Researches On The Apoptosis-Reducing Effects Of Alcaine And Oxybuprocaine On The Human Corneal Endothelial Cells

Along with the development of ophthalmic operating technology and equipments, topicalanesthetics are more and more widely used. The influence of topical anesthetics to cornea isof particular interest now. The most frequently used topical anesthetics are alcaine andoxybuprocaine. Cornea mainly consists of endothelial, substrates, epithelial and theirderivatives. Human corneal endothelial cell (HCEC) will lost split ability in adult and theinjury to it is beyond repair, which making it the most studied. Up to now, the effect of topicalanesthetics on HCEC has been investigated mainly by animal experiments, which cost a lot oftime and financing, and the broadness and advances of this kind of researches have beengreatly limited, and the research of HCEC has not been reported yet. In this study, HCECobtained from the untransfected human corneal endothelial cell line was used as experimentalmaterial to investigate the effects of alcaine and oxybuprocaine to HCEC. In addition, catswere used in the further study of effects to corneal endothelial cell caused by alcaine surfaceanesthetization. According to the results of in vitro and in vivo experiments, the accurateevaluation of the two kinds of topical anesthetic’s effect on HCEC and the experimental basisfor clinical use is given.After treated with156.25mg/L~5g/L gradient concentration of alcaine respectively,under light microscope, the cultured HCEC treated with alcaine at a concentration above312.5mg/L showed cell shrinkage, plasma membrane blebbing, detachment and quantitydecrease; but no obvious changes were found at a concentration lower than156.25mg/L. Theresults indicated that alcaine had significant cell toxicity to HCEC at a concentration above312.5mg/L. AO/EB double-fluorescent staining of156.25mg/L~5g/L treated cells showedthe apoptotic rate varies in a dose-and time-dependent manner. The apoptotic rate of HCECtreated with2.5g/L alciane for24h and5g/L alcaine for16h were both100%. The apoptoticrates of HCEC treated with312.5mg/L,625mg/L and1.25g/L alcaine for12h showed thehighest apoptotic rates of17.9%,49.2%and58.5%, respectively. The results proved thatalcaine at a concentration above312.5mg/L could induce membrane permeability increasing.Agarose gel electrophoresis of DNA samples of HCEC treated with312.5mg/L~5g/Lalcaine showed that, DNA ladder were observed at a concentration of625mg/L~5g/L, illustrating that alcaine at a concentration above625mg/L could lead to DNA fragmentationsin HCEC, while312.5mg/L alcaine showed no DNA ladder. Transmission electronmicroscope (TEM) of1.25g/L alcaine treated HCEC showed some characteristic features ofapoptosis, including cell structure disorder, plasma membrane blebbing, chromatincondensation, and the presence of apoptotic bodies, indicating that HCEC treated with1.25g/L alcaine had ultrastructural characteristics of apoptosis. Summarize all the above studies, itwas proved that alcaine at a concentration above312.5mg/L could induce HCEC apoptosis.After treated with7.8125mg/L~4g/L oxybuprocaine respectively, cultured HCECshowed cell shrinkage, plasma membrane blebbing, detachment and quantity decreaseobserved by light microscope when treated with oxybuprocain at a concentration above62.5mg/L. No obvious changes were found at a concentration lower than31.25mg/L, indicatingthat oxybuprocaine had significant cell toxicity to HCEC at a concentration above62.5mg/L.AO/EB double-fluorescent staining showed the apoptotic rate varies in a dose-andtime-dependent manner. The apoptotic rates of125mg/L~4g/L oxybuprocaine treated cellswere all reached100%after8~24h. The apoptotic rate of HCEC treated with62.5mg/Loxybuprocaine showed the highest apoptotic rate after12h, proving that oxybuprocaine at aconcentration above62.5mg/L could induce membrane permeability increasing. RemarkableDNA ladder was found at a concentration of125mg/L~4g/L, illustrating that concentrationsabove125mg/L could lead to DNA fragmentations in HCEC; concentrations lower than62.5mg/L showed no DNA ladder. TEM of125mg/L oxybuprocaine treated HCEC showed somecharacteristic features of apoptosis, including cell structure disorder, plasma membraneblebbing, chromatin condensation, and the presence of apoptotic bodies, indicating thatHCEC treated with125mg/L oxybuprocaine had ultrastructural characteristics of apoptosis.Based on the results above, it was demonstrated that oxybuprocaine at a concentration above62.5mg/L could induce HCEC apoptosis.In order to further prove that the two kinds of topical anesthetics haveapoptosis-inducing effect on HCEC, cats were used as experimental animals in alcaine studiesin vivo and in vitro identification. After surface anesthetization with5g/L alcaine, theendothelial specular microscope observation was taken every5d. The result showed that, theendothelial cell density reduced, average cell area increased, and there was a significantdifference beween experimental eyes and controls in5d (P<0.05). The coefficient ofvariation of cell area (CV) increased and the ratio of hexagonal cells decreased, but showedno significant difference (P>0.05). After surface anesthetization in25d，the four quantitative indexes changes tend to be gental.40d after surface anesthetization, the cats’ corneas wereidentified in vitro. According to alizarin red staining, pleomorphic cell number and cell areaof the experimental eyes increased obviously. SEM showed a small number of apoptotic cellsin the corneal endothelium of experimental eyes. According to the studies above, alcaine inclinical used concentration could lead to corneal endothelial cell apoptosis was proved.With the untransfected human corneal endothelial cell line as an ideal study system invitro, the effects of alcaine and oxybuprocaine on HCEC had been investigated in this study.The results showed that alcaine and oxybuprocaine had apoptosis-inducing effect on HCECindeed. In the animal experiments, the concentration alcaine used in clinical applicationscould lead to HCEC apoptosis was confirmed. Consequently, in clinical applications, the useof alcaine and oxybuprocaine should be avoided and other topical anesthetics free of toxinsshould be taken into consideration. In addition, these findings using the cornea endothelialcell line cells provided important experimental bases for the research and development ofinnocuous topical anesthetic.