| BackgroundRetinal photocoagulation has been the standard care for the treatment of diabetic retinopathy(DR) for decades. The effect of continuous wave(CW) laser for retinopathy due to its thermal damage has been demonstrated in many clinical trial. However the progressive enlargements of laser scars, subretinal fibrosis and subretinal neovascular membranes have been reported as complications from laser tissue simultaneously, resulting in the occurrence of scotomas or color vision loss. Less invasive treatment strategies have been advocated in order to reduce the amount of laser energy applied and thus avoid tissue damage. Advances in laser technology have led to the development of selective photocoagulation. It was firstly reported micropulse diode laser(MPDL) photocoagulation didn’t damage retina by Pankratov in 1990. In 1997, Friberg et al reported on the clinical application of 810-nm SMLP (810-nm MP) in the treatment of DME and several clinical studies demonstrated that SMLP achieves effects similar or superior to those of modified ETDRS photocoagulation DR is the most common complication of diabetes and one of the leading causes of preventable blindness, population-based studies suggest that one-tenth have vision-threatening states of retinopathy, such diabetic macular edema (DME) and proliferative diabetic retinopathy (PDR). Anti-VEGF are often expensive and require repeated injections over months to years to achieve and maintain an optimal effect. All intravitreal injections have a small but inherent and cumulative risk of potentially visually catastrophic endophthalmitis.The most leading factor of DME and PDR is damage of blood retinal barrier(BRB) and neovascularization, respectively. Thus VEGF which is related with BRB and neovascularization is the pathogenic factor for DME and PDR. Therefore Vascular endothelial growth factor antagonist(anti-VEGF) has been the most important therapy for DME and PDR. RPE cell can produce VEGF which promotes angiogenesis and PEDF which has inhibition of endothelial cell proliferation and stabilize function of vascular endothelia cells. So far, it is believed cytokines produced by RPE has primary modulation effect on retinopathy.810 nm-MP is selectively absorbed by the retinal pigmented epithelium (RPE) and therefore it does not cause retinal damage. A series of clinical trial had reported 810 nm-MP was effective in treatment of DME and PDR,and our previous study had showed biological modulation of mouse RPE cells in response to subthreshold MPLD treatment. Based on molecular mechanism research and clinical trial results as mentioned above, we will try to investigate the modulation effect of 810-nm MP photocoagulation on VEGF and PEDF of RPE of Brown Norway (BN) rats and the change of fluorescein angiography (FFA) of retina. And then molecular mechanism of subthreshold micropulse of treatment for diabetic rats will be explored.AimTherefore, the aims of the present study were:(1) to explore 810nm-MP laser parameters without visible burn; (2) to investigate the modulation effect of 810nm-MP on RPE to produce cytokines; (3) and to explore the effect of 810nm-MP on retina of diabetic rats.MethodsExperimental designTen weeks adult male Brown Norway rats weighing 280 to 320 g were obtained from the Beijing Weilitonghua limited liability company (Product License No. SCXK(jing)2012-0001). Six rats were randomly extracted to explore 810nm-MP laser parameters without visible burn. The remaining 32rats were randomly divided into the following 4 groups, including normal control group (Al,n=2), normal MPL group (A2,n=10), diabetic control group (B1,n=3), diabetic MPL group (B2,n=15). Group B1 and B2 rats were peritoneal injected with streptozotocin (STZ) to build diabetic model and had high-carbonhydrate diet for 2 months while group A1 and A2 were peritoneal injected with 0.1% Sodium citrate solution and had euphagia for 2 months. Then Group A2 and B2 were performed with 810nm micropulse diode laser pan-retinal photocoagulation, but group A1 and A2 with no intervention.Explore 810nm-MP laser parameters without visible burnThree rat were under intraperitoneal anesthesia with chloral hydrate(10%,3.5ml/kg), six eyes of all the rats were dilated with Compound Tropicamide Eye Drops. One eye receive 532nm continuous wave laser photocoagulation(laser parameter:spot size is 100μm, exposure time is 0.2ms, laser power is 200mW, laser points is 9), the remaining 5 eyes receive 810nm-MP photocoagulation(laser