| Mucociliary transport is a major defence mechanism of the respirator y tract.An efficient mucociliary transport system depends on adequate cili ary function(ciliary beat frequency,structure,coordination),and its efficienc y is deter-mined by the tip velocity of the cilia and the degree of co-opera tive(or meta-chronal)activity between cilia.Both tip velocity and co-oper ative activity are increased by increases in ciliary beat frequency(CBF)an d,as a result a key parameter controlling mucus clearance is the regulatio n of CBF.Allergic rhini-tis(AR)produce multifarious autacoids that dest roy the ciliary function.Auta-coids is a major cause of morbidity after AR.I mpairment of respiratory ciliary function produces a decreased mucus transp ort rate.Subsequently,Chronic sinu-ses may occur in these patients.The d epressant effects of different autacoids on human nasal CBF has recently been reported.However,the early effects of histamine on ciliary function ha ve not been evaluated.Therefore we investigated the effect of different hista mine concentrations after short exposure times on CBF using an in vitro m odel.Part one:Manual microdissection in isolation of ciliated cells from guine a pig's nasal epithelium and technique of cell's fixationObjective①To apply manual microdissection to isolate cilia ted cells from guinea pig's nasal epithelium in order to offer pure cells to further reseach;②To fuond a technique of ciliated cell's fixation.Methods①The experiment was fininshed at institute Otolaryngology Head and Neck Surgery from Mar.of 2007to Nov.of 2007.Kill guinea pig humanely following institu-tional and national guidelines.Remove the nasal epitheli um(NE)either side of the nasal septum by Dissection.Isolate ciliated cell s layer from submucous layer by instruments for microsurgery under the li ght microscope(×15 magni-fication),ciliated cells layer and mucous layer were stained with HE in order to demonstrated the result of microdissecti on.The protease-treated NE was viewed under phase-contrast optics(×100),to count number of single ciliated cells and other cells and size of group of ciliated cells.Evaluate ciliated cells morphology under Inverted M icroscope(×400 magnification).②Glass pole fixup ciliary cell that w as obtained in part one.Phase-contrast images were detected with a digita l high-speed camera.This camera is a progressive scan CCD(charge-coup led device)that provides images(648 pixels×200 lines)at 120 f.p.s.The camera is used with a frame grabber and recording software running und er Windows 2000.The high signal-to-noise ratio of grey waveform is obtai ned by interactively selecting the optimal ROI(Region of interest).Results①Result of dying confirm that microdissection is correct.After microdissect ion,density of single ciliated cells were larger,number of other cells were l ittle and size of groups of ciliated cells were smaller.To compare manual microdissection with conventional isolation,there are distinct differences in purity and activity of cells.P＞0.05.②Glass pole fixup ciliary cell is avail ability,when diameter of glass pole is 2～5μm and negative pressure is 1 0～20cm H2O;Conclusion①manual microdissection is a feasible and pr actical technique;②glass pole fixup ciliary cell is availability.Part two:Effects of histamine on guinea pig's nasal ciliated cells movem ent with high-speed digital microscopyObjective①To test the availability that glass pole fixup cili ary cell;②To investigate the effects of histamine on guinea pig's nasal cilia ted cells movement with high-speed digital microscopy;③To investigate the effects of extracellular calcium on ciliated cells.Methods Glass pole fi xup ciliary cell that was obtained in part one.Phase-contrast images were detected with a digital high-speed camera.This camera is a progressive sca n CCD(charge-coupled device)that provides images(648 pixels×200 line s)at 120 f.p.s.The camera is used with a frame grabber and recording s oftware running under Windows 2000.The high signal-to-noise ratio of gre y waveform is obtained by interactively selecting the optimal ROI(Region of interest).The frequency of each ciliary beat cycle is determined from t he period of each cycle of the grey-intensity waveform.Histamine solution o f different concentrations from 10-6mol/L tol0-1mol/L,was used to stimulat ed the ciliated cells respectively.Results①CBF increased signigicantly in 1-2minutes and reached the apex;②after apex,group 10-6mol/L and group 10-5mol/L decrease abruptly,a sustained elevation was observed after the i nitial rise from group 10-4mol/L to 10-2tool/L;③CBF is not coordinated from group 10-4mol/L to 10-2mol/L;④ATP can induced an elevation of CBF after histamine administration Conclusion①Histamine of concent rations from 10-6mol/L to10-3mol/L can stimulated a rise of CBF respecti vely,and do not decrease activity of ciliated cell;②Histamine of concentra tions from 10-2mol/L can stimulated a rise of CBF while decrease activity of ciliated cell;③Histamine of concentrations from 10-1mol/L can kill ci liated cell;④Activity of ciliated cells in Ca2+-free HBS is not good.Part three:Effects of Promethazine on guinea pig's nasal ciliated cells movement with high-speed digital microscopyObsjective To investigate the effects of Promethazine(a speci tic H1 receptor antagonist)on guinea pig's nasal ciliated cells movement.Methods Measurement of CBF with high-speed digital microscopy is si milar to part two.Promethazine solution of different concentrations from 10 -6mol/L to10-3mol/L,was used to stimulated the ciliated cells respectively. After 20 minutes,histamine of same concentration was administrated.Result s①CBF increased moderately inlminutes and reached the apex that is 50 %of maximum;②after apex group 10-6mol/L PML decrease abruptly,at t he time of 5 minute CBF decrease blow baseline,then recover slowly;③aft er apex group 10-5mol/L PML decrease,at the time of 10 minute CBF d ecrease blow baseline,then recover slowly;④after apex group 10-4mol/L P ML decrease slowly,but CBF is not coordinated;⑤Histamine induced an ele vation of CBF after Promethazine administration,but apex is lower then pa rt two;⑥ATP can induced an elevation of CBF after histamine administrati on,but apex is lower then part two.Conclusion①Promethazine increase d CBF moderately;②the process of PML effect CBF is:excitation-depression -recovery;③depression of PML is dose-dependent;④PMZcan depress h istamine partly;⑤This mechanism of depression should be further studie d.
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