Objective To investigate the effects of lights with different wavelength on the retina of rd12 and C57BL/6J mice. Methods Thirty two rd12 mice and C57BL/6J mice were randomly divided into the control group, white light group, midwavelength light (505 nm) group and shortwavelength light (405 nm) group, with eight mice in each group. Besides the control group, other groups were exposed to cycle illuminations ［12 hours dark, 12 hours (800plusmn;130) Lux］ for seven days to establish the model of retinal light damage. Electroretinogram (ERG) responses of all mice were recorded at the day before illumination and 1st, 4th and 7th days after illumination. The eyes were enucleated at 7th days after illumination to assess levels of reactive oxygen species (ROS), expression of peroxiredoxin 6 (PRDX6), and activity of caspase-3. Results ERG amplitudes of all groups declined gradually in C57BL/6J mice, and the most significant effects was found in the short-wavelength light group. The amplitudes of photopic b-wave were significantly different at 1st, 4th and 7th days (F=4.412, 5.082, 9.980；P＜0.01). The amplitudes of cone b-wave of the four groups decreased to (85plusmn;10) %, (70plusmn;19) %, (57plusmn;22) % and (46plusmn;19) % at 7th days, respectively, and were significantly different between white light group and short-wavelength light group(t=3.19,P＜0.01). The levels of ROS were significantly different in rd12 mice (F=16.08,P＜0.01), and elevated obviously in shortwavelength light group. The expressions of PRDX6 of retina were significantly different in rd12 mice (F=7.214,P＜0.05), and were decreased obviously in short-wavelength light group. The caspase-3 relative activity was significantly different in rd12 retina (F=7.530,P＜0.05); but there was no significant difference in C57BL/6J mice (F=3.625, 1.993, 1.133; P＞0.05).The caspase-3 relative activity were significant different between rd12 mice and C57BL/6J mice in short wavelength light group (t=5.474,P＜0.05). Conclusions Short-wavelength light can induce retinal damage of mouse retina, especially in rd12 mouse. The retinal light damage possibly relates to the oxidative damage.
Objective To investigate the role of bone marrow-derived cells (BMC) plays in choroidal neovascularization (CNV).Methods Green fluorescent protein (GFP) chimeric mice were built by transplanting BMC from GFP transgenic mice to adult wild type C57BL/6J mice. Retinal laser photocoagulation was used to induce CNV in the chimeric mice (treated group) and adult wild type mice (control group). Four weeks later, choroidal flatmount was prepared to detect GFP positive BMC expression in the CNV lesions, and immunofluorescence stain was used to determine the expression of vascular endothelia growth factor (VEGF) and basic fibroblast cell growth factor (bFGF).Results Twenty-nine days after photocoagulation lots of GFPpositive BMC presented in the CNV area, which accounted approximate 16.22% of the total CNV area. Some BMC in the CNV area expressed VEGF and bFGF. Conclusions BMC may play an important role in CNV by forming new vessles and secreting angiogenic factors.
Objective To investigate the expression of hypoxia inducible factor 1(HIF1alpha;) in ratsprime; retinae during the embryonic and earlier postnatal period. Methods The retinal expression patterns of HIF-1alpha; protein and mRNA of embryonic day 12 (E12), E16, E20, and postnatal day 1(P1) and P5 rats were determined by immunohistochemical staining and reverse transcriptionpolymerase chain reaction (RT-PCR). Results HIF-1alpha; protein was detected in the neural epithelial layer and the pigment epithelial layer at all those 5 timepoints, with higher expression in the ganglion cell layer and the inner plexiform layer, and seems limited to the ganglion cell layer when re tina became more mature. Embryonic rat retina had higher expression of HIF-1alpha; protein and mRNA than postnatal retina, the difference was significant (P＜0.01）. Conclusion The expression of HIF1alpha; in ratsprime;retina e differs from embryonic to earlier postnatal stages.
