动物造模 z-bio 2022-01-31 1231

　　Introduction: The development of animal models of choroidal neovascularization (CNV) and retinal neovascularization (RNV) helps to understand biology in this situation. These models have also been developed to test new treatment methods. This model summarizes many clinical manifestations of human CNV and RNV, but the timing of model development, disease progression, lesion size and appearance are all different. We will specifically introduce the advantages and disadvantages of various CNV and RNV animal models. This review is comprehensive, not an encyclopedia. Choroidal animal model: Choroidal neovascularization (CNV) is a non-specific response to specific stimuli. In many chorioretinal diseases, CNV is a dynamic process involving initiation, maintenance and degeneration stages. The degenerative phase is characterized by reduced production of cytokines associated with scar formation and fibrosis. CNV may appear as new or reactivated in an inactive CNV area. CNV patients with age-related macular degeneration increase by about 0.2 tablets per month (DA). The onset of CNV begins when the glass membrane is broken or defective. This may be secondary trauma rupture, degenerative processes, tissue stretch and/or inflammation. When this happens, choroidal capillary endothelial cells, pericytes, fibroblasts and inflammatory cells enter the retinal pigment epithelium (RPE) and/or subretinal space. CNV has inflammation, angiogenesis and extracellular matrix components. Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) are mediated by the surrounding extracellular matrix and the growth and envelope (ECM) of endothelial cells, and seem to be essential for the formation of CNV. The formation of CNV can be divided into three categories: inflammation, angiogenesis and proteolysis. These systems are interrelated. CNV is involved in several inflammatory systems, including the complement system, cytokines and chemokines. Angiogenesis promoting factors stimulate the proliferation of CNV vascular endothelial cells, including vascular endothelial growth factor, basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF). The proteolytic components include temporary fibrin matrix rays, which are the physical location of CNV. Fibrin acts as a scaffold for CNV growth. Fibrin is derived from the conversion of circulating fibrinogen. Tissue factor (TF) receptor is a co-activator of serine, a protease that promotes thrombin and fibrin. TF is also related to inflammation, cell adhesion and vascular endothelial growth factor secretion. Thrombin sensitive protein has anti-angiogenesis properties, but it also promotes the release of angiogenesis-promoting molecules. Cells important for the pathogenesis of CNV include macrophages and RPE macrophages, which are believed to play an important role in this process because they express angiogenic cytokines (VEGF) and tumor necrosis factor. PE expresses VEGF, MCP-1 and IL-8 and is therefore an important regulator of this process, all of which regulate the recruitment of monocytes and endothelial cells. The dynamic phases of CNV (startup, maintenance and degradation) depend on whether the unit signal is turned on or off during this process. These conflicting signals include pro- and anti-inflammatory, angiogenesis and proteolysis. Oxidative damage and inflammation stimulate age-related macular degeneration, while CNV angiogenesis has immune-related mechanisms. The most widely studied inflammatory cascade in age-related macular degeneration is the complement pathway, where competitive proteins enhance or block these pathways. VEGF is the main initiator of CNV angiogenesis. Angiogenesis factors include angiopoietin 1 and 2, basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and KDR/Flt-1, endogenous angiogenesis inhibitors include PEDF, angiostatin and Endostatin. Retinal pigment epithelial factor (PEDF) is a natural anti-angiogenic protein. By regulating the activity of macrophages, PEDF also has anti-inflammatory effects. Laser and light-guided CNV models: rats and mice: After Dobi constructed the first CNV model in primates, he created a rat CNV model in 1989. Through dilated pupils and corneal coverslips, the author created an argon laser photocoagulation spot (647m, 100μM 50-100 MW, 0.1 second). By breaking the glass membrane, a central bubble is formed or a spot is formed where there is no intraretinal or choroidal bleeding. Create evidence of 24% CNV fluorescein angiography. Examination of the removed eyes with an optical microscope and an electron microscope showed pathological evidence of 60% of CNV lesions. Frank and his colleagues also developed a rat model in 1989. This model uses a laser (turquoise or green wavelength, 500μM50 MW, 0.02 s) to destroy the glass film and CNV. Instruct C57BL6 mice to use k laser and change the settings to reduce the spot size, increase the intensity, and shorten the duration (turquoise or green wavelength, 50um50-350μ400mW0.05s), and increase the incidence of CNV (100%). The rat laser-induced CNV model was used to study the role of tissue inhibitor of metalloproteinase 3 and tissue factor in the formation of CNV. It is also used in drug intervention experiments. In mouse models, vascular endothelial growth factor has been shown to be the main stimulator of CNV. This model, especially the immunohistochemical staining of transgenic mice, has been used to study various components of CNV, including basic fibroblast growth factor 2 (FGF2) and matrix metalloproteinases. Albert and his colleagues used circulating light to guide the CNV rat model. In this model, rats are exposed to a photoperiod of 3000 lux for 1, 3, or 6 months. The results showed that RPE angiogenesis under the microscope appeared at 1 month, retinal angiogenesis spread outside the retina at 3 months, and multiple local tissues related to retinal blood vessels appeared at 6 months. The immunohistochemical staining of animal retina under light is HNE, which can measure the peroxidation of omega-6 polyunsaturated fatty acids and indicate oxidative stress. Primates, rabbits, pigs: Ryan et al. have developed the first CNV animal model, the experimental CNV primate model "subretinal angiogenesis". The initial working chamber injects collagenase into the subretinal space through the sclera and vitreous. This can cause mechanical or enzymatic damage to the glass membrane. These minimally invasive techniques have reduced the incidence of CNV and increased the incidence of laser damage to the glass film. Ryan used argon lasers (488m and 514m, 50200μm, 200-950 mw, 0.1-0.5s) to rupture the glass membrane and induce CNV in cynomolgus monkeys. Approximately 40% of monkeys with CNV have evidence of angiography. Approximately 90% of the evidence indicates that the ultrafine structure has changed. These studies provide experimental evidence of early ischemic effects (subsequent studies have shown that vascular endothelial growth factor is in an ischemic state). Inflammation involves the role of macrophages and RPE in the formation of CNV. Several primate models of CNV play an important role in the development of photodynamic therapy. Criswell and his colleagues used the "New World" monkey to create a CNV model and compared it with the CNV model created by the "Old World" cynomolgus monkey. The optimized laser parameters show that there are 65% laser spots in the New World monkeys and 37% laser spots in the Old World monkeys. These CNV lesions extend beyond the original photocoagulation point and contain diffuse subretinal fibrous tissue. The benefits of primate models include monkey retina and macula, and research on drug delivery in primate eyes is similar to humans. Disadvantages include the cost of animal use and reproduction, and the ethical issues of using primates when mice and rats are reliable models. A model between rodents and primates, the laser-induced CNV rabbit model, has been developed. This model avoids the cost and ethical issues of primate models. The rabbit does not have a macula. Unlike primates and rodents that have blood vessels, rabbit retinas are provided by bone marrow rays. This model uses subretinal laser photocoagulation to create CNV. Another intermediate laser CNV model is a pig. Saitama et al. used laser diodes (75μm, 400 MW, 0.1 s) to create a pig model of glass membrane defects and established histological evidence of 100% CNV lesions. The histological analysis part is used to evaluate the treatment of CNV by drugs.

