Objective To observe the effect of epithelial-mesenchymal transdifferentiation (EMT) of human retinal pigment epithelial (RPE) cells induced by vitreous humor in vitro. Methods The third to fifth passage cultured RPE cells were divided into two groups of treatment by 10% serum containing Dulbecco minimum essential medium (DMEM)/F12 medium (group A), or the same medium supplemented with 25% human vitreous (group B). The morphological changes were observed with a phase contrast microscrope. Cell migration, invasion and contractility were tested using a scratch wound assay, Transwell invasion assay and collagen gel contraction analysis. The expression levels of alpha;-smooth muscle actin (SMA) and Snail1 were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The RPE cells in group A were flat and gathered together. The RPE cells in group B grew as a fan-shaped projection at one edge and cone-shaped tail at the opposite edge, or spindle-shaped, and appeared to separate. In group A, filamentous actin distributed mainly at the margin of the cells with the distribution an oval shape. In group B, filamentous actin reorganized and formed fan-like flat pseudopodia at one edge of the cells. Compared to group A, the migration and invasion of the cells increased significantly (t=14.190, 22.630; P＜0.05), but contractility decreased remarkably (t=6.221, P＜0.05) in group B. Compared to group A, the expression level of Snail1 mRNA increased significantly (t=3.218, P=0.032), but the expression level of alpha;-SMA mRNA decreased (t=3.990, P=0.016). Conclusions Vitreous humor can induce the EMT of RPE cells. Increasing cell migration, cell invasion, and expression of Snail1 mRNA as well as up-regulated cellsprime; contractility and expression of alpha;-SMA mRNA may be the mechanism.
Objective To investigate the expression of eotaxin-1, eotaxin-2 and eotaxin-3 in ARPE-19 human RPE cells after exposure to light. Methods Cultured human RPE cells (5th~10th generations) were divided into lightinduced group and control group. Cells light-induced group were exposed to the blue light at the intensity of (600plusmn;100) Lux for 12 h to establish the light damaged model. Eotaxin-1, eotaxin-2 and eotaxin-3 mRNA and protein were determined by real time polymerase chain reaction and Western blot at 0, 3, 6, 12, 24 hours after light-induced. Results In light-induced groups, mRNA levels of eotaxin-1 and eotaxin-2 were increased at 0 h (t1=6.05.t2=12.561) and 3 h （t1=2.95.t2=3.67） significantly(P＜0.05), but the mRNA level of eotaxin-3 had not changed （t3=1.57 and 1.00 respectively，P＞0.05） at that time. At 6 h （t1=4.73，t2=18.64，t3=28.48）, 12 h （t1=3.11，t2=20.62，t3=18.50）, 24 h （t1=8.25，t2=38.27，t3=18.60）, mRNA levels of eotaxin-1, 2, 3 were increased significantly （P＜0.05）. Except for the eotaxin-3 protein had not changed at 3 h （t3=1.28，P＞0.05）, protein expression of eotaxin-1, 2, 3 were increased significantly （P＜0.05） at 0 h （t1=4.85，t2=5.45，t3=6..21）, 3 h （t1=5.64，t2=4.55）, 6 h （t1=31.60，t2=6.63，t3=7.15）, 12 h （t1=14.09，t2=18.22，t3=15.76）, 24 h （t1=6.96，t2=10.47，t3=12.85）. Conclusion Eotaxin-1, eotaxin-2 and eotaxin-3 expression were increased after Light-damage, corresponding to the time after light exposure. Eotaxin-3 was the most prominent isoform.
