Effects Of Tetracaine On Isolated Guinea Pig Nasal Cilia Movement Measured With High-speed Digital Microscopy

Topical anesthestics are widely used in the examination,treatment and operation of nasal and paranasal sinus diseases.As characterized of its potency, rapid onset,long duration of action,and low price,tetracaine is still the most frequently used topical anesthestic in China nowadays.However,tetracaine not only can cause central nervous cardiovascular system suppression and cardiovascular system depression,but also has nasal cillotoxicity.Mucociliary clearance is a critical airway host defense mechanism.Ciliary beat frequency(CBF) is one of the most important indices to evaluate the function of mucociliary transport system.High-speed digital microscopy is a objective and precise method developed in recent years to measure ciliary beat frequency.According to the above-mentioned backgrounds,we carried on some studies as follows:①Observation on isolated healthy guinea pig nasal cilia movement measured with high-speed digital microscopy;②Effects of tetracaine on isolated guinea pig nasal cilia movement(in vitro);③Effects of tetracaine intranasal administration on guinea pig nasal cilia movement.Objective1.To observe isolated healthy guinea pig nasal cilia movement measured with high-speed digital microscopy.2.To explore effects of different concentrations of tetracaine on isolated healthy guinea pig nasal cilia movement in the cell level.3.To explore effects of different concentrations of tetracaine on isolated healthy guinea pig nasal cilia movement in the whole.Methods1.The healthy guinea pig nasal ciliated epithelial cells were isolated using a combinatorial approach of enzymatic digestion(collagenaseⅠ)and mechanical separation,and then observed at a magnification of 1000 folds using an inverted microscope.Cilia movement images of different sides and regions(nasal septum and inferior turbinate)of nasal cavity within 5 hour were captured by a high-speed digital camera with a sampling rate of 120 frames per second.In addition,the lasting time of ciliary movement(LTCM)was recoded.CBF was calculated using computerized data analysis.2.Cilia movement images were captured before and after(1～10 minutes) administration of respectively different concentrations of tetracaine(0.02%,0.1%, 0.2%,1%and 2%).And then CBF was calculated respectively.Ciliated epithelial cells with ciliostasis were observed 30 minutes after tetracaine was eluted by Hanks balanced salt solution.3.Fifteen Guinea pigs were randomly divided into five groups.In each group, one tetracaine concentration(0.1%,0.5%,1%,2%and 2%tetracaine containing 1% adenosine triphosphate)was administrated into one side of nasal cavity randomly, while the other side of nasal cavity was administrated of Hanks' balanced salt solution as a comparison.The guinea pigs were killed 10 minutes after intranasal administration.And then the nasal ciliated epithelial cells were isolated,cilia movement images were captured and CBF calculated as mentioned before.Results1.①The LTCM of healthy guinea pigs nasal mucosa is 536.8±58.2min,and 537.0±62.0min in left nasal septum,536.0±59.7min in right nasal septum, 536.0±56.2min in left inferior turbinate and 535.0±64.2min in right inferior turbinate.②The CBF of healthy guinea pigs nasal mucosa is 11.1±1.12Hz,and 11.01±1.08 Hz in left nasal septum,11.27±1.21 Hz min in right nasal septum, 11.06±0.99 Hz in left inferior turbinate and 11.15±1.17 Hz in right inferior turbinate.③The CBF of healthy guinea pigs nasal mucosa after exposed in room temperature for 1 h is 11.21±0.75Hz,2 h:11.11±0.78Hz,3h:11.08±1.48Hz,4h 10.44±1.16Hz and 5 h:9.46±0.79Hz.There is no statistical significant difference of CBF of healthy guinea pigs nasal mucosa in 4 hours.2.The ciliary beat frequency of healthy guinea pigs nasal mucosa decreases with an increase of concentration and action time of tetracaine.0.02%tetracaine solution can reduce the CBF in 5 minutes and 0.1%tetracaine solution can lead irreversible ciliostasis in 10 minutes.3.When tetracaine was intranasal adminstrated,CBF decreases only on the concentration of 1%or above it.The CBF didn't decrease in 2%tetracaine containing 1%adenosine triphosphate.Conclusion1.The guinea pig nasal ciliated epithelial cells can be isolated well by using a combinatorial approach of enzymatic digestion and mechanical separation,and can be applied to the acute experiments of single isolated cells within 4 hours.2.Single isolated nasal ciliated epithelial cells were fixed on the tip of glass microelectrode,the influence of whole cell movement on the observation of CBF was eliminated,establishing a method to observe CBF of single acute-isolated ciliated epithelial cells.3.High-speed digital microscopy for observation of acute-isolated ciliated epithelial cells is objective and precise,data-collecting convenient and advanced in data-analysis.It is an objective and accurate method to evaluate the function of mucociliary transport system.4.The differences from CBF and LTCM of healthy guinea pig nasal mucosa of different sides and regions(nasal septum and inferior turbinate)of nasal cavity have no statistical significance.5.The inhibition effect of tetracain on cilia movement increases with an increase of concentration and action time of tetracain,and the inhibition effect is much greater when tetracain directly adminstrated to cells in vitro.6.Adenosine triphosphate intranasal administration can alleviate the inhibition of tetracain on cilia movement.