Objective
To investigate the effect of exogenous Rb gene on the cell cycle of vitreous retinoblastoma (RB) transplantation tumor in nude mouse.
Methods
Based on establishing vitreous RB transplantation tumor in nude mouse,constructing retrovirus vector of Rb gene PBabe-Rb and transfecing it into the RB transplantation model by liposome Dosper,the change of cell cycle of the RB transplantation tumor by flow cytometry(FCM)was analysed．
Results
FCM showed that the cells of G1phase of the treated eyes were obviously more than the control eyes with the value of DNA index(DI)and S phase fraction(SPF) decreased by the Rb gene expression.
Conclusion
The exogenous PBabe-Rb gene can partially suppress the progress of the cell cycle of RB transplantation tumor in vivo.
(Chin J Ocul Fundus Dis，2000，16：1-70)
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）
Objective
To determine the effect of methimazole (MMI) on retinal vascular development in neonatal rats, and to investigate the relationship between the concentration of insulin-like growth factor-I (IGF-I) in serum and the development of normal blood vessels and between the concentration of IGF-I and the formation of abnormal blood vessels.
Methods
There were 75 neonatal SpragueDawley rats in experimental group whose mothers were raised with water with 0.1% MMI at the first day of parturition. Another 50 neonatal rats were in the control group whose mothers were raised with normal water. The rats in the two groups were sub-divided into 4day and 10day subgroup, respectively. The retinal flatmount of the right eyes were stained with adenosine diphosphatase (ADPase); with the paraffin section of the left eyes, the number of nucleolus breaking through retinal inner limiting membrane was counted and the retinal blood vessels were evaluated. Serum IGF-I levels were detected by radioimmunoassay, and the weight of the neonatal rats in each group were observed and recorded.
Results
The incidence of retinal neovascularization in 10 day MMI group was 27%, and 0% in 4-day MMI group and control group. The serum IGF-I level in 4-day and 10-day MMI group (73.07 ng/ml, 175.13 ng/ml) was obviously lower than which in the 4-day and 10-day control group (168.73 ng/ml,306.38 ng/ml) （P=0.00）. Obvious slow growth of the neonatal rats was found in MMI group compared with which in the control group.
Conculsions
MMI may inhibit the normal growth of retinal blood vessels and lead neovascularization, which may relate to the initial decrease of the serum IGF-I level.
(Chin J Ocul Fundus Dis, 2007, 23: 198-201)
Objective
To set up a new animal model of branch retinal vein occlusion (BRVO), which was quite similar to the clinical features and pathogenesis of this disease.
Methods
The animal model was set up by laser (krypton green 90 ～150 mW) irradiating a branch of central retinal vein after intravenous injection of photochemical drug (3% rose bengal) to 5 pigmented rabbits, and the model was confirmed by fundus fluorescein angiography (FFA) and pathological examination.
Results
The model of BRVO was successfully set up, which was confirmed by clinical examination and FFA. Pathological examination showed that the occlusion was caused by intra-venousthrombosis.
Conclusion
An experimental BRVO model, which has the similar pathological processes of occlusion of central retinal vein and intra-venous thrombosis as those in clinic can be set up by using photochemical method. The method is quite simple, and it offers a better animal model for clinical therapeutic research.
(Chin J Ocul Fundus Dis,2002,18:23-25)
Objective
To determine the effect of thiazolidinediones (TZDs) on early retinopathy in rats with experimental diabetes.
Methods
In 40 rats, diabetic models were set up in 36 by one-off intraperitoneal injection with streptozotocin (STZ), and other 4 were in the normal control group. Twenty-four diabetic rats with the disease-duration of more than 6 months underwent intravitreous injection (with rosiglitazone or pioglitazone in 10 rats, respectively), and the rest 4 rats werenprime;t injected with drugs as the diabetic positive control group. Immunohistochemical treptomycin-avidin-biotin-complex (SABC) method, in situ hybridization of retinal vascular endothelial growth factor (VEGF) mRNA, and TdT-dUTP terminal nick-end labelling (TUNEL) were performed on the ocular paraffin section to detect the cellular apoptosis. The difference of VEGF expression and cellular apoptosis between TZDs and control group was observed and analyzed.
Results
The results of immunohistochemical staining and hybridization in situ were negative in the normal control group. The positive expression rate of VEGF was lower in rosiglitazone and pioglitazone group than which in the diabetic positive control group, and there was no obvious differences of positive expression of VEGF mRNA and cellular apoptosis between the 2 groups.
Conclusion
TZDs (rosiglitazone and pioglitazone) may inhibit the positive expression of VEGF protein in retina of STZ-induced diabetic rats to some extent, but not affect the growth of VEGF in retina.
