Objective：To observe the protective effect of ginkgo bilo ba extrac t (EGb 761), a free radical scavenger, on the photoreceptor cells after lighti nduced retinal damage.
Methods：Seventytwo female SpragueDa wley (SD) rats we re randomly divided into 4 groups: normal control group, lightinduced retinal da m age model group, model+physiological saline group, and model+EGb 761 group, with 18 rats in each group. All of the rats except the ones in the control group were exposed to white light at (2740plusmn;120) lx for 6 hours after the dark adap tation for 24 hours to set up the lightinduced retinal damage model. Rats in m o del + physiological saline group and model+EGb 761 group were intraperitoneall y injected daily with physiological saline and 0.35% EGb 761 (100 mg/kg), respec tively 7 days before and 14 days after the light exposure. Apoptosis of photorec eptor cells was detected 4 days after light exposure; 7 and 14 days after light exposure, histopathological examination was performed and the layer number of ou ter nuclear layers (ONL) on the superior and inferior retina was counted.
Results：Four days after light exposure, the apoptosis of photorecep tor cells was fou nd on ONL in model, model+ physiological saline and model+EGb 761 group, and w as obviously less in model + EGb 761 group than in model and model+physiologic al saline group. Seven days after light exposure, the layers of ONL on the super ior retina were 3 to 4 in model and model+physiological saline group, and 7 to 8 in model+EGb 761 group; the mean of the layer number of ONL in model+EGb 761 group (6.92plusmn;0.82) was less than that in normal control group (8.40plusmn;0.95) (t=-1.416, P＜0.05), but significantly more than that in model (5.96 plusmn;1.36 ) and model+physiological saline group (5.90plusmn;1.40）（t=1.024, 1.084; P＜0.05). Fourteen days after light exposure, the layers of ONL on the superior retina were 0 to 1 in model and model+physiological saline group, and 3 to 4 i n model+EGb 761 group. The mean of the layer number of ONL in model+EGb 761 group (5.5 2plusmn;1.06) was significantly more than that in model (3.44plusmn;2.15) and model + physiological saline group (3.37plusmn;1.91) (t=2.082, 2.146, P＜0.05).
Conclusion：EGb 761 can partially inhibit the apoptosis of pho toreceptor cells, thus exert protective effect on photoreceptor cells.
PURPOSE:The changes of expression level of rhodopsin mRNA and its relationship with the morphology in light damaged rat retinas were studied. METHODS:The changes of expresson level of rhodopsin mRNA in light damaged rat retinas and the changes on retinal morphology were observed through the technique of in situ hybridization and electron microscopy.
RESULTS:The hybridization signals of rhodopsin mRNA mainly distributed in the photoreceptor layer of retina,relatively b in the inner and outer segments. As the increase of light exposure time,the expression level of rhodopsin
mRNA in retinas greatly decreased before the changes on morphological injury of retina. For the same eye globe of the same rat at the same time,the hybridization signals at the upper and posterior region of the retina decreased more obviously than the lower and peripheral region of the retina. CONCLUSIONS:It was demonstrated for the first time that the expression of
rhodopsin mRNA was located at the photoreceptor layer of the retina. Continuous exposure to light could greatly decrease the expression of rhodopsin mRNA and the decreases differ regionally. It might be the early signals of retinal photic injury.It is a good method to study the expression level of retina mRNA through the in situ hybridization.
（Chin J Ocul Fundus Dis,1997,13: 228-210）
Objective
To further investigate pathologic mechanism of retinal phototrauma.
Methods
Twenty Wistar rats were divided into control and experimental groups.Their eyes were extracted in 12，24 and 36 hours after light exposure.HE stained retina samples were examined and TDT-mediated dUTP nick end labelling(TUNEL)method was employed to distinguish apoptotic cells.
Results
After 12-hour light exposure,slight vesiculation was observed in the rod outer segment of the retinas.After 24-hour light exposure,the outer nuclear layer showed predominant fractured and condensed nuclei and fragmented DNA.After 36-hour light exposure,the rod outer and inner segments were lysed and most of the nuclei in the outer nuclear layer were disappeared.
Conclusions
Apoptosis of photoreceptor cell is one of the important mechanisms which cause experimental retinal photoinjury of rats.
(Chin J Ocul Fundus Dis, 1999, 15: 167-169)
Objective To observe the expression of proteins in light-injured retinal pigment epithelial (RPE) cells. Methods ARPE19 cells were exposed to the cool white light at the intensity of (2200plusmn;300) Lx for 6 hours to set up the light injured model. Cellular soluble proteins was extracted and analyzed by means of twodimensional electrophoresis to find out the changes of protein map of lightinjured RPE cells. Results Cellular soluble proteins had (390plusmn;10) spots on the map, in which 11 spots had obvious difference between the light injured group and the normal control group. In the lightinjured cells, the expressio of 8 proteins increased, 1 decreased, and 2 disappeared. Conclusion Twodimensional electrophoresis can find out the difference of expression of proteins in lightinjured and normal RPE cells.
Purpose
To observe the pathologic changes of retinal photic injury in mice.
