动物实验,早产小鼠视网膜病变 z-bo 2020-08-26 499

　　Purpose: To determine whether brimonidine (BRI) can chronically treat neonatal mice with retinal vascular leakage and neovascularization after hyperoxia exposure (mouse model of premature retinopathy (ROP)) and choroidal neoplasia after laser treatment Blood vessel (CNV).

　　Method: Laser treatment can induce experimental CNV in BN rats, BRI or excipient (VEH) is administered by osmotic micropump, and CNV formation is measured 11 days after laser treatment. Neonatal mice were exposed to 75% oxygen to induce retinopathy of newborn mice on 7-12 days after birth. BRI or VEH was administered by gavage. On day 17, the concentration of vascular endothelial cell growth factor (VEGF) in the vitreous retina, retinal vascular leakage, neovascularization and vascular occlusion were measured. Experimental CNV was induced by subretinal injection of lipopolysaccharide/fibroblast growth factor-2 in rabbits.

　　Result: Systemic BRI treatment significantly attenuates the CNV formed in the first 3 days of BN rats or the CNV formed by laser treatment for 1 hour. On the 17th day, BRI initiated treatment significantly reduced VEGF concentration, retinal vascular leakage and retinal neovascularization in mice with retinopathy induced by hyperoxia exposure. BRI intravitreal treatment has no effect on the formation of ischemic choroidal neovascularization in rabbits.

　　Conclusion: BRI treatment significantly reduced the vitreoretinal VEGF concentration, retinal vascular leakage, and retinal choroidal neovascularization in animal models of ROP and CNV. BRI can inhibit the expression of vitreoretinal VEGF caused by ischemia, thereby reducing vascular leakage and retinal choroidal neovascularization.

　　Background: Ischemia is the main cause of retinal neovascularization associated with ocular diseases, including retinopathy of prematurity (ROP) and proliferative diabetic retinopathy (PDR). During the normal growth and development of ROP2 or the loss of local capillaries in PDR3, vascular occlusion and retinal ischemia caused by the stop of the vasculature lead to abnormal blood vessel proliferation on the surface of the retina. In ROP, new blood vessels often decline. If the blood vessels cause retinal detachment or blood vessel leakage causes scars, it can cause irreversible vision loss. Ischemia may cause choroidal neovascularization (CNV), which occurs in wet (exudative or neovascular) and age-related macular degeneration (AMD). Wet AMD, fragility, vascular leakage, grows from the choroid through Bruch's membrane to retinal pigment epithelial cells (RPE) and proliferates on the RPE or subretinal space. Wet AMD, vascular leakage and bleeding associated with CNV, can quickly lead to severe vision loss. Hypoxia up-regulates vascular endothelial growth factor (VEGF), which plays a major role in stimulating retinal neovascularization in ischemic retinopathy. It is confirmed that the concentration of vascular endothelial growth factor in the vitreous of PDR patients is elevated. In addition, treatment with anti-vascular endothelial growth factor drugs has been shown to reduce retinal neovascularization and PDR and proliferative ischemic retinopathy in animal model patients. In a well-studied ROP neonatal mouse animal model, from birth (P) 7 to P12, exposed to 75% oxygen and then returned to the room (containing normal oxygen concentration) to develop oxygen-induced retinopathy (OIR), characterized by Central retina ischemia during hyperoxia exposure, followed by neovascularization at the junction. From P17 to P21, the formation of neovascular plexus of neovascularization reaches the maximum.

　　In wet AMD, VEGF is also an important mediator of CNV. The CNV tissue of patients with wet AMD was surgically removed, and vascular endothelial growth factor was located by immunohistochemistry. Intravitreal injection of anti-VEGF drugs is used for clinical treatment of wet AMD. In animal models, laser photocoagulation of the choroidal retina and destruction of the glass membrane can reliably produce CNV. Experimental study of laser-induced CNV model in experimental rats: increase gene expression of retinal pigment epithelium and choroidal vascular endothelial growth factor. The study of kinase inhibitors inhibiting vascular endothelial growth factor receptor signal transduction has been proved to almost completely eliminate CNV in laser-induced CNV animal models.

　　Method: Rat CNV experimental model: male BN rat, weighing 250-300g. The animals have a normal diet and are domesticated for at least one week before the experiment. After acclimation, the rats were weighed and assigned to the treatment group. The weight of this group was evenly distributed. Rats were anesthetized with 1:1 ketamine hydrochloride injection (65 mg/ml) and xylazine (11 mg/ml) mixed solution intramuscular injection 1ml/kg, dilate pupils with 1% tropikamide and 10% phenylephrine hydrochloride. The experiment uses laser to induce CNV, and uses argon ion laser to burn 3-4 laser spots around the optic disc of each eye. Each photocoagulation uses a wavelength of 514 nanometers (green), a spot size of 100 microns, a power of 110 MW, and an exposure time of 100 milliseconds.

