| BACKKGROUNDRetinopathy of prematurity is a proliferative retinopathy in preterm infants, in the retinal diseases such as diabetic retinopathy and retinopathy of prematurity, the retinal vascular disease is the leading cause of visual impairment or blindness. In the past10years with the rapid development of perinatal medicine and pediatrics, the survival rate significantly improve between premature and low birth weight infants, the occurrence of retinopathy of prematurity rate also increased, that seriously affecting the survival of preterm children quality, but also to medical malpractice in recent years due to problems caused by retinopathy of premature children showed a gradually increasing trend. The pathogenesis of ROP has not been elucidated, immature retinal vascular is very sensitive to oxygen, high concentrations of oxygen caused the retinal blood vessel constriction or obstruction, leaded to retinal hypoxia. Due to the lack of oxygen to produce blood vessel growth factor, stimulate the retinal neovascularization. ROP occurs in the peripheral portion of the retina, especially the temporal side, first inner retinal neovascularization occurs, blood vessels grow gradually from the retina to the surface, thereby extending into the vitreous body. Neovascularization are accompanied by fibrosis, vascular membrane along the front of the vitreous fibers grow behind the lens in the lens fiber membrane formation, shrink film will be pulled to the peripheral retina of the eye center, causing traction retinal detachment, leading to ocular atrophy, blindness. In the development of ROP, the formation of new blood vessels has played a dominant role. Angiogenesis. is a complex interaction and mutual adjustment between the numerous vascular factors. When the balance of angiogenic and anti-angiogenic substances are broken, produce new blood vessels.Currently, the treatment of the disease mainly with operation treatment, such as retinal photocoagulation, vasectomy, condensation, but the poor treatment effect. Based on the pathogenesis of retinal neovascularization in ROP and retinal neovascularization medication will surely become a promising treatment. Actively explore the role of medical treatment by angiogenesis and inflammation is urgent priority in the early non-surgical treatment of ROP.RAAS is not just a typical endocrine system, and is a autocrine and paracrine system,composed of renin,angiotensin and its receptor. A complete RAS system exists in humans and a variety of biological retina, and are found in the developing retina. The study found that AT1and AT2in the neural retina and pigment epithelium layer and uveal tissue5-100times higher than in plasma, high levels of AT1and AT2in ocular tissues is not originated from circulation, but independent of blood circulation. RAS is abundant in the inner retina、retinal vascular neurons and glial cells. Kida T et al found that the presence of the renin angiotensin and amacrine cells in the retina and ganglion cells, which are also present in the Miiller cells. Renin angiotensin inhibitors include ACE inhibitors, ARB, aldosterone antagonists, it with anti-hypertensive, anti-heart failure, renal insufficiency resistant, anti-diabetic and other biological effects. Recent studies have found that ACE inhibitors and ARB prevent ROP retinal neovascularization and reduced angiogenesis, but less for aldosterone antagonists for ROP retinal neovascularization reports. Aldosterone secretion is mainly controlled by the renin angiotensin regulating, so whether the existence of MR-aldosterone system in ocular tissue independent of the expression, in retinal vascular development, whether aldosterone plays a role, and there is no relative reports. The need for a fixed retina can better and avoid stationary liquid retinal detachment eyeball fixing solution based on experimental and clinical pathological study. Existing eye fixative reported mostly used to observe the cornea and other parts of the lens, and retina fixative for no clear reports.Based on the above analysis, this study consists of two parts. Firstly, observe the four different effects of compound fixed liquid in mice retina, and comparative analysis; Secondly observe whether aldosterone inhibitors can inhibit the formation of new blood vessels in the retina like inhibitors of angiotensin Ⅱ, and to explore its possible mechanism inhibition on the basis of the first portion.Part1Comparison of four different compound fixed liquid in mice retina fixationObjective:Observation of four fixed effects of different composite fixed liquid on the mouse retina, and comparative analysis, choose safe and effective fixed liquid for the second part of the experiment, provide experimental evidence and clinical application for the fixed retina.Methods:The40Kunming mice were sacrificed, removal of the eyes immediately, and randomly assigned to A-D four groups,20eyes in each group. Group A immersed in the fixative1(Paraformaldehyde4g, add80mL0.1mol/L phosphate buffer, heated to around60℃, stirring, the powder is completely dissolved, add a few1mol/L NaOH is clarified, after being cooled, add5mL glacial acetic acid,10mL acetone, with0.1mol/L phosphate buffer up to100mL);Group B immersed in the fixative2(5%potassium dichromate40ml,10%formaldehyde10ml,10ml glacial acetic acid,,10%trichloroacetic acid,10mL10%sodium acetate);Group C immersed in the fixative3(acetic acid10mL,40%formaldehyde solution20mL, saline70mL,10mL75%ethanol blend mixed fixed); Group D immersed in the fixative4(ethanol60mL,40%formaldehyde solution10mL, acetic acid lOmL,20mL chloroform)。 