hours (group (1) ) remained almost complete viability. The group(5) with united application of two protective drugs is similar with it. There was no significant difference of inner\ outer hair cell livability between these two groups. Hair cell loss in the groups given one protective drug only ( group (3) \(4)) was increased clearly compared with group (1) and group(5) (P < 0.01, P < 0.01). Hair cells in group ( 2 ) given GM only almost disappeared entirely, remained lots of striation of scar, cell survival was significantly worse than that in cells treated with one protective drug alone(P < 0.01, P < 0.01).DISCUSSIONEvidence is accumulating to suggest that NO is an important contributory factor in inner ear damage. Inoculation to animals with GM induced expression of nitric oxide synthase (NOSII ) , with consequent functional disorders in cochlea and vestibule, which could be blocked using the NOS inhibitor L - NG - ni-troarginind methylester (L-NAME) ] and ebselen, a scavenger of peroxyni-trite.Some inner ear cells in vestibule and Corti's organ of Guinea pig explosed to GM can be stimulated and express iNOS, a great quantity of NO ROS and products of nitrogen peroxide are produced. All of them destroy cells and play an important role in GM ototoxicity. Being treated with L - NAME and ebselen in advance can reduce the GM ototoxicity because the former play a competitive antagonistic action and dicrease the generation of NO and the latter can clear products of nitric peroxide.Intracellular calcium and calcium buffering mechanisms have been implicated in aminoglycoside ototoxicity. In tissue cultures of the chick sensory epithelium , GM caused a dose - dependent increase in intracellular calcium levels in hair cells. Aminoglycoside antibiotics also caused a large transient increase in intracellular calcium in isolated OHCs. This cell death can be .attenuated by cal-pain inhibitors, such as leupeptin.Previous studies have shown that aminoglycoside antibiotics cause a dose -dependent increase in intracellular calcium levels in avia...
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