Objective To investigate the protective effects of riluzole, a sustained activator of K2P subfamily member TRAAK potassium channel, in human retinal pigment epithelium (hRPE) cells with oxidative induce by tert-butyl hydroperoxide (t-BHP) in vitro, and to evaluate the possible involvement of K2P in the cytoprotective function of retina degeneration diseases. Methods The third to fifth passage of the primary cultured hRPE cells were used in the following experiments．hRPE cells were divided into seven groups: normal control group.t-BHP (300 mu;mol/L) group.t-BHP with riluzole (2, 5, 10, 20 mu;mol/L) group and riluzole (10 mu;mol/L) group. The apoptosis was measured by the 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, annexinV/PI double staining flow cytometry. Changes of cells and nuclei morphology were observed under a phase contrast microscope and a fluorescence microscope after 4prime;, 6-diamidino-2-phenylindole (DAPI) staining. Immunofluorescence 1abelling was carried out to analysis the expression of TRAAK. Results After 24 hours incubation with 300 mu;mol/L t-BHP, the cells viability decreased to (58.7plusmn;12.2)% as compared to the normal control groups. The cell viability of t-BHP with riluzole group at different concentrations was higher than the t-BHP group, while 10 mu;mol/L riluzole showed maximally protective effect on hRPE death induced by t-BHP（t=4.84.P＜0.05）. Riluzole remarkably decreased pyknotic nucleus and cell swelling when compared with t-BHP group. Morphology of cells was fusiform with the uniform elliptic nuclei in normal and riluzole group. The Results of annexinV/PI double staining flow cytometry showed that ratio of normal cells were （97.6plusmn;1.3）%, （70.3plusmn;7.0）%, （86.9plusmn;5.2）%, （93.9plusmn;1.5）% in normal group.t-BHP group.t-BHP with riluzole group and riluzole group respectively. The ratio significant decreased in t-BHP group when it was compared with the other groups (t=7.53, 4.59, 6.49, respectively.P＜0.05). By contrast with normal group and riluzole group, the ratio of normal cells in t-BHP with riluzole group had no statistical significance（t=2.94, 1.91, respectively.P＞0.05）. Riluzole (10 mu;mol/L) also significantly decreased the ratio of early stage apoptotic cells from (25.50plusmn;8.02)% to (1.20plusmn;0.72)% in t-BHP injured groups （t=7.13，P＜0.05）. The ratio of early stage apoptotic cells significant decreased in t-BHP group when it was compared with the normal group and riluzole group （t=7.07, 5.94, respectively.P＜0.05）. By comparison with normal group and riluzole group, there are no statistical significance in t-BHP with riluzole group（t=0.06, 1.18, respectively.P＞0.05）. The mean gray values of TRAAK expression were 0.040plusmn;0.003, 0.041plusmn;0.001, 0.049plusmn;0.001, 0.055plusmn;0.001 in normal group.t-BHP group.t-BHP with riluzole group and riluzole group respectively. TRAAK density was significantly higher in t-BHP with riluzole group and riluzole group（t=7.40, 12.70, respectively.P＜0.05）. Conclusions Riluzole can protect hRPE cells against oxidative injury-induced cell death at early apoptosis stage. The mechanism may relate to that riluzole can promote the expression of K2P TRAAK potassium channel.
Objective To investigate the effect of blue light on mRNA expression of L-type calcium channel subtypes of human retinal pigment epithelial (RPE) cells in vitro. Methods The fourth-generation of human RPE cells were randomly divided into four groups including control group (no light group), light group, light + nifedipine group, and light + (-) BayK8644 group. The cells were exposed to blue light (2000plusmn;500) lux for 6 hours, and then cultured for another 24 hours. Reverse transcription polymerase chain reaction real time (RT-PCR) and fluorescence quantitative PCR technologies were used to analyze mRNA expression of L-type calcium channel subunit of cardiac subtype ( 1C or CaV1.2), neuroendocrine subtype ( 1D or CaV1.3) and retinal subtypes ( 1F or CaV1.4) in each group. Results The length of PCR product of 1C, 1D, 1F subunit and actin was 68, 157, 125 and 186 base pairs respectively. (1) 1C mRNA expression in light, light + nifedipine and light + (-) BayK8644 group was higher than that in control group, the difference was statistically significant (P＜0.05). 1C mRNA expression in light +nifedipine group and light + (-) BayK8644 group was higher than in light group (P＜0.05). 1C mRNA expression in light + (-) BayK8644 group was higher than that in light + nifedipine group (P＜0.05). (2) Comparing with control group, 1D mRNA expression was higher in light, light +nifedipine and light + (-) BayK8644 group, the difference was statistically significant (P＜0.05). Light + (-) BayK8644 group was higher than light group and light + nifedipine group (P＜0.05), light group and the light + nifedipine group was not statistically significant (P＞0.05). (3) 1F mRNA expression in light, light + nifedipine and light + (-) BayK8644 group was higher than those in control group, there was statistically significant (P＜0.05), light +nifedipine group and light + (-) BayK8644 group was higher than light group (P＜0.05), light + nifedipine group and the light + (-) BayK8644 group was not statistically significant (P＞0.05). Conclusions The human RPE cells mRNA expression of L-type calcium channel 1C, 1D and 1F subunit was increased after exposing to blue light. Application of the 1times;10-5 mmol/L (-) BayK8644 can increase mRNA expression of 1C, 1D and 1F subunit.
