ObjectiveTo investigate the inhibitory effect of lentivirus-mediated polypyrimidine bundle binding protein-associated splicing factor (PSF) on retinal neovascularization (RNV) in mice model of oxygen-induced retinopathy (OIR).MethodsOne hundred and twelve 5-day-old C57BL/6J mice were randomly divided into normal control group, simple OIR model group, OIR model + lentivirus empty vector treatment group (Vec group) and OIR model + PSF lentivirus treatment group (PSF group), with 16, 32, 32 and 32 mice, respectively. When the mice were 7 days old, the mice in the normal control group were fed in a routine environment, and the mice in the OIR model group, Vec group and PSF group were established OIR model. The mice in the Vec group and PSF group were given an intravitreal injection of 1 μl of lentiviral vector and PSF lentivirus (titer 1×1011 TU/ml) at the age of 12 days. No injection was performed in the normal control group and simple OIR group. RNV was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area by immunofluorescent staining of the mouse retina. Real-time quantitative PCR was applied to detect the mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1). Western blot analysis was applied to detect the protein expression of Nrf2, HO-1 and PSF. Results Of the normal control group, simple OIR model group, Vec group and PSF group, the number of pre-retinal neovascular cell nuclei were 0.00, 14.36±5.50, 15.67±4.96, 8.13±2.09, the non-perfusion area were 0.00%, (35.71±2.81)%, (36.57±4.53)%, (15.33±4.75)%, respectively. The differences of the number of pre-retinal neovascular cell nuclei and non-perfusion area among 4 groups were significant (F=24.87, 165.70; P＜0.05). Compared with the normal control group, there were more pre-retinal neovascular cell nucleis and larger non-perfusion area in the simple OIR model group and Vec group (P＜0.05). Compared with the simple OIR model group and Vec group, there were lower pre-retinal neovascular cell nucleis and smaller non-perfusion area in the PSF group (P＜0.05). Real-time quantitative PCR and Western blot showed that the mRNA expression of Nrf2, HO-1 (F=53.66, 83.54) and protein expression of Nrf2, HO-1 and PSF (F=58.38, 52.69, 24.79) among 4 groups were significant (P＜0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the simple OIR model group and Vec group decreased significantly than those in the normal control group (P＜0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the PSF group were increased significantly than those in the simple OIR model group and Vec group (P＜0.05). model group and Vec group (P＜0.05).ConclusionIntravitreal injection of lentivirus-mediated PSF inhibits RNV in mice model of OIR possibly through up-regulating the expression of Nrf2 and HO-1.
Objective To observe the effects on rabbit corneas and retinas after single intravitreal injection of voriconazole at different doses. Methods According to the randomization table, 25 healthy rabbits were randomly divided into control group, and voriconazole 50, 100, 200, and 400 μg groups. Therefore, there were 5 rabbits in each group. The eyes of control group received intravitreal injection of 0.1 ml balanced saline solution, and those treatment groups received 0.1 ml voriconazole injection of corresponding dose. Before the injection and 1, 7, and 14 days after the injection, endothelial cell counts and corneal thicknesses were measured; full-field electroretinogram were performed and b-wave amplitudes in maximal combined reaction (Max-R) were recorded. On 14 days after the injection, histologic structures were observed by light microscope and transmission electron microscope. Results There was no significant difference in endothelial cell counts (F=0.320, 0.291, 0.467, 0.649) and corneal thicknesses (F=0.214, 0.284, 0.360, 0.225) with those of control group at any time points (P > 0.05). Before and 1 day after the injection, b-wave amplitudes of each voriconazole group had no significant difference compared with those of control group (F=0.220, 0.106; P > 0.05). On 7 days after the injection, b-wave amplitudes decreased significantly at doses of 200 μg and 400 μg (P < 0.05). On 14 days after the injection, there was no significant difference between the the amplitude of 200 μg group and that of control group (P > 0.05). However, the amplitude of the 400 μg group decreased continuously and there was still significant difference (P < 0.05). Light microscopy did not reveal any corneal abnormality in both control group and voriconazole groups. The retinas were normal except that of the 400 μg group, which hadathinner and degenerated inner nuclear layer and disordered photoreceptor layer. Under transmission electron microscope, there were no ultrastructure damages of corneas in both control group and voriconazole groups, either. The rabbit retinas of the 50 μg and 200 μg group have normal inner nuclear layer and photoreceptor layer, but degrees of changes in both layers were observed in the eyes of 200 μg and 400 μg group. Conclusions There is no obvious effects on rabbit corneas and retinas after single intravitreal injection of voriconazole at he dose less than or equal 100 μg. There are no obvious effects on rabbit corneas at the dose of 200 μg and 400 μg, while there are damages to the retinas in both functions and histological structures.
