复发性实验性自身免疫性葡萄膜炎小鼠模型的诱导及其特点

目的 诱导复发性实验性自身免疫性葡萄膜炎(experimental autoimmune uveoretinitis,EAU)小鼠模型,评价其发生过程及特点.方法 完全弗氏佐剂(CFA)乳化的光感受器间维生素A类结合蛋白(IRBP) 161-180肽段免疫B10.RⅢ小鼠作为诱导组(35只),用CFA-PBS免疫小鼠作为对照组(6只).小鼠免疫后7、14、21、28、35 d裂隙灯显微镜和光学显微镜观察及评价眼部炎症发生、眼部表现和病理学变化;待炎症完全消失时,再次免疫小鼠,评价小鼠眼部炎症复发.结果 诱导组首次免疫后7d出现初发炎症,14 d炎症达高峰,随后炎症消退,至35 d完全消失.第35天进行再次免疫,36 d出现复发炎症,42 d达炎症高峰,随后炎症消退,但至观察期第96天部分鼠(1/5)仍维持炎症.眼部表现为睫状充血、前房渗出、瞳孔受损.病理学表现为视网膜和脉络膜炎性细胞浸润,血管炎、肉芽肿性炎,光感受器细胞层破坏,视网膜下渗出.对照组未见明显炎症.结论 建立的EAU呈现慢性复发性病程,其眼部表现和病理学特征与人类葡萄膜炎相似,可作为葡萄膜炎研究的新型动物模型.     

雷公藤内酯醇对试验性色素膜视网膜炎IL-12mRNA表达以及NF-AT活性的抑制作用

目的：探讨雷公藤内酯醇对试验性色素膜视网膜炎IL－12mRNA表达的影响及其机制。方法：采用原位杂交染色法和电泳迁移率改变试验观测雷公藤内酯醇对试验性色素膜视网膜炎的TI林巴细胞IL－12mRNA的表达及NF－AT活性。结果：雷公藤内酯醇能有效地降低S－抗原诱导产生的试验性色素膜视网膜炎，能抑制淋巴细胞IL－12mRNA的表达及NF－AT活性。结论：雷公藤内酯醇对EAU的免疫抑制作用可能是通过抑制与Th1反应相关的调节因子使Th1反应减弱，而使EAU的发病率及疾病严重程度明显降低。     

TGF-β2玻璃体腔注射对EAU大鼠Foxp3表达的调控作用

目的:利用RT-PCR的方法检测EAU时Lewis大鼠视网膜和葡萄膜Foxp3的mRNA表达情况,探讨EAU时转录因子Foxp3的变化及TGF-β2对其的诱导调控作用,为研究葡萄膜炎的病变机制提供理论支持,为临床治疗葡萄膜炎探索新方向。方法:将IRBPR16多肽注入Lewis大鼠左后足底部制备EAU动物模型。按照随机数字表法将36只Lewis大鼠分为正常对照组、EAU未治疗组和EAUTGF-β2治疗组。TGF-β2治疗组于EAU动物模型制备后第1,4,7,10,13,16,19d给予TGF-β210μL(浓度为5mg/L)玻璃体内注射治疗。分别于IRBP免疫后7,10,14,21d处死Lewis大鼠,刮取视网膜和葡萄膜,利用RT-PCR法检测Foxp3的表达,所得数据应用SPSS10.0统计分析软件进行处理。结果:随着EAU病程的进展,Foxp3的表达逐渐增加,但同正常对照组相比,差异不显著,各时间组之间差异不显著。随着EAU病程的进展,TGF-β2治疗组7dFoxp3的表达增加,同正常对照组和未治疗组相比无显著差异。TGF-β2治疗组10,14,21d Foxp3的表达增加,同正常对照组相比有显著差异(P<0.01),同TGF-β2未治疗组相比Foxp3的表达增加,差异显著(P<0.05)。结论:探讨Foxp3在EAU疾病过程中的表达变化,为进一步研究Foxp3在EAU中的作用机制提供了理论支持。研究了TGF-β2在EAU病程中对Foxp3的表达调控,发现TGF-β2能够增加Foxp3的表达,从而调控Treg细胞,启动免疫抑制功能,为临床治疗葡萄膜炎开辟新方向。     

Circulating immune complexes may play a regulatory and pathogenic role in experimental autoimmune uveoretinitis.