Objective To observe the characteristics of spectraldomain optical coherence tomography (SD-OCT) and fundus autofluorescence (FAF) in acute and chronic central serous chorioretinopathy (CSC).Methods Seven-three eyes of 67 patients with CSC diagnosed by slit-lamp microscopy, fundus photochromy, fundus fluorescein angiography (FFA) and indocyanine green angiography were enrolled in this study. All the patients were examined for FAF and SD-OCT. The patients were divided into acute CSC group (37 patients, 37 eyes) and chronic CSC group (30 patients,36 eyes) according to the clinical features and FFA images. According to the OCT feature in retinal detachment area, they were divided into three categories, which including intact, non-intact and atrophy outer segment, respectively. According to the FAF characteristics, they were divided into hyper-FAF, hypo-FAF and mixed type, respectively. The characteristics of SD-OCT and FAF of both acute and chronic CSC patients were evaluated and analyzed. Results In acute CSC group, 19 eyes (51.35%) were hypo-FAF, 18 eyes (48.65%) were hyper-FAF. In chronic CSC group, two eyes (5.56%) were hypo-FAF, 16 eyes (44.44%) were hyper-FAF, and 18 eyes (50.00%) were mixed type. There was significant difference between both groups (chi;2=31.872,P=0.000). The SD-OCT results showed that in acute group, 15 eyes (40.54%) were intact outer segment, 18 eyes (48.65%) were non-intact outer segment, and four eyes (10.81%) were atrophy outer segment. In chronic group, five eyes (13.89%) were intact outer segment, 17 eyes (47.22%) were non-intact outer segment, and 14 eyes (38.89%) were atrophy outer segment. There was significant difference between both groups (chi;2=10.572,P=0.005). Conclusions The FAF characteristics of acute and chronic CSC mainly manifests hypo-FAF and mixed type, respectively. The OCT characteristics of acute CSC mainly manifests intact outer segment and non-intact outer segment, but non-intact outer segment and atrophy outer segment in chronic CSC.
Objective To observe the clinical features of polypoidal choroidal vasculopathy (PCV) with retinal pigment epithelium (RPE) tears. Methods Twelve patients of PCV with RPE tears (12 eyes) were enrolled in this study. The patients included eight males and four females, with a mean age of 58.6 years (from 39 to 71 years old). All the patients were affected unilaterally, including eight right eyes and four left eyes. There were one eye with serous RPE detachment and 11 eyes with hemorhagic RPE detachment. All the patients were examined for fundus photography, fundus fluorescein angiography (FFA) and indocyanine green angiography (ICGA), three patients were examined for optical coherence tomography (OCT). The location of RPE tear was classified as within vascular arcade, on vascular arcade, and outside vascular arcade. The shape of tear was classified as crescent, semilunar, or irregular. The features of fundus, FFA, ICGA and OCT were observed. Results Fundus examination presents a gray lesion in all eyes. The location of tear were within vascular arcade in four eyes (33.3%), on vascular arcade in five eyes (41.7%) and outside vascular arcade in three eyes (25.0%). The shape of tears were crescent (one eye, 8.3%), semilunar (ten eyes, 83.3%) or irregular (one eye, 8.3%). The RPE tear region present transimitted fluorescence of at the early stage of FFA and hyperfluorescence with a clear border at late stage. There was no leakage, and at the border of hyperfluorescence, blockage fluorescence of rolled and contracted RPE was present. In ICGA manifestation, transimitted fluorescence was found in RPE tear region at early stage, and a clear border was seen in nine eyes at late stage. There was also blockage fluorescence in ICGA of contracted RPE. In OCT manifestation, the RPE reflections were disappeared, and at the margin of tear, the contracted RPE present a dense rolled b reflection. Conclusions In PCV patients, RPE tears are semilunar and usually located within or around the vascular arcade. Fundus angiography shows transimitted fluorescence at the RPE tear region, and curl blockage fluorescence at the edges. OCT shows RPE reflection is disappeared in the tear region and a b reflection at the edges.