(Chin J Ocul Fundus Dis, 2006, 22: 7-10)
ObjectiveTo observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR).MethodsThe conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Müller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF-1α) was detected by immunofluorescence analysis. ResultsDuring the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x2=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59) , the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer.ConclusionsThe local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability.
Objective To explore the effect of oxygen inhalation on the retinae of newborn rats and its mechanism.Methods We mimicked the retinopathy of prematurity(ROP) by putting the newborn rats in high concentrated oxygen. One-day old rats were put into the oxygen box with the oxygen concentration of 80% for continuous 7 days; then in air condition for 7 days. The arterial blood oxygen pressure, retinal superoxide dismutase (SOD), and malondialdehyde (MDA) of the rats (1,2,4,7,8,9,11,14 days old) were examined. The diameter of retinal vessels′main branch and the coverage rate of peripheral vessels were measured in 7- and 14-day-old rats by ink perfusion. The retinal neovascularization of rats (8,9,11, 14 days old) were observed by HE staining. The rats of the same age fed in air condition were in the control group.Results The differential pressures of blood oxygen of rats (1,2,4,7 days old) in study group were significantly higher than those in the control group (P＜0.01), while the differential pressures of blood oxygen of rats (8,9,11,14 days old) in study group were lower than those in the control group (P＞0.05). The contents of SOD of the retinae in the rats ( 1,2,4,7,8 days old) were significantly lower than those in the control group(P＜0.01, P＜0.05 ), while the contents of MDA were significantly higher than those in the control group (P＜0.01,P＜0.05). The diameter of retinal vessels′main branch in 7-day rats was 75% of the control group, and the coverage rate of peripheral vessels was 22% of the control group; and was 61% and 73% respectively in 14-day-old rats. The neovascularization could be seen in 16.7% of the rats in the study group and nought in the control group.Conclusion The damage of free radical of the retina in high concentrated oxygen and hypoxia situation after oxygen supply may be one of the most important mechanism of ROP. (Chin J Ocul Fundus Dis，2003，19：269-332)
Purpose
To investigate nucleoside diphosphate kinase (NDPK ) expression of tumor metastasis suppressor gene nm23 in heterotransplanted model of retinoblastoma(RB) in nude mice，and analyse the correlation between the expression of nm23 gene and the formation and progression of heterotran splanted RB．
Methods
SP immunohistochemical method was used to detect the expression of nm23 gene product NDPK in 20 tumors of heter otransplanted RB model and normal retinal tissue．
Results
The negative staining of nm23/ NDPK was found in normal retinal tissue , whereas 100% expression rate in RB tumors with positive number of 48.73plusmn;2.37． No statistical significance of the expression of nm23/ NDPK was observed between the intraocular growth phase （I～Ⅲ grade） and invasive phase （ Ⅳ～Ⅴ grade）in heterotransplantedRB tumors．
Conclusion
The function of nm23 gene as a tumor metastasis suppressor in heterotra nsplanted RB tumors was less prominent ,but it may play a role in carcinogen esis and progrssion of RB and may predict poor prognosis．
(Chin J Ocul Fundus Dis, 2001.17:47-49)
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)
Objective
To investigate the effect of hepatocyte growth factor (HGF) on the barrier function of retinal peigment epithelium (RPE) and to detect the pathological mechanism of retinal detachment (RD) induced by over expression of HGF in RPE.
Methods
Sub-retina injection of E1/E3deleted adenoviral vectors encoding HGF (Ad CMV.HGF) and green fluorescent protein (Ad CMV.GFP) in adult pigmented rabbits [5times;104 plaque-forming units (pfu)/eye] to set up the model of retinal detachment. The ocular fundus and pathological changes were observed 3, 7, 14, and 28 days after injection. The expression level of HGF in retina and vitreous body was detected by immunohistochemistry and enzyme linked immunosorbent assay (ELISA).
Results
In the control eyes injected with AdCMV.GFP, expression of GFP only detected in RPE monolayer. The eyes injected with AdCMV.HGF had b HGF immune positive action in RPE cells at the injection site. The expression level of HGF in vitreous body reached the peak 7 days after injection and decreased to the basic level 28 days after injection. Chronic RD and chronic choroidal inflammation were found in the eyes injected with AdCMV.HGF within the time frame of HGF expression. Proliferative RPE cells were found in subretinal space in the region of RD, and multilayered cellular membranes developed in some eyes.
Conclusion
Over expression of HGF in RPE may induce chronic serous RD with subretinal proliferation of RPE, which suggests that HGF should be further studied as a target for therapeutic intervention in RD.
(Chin J Ocul Fundus Dis, 2007, 23: 193-197)
