Endotoxin-induced uveitis in mice. 1. Induction of uveitis and role of T lymphocytes.

Endotoxin-induced uveitis (EIU) with a high frequency of posterior iris synechiae was induced by the systemic injection of 200 micrograms of endotoxin into C3H/HeN mice, an endotoxin-responsive strain. The cell number and the protein concentration within the aqueous humor began to increase 6 hours after the injection, achieving a peak at 24 hours, and decreased gradually thereafter. Inflammatory cells were observed in the anterior chamber, the vitreous body and near the iris-ciliary body histologically. Most of the inflammatory cells were polymorphonuclear cells. On the other hand, C3H/HeJ mice, an endotoxin-unresponsive strain, showed no increase in either cell number or protein concentration in the aqueous humor after endotoxin administration. Pretreatment of C3H/HeN mice with anti-Thy-1.2 antibody significantly decreased both the cell number and the protein concentration in the aqueous humor and the incidence of the posterior synechiae, as compared with the control group. Anti-CD4 antibody also significantly reduced the severity of EIU, while anti-CD8 antibody had no influence on the disease. Anti-IFN-gamma antