血管渗漏,慢性视网膜新血管形成,动物模型 z-bo 2020-07-23 706

　　Retinal vascular diseases, such as exudative age-related macular degeneration (eAMD), diabetic macular edema (DME), and proliferative diabetic retinopathy (PDR), all cause persistent and progressive vascular leakage. The disease indicates leakage and abnormal angiogenesis. Choroidal neovascularization (CNV) is an important function of eAMD. Pathological neovascularization originates from choroidal blood vessels, destroying Bruch's membrane and penetrating the subretinal space. CNV vascular development is immature, the permeability of endothelial cells is enhanced, the tight junction of endothelial cells and the integrity of pericytes are lost, which can cause fluid accumulation and bleeding, leading to blindness. In DME and PDR, the damage caused by hyperglycemia can cause ischemia and inflammation, leading to continuous fluid accumulation in the vitreous and retina and retinal neovascularization (RNV), leading to retinal detachment and blindness. Is connected. In these common angiogenic and ischemic diseases, vascular endothelial growth factor (VEGF) is the main mediator of endothelial instability, which can lead to continuous vascular permeability and the formation of choroidal or retinal new blood vessels. At present, the standard of nursing intervention for CNV, DME and PDR is bevacizumab, ranibizumab and aflibercept combined to neutralize VEGF, thereby limiting its pathological effects. Traditional prescriptions have obvious therapeutic effects, but there are usually options: sexual and short-term therapeutic effects. Most patients benefit from anti-vascular endothelial growth factor treatments, but due to the limited duration of these treatments, monthly intravitreal injections (IVT) are required for best results. Occasional and suboptimal dosing schedules are often used as a compromise. Therefore, patients and health care providers are in great need of new and/or anti-vascular endothelial growth factor therapies that have a long duration, can effectively stabilize the vasculature and reduce pathological angiogenesis. .. For retinal vascular disease models that are very similar to the human eye (such as non-human primates (NHP)), one challenge is the lack of chronic vascular leakage and/or the angiogenic response characteristics of human eAMD, DME, and PDR. .. In the CNV model of rodents and NHP laser-induced eAMD, transient vasculature vasculature leakage and angiogenic response lasted for 2-3 weeks after the laser destroyed Bruch’s membrane, and then subsided spontaneously. Anti-VEGF treatment accelerates the regression of this induced pathology, but in humans, CNV pathology recovers and persists after removal of anti-VEGF drugs. Therefore, the establishment of a model of persistent and recurring vascular leakage and angiogenesis greatly helps and accelerates the evaluation of long-term interventions to address the multiple clinical manifestations of pathological vascular instability and angiogenesis. The DL-α-aminoadipate (DLAAA) model is a chronic leakage model reported in rats and rabbits, in which common drug candidates have been screened. DLAAA is a selective glial cytotoxin. According to reports, it inhibits the action of glutamine synthetase, impairs the homeostasis of the retinal function of a wider range of Muller cells, leads to glial dysfunction and death, and leads to the destruction of the blood-retinal barrier. Two months after DLAAA injection, the blood-retinal barrier under the retina was destroyed, and blood vessel leakage and tortuosity increased. Recent studies have shown that vascular leakage and RNV will increase 12-18 months after IVT administration of DLAAA to rabbits. Anti-VEGF drugs bevacizumab, ranibizumab, aflibercept and DARPins target VEGF. It has been shown that -A165 inhibits such pathologies. Rats, rabbits and humans have different anatomical structures of retinal blood vessels and neurons, but monkeys and humans are basically the same. The monkey’s vasculature, retinal segmentation, the basic boundary between monkeys and humans, the ratio of retinal neurons to glial subtypes, and the presence of the macula are homologous. Here, we report a new NHP model of chronic vascular leakage caused by DLAAA. Preclinical models of chronic retinal vascular leakage and neovascularization can screen the efficacy of short-acting and long-acting anti-angiogenic compounds in multiple stages of disease development. Method: Animals: A total of 8 adult males and 9 female African green monkeys (male: 5.2-6.9 kg, female: 3.1-4.1 kg) were used. Eye examinations before screening include slit lamp (SL) examination, color fundus photography (CFP), fluorescein angiography (FA) and optical coherence tomography (OCT) to confirm that there are no eye abnormalities. Observe the cage twice a day and conduct additional monitoring during the planned eye examination. The animals were recruited, grouped 6-9 weeks after the administration of DLAAA, and raised in pairs.

