动物造模,干眼模型 z-bio 2021-12-27 1070

　　Background: Dry eye (DE), also known as keratoconjunctivitis sicca (KCS), is a multifactorial tear disease that can cause discomfort, visual disturbances, tear film instability, and ocular surface damage, accompanied by sex behavior. KCS is related to increased tear layer osmotic pressure and ocular surface inflammation. The prevalence of DE is between 5% and 35%, is more common in women, and increases with age. Clinical symptoms of DE include mild discomfort, dryness, foreign bodies, eye fatigue, photophobia, pain, and other symptoms such as difficulty in opening eyes and poor vision. For complex pathogenesis, different animal models can simulate the different pathophysiological mechanisms of DE. These models include genetic mouse models similar to SJ Sjogren syndrome, rat models of induced lacrimal gland inflammation, anticholinergic drugs, and rabbit models induced by meibomian gland sealing. The rabbit eye is convenient for slit lamp microscopy and is suitable for research due to its gentle nature and relatively low maintenance cost. The common method of establishing KCS in rabbits is to disable the lacrimal gland and surgically remove the harder glands and nerve membranes. In another experiment, 50% trichloroacetic acid was used to burn rabbit eyeball conjunctiva, and the lacrimal gland, harder gland and nitrifying membrane were surgically removed.

　　Method: Experimental animals and ethics statement: 15 New Zealand female rabbits, weighing 2.0-2.5 kg. Raise rabbits under standard laboratory conditions (22±2°C, relative humidity 60%±10%, light and dark cycle for 12 hours). All rabbits can eat and drink freely. This study strictly complies with scientific statements regarding the use of animals in ophthalmology and vision research. All operations were performed under general anesthesia, and 40 mg/kg ketamine hydrochloride was injected intramuscularly to minimize pain. DE model grouping and induction: 15 rabbits were randomly divided into 3 groups. In group A (n = 5), the left lacrimal gland, harder gland and nitrifying membrane were surgically removed. In group B (n = 5), 50% trichloroacetic acid was used to burn the conjunctiva of the left eye. In group C (n = 5), the left eye conjunctiva was burned with 50% trichloroacetic acid, and the lacrimal gland, Harder’s gland and nitrifying membrane were surgically removed. The right eye is a normal control group. Rinse the surface of the conjunctiva with sterile saline, disinfect the area around the surgical eye before using iodine, and perform the operation in the operating room under aseptic conditions. Instill 0.5% propakin hydrochloride eye drops, and make a curved incision in the left eye conjunctiva 5 mm long and 2 mm from the edge of the eyelid. The orbital tissue was bluntly separated, the marginal fascia of the was arch was cut, and the lacrimal gland was gently cut with curved smooth forceps. The removed gland has a complete envelope and is similar in volume and length. Cut the nitrifying membrane from the bottom between the medial rectus muscle and the anterior orbital wall of Harder’s gland. Both are removed from the incision by septumectomy and capsule. Our method of removing lacrimal glands and harder glands is simple, minimally invasive and feasible. The lacrimal glands and harder glands are reserved for routine pathological examinations. Cauterize the bulbar conjunctiva, dip a cotton swab into the freshly prepared 50% trichloroacetic acid, and act on the left eye conjunctiva 2-3 mm away from the corneal annulus for 5 seconds. Immediately flush the conjunctival sac with 100 ml of 0.9% sterile normal saline. Three days before the operation, eye drops containing 0.3% tobramycin were applied 3 times a day. The eye drops containing 0.3% tobramycin and 0.1% dexamethasone were used four times a day, and the eye drops containing 0.3% tobramycin and 0.1% dexamethasone were used once a day for 7 consecutive days. All topical drugs are applied to both eyes. On the 56th day, all animals were killed with an overdose of anesthetic (chlorpromazine). Test: Schirmer I test (SIT), corneal fluorescein staining and conjunctival blot cytology were performed on the 14, 28, 42 and 56 days after the model was established. All tests are performed by the same person using standard techniques under the same environmental conditions.

