ABSTRACT
Background Selective laser trabeculoplasty (SLT) can increase the outflow of aqueous humor and reduce the intraocular pressure of patients with open angle glaucoma,but its mechanism is unknown.To investigate the mechanism of SLT would improve the therapeutic effect of SLT.The aqueous outflow resistance in trabecular meshwork was affected by the extracellular matrix (ECM).Matrix metalloproteinase-3 (MMP-3) and MMP-9 were closely related to ECM degradation in trabecular meshwork.Objective This study was to observe the effects of SLT on the expressions MMP-3 and-9 in human trabecular ceils in vitro.Methods Immortallized human trabecular cells were cultured with pigment particles mixed suspension for 16 hours and incubated overnight.Then the cells were irradiated with Q switch frequency doubling 532 nm Nd:YAG laser to establish SLT-effective cells with the energy of 0.2 mJ,spot diameter of 400 μm and pulse duration of 3 ns.The expressions of MMP-3 mRNA and MMP-9 mRNA in the cells were detected by fluorescence quantitative real time PCR before and 1 hour and 4,8,12,28 hours after exposure of laser.The concentrations of MMP-3 and MMP-9 in the medium were assayed using ELISA 1 hour and 4,8,12,28 hours after exposure of laser and compared between the non-irradiation group and the irradiation group.Results The relative expressing levels of MMP-3 mRNA were 1.00,1.32±0.12,2.08±0.05,2.34±0.04,2.77± 0.05 and 2.49±0.27 in the non-irradiation group and irradiation for 1 hour and 4,8,12,28 hours after exposure of laser SLT,showing a significant difference among the groups (F =15.966,P=0.007),and relative expressing levels of MMP-3 mRNA were significantly higher in various time points after laser irradiation than those of the non-irradiation group (all at P＜0.05).The relative expressing levels of MMP-9 mRNA were 1.00,0.91 ±0.10,1.27 ± 0.07,1.46±0.07,1.69±0.09 and 0.87±0.09 in the non-irradiation group and irradiation for 1 hour and 4,8,12,28 hours after exposure of SLT,which was considerably different among the groups (F =30.526,P =0.005),and significant increased values were seen in the 4,8 and 12 hours after irradiation compared with the non-irradiation group (all at P＜0.05),with highest expression in the irradiation for 12-hour group.The concentrations of MMP-3 and MMP-9 proteins in medium were significantly increased in various time points after laser exposure in comparison with the control group (all at P＜0.05).Conclusions The expressions of MMP-3 and MMP-9 in human trabercuolar cells upregulate and the secretion ability of human trabecular cells to MMP-3 and MMP-9 proteins improves in early stage of SLT in vitro.However,these procedures gradually diminish with the lapse of time.
ABSTRACT
Background Selective laser trabeculoplasty (SLT) plays lowing-intraocular pressure (IOP)effect by irradiating pigmented trabecular cells selectively using 532 nm Nd:YAG laser.However,its affect pattern is not fully known.Objective This study was to establish a human trabecular cells (HTCs) model of SLT effect,and to observe the morphological changes of HTCs after they were exposed to different energies of SLT.Methods Immortalized HTCs were incubated in DMEM/F12 with pigmental granules for 16 hours (overnight) to prepare pigmented trabecular cells.The cells were irradiated with modified laser lens with the spot of 400 μm and emitting duration of 3 ns.The energy scale of 0.2 mJ was set for standardized energy and 0.1 mJ was used as the low energy.The morphology and ultrastructure of the cells were examined under the light microscope and transmission electron microscope 1 hour,4,8,12,24 hours after irradiation.Trypan blue staining and TUNEL fluorescence staining were used to evaluate the death and apoptosis of the cells after laser irradiation.Results The brown pigment particles were seen in cytoplasm around nuclei of trabecular cells after cultured for 16 hours.After laser irradiation,there was no obvious change in the shape of nonpigmented trabcular cells,but a 400 μm spot was found in the pigmented trabecular cells under the light microscope,and the pigmented particles released as the cell disruption.After 0.2 mJ laser irradiation,cell loss zone was seen at the center of the laser spot,and the positive cells for trypan blue and TUNEL staining were found;while the abnormal manifestations were slight after the 0.1 mJ irradiation.With the lapse of the time,the number of positive staining cells was gradually decreased.Twenty-four hours after laser irradiation,proliferative trabecular cells emerged and migrated to the center area of laser spot.Statistical analysis showed that the numbers of the positive cells for trypan blue and TUNEL staining were significantly reduced in the lower energy group in comparison with the standardized energy group at various time points (all at P＜0.001),and those of both energy groups were gradually reduced with lapse of time with the significant differences between any two time points (all at P＜0.01).Conclusions An in vitro laser effect model of HTCs can be established by exposing pigmented HTCs to SLT laser.After exposed to SLT,the gasification,necrosis-like death,apoptosis-like change appear at the laser center.The laser destroyed cells can be replaced by proliferative cells with the lapse of time.Low-energy laser irradiation cause a similar morphological change of the cells to high-energy irradiation,but the number of abnormal cells is much less.