Objective To investigate the protective effect of Niacin on blood-retina barrier (BRB) in diabetic rats and related mechanism. Methods The male Wistar rats (60) were divided into control (CON) group, diabetes (DM) group and Niacin-treated (NA) group, 20 rats in each group. Rats diabetes models were induced with streptozotocin injection. Niacin (40 mg/kg·d) was administrated orally everyday in Niacin-treated group until sacrificed after 3 months. Pathological outcomes, total cholesterol (TC) and high-density lipoprotein (HDL) were evaluated at month 3. Optical microscopy was used to observe the retinal structure. The integrity of BRB and the vascular permeability was quantified by analyzing albumin leakage using Evans blue (EB) method. The relative expressions of Claudin-5, Occludin, zonula occluden (ZO)-1 and GPR109A mRNA in rat retinas were detected by reverse transcription PCR (RT-PCR) and relative expression of GPR109A, tumor necrosis factor (TNF)-α and interleukin (IL)-6 by Western blot. Results Compared to CON group, the TC content was increased and HDL content was decreased in DM group (t=4.034, 5.831; P < 0.05). Compared to DM group, the TC content was decreased and HDL content was increased in NA group (t=6.868, 3.369; P < 0.05). The retinal structure of CON group was normal. Pathological changes were found in the DM group, such as tumescent nuclei and disorganized structures. The retinal structure of NA group was similar to the control group. Evans blue dye that the microvascular leakage in DM group was increased compared with CON group (t=24.712, P < 0.05), while in NA group was decreased compared with DM group (t=16.414, P < 0.05). The mRNA expression of Occludin, Claudin-5, ZO-1 in DM group were decreased compared with CON group (t=11.422, 12.638, 12.060; P < 0.05), while in NA group were increased compared with DM group (t=5.278, 3.952, 8.030; P < 0.05). The mRNA expression of GPR109A in NA group were increased compared with DM group (t=5.053, P < 0.05). The protein expression of GPR109A, IL-6, TNF-αin DM group were increased compared with CON group (t=4.915, 11.106, 6.582; P < 0.05). Compared to DM group, the protein expression of GPR109A was increased (t=5.806, P < 0.05), while the protein expression of IL-6 and TNF-α were decreased (t=10.131, 5.017; P < 0.05). Conclusion Niacin has the protective effect for BRB by up-regulating GPR109A expression which may suppress inflammation.