　　CNV drug treatment and evaluation: BRI or excipients were administered systemically 3 days before laser induction, and at different times after laser induction. Through an osmotic pump, continuous subcutaneous administration of BRI (1mg/kg/d) or excipient (distilled water). Eleven days after laser treatment, the animals were sacrificed by the CO2 asphyxiation method, and CNV formation was determined according to the previously described method. Eyeballs were fixed in 4% paraformaldehyde-phosphate buffered saline (PBS; 9 g/L NaCl, 0.232 g/L potassium dihydrogen phosphate and 0.703 g/L disodium hydrogen phosphate [pH 7.3]) for 1 hour. The anterior segment of the eye, the lens and retina were removed, and the remaining part was washed with PBS buffer (containing 0.5% bovine serum albumin, 0% Tween 20, and 0.05% sodium azide) at 4°C, and the same at 4°C. Incubate for a total of 4 hours with 1:100 diluted 1mg/ml lectin IB4 solution. After incubation, rinse with ICC buffer.

　　Rabbit CNV animal model: 24 Dutch rabbits, 6-7 months old, weighing 2-2.5kg were selected for the experiment. After intramuscular injection of ketamine (50 mg/kg) and xylazine (5 mg/kg) anesthetized the animals, CNV was induced by intraocular surgery. One eye of each animal is used for research. Before the internal eye surgery, the pupils were dilated with 1% prop and 2.5% phenylephrine hydrochloride, and the fundus and fluorescein angiography were performed. CNV was induced by subretinal injection of 50L of angiogenic agent containing 100ng of recombinant human growth factor and 100ng of lipopolysaccharide. A 30-gauge needle is used for injection through the retina, and the damaged glass membrane is visible. Six rabbits were injected with vehicle under the retina, but did not damage the glass membrane as a control group. After the operation, topical mydriatic ointment (1% atropine) and antibiotic ointment (bacitracin/neomycin/polymyxin) are used to prevent inflammation-related iris adhesions and other complications.

　　CNV drug treatment and evaluation: subretinal injection of FGF-2 / LPS for 1 hour, 3 days, 7 days and 10 days, BRI or excipients were injected into the vitreous cavity of rabbit eyes. 14 days after the subretinal injection, an eye examination was performed, and changes in the vitreous, retina, choroid, and blood vessels were recorded with a fundus camera. Intravenous injection of 0.2ml 5% fluorescein-dextran and 0.25ml 10% fluorescein sodium was used to evaluate choroidal neovascularization and vascular leakage. Quantitative analysis of CNV lesion area from digital images.

　　Experimental oxygen-induced retinopathy mouse model: Using C57B6 mice reported by Smith et al. to induce OIR, newborn mice P7-P12 were placed in a 75% oxygen box with sufficient food and water for 5 days. The box is only allowed to be opened for administration. On day P12, the mice returned to normal oxygen levels. P10, P12 and P16 were administered intragastrically once a day with BRI or excipient (water) dissolved in water. Five days after the animals were exposed to indoor air, retinal neovascularization and vascular leakage were evaluated.

　　Retinal Angiography and Quantification: As mentioned above, angiography was used to evaluate retinal neovascularization and vascular occlusion in OIR mice. P17 mice were deeply anesthetized and perfused with PBS buffer containing 50 mg of 1 ml fluorescein dextran through the left ventricle. The eyeballs were removed and fixed with 4% paraformaldehyde for 24 hours. After the lens is removed, the retina is detached and embedded. Quantify vascular occlusion and retinal neovascularization. Observe the overall image of the retina under a 4x fluorescence microscope. The area of neovascularization and avascular area are expressed as a percentage of the entire retina area.

　　Western blot analysis: Vitreoretinal VEGF expression and Western blot analysis confirmed the leak. At P17, the animals were killed by carbon dioxide asphyxiation, the retina and vitreous tissue were separated and homogenized by ultrasound in lysis buffer at 4°C (5mM HEPES [pH 7.5], 50 mM NaCl, 0.5% Triton X-100, 0.25% sodium deoxycholate) , 0.1% SDS, and 1 mM EDTA). The insoluble particles were separated by centrifugation at 4°C and the protein concentration of the supernatant was determined using a protein detection kit. The proteins were separated by SDS-PAGE and electrotransferred to PVDF membrane. The membrane was incubated with 1:1000 polyclonal goat anti-albumin antibody, 1:1000 monoclonal mouse anti-actin antibody, and 1:500 polyclonal rabbit anti-VEGF antibody, and used anti-goat, anti-mouse, and anti-rabbit IgG related antibodies. Oxidase acts as a secondary antibody. The immunoreactive band was detected by chemiluminescence method. The intensity of the α-actin signal was used as an endogenous control.

