动物造模,2型糖尿病大鼠模型 z-bo 2021-01-08 329

　　Background: The prevalence of diabetes is increasing worldwide. The rat model of type 1 diabetes has been widely used by vision scientists to analyze the molecular mechanisms related to diabetic retinopathy. These animals are very fragile and die before retinal angiogenesis. In type 2 diabetic rats, proliferative retinopathy has not been reported.

　　 Method: Diabetic rat model: Purchase pregnant Wistar rats and raise them in an animal facility (21±1°C, light and dark for 12 hours), and observe carefully every day until delivery. Young rats 2 days after birth were intraperitoneally injected with streptozotocin (SZT) (45 mg/kg), citrate buffer containing 154 mM NaCl 0.1 ml 0.1 M solution, pH 4.5. The young rats were kept together with the female rats until the 21st day. After 8 weeks, SZT treated animals were fed a high-fat diet. The table shows the formula of a high-fat diet and is stored at -7°C. Blood glucose levels are measured for the first week of each month. Cut the tail to collect 32 μL of blood sample and test. High blood sugar levels (160-220mg/dl) were found in the high-fat diet group. In the two groups, 12 animals were euthanized at 57 weeks and 5 animals were euthanized at 110 weeks.

　　Control rats: non-diabetic animals without SZT injection, fed with standard feed with 2.71% fat content. Their blood sugar is normal (80-95 mg/dL). In the control group, 12 animals were sacrificed at 62 weeks of age and 5 animals were sacrificed at 115 weeks of age.

　　Clinical parameters: Each animal was weighed at the 8th week and before death. The reflotron system is used to determine abnormal blood lipid levels every month and before the animal is euthanized.

　　 Fatty acid: See the table for the formula of high fat feed. The high-fat diet contains 25.6% fat (46.4% saturated, 47.5% monosaturated and 6.3% supersaturated), while the standard diet contains 2.71% fat (24.2% saturated, 30.2% 45.6% monosaturated and supersaturated) .

　　 Clinical photos: Take clinical photos before the animals are alive and euthanized. Take out the eyeball and inject air into the anterior chamber through the cornea with a 30G needle to make the corneal blood vessels more obvious.

　　 Histological examination: The rats were anesthetized with 350 mg/kg intraperitoneal injection of chloral hydrate. The eyes were removed and fixed in 4% paraformaldehyde. The rats were euthanized with excess chloral hydrate. The eyeballs are fixed within one day, then frozen and immersed in four concentrations of glucose solution 5% overnight and 7.5%, 10% and 20% for two hours respectively), and resin mounts. Obtain ten-micron sections and microscopically inspect with hematoxylin-eosin staining (HE) and periodic acid Schiff staining (PAS). At least 5 parts of each animal’s eyes are examined.

　　Immunohistochemistry and immunofluorescence analysis: the eyes were removed and fixed with 4% paraformaldehyde for 48 hours. They were then frozen and immersed in four concentrations of glucose solutions (5% overnight and 7.5%, 10% and 20% for two hours each), and then mounted with resin. Make slices. The immunohistochemical section was first incubated with biotin-labeled goat anti-mouse IgG, then placed in an avidin-biotin-peroxidase complex kit, and finally treated with 3.3-diaminobenzidine (DAB)/nickel Solution. For immunofluorescence, a secondary goat anti-mouse antibody and fluorescein were used to display axial sections. Fluorescence immunoassay was performed using Eclipse Nikon microscope. GFAP expression was analyzed using primary monoclonal antibody against glial fibrillary acidic protein. Use anti-vascular endothelial growth factor polyclonal antibody to check the immunoreactivity of vascular endothelial growth factor. Anti-human vWF is used to detect Von Willebrand factor (vWF).

　　 Trypsin digestion technique: After the cornea is incised, the eyeball is infiltrated and fixed for at least four hours in 4% formalin solution -50mM sodium potassium phosphate buffer (pH 7.2). The retina was dissected and then placed in a 4% formaldehyde buffer solution for several hours. The retina was cut and rinsed with running water overnight. Incubate at 37°C with 3% trypsin solution and 0.1 M Tris buffer (pH 7.8) for one to three hours. Upon completion, the medium becomes turbid and the tissues show signs of digestion. The inner limiting membrane is peeled off. Gently, the blood vessels are detached from the retinal tissue. Place on the slices and air dry. Stain with PAS and eosin.