ObjectiveTo investigate the effect of triamcinolone acetonide (TA) with different dosage and excipient on retina.MethodsThirty-two purebred New Zealand white rabbits randomly divided into 4 groups underwent intravitreous injection with TA. Group 1:4 mg TA without excipient; group 2:25 mg TA without excipient; group 3:4 mg TA with excipient; group 4:25 mg TA with excipient. Electroretinography (ERG) was performed on each rabbit before intravitreal injection, 1 week, 1 and 2 months after the injection. All the animals were killed and the eyeballs were extirpated 2 months after the injection, and pathological examinations including light and electron microscopy were performed.ResultsNo significant difference was found in the latent period of ERG at the points of time before and after the injection in all the groups, but the amplitudes of ERG waves was lower in groups containing excipient than that before the treatment (Plt; 0.01). The results of light and electron microscopy showed damages of tissue or structures of retina in various degrees in groups containing excipient.ConclusionIntravitreous injection of TA with the dosage of ≤25 mg without excipient does no harm to the retinal configuration and function, and excipient may lead to the change of retinal configuration and function.(Chin J Ocul Fundus Dis, 2005,21:229-232)
Objective To observe the effects of high concentr at ion glucose on the calcium-activated potassium channel of rabbits′ retinal Müller cells. Methods The rabbits′retinal Müller cells were cultured in vitro under the condition of high concentration glucose, and identified by immunohistochemical staining and transmission electron microscopy. Patch-clamp technique was used to observe the changes of the calcium-activated potassium channel of retinal Müller cells caused by high concentration glucose at different time.Results High concentration glucose could inhibit the calcium-activated potassium channel of cultured retinal Müller cells in a time-dependent manner. Conclusion High concentration glucose may reduce the biological functions of Müller cells by inhibiting calcium-activated potassium channel. (Chin J Ocul Fundus Dis，2003，19：164-167)
Objective To inverstingate the effect of perfluorohexyloctane(F6H8)to the retina of rabbit eyes. Methods Fifteen vitrectomized New Zealand white rabbits were injectedF6H8(experiment group,12 rabbits ) and BSS(control group,3 rabbits) into vitreous cavity.Slit-lamp biomicroscopy and indirect ophthalmoscopy were performed pre- and postoperatively in all the eyes.Histopathological examination was done after the rabbits were sacrificed at the end of the study. Results A large clear balb was formed after intravitreal injection of theF6H8 in the vitreous was injected and no retinal detachment and cataract were found.The OPL was edematous and then thinned out in 4th week in experimental group.Degenerating cells was found in inner and outer nuclear layers.Cellular vaculoar degeneration was present in TEM. ConclusionF6H8 in vitreous cavity may cause significant side effects on retina,we could not recommend it to be used as an intraocular temponade.
OBJECTIVE:To verify the safe dose of cephradine in intravitreal injection. METHODS:After injecting different doses of cephradine(100mu;g,200mu;g,250mu;g,300mu;g,400mu;g)into vitreous cavity of different group of rabbits the activities of the retinal enzymes (SDH,LDH )on different time (Id,3d, 7d ) were determined respectively, and the histological and ultrastructural changes of retinas were also observed simuhaneously. RESULTS:The activity of rellnal SDH and LDH was found to be decreased gradually with tbe icreasing of the dosage of intravitreal cephradine. The activities of SDH and LDH were found in the lowest level on tile 3rd and lsl day,but they recover to normal levels on tile 7th day after intravitreal in}eetion in 100mu;g,200mu;g groups,and still lower tban normal in the other groups. Histologically,retinal edema was found both in 100mu;g and 200mu;g groups,but degradation of retinal cells,and loss of cones and rods were round in the 250mu;g, 300mu;g and 400mu;g groups. CONCLUSION: The safe dose of intravitreal injection of cepbradlnc is 200mu;g. （Chin J Ocul Fundus Dis,1997,13:139-142 ）
