Objective To determine the expression of the growth factors and the receptors related to angiogenesis in the intraocular tissues incarcerating in the sclerotomy sites. Methods Ten specimens from prolapsing intraocular tissues in sclerotomy sites during vitrectomy were obtained and serially sectioned in cryostate and were stained with a group of polyclonal antibodies against vascular endothelial growth factor(VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor-A(PDGF-A) and transforming growth factor-β1(TGF-β1) as well as their receptors by using a streptavidin peroxidase system. Results The tissues prolapsed from the sclerotomy sites were identified as retina(3 cases), vitreous tissues(3 cases), degenerated red blood cell components(2 cases), ciliary body(one case) and fibrous tissue(one case). All specimens expressed VEGF and bFGF as well as their receptors. PDGF-A, TGF-β1 and their receptors expressed in the most of specimens. The positive cells included retinal cells, ciliary non-pigmented epithelial cells and pigmented epithelial cells, fibrous cells and the cells in vitreous. Conclusions The intraocular tissues incarcerated in the sclerotomy entries express the growth factors and receptors related to angiogenesis. This might be one of the potential factors of developing anterior proliferative vitreoretinopathy. (Chin J Ocul Fundus Dis, 2002, 18: 34-37)
Purpose
To evaluate the efficacy of vitreous surgery and endolaser in a series of patients with retinal vein occlusion(RVO)with vitreous hemorrhage,neovascular membranes(NVM) and/or traction retinal detachment(TRD).
Methods
Clinical records were reviewed on 37 consecutive patients(38 eyes)who underwent vitreous surgery and endolaser for RVO with persistent vitreous hemorrhage,NVM and/or TRD.There were 19 patients(20 eyes)with retinal branch vein occlusion (BRVO)and 18 patients(18 eyes)with central retinal vein occlusion(CRVO).
Results
NVM and TRD were confirmed during operation in 27 and 23 eyes,respectively.Visual acuity improved postoperatively in 34 eyes(89.5%)including 22 eyes with 0.1 or better vision,and 4 eyes remained unchanged.CRVO group had longer history and less visual improvement after surgery.
Conclusions
Vitreous surgery and endolaser photocoagulation can improve the outcome in the majority of patients with RVO with vitreous hemorrage,NVM and/or TRD.
(Chin J Ocul Fundus Dis,1998,14:3-6)
ObjectiveTo observe the effects of NDRG1 on proliferation, migration and lumen formation of retinal vascular endothelial cells (RF/6A cells) in monkeys under high glucose condition. MethodsRF/6A cells were divided into normal group, mannitol group, high glucose group, small interfering RNA (siRNA) negative control group without target gene (siRNA group), 30 nmol/L siRNA down-regulated NDRG1 genome (siNDRG1 group) and 50 nmol/L siNDRG1 group. Normal group cells were cultured conventionally. The mannitol group was added with 25 mmol/L mannitol, and the high-glucose group was added with 25 mmol/L glucose. In the siRNA group, 25 mmol/L glucose was added, and then blank siRNA was added for induction. The 30 and 50 nmol/L siNDRG1 groups were added with 25 mmol/L glucose and induced with 30 and 50 nmol/L siRNDRG1, respectively. All cells were incubated for 24 h for follow-up experiments. Cell proliferation was observed by 4', 6-diaminidine 2-phenylindole staining. Cell counting kit-8 staining was used to detect cell activity. The expression level of NDRG1 mRNA and protein was detected by Western blot and real-time quantitative polymerase chain reaction. Cell migration was observed by cell scratch assay. Cell lumen formation assay was used to detect lumen formation. The two-tailed Student t test was used to compare the two groups. One-way analysis of variance was used to compare groups. ResultsThere were significant differences in cell proliferation rate (t=36.659, 57.645) mobility rate (t=24.745, 33.638) and lumen formation number (t=41.276, 22.867) between high glucose group and normal group and mannitol group (P＜0.01). Compared with normal group and mannitol group, the relative expression levels of NDRG1gene mRNA and protein in high glucose group were significantly decreased, with statistical significance (t=46.145, 21.541, 36.738, 32.976; P＜0.001). Compared with the siRNA negative group, the relative expression levels of NDRG1gene mRNA and protein in 30 nmol/L siNDRG1 group and 50 nmol/L siNDRG1 group were significantly decreased, and the differences were statistically significant (t=44.275, 40.7577, 57.167, 25.877; P＜0.01). Compared with normal group and siRNA group, cell mobility in 30 nmol/LsiNDRG1 group was increased, and the difference was statistically significant (t=57.562, 49.522; P＜0.01). Compared with normal group and siRNA group, the number of cell lumen formation in 30 nmol/LsiNDRG1 group was significantly increased in the same field of vision, and the difference was statistically significant (t=63.446, 42.742; P＜0.01). ConclusionDown-regulation of NDRG1 gene can improve the activity, migration and lumen formation of RF/6A cells under hyperglycemia.
