| BackgroundDry eye is a class of diseases due to the tear film instability and (or) ocular surface damage caused by abnormal tear quality and (or) the amount or abnormal fluid dynamics, leading to visual impairment and eye symptoms. Is one of the highest incidence of ocular surface disease present, but its pathogenesis is more complex Dry eye is currently being considered with inflammation, the level of sex hormone imbalance, abnormal neural regulatory mechanisms, apoptosis and other factors. Sex hormone which plays in an important role in the pathogenesis of dry eye.Recent studies show that androgens have a more positive role in the pathogenesis of dry eye which can improve the lacrimal gland atrophy,to promote the lacrimal gland cell proliferation, alleviate ocular surface damage, promote tear secretion, improve tear film stability, protect lacrimal gland from inflammation. It has confirmed that androgen receptors are present in the meibomian glands, lacrimal gland and cornea and conjunctiva, etc.Flutamide ia s non-steroidal anti-androgen drug, in addition to anti-androgen effect, this product no other effect. Its main active form is metabolites small hydroxy-flutamide, capable of binding with the androgen receptor in the target tissue, blocking dihydro-testosterone (androgen active form) and androgen receptor binding, inhibition of target tissue up take testosterone, which play a role in anti-androgen.Animal models of dry eye an important means to research the incidence and development of dry eye disease, the establishment of an appropriate animal model according to the different pathogenesis of dry eye is the basis of experimental studies, In domestic and foreign there is few studies about the relationship between dry eye application of anti-androgen drugs and ocular inflammatory factors, and also no report about making dry eye model induced by androgen receptor antagonist.Thus,the present study is to establish rat dry eye model induced by androgen receptor antagonist flutamide, observe the tear volume (S I t), tear film break-up time (BUT test), pathological changes of ocular surface; detect the expression of proinflammatory factors TNF-a and IL-la of the cornea, conjunctiva, lacrimal and meibomian gland epithelium;explore the main pathogenesis of androgen receptor antagonist flutamide leads to dry eye, analysis the science and practicality of the model, and provide a theoretical basis for the treatment of dry eye.Part 1 Establishment of rat dry eye model induced by flutamideObjectiveTo establish rat animal models of dry eye induced by androgen receptor antagonist flutamide,provide a basis for further study of the pathogenesis about androgen and dry eye.MethodClean healthy adult female SD rats were randomly divided into high concentration group,medium concentration group, low concentration group and control group,8 cases in each group. high,medium, low concentration group were respectively irrigated stomach with flutamide solution on concentration of 16mg·kg-1,13mg·kg-1,10mg·kg-1, control group were irrigated stomach with lml saline solution every day. Each group before and after administrate on week 2,4,8,16,20 were evaluated dry eye clinical indicators:the tear volume(Schirmer I test, S I t),tear break-up time (BUT) and corneal fluorescence staining score. Successful model standard:on week 20, each index of high,medium, low concentration group compared with the control group differences were statistically significant. Each group before and after administrate on week 10,20 were measured serum testosterone and estradiol concentrations by radioimmunoassay.Use SPSS 19.0 statistical software data analysis, descriptive statistics values X±S, P<0.05 was considered statistically significant.Result1. Schirmer I test:On week 4, the S I t of high(8.20±0.58mm),medium (8.53±0.34mm)concentration groups began to decrease,compared with control group(9.84±0.54mm), difference was significant(P<0.05);on week 8, the S I t of high(7.13±0.25mm),medium(7.04±0.47mm), low(8.43±0.67mm) concentration groups were shorter than control group(9.94±0.43mm) (P<0.05); With time, on week 20, the SIt of high(3.23±0.31mm),medium(3.98±0.36mm), low(4.73±0.18mm) concentration group further shortened, there were significant differences (P <0.05),respectively compared with control(9.60±0.60mm) group decreased more than 50%(P<0.05).2. BUT:On week 4,BUT of high concentration group(6.80±1.05s) began to shorten, respectively compared with low concentration group(8.63±0.65s), control group(9.25±1.03s), there were significant difference (P<0.05); on week 8,BUT of high(5.20±0.58s),medium(7.21±0.56s), low (8.32±0.37s)concentration groups were shorter than control group(10.70±1.07s) (P<0.05); on week 20,BUT of high(2.40±0.61s),medium(4.32±0.68s), low(6.10±0.81s) concentration groups were shorter than control(9.12±0.73s) group, there were statistical differences among each group (P<0.05).3. Corneal fluorescein staining sodium:On week 4,high,medium concentration groups appeared corneal epithelial punctate fluorescein coloring; on week 8, the sodium fluorescein corneal staining scores of high(6.80±0.79), medium (4.13±0.53)concentration groups respectively compared with low concentration group(2.50±1.08) and control group(0.56±1.30) were statistically significant differences (all P<0.05).on weekl6,high concentration group with diffuse large sheet coloring and medium, low concentration groups had small pieces of corneal epithelial coloring; with time the sodium fluorescein corneal staining scores of high(9.05±0.49), medium (7.21±0.57)and low (4.82±0.47)concentration groups gradually increased, on week20, there were statistical differences among each groups (all P<0.05).4. Sex hormone levels:On week 10, the serum testosterone concentration of high (4.38±0.68pg·ml-1), medium (3.56±0.53pg·ml-1) concentration groups started to rise,compared with control group (2.53±0.24pg·ml-1) respectively, the differences were statistically significant (P<0.05). On week 20, the serum testosterone of high (5.08±0.21pg·ml-1), medium (4.23±0.16pg·ml-1) and low (3.58±0.24pg·ml-1) concentration groups compared with the control group (2.24±0.15pg·ml-1) increased to varying degrees, where high concentration group increased most significantly, pairwise comparison, the differences were statistically significant (P<0.05). On week 10,20, the differences