动物造模,葡萄膜炎模型 z-bio 2024-01-29 717

　　1. Model making method: Wistar rats, weighing around 200g.

　　(1) Preparation of endotoxins: 18001 strains of Vibrio cholerae, classical biotype, and Ogawa serotype The purified Vibrio cholerae strain that has been identified will be first transferred from the inclined surface of the Hough's tube to the Hough's broth culture medium and incubated overnight at 37 ℃. On the second day, expand the culture of thick flat culture to 20 bottles and incubate at 37 ℃ for more than 4 hours. After the pH stabilizes, inactivate with formaldehyde and centrifuge to harvest the bacteria. Store at 20 ℃. ② The separation and purification of LPs were carried out in accordance with the steps of extracting bacterial lipopolysaccharides using West2phal and Jann's phenol water method. Take 200g of inactivated bacterial cells, add non pyrogenic double distilled water (PFW) and stir to form a bacterial suspension. Add an equal volume of 90% (W/W) re distilled phenol (preheated to 68 ℃, pH adjusted to 7-10), and the bacterial concentration in the mixture reaches 20ml per gram of bacterial body. Stir the mixture in an ice bath for 30 minutes, then cool it in the ice bath (continuously stirring) to around 10 ℃. Centrifuge at 7 300g and 10 ℃ for 1 hour, carefully remove the upper phase liquid, merge the middle layer and lower layer liquid, measure the total volume, add PFW to the total volume of the original mixture, and repeat the extraction once more. Merge the two phases, add ethanol to achieve a final concentration of 25% (V/V), and add NaAc (final concentration of 10mmol/L) and CaCl2 (final concentration of 1mmol/L) to stand overnight at 3-8 ℃. Centrifuge at 7300g for 1 hour on the third day, and collect the precipitate to obtain crude LPS. Suspend this crude extract in 200ml 50mmol/L Tris HCl (pH7.2) buffer, add it to a dialysis bag (Mwco=12000), and equilibrate it in 50mmol/L Tris HCl (pH7.2) buffer to 37 ℃. Then, add 50mg RNase and 50mg DNase I to the dialysis bag, and replace the dialysis solution. Stir the dialysis solution at 37 ℃ for 6 hours. Add 100mg of protease K to the dialysis bag and replace the dialysate. Stir the dialysate at 37 ℃ for 24 hours. Then dialyze at 4 ℃ in PFW for 2 days, and change the dialysate twice a day. Suck out the dialysate at 64000 g, centrifuge at 3-8 ℃ for 5 hours, discard the supernatant, suspend and precipitate with PFW, then add an equal volume of 90% (V/V) re distilled phenol (preheated to 68 ℃ at pH 7.0) and re extract once (the same method as before). Carefully remove the supernatant obtained by centrifugation after extraction, and dialyze it in PFW at 3-8 ℃ for 2 days. Remove the dialysate, scan and analyze its concentration under ultraviolet light within the wavelength range of 190-340nm, vacuum freeze dry it, and store it at T-20 ℃ Preparation of LPS. Dilute LPS with sterile physiological saline to 16g/L, with a set of injection formulas of 13 μ Dilution solution+500 μ PBS+500 μ Complete Freund's adjuvant; One group is 13 μ Dilution solution+400 μ PBS+5 μ Pertussis stock solution.

　　(2) Animal sensitization: Healthy adult Wistar rats were sensitized by subcutaneous injection of 200g/kg liquid paraffin into the abdomen at a dose of 1ml per animal one week ago. Take individuals with no abnormal reactions for LPS injection.

　　(3) LPS was injected into sensitized rats. The first formulation of LPS was used for foot pad injection, with a volume of 0.4ml per animal. The second formulation was used for intraperitoneal injection, with a volume of 0.15ml per animal.

　　2. Observation indicators and analysis

　　(1) Eye symptoms: LPS injection causes severe dilation and congestion of iris blood vessels, as well as adhesion of the anterior chamber angle; Choroidal vascular hyperplasia with diffuse infiltration of blood cells, accompanied by increased white blood cells and local edema with wrinkles; Retinal and choroidal detachment, local edema and proliferation, proliferation of pigment epithelial cells in the retinal pigment layer; The local protein like exudate in the retina affects the adjacent ganglion cell layer, and the fibrous layer is masked or tends to be missing by the exudate. The protein like exudate exhibits heterogeneity, with disordered arrangement of cells in the connected ganglion layer, and fibrosis occurring in the vascular wall and surrounding tissues; The fibers in the local fibrous layer are clustered into bundles and scattered throughout, some of which experience severe fibrosis, while thrombus like substances can be seen in the blood vessels between. A series of clinically visible inflammatory reactions, with symptoms consistent with uveitis. At 4 hours, dilation and congestion of the iris blood vessels can be observed; At 8 hours, corneal edema, anterior chamber opacity, Thydull sign (+), slight exudation, blood vessels still congested, iris edema worsened, and the scope expanded; At 12 hours, the reaction reached its heaviest, with high iris edema, severe vascular dilation and congestion, visible exudation, anterior chamber hemorrhage, and visible floating flocculent material in the anterior chamber, Thydull sign (+), and reduced pupil size; At 24 hours, the above symptoms alleviate.

　　(2) Pathological examination: visible corneal epithelial edema, loose arrangement of corneal stroma, increased and congested ciliary vessels with protein like exudation, severe dilation and congestion of iris blood vessels, and anterior chamber angle adhesion; Choroidal vascular hyperplasia with diffuse infiltration of blood cells, accompanied by increased white blood cells and local edema with wrinkles; Retinal and choroidal detachment, local edema and proliferation, proliferation of pigment epithelial cells in the retinal pigment layer; The local protein like exudate in the retina affects the adjacent ganglion cell layer, and the fibrous layer is masked or tends to be missing by the exudate. The protein like exudate exhibits heterogeneity, with disordered arrangement of cells in the connected ganglion layer, and fibrosis occurring in the vascular wall and surrounding tissues; The fibers in the local fibrous layer are clustered into bundles, with some experiencing severe fibrosis, and thrombus like substances can be seen in the blood vessels between them. In addition, in the slice, it is also evident that the blood vessels in the retina are dilated and congested, especially the capillaries in the inner and outer layers, as well as a large amount of fiber wrapping around the retinal aortic wall in the optic disc. The tightly connected part of the retina shows proliferation or enlargement, and the fiber layer is fibrotic.