| Background:As one kind of common eye diseases, glaucoma has become one of the main eye diseases which cause human blindness. The main feature of glaucoma is that elevated intraocular pressure makes the optic nerve damage. It is very difficult to control intraocular pressure only with drug treatment. Glaucoma filtration surgery has becomes an important way to control intraocular pressure. Although great progress has been made in glaucoma surgery, an operation failure rate in 2 years remains as high as 20%. The main reason is that scar is formed the filter corridor and filtering bleb cannot work well. Therefore, how to effectively reduce scarring after surgery has become the hotspot and difficulty of ophthalmic research.Although a lot of anti-scarring drugs have come to market, and have achieved some effects have been acquired, such as improving filtering bleb function and higher success rate of surgery. However, the use of these drugs also brought a series of complications and side effects, such as low pressure. Therefore, it is difficult for these agents to go into the clinical application. Consequently, it is particularly important to choose anti-scarring agents with good curative effect and less complications and side effects.As one of macrolide antibiotics, rapamycin was used as immunosuppressive drugs in the first. At present, it has been applied in the study of anti-scarring by many researchers in the world. However, the mechanism of anti-scarring has not been fully elucidated. This topic mainly studies the effect of rapamycin in anti-scarring after rabbit glaucoma filtering surgery, which can provide certain theory basis for its clinical application.Objective:To assess the function of rapamycin in anti-scarring and eye poisoning after filtering surgery by comparing intraocular pressure, ocular reaction, filtering bleb morphology, histopathology, cell proliferation index PCNA and α-SMA, and supply a certain theoretical basis for its application in clinic, and improving the surgical success rate. The anti cicatricial effect of rapamycin was also discussed through detecting RTFs cells apoptosis caused by different concentrations of rapamycin.Methods:Ninety-six chinchilla rabbits were employed auto-control, weight two kilometers to two point five kilometers. All rabbits underwent trabeculectomy. They were randomly divided into four groups, three experimental groups and one control groups, each group had twenty-four bruising Blue Rabbits.The treated left eyes were injected with 1% rapamycin,3% rapamycin,5% rapamycin 4 times a day for 7 days.while the control groups were treated with castor oil 4 times a day for 7 days. operation organization by gradient ethanol dehydration, transparent, paraffin embedding, serial section, conventional HE staining, after being dry gum, light microscope observation.In 7 days,14 days,21 days,28 days with Schiotz tonometer intraocular pressure measurement, the measured average three times.Postoperative day observed under slit-lamp conjunctival congestion, transparency of cornea, anterior chamber reaction and the transparency of the lens.In accordance with the Krofeld classification method to compare filtering bleb morphology and function. The intraocular pressure (IOP) was measured after surgery. In the meanwhile, conjunctiva, cornea, anterior chamber and lens were observed. Simultaneously, number of functional filtering bleb was recorded. Finally, histopathological HE observations were conducted following by killing the rabbits. Proliferation cell nuclear antigen (PCNA) and smooth muscle actin antibodies (a-SMA) were marked by using immunohistochemical method. The results were tested by SPSS 13.0 statistics software, P< 0.05 as a significant standard.RTFs for in vitro culture, and identified by immunofluorescence observations fibroblasts. With Hoechst staining method to detect different concentrations of rapamycin(Omg/L、0.06mg/L、0.25mg/L、1mg/L、4mg/L) in presence of inducing apoptosis RTFs cells. Determined by MTT method is used to detect different concentrations of rapamycin after processing the OD value of the fibroblasts, the difference between spectral caspase broad spectrum and join blocker comparison, analysis the mechanism of action of rapamycin. Western blot method to detect different concentrations of rapamycin fibroblasts, caspase 3,8,9 the amount of change. By adopting the method of semi-quantitative PCR detection cell mRNA level of rapamycin processing caspase 3,8,9 quantity change.Results:1. For experimental groups with 1% rapamycin,3% rapamycin and 5% rapamycin treatment, the control groups with castor oil, four groups of intraocular pressure were reduced, and each point in time, postoperative experimental intraocular pressure was lower than control group, and 