　　Iris thickness measurement: All long-term diabetic animals (LT-DBT) and the control group 110-week-old animals were subjected to iris thickness measurement. From the pupil to the bottom of the iris, it is distributed between 300μm. 10 samples are used for each iris. Results: Compared with age-matched control rats (range 80-120mg/dl, average 100.5 mg/dl, SD 18.8), diabetic rats had higher blood glucose levels (range 140-416 mg/dl, average 232 mg) /dl, SD 81.9). Compared with control animals, diabetic animal models have higher levels of triglycerides. Observing the anterior segment of the eye, the clinical photos showed that 5 long-term diabetic animals (LT-DBT) (110 weeks old), 2 110-week-old control groups and cataracts, while short-term diabetic rats and 62-week-old control rats did not appear cataract. The difference between LT-DBT mice and other animal groups was statistically significant (P<0.05). Corneal neovascularization was observed in 3 out of 5 long-term diabetic animals. Neovascularization was observed at the periphery and center of the cornea, and there was at least one quadrant extension in each animal. Due to the presence of abnormal blood vessels, all the diabetic animals had significantly thicker iris than the control group. Find at least five new blood vessels in each part of the iris. Corneal histology: shows neovascularization in epithelial cells and anterior stroma. No corneal and iris neovascularization were found in the control group and ST group animals. Observing the posterior segment of the eye, all LT diabetic animals found new blood vessels in the retina. Vasodilatation or aneurysm was found in the four animals, and none of the above results were found in the control group and ST group animals. The pre-optic retinal blood vessels were found in two LT diabetic animals. Retina VEGF, vWF immune response, analysis of anti-vWF antibody and anti-vascular endothelial growth factor of iris and cornea. Only in LT diabetic animals, the expression of VEGF and vWF was up-regulated. Retina display: The expression of vWF in blood vessels located in the fibrous layer (FL) and in the outer plexiform layer (OPL) is up-regulated. The upregulation of EGF was found in OPL. In this layer, co-localization of vWF and VEGF was observed. Similar co-expression of two proteins (vWF and VEGF) was found in the blood vessels of the iris stroma. In the corneal stromal blood vessels, the expression of vWF and VEGF was observed. There was no positive for vWF and VEGF in the retina, iris and cornea of ST diabetes group and control group. It was found that the number of cells measured by trypsin digestion method was significantly lower in the LT diabetes group than in the control group. In FL, a positive GFAP immunoreaction was found, similar in shape to Müller cells. Diabetic animals stained more extensively than the control group and ST group.

　　Discussion: An animal model of diabetic retinopathy was established by feeding a high-fat diet to female type 2 diabetic rats to carry out experimental research. After 110 weeks of diabetic animals, the animals developed retinal and corneal neovascularization and red iris. As far as we know, this is the first report of neovascularization in the eyes of type 2 diabetic rats fed a high-fat diet. It is worth mentioning that we were fed a high-fat diet in male type 2 diabetic rats, and the 90-week study did not find changes in proliferative retinopathy. The potential effects of estrogen on the progression of diabetes in rats are well known. Female rats of all age groups are less sensitive to insulin than males, and are more likely to develop severe diabetes quickly. It is described in scientific literature that the morphological changes of the retina of diabetic rats include the loss of pericytes and the presence of decellularization and capillaries, which cause retinal ischemia. The pericytes of diabetic rats were significantly lower than those of the control group. Can cause corneal endothelial dysfunction are bullous keratopathy, corneal edema, epithelial erosion, inflammatory cytokine release, lack of limbal stem cells and corneal neovascularization. In this study, corneal neovascularization was also found in 3 diabetic animals with LT.

　　 Conclusion: The animal model mimics human diseases, and it can be regarded as a platform for the study of mechanisms and therapeutic drugs. To this end, several ophthalmological disease models have been developed, such as oxygen-induced retinopathy models and proliferative diabetic retinopathy models using gene therapy. In our model, streptozotocin injection, HFD, early onset and longer course of diabetes, female animals are considered to be the main factor in the growth of the disease. Future investigations will confirm the utility of this animal model of diabetic retinopathy.