　　Result: The effect of BRI on the formation of CNV in BN rats induced by laser: To determine the effect of BRI on the formation of CNV in animal models. The two eyes of BN rats were treated with laser to induce CNV. 11 days after stimulating the formation of CNV, continuous systemic treatment with 1 mg/kg/d BRI significantly reduced CNV focal areas. Three days before laser induction, the CNV area of animals treated with BRI was 11919+1128 square microns, while the vehicle-treated group was 19185+1522 square microns. The treatment started 1 hour after laser treatment, the CNV area was 10382+864 square microns, and the excipient treatment group was 17101+1407 square microns.

　　Time dependence of BRI on laser-induced CNV treatment in BN rats: To determine the time dependence of BRI treatment on the formation of CNV in BN rats induced by laser, 1h, 1d, 3d or 5d after laser treatment, BRI (1mg/kg/d) ) Or excipients systemic treatment through osmotic micropumps. 1mg/kg/d BRI, systemic treatment within 1 hour of laser induction can significantly reduce CNV lesions. There was no significant difference between the BRI systemic treatment and the excipient treatment group when the laser was induced for 1 day or longer.

　　Effect of BRI on retinal vascular leakage in a mouse animal model: At P17, retinal vascular leakage was determined by Western blot analysis of albumin concentration in the retina/vitreous body. The vitreous retin-actin ratio of BRI-treated OIR mice was 1.32+0.10, and the retin-actin ratio of vehicle-treated OIR mice was 2.09+0.12. Control group, OIR mice not exposed to hyperoxia The retinal protein-actin ratio was 1.23+0.11, indicating that from P10-P16, treatment with BRI reduced about 90% of retinal vascular leakage caused by previous exposure to hyperoxia in mice.

　　Effect of BRI on retinal vascular occlusion and neovascularization in mouse OIR animal model: To determine the effect of BRI on retinal neovascularization in OIR mice, newborn mice P7-P12 were placed in 75% oxygen and P12-P17 were placed in indoor air. Gavage from P10-P16BRI (3 mg/kg) or excipients once a day. In P17, gavage with high molecular weight fluorescein dextran. The retinal neovascularization was measured by angiography. Daily BRI treatment from P10-P16 can reduce retinal neovascularization. The area of retinal neovascularization of mice in the BRI treatment group was 5.83% (+0.81%), while that of the vehicle treatment group was 10.80% (+0.71%). The mice in the control group that were not exposed to hyperoxia did not develop retinal neovascularization. Retinal vascular occlusion was used to evaluate and determine the effect of BRI treatment on the degree of ischemic injury in OIR mice.

　　The effect of brimonidine on the dose and time dependence of retinal neovascularization in a mouse model: P17, using high molecular weight fluorescein-dextran to determine retinal neovascularization by angiography. The effect of BRI treatment on retinal neovascularization is dose-dependent. From P10-P16, the daily dose of BRI is 1, 2, 3 mg/kg, respectively, and the treatment of BRI on retinal neovascularization is also time-dependent. Daily treatment can effectively reduce retinal neovascularization. When 3mg/kg BRI was given from P12-16, it had no effect on retinal neovascularization.

　　"The effect of brimonidine on the concentration of VEGF in the vitreous retina of a mouse animal model: the molecular weight of VEGF is approximately 42Kda. P10-P16, daily BRI administration can effectively prevent the increase of vitreoretinal VEGF concentration. To determine the effect of BRI treatment on non-ischemic CNV animal models, fundus fluorescein angiography was used to evaluate corneal neovascularization 14 days after induction. 14 days after CNV induction, repeated administration of 10 or 100 g BRI into the vitreous cavity had no significant effect on CNV lesions. The CNV area of 10ug BRI treated animals is 15.8+2.7 square millimeters, the CNV area of 100ug BRI treated animals is 16.7+4.6 square millimeters, and the CNV area of vehicle-treated animals is 14.8+2.5 square millimeters.

　　Conclusion: This study shows that BRI treatment can significantly reduce the formation of retinal neovascularization in neonatal mice, retinopathy of prematurity, and significantly reduce the CNV of laser-induced vitreous membrane rupture rats. The effect of BRI treatment on retinal and choroidal neovascularization is time-dependent, and only when the treatment occurs when myocardial ischemia occurs, vascular endothelial growth factor stimulation is the main factor for neovascularization in this case. These results indicate that BRI can be used to treat diseases related to retinal and choroidal neovascularization.