7(F=240.379,P<0.001),14 (F=127.221,P<0.001),21 (F=56.036,P<0.001),28 (F=85.413,P<0.001) day the difference between the three experimental groups and control group with significant statistical significance.2. The experimental group and control group were slightly conjunctival congestion and anterior chamber benefly, no inflammatory cells (0-1), in 7 days after the basic disappeared and there was no evident difference in the two groups, did not see the corneal edema and lens opacity.3.28 days after the operation, the control of the filtering bleb formation rate drop to zero, and the experimental 1% rapamycinfiltering bleb formation rate is 33.33%,3% rapamycinfiltering is 50%,5% rapamycinfiltering is 66.67%. Experimental 1% rapamycin,3% rapamycin,5% rapamycin) group functional filtering bleb formation rate in 7,14,21 days after surgery, were higher than the experimental group, and with the increase of the concentration of rapamycin, functional filtering bleb formation rate showed a trend of rising.4.7,14,21 days after the surgery, number of PCNA positive cells in the experimental group is much less than the control group(P<0.05), and with the extension of operation time, the number of PCNA positive cells showed a trend of decrease(P<0.05).And PCNA positive cells in the control group were higher than the PCNA positive cells in the experimental group(P<0.05), shows that the control of fibroblast proliferation degree higher than that of the experimental group, and therefore a scarring in the control group was significantly higher than that of the experimental group, meaning that rapamycin can resist scar effect well.5.7,14,21 days after the surgery, the experimental group of a-SMA positive cells number average is much less than the control group, and longer duration of surgery, a-SMA positive cells number showed a trend of decrease.And a-SMA positive cells in the control group were higher than the a-SMA positive cells in the experimental group, shows that the control of fibroblast proliferation degree higher than that of the experimental group, and therefore a scarring in the control group was significantly higher than that of the experimental group, meaning that rapamycin can resist scar effect well.6. Intraocular pressure values of the experimental group and control group were counted respectively. At the same time, the number of filtering bleb, PCNA and a-SMA positive fibroblasts was added up and tested (AVONA and chi-square test) by using SPSS 13.0 statistics software and P< 0.05 as standard significantly.7. Pathological specimens were conducted the histological examination by specialized technical personnels. Tan particles was observed in the nucleus in the PCNA staining observation, which significantly higher than the background. In the a-SMA dyeing observation cytoplasm was dyed brown, which significantly higher than the background. Both of the two dye tests were successful.8. During tissue culture, cultivate commonly 2 days later, the group will adhere to the vessel wall, and 4 to 7 days, you will find the place not far away from the group with irregular cells. Under the condition of the growth space is enough, the cells present radioactive growth; In a narrow space mainly presents the lateral growth. When the organization is fixed, the cultivation of cells after 1 week can cover 70% of the bottle.9. Waveform after protein antibody staining, the cytoplasm green fluorescence positive reaction.Keratin antibody staining cytoplasm were negative staining.The nuclei are DAPI aizen.To prove to fibroblasts cultured cells.10. With different concentrations of rapamycin, caspase 3, and 9 mRNA amount (F>116.572,P<0.001) and protein expression (F>28.564, P<0.001) increases with increasing concentration of rapamycin, show the concentration dependence.But the mRNA amount (F=0.671, P=0.618) and protein expression (F=0.300,P=0.875) of caspase-8 and the level in the control group is not present the more obvious differences.11. The results showed that after dealing with the broad spectrum of caspase blockers, caused by rapamycin mRNA and caspase 3,9 protein leels of blocking phenomenon effectively, and the broad spectrum of caspase blockers for cell based caspase 3-9 mRNA (t>5.209,P<0.001) and protein expression (t>5.357,P<0.001) has obvious inhibitory effect.Broad spectrum of caspase blockers can also inhibit caspase-8 mrna and protein expression.Conclusion:Rapamycin could inhibit excessive proliferation of fiber cells in the filter corridor after rabbit glaucoma filtration surgery. Meanwhile, it could reduce scar formation and prolong the survival time of functional filtering bleb. Rapamycin can cause RTFs apoptosis by increaseing the expression of caspase 3 and 9. |
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