Study On The Nature Of ATP-containing Vesicles In Marginal Cell Cytoplasm Of The Stria Vascular In Neonatal Rat Cochlea

PART 1: Primary Culture of Marginal Cells of the Stria Vascular in Neonatal RatsObjective: This part of study was to modify primary culture of marginal cells of the stria vascular in neonatal rat cochlea.Methods: Healthy male and female neonatal Sprague-Dawley rats aged 1-3 days were used for primary culture.The stria vascular and spiral ligament were isolated by microdissection and the explants were cultured in vitro.The marginal cells were purified by selective trypsin digestion combined differential adhesion methods at the fourth day of tissue culture.Cultured marginal cells were verified by flow cytometry.Results: Proliferated marginal cells grew outside the stria vascular explant and were arranged like paving stones,displaying a polygonal shape.The purified marginal cells were obtained at tenth day of tissue and cell culture by selective trypsin digestion combined differential adhesion methods.Flow cytometry revealed 85.3 per cent of the cells were cytokeratin18-positive cells.Conclusion: Marginal cells of the stria vascular of neonatal rats,purified by selective trypsin digestion combined differential adhesion methods,can be used for further experimental research at tenth day of tissue culture combined with cell culture.PART 2: Study on the Nature of ATP-containing Vesicles in Marginal Cell Cytoplasm of the Stria Vascular in Neonatal Rat CochleaObjective: This part of study was to investigate ATP(adenosine triphosphate)-containing vesicles in cultured marginal cell isolated from the stria vascular of neonatal rat cochlea and to illustrate these vesicles are lysosomes.Methods: Healthy male and female neonatal Sprague-Dawley rats aged 1-3 days were used for isolation,culture in vitro,and purification of marginal cells.The double staining of marginal cells with Quinacrine/LAMP1(lysosomal-associated membrane protein 1),quinacrine/Lyso-tracker or Mant-ATP[2'-/3'-O-(N'-Methylanthraniloyl)adenosine-5'-O – triphosphate]/Lyso-tracker were identified by confocal laser scanning microscope.Marginal cells were loaded with quinacrine,Lyso-tracker or Mito-tracker at room temperature in the dark,and then cells were treated with 200 ?M glycyl-L-phenylalanine-?-naphthylamide(GPN)or p-trifluoromethoxyphenylhydrazone(FCCP,1 ?M)and oligomycin(10 ?M).The fluorescence intensity was observed under confocal laser scanning microscope.The ultrastructure of the cultured marginal cells and quinacrine-labeled organelles were observed by means of transmission electron microscope(TEM).Results: Incubation with quinacrine for 30 min at room temperature in the dark resulted in numerous granule-like fluorescent puncta in the cytoplasm in cultured marginal cells under confocal laser scanning microscope.And fluorescent puncta in the cytoplasm in 3T3 cells,as a negative control,was not observed at same background fluorescence.Average 89.8% of the quinacrine-stained granules were immunopositive for LAMP1,where quinacrine and LAMP1-positive puncta showed co-localization.Quinacrine-stained granules within marginal cells could be labeled by Lyso Tracker,the lysosome tracer,but not labeled by Mito Tracker,the mitochondria tracer.The co-locolization fraction of Quinacrine/Lyso Tracker is about 91.3%,while Quinacrine/Mito Tracker is 25.3%.Lyso Tracker-labelled puncta showed accumulation of Mant-ATP,an ATP analog.Treatment with 200 ?M GPN largely quenched the fluorescence of labeled puncta after incubation with Lyso Tracker or quinacrine,but not the case with Mito Tracker.In contrast,1 ?M FCCP and 10 ?M oligomycin disrupted the labeled puncta after pre-incubation with Mito Tracker,however,they did not affect Lyso Tracker or quinacrine staining.The ultra-structure of the cultured marginal cells was observed by TEM.TEM microphotographs showed many microvilli-like extensions,numerous coated and uncoated vesicles,coated omega-shaped invaginations,quinacrine labeled lysosomes and unlabeled mitochondria.Neighboring cells were connected each other with tight junctions.Exoor endocytosis was found in the apical plasma membrane.Conclusion: We confirmed the vesicular storage of ATP in the marginal cells of the stria vascular.This is the first study to demonstrate that the ATP-containing vesicles in the marginal cells of the stria vascular in the cochlea of neonatal rats were lysosomes by immuno-staining under confocal laser scanning microscope and TEM.ATP release from marginal cells may be via lysosomal exocytosis.PART 3: Experiment of ATP Release from Cultured Marginal Cells of the Stria Vascular in Neonatal Rat CochleaObjective:This part of study was to investigate ATP releasing from cultured marginal cells isolated from the stria vascular in neonatal rat cochlea.Methods: Primary culture in vitro of marginal cells of Sprague-Dawley rats aged 1-3 days was established.ATP releasing from marginal cells was measured using a bioluminescence assay after treated by different methods.3T3 cells were as a negative control group.Luminescence of 3 parallel replicates was recorded in each cell group and every experiment repeated more than four times.200 ?M GPN was added to 5 different dilutions of marginal cells,luminescence was measured before or 5 min,10 min and 15 min after stimuli,respectively.Data analysis was carryed out with SPSS 20.0 software.Results: A direct linear relationship(r12=0.9996,3T3 cell,r22=0.9997,marginal cell)existed between luminescence measured with the assay kit and the fivefold dilutions of cells varied from 0 to 50000.200 ?M GPN treatment resulted in an average increase of 25.7% in luminescence in serial fivefold dilutions of marginal cells compared with 3T3 control(n=12,P<0.01,independent samples t-test).Different treatment with 200 ?M GPN,1%Triton X-100,10 ?M thapsigargin(TG),200 ?M GPN +5 ?M TG also evoked an ATP releasing compared with 3T3 control(P<0.01).However,GPN was unable to increase ATP releasing following 5 ?M TG treatment for 5 min.Compared with the 3T3 cells,there was no statistically significant difference(P>0.05).Conclusion: Marginal cells isolated from the stria vascular in neonatal rat cochlea can release ATP which was contained within membrane-enclosed lysosomal compartments.PART 4: Preliminary Experiment of the Mechanism of ATP Release from Stria Vascular Marginal Cell in Neonatal Rat CochleaObjective: The aim of this part of study was to investigate the mechanism of the release of ATP from the primary cultured stria vascular marginal cell in neonatal rat cochlea.Methods: Healthy male and female neonatal Sprague-Dawley rats aged 1-3 days were used for isolation,in vitro culture,and purification of marginal cells.Marginal cells were incubated with AM1-43 and immunostained with anti-LAMP1 antibody or anti-EEA1 antibody,then fluorescent images of co-locoalization was observed under confocal laser scanning microscope.We incubated marginal cells with 2.5 ?M FM1-43 dyes,then the fluorescence intensity was monitored after 2 min,5 min,10 min,15 min,20 min of 200 ?M GPN stimulation,respectively.Results: AM1-43,a fixable FM1-43 analogue,labeled average 87.5% population of vesicles that were immunopositive for the lysosomal membrane protein LAMP1,but not for the early endosome marker EEA1.Fluorescent FM1-43 dye was incubated with isolated cells,then increased fluorescence was observed after 2 minutes of GPN stimulation,following with attenuated staining.Conclusion: ATP-containing vesicles in cochlear marginal cells of the stria vascular in neonatal rats are likely lysosomes.ATP release from marginal cells may be via Ca2+-dependent lysosomal exocytosis.