Methods
A light damaged trunk was designed by ourselves.The mice were given an intermitent light exposure for 3 days,12 hours light exposure every day and 12 hours dark adaption before every exposure.Experimental animals were sacrificed on the 1st,6th,12th,18th and 30th day after light injury,and the eyes were enucleated for light and electronic microscopy observation.
Results
The early pathologic changes including disc membrance swelling,disorganization in outer segments,and mitochondrial swelling,spherical change in inner segments．Then the chromatin densification,liquefaction and margination,and the shrinkage of nuclear membrance were found in the nuclear layer.Finally the outer nuclear layer became thin and disappeared.The apical microvill of RPE cell disappeared and basic fold became flat in some samples.
Conclusion
The photoreceptor degeneration was the pathologic characteristic of retinal photic injury in mice.
(Chin J Ocul Fundus Dis,1998,14:215-218)
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.
ObjectiveTo observe the influence of down-regulation of HtrA1 expression by small interfering RNA on light-injured human retinal pigment epithelium (RPE) cells.
MethodsCultured human RPE cells(8th-12th generations)were exposed to the blue light at the intensity of (2000±500) Lux for 6 hours to establish the light injured model. Light injured cells were divided into HtrA1 siRNA group, negative control group and blank control group. HtrA1 siRNA group and negative control group were transfected with HtrA1 siRNA and control siRNA respectively. The proliferation of cells was assayed by CCK-8 method. Transwell test was used to detect the invasion ability of these three groups. Flow cytometry was used to detect the cell cycle and apoptosis. The expression of HtrA1 and vascular endothelial growth factor (VEGF)-A was detected by real time-polymerase chain reaction and Western blot respectively.
ResultsThe mRNA and protein level of HtrA1 in the light injured cells increased significantly compared to that in normal RPE cells (t=17.62, 15.09; P<0.05). Compared with negative control group and blank control group, the knockdown of HtrA1 in HtrA1 siRNA group was associated with reduced cellular proliferation (t=6.37, 4.52), migration (t=9.56, 12.13), apoptosis (t=23.37, 29.08) and decreased mRNA (t=17.36, 11.32, 7.29, 4.05) and protein levels (t=12.02, 15.28, 4.98, 6.24) of HtrA1 and VEGF-A. Cells of HtrA1 siRNA group mainly remained in G0/G1 phase, the difference was statistically significant (t=6.24, 4.93; P<0.05).
ConclusionKnockdown of HtrA1 gene may reduce the proliferation, migration capability and apoptosis of light-injured RPE cells, and decrease the expression of VEGF-A.
Objective
To observe the effect of visible light on apoptosis of cultured human retinal pigment epithelium (RPE) cells.
Methods
Being the light source,500lx,(2 000±500)lx and (3 400±200)lx cold white light were used. The duration of exposure was 0,6,12 and 24 hours respectively. Apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling, Annexin V-flunorescein isothiocyanate/Propidium iodium labelling and flow cytometry.
Results
Apoptosis and necrosis were found in cultured human RPE cells which were exposed to visible light.(1)A significant increase in apoptotic and necrotic percentages was consistent with a higher light intensity.(2)Apoptosis was the main response to shorter (6 h and 12 h) exposure duration,while necrosis was more pronounced correlated to the prolongation of post-exposure culture (P＜0.05),and the longer the post-exposure period was, the more apoptotic necrosis were seen.Thirty-six hours after exposure the necrotic percentages were more pronounced (P＜0.01).
Conclusions
Visible light (＞500 lx) increases the proportion of apoptosis and necrosis of human RPE cells in vitro.The extent is related to exposure intensity and duration. It demonstrates that the lower intensity and the shorter duration of exposure to light are, the more pronounced apoptotic percentages are observed,otherwise necrosis.
(Chin J Ocul Fundus Dis, 2002, 18: 227-230)
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.
Purpose
To evaluate the prostag landins(PG) levels and to identify the effect of dexamethasone(DXM) on PG in response to photochemical insult in rat retina.
Methods
The experiments were performed on 36 SD rats which were separated into two groups,control and treated groups,and the latter received daily intraperitoneal injections of DXM (1 mg/kg) for 5 consecutive days,starting 3 days before light exposure.The animals were continually exposed to green fluorescent light(510-560 nm)with an illuminance level of (1900plusmn;106.9)lx for 24 hrs.The retinal concentration of PGE 2 and 6-keto-PGF1alpha; were tested at 6hrs,1,3,7 and 14 days after light exposure.
Results
The PGE2 and 6-keto-PGF1alpha; levels of the control groups (37.50plusmn;2.75,48.06plusmn;4.0 4,81.90plusmn;4.89) pg/mg and (4.68plusmn;0.69,7.50plusmn;0.57,10.40plusmn;0.71) pg/mg had significantly higher values than those of the treated rats(20.60plusmn;4.28,37.36plusmn; 3.34,54.85plusmn;4.57) pg/mg and (2.50plusmn;0.59,4．68plusmn;0.81,6.87plusmn;1.10)pg/mg (Plt;0.01) after 6 hrs,1 and 3 days light exposure respectively.
Conclusion
By inhibition of PG synthesis,the DXM may play an ameliorative effect on retinal photochemical injury of rats.
(Chin J Ocul Fundus Dis,1999,15:94-96)