Fixed24hours, conventional dehydrated, embedded in paraffin fixed, HE staining. Using SPSS13.0statistical software for data chi-square test, P<0.05was considered statistically significant.Results:Visible in group A, B mice eyeballs part of the optic cup shrinkage, can not maintain the original shape; Groups C, D were rounded eye shape, color white and transparent, eye cup no obvious shrinkage concave shape, eye shape remained in their original state; When sliced,,group A and B are difficult to come to a complete slice; Group C section looser; Group D slice is relatively complete.HE staining of retinal showed that A group of mice have different levels of retinal layers of tissue rupture, missing part of the organization structure, the retinal tissue gap bigger, tissues structure was loose, color bright, uniform chromatin, clear nuclei, each layer of color contrast is obvious; Group B mice retina each layer separation, fracture, lack of organization, The crack in all layers of the retina structure between cells, tissue deformation, the organization gap increased, cell shrinkage, the coloring to deepen the color contrast, intracellular organization structure is not obvious; Group C mice retina each layer structure is clear, colour and lustre, the layers of organization structure is complete, the cellular structure is clear but visibility fixative retinal separation; Group D mouse retina layers of structural integrity, no significant structural changes, no detachment and separation of retinal, the layers of eye clear organizational structure without cracks, nucleolus, nuclear membrane clear chromatin uniform, bright color, contrasting colors.Each group of data using the chi-square test, each group was significantly different than the contraction (chi-square value of34.687, P=0.00).There are significant differences between the groups hardening ratio (chi-square value of34.972, P=0.00).Fixative of each group detachment to occur than there are significant differences (chi-square value of49.048, P=0.00).Conclusion:After fixed liquid the tissue of the retina can clearly see the layers of retina and rare fixatives retinal detachment. Join chloroform that can be dissolved in ethanol and acetic acid can effectively promote the stationary liquid eye penetration, reducing the fixed liquid retinal detachment.Part2Observed with valsartan spironolactone model of oxygen-induced retinopathy in the mouse retina and mechanism of inhibition of neovascularizationObjective:Observed with valsartan spironolactone model of oxygen-induced retinopathy in the mouse retina neovascularization and investigate the effect of changes in the expression of VEGF, MCP-1protein and mRNA.Methods:60newborn C57BL/6J mice were randomly divided into six groups AF, each10. A,B Group newborn mice were placed in a normal air environment with their maternal feeding. C, D, E, F four groups of7-day-old newborn mice with manufacturing OIR. In the age12-18days, A, C group newborn mice not treated, B, D group newborn mice injected solvent2.5ml/(Kg·d), E group of newborn mice by intraperitoneal injection of spironolactone25mg/(Kg· d), F group of newborn mice injected with valsartan10mg/(Kg· d). A week after the intervention to the retina specimens in each group of mice, retinal neovascularization HE staining observed growth, Western-Blot and RT-PCR detection of VEGF, MCP-1expression, and to explore the possible mechanism of inhibition of OIR.Results:HE staining of retinal A, B group layers of cells in the retina neatly arranged, out of shape smooth inner limiting membrane, missing or rare vascular endothelial cells break through the internal limiting membrane; C, D groups seen a lot of the internal limiting membrane of vascular endothelial cells even visible group of vascular endothelial cells, vascular dilation, visible intravitreal neovascularization and bleeding; E group compared with the retinal cells arranged in neat, visible vascular endothelial cell breakthrough moderate internal limiting membrane; F retina layers of cells arranged in neat This shows a small amount of vascular endothelial cells to break the internal limiting membrane.Breakthrough internal limiting membrane endothelial cell count:Group A was3.23±2.192, group B was2.7±1.915, group C was49.90±4.809, D group was50.17±5.140, E group25.70±3.415, F group7.20±3.478; each group there were significant differences (F=1109.859, P=0.00); between the two groups group A and Group B (P=0.995), no statistically significant difference between group C and group D (P=1), the rest of the visible obvious statistical difference between the groups (P=0.00<0.05). Western blot and RT-PCR detected the expression of the six groups in both the retina MCP-1protein and mRNA, and its fluctuations show consistency. Two indicators of differences between groups was statistically significant. VEGF in normal control group (A, group B) weak expression in the retina, and in the OIR model group (group C, D group) was significantly up regulated in the retina, and normal controls were statistically significant difference (P=0.00<0.05); E group, F group and the OIR model group down regulation of VEGF, E and F group was significantly lower than that, the difference was statistically significant (P=0.00<0.05). The same detection methods are found in the retina of six groups of VEGF protein and mRNA expression, and its volatility presents consistency. Two indicators of differences between groups was statistically significant. VEGF in normal control group (group A and group B) weak expression in the retina, and in the OIR model group (group C, group D) was significantly up regulated in the retina, and normal controls were statistically significant difference (P=0.00<0.05); group E, group F and the OIR model group down regulation of VEGF, F group was significantly lower than the E group (P=0.00<0.05).Conclusion:1. There was independent renin-angiotensin—aldosterone system in the retina of mouse;2. Both spironolactone and valsartan had a retinal neovascularization inhibiting effect;3. At the does of drugs in this experiment, valsartan had a stronger effect, which could be the result of angiotensin’s different ways to inhibit neovascularization. |
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