Objective
To investigate auto-cortex of crystalline lens-induced neovascular epiretinal membrane（NVERM）by micro-injuring posterior c apsule of crystalline lens.
Methods
twenty four C57BL/6 mouse between 4-6 weeks were selected, and divided into two groups randomly: auto-cortex of crystalline group and the control group. The auto-cortex of crystalline group was treated by penetrating the posterior capsule of lens and washing out the lens cortex into the mouse vitreous using PBS (phosphate buffered solution), while the control group were injected PBS into vitreous merely. Clinical change s were followed by slit-lamp examination and photograph. The eye balls were enu cleated at the day of 3, 7, 14 and 28 after operation. Both HE and immunohistoch emistry were used to detect the pathological changes.
Results
postoperative one to three days, 11 of 12 mouse in autocortex of crystalline g roup, lens appear to alba turbid at different levels one after another, and then develop into highdensity chinaware white. Postoperative (po) three days, HE s taining shows cortex of lens debris transmigrated in vitreous cavity, and some o f which approached to internal limiting membrane and lead it to rough and discon tinue; Po7-14 days, the capillary in retina expanded, migrated and broke though t internal limiting membrane which got to the pro retina and became the new ves sels. And typical NVERM were observed. Po28 days, some vascularslike structure formed in vitreous cavity. None of mouse in control group developed NVERM.
Conclusion
Auto-cortex of crystalline lens can induced neovascular epiretinal membrane in C57BL/6 mouse.
(Chin J Ocul Fundus Dis,2008,24:118-121)
Purpose
To investigate the roles of vascular endothelial growth factor (VEGF),glutamate and gamma;-
aminobutyric acid (GABA) in neovascularization of proliferative diabetic retinopathy (PDR).
Methods
Vitreous samples were collected from 25 patients (27 eyes)with PDR and 14 patients (14 eyes)
with idiopathic macular hole.VEGF levels were determined by ELISA.Amino acids analyses were performed by high-performance liquid chromatography.
Results
Patients with PDR had significantly higher concentrations of VEGF (median 0.41 ng/ml,quartile 0.54 ng/ml) than controls (median 0.017 ng/ml,quartile 0.01 ng/ml)(Plt;0.001).The levels of glutamate [(11.7plusmn;3.0) mu;mol/L] and GABA [(7.2plusmn;3.9mu;mol/L)] were also higher in patients with PDR than glutamate [(5.8plusmn;0.7) mu;mol/L] and GABA [(3.3plusmn;2.9) mu;mol/L) in controls (Plt;0.05).The glutamate level and GABA level had a significantly positive correlation with VEGF level.
Conclusions
The increased levels of glutamate and GABA indicate retinal ischemia.The correlations of glutamate and GABA levels with an elevated VEGF level provide biochemical support for ischemi induced neovascularization in patients with PDR.The findings present novel modalities in treatment of PDR.
