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
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)
Objective To assess the effects of 670nm LED (lightemitting diode) to protect the photoreceptor from the lightinduced damage in a rat model. Methods 32 SD rats were randomly assigned to one of eight groups: untreated control group, the LEDtreated control group, three groups of lightinduced damage，and three groups of lightinduced damage treated with LED. Lightinduced damage result from exposing to constant light for 3 hours of different illuminations of 900，1800 and 2700 lx, respectively. The LED treatment (50 mW) was delivered for 30 minutes at 3 hours before the light damage and 0，24 and 48 hours after the light damage. Retinal function and morphology were measured by electroretinogram (ERG) and histopathology assay. Results The illumination of 900 lx for 3 hours did not damage the rat retina. The illumination of 1800 lx for 3 hours resulted in thinner ONL and no OS and IS. The ratio of damaged area/total retinal area was 048plusmn;012， the damaged thickness of ONL/normal ONL (L5 ) was 039plusmn;007，and the amplitude of ERG b wave was (431plusmn;120) mu;V. With the LED treatment the ratio of damaged area decreased (M6=017plusmn;0.12, P5/6=0.002), and the ratio of the damaged thickness of ONL also decreased (L6=0.22plusmn;0.09, P5/6lt;0.01), and the amplitude of ERG b wave increased to (1011plusmn;83) mu;V（P5/6lt;0.001）. The illumination of 2700 lx for 3 hours caused severed damage to the rat retina and the LED could not protect them significantly. Conclusions 670 nm LED treatment has an evident protective effect on retinal cells against light-induced damage, which may be a simple and effective therapy to prevent or to delay agerelated macular degeneration.
Objective
To observe the pathological and functional changes of retinal photochemical damages exposed to green flurescent light.
Methods
The Sprague Dawley rats were continually exposed to green fluorescent light with an illuminancem level of (1 900plusmn;106.9) Lx for 24 hours.The changes of retinal morphology and morphometrics and flash electroretinogram were studied before light exposure and at the 6th hour,6th day and 14th day after light exposure.
Results
At the 6th hours after light exposure,the outer nuclear layer(ONL)of retina becoma thinner compared with that bfore light exposure.The thickness of ONL decreased by 23.91% and the inner and outer segments appeared disorderly arranged.At the 6th day after light exposure the thickness of ONL is thinner than that at the6th hour,i.e.decreased by 46.6%. At the 14th day after light exposure the thickness of ONL decreased by 42.40%.Flash electroretinogram showed that the amplitudes of a and b wave decreased continuously at the 6th hour and 6th day and unrecovered at the 14th day after light exposure.
Conclusion
This model might be an ideal one for research on retinal photochemical damage.
(Chin J Ocul Fundus Dis,1998,14:101-103)
Objective To assess the protective effect of recombinant human erythropoietin (EPO) on human retinal pigment epithelial (RPE) cells injured by light. Methods Cultured human RPE cells were exposed to light for 12 hours, and the culture was stopped 24 hours later. The 3(4,5dimethylthiazole2y1)2,5diphenyl tetrazolium bromide (MTT) cell viability assay and annexin V flunorescein isothiocyanate/propidium iodium labeling and flow cytometry were used to assess the effects of EPO with different concentration on the cellular viability and apoptosis of human RPE cells. The protective effect and mechanism of EPO on RPE cells injured by light was detected by adding AG490. Results EPO, especially with the concentration of 40 IU/ml, obviously increased the cellular viability of RPE cells and apparently decrease the cellular apoptosis induced by light injury. After adding AG490, the effects of EPO on cellular viability and apoptosis were inhibited. Conclusion It is suggested that EPO can protect the human RPE cells from lightinduced injures, and its protective mechanism works after the combination of EPO and its receptor.
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)
ObjectiveTo investigate the effect of blue light on Ca2+-protein kinase C (PKC) signaling pathway in human retinal pigment epithelial (RPE) cells in vitro.
