Objective To evaluate the inhibited effects of small interfering RNA targeting Rac1 (Rac1-siRNA) on rat retinal neovascularization in retinae. Methods Retinal vein occlusion was induced by retinal photodynamic medthod in 25 Sprague-Dawley rats. Rac1-siRNA vector DNA was injected into the vitrous of one eye of those rats (gene intervention group), and empty vector DNA was injected into the fellow eye (blank control group). Rac1-siRNA vector was injected in other 25 SD rats without retinal vein occlusion (blank intervention group). Two weeks after injection, fluorescein isothiocyanate (FITC)-dextran was perfused into the hearts of all the rats, and the retinal wholemount was made to observe the neovascularization. The numbers of endothelial cells which break through the internal limiting membrane were counted after hematoxylin-eosin staining. Results A massive of neovascularization and FITC leakage were found in blank control group. Small part of neovascularization and a little FITC leakage were observed in the gene intervention group. Retinal vessels were normal in blank intervention group. Compared with blank contrast group and blank intervention group, the difference of the mean numbers of endothelial cells which broke through the internal limiting membrane in the gene intervention group was significant（t=? P=0.000??lt;0.05）. Conclusion Rac1-siRNA can inhibit retinal neovascularization induced by retinal vein occlusion in rats.
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 Then the needle was withdrawn and the sheath was indwelling.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 observe the morphological changes of dendrite and soma in retinal ganglion cells (RGCs) which subsisted in early diabetic rats. Methods The RGCs of 3-months-course diabetic rats and coeval normal rats were marked by gene gun techniques. To collect RGCs photographs by Leica microscope with Z axis and CCD camera；to observe the changes of diameter, variance of structural features in dendritic field and somata after classification which according to the size and morphology. Thy-1 antibody marks on the retinal RGCs, taking a photograph under fluorescent microscope, counting the changes of retinal RGCs density in early diabetic rat. Results In three-month diabetic rats,the density of retinal RGCs was decreased obviously. Morphological changes of RGCs in the dendritic fields were observed with gene gun technique. There was no severe variation in all kinds of the bole of cell dendrite, in which some only showed crispation partially and sparseness also twisting in the dendritic ramus. The mean diameter of dendritic field and soma in class A of diabetic rats was (401plusmn;86) mu;m, the mean diameter of dendritic field in control group was (315plusmn;72) mu;m,compared with each other, there is statistically significant differences (t=21.249，Plt;0.001); the mean diameter of soma in class A of diabetic rats was (24plusmn;6) mu;m, the mean diameter of soma in control group was (22plusmn;5) mu;m, compared with each other, there is no statistically significant differences (t=0.927，Pgt;0.05); the mean diameter of dendritic field and soma in class B of diabetic rats were (170plusmn;36)、(14plusmn;2) mu;m respectively, in control group were (165plusmn;36)、(16plusmn;2) mu;m, the mean diameter of dendritic field and soma in class C of diabetic group were(265plusmn;78)、(17plusmn;5) mu;m respectively, in control group were (251plusmn;57)、(17plusmn;4) mu;m , compared with each other, there are on statistically significant differences(t=1.357，0.798，0.835，1.104，Pgt;0.05). Conclusions In short-term diabetes, the survived RGCs show good plasticity in adult diabetic rats, especially in class A. The changes of dendrites were more sensitive than the soma, which could be the leading index of the morphologic changes of RGCs in the early stage. The good plasticity showed by the RGCs and the time window from changing in dendrite to cell death provide us many evidences not only for the research but also for the nerve protection in clinic. (Chin J Ocul Fundus Dis,2008,24:249-254)
Objective To observe the effect of intravitreal injection of mouse nerve growth factor (NGF) on interphotoreceptor retinoid-binding protein (IRBP) in the vitreous of diabetic rats at early stages. Methods Ninety-six male Sprague Dawley (SD) rats were divided into control group (group A, 24 rats) and experimental group (72 rats). The rats in experimental group were induced with streptozotocin injection for diabetic retinopathy model, and then randomly divided into positive control group (group B), normal saline group (group C) and NGF group (group D), 24 rats in each group. The rats in the group A and B were not intervened. The rats were received intravitreal injection with 4mu;l normal saline (group C) or 4 mu;l (0.5 mu;g/mu;l) NGF (group D). At 2, 4, 6 and 8 weeks after injection, IRBP levels were detected by enzymelinked immunosorbent assay (ELISA); hematoxylin-eosin (HE) staining and light microscope were used to observe the morphological changes of the retina; transmission