动物造模,药效评价,变应性结膜炎 z-bio 2024-08-02 392

　　The main mechanism of ocular hypersensitivity is type I hypersensitivity mediated by mast cells. Hericidal conjunctivitis, vernal keratoconjunctivitis, atopic keratoconjunctivitis, and macromastic keratoconjunctivitis are mainly involved in type I allergic reactions. There are many methods used to induce type I allergic reactions, and here we mainly introduce the active allergic conjunctivitis induced by ovalbumin. Inject ovalbumin into the abdominal cavity of experimental animals to produce IgE antibodies, which actively sensitize them by binding to Fe receptors in mast cells in conjunctival tissue. When antigens are used for local attacks, activated mast cells degranulate and release allergenic mediators, leading to increased permeability of local blood vessels and accompanied by infiltration of local eosinophils. Injecting Evans blue can infiltrate into the eye tissue, causing tissue edema. Based on the degree of conjunctival edema, eosinophil infiltration, degranulation of mast cells, and optical density of conjunctival tissue extract, changes in vascular permeability are determined to reflect the degree of conjunctival hypersensitivity. DL dithiothreitol (DDT) is a mucolytic agent that can disrupt the mucosal barrier function of the conjunctiva, allowing large molecular substances such as ovalbumin to penetrate the conjunctiva and bind to IgE antibodies in the tissue, enhancing ocular hypersensitivity reactions.

　　[Modeling Method] Rats or guinea pigs were immunized by intraperitoneal injection of 1ml of ovalbumin phosphate buffered saline (PBS) (containing 100 μ g of ovalbumin, 10-20mg of potassium aluminum sulfate, pH 7.4). After 14 days, administer 10-20 μ l of 1mol/L DDT to each eye to eliminate the conjunctival mucus barrier and enhance the effectiveness of the attack. After 15 minutes, inject 1ml of (0.125mg/100g) solution intravenously into the animal for assessing changes in vascular permeability. Immediately administer 10 μ l of 5% ovalbumin PBS solution for eye attack; For animals with clinical and histological changes, only 10 μ l of 5% ovalbumin PBS solution was used for eye attack without injection of Evans blue. 30 minutes after local antigen attack, tissue response was assessed under slit lamp and quantified using the following scoring method (up to 10 points per eye):

　　1. Congestion (eyelid conjunctiva and conjunctiva) 0: normal; 1: The conjunctiva is pink in color; 2: The conjunctiva is red in color; 3: The conjunctiva is dark red with petechiae and ecchymoses.

　　2. Edema 0: None; 1: Only lower eyelid edema; 2: Both upper and lower eyelids are swollen, with partial closure of the eyelids; 3: Eyelid eversion, obvious edema, eyelids at least partially closed; 4: Both the upper and lower eyelids, as well as the eyelid margin, are swollen.

　　3. Secretion 0: None; 1: Dilute viscous liquid; 2: The eyelids and surrounding hair are damp; 3: The eyelids and surrounding hair are damp and sticky.

　　Determination of vascular permeability: 20-30 minutes after antigen attack, animals are euthanized, eyelids, conjunctiva, and eyeballs are removed, adipose tissue is removed, and weighed. Place the tissues in a mixture of 0.5% sodium sulfate and acetone (3:7 mixture) of 5-10ml, shake at room temperature, and after 24 hours, centrifuge the solution at 300r/min for 10 minutes. Measure the optical density of the supernatant at 620nm using a spectrophotometer, and convert it into Evans blue content μ g/g based on the weight of the tissues.

　　Histological examination: 30 minutes after antigen attack, the upper conjunctiva was excised to prepare a light microscopy specimen. The tissue was fixed in Karnovsky fixative, dehydrated with ethanol gradient, embedded with isobutene glycol, sliced to a thickness of 2 μ m, stained with H&E, PAS, and alkaline Giemsa, and observed under light microscopy. Count mast cells and eosinophils in conjunctival epithelium, subepithelial tissue, and stromal tissue using a 0.25mm × 0.025mm mesh counting ruler in three different fields of view.

　　【 Model Characteristics 】 Positive histological examination shows a large infiltration of eosinophils in the conjunctival epithelium and stroma, and a significant increase in degranulated mast cells; The ratio of degranulated mast cells to the total number of mast cells significantly increased.

　　[Model Evaluation and Application] This method is a commonly used screening method for anti type I hypersensitivity drugs. The method is objective, simple, rapid (obvious conjunctivitis symptoms appear 10 minutes after attack, peak at 30 minutes, and disappear after 1 hour), and quantitative. The screening results are basically consistent with clinical efficacy. The administration route and administration time are determined by the pharmacological and pharmacokinetic characteristics of the test drug.