Objective To investigate the effects of QUE on proliferation and DNA synthesis of cultured retinal pigment epithelium(RPE) cells with or without EGF. Methods With or without EGF, cultured RPE cells were treated with QUE by various concentrations(200,100,50,1mu;mol/L) and with QUE 200mu;mol/L at different times(24-168 hr), cells proliferation and DNA synthesis were evaluated by cell count method and the uptake of thymidine. The viability of cells was determined by trypanblue exclusion. Results The best concentration of QUE which inhibits proliferation and DNA synthesis of PRE cells was 200mu;mol/L. The significant inhibition effect of QUE occurred at 48hr, and the best inhibition of QUE occurred at 96hr. QUE had more powerful effect of antiproliferation on RPE cells, and the viability of RPE cells was over85%. Conclusion The results suggested that QUE could inhibit the proliferation of RPE cells in a dose-dependent and time-dependent manner, especially inhibit the proliferation induced by EGF stimulating. QUE had no cyto-toxic effect on RPE cells cultured in vitro. (Chin J Ocul Fundus Dis,1999,15:27-29)
Objective To observe the change of diffusion upper limit of macromol ecules through pathological retina and the difference between the layers of retina. Methods Retinal edema was emulated by establishing branch retinal vein occlusion (RVO) model in miniature pig eyes under photodynamic method. Two days later, the retinas of both eyeballs were peeled off. The diffusion test apparatus was designed by ourselves. FITC-dextrans of various molecular weights (4.4, 9.3, 19.6, 38.9, 71.2 and 150 kDa) and Carboxyfluorescein (376 Da) were dissolved in RPMI1640 solutions and diffused through inner or outer surface of retina. The rate of transretinal diffusion was determined with a spectrophotometer. Theoretical maximum size of molecule (MSM) was calculated by extrapolating the trend-linear relationship with the diffusion rate. In separate experiments to determine the sites of barrier to diffusion, FITC-dextrans were applied to either the inner or outer retinal surface, processed as frozen sections, and viewed with a fluores cence microscope. Results FITC-dextrans applying to inner retinal surface, 4.4 kDa dextrans were largely blocked by inner nuclear layer (INL); 19.6,71.2 kDa dextrans were blocked by the nerve fiber layer (NFL) and inner plexiform layer; 15.0 kDa dextrans were blocked by NFL. FITC-dextrans applying to outer retinal surface, most dextrans with various molecular weights were blocked before outer nuclear layer (ONL). No matter applying to the inner or outer surface, Carboxyfluore scein can diffuse through the whole retina and aggregate at INL and ONL. After RVO, the inner part of retina became edema and cystoid, loosing the barrier function. Compared with the normal retina, the MSM in RVO tissues increased (6.5plusmn;0 39nm Vs 6.18plusmn;0.54nm, t=4.143, P=0.0001). Conclusions A fter RVO, the barrier function of inner part of retinal is destroyed and the upper limit of diffusion macromolecule size increased, which is nevertheless limited. ONL acts as bottle-neck barriers to diffusion, if the outer part of retina is damaged, the change of the diffusion upper limit will be prominent. （Chin J Ocul Fundus Dis，2008，24：197-201）
ObjectiveTo observe the expression in vitro and the influence of adenovirus-mediated recombinant Tum5 gene to the proliferation, migration and tubing of Rhesus RF/6A cell under high glucose. MethodsTo construct the adenovirus vector of recombinant Tum5 gene (rAd-Tum5), and then infected RF/6A cell with it. The Flow Cytometry was used to detect the infection efficiency. RF/6A cells were divided into normal group, high glucose (HG)-control group (HG group), empty expression vector group (HG+rAd-GFP), and HG+rAd-Tum5 group. Western blot was used to detect the expression of Tum5. The CCK-8 test was applied to detect the proliferation of RF/6A cell, the Transwell test was applied to detect the migration and the Matrigel test was applied to detect the tubing of RF/6A cell under high glucose. The proliferation, migration and tubing of RF/6A were tested respectively by CCK-8 test, Transwell test and Matrigel test. ResultsThe adenovirus vector of recombinant Tum5 gene was successfully constructed. The infection efficiency of rAd-Tum5 in RF/6A cell was 50.31% and rAd-GFP was 55.13% by the Flow Cytometry. The results of Western blot indicated that Tum5 was successfully expressed in RF/6A cell. The result of CCK-8 test, Transwell test and Matrigel test indicated that there were statistical differences between all groups in proliferation, migration and tubing of the RF/6A cell (F=44.484, 772.666, 137.696;P < 0.05). The comparison of each group indicated that the HG group was higher than normal group (P < 0.05). There were no statistical differences between HG group and HG+rAd-GFP group (P > 0.05). However, the HG+rAd-Tum5 group was less than HG group (P < 0.05), and the same to HG+rAd-GFP (P < 0.05). ConclusionThe adenovirus vector of recombinant Tum5 gene can inhibit the proliferation, migration and tubing of RF/6A cell under high glucose.