The Research In Establishment Of Rabbit Tear-Deficient Dry Eye Model And Treatment Mechanism Of FK506

PartⅠAn experimental of rabbit tear-deficient dry eye model by subcutaneous injection of scopolamine hydrobromideObjective To evaluate the characteristic of rabbit tear-deficient dry eye model by subcutaneous injection of scopolamine hydrobromide and explore the mechanism of ocular surface.Methods The rabbits were randomly divided into three groups, 6 rabbits in each group. Subcutaneous injection of scopolamine hydrobromide was made in experimental group including 1.0mg in low dose group, 2.0mg in high dose group and normal saline in control group, at 8am, 11am, 14pm and 18pm each day for 30 days. Schirmer I test, tear membrane break-up time( BUT), corneal fluorescein staining and conjunctival impression cytology test with PAS staining to evaluate the phenotype of epithelia cells were performed before and 7,14,21,30 days after injection. On the thirtieth day, cornea, conjunctiva and lacrimal glands tissues biopsy were carried out for H-E staining and electron microscope observation, and expression of cytokine Fas BCL-2 and NFκB were detected by immuno- histochemistry.Results Schirmer I test by phenol red thread test of H group was 16.25±2.299mm before injection and decreased to 6.75±1.982mm on the 21st day, with a statistically difference(p<0.05) compared to 16.50±2.619mm of the control group ,however, it was 15.00±2.390mm of L group with no statistically difference .BUT decreased at the 3rd day in high dose group and the 7th Day in low dose group after which it kept shorter than 5s, BUT kept greater than 20s in control group .Flurescein staining of corneal were found in two experimental groups. It was initially observed in H group at the 3rd day and last for a long time. Compared with the control group, the morphology of goblet cells were irregular and nucleus were slightly stained, some of which were pyknotic in two experimental groups. There was no significant difference between H and L group. H-E stainning showed that lymphocyte dispersedly distributed around lacrimal gland,apoptosis in glandular cavity, severe tumor-like hyperplasia in corneal epithelium, degeneration with edema in corneal stroma layer, conjunctival goblet cell increased reactively and increased the number of cell infiltration in lamina propria in H group, Slight hyperplasia, cell disarrangement in corneal and mild histopathology changes in conjunctiva and lacrimal gland. The decreased microvilli of lacrimal glandular and corneal, conjunctival epithelium cell with cell necrosis and nucleus pyknotic compared with the control group were observed by transmission electron microscope. BCL-2 was seldomly expressed in the tissues biopsys of experimental group and fas and NFκB was in a high level compare with he control group. Conclusions Subcutaneous injection of scopolamine hydrobromide, 2mg every time for 4 times a day can effectively resulting in lacrimal secretion decrease and damage to epithelium of coneal and conjunctiva, which mainly caused by tear deficient and apoptosis and inflammation may also participate in.PartⅡthe effect of FK506 in the treatment of rabbit tear-deficient dry eye model by subcutaneous injection of scopolamine hydrobromideObjective To observe the therapeutic effect of FK506 eye drops to rabbit tear-deficient dry eye model by subcutaneous injection of scopolamine hydrobromide .Methods The rabbits were randomly divided into experimental group 18 and control group 12. Subcutaneous injection of scopolamine hydrobromide 2.0mg was made in experimental group at 8am, 11am, 14pm and 18pm each day .And since the day 4, the left eyes of the experimental group rabbits were treated with 0.5%FK506 eye drops four times each day,and,, the control group was injected with physiological saline.Methods Schirmer I test, lacrimal membrane break-up time( BUT) and corneal fluorescein staining were measured in 3,7,14,21,35 days after injection. The level of IL-1β,TNF-αand MMP-9 mRNA was evaluated by real-time polymerase chain reaction in corneal, conjunctival and lacrimal gland. At day 35,we used impression cytology to obtain corneal, conjunctival epithelium cells and detected NFκB expression in which by immunohistochemistry.Results The corneal fluorescein staining were observed in both eyes of model rabbits, there is no statistically difference at day 3,7 but the level of corneal fluorescein staining was much lower in the eyes treated with 0.5%FK506 than the other side since day 14, with a statistically difference(p<0.01). At day 35, NFκB expression in untreated side was significantly positive with high levels of IL-1β,TNF-αand MMP-9 mRNA in corneal and conjunctival,compare to the treated eye which NFκB was seldomly expressed with low level of cytokines , with a statistically difference(p<0.05).The expression of MMP-9 was increased in both side of rabbit eyes in day 14,with no statistically difference.Conclusions Subcutaneous injection of scopolamine hydrobromide, 2mg every time for 4 times a day can effectively resulting in rabbit dry eye model that with high level of IL-1β,TNF-αand MMP-9 mRNA and significantly positive expression of NFκB in corneal, conjunctival and lacrimal gland.FK506 can probably inhibit activation of inflammatory cytokines and improve the symptoms of dry eye.