Research On The Expresstion Of IL-6, TNF-a, ICAM-1 And The Treatment On Experimental Evaporative Rabbit Dry Eye

Objective:The Dry Eye Syndrome is a group of dieases which is characterized by a set of alterations of the eye surface which could relate to tear quality and quantity,which entail a reduction of the stability of the tearfilm and the alteration of the eye surface.The patients always feel fatigue,foreign body sens- ation,burning,stinging,sensitivity to light,weeping,redness and so on.With the development of social economy,Current demographic changes and lifestyle factors indicate that the dry eye syndrome patient population increase significantly.Dry eye was divided into decreased tear production dry eye and over evaporative dry eye by National eye institute of American.The former include Sj?gren's syndrome and kerato- conjunctivitis sicca (KCS),while the latter include Meibomian gland dysfunction (MGD)and other dry eye caused by exposture and contact lens.Recently with the development of immunology and molec- ular biology,we have a new recognition to the cause of dry eye. Cytokine is a kind of heperactive protein molecule secreted by immune cell.Its main role is to adjust cellar function and take part in immune and inflammation reaction.A growing body of evidence suggest that chronic KCS is a complex multifactorial disease characterized by an immune and inflammatory process that affects the lacrimal glands and ocular surface.It has been confirmed that dry eye is related to the inflammation of ocular surface,is based on immune response and induced by many cytokines.The evidence for this inflammation includes increased expression of immune activation markers, such as IL-6,TNF-αand intercellular adhesion molecule (ICAM-1 ) and so on.But the pathogenesis of evaporative dry eye is still under discussing.γ-linolenic acid (GLA) is an essential fatty acids which has extensive physiological action and conspicuous pharmacological action.Recent reports show that it is possibly useful therapies for KCS,but the mechanism is unknown.Animal model is a kind of important carrier to develop clinical and basic research.In this exprimient,we establish a rabbit model in which the Meibomian gland orifices were individually closed by cauterization.We detect the concentrat- ion.of the IL-6,TNF-αin tears,corneal and conjunctival hom- ogenate fluid and the expression of ICAM-1 in cornea and conjunctiva before and after treatment.To observe the changes of the three cytokine,then identify its contribution to pathog- enesy of evaporative dry eye.To investigate GLA's effection and mechanism through comparing the variation of cytokine concentration before and after treated by GLA,thereby to provide the theoretic foundation for the application in clinic.Methods 1 Thirty New Zealland white rabbits that are 2-2.5kg in weight and sex undiscriminating were randomly divided into three groups.The first goup (10 rabbits) is normal group;the second group (10 rabbits) is control group;the third group (10 rabbits) is treated group which were treated with GLA.2 Dry eye model building:Dry eye model was established by cauterizing the meibomian gland orifices on control and treated group.Fluorescence stain,SchirmerⅠtest and break up time (BUT) were performed scheduledly before and after opera- rtion.Dry eye model was established successfully on sixth week after operation according to diagnostic criteria.3 After dry eye model was established,the treated group were treated with GLA by intragastric administration from the second day on.50mg,twice a day,5weeks.Fluorescence stain,SchirmerⅠtest and break up time (BUT) were observed scheduledly after GLA was used.4 Collecting the tears of rabbits:All tear samples were collected in graduated disposable microcapillaries from the conjunctival cul-de-sac to detect the concentration of IL-6,TNF-αon 1, 3, 6, 9,11week.5 Thirty rabbits of three groups were killed to get cornea and Conjunctiva biopsy tissue,which was made pathological secti- on.ELISA and Radioimmunity were used to detect the concentr- ation of IL-6,TNF-αin the tissue.Cornea and conjunctival epithelium was examined by immunohistochemical staining to detect the expression of ICAM-1. 