We compared the time course of changes in serum levels of circulating immune complexes (CICs) and of IgG antibody after sensitization of albino Lewis and pigmented Lister strain rats with uveitogenic (retinal S-antigen) and non-uveitogenic (ovalbumin) protein antigens of comparable molecular weight. Normal levels of CICs were far lower in Lewis rats in which experimental autoimmune uveoretinitis (EAU) takes the form of a severe panuveitis, than in Lister rats, in which the disease is mild, focal, confined to the posterior segment, and of lower incidence. After sensitization with either S-antigen or ovalbumin, polyethylene-glycol-precipitable CIC (PEG-CIC) peaked and fell as IgG antibody levels rose in both rat strains. However, peak levels of PEG-CIC were lower and subsequent IgG antibody levels were higher in the Lewis strain than in the less susceptible Lister strain. In both strains of rat these linked PEG-CIC/IgG antibody responses occurred earlier after sensitization with uveitogenic (S-) antigen than with ovalbumin, whether or not individual S-antigen-sensitized Lister rats developed EAU. In contrast, complement-binding CIC rose substantially only in those rats of both strains displaying EAU in response to S-antigen and not in response to ovalbumin. We suggest that immune complex (idiotypic) regulation of IgG antibody responses may be more readily perturbed by a pathogenic autoantigen (S-antigen) than by a bland antigen (ovalbumin). We also suggest that differences between the balance of regulatory and pathogenic CIC responses to uveitogenic retinal antigen may underlie or reflect strain differences in susceptibility to and severity of EAU.     

大鼠实验性自身免疫性葡萄膜视网膜炎模型

目的 :建立大鼠实验性自身免疫性葡萄膜视网膜炎 (EAU)模型 ,为探讨人类葡萄膜炎的发病机制奠定基础。方法 :18只Wistar大鼠用 3只不同剂量牛视网膜可溶性抗原 (S Ag)免疫后 ,每日扩瞳进行EAU临床观察 ;当大鼠出现中度以上EAU临床表现时处死、其他大鼠第 3～ 4w时处死后眼球摘除 ,行组织学观察。结果 :3组不同剂量S Ag免疫大鼠EAU发病率分别为 :10 0 μgS Ag组 6只大鼠 (12只眼 )为 2 12、2 0 0 μgS Ag组 6只大鼠 (12只眼 )为 6 12、30 0 μgS Ag组 6只大鼠 (12只眼 )为8 12 ;组织学炎症评分分别为 :0、16 76± 11 0 2、17 5 6± 9 96。结论 :使用中等纯度的S Ag ,以及中等敏感度的Wistar大鼠 ,可成功诱发出EAU模型     

Correlation of optical coherence tomography with clinical and histopathological findings in experimental autoimmune uveoretinitis

Optical coherence tomography (OCT) is becoming the state-of-the-art method for the non-invasive imaging of a variety of ocular diseases. The aim of this study was to assess the application of OCT for the in vivo monitoring and follow-up of pathological changes during experimental autoimmune uveoretinitis (EAU) in rats. Initially we established OCT imaging in healthy brown Norway rats and correlated it with retinal histology. Subsequently, we induced EAU and imaged animals by OCT throughout the pre-peak, peak, and post-peak phases of the disease. The sensitivity of OCT imaging was determined by comparison with clinical EAU and histopathology scores obtained ex vivo at several time points throughout the disease course. Our data demonstrate that OCT imaging of the healthy rat retina closely correlates with histological observations and allows the clear visualization of all retinal layers. After induction of EAU, the first pathological changes could be detected by OCT at day (d) 8 post-immunization (p.i.) which corresponded to the time point of clinical disease onset. An increase in retinal thickness (RT) was detected from d10 p.i. onwards which peaked at d16 p.i. and decreased again to near control levels by d20 p.i. We introduce a novel semi-quantitative OCT scoring which correlates with histopathological findings and complements the clinical scores. Therefore, we conclude that OCT is an easily accessible, non-invasive tool for detection and follow-up of histopathological changes during EAU in rats. Indeed, significant differences in RT between different stages of EAU suggest that this OCT parameter is a sensitive marker for distinguishing disease phases in vivo.     

