ObjectiveTo evaluate the inhibitory effect of small interfering RNA (siRNA) targeting peroxisome-proliferator-activated receptor-γcoactivator-1α(PGC-1α) on retinal neovascularization in the mouse.
MethodsEighty seven-day-old C57BL/6J mice were divided into normal group, model blank group, model control group and PGC-1αsiRNA group, twenty mice in each group. Mice in the normal group were kept in normal room air. Mice in the model blank group, model control group and PGC-1αsiRNA group were induced for retinal neovascularization by hypoxia. Liposome with PGC-1αsiRNA (1 μl) and liposome with negative control siRNA (1 μl) were injected into the vitreous in the PGC-1αsiRNA group and model control group respectively when mice were moved out to room air from the cabin (Postnatal 12). No injection were performed in the model blank group. At postnatal 17, fluorescein angiography was used to assess the vascular pattern.The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections. PGC-1αand vascular endothelial growth factor (VEGF) level in retina were measured by real-time polymerase chain reaction (real-time PCR) and Western blot. Inhibition efficiency of PGC-1αsiRNA on PGC-1αand VEGF was calculated.
ResultsMice in the normal group showed reticular distribution of retinal blood vessels. Central nonperfused retina, neovascular tufts and fluorescein leakage were seen in the model blank group and model control group. Neovascular tuft and fluorescein leakage were decreased in the PGC-1αsiRNA group compared to the model blank group and model control group. The neovascular nuclei were increased in the model blank group and model control group compared to the normal group (P < 0.05). The neovascular nuclei were decreased in the PGC-1αsiRNA group compared to the model blank group and model control group (P < 0.05). The expression of PGC-1αmRNA and protein in retina was increased significantly in the model blank group and model control group as compared with normal group, while decreased 54% and 53% respectively in the PGC-1αsiRNA group as compared with model blank group and model control group (P < 0.05). The expression of VEGF mRNA and protein in retina was increased significantly in the model blank group and model control group as compared with normal group, while decreased significantly in the PGC-1αsiRNA group (decreased 48% and 40% respectively) as compared with model blank group and model control group (P < 0.05).
ConclusionsIntravitreal injection of PGC-1αsiRNA mediated by liposome can inhibit retinal neovascularization in the mouse effectively.
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 observe the regulation effect of transforming growth factor alpha (TGFalpha;) on expression of glutamate transporter（GLAST）and ingestion activity of retinal Muuml;ller cells in mice. Methods To take the retinal tissue of Kunming mouse at postnatal 7～10 day, and then cultured Muuml;ller cells according to literature. The 3～4 generation cultured cells of the same primary cell were divided into two groups at random: ① TGFalpha; group: maintained in different concentrations of TGFalpha; as 50, 75, 125 and 150 ng/ml, 3 holes in each concentration;② Control group: cultured by Eagle culture medium which improved from Dulbeccon and contained 20% fetal calf serum. The influence of different concentrations TGFalpha; on GLAST activity in Muuml;ller cells were observed by L-3H-glutamate uptake detection; the expression of GLAST mRNA in Muuml;ller cells was determined by RT-PCR; the expression of GLAST protein was detected with immunocytochemical staining. Results With the increase of TGFalpha; concentration, both L3H glutamate uptake and GLAST mRNA expression were increased. The L-3H-glutamate accumulation had got to the maximum uptake at concentration of 125 ng/ml, which was 266% of that in control group, meanwhile, the expressions of GLAST mRNA also got to the maximum as 4 times of control group. Immunocytochemical staining indicated that the effect of 125ng/ml TGFalpha; on expression of GLAST protein was higher than that in the control group, the differences between two groups were statistically significant (Plt;0.05). Conclusion TGF-alpha; can increase GLAST activity through up-regulating the expression of GLAST mRNA and protein.
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 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 observe the safety and efficacy of posterior vitreous detachment (PVD) induced by combined intravetreal injection of lysineplasminogen and reteplase in rabbits.Methods Fifteen healthy New Zealand rabbits were divided into three groups with five rabbits in each. Take the right eyes as experimental eyes，while the left eyes as the control. The experimental eyes of three groups received combined intravetreal injection of 1250 mu;g/ml lysineplasminogen at 0.1 ml dose and 104U，3times;104U，105U reteplase at 0.05 ml dose recpectively, while the control eyes were injected intravetreally with 0.15 ml balanced salt solution. The conjunctiva, anterior chamber, lens, vitreous body, and retina were examined by slit lamp microscope and +120D preset lens. The retinal function was examined by electroretinogram (ERG).Results All the experimental eyes had PVD. The results of optical microscope showed that no change in retinal structure was found in the control group and 104 U reteplase group, clear retinal hierarchical but decreased ganglion cells and kernel layer cells were found in 3times;104 U reteplase group, only retinal pigment epithelium layer but no normal retinal structure was observed in 105U reteplase group. The results of ERG showed that compared the maximum mixed reaction of a and b wave amplitude in control group and reteplase group respectively, the difference was not statistically siginificant between 104U reteplase group and control group(a wave：t=0.881，-1.773，0.809；b-wave：t=-0.223，-0.441，1.400；P＞0.05),the differences were statistically siginificant between 3times;104 U(a wave：t=-3.20，b-wave：t=-4.182，-4.103),105 U reteplase group(a wave：t=-0.737，b wave：t=-15.150，6.597)and control group(P＜0.05). The control eyes didnprime;t had PVD.Conclusion Combined intravetreal injection of lysine-plasminogen and reteplase can induce complete PVD, and no damage to the retinal structure in rabbits.