of the serum estradiol concentration of high, medium and low concentration groups compared with control group had no significant differences (P> 0.05), no statistically significant differences between each groups (P> 0.05).ConclusionsIrrigated stomach with AR antagonist flutamide solution can successfully established mixed type dry eye model and the the severity of dry eye in a time and dose-dependent manner.Part 2 Study on mechanism of rat dry eye model induced by flutamideTo explore the mechanism of rat dry eye model induced by flutamide.MethodExperimental animals and groups the same as part1.On week 20, all animals were sacrificed under anesthesia, take the cornea, conjunctiva, lacrimal and meibomian gland epithelial tissue HE staining to observe the histopathological changes and detect the expression of proinflammatory factorsTNF-a, IL-la in the epithelial tissue use immunohistochemical methods.Each slice randomly selected five horizons 200 times photographed,use Image-Pro Plus software for count the positive cells,using SPSS 19.0 statistical software data analysis, descriptive statistics expressed the X±S, expression of TNF-a, IL-la analysis with one-way ANOVA, P<0.05 was considered statistically significant.Result1.Corneal tissue pathological observation:High, medium and low concentration groups with varying degrees of corneal squamous cell levels increased significantly, disorganized and irregular visible surface cells missing, wing-shaped cell and basal cell disorder, stroma edema, high concentration group the most significant changes. Corneal epithelial cells in the control group compared with the normal level flat surface cells without keratosis, wing-like epithelial cells were polygonal, basal cells arranged in neat rows.2. Lacrimal gland pathological observation:High, medium and low concentration groups with varying degrees of lacrimal gland epithelial atrophy flat small and dense, lightly stained cytoplasm, vacuoles, cell disorder, glandular expand. Acinar atrophy fusion, sizes, disordered structure, high concentration group the most significant changes. The control group showed the substance of connective tissue depth is divided into lobules glandular tissue boundaries clear and uniform rules acinar cell size, inner layer of columnar epithelial cells, located at the bottom of the core group.3. Meibomian gland pathological observation:High, medium and low concentration groups varying degrees meibomian gland atrophy fusion, squamous metaplasia, glandular wall thickening, thinning basal cells, fibrosis around the duct and pit expansion. the control group meibomian gland visible clear boundaries and glandular cells, the size of the uniform rules of neat arrangement, duct form a concentric wall thickness is normal.4. Conjunctival tissue pathological observation:High, medium and low concentration groups showed an increase in varying degrees of conjunctival epithelial cell layers between the level fuzzy, like derangement was keratosis, basal cells irregular arrangement, infiltration of inflammatory cells. The control group conjunctival epithelial cells arranged regularly, without keratinization and inflammatory cell infiltration.5. Expression of TNF-a, IL-la in cornea:Mmunohistochemistry showed TNF-α, IL-1α of high, medium and low concentration groups expressed large amount in corneal epithelial cells and little expressed in stroma, expressed micro in the control group. The counts of positive cells of TNF-a, IL-1α of per 1000 corneal epithelium expressed in high, medium and low concentration groups significantly higher than control group (P<0.05). The expression of TNF-a, IL-la of high, medium concentration groups was relatively higher than low concentration group (P<0.05).6. Expression of TNF-a, IL-la in lacrimal gland:Mmunohistochemistry showed TNF-a, IL-1α of high, medium and low concentration groups expressed large amount in lacrimal gland acinar epithelial cells, expressed small amount in the control group. The counts of positive cells of TNF-a, IL-la of per 1000 lacrimal gland cells expressed in high, medium and low concentration groups significantly higher than control group (P<0.05). the positive cells counts of low concentration group of TNF-a less than high, medium concentration groups (P<0.05).The expression of IL-la between high, medium and low concentration groups the differences was statistically significant (P<0.05).7. Expression of TNF-a, IL-la in meibomian gland:Mmunohistochemistry showed TNF-a, IL-1α of high, medium and low concentration groups expressed large amount in the cytoplasm of meibomian gland acinar cells, TNF-a, IL-la expressed small amount in the control group. The counts of positive cells of TNF-a, IL-la of per 1000 meibomian gland acinar cells expressed in high, medium and low concentration groups significantly higher than control group (P<0.05). The expression of TNF-a, IL-la of high, medium concentration groups was relatively higher than low concentration group (P<0.05).8. Expression of TNF-a, IL-la in cornea:Mmunohistochemistry showed TNF-a, IL-la expressed in the conjunctiva of each groups, TNF-a, IL-la of high, medium and low concentration groups expressed large amount in conjunctival epithelial cells, micro-expression in the lamina propria, small amount expressed in the conjunctival epithelial cells of control group. The counts of positive cells of TNF-a, IL-la of per 1000 conjunctival epithelial cells expressed in high, medium and low concentration groups significantly higher than control group (P<0.05). The expression of TNF-a between high, medium and low concentration groups the difference was statistically significant (P<0.01).The expression of IL-la of high concentration group was significantly higher than low concentration group (P<0.05). high concentration group compared with medium concentration group and medium concentration group compared with low concentration group,the differences were not significant (P>0.01).Conclusions1.Flutamide can cause pathological changes in rat cornea, lacrimal gland, meibomian gland, conjunctiva tissue,and the pathological changes and drug concentration were positively correlated.2. Flutamide induced dry eye were related with the expression of TNF-a, IL-la increased in cornea, lacrimal gland, meibomian gland, conjunctival tissue. |
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