Objective To observe the characteristics of fundus autofluorescence （AF） distribution at the posterior pole in normal subjects. Methods Seventy-nine normal subjects （156 eyes）were studied. Confocal scanning laser ophthalmoscope (cSLO) HRA2 was used to obtain the AF average image at the posterior pole. The distance was calibrated by Digimizer image analysis system. With umbo as the center, the macula was divided into foveola, fovea, parafovea and perifovea areas which with the radius 175, 750, 1250 and 2750 mu;m respectively. Each area was further divided into inferior, superior, temporal and nasal quadrants by two radial lines angle of 90deg;, except for foveola. The AF intensity in four quadrants of different macular regions and optic disc were measured. The AF intensity in vertical and horizontal direction of umbo was also measured. Then the effects of age, eyes, and gender on AF intensity in four quadrants of different macular regions were analyzed. Results There were statistically significant differences in AF intensity among optic disc and four quadrants of macular regions （F=528.648, P＜0.05）. AF distribution was V-type in vertical direction and M-type in horizontal direction. There were statistically significant differences between age groups in foveola, inferior parafovea, temporal parafovea, inferior perifovea, superior perifovea and temporal perifovea （P＜0.01）. There were no statistically significant differences between the two eyes （P＞0.05）. Between genders group, there were statistically significant differences in foveola, superior fovea, inferior fovea, nasal fovea and temporal perifovea （P＜0.05）; no statistically significant differences in the other quadrants （P＞0.05）. Conclusions The distribution of AF intensity is inhomogeneous in macular regions and four quadrants of each region in normal subjects. AF intensity increases with aging. AF distribution is symmetrical in both eyes. There is probably no correlation between gender and AF intensity distribution.
Objective To observe the autofluorescence （AF） manifestation in related lesions of periphery retinopathy.Methods Sixty eyes of 42 patients with periphery retinopathy underwent the examination of Optomap fundus photograph (200deg;) and fundus fluorescein angiography (FFA). The HRAⅡ melaninrelated nearinfrared fundus autofluorescence (NIA, excitation 795 nm) and lipofuscinrelated fundus autofluorescence (FAF, excitation 488 nm) were measured for all the patients. The AF was recorded with nine images per second, and then a final AF image with 55deg; view and 822times;768 pixel was generated by the HRA. AF images can be valuable or valueless if there was or was not visible blood vessels and related retinal tissues on the image. AF from lesion regions can be normal or abnormal fluorescence comparing to the normal vascular and retinal tissue AF. The abnormal fluorescence was divided into no AF, weak AF and b AF relative to the background grayscale. The grading consistency of abnormal fluorescence based on FAF and NIA examination was comparatively analyzed. Results Valuable AF images were captured in 53/60 eyes （88.33%）and valueless AF images were captured in 7/60 eyes （11.67%）. Among 53 eyes with valuable AF image, NIA showed normal fluorescence in 28 eyes （52.83%），abnormal fluorescence with sheetlike, dotshaped or stripped in 25 eyes （47.17%）; FAF showed normal fluorescence in two eyes （3.77%）, abnormal fluorescence with sheetlike, scattered along vessels or pigments in 51 eyes （96.23%）. Twentyfive eyes with abnormal fluorescence were observed both in two examinations, including same grades in 18 eye （72.00%） and different grades in seven eyes （28.00%）. Conclusion The AF manifestation with different levels exists in related lesions of periphery retinopathy.
To investigate the characteristics of fundus autofluorescence (AF) in the leakage site of central serous chorioretinopathy (CSC). Methods Sixty-seven CSC patients (67 eyes) underwent fundus fluorescein angiography (FFA) examination with a confocal scanning angiography (HRA2). Autofluorescence was elicited by the wavelength of 488 nm. The patterns of autofluorescence corresponding to the leakage site on FFA were observed. All the enrolled patients were grouped by age (agele;45 in 47 eyes and age ＞45 in 20 eyes) and courses (acute CSC in 25 eyes and chronic or recurrent CSC in 42 eyes), the patterns of autofluorescence were analyzed respectively. Results There are 4 patterns of AF in the leakage site on FFA of CSC patients: no AF changes, punctuate hypo-AF, expanded hypo-AF or speckled AF, hyper-AF. The percentages of those patterns in all 67 eyes are 52.2%, 23.9%, 14.9% and 9.0% respectively. The percentages of those patterns in the group of age le;45 (n=47) are 55.3%, 23.4%, 14.9% and 6.3% respectively. The percentages of those patterns in the group of age ＞45 (n=20) are 45.0%, 25.0%, 15.0% and 15.0% respectively. The percentages of those patterns in acute CSC (n=20) are 80.0%, 16.0%, 4.0% and 0% respectively. The percentages of those patterns in chronic or recurrent CSC (n=42) are 35.7%, 28.6%, 21.4% and 14.3% respectively. Conclusion There are different patterns of fundus autofluorescence in different age and courses of CSC patients.