electron microscope was used to observe the retinal ultrastructure.Results At 2 weeks after injection, there was no significant difference in IRBP expression between group A,B,C and D (F=2.833，P=0.052). At 4, 6, 8 weeks after injection, the differences of IRBP expression between group A, B, C and D were significant (F=22.252, 108.459, 105.726; P=0.000). At different time points after injection, there was no significant difference in IRBP expression of group A (F=1.462, P=0.241), but there were significant differences in IRBP expression of group B, C and D (F=150.98, 63.519, 64.604; P=0.000). Light microscope found that the retinal structure was clear in group A and in group B, C, D at 2, 4 weeks after injection; the retinal thickness were thinner in group B, C, D at 8 weeks after injection. Transmission electron microscope displayed that the structure of rod outer segments was clear in group A and in group B, C, D at 2 weeks after injection; partly unclear structure of rod outer segments and slightly enlarged gap were observed in group B, C, D at 4, 8 weeks after injection. Conclusion Intravitreal injection with NGF can stabilize the IRBP expression in the vitreous of diabetic rats at early stages effectively.
Objective To investigate the effects of knocking down Rac1 gene (ras-related C3 botulinum toxin substrate 1) by small hairpin RNA (shRNA) on retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Methods One hundred and eight 7-day-old C57BL/6J mice were divided into three groups randomly.The OIR was induced by Smith protocol in 2 groups. OIR mice received an intravitreal injection of Rac1-shRNA plasmid or the nonsense plasmid in the geneintervention group and control group respectively at the age of postnatal day 11 (P11). Non-OIR mice also received an intravitreal injection of Rac1-shRNA plasmid at P11 as the blankintervention group which lived in the normoxic environment.Retinal neovascularization was investigated on flat-mounts after fluorescence angiography at P15 and P17. Endothelial cell nuclei breaking through the internal limiting membrane were counted on pathological section at P17.The expression of Rac1 and NF-kappa;B p65 subunit was measured by immuohistochemistry, Western blot, real-time polymerase chain reaction (RT-PCR) and in situ hybridization. Results Compared with the blank-control group,the level of Rac1 mRNA in the gene-intervention group decreased obviously（t=4.500，P=0.001）;the retinal non-perfusion areas,fluorescence leakage, neovascularization and the number of endothelial cell nuclei breaking through the internal limiting membrane were reduced significantly（t=6.521,P＜0.001）; the level of NF-kappa;B p65 nuclear translocation decreased（t=16.008,P＜0.001）while the expression of NF-kappa;B p65 mRNA was reduced obviously（t=3.354,P=0.006）, which was positively correlated with the expression of Rac1-mRNA (P=0.012).Conclusion Intravitreal injection of Rac1-shRNA with liposome in mice can effectively inhibit the expression of Rac1,and inhibit the retinal neovascularization under relative hypoxia via blocking the ROS-NF-kappa;B pathway.
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)
Natural nonorganic bone(NNB) was obtained after the fresh bone of pig was heated to 100℃. The NNB was white and in a shape as its original bone.The tensile strength of the compact bone was 200kg/cm3 and that of the cancellous bone was 25kg/cm3. The ratio of calcium to phosphorus was 10∶6. The main componentwas hydroxyapatite. The material was composed of trabeculae and intertrabecular spaces. Three experiments were performed. Experiment 1: 18 pieces of NNB in a size of 0.5×0.5×0.5cm3 were implanted intothe back muscle of 18 rabbits. At 4, 8 and 12 weeks after the operation, 6 specimens were obtained seperately and were stained by HE, and then examined under microscope. The result showed that the mesenchymal cells had no regeneration and differentiation, and the NNB and the surrounding tissues had no evidence of formation of new bone or chondrosynthesis. This NNB did not produce rejection reaction between tissues but the new blood vessels could easily grow into the space of the NNB. The fibrils had intimate contact with NNB. Experiment 2: The NNB and hydroxyapatite(HA) were mixed to make a cylindroid body with 2mm in diameter and 4mm in length and was implanted in the bilateral tibias of 40 rabbits, respectively. The roentgenography, fluroscent microscopic examination and histological observation were carried out at 2, 3, 4 and 8 weeks after the implantation. Experiment 3: In 50 rabbits, a defect of 2.5cm was made on both radius, and in one sideNNB was implanted and the other side was served as the control. Another 50 rabbitsHA was implanted in the defect in one side and the other side was served as the control. The results showed that in the NNB group at the 16th week, the bone united in 16 of the 30 cases, while in the HA group, in the 30 cases,only 2 had the bone united, while those of the controls no union had occurred.It was suggested that NNB had more formation of new bone than HA did.