parameter:spot size is 100μm, exposure time is 0.2ms, loading factor is 10%, laser power is from 400-1200mW, laser points is 9). Laser photocoagulation was performed surrounding the video disk equidistantly. Immediately fundus photograph was performed after laser photocoagulation.diabetic animal modelsRats of group B1 and B2 were peritoneal injected with STZ (0.1% Sodium citrate solution, PH=4.5) and had high-carbonhydrate diet for 2 months to build diabetic model. Rats of group Al and A2 were peritoneal injected with equal Sodium citrate solution. Fasting blood glucose was tested after injection, which was above 16.7mmol/L was defined as diabetic model rats.Experimental intervention with 810nm-MP laser photocoagulationGroup A2 and B2 were randomly devided into 5 groups according to laser power, respectively and performed with 810nm micropulse diode laser pan-retinal photocoagulation after dialation(laser parameter:spot size is 100μm, exposure time is 0.2s, loading factor is 10%, laser power is from 400-1200mW), but group A1 and A2 with no intervention. The expression of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF) of RPE-choroid- sclera complex were analyzed in retinal tissue homogenates by western blot 24 hours after laser photocoagulation.Fluorescein angiography examination performed after 810nm-MPThe remaining rats were under intraperitoneal anesthesia 2 weeks and one month after 810nm-MP laser photocoagulation. They were peritoneal injected with 10% fluorescein and immediately performed fluorescein angiography examination.Statistical analysis All analyses were performed with SPSS 19.0(American IBM Statistic Company). Data are expressed as mean±SD. The statistical analysis of the data was conducted using independent sample T-test and the one-way analysis of variance (ANOVA) followed by LSD. Statistical significance was defined as P<0.05.ResultsThe number of Visible spots with different power of laser photocoagulationThere are 9 visible laser spots surrounding the video disk with 532nm continuous wave laser photocoagulation, but no visible spot with 810nm-MP below 600mW. The visible burn began to appear when the power was above 600mW. AS increasing with the rise of laser power, the number of Visible spots increased.Condition of diabetic model ratsUrine of rats increased obviously one day after peritoneal injection with STZ. Fasting blood glucose of fourteen rats was above 16.7mmol/L, including 2 rats in group B1 and 12 in group B2. The successful rate of model was 77.8%. Fluorescein angiography examination showed 8 rats with diffuse retinal fluorescence leakage after two months’s high-carbonhydrate diet. However retinal microaneurysm, retinal hard exudates and macular edema as DR paitients have not been seen in none of retina in diabetic rats.Difference of expression of VEGF and PEDF between group A1 and A2There was statistically significant difference between group Al and A2, whose VEGF/β-actin was 0.153±0.011 and 0.429±0.051, respectively.Expression of VEGF and PEDF with different power of 810nm-MP laser photocoagulationVEGF-A was statistically significant decreased after 810nm-MP laser photocoagulation both in normal or diabetic rats compared with on laser intervene (F=38.373, p=0.000; F=13.69,p=0.000) but PEDF was statistically significant increased(F=27.771, p=0.000; F=5.966,p=0.001). AS increasing with the rise of laser power, the number of Visible spots increased, the express of VEGF-A gradually decreased but PEDF increased. When the power below 600-800mW, there was no visible spot, the expression of VEGF-A was least but PEDF was most. However, VEGF-A would rise and PEDF decend when the power kept on rising.Difference of FFA between group Bl and B2 after experimental interventionFluorescein leakage of fundus posterior pole has been seen in group B1 and B2 before 810nm-MP intervene. Fluorescein leakage was aggravated in group B1 without 810nm-MP intervene, but was obviously alleviate in group B2 with 810nm-MP intervene 2 weeks and 1 month after experimental intervention.Conclusion1. There was no visible laser burn with 810nm-MP laser photocoagulation with 10% load coefficient and below 600mW laser power.2. The expression of VEGF will decrease and PEDF will increase after intervention of RPE with 810nm micropulse laser photocoagulation, and there is no damage when power behind 600mW.3. Micropulse laser photocoagulation can relieve fluorescein leakage. |
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