MethodsPrimary human RPE cells were cultured in vitro and characterized. The experiments were carried out using the 4th generation of human RPE cells. The PKC protein level was measured by Western blot to determine the most appropriate concentration of phorbol ester (PMA) and calcium phosphate binding protein (calphostin C) on PKC expression. Non-radioactive isotope method was used to determine the effect of blue light on PKC expression of cultured cells. Blue-light damage model of human RPE cells was established by 6 hour irradiation of medical blue-light lamp [20 W, 450-500 nm wavelength, (2000±500) Lux], and 24 hours prolongation of post-exposure culture. The human RPE cells were randomly divided into 5 groups. Group A did not receive light irradiation, group B only received blue light irradiation, group C was blue light irradiation and 0.1 mmol/L nifedipine treatment, group D was blue light irradiation and 100.0 nmol/L calphostin C treatment, group E was blue light irradiation and 100.0 nmol/L PMA treatment. Intracellular Ca2+ concentration was measured by acetoxymethyl ester (Fluo 3-AM) labelling and confocal microscope imaging.
ResultsThe PKC protein expression in 100.0 nmol/L or 200.0 nmol/L PMA-treated groups was higher than 0.1, 1.0, 10.0, and 50.0 nmol/L PMA-treated groups, the difference was statistically significant (F=217.537, P<0.05), but there was no statistically difference between 100.0 nmol/L and 200.0 nmol/L PMA-treated groups (P=0.072). The PKC protein expression in 100.0 nmol/L or 200.0 nmol/L calphostin C-treated groups was lower than 5.0, 25.0, 50.0, and 75.0 nmol/L calphostin C-treated groups, the difference was statistically significant (F=164.543, P<0.05), but there was no statistically difference between 100.0 nmol/L and 200.0 nmol/L calphostin C-treated groups (P=0.385). PKC level in blue light group was higher than non-light group, the difference was statistically significant (t=-9.869, P<0.05). The Ca2+ fluorescence intensity values in group B, C, D and E was higher than group A, the difference was statistically significant (F=26 764.92,P<0.05). The Ca2+ fluorescence intensity values in group E was higher than group B, C and D (P<0.05), and that in group B was higher than group C and D (P<0.05).
ConclusionsThe PKC activity and intracellular Ca2+ concentration in human RPE cells increase after blue-light irradiation. Both calcium channel inhibitor nifedipine and PKC inhibitor calphostin C can reduce intracellular Ca2+ concentration in human RPE cells. PMA can induce intracellular Ca2+ concentration in human RPE cells after blue light irradiation.
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 observe the expression of vascular endothelial growth factor A (VEGFA) and its receptors sFlt-1, kinase insert domain receptor (KDR) in lightinjured human retinal pigment epithelial (RPE) cells. Methods Cultured human RPE cells (8th - 12th generations) were divided into normal control group and light damage group. The cells of two groups were exposed to the 18 W cold white light (2200±300) Lux for 12 hours to induce light damage responses, but the cells of normal control group were packed by tinfoil with doubledeck high pressure disinfection. The VEGF-A, sFlt-1 and KDR mRNA and protein expressions were detected by reverse transcriptionpolymerase chain reaction (RT-PCR) and Western blot at 0, 6, 12, 24 hours after light damage. Results The VEGF-A mRNA and protein expressions in light damage group were significantly increased at 6 hours, and reached its peak at 12 hours after light damage which obviously higher than that in normal group (t=2.74, 2.93; P＜0.05), and then went down gradually. The sFlt-1 mRNA and protein expressions in light damage group reached its peak at 12 hours after light damage which obviously higher than that in normal group (t=4.32, P＜0.01), but obviously lower than that in normal group at 24 hours after light damage (t=2.41, P＜0.05). The KDR mRNA and protein expressions in light damage group were obviously higher than that in normal group at 24 hours after light damage （t=2.89, P＜0.05）,but there was no changes at 6, 12 hours after light damage (t=1.84, P＞0.05). Conclusions At 6, 12 hours after light damage, the expressions of VEGF-A and sFlt-1 increases significantly and KDR expression is stable in lightinjured RPE cells. At 24 hours after light damage, the expression of VEGF-A and sFlt-1 decreases, but KDR expression increases in light-injured RPE cells.
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.