　　DLAAA model: On day 0, both eyes (OU) of the two monkeys in this group received IVT injection of DLAAA (5 mg). Dissolve DLAAA in 1M hydrochloric acid to make a 100 mg/mL stock solution, dilute with phosphate buffered saline (PBS), adjust the pH to 7.4, and then filter through a 0.2 micron filter. Prepare a small amount of DLAAA solution (25 mg/mL) before administration and store it at -80°C. All aliquots were prepared from DLAAA batches. Before administering IVT, 1% atropine was applied topically to both eyes to achieve complete pupil dilation. Anesthetize the surface of the eye with 1-2 drops of 0.5% Proparacaine. Use a 1 mL syringe connected to a 27-gauge needle to perform vitreous puncture, remove 100 μL of vitreous humor and store at -80°C. Before administration, a vitreous puncture is performed to suppress the increase in intraocular pressure. The success rate of vitreous puncture is 70%. Use a 0.3 cc insulin syringe and a 31G 0.5 inch needle to deliver the DLAAA solution (5 mg/200 μL) 3 mm behind the center of the vitreous. Use 3 kinds of antibiotic ointment (neomycin/polymyxin B sulfate/zinc bacitracin) and 1% atropine ointment immediately after injection. If the pathology caused by DLAAA is severe or retinal detachment is observed, the eyes are excluded. The pre-defined exclusion criteria include: 1) severe pathological development, massive or extensive leakage beyond the vascular arcade or surrounding retina, 2) severe hemorrhagic eye disease, or 3) edema and fibrosis. Retinal detachment caused by serous or complete retinal detachment and tissue remodeling caused by traction. The application of these criteria resulted in the exclusion of 25% of animals receiving DLAAA. Anti-VEGF injection: Eight or nine weeks after administration of DLAAA, fluorescein angiography images are graded to assess the severity of DLAAA-induced retinal vascular leakage. Please refer to the standard leak score sheet. FA imaging was repeated 10 weeks before treatment to confirm animal tasks and capture baseline FA images. Before the administration of anti-VEGFIVT, 1-2 drops of 0.5% procacaine were used to anesthetize the ocular surface. Use a sterile 0.3 mL insulin syringe pre-connected with a 31G5/16 inch needle to inject the anti-VEGF drug into the vitreous cavity. Place the needle 2 mm behind the edge of the inferior temporal quadrant and point it toward the center of the vitreous. The eyes receive a single IVT injection of vehicle (0.9% saline), 50 μL) or aflibercept. The dose level of the anti-VEGF agent was selected according to the relative volume of the vitreous of the African green monkey (approximately 2.7 mL) and the relative volume of the human vitreous 4.4 mL. All contralateral eyes received the same treatment. After injection, neomycin/polymyxin B sulfate/bacteriocin antibiotic ointment was applied locally. Dosing within 2 days and follow up during the study period.

　　Eye examination: Before taking the medicine, once every two weeks after the DLAAA management intervention, and once a week at the end of the study, ocular surface integrity, general eye health, extensive eye response to DLAAA management, and a slit lamp microscope to examine the eyes until Tolerance to dilated pupils was confirmed, confirmed to have 1% HCl ring, and normal response to pentose salt. A modified Hackett-McDonald scoring system was used to score the results of eye examinations.

　　Color fundus photography: Use Topcon TRC-50EX retina camera with Canon 6D digital imaging hardware and new visual fundus image analysis system software. Once a week, and every two weeks after the intervention, obtain bilateral color fundus images of the retina until the end of the study.

　　Fluorescence contrast: After intravenous injection of 0.1 mL/kg of 10% fluorescein sodium, using Topcon-TRC-50EX retinal camera or Heidelberg HRA + OCT, high resolution fluorescein angiography (FA) has a fixed gain and flash intensity. )Obtain. Collect images in minutes. Use the scoring system to evaluate and score the retinal area to show the vascular leakage during the entire angiography. Use the ImageJOI tool to measure the total fluorescence intensity of the original angiography leakage area within 1 minute. Quantitative evaluation (from the 10th week to the end of the study)

　　Leak analysis: Use a custom grading scale and multiple ROI automated analysis to evaluate the leakage caused by DLAAA. The analysis shows that the maximum therapeutic effect is limited to the choice of showing leakage and time point recurrence: check "lesion size" and "severe lesions" "Extent" scale to perform relative FA image series inspection (about 5 seconds to 6 minutes after fluorescein administration) to evaluate the size and severity of the leak. Use fluorescein injection. The absolute fluorescence intensity was measured on the FA image acquired after 1 minute. Each grid defines 14 regions of interest (ROI), representing the upper (S), nasal (N), inferior (I) and temporal (T) regions, as well as the central retina (1) inside the fovea and optic disc, and the middle (2) and external (3) part. From each baseline image of each eye (before the DLAAA injection), determine the position of the ONH relative to the fovea and add the ROI grid. The covering layer can be used in all positions of the eyes. Results: The use of DLAAA can damage the retina, leading to structural integrity, fluid accumulation, blood vessel tortuous, pathological vascular leakage and remodeling. In FA imaging, from the second week, hyperfluorescence leakage was mainly observed in and around the macula, and in some eyes, leakage was observed in and around the vascular arcade. The leak developed rapidly in the first 4 weeks, stabilized in 8-10 weeks, and lasted more than 18 weeks. In addition, enlargement of the avascular area of the fovea was observed in some eyes, and the capillary network around the fovea disappeared. Early FA assessments at 4, 6 and 9 weeks showed signs of neovascularization in the macula. In addition, after the administration of DLAAA, the capillaries and capillaries around the fovea often show abnormalities of dilation and leakage. This is a very obvious sign of MacTel in early FA imaging. The color fundus photos also showed vascular lesions similar to MacTel. Fundus microscopy and color fundus images showed that the macular vascular obstruction was consistent with angiography data and clinical correlation. In addition, the whitening of the retina, especially the edema of the nerve fiber layer, is consistent with hypoperfusion.