The Role Of Anti-TNF-α Antibody In The Treatment Of Experimental Endotoxin Induced Uveitis In Rats

ObjectiveBecause the studies of human uveitis are limited by many factors, so the animalmodels of uveitis are used widely for studing. We established the endotoxin induceduveitis (EIU) disease model in Wistar rats with lipopolysaccharides (LPS) in presentstudy and this afford for a worthy experimental study of pathogenesis and therapy ofuveitis. Recently, anti-tumor necrosis factor (TNF) has been used to treat manyautoimmune diseases, and got an exciting outcome. The aim of present study was toinvestigate the effect of systemic injections of anti-TNF-αantibody on the levelTNF-α, IL-6 and IL-10 in sera and eye fluid of EIU in the rats, and to evaluate thetherapeutic role of this agent against EIU and observe the disease scores andhistopathologic levels after inhibiting TNF-α.Methods1. The establishment of endotoxin induced uveitis disease model: 15 Wistar ratswere divided into healthy group and experimental group. Lipopolysaccharides (LPS)were dissolved in sterile, pyrogen-free 0.9% saline at a concentration of 2 mg/ml, and75μl was injected into each hind footpad of Wistar rats in experimental group (300μgtotal dose per rat). The healthy group was injected with 0.9% saline in same volume asLPS. Carefully following-up, the clinical evaluation and histopathologic study wereevaluated. At the end of 24 hours and the third day after immunization, the animalswere killed, and the eyeballs were excised to make pathological sectionconventionally. 2. The role of anti-TNF-αantibody in the treatment of experimental endotoxininduced uveitis in rats:(1) Additional 54 Wistar rats are used to study the role of anti-TNF-αMcAb. Therats were divided into three groups, including the healthy control group, EIU groupand anti-TNF-αMcAb therapy group.(2) Healthy control group was injected same volume of 0.9% saline as LPS, LPSwas injected into each hind footpad of Wistar rats in EIU group and anti-TNF-αMcAb therapy group. Anti-TNF-αMcAb at 1μg/kg were administered one time viaintravenous injection through the tail vein at 0.5 hour before LPS injection inanti-TNF-αMcAb therapy group.(3) Clinical presentation and histopathologic observation: Animals were examinedevery 2 hours using slit lamp biomicroscope and ophthalmoscope after immunization,the onset time, peak time, clinical manifestation and grades, durations of theinflammation were noted particularly. We extracted blood from angular vein at forthhours, 24ed hour and the third day after immunization, killed the animals, excisedeyeballs. One eye was used to extract eye fluid, the other to make pathological sectionconventionally, examine the sample with light microscope and grade the pathologicalchanges. The serum and intraocular fluid TNF-α, IL-6 and IL-10 levels weremeasured by ELISA method.3. Statistical method: All data in present study denoted mean±standard deviation((?)±s), and all management was finished with SPSS 11.5 softwire. The statisticalmethods included independent-simple t test, Pearson correlation, assume equalvariance and one-way ANOVA(analysis of variance).Results We successfully established the EIU disease model in Wistar rats, the prevalencerate was 100%, clinical and histological features were significant and stable. The irisblood vessels hyperemia developed within four hours of the endotoxin injection. Thepeak time of the inflammation was 24 hours. This maximal response was at 24 hoursafter LPS injection and was characterized by the vasodilatation of iris vessels,infiltrate of polymorphonuclear cells in the anterior segment, protein extravasationinto the aqueous humor of the eye, occlusion of pupil, and vitreous opacity.Whereafter the inflammation subsidized gradually. Histologic examination of the eyeshowed heavy infiltration by leukocytes in the anterior chamber and the iris-ciliarybody.Inflammation was not seen in the healthy control group. Clinical presentation andhistopathologic observation were ameliorated markedly in anti-TNF-αMcAb group.Disease scores and histopathologic levels in anti-TNF-αMcAb group were lower thanthose in EIU group, and the onset time of disease was delayed. Side effects were notfound in present study.Serum and intraocular fluid TNF-α, IL-6 and IL-10 levels were measured byELISA method. The serum and intraocular fluid TNF in healthy group were 51.03±9.59 pg/ml and 26.8 pg/ml. High level of TNF-α(212.76±64.26 pg/ml) was detectedin sera of rats early after LPS injection in EIU group, and it was higher than that inhealthy control group. Serum TNF in anti-TNF-αMcAb group decreased to the levelin heathy group. Serum IL-6 level was 3.23±1.40 pg/ml in healthy group, but theintraocular fluid IL-6 was not detected. High levels of IL-6 were detected in sera andintraocular fluid of rats early after LPS injection in EIU group, and they were higherthan those in healthy control group; they decreased in anti-TNF-αMcAb group.Serum IL-10 levels in anti-TNF-αMcAb group were higher than those in EIU group,and they were highest in 4 hours. TNF-α, IL-6 and IL-10 levels in intraocular fluid were higher than those in sera. There were positive correlations between levels ofserum and intraocular fluid IL-6 and serum TNF-αand disease severity(r=0.777,p＜0.01; r=0.678, p＜0.01; r=0.607, p＜0.01). We also found a positive correlationbetween TNF and IL-6 (p＜0.05).ConclusionWe established the EIU disease model in Wistar rats with LPS successfully. Thisdisease model is similar with human uveitis, and it can be used to study pathogenesisand therapy of uveitis. This method is simple and stable; there was a high prevalencerate and perfect repetition. This established an experimental foundation for the furtherstudy of uveitis.Anti-TNF-αMcAb was effective in the treatment of EIU, leading to significantimprovement of the clinical manifestation, and the onset times were delayed. Theresults of this study confirmed that both TNF-αand IL-6 might play a role in thepathogenesis of EIU. Intraocular fluid of TNF-α, IL-6 and IL-10 levels were higherthan those in sera, indicating that they were produced locally during EIU in the eye.Serum TNF-αwas completely neutralized by anti-TNF-αMcAb. After therapy withanti-TNF-αMcAb, the levels of TNF-αand IL-6 in serum and eye fluid weredecreased, but the levels of IL-10 were increased, especially in early inflammation,revealing that IL-10 induced the improvement of EIU. In summary, anti-TNF-αtherapy can be served as an effective method to uveitis.There was a positive correlation between TNF-αand IL-6, indicating that TNF-αcan upregulate the production of IL-6, and TNF is one of determining factor ofproducing IL-6.