　　Surgery-induced CNV models: Rats and mice: Rat and mouse models of retina or choroidal neovascularization are mainly immune by injecting synthetic peptides, vascular endothelial growth factor, cells and viruses into inactive synthetic carriers. Spilbury et al. injected a recombinant adenovirus vector expressing rat VEGF164 into the retina and was driven by the CMV promoter. Although fluorescein angiography and pathological evidence were found to be as high as 80%, the possibility that puncturing the glass membrane can help induce CNV cannot be ruled out. Baffi et al. injected 5-10 μl of an adenovirus vector expressing VEGF165 into the subretinal space of rats. Fluorescein angiography, histology and electron microscopy revealed CNV in most eyes. The authors used FITC-labeled dextran to expand the retina. This is now the standard procedure. Wang et al. conducted a similar study using a 2μL adenovirus vector expressing human vascular endothelial growth factor (or GFP) and a CMV promoter injected under the rat retina. Extensive studies of PE vascular endothelial growth factor expression, angiography and histological evidence of CNV indicate that approximately 90% of rats have CNV. Compared with the control group, the electroretinogram (ERG) and b waves of CNV rats were reduced by about 50%. Vitreous trauma caused by subretinal injection itself may be sufficient to cause CNV. Rabbit: Early studies have shown that subretinal injection can cause the proliferation of retinal pigment epithelial cells, and CNV can be observed in rabbit eyes under a microscope. Tamai et al. found that subretinal injection of 12.5-25 ul (LHP) into the subretinal space induces CNV in rabbit eyes. Angiography showed that 46% of rabbits had CNV, and TNFα, PDGF, VEGF, TGFβ, bFGF and IL1α were up-regulated. i and his colleagues injected 50 uL of endotoxin, growth factor, heparin beads, FGF-2 and 100g LPS into the subretinal space through the vitreous body with 50μg heparin agarose. Fluorescein angiography, ocular coherence tomography (CT) and histological examination results showed 100% increase in CNV in the eye. Primary choroidal neovascularization grows into the subretinal space and damages the glass membrane. It was visible at the injection site for 2 weeks and was stable for 3 months after injection. Eight months after injection, secondary CNV extended from the injection site to the subretinal space of both eyes, while CNV lasted for 3 years. The characteristic phases of CNV are startup, growth and maintenance. Similar to human CNV, it can inhibit subcutaneous dexamethasone. Qui and his colleagues injected 10 μl of Matrigel and 750 μg of VEGF through the vitreous body into the subretinal space of rabbit eyes. In addition to fluorescein angiography and histological examination, they also use OCT to show that 100% of the eyes have CNV within 1-9 weeks after vaccination. Immunohistochemistry and electron microscopy. ICG imaging and electron microscopy showed changes in the choroid in the model.