ObjectiveTo explore the effect and mechanism of netrin-1 on blood-retinal barrier permeability in diabetes mellitus (DM) rats. MethodsEighty Sprague-Dawley rats were randomly divided into the normal control group, DM+balanced salt solution (BSS) group, DM+netrin-1 low dose group and DM+netrin-1 high dose group, with 20 rats in each group. DM rats were induced by intraperitoneal injection of streptozocin (STZ). These rats were feed with high sugar and fat for 3 months after STZ injection. All rats were sacrificed at 1 month after intravitreal injection. Retinal vascular permeability was measured by Evans blue. The expression level of occludin was determined by immunohistochemistry. Hematoxylin-eosin (HE) staining of retina was used to observe the pathological change of DM and the level of occludin mRNA was analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR). Five rats of each group. ResultsHE staining of retina showed that the degree of edema and vascularization in DM+netrin-1 high dose group was better than DM+BSS group. Staining of occludin in retina was limited to nerve fiber layer, ganglion cells, inner plexiform layer and inner nuclear layer in normal rats, but in DM+BSS group, the color of staining positive of occludin was lighter and more reduced. However, DM+ netrin-1 group occludin staining was deepen and enlarged. The result of RT-PCR showed that the expression of occludin mRNA in other three groups was less than normal control group (P < 0.05). The significant difference during DM+BSS group, low dose group and DM+netrin-1 high dose group (F=177.13, P=0.00), and the more concentrate of netrin-1 the higher expression of occluding. Compared the DM+netrin-1 low dose group with DM+BSS group, there was significant difference expression of occludin (t=-13.98, P=0.00). There was significant difference between the DM+netrin-1 high dose group and normal control group (t=12.87, P=0.00). There was statistically significant difference in DM+BSS group, DM+netrin-1 low dose group and DM+netrin-1 high dose group (F=179.69, P=0.00). Compared the two group of different concentration netrin-1, the quantification of vascular permeability in DM+netrin-1 high dose group reduced more (t=12.73, P=0.00). ConclusionsNetrin-1 can protect the blood-retinal barrier in DM rats. Netrin-1 may decrease BRB leakage in DM rats by protecting the expression of occludin.
Objective To evaluate the inhibited effects of small interfering RNA targeting Rac1 (Rac1-siRNA) on rat retinal neovascularization in retinae. Methods Retinal vein occlusion was induced by retinal photodynamic medthod in 25 Sprague-Dawley rats. Rac1-siRNA vector DNA was injected into the vitrous of one eye of those rats (gene intervention group), and empty vector DNA was injected into the fellow eye (blank control group). Rac1-siRNA vector was injected in other 25 SD rats without retinal vein occlusion (blank intervention group). Two weeks after injection, fluorescein isothiocyanate (FITC)-dextran was perfused into the hearts of all the rats, and the retinal wholemount was made to observe the neovascularization. The numbers of endothelial cells which break through the internal limiting membrane were counted after hematoxylin-eosin staining. Results A massive of neovascularization and FITC leakage were found in blank control group. Small part of neovascularization and a little FITC leakage were observed in the gene intervention group. Retinal vessels were normal in blank intervention group. Compared with blank contrast group and blank intervention group, the difference of the mean numbers of endothelial cells which broke through the internal limiting membrane in the gene intervention group was significant（t=? P=0.000??lt;0.05）. Conclusion Rac1-siRNA can inhibit retinal neovascularization induced by retinal vein occlusion in rats.
Objective To observe the therapeutic effects of ganciclovir (GCV) with different injection methods on experimental acute retinal necrosis (ARN). Methods The right eyes of 41 pigmented rabbits were infected by herpes simplex virus (HSV-1) (COS strain) to establish ARN animal model. After 24 and 72 hours, GCV was given by intravitreal injection (10 eyes), intravenous injection (11 eyes) and the intravitreal+intravenous injection (10 eyes); intravitreal injection of GCV and dexamethasone (6 eyes) was also included. Four eyes were not treated as the control. The dosage of GCV in intravitreal and intravenous injection was 800mu;g and 5mg/kg weight, respectively. Retina necrosis was observed and the grade was recorded 1-21 days after injection according to the grade standard of retinopathy. The maximum grades of retinal necrosis in different groups were compared. Results The grade of retinal necosis was 3.8 in the control group, and 0.2, 0.4, 0.8, and 2.2 in intravitreal injection, intravitreal+intravenous injection, intravitreal injection with GCV and dexamethasone, and intravenous injection, respectively, 24 hours after the model was set up. The effects of the first 3 groups were obviously better than the last group (P=0.003, 0.011, 0.045); while the difference among the first 3 groups were not significant (P=0.881、0.054、0.107). Seventy-two hours after the model was set up, the grades of retinal necrosis were above 1.4 in 4 groups, and the differences among the 4 groups were not apparent (P=0.214). Conclusions In the animal model of ARN, intravitreal injection with GCV can effectively decrease the grade of retinal necrosis. The difference among intravitreal injection, intravitreal+intravenous injection, intravitreal injection with GCV and dexamethasone, and intravenous injection is not significant.
