The therapeutic effects of panretinal cryotherapy(PRC)on proliferative diabetic retinopathy(PDR)were prospectively investigated in 80 eyes with PDR of 44 patients.Forty eyes with PDR(20 of them stage Ⅳ and 20 stage Ⅴ)received PRC operation.Of the 80 eyes,the other 40 ones with stage Ⅳ and Ⅴ similar to the formers were observed conservatively as controls.The follow up duration was 2 years.We found that in the cases of stage Ⅳ,no more remarkable visual loss was found after operation.There was a significant difference comparing with the control（P＜0.002）,and the retinal neovascularization regressed more noticeably than the control group（P＜0.001）.In the cases of stage Ⅴ,the incidence of the traction retinal detachment was 55% in the operated group,and was 20% in the control group.There was a statistic difference between them（P＜0.05）.The clearance of the vitreous hemorrhage was more rapid in the operated group than the control（P＜0.025）.The above results suggest that cryotherapy is suitable for the cases of earlier stage which cannot be performed with photocoagulation for any reasons,but not for the patients with advanced retinal proliferation.Photocoagulation for any reasons,but not for the patients with advanced retinal proliferation. （Chin J Ocul Fundus Dis,1993,9:148-151）
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)
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.
ObjectiveTo analyze the regulative rule of mRNA of vascular endothelial growth factor (VEGF) in mice with oxygen-induced retinopathy, and to elucidate the possible mechanism of occurrence of neovascularization in retinopathy of prematurity (ROP).MethodsSixty 7-day-old C57BL/6J mice were divided into oxygen-induced retinopathy group and control group. In oxygen-induced retinopathy group, 36 mice were exposed to 75% oxygen for 5 days and then to room air for 5 days; in control group, 24 mice were raised in room air. Vascular perfusion of fluorescein and retinal stretched preparation were used to observe the morphologic changes of retinal vessels. Reversal transcriptionpolymerase chain reaction (RT-PCR) was used to observe changes of VEGF mRNA in each group. ResultsIn oxygen-induced retinopathy group, the morphologic characteristics of retinal vessels were the unperfused area at the center of superficial and deepseated vessels, and the neovascularization appeared at mid-peripheral retina after 2 days in relative hypoxia condition. The results of RT-PCR showed space-time corresponding relation between expression of VEGF and neovascularization, which meant that the transcription of VEGF mRNA decreased in hyperxia conditionand increased in relative hypoxia condition. ConclusionHypoxia is the main reason of occurrence of retinal neovascularization; increased expression of VEGF caused by relative hypoxia after hyperxia might be effective in reducing the occurrence of neovascularization in ROP.(Chin J Ocul Fundus Dis, 2005,21:292-295)
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.
Purpose To investigate the status of proliferation and activation of vascular endothelial cells in preretinal neovascular membranes from patients with insulin dependent diabetetes mellitus(IDDM)by means of immunohistochemical techniques. Methods Status of vascular endothelial cells in 18 preretinal neovascular membranes from 18 patients with IDDM was studied by double-immunofluorescence technique and the alkaline phosphataes-anti-alkaline phosphatase(APAAP)technique and compared the findings with the main clinical features of the patients. Results Of 18 vascularized membranes,16(88.9%)contained proliferating endothelial cells (positive for proliferating vascular endothelial cell marker EN 7/44) and 14 (77.8%) were positive for endothelial cell activation marker anti-VCAM-1;furthermore,by using a double staining technique,we found that in 14 of the 16 cases(87.5%) the proliferating vascular endothelial cells were activated (expressing VCAM-1). Conclusion The proliferation and activation of the vascular endothelial cells of the newly formed vessels in preretinal neovascular membranes suggests the significance of the vascular endothelial cells in the pathophysiology and the progress of proliferative diabetic retinopathy. (Chin J Ocul Fundus Dis,1998,14:141-143)
