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 study the response of the retinal neuronal adaptive system to changes of background illumination (BG) by measuring the oscillatory potentials (OPs) and the a- and b-waves of the electroretinogram (ERG) in different BG illuminations. Methods The a- and b-wave and the digitally filtered OPs were simultaneously recorded from Wistar Fu rats aged from 25 to 29 days during dark adaptation (DA) and during 6~8 minutes of BG illuminations at four levels increased successively by steps of two log units, i.e., ldquo;low scotopicrdquo; level of 1.43times;10-6cd/m2, ldquo;high scotopicrdquo; of 1.43times;10-4cd/m2 , ldquo;low mesopicrdquo; of 1.43times;10-2cd/m2 and ldquo;high mesopicrdquo; of 1.43times;10-2cd/m2. Full field stimulus flashes of 75 msec duration and 1.43times;10-2cd/m2intensity was delivered at an interval of 1 minute. Results Five OP wavelets were recorded in DA and during scotopic BG illuminations. The number of wavelets was reduced to three as the eyes were exposed to mesopic BG levels. However, the sum of OPs amplitudes (SOPs) increased as the BG was intensified, except at ldquo;high mesopicrdquo; level, by which a significant decrease of SOPs occurred. The amplitudes of the a-and b-waves remained unchanged at the two scotopic BG and decreased as the BG intensity increased to mesopic levels. Conclusion The response of retinal neural adaptive system of the Albino rat to changes of BG light is more sensitive and robust than the slow components of the ERG. The enhancement of the oscillatory responses at ldquo;low mesopicrdquo; illumination level suggests that using proper BG light may be conducive to reducing the variation of OPs. (Chin J Ocul Fundus Dis, 2001,17:286-288)
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)
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）
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 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 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.