Objective To investigate the temporal and spatial expression pattern of Caspase3、Bax and Bclxl in NmethylNnitrosourea (MNU) damaged rat retina. Methods Twenty-four 50 dayold female Sprague-Dawley rats (n=24) received single intraperitoneal injection of MNU 40 mg/kg and were examined at 1, 3, 7 and 10 days after MNU treatment (6 rats sacrificed at each timepoint). As control, six rats were injected with saline (5 ml/kg) and sacrificed 3d after injection. Expressions of Caspase-3 and bax and bcl-xl were detected by RTPCR and immunofluorescence assays, photoreceptor cell apoptosis was measured by terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphatedigoxigenin nick-end labeling (TUNEL). Results Animal models were successful established and confirmed by pathological studies. RTPCR results indicated that caspase3 and bax upregulated at 1 d (caspase-3 RA =83.23plusmn;8.11，P= 0.009; bax-RA=72.73plusmn;9.46，P=0.004) and peaked at 3 d （caspase-3 RA=140.48plusmn;18.40，P=0.000；bax-RA=102.36plusmn;13.97，P=0.001）compared with control (caspase-3 RA=62.45plusmn;7.65; bax-RA =46.53plusmn;4.41). Bcl-xl expression increased and peaked at 3d (3d RA=79.83plusmn;5.58, P=0.000 vs control 45.98plusmn;3.06). It was noted that the ratios of bax / bclxl expression at 1 d, 3 d and 7 d after MNU injection were enhanced (1 d 1.15plusmn;0.14, P= 0.143; 3 d 1.28plusmn;0.16, P=0.001; 7 d 1.17plusmn;0.08, P= 0.079, vs control 1.01plusmn;0.09), and at 3 d the ratio reached the peak, whereas at10 d bax / bcl-xl ratio (0.73plusmn;0.07, P= 0.001) was decreased compared with the control. Immunofluorescence assays demonstrated that the changes of bax, bclxl and caspases3 protein expressions coincided with their RTPCR results respectively. The Bax positive cells were detected in the outer nuclear layer; while caspase3 and bclxl positive cells emerged in several layers of retina included the pigment epithelium layer, the photoreceptor cell inner segments, the outer nuclear layer, the outer plexiform layer, the inner plexiform layer and the ganglion cell layer. Photoreceptor cell apoptosis was only detected in the outer nuclear layer and peaked at 3 d in MNU treated rats (AI= 76.97plusmn;5.83, P= 0.000 vs control 0.00 plusmn; 0.00). Conclusions These data suggest that bax and bcl-xl and caspases3 may involve in the MNUinduced rat photoreceptor cell apoptosis.
Objective To investigate the effects of recombinant adeno-associated virus type-2 (rAAV2) mediated delivery of pigment epitheliumderived factor (PEDF) on oxygen-induced retinal neovascularization (OIRNV) in mice. Methods A total of 22 C57/BL6 mice at the age of 3 days received intravitreal injections of 1 mu;l rAAV2-PEDF and rAAV2EGFP into the left eyes (experimental group) and the right eyes (control group). All mice were put into the oxygen box right after the injection to induce the OIRNV model.4 mice were sacrificed and PEDF protein in retina was measured by western blot at postnatal days 13 (P13). Twelve mice underwent retinal angiography with high molecular weight fluoresceindextran, and another 6 mice were sacrificed for retinal lectin immunohistochemistry staining at P17. Absolute and relative nonperfusion areas of retinal neovascularization were analyzed by Image-Pro Plus 5.1 software. Results The expression level of PEDF protein was higher in the experimental group than that in the control group.The absolute nonperfusion area was (0.96plusmn;0.22) mm2 in the experimental group and (1.96plusmn;0.34) mm2 in the control group; the difference between the two groups was significant（t=-8.554, P＜0.01）. The relative nonperfusion area was (8.64plusmn;1.52)% in the experimental group and (17.27plusmn;2.98)% in the control group with a significant difference between the two groups (t=-8.97, P＜0.01).The absolute area of retinal neovascularization was (0.37plusmn;0.11) mm2 in the experimental group which was obviously higher than (1.26plusmn;0.38) mm2 in the control group (t=-7.8, P＜0.01); the relative areas in experimental and control groups was (3.96plusmn;0.66)% and (11.45plusmn;2.06)%, respectively, whose difference is apparently(t=-8.51, P＜0.01).The areas of retina neovascularization were (0.11plusmn;0.003) mm2 and (0.41plusmn;0.02) mm2 in the experimental and control groups, respectively, and the difference between the two groups was significant（t=-5.14, P＜0.01）.Conclusions PEDF protein can stably express in the mice retina after rAAV2-PEDF transfetion. rAAV2-PEDF can decrease the retinal non-perfusion areas and inhibit the retinal neovascularization in OIRNV mice.