Objective To demonstrate if apoptosis is one of the mechanisms of siderotic retinopathy. Methods Autoclaved iron particles were implanted in the vitreous cavities of 32 eyes of SD rats.Glass chips were implanted in 10 control eyes.The experimental eyes were enucleated at various time intervals from days 1 to 15.Retinal degeneration was examined using the TdT-mediated,dUTP-biotin nickend labeling(TUNEL)method.Electrophoresis on agarose gel was used to detect internucleosomal DNA fragmentation.Results TUNEL-positive nuclei were observed only in the outer nuclear layer beginning on day 2.The nuclei spread throughout the outer nuclear layer by the end of day 3.No TUNEL-positive nuclei were observed in other layers throughout the experimental perios.Analysis of DNA,extracted from the retinas by electrophoresis on agarose gel,revealed a typical ladder pattern of internucleosoma DNA cleavage in the experimental eyes.ConclusionApoptosis of photoreceptors occurs at the early phase of iron-induced retinopathy in the rats.
Objective To investigate the cl inical appl icabil ity and value of internal fixator for the reconstruction of lumbar isthmus in the treatment of lumbar vertebral spondylolysis and to lay a fundation for its cl inical appl ication. Methods Sixteen healthy goats weighing 22.65-31.22 kg were selected to establ ish the models of vertebral spondylolysis at L5, which thereafter were randomized into two groups (n=8): bone graft group in which 0.8-1.1 g fresh autogenous bone was transplanted into the isthmus spondylolysis area, and internal fixation with bone graft group in which internal fixator was installed before transplanting 0.8-1.1 g fresh autogenous bone into the isthmus spondylolysis area. All animals were killed 8 weeks after operation to receive imaging, topographic anatomy and histology detection. Meanwhile, biomechanics test was performed by using 5 donated vertebral body specimens (4 males and 1 female aged 35-51 years old). The left isthmus of L5 vertebra was transected to serve as lumbar vertebral spondylolysis model. A mini-displacement sensor was put at the transected ends of the isthmus. Then loading was conducted with a constant velocity of 2 mm/min by electronic omnipotent tester simulating the direction of fixation force of the internal fixator, and the deformation value of the transected ends was collected by a dynamic data collector and analyzer. The loading wascontinued until the vertebra specimens were damaged. The deformation of displacement sensor and the closure of transected ends of the lumbar isthmus were observed. Results All the goats behaved normally shortly after operation, and no nerve injury induced by operation and no wound infection occurred. Bilaterally obl ique X-ray films of lumbar vertebra and topographic anatomy 8 weeks after operation showed the fusion rate of the internal fixation and bone graft group and the bone graft group was 100% and 62.5%, respectively, indicating there was a significant difference (P lt; 0.05). Histology observation showed 3 goats in the bone graft group presented empty bone trabecula, empty bone lacuna and the disappearance of osteocytes at the transected ends of lumbar isthmus; while in the internal fixation and bone graft group, the bone trabecula grew into cancellous structures with hematopoietic and fatty bone marrow tissue inside, and parts of the bone trabecula had various degrees of mosaic-l ike pattern. During the upload, the biomechanics test and data processing results showed when the external load was 40 N, the deformation of displacement sensor was identified and the gap between the transected ends of lumbar isthmus started to close; then with the increase of external load, the displacement sensor tended to ascend in a l inearity manner; while when the external load was 212 N, the displacement sensor had no further deformation, the gap between the transected ends of lumbar isthmus wascompletely closed, and the pressor effect appeared. Conclusion The internal fixator for the reconstruction of lumbar isthmus has mechanical effects of stabil izing and elevating pressure with a high fusion rate.