Effect Of Estrogen And Androgen On The Expresstion Of IL-6 In Lacrimal Gland, Cornea And Tear And Histological Change In Ovariectomized Rat

Objective: Dry eye is one of the leading reasons for patient visits to ophthalmologists, and it occurs frequently in peri-menopausal women. But we still don't know its pathogenesis so far. Most researchers believe that dry eye in peri-menopausal women is related to the change of the serum estrogen and androgen levels. So we made the model of ovariectomized rat to simulate artificial menopause state, and treated the rats with systemic or eyedrop application of estrogen and androgen. Meanwhile we examined the rats'serum estrogen and androgen levels,tear fluid secretion,the stability of tear-film and the content of Interleukin-6(IL-6)in different tissues to investigate the effect of estrogen and androgen on the pathogenesis and therapy of dry eye in peri-menopausal women, and to make experiment evidence and rationale for the treatment of estrogen and androgen to dry eye.Methods: 64 female healthy Wistar rats, BW:230-250g, were divided into normal group(8 rats), sham operation group(8 rats) and experimental group(48 rats) randomly. All the rats in the experimental group had been done with ovariectomy(OVX), and 5 months after OVX, they were divided into 6 groups randomly [Group C: with no treatment,Group D: with corn oil injected hypodermically every 3 days (100ul/day),Group E: with Estradiol Benzoate injected hypodermically every 3 days (200ug/kg),Group F: with Estradiol Benzoate treated locally 4 times/day,Group G: with 2.5% testosterone intramuscular injected every 3 days (3.75mg/kg) and Group H: with 2.5% testosterone treated locally 4 times/day]. Each group had 8 rats. Tear fluid secretion (Schirmer test),tear film break-up time(BUT) and fluorescence staining of the cornea were measured in all groups before and 1,2,3,4 and 5 months after the operation. Then six weeks later, Schirmer test, BUT and corneal fluorescence staining were measured again. Then all rats were sacrificed, lacrimal gland and cornea were collected,and their morphosis was observed by microscope and electron microscope. The expression of IL-6 of lacrimal gland and cornea were detected by ELISA in different groups. The serum estrogen and androgen levels were examined before and 5 months after OVX and after being treated for 6 weeks. All statistical analyses were performed with SPSS 13.0. One-way ANOVA and two-sample T test were used for statistical evaluations. A p value of <0.05 was considered statistically significant.Results :1 The result of BUT in the experimental group 1 month after OVX was shortter than that before OVX,P<0.01. The result of BUT in the group with Estradiol Benzoate injected hypodermically for 6 weeks was shortter than that before injection,P<0.01. While the BUT of the group with testosterone intramuscular injected for 6 weeks was longer than that before injection,P<0.05. There was no difference in other groups (P>0.05).2 The result of Schirmer test 3 months after OVX decreased to 50% of the result before OVX,P<0.01. The result of Schirmer test in Group E was shorter than that before injection. P<0.05.The result of Schirmer test in Group G was longer than that before injection, P<0.05. There was no difference in other groups (P>0.05).3 The score of corneal fluorescence staining 4 months after OVX increased compared with that before OVX,P<0.05. The score of corneal fluorescence staining in Group E increased compared with the score before injection. P<0.05. The score of corneal fluorescence staining in Group G decreased compared with that before injection. P<0.05. There was no difference in other groups (P>0.05).4 Histopathology observation of the lacrimal gland: The lacrimal gland epithelium of Group C was small with HE stained cytoplasm lightly and some vacuolus. The size of nucleolus was unequal. Vasodilatation and a lot of coalesced vacuolus could be seen in the lacrimal gland of Group E .Many gland alveolus coalesced and the ultrastructure became disordered. In Group G, the size of lacrimal gland epithelium and their nucleolus was equal, and vacuolus could hardly been seen. The other experimental groups didn't have marked change compared with Group C.5 Histopathology observation of the cornea: Hyperplasia of disordered corneal epithelium