6 One-way ANOVA and two-sampal T test was used to compare the differences between three groups.The statistical analysis was described by SPSS 12.0 software.Results1 According to the change of SchimerⅠtest,BUT and fluo- resceence stain before and after operation and diagnostic criteria,we can found the dry eye model was successfully made.After treated,SchirmerⅠtest wetting and BUT time in the treated group was longer than that of control group signify- ciantly.(p<0.01),The score of cornea fluorescence stain decreased significiantly comparing with control group(p<0.01).2 On six weeks after operation,The concentration of IL-6,TNF-αin control group was higher than that of nomal group (t=43.656;t=52.117),in treated group was higher than that of nomal group (t=52.503;t=51.692),there was significant differ- ence through statistical treatment(p<0.001).Comparing with treated group,there was no significant difference through statis- tical treatment(t=0.628,p>0.05).3 Tissue histopathology observation:In control group,corneal epithelium cell layer was increased,the surface flat cell was absence irregularly,substantia propria layer was edematous and thickening,cell component was increased and inflammatory cell infiltrate was observed.The arrangement of conjunctival was not regular and epithelium cell layer was increased,the conjunctival goblet cells was reduced and and inflammatory cell infiltrate was observed.After treated,the number of the conjunctival goblet cells was more than that of control group,but fewer than that of nomal group.The arrangement of cell was more regular than before and inflammatory cell infiltrate was reduced.4 The compartion of the concentration of IL-6,TNF-αon eleven weeks after operation:In rabbit tears,the concentration in control group was higher than that of nomal group (t=19.815;t=19.313),the concentration in treated group was lower than that of treated group(t=16.794;t=16.834),it had significant difference (p<0.001).The concentrations of IL-6, TNF-αin treated group was higher comparing with nomal group,there was no significant difference through statistical treatment(t=2.263;t=0.334,p>0.05).In corneal homogenate fluid, the concentration in control group was higher than that of nomal group(t=27.051;t=21.613),the concentration in treated group was lower than that of treated group(t=24.730;t=19.788),it had significant difference (p<0.001).The concentrations of IL-6, TNF-αin treated group was higher comparing with nomal group,there was no significant difference through statistical treatment(t=1.368;t=3.499,p>0.05).In conjunctival homogenate fluid, the concentration in control group was higher than that of nomal group(t=36.087; t=22.456),the concentration in treated group was lower than that of treated group(t=42.925;t=28.574), it had significant difference (p<0.001).The concentrations of IL-6,TNF-αin treated group was higher comparing with nomal group,there was no significant difference through statistical treatment(t=2.845;t=0.474,p>0.05).5 The expression of ICAM-1 was observed in three groups. Conjunctival epithelium cells appear a strong brown-yellow color in cytoplasm for ICAM-1 in three group.The expression of ICAM-1 in cornea in nomal group was 50.24±5.88/1000 cells,in control group was 272.12±46.76/1000cells, 60.34±10.21/1000cells.In conjunctival epithelium in nomal group was 60.36±15.34/1000cells,in control group was 302.67±56.28/ 1000cells,in treated group was was 74.32±21.45/1000cells.The expression of ICAM-1 in control group was stronger than that of nomal group(t=26.384,;t=30.572,p<0.001).The expression of ICAM-1 in treated group was weaker than that of control group. there was significant difference through statistical treatment (t=25.021;t=29.894,p<0.001).Conclusions1 The concentration of IL-6,TNF-αin corneal,conjunctival homogenate fluid and tears of evaporative dry eye rabbit model was higher than that of nomal rabbits.It is showed that the cytokine play an important role in the pathogenesis of evaporative dry eye.2 The expression of ICAM-1 in cornea,conjunctiva of evapor- ative dry eye rabbit model was stronger than that of nomal rabbits.It suggests that the pathogenesis of evaporative dry eye is related to T lymphocyte infiltration.3 It is showed thatγ-linolenic acid can alleviate the inflamm- ation markers on the rabbit ocular surface and restrain the action of T lymphocyte,which suggest it is effective on the treatment of over evaporative dry eye.