The Study Of Rabbit Lens Epithelial Cell Apoptosis In The Early Period After The Operation Of Vitrectomy With Nonexpansile Perfluoropropane Gas

PART1 Detectation of apoptosis correlated protein Bcl-2 and Bax and its significance of Rabbit Lens after the Operation of Vitrectomy with Nonexpansile Perfluoropropane GasObjective:In this part,we observed the microstructure changes and opacity degree of rabbit lens with slit-lamp microscope and transmission electron microscope,detected the apoptosis rate and examined the expression of apoptosis correlated protein Bcl-2 and Bax of lens epithelial cell in the early period after the operation of vitrectomy with nonexpansile perfluoropropane gas,and tried to study the relativity between the microstructure changes,lens opacity,apoptosis rate,protein expression and perfluoropropane gas.Methods:50 new zealand health rabbits(100 eyes)were assigned randomly to three groups:the A group-6 rabbits(12 eyes)was normal group;the B group-44 eyes were control group,performed the operation of simple vitrectomy with BSS;the C group-44 eyes were experimental group,performed the operation of vitrectomy with nonexpansile perfluoropropane gas.The intraocular pressures were measured with Schiotz indentation tonometer,and the lens shape were observed with slit lamp microscope on the 1st,3th,8th,35th,45th day after operation.The electron microscope specimens were made with rabbit lens only on the 45th day after operation.The ultrastructural changes of LEC were observed with transmission electron microscope.The apoptosis rate of lens epithelial cell was detected under light microscope after the treatment of TUNEL method and the expression of apoptosis correlated protein Bcl-2 and Bax was examined with immunohistochemical method on the 1st,3th,8th,35th,45th day after operation.Results:1.IOP:There was no significant difference(P＞0.05)of IOP among the three groups on the 1st,3th,8th,35th,45th day after operation.2.The lens opacity degree:There was no distinct opacity on the rabbit lens of normal group and control group on the 1st,3th,8th,35th,45th day after operation.The opacity rate of experimental group was 92.36%.There was significant difference(p＜0.05)between experimental group and the control group,normal group on the 1st,3th and 8th day after operation.The occurrence rate and degree of cataract was positively correlated with the area and time of contact between perfluoropropane gas and lens,but the opacity alleviated or dead away gradually with the disappearance of perfluoropropane gas.3.The ultrastructural changes of LECs:There was no evident abnormal of control group and normal group LECs on 45th day after operation.But the myeloid body was finded in experimental group LECs,the nuclears deformed distinctly and there were a lot of mitochondrias collected around the nuclear,they swelled and vacuolized apparently.4.The detection of apoptosis rate after the treatment of TUNEL method:There was significant difference(p＜0.05)on apoptosis rate of lens epithelial cell between experimental group and the control group,normal group on the 35th and 45th day after operation.The percentage of apoptotic cells were gradually increased as the incubation time prolonged.5.The examination of the expression of Bcl-2 and Bax:In experimental group,the expression of Bcl-2 protein was down-regulated and that of Bax protein was up-regulated on the 35th and 45th day after operation and there was significant difference(p＜0.05)between experimental group and the control group,normal group,so the ratio of Bcl-2/Bax was lower.The expression of Bcl-2 protein showed a negative correlation with the percentage of apoptosis cells(r=-0.85,P＜0.05).While the expression of Bax protein showed a positive correlation with the percentage of apoptosis cells(r=0.99,P＜0.05).Conclusions:1.The perfluoropropane gas was one of the main reasons of the early cataract after the operation of vitrectomy with nonexpansile perfluoropropane gas. 2.The permanent damage of LECs could be induced by C₃F₈.2.LECs apoptosis could be induced by C₃F₈.LECs apoptosis was a cytology foundation of the cataract in the early period after the operation of vitrectomy with nonexpansile perfluoropropane gas.3.The variational ratio of Bcl-2/Bax may play an important role in the cataract in the early period after the operation of vitrectomy with nonexpansile perfluoropropane gas.PART2 The effect of multiple sequential fiuid-gas exchange of Rabbit Lens after the operation of vitrectomy with nonexpansile perfluoropropane gas on Lens epithelial cell apoptosisObjective:In this part,we observed and detected the apoptosis rate of lens epithelial cell of undertaking no one or multiple sequential fiuid-gas exchange of Rabbit Lens after the operation of vitrectomy with nonexpansile perfluoropropane gas,and tried to study the relativity between the frequency of fiuid-gas exchange with nonexpansile perfluoropropane gas and the apoptosis rate.Methods:24 new zealand health rabbits(48 eyes)were assigned randomly to two groups:the A group-24 eyes were control group, performed the operation of vitrectomy with nonexpansile perfluoropropane gas;the B group-24 eyes were experimental group, performed the operation of vitrectomy with nonexpansile perfluoropropane gas and fluid-gas exchange with nonexpansile perfluoropropane gas on the 10th,20th,30th,40th day postoperation.The intraocular pressures were measured with Schiotz indentation tonometer, the lens shape were observed with slit lamp microscope,the optical microscope specimens were made and the apoptosis rate of lens epithelial cell was detected under light microscope after the treatment of TUNEL method on the 15th,25th,35th,45th day postoperation.Results:1.IOP:There was no significant difference(P＞0.05)of IOP among the two groups on the 15th,25th,35th,45th day postoperation.2.The lens opacity degree:The opacity rate of control group and experimental group was 91.35%and 100.00%.There was significant difference(p＜0.01)between experimental group and the control group on the 15th,25th,35th,45th day postoperation.3.The microstructure changes:The TUNEL masccline cell was up-regulated in experimental group when compared with the control group on the 15th,25th,35th and 45th day postoperation.4.The detection of apoptosis rate after the treatment of TUNEL method:There was significant difference(p＜0.05)on apoptosis rate of lens epithelial cell between experimental group and the control group on the 15th,25th,35th and 45th day postoperation.The percentage of apoptosis cells showed a positive correlation with the lens opacity degree (r=+0.910,P＜0.001)Conclusions:1.The lens opacity degree after the operation of vitrectomy with nonexpansile peffluoropropane gas was gradually increased with the postoperative gas maintenance dose.2.The apoptosis rate of lens epithelial cell after the operation of vitrectomy with nonexpansile perfluoropropane gas was gradually increased with the postoperative gas maintenance dose.3.The lens opacity degree after the operation of vitrectomy with nonexpansile perfluoropropane gas and multiple sequential fiuid-gas exchange was gradually increased with the percentage of apoptosis cells.