ObjectiveTo investigate the ability of autologous peripheral blood endothelial progenitor cells (EPCs) in promoting neovascularization of tissue engineered bone and osteogenesis of bone marrow mesenchymal stem cells (BMSCs). MethodThe peripheral blood EPCs and BMSCs from No. 1-9 New Zealand rabbits were isolated, cultured, and identified. According to the cell types, the third generation of cells were divided into 3 groups:EPCs (group A), BMSCs (group B), and co-cultured cells of EPCs and BMSCs (group C, EPCs:BMSCs=1:2) . Then cells were seeded on the partially deproteinised bone (PDPB) packaged with fibronectin to construct tissue engineered bone. After 4 days, autologous heterotopic transplantation of tissue engineered bone was performed in the rabbit's muscles bag of groups A, B, and C (the right arm, left arm, right lower limb respectively, 2 pieces each part). At 2, 4, and 8 weeks after transplantation, the growth of tissue engineered bone was observed, and the rate of bone ingrowth was calculated by HE staining; the expressions of CD34, CD105, and zonula occludens protein 1(ZO-1) were compared by immunohistochemical staining at each time point in tissue engineered bone among 3 groups. ResultsThe EPCs and BMSCs were isolated and identified successfully; immunofluorescent staining showed that EPCs were positive for CD34, CD133, and von Willebrand factor (vWF), and BMSCs were positive for CD29 and CD90 and were negative for CD34. The tissue engineered bone constructed in 3 groups was transplanted successfully. At 2, 4, and 8 weeks after autologous heterotopic transplantation, the general observations showed that the soft tissue around the tissue engineered bone increased and thickened gradually in each group with time passing; the boundary between bone and soft tissue was not clear; the pore space of tissue engineered bone gradually was filled, especially in group C, the circuitous vascular network could be seen in the tissue engineered bone. HE staining showed capillaries and collagen fibers increased gradually, tissue engineered bone ingrowth rate was significantly higher in group C than groups A and B at 4 and 8 weeks (P<0.05) , and group B was significantly higher than group A (P<0.05) . Immunohistochemical staining showed that the expressions of CD34, CD105, and ZO-1 in tissue engineered bone of 3 groups all increased with the extension of time, showing significant differences between groups at each time point (P<0.05) . At 2 weeks after transplantation, the expression of CD105 in group C was significantly higher than that in groups A and B (P<0.05) ; at 4 and 8 weeks, CD34, CD105, and ZO-1 expressions showed significant differences between 2 groups (P<0.05) ; the expression was the highest in group C, and was the lowest in group B. ConclusionsAutologous peripheral blood EPCs and BMSCs have synergistic effect, and can promote neovascularization and osteogenesis of tissue engineered bone in vivo.
Purpose To estabalish a quantifying model of retinal neovascularization suitable for the study of pathogenesis and therapeutic intervention for the retinal neovascularization． Methods Sixteen one-week-old C57BL/6 mice were exposed to 75% oxygen for 5 days and then to room air and 16 mice of the same age kept in room air as controls.Ink-perfused retinal flatmount was examined to assess the oxygen-induced changes of retinal vessels.The proliferated neovascular response was quantitated by counting the nuclei of endothelial cells of new vessels extending from the retina into the vitreous in 6 mu;m sagittal cross sections． VEGF and bFGF were determined on the cross-sections after immunohistochemcal stain. Results Constriction and closure of the blood vessels were found under the hyperoxia condition,and dilation and proliferation were found under the relatively hypoxia status.There was a mean of 24 neovascular nuclei per cross-section in the oxygen-treated retina and less than 1 nucleus in the control group (P＜0.001).VEGF stain was found ber in the inner retinal layer of oxygen-treated mouse than in that of the controls. Conclusion The quantifying model of retinal neovascularization may fascilitate the further researches of medical intervention and pathogenesis of retinal neovacularization. (Chin J Ocul Fundus Dis，2000，16：213-284)
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)
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.