(Chin J Ocul Fundus Dis,2000,16:162-165)
Objective To compare two kinds of myofascial flap encapsulating adi pose-derived stromal cells (ADSCs) in adi pogenic efficacy in vivo, and to provide experimental basis for the efficient transplantation of free adi pose tissue. Methods ADSCs were isolated from the subcutaneous adipose tissue in the neck of 10 New Zealand rabbits (aged 3-4 months old, male and female, weighing 2.0-2.5 kg), and primary culture and subculture of ADSCs were conducted. When the cells at passage 3 covered 70%-80% of the bottom of the culture flask, BrdU (10 μg/mL) was appl ied to label the cells for 48 hours before performing immunofluorescence staining. Oil red O staining observation was conducted to thosecells 2 weeks after being induced towards adi pocyte, al izarin red staining observation was performed 3 weeks after being induced towards osteoblast, and alcian blue staining was conducted 2 weeks after being induced towards chondrocyte. Besides, after being induced towards adipocyte for 2 weeks, 1 × 107 ADSCs/piece at passage 3 labeled by BrdU was seeded into Col I (10 mm × 10 mm × 5 mm/piece) to prepare cell carrier complex. The experiment was divided into two groups: group A in which vascular pedicled dextral latissimus dorsi fascial flap was adopted to encapsulate the complex; group B in which dextral gluteus maximus fascial flap with no specific vessel pedicle was appl ied to encapsulate the complex. Rabbits in each group went through autogenous ADSCs transplant and self control. The implants were dislodged 8 weeks after operation, HE staining and immunohistochemistry staining were performed to testify cambium, the wet weight and micro vessel count of the cambium in each group were tested, immunofluorescence staining was performed to determine the origin of cambium and microvascular endothel ium. Results The nucleus of ADSCs positive for BrdU label ing showed green fluorescence under fluorescence microscope, with the positive label ing ratio of ADSCs above 90%. For ADSCs at passage 3, the formation of red l ipid droplets within cells was observed 2 weeks after being induced towards adipocyte, red calcium nodules were evident 3 weeks after being induced towards osteoblast, and highly congregated cell mass positive for alcian blue staining appeared 2 weeks after being induced towards chondrocyte. Eight weeks after operation, neogenetic blood vessel grew into scaffolds and no obvious fibreencapsulation was observed in group A, while few blood vessel grew into scaffolds in group B. The wet weight of cambium in group A and B was (0.149 5 ± 0.017 3) g and (0.095 3 ± 0.012 7) g, respectively, indicating there was a significant difference between two groups (P lt; 0.01). HE staining showed the formation of neogenetic adipose tissue and the growth of micrangium in the implant, and the degradation and absorption of scaffold. The micro vessel count of group A and B was 31.2 ± 4.5 and 19.3 ± 2.6, respectively, indicating there was a significant difference between two groups (P lt; 0.01). Eight weeks after operation, the immunofluorescence staining of cambium showed that the cell nucleus of regenerated adi pocytes and partial capillary endothel ium in groups A and B presented green fluorescence. Conclusion ADSCs encapsulated by vascular pedicled latissimus dorsi fascial flap and collagen protein scaffold complex has a higher adi pogenic efficacy in vivo than the gluteus maximus fascial flap with no specific vessel pedicle.
Objective To investigate the effect of Nodal protein on retinal neovascularization under hypoxia. MethodsIn vivo animal experiment: 48 healthy C57BL/6J mice were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+dimethyl sulfoxide (DMSO) group and OIR+SB431542 group, with 12 mice in each group. Retinal neovascularization was observed in mice at 17 days of age by retina flat mount. Counts exceeded the number of vascular endothelial nuclei in the retinal inner boundary membrane (ILM) by hematoxylin eosin staining. In vivo cell experiment: human retinal microvascular endothelial cells (hRMEC) were divided into normal group, hypoxia group, hypoxia+DMSO group and hypoxia +SB431542 group. The cell proliferation was detected by thiazolyl blue colorimetry (MTT). The effect of SB431542 on hRMEC lumen formation was detected by Matrigel three-dimensional in vitro molding method. Cell migration in hRMEC was detected by cell scratch assay. The Seahorse XFe96 Cell Energy Metabolism analyzer measured extracellular acidification rate (ECAR) of intracellular glycolysis, glycolysis reserve, and glycolysis capacity. One-way analysis of variance was used to compare groups. ResultsIn vivo animal experiment: compared with normal group, the neovascularization increased in OIR group (t=41.621, P＜0.001). Compared with OIR group, the number of vascular endothelial nuclei breaking through ILM in OIR+SB431542 group was significantly reduced, and the difference was statistically significant (F=36.183, P＜0.001). MTT test results showed that compared with normal group and hypoxia+SB431542 group, the cell proliferation of hypoxia group and hypoxia+DMSO group was significantly increased, and the difference was statistically significant (F=39.316, P＜0.01). The cell proliferation of hypoxia+SB431542 group was significantly lower than that of hypoxia+DMSO group, and the difference was statistically significant (t=26.182, P＜0.001). The number of intact lumen formation and migration cells in normal group, hypoxia group, hypoxia+DMSO group and hypoxia+SB431542 group were statistically significant (F=34.513, 41.862; P＜0.001, ＜0.01). Compared with the hypoxia+DMSO group, the number of intact lumen formation and migrating cells in the hypoxia+SB431542 group decreased significantly, and the differences were statistically significant (t=44.723, 31.178; P＜0.001, ＜0.01). The results of cell energy metabolism showed that compared with the hypoxia +DMSO group, the ECAR of intracellular glycolysis and glycolysis reserve in the hypoxia +SB431542 group was decreased, and the ECAR of glycolysis capacity was increased, with statistical significance (t=26.175, 33.623, 37.276; P＜0.05). ConclusionSB431542 can inhibit the proliferation, migration and the ability to form lumens, reduce the level of glycolysis of hRMECs cells induced by hypoxia.