　　Tear secretion test (SIT): Without anesthesia, insert Schirmer's test strip into the subconjunctival sac and close both eyes tightly. Record the wet length (in millimeters) after 5 minutes. After all tests, the rabbit was fixed in the holding device and kept quiet. Fluorescein staining: Inject a drop of 1% fluorescein solution, and observe the eyes of all animals with a slit lamp microscope and a cobalt blue filter at 16 times magnification. According to the grading criteria described for corneal fluorescein staining. The cornea is divided into four quadrants. The intensity of staining in each quadrant ranges from 0 to 3 (the highest score is 12). The following criteria are used for scoring: 0 points for no corneal punctate staining, 1 point for 1-10 punctate staining, 2 points for 11-30 punctate staining, 30 points for punctate staining or 3 for cluster staining Minute. Scores 1 to 4 are defined as mild, scores 5 to 8 are considered moderate, and scores 9 to 12 are considered severe.

　　Conjunctival imprinting cytology: Inject 0.5% Propakin hydrochloride eye drops to wipe away excess tears, and then gently place 0.45 micron nitrocellulose filter paper on the outer surface of the 2 mm corneal ring of the temporal bulbar conjunctiva. Apply light pressure for 5-10 seconds. Then remove the filter paper and immediately put it in freshly prepared 4% formaldehyde for at least 30 minutes. The filter paper will change color due to periodic periodic acid changes: (1) Periodic acid is oxidized for 10 minutes, (2) Rinse with tap water for 5 minutes, (3) Rinse once with distilled water, (4) Rinse with magenta for 25 minutes. Dyeing, (5) reduce sodium metabisulfite for 5 minutes, (6) rinse with tap water for 5 minutes, (7) dye with hematoxylin for 5 minutes, (8) decolorize with 1% hydrochloric acid-alcohol for 30 seconds to 1 minute, (9) use Rinse with tap water for 5 minutes, (10) dehydrate 95% of the substance with 100% ethanol for 1 minute, (11) wash with xylene for 20 minutes, and (12) permanently install the slide. Observe the cell morphology with a 40X microscope. All samples will be evaluated by the same person. Average number of different parts of goblet cells/10. Expressed as the density of goblet cells. Three samples of each rabbit are evaluated. Optical microscope: After the animals were euthanized, a 5 mm wide cornea and upper conjunctiva were taken out of the rabbit’s eyes and fixed in a 10% formaldehyde solution. After dehydration, the specimens were embedded in conventional paraffin, sectioned and stained with HE. Observe the morphology of the cornea and conjunctiva under a 40X microscope. In the established DE model, the lacrimal gland and harder glands were also removed by the above-mentioned method, and checked by the above-mentioned method.

　　Statistical analysis: The data results are based on the mean standard deviation (SD). Use SPSS16 version to perform statistical analysis on the data.

　　Result: tear secretion test: there was no significant difference in the wet length between the left eye and the normal control group before the operation (P\→0.05). In the 28-day, 42-day, and 56-day studies, the SIT of the AIT and B groups was significantly lower than the preoperative measurement. Similarly, SIT in group C was significantly lower than before surgery at all time points (14, 28, 42, 56 days). However, there was no significant difference in SIT between the two groups at 28, 42 and 56 days. On days 28, 42, and 56, the SIT of the left eye of each group was significantly lower than that of the control group (right eye). The SIT of group C is also very low, only 14 days. On the 28th, 42nd and 56th days, the SIT of the left eye of group C was significantly lower than that of the left eye of group B. However, at any given time, there was no significant difference between group A and group B.

　　Corneal fluorescent staining: Perform slit lamp test at baseline, and then check again every 14 days. It was pointed out that in all three groups, the corneal fluorescein staining of the operated eyes was abnormal. Before the model was established, none of the eyes had corneal punctate staining, and there was no significant difference in the scores of the left and right eyes before the operation. Compared with baseline, corneal fluorescein staining in group A showed significantly higher scores at 28, 42 and 56 days. In groups B and C, the fluorescein staining score was higher than baseline on days 14, 28, 42 and 56. On day 42 and day 56, there was no difference in fluorescein staining scores between the left eye groups. Compared with the eyes of the contralateral control group (right), the eyes of group A had higher fluorescein staining scores at 28, 42 and 56 postoperatively. The 14-day scores of groups B and C were also significantly higher than those of the control group. At 14 days, 28 days, 42 days and 56 days after operation, the score of group A was significantly lower than that of group C, while the score of group B was significantly lower than that of group C on 42 and 56 days. At 42 days and 56 days after surgery, the fluorescein staining score of the eyes of group A was also significantly lower than that of group B. Therefore, 56 days after the operation, the average scores of corneal fluorescein staining in group A (3.8±1.30), group B (7.4±1.14) and group C (10.8±1.30) were light to moderate.