　　Boar: The mechanically induced pig CNV model mainly appeared in Denmark, a developed country. Previous research by Sourbane and colleagues showed that intrachoroidal blood production caused by injection of basic fibroblast growth factor instead of CNV into the suprachoroidal space does not damage the glass membrane. Kiilgaard et al. found that swine CNV can occur due to xenon laser coagulation. PE laser diode laser photocoagulation and cardioversion caused 54%, 83% and 100% damage to the glass membrane, respectively. It has been confirmed that the development of CNV requires the destruction of the glass membrane. The mechanical model is a retinal curette after injecting saline into the retina to induce wound resection and retinal detachment under the vitreous retina. Through fundus examination, fluorescein angiography and histological examination, the mechanically induced model was further examined to indicate the location of fibrovascular tissue (CNV). Immunohistochemistry showed that the cytokine production and composition of porcine CNV is similar to that of human CNV. In addition, like rabbit eyes, pig eyes are large enough for drug delivery experiments. The disadvantage is similar to the mechanically induced CNV animal model.

　　Non-human primates: Ryan established an early primate model by injecting collagenase and hyaluronidase into the subretinal space. Cui injected 80ul of VEGF gelatin microspheres into the subretinal space of rhesus monkeys through a retinal incision. Observation of fundus and angiography showed that 92% of monkey eyes have CNV.

　　Transgenic and knockout mouse choroidal angiogenesis model:

　　VEGF164RPE65 age-related macrophage transgenic mice, few animals in this model develop spontaneously in CNV

　　CCR2/CCL2-deficient mouse model: In CCR2-deficient mouse models, CCL2 cannot recruit macrophages in this area in the RPE and choroid. This allows the accumulation of C5a and IgG and induces the production of vascular endothelial growth factor. ApoE overexpression model: There are AMD-like changes in the RPE area and glass membrane of hypercholesterolemia mice. Malek et al. demonstrated that ApoE4 overexpressing transgenic mice fed a high-fat and high-cholesterol diet produce CNV between 65 and 127 weeks.

　　Cp/Heph-/Y knockout mice: The deletion of ceruloplasmin and iron transport accessory protein leads to AMD-like changes in transgenic mice. These mice developed RPE and photoreceptor cell degeneration. Mice with spontaneous mutations in BST chromosome 16: These mice develop retinopathy spontaneously.

　　Summary: There is no ideal CNV animal model. The overexpression of vascular endothelial growth factor is not enough to support the development of CNV. Requires glass membrane, mechanical or biological induction methods to produce CNV. All models require glass membrane-mediated, vascular endothelial growth factor and inflammatory cytokine expression. Animal model of retinal neovascularization: The etiology of retinal neovascularization is better than CNV. A large number of clinical and experimental observations have shown that ischemia is the main cause of retinal neovascularization. Retinal ischemia caused by weakened retinal vessels is related to all diseases, including diabetic retinopathy, retinopathy of prematurity (ROP), central retinal vein occlusion and branch retinal vein occlusion (called ischemic retinopathy). Common feature. Retinal neovascular disease is caused by retinopathy of prematurity. Oxygen-induced retinal neovascularization model: Oxygen-induced retinopathy animal model (OIR) has become the main model of ischemic pathological neovascularization. This pattern first appeared in cats and then extended to other animal species, such as rats, mice, dogs, and zebrafish. A common feature is that high oxygen exposure in the early stages of retinal development can lead to the stagnation or delay of normal retinal vascular development. This ischemia-mediated pathological angiogenesis makes the OIR model useful for studying other ischemic retinal diseases, such as Diabetic retinopathy. Conclusion: The two main types of retinal neovascularization are CNV and RNV. CNV comes from the capillary circulation of the choroid, passes through the glass membrane and grows in the subretinal space. NV is produced by the expansion of the retina to the posterior interface of the vitreous or vitreous. CNV is usually restricted, cell proliferation is restricted, and RPE controls the size and extent of the lesion. NV grows casually, and diffuse angiogenic cytokines enter the vitreous.