A clinical grading system for retinal inflammation in the chronic model of experimental autoimmune uveoretinitis using digital fundus images

Experimental autoimmune uveoretinitis (EAU), a widely used animal model of human posterior/pan-uveitis, is extremely valuable in allowing understanding of the pathogenesis of uveitis as well as in developing new treatments. Depending on the animal strain and immunization protocol used, the clinical course of EAU can be acute, severe and involving the anterior and posterior part of the eye, or chronic, mild and involving only the posterior part of the eye. Clinical signs of EAU can be examined by bio-microscopy. Using appropriate criteria EAU can be quantitatively evaluated clinically in living animals. However, correlation of research within different laboratories is difficult since clinical grading systems are subjective and susceptible to considerable variability. In this study, we have developed a recordable, image-based clinical grading system for the chronic models of EAU. Fundus images were taken from EAU mice using an endoscopic imaging system. Fundus changes were classified as (1) inflammatory changes (including optic disc inflammation, vasculitis and retinal tissue inflammation) and (2) retinal structural damage. Each element was scored separately based on the severity of the lesions, and the average score of the three inflammatory elements was used as the overall EAU clinical inflammation grade of the eye. The validity and reproducibility of the grading system was tested using a set of images scored independently in a masked manner by 5 individuals. The grading system proved robust, easy to use and reliable. We offer this image-based EAU clinical grading system as a useful quantitative evaluation method for clinical grading of the severity of inflammation in the chronic EAU model, in which the inflammation can be mild and mainly involves posterior part of the eye.     

视网膜S抗原在不同动物诱发的实验性自身免疫性葡萄膜视网膜炎

本研究从牛视网膜提纯了S抗原,将其兔疫Hartley豚鼠、SD大鼠和Lewis大鼠,诱发的实验性自身免疫性葡萄膜视网膜炎(EAU)。但其发生率、临床及组织学表现则有很大不同。根据本文的结果及文献报道的结果,作者就其差异的原因及与人类葡萄膜视网膜炎的关联性等方面进行了有关讨论。     

Role of T-cell receptor V beta 8.3 peptide vaccine in the prevention of experimental autoimmune uveoretinitis

Background T-cell receptor （TCR） plays an important role in the development of autoimmune diseases. Recently, it was reported that immunization of animals with TCR peptide derived from the pathogenic cells could prevent autoimmune diseases. The aim of this study was to investigate whether vaccination with a synthetic peptide from the hypervariable region of TCR Vβ 8.3, an experimental autoimmune uveoretinitis （EAU）-associated gene, was able to prevent the disease. Methods EAU was induced in Lewis rats by immunization with IRBP R16 peptide emulsified in complete Freund＇s adjuvant （CFA）. The clinical and histological appearances were scored. Delayed type hypersensitivity （DTH） and lymphocyte proliferation were detected. Cytokine levels of aqueous humour, supernatants of cells from spleen and draining lymph nodes were measured by enzyme linked immunosorbent assay （ELISA）. Gene expression of TCR Vβ8.3 on CD4^＋ T cells was examined by real time quantitative polymerase chain reaction （PCR）. Results After vaccination, the intraocular inflammation was significantly mitigated, antigen specific DTH and lymphocyte proliferation responses were suppressed, interleukin （IL）-2 in aqueous humour, interferon （IFN）-γ and IL-2 produced by the spleen and draining lymph node cells were significantly decreased, whereas the production of IL-4 and IL-10 were increased. The response of draining lymph node cells to TCR Vβ 8.3 peptide was enhanced after vaccination. Inoculation with CFA alone did not affect the severity of EAU and the above parameters. The suppression of EAU was much stronger in the group of four fold inoculations than the group of two fold inoculations. The expression of TCR Vβ 8.3 gene was significantly reduced in the group of fourfold inoculations. Conclusion Vaccination with the synthetic TCR Vβ 8.3 peptide could remarkably inhibit the development of EAU.     

SOCS1反义寡核苷酸对实验性自身免疫性葡萄膜视网膜炎的作用

目的 探讨细胞因子信号抑制因子1(SOCS1)反义寡核苷酸对预防实验性自身免疫性葡萄膜视网膜炎(EAU)的可行性和有效性.方法 制作大鼠EAU模型,于免疫后10 d前房内分别注射SOCS1反义寡核苷酸脂质体(A组)、SOCS1正义寡核苷酸脂质体(B组)、脂质体(C组)和生理盐水(D组).于14、21、28 d观察其炎症反应,检测外周血单个核细胞(PBMC)、视网膜内SOCS1mRNA及蛋白的表达和血清中IL-4、IL-12和IFN-γ的含量.结果 免疫后14 d、21 d,B、C、D组可见明显的虹膜睫状体炎,A组炎症反应不明显;与D组相比,A组IL-4升高,IL-12和IFN-γ降低(P<0.05),B组、C组细胞因子无明显改变(P>0.05).与D组相比,14d、21d A组视网膜SOCS1mRNA及蛋白表达降低(P<0.05),PBMC内SOCS1mRNA及蛋白无明显改变,B组、C组PBMC和视网膜内SOCS1mRNA及蛋白无明显改变.结论 SOCS1反义寡核苷酸脂质体对EAU在眼部的炎症可能有一定的抑制作用.