Biological Modulation Of Mouse RPE Cells In Response To Subthreshold Diode Micropulse Laser Treatment

Background:Macula is the most sensitive and critical part in the formation of human vision. Macular edema secondary to Retinal vein occlusion, diabetic retinopathy, retinal vasculitis, etc. and macular choroid neovascularization (CNV) due to central exudative retinopathy, wet age-related macular degeneration, pathological myopia, etc. are the basic pathological changes of many serious blinding fundus diseases that can cause severe vision loss. They are related to lots of fundus diseases which are lack of effective means to be prevented and controlled and bring heavy burden to society and family. Retinal laser photocoagulation appeared just one year after laser was invented in 1961, which bringed revolutionary change for the treatment of retinal diseases. Most of the fundus diseases above can only be controlled or eased by laser which has become a strong treatment for ophthalmic clinical and. saved the vision in numerous patients with fundus diseases. However, with the development of laser technology, many clinical doctors also gradually realized its role to the destrution of the retinal tissue. For a long time, most of us accepted the notion that when laser radiated to the fundus, most of its energy will be absorbed by pigment tissue such as melanin, hemoglobin and lutein, etc. and then transformed into heat. In general, the laser light is applied across the pupil, then after approximately 1 msec of starting the irradiation, heat generated is spread to the adjacent pigmented subretinal structures which were not directly targeted by the laser. The spread of released thermal energy causes a gradual decrease of the temperature to baseline body temperature [20]. In case of the conventional threshold photocoagulation, the appearance of grayish endpoint indicates that the thermal wave initiated in the RPE/choroid interface has arrived at the overlying neurosensory retina. This endpoint or laser lesion is a sign that the temperature is sufficiently high (20 to 30℃ above the baseline of 36℃) to affect natural transparency leading to the scatter of the slit lamp illumination light. The overlying neural retina which is normally transparent to laser wavelength is damaged and loses its transparency causing scatter of the white ophthalmoscopic light back and therefore making the lesion visible. Light damage to the organization can be temporary or permanent, but its progress is slow. Traditional laser treatment can destroy high oxygen consumption of photoreceptor cells and RPE cells, reduce the metabolic requirements, remove the pathological tissue of retina, increase oxygen tension within the eye and improve production of vascular factors including vascular endothelial growth factor. However, retinal injury and inflammation caused by this threshold retinal photocoagulation increase the risks and side effects of treatment. Compared with on treatment, threshold photocoagulation has definite benefits, but also is accompanied by severe destruction of photoreceptors and choroid blood capillary. Therapeutic laser photocoagulation abolishes oxygen utilizing photoreceptor cells and pigment epithelium that leads to retinal hypoxia. However, the use of conventional suprathreshold laser photocoagulation is associated with many complications including reduced visual acuity, visual field, color vision, night vision and contrast sensitivity. In addition, risks of CNV, hemorrhage, epiretinal fibrosis and serous detachment of the peripheral retina are increased with conventional laser treatment. Although the mechanism underlying the pathogenesis of CNV is not completely understood, RPE is considered to play a pivotal role in the genesis of this disorder. Implication of cytokines produced by the RPE in the pathogenesis of retinal vascular disease such as DME has also been evidenced. The highly specialized cells of RPE function as a blood-retinal barrier and allow selective transport of angiogenesis factors such as VEGF between choriocapillaries and neural retina. A dynamic balance between a pro-angiogenic (VEGF) and anti-angiogenic (PEDF) factors has been evidenced to play a critical role in maintaining the normal physiological functions of retinal choroidal endothelial cells.In order to understand the mechanism of SDM in treating retinal vascular disease, we aimed to investigate response of mouse RPE cells to 810nm diode laser exposure, focusing on the characteristics of the cell defense system against CNV. We measured apoptosis of RPE cell and expression of the angiogenesis stimulating cytokines (vascular endothelial growth factor A (VEGF-A), transforming growth factor beta (TGF-β) and basic fibroblast growth factor (bFGF)), and pigment epithelium derived factor (PEDF) the inhibitor of angiogenesis.Methods:1 Cell culture:Method of one-step enzymatic digestion was used to isolate and gain RPE cells of mice. Viability and proliferation of cells were observed using growth curve, time of cell division and other indicators. The characteristics of cells were observed and identified using light microscopy and immunofluorescence assay. RPE cells in logarithmic growth phase were chosen for the experiment.2 select appropriate duty cycle of laser which reduces damage of RPE cells to the minimum: RPE cells cultivated in a culture dish (3×106 cells/well in a 6-well plate) with DMEM/F12 were treated with 810nm diode laser radiation as a heat source. Both 5% duty cycle (DC) and 10% DC have been tested. Radiation intensity ranged from 0 to 400mW with beam diameter of 