Objective To observe the therapeutic effect of ultrasonic microbubble combined with bevacizumab (Avastin) on choroidal neovascularization induced by photocoagulation in rabbits.Methods CNV was induced by photocoagulation with argon laser in 30 rabbits (60 eyes).All of the rabbits underwent fundus fluorecein angiography (FFA) 21 days after photocoagulation; 6-8 hours later, 3 rabbits were randomly chosen to be executed to having the immunohistochemical examination.Twenty one days after photocoagulation, 27 rabbits were divided randomly into 3 groups: bevacizumb, ultrasonic microbubble + bevacizumb,and control group; each group has 9 rabbits (18 eyes).The rabbits in control group had no interference treatment; while the rats in bevacizumb and ultrasonic microbubble + bevacizumb group underwent injection with bevacizumb or ultrasonic microbubble + bevacizumb respectively.FFA was performed on all of the rabbits 7,14,and 28 days after photocoagulation to observe the inhibition of CNV; immunofluorecence and Western blot were used to detect the expression of VEGF in retina and choroid.Twentyeight days is the time point to determine the therapeutic efficacy. The expression of VEGF and the results of FFA were the sdandards of the judgement of therapeutic efficacy.Results Proliferaion of CNV to the retinal inner layer and the obvious leakage of fluoresein in the photocoagulation area indicated that the model of CNV was set up successfully. Twenty eight days after injection,obvious fluorescent leakage was found in the control group, and the average fluorescent leakage in bevacizumab group differed much from the control group（t=16.2952，Plt;0.05）; while the difference between ultrasonic microbubble + bevacizumb group and bevacizumab group was also significant (t=4.7955，Plt;0.05) . At the same time point, the expression of VEGF in bevacizumab group detected by immunofluorecent assay and Western blot differed much from the control group (t=7.0327，9.2596；Plt;0.05),and the difference of VEGF between ultrasonic microbubble + bevacizumb group and bevacizumab group was significant（t=2.9724,17.1937；Plt;0.05）. this experiment show that ultrasound combined bevacizumab intravitreal injection of the therapeutic effect of CNV superior to other groups(Plt;0.01).Conclusion Ultrasound microbubble combined with bevacizumab injection may improve the therapeutic effect on CNV by inhibiting the expression of VEGF.
Objective To observe the effect of Twist gene interference on the migration and pAkt protein expression of Rhesus retinal vascular endothelial cell line. Methods The Rhesus retinal vascular endothelial cells (RF/6A) were divided into Twist interference plasmid group, negative control group, and phosphate buffered solution (PBS) group; plasmid vectors were transfected via liposome gene transfection method. Migrated endothelial cells was detected and counted by Transwell chamber assay. Matrigel was used in endothelialcell tube formation; the inhibitory effect of Twist gene interference on endothelial cell tube formation was observed.The effect of Twist gene interference on the expression of pAkt protein in RF/6Acells was measured by Western blot. Results The number of migrated endothelial cells in Twist interference plasmid group was lower than that in the negative control and PBS group (F=23.786，P=0.000).The number of endothelial cell tubes in Twist interference plasmid group was apparently less than that in the negative control and PBS gorup (F=7.159，P=0.014). The expression of pAkt protein in Twist interference plasmid group decreased markedly.Conclusion Twist gene interference can suppress the migration of retinal endothelial cells via inhibiting the expression of pAkt protein.
The effect and opportunity of argon laser photocoagulation for the retinal neovascularization in branch retinal vein occlusion in 30 patients were investigated with a control group of 34 patients received nonlaser but routine treatment. The results of the therapeutic effect demonstrated that the neovascularization disappeared completely in 23 cases and became smaller in 7 cases after laser photocoagulation. The incidnce of vitreous hemorrhage in laser group was 43.3% before laser treatment and none after treatment in the duration of observation,and 70.6% in control group. The progression of visual acuity after treatment in laser group was much better than in control group(P＜0. 005)at the time of the latest examination. We found the therapeutic effect was relation to the area, location of the neovascularization in retina,as well as whether the new vessels protruding into vitreous or not.
（Chin J Ocul Fundus Dis,1994,10:195-198）