Objective To establish a rat model of blood ocular barrier breakdown induced by anterior segment intraocular analogic surgery. MethodsOne hundred and fifty healthy adult male rats were randomly divided into control group and model group, 75 rats in each group. The rats were anesthetized with 1 ml/kg ketamine hydrochloride/xylazine hydrochloride solution. Three way pipes were attached to a phosphate buffer infusion bag and two intravenous catheters. One catheter was inserted 30° obliquely through the transparent cornea anterior to the limbus into the rat's anterior chamber. Then the needle was withdrawn and the sheath was indwelling. Another catheter was connected with a manometer. Intraocular pressure was varied from 0 to 12 mm Hg (1 mm Hg=0.133 kPa) 60 times, 30 times per min. The catheter was removed. The eyes were treated with ofloxacin ophthalmic solution after surgery. The 1st, 2nd, 3rd, 5th and 7th day after surgery, the integrity of the blood ocular barrier was assessed by immunohistochemical staining for albumin and quantitative measurement using Evan′s blue as a tracer. ResultsAlbumin immunohistochemical staining of the control group was confined to the iris and retinal blood vessels. The choroid was stained at each time point after surgery. Albumin immunohistochemical staining of the model group was abundant around the iris and the retinal vasculature on the 1st day after surgery. The albumin diffused throughout the iris and the retina on the 2nd and the 3rd day after surgery. The albumin reached the retinal vessels on the 5th and 7th day after surgery. The aqueous humor Evans blue leakages of the model group were higher than those of the control group on the 1st, 2nd, 3rd and 5th day after surgery. The differences were statistically significant (t=25.781, 37.433, 25.150, 19.171； P<0.01). The Evans blue leakage of the model group was close to that of the control group on the 7th day after surgery. The difference was no statistical significant（t=1.303, P=0.209）. The retinal Evans blue leakages of the model group were higher than those of the control group on the 1st, the 2nd and the 3rd day after surgery. The differences were statistically significant (t=11.997, 14.622, 23.014; P<0.01). The Evans blue leakage of the model group was close to those of the control group on the 5th and 7th day after surgery. The differences were not statistically significant（t=2.027, 0.756; P=0.058, 0.459）. Conclusion This study establishes a rat model of blood ocular barrier breakdown induced by imitating the injury to the anterior segment during intraocular surgery.
Objective To investigate the expression and prolyl hydroxylase (PHD)2 in retina of diabetic rats．Methods Wistar rats were randomly divided into the control group (n=48) and the diabetes group (n=60). The rats in diabetes group were induced with streptozotocin (STZ) injection creating a diabetic retinopathy model. The same volume of citric acid buffer was injected into the rats in the control group. Fluorescence microscope was used to observe the retinal vasculature at one, three and six months after injection. Evans blue perfusion was used to detect the bloodretinal barrier (BRB) permeability. Immunohistochemical staining was used to observe the distribution of PHD-2 positive staining. Western blot was used to measure the protein expression of PHD-2, hypoxia inducible factor（HIF）-1alpha; and vascular endothelial growth factor (VEGF) every month from one to six months after injection. Results The vascularization was normal and form was clear in retina of rats in control group. The retinal blood vessels of rats in diabetes group showed significantly increased fluorescence. Compared with the control group, the BRB permeability was significantly increased in diabetes group (P＜0.05). Abundant expression of PHD-2 protein was detected in the inner layers of retina in control and diabetes group．Compared with the control group, the PHD-2 expression was decreased in diabetes group at one and two months after injection (t=16.230, 16.390;P＜0.05). The HIF-1alpha; expression was significantly increased in diabetes group at one, two and three months after injection (t=27.073, 36.709, 10.176; P＜0.05). The VEGF expression was significantly increased in diabetes group every month from one to six months after injection (t=13.547, 31.984, 21.897, 8.912, 9.019, 14.046; P＜0.05). Conclusions There is abundant expression of PHD-2 in the inner layer of retina in diabetic rats. PHD-2 may play an important role in diabetic retinopathy, which is correlated with VEGF．
Objective To observe the effect of medicineinduced posterior vitreous detachment (PVD) on proliferative vitreoretinopathy (PVR). Methods PVR was induced in the left eyes of 24 pigmented rabbits by intravitreal injection with platelet rich plasma. The rabbits were randomly divided into two experimental groups (group A and B) and one control group with 8 eyes in each group. Three hours later, the eyes in group A and B and the control group underwent intravireal injection with 1 U plasmin 0.05 ml+20 U hyaluronidase 0.05 ml, plasmin 0.1 ml, and balance salt solution 0.1 ml, respectively. The grade of PVR was recorded 1, 7, and 28 days after the intravitreal injection, and the eyes were examined by flash electroretinogram (FERG), B-scan, and retinal histopathological examination. Results The PVR models of rabbit eyes were induced successfully. On the 7th day after injection, complete and partial PVD was found in 5 and 3 eyes respectively in group A; partial PVD in 5 eyes and no complete PVD was observed in group B; there was no PVD in the other 3 eyes in group B and also in the eyes in the control group. On the 28th day after intravitreal injection, PVR grade of group A and B were both obviously lower than that of the control group（D=75.6, 98.9；P=0.003，P=0.011）; On the 7th and 28th day after injection, the b-wave amplitude in group A and B was significantly higher than that in the control group; PVR grade of the PVD eyes was lower than that of nonPVD eyes; PVR grade of the complete PVD eyes was only 0～1. Conclusions Three hours after the PVR models of rabbit eyes were induced, complete PVD induced by intravitreal injection of plasmin combined with hyaluronidase could prevent the development of PVR of rabbit eyes in some degree; partial PVD induced by plasmin alone or combined with hyaluronidase could relieve the development of PVR.