75μm, and 100 msec of exposure were applied to different groups. After the laser radiation treatment, MTT method was used to detect the OD value of each group. The cell growth inhibition rate%= (1-OD of the experimental group/OD of the control group)×100%.3 Detection of apoptotic rate of RPE cells by flow cytometry:RPE cells incubated in serum free DMEM/F12 were treated with laser induced radiation with intensity ranged from 0 to 400mW,5% DC, with beam diameter of 75μm and exposure duration of 100 m sec. After 24 h of laser irradiation, Flow cytometry method was used to detect the apoptotic rate of RPE cells in each group. The data was analyzed using Cell Quest software. Apoptotic cells were characterized by Annexin V-FITC+PI+ and Annexin V-FITC+PI-. Apoptotic rate was calculated after 10000 cells were sorted.4 The expression of VEGF-A, TGF-β, bFGF, PEDF proteins in RPE cells by western blot method:RPE cells incubated in serum free DMEM/F12 were treated with laser induced radiation with intensity ranged from 0 to 400mW,5% DC, with beam diameter of 75μm and exposure duration of 100 m sec. After 24 h of laser irradiation, Western blot method was used to detect the expression of VEGF-A, TGF-P, bFGF, PEDF and P-Actin which was taken for internal reference. The data was analyzed by AlphaEase FC software to determine the gray levels of each protein in each group, and the relative content of proteins was calculated by the ratio of grey levels of target proteins and the one of the reference.5 mRNA expressions of VEGF-A, TGF-β, bFGF and PEDF were analyzed by RT-PCR: RPE cells incubated in serum free DMEM/F12 were treated with laser induced radiation with intensity ranged from 0 to 400mW,5% DC, with beam diameter of 75um and exposure duration of 100 m sec. After 24 h of laser irradiation, RT-PCR method was used to detect the expression of VEGF-A, TGF-p, bFGF, PEDF and P-Actin which was taken for internal reference.The data was analysed by 2-△△CT detection method.6 Statistical analyses:SPSS19.0 software was used to analyze the data. The data was revealed by mean ± standard deviation (x±s). Single factor variance analysis was used to analyze the data among multi-groups, and the significance between two specific groups was detected with q-test. Statistic difference was considered to be significant if P<0.05.Results:1 Morphological change of RPE cells under the inverted phase contrast microscope:The black and round primary RPE cells started to stick to the bottle after 24 hours. After 3 days, cells were black and short spindle like and the number was double. Cells gathered in monolayer to cover 80% of the bottom of bottle in 5 days. After 7 days, the whole bottom of bottle was covered by cells which symbolized as the typical cobble-stone modality. Passaged as 1:2 ratios, the viability of cells was active, the nucleus was transparent and cytoplasm was rich of black particles. Cells began to be fused which were symbolized as sprindle and irregular shape after 2 or 3 days. The result of anti-Keratin immunofluorescence revealed that 99% of cells were of epithelial origin.2 Laser-induced reductions in RPE viability:We tried to determine RPE viability subjected to laser induced radiation of different intensity at 5% and 10% DC respectively. Cell viability of 5% DC group was significantly higher compared to 10% DC group (P<0.05). In 5% DC group, 100mW and 200mW of laser radiation didn’t have any significant effect compared to the control (P> 0.05). Cell survival was significantly reduced in a power-dependent manner (P< 0.05) in both 5% DC and 10% DC group.3 Laser application with low DC and power did not induce apoptosis in RPE cells:The annexin V flow cytometry analysis was used to determine the apoptosis of RPE cells irradiated with 810nm diode laser at 5% DC. At the power of 100 and 200mW with 5% DC, the laser-induced apoptosis was low (3.52%±0.36% and 3.55%±0.29%, P>0.05 respectively) and it was not different from that of the control Group (3.68%±0.27%, P> 0.05). At the power of 300 and 400mW, laser-induced apoptotic rate was increased in a power-dependent manner (9.31%±0.59% and 14.24%±0.45%, respectively P<0.05) and it was significantly higher than that of the control group (P<0.05).4 Angiogenic stimulators were down-regulated and inhibitor was up-regulated with laser treatment:Based on the results of western blot, laser treatment induced a significant down regulation of angiogenic stimulators (VEGF-A, TGF-β and b FGF) (P<0.05), and up-regulation of the inhibitor (PEDF) (P<0.05). This response of the cells treated with different doses of laser was not significantly different (P>0.05). Consistent with the Western results, compared to the control group, laser treatment induced a significant (P<0.05) decrease in the mRNA expression of for angiogenic stimulators (VEGF-A, TGF-P and b FGF), whereas that of the inhibitor (PEDF) was significantly increased (P<0.05). No significant difference was found between the groups treated with different intensities of laser (P>0.05).Conclusion:Our results showed that compared with woking in 10% DC, Subthreshold Diode Micropulse Laser (SDM) causes inhibition to RPE cells activity in 5% DC. At the power of 100 and 200mW with 5% DC, the laser-induced apoptosis was low and it was not different from that of the control Group. At the same time, SDM treatment of the RPE cells suppressed the expression of CNV promoting cytokines and up-regulated the angiogenic inhibitor, PEDF. Further investigation is needed to understand the mechanism and to optimize the use of SDM as a novel method of treatment for retinal vascular diseases.