Objective To investigate if lactic acid can promote the expression of vascular endothelial growth factor (VEGF) in the rat retinal explants.Methods The retinas of two-week neonatal SD rats were placed onto the culture plate inserts and incubated with Dulbeccoprime;s modified Eagleprime;s medium （DMEM） plus 2% fetal bovine serum (FBS) containing 10,20,30 mmol/L of lactic acid, respectively. Each group had 24 retinas. At 24 hours after incubation, the retinas were sectioned for light microscopy and the expression of VEGF was measured by real time PCR and Western blot. Results The cultured retinas maintained intact construction, and no cytolysis and apoptosis were observed under light microscope. RT-PCR showed the levels of VEGF mRNA were 0.74plusmn;0.06 for 10 mmol/L lactic acid group, 0.99plusmn;0.12 for 20 mmol/L group, and 1.45plusmn;0.17 for 30 mmol/L group respectively. VEGF expression was 0.34plusmn;0.15 for 10 mmol/L, 0.54plusmn;0.16 for 20 mmol/L, and 0.93plusmn;0.23 for 30 mmol/L group respectively by Western blot. Both PCR and Western blot showed 30 mmol/L of lactic acid significantly increased the levels of VEGF mRNA and VEGF expression. Conclusion The induction of retinal VEGF by lactic acid is concentration-dependent.
Objective To observe the expression of cyclin dependent kinase 5 (Cdk5) and p25 in the pathogenesis of retinitis pigmentosa (RP) in Royal College of Surgeon (RCS) rats and its relationships with apoptosis. To explore the mechanism of Cdk5 and p25 induced photoreceptor apoptosis in the pathogenesis of RP. Methods Retinas of RCS and RCS-rdy+ rats were obtained at the ages of postnatal day 17, 25, 35, 60. The retinal structure and thickness of outer nuclear layer were measured by optical microscopy. The expression of Cdk5, p25, cleave-caspase 3 in the retina was evaluated by immunohistochemistry. The protein expression of cleave-caspase 3 in the retina was determined by Western blot. The apoptosis of retinal cells was examined by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL). The mean absorbance value of apoptotic cells was analyzed by SPSS 17.0 software. Results The retinal thickness of the RCS rats was significantly reduced in comparison to the RCS-rdy+ rats as the postnatal days progressed, particularly in the layer of rods and cones and the outer nuclear layer. The expression level of Cdk5, p25, cleave-caspase 3 of RCS rats increased from postnatal 17 days to postnatal 35 days, while decreased on postnatal 60 days; but there was no obvious change of above indexes in RCS-rdy+ rats. The protein expression of cleave-caspase 3 in the RCS rats was significantly increased with progression of postnatal days to postnatal 35; but there was no obvious similar change in RCS-rdy+ rats. The results of TUNEL showed that the apoptotic cells significantly increased in the outer nuclear layer of RCS rats from postnatal 17 days to postnatal 35 days, while decreased on postnatal 60 days; but there was no obvious change of above index in RCS-rdy+ rats. This study showed that there were significant correlations between the following variables: Cdk5 expression and p25 expression, Cdk5 expression and cleave-caspase 3 expression, Cdk5 expression and apoptotic cells, p25 expression and cleave-caspase 3 expression, p25 expression and apoptotic cells, cleave-caspase 3 expression and apoptotic cells. The partial correlation coefficients were 0.949, 0.808, 0.959, 0.887, 0.979, 0.852, respectively and the P value was 0.000. Conclusions The apoptotic cells significantly increases and the expression level of Cdk5, p25, cleave-caspase 3 of RCS rats increases from postnatal 17 days to postnatal 35 days. The tendency of apoptotic cells to increase is consistent with the change of Cdk5, p25, cleave-caspase 3 expression. The apoptosis of photoreceptor cells is related to increasing expression of Cdk5 and p25 in RCS rats. Cdk5 may be involved in the development of RP in RCS rats.