could be seen in Group C, The superficial cells were absent irregularly and the wing cells and basal cells became disordered. The stroma was a little dropsica. Hyperplasia of disordered corneal epithelium became serious and more superficial cells were absent in Group E. The wing cells and basal cells became disordered significantly. The stroma was seriously dropsica. From Group G, the corneal epithelium and basal cells aligned regularly, with less epithelium absent. the dropsy of stroma disappeared and the thickness of the dropsy became normal. The other experimental groups didn't have marked change compared with Group C.6 Histopathology observation of the conjunctiva: Hyperplasia of disordered conjunctival epithelium could be seen in Group C, and the cells of basilar membrane became disordered too. Hyperplasia of disordered conjunctival epithelium became serious and the cells of basilar membrane became more disordered, and inflammatory cell infiltrating could be seen in Group E. In Group G, the conjunctival epithelium and the cells of basilar membrane aligned regularly, hyperplasia of the conjunctival epithelium lessened.and less inflammatory cells could be found. The other experimental groups didn't have marked change compared with Group C.7 The ultrastructure of lacrimal gland detected by electron microscope: Broadened perinuclear space could be seen in Group C, and mitochondria was swollen with no mitochondrial membrane and crista coalesced. Some lipofuscin could also be seen. While in Group E , perinuclear space broadened significantly, and rough endoplasmic reticulum expanded seriously. there was a lot of vacuolization in mitochondria, and there was more lipofuscin. No broadened perinuclear space could be seen in Group G. Rough endoplasmic reticulum arranged parallelly expanded slightly. Mitochondrial membrane and crista coalesced partly, and relatively normal mitochondria and mitochondrial crista could be seen. The other experimental groups didn't have marked change compared with Group C.8 The serum estrogen and androgen levels after OVX decreased compared with those before OVX.After estrogen or androgen injected, both the serum estrogen level in Group E and the serum androgen level in Group G were elevated.9 The expression of IL-6 in Rats'lacrimal gland of Group C was more than that in Group A (P<0.01). The expression of IL-6 of lacrimal gland in Group E was more than that in Group C (P<0.05). The expression of IL-6 of lacrimal gland in Group G decreased significantly compared to Group C (P<0.05). There was no difference between the other groups and Group C (P>0.05). 10 The expression of IL-6 in Rats'cornea of Group C was more than in Group A (P<0.01). The expression of IL-6 of cornea in Group E was more than in Group C (P<0.01). The expression of IL-6 of cornea in Group G decreased significantly compared to Group C (P<0.01). There was no difference between the other groups and Group C (P>0.05).11 The expression of IL-6 in Rats'tear of Group C was more than Group A (P<0.01). The expression of IL-6 in Rats'tea in Group E was more than in Group C (P<0.01). The expression of IL-6 of cornea in Group G decreased significantly compared to Group C (P<0.05). There was no difference between the other groups and Group C (P>0.05).Conclusion:1 The experimental dry eye model of female rat is made successfully by ovariectomy. Both the levels of serum estrogen and androgen of the rats with dry eye by OVX decrease compared with that before OVX. Androgen injection increase tear fluid secretion and stabilize tear film. While estrogen injection makes reverse effect. So it infers that androgen deficiency will result in dry eye.2 The concentration of IL-6 in lacrimal gland, cornea and tear of the ovariectomized rat is higher than the normal. It indicates that the pathogenesis of the Rats'dry eye by OVX relates to inflammation,and the Interleukin-6 participates in it.3 The concentration of IL-6 in lacrimal gland ,cornea and tear decreases after androgen injection. It shows that androgen may be effective on the treatment of dry eye by palliating the inflammation of ocular surface.4 Estrogen injection can aggravate the inflammation of dry eye, reduce tear fluid secretion and break the stability of tear film, so the use of hormone replacement therapy should be careful in the clinical work.