动物造模,药效评价,角膜炎 z-bio 2024-09-23 350

　　Acanthamoeba keratitis is a rare and severe chronic progressive corneal disease. The infection of this disease is originally caused by Acanthamoeba, and more than 80% of patients are caused by wearing contaminated corneal contact lenses. Deng Xinguo et al. selected healthy New Zealand rabbits, injected dexamethasone under the conjunctiva of the rabbits three days before the experiment, and applied hormone eye drops. Then, the trophozoites and capsule suspension of Acanthamoeba were injected into the corneal stroma of the rabbits, successfully establishing an animal model of Acanthamoeba keratitis in rabbits. This model has the advantages of fast construction, simple technology, and easy operation, and is an important tool for experimental research on Acanthamoeba keratitis.

　　1. Experimental animal: New Zealand white rabbit.

　　2. Cultivation of Acanthamoeba: Acanthamoeba was isolated from corneal material surgically removed from patients with keratitis. The culture medium is mainly composed of peptone yeast glucose (PYG), and contains penicillin (500U/ml) and streptomycin (500U/ml). After culturing Acanthamoeba for 3-5 days, wash the cultured Acanthamoeba three times with physiological saline (centrifuge at 300r/min for 5 minutes each time), and finally adjust the concentration of protozoa to 3 × 105 cells/ml with physiological saline (80% -90% trophozoites, 10% -20% cysts); And the vitality of the protozoa was determined by staining with trypan blue, and the vitality of the protozoa was>90%.

　　3. Model making method: Before the experiment, the 3D animal model eye was first subconjunctival injected with 2mg of dexamethasone, 1/d, and at the same time, tobramycin dexamethasone (Dianbishu) eye drops were applied 3-4/d. After the experiment began, the hormones were stopped. After anesthetizing the animals, 0.2ml of Acanthamoeba suspension was injected into the corneal stroma of the model eye using a 0.5ml insulin syringe with a 28 or 30 gauge needle.

　　4. Observation of Results

　　(1) Clinical observation: The infected model eye and left eye all developed corneal stromal inflammation. Secretions appeared in the conjunctival sac within 12 hours, and there was an inflammatory reaction at the injection site; There is a significant amount of external eye discharge within 24 hours, with the formation of inflammatory plaques in the local area and congestion of the conjunctiva and conjunctiva; Symptoms gradually worsen from 3 to 14 days, inflammatory plaques gradually increase, the entire cornea presents as diffuse punctate opacities, there is more secretion from the outer eye, and congestion of the conjunctiva and eyelid conjunctiva becomes more severe. Asymptomatic cessation of development within 15-30 days, gradual reduction of external eye secretions, slight alleviation of congestion in the conjunctiva and eyelid conjunctiva, and initiation of neovascularization into inflammatory plaques.

　　(2) Corneal scraping and light microscopy examination: In the infected model eye, double walled wrapping of Acanthamoeba parasites can be seen.

　　(3) Cultivation examination of Acanthamoeba: Place the cornea of the model eye into the central surface of 2% nutrient free agarose culture medium, drop 1-2 drops of live Escherichia coli broth onto the inoculum, seal with tape, and incubate in a 35 ℃ incubator for Acanthamoeba cultivation. Infection with the model eye can cultivate Acanthamoeba.

　　(4) Corneal pathological examination: Giemsa staining was performed on corneal pathological sections of the model eye, and a large number of inflammatory cell infiltration (mainly neutrophils) was observed in the model eye; PAs staining showed a large number of cysts and trophozoites of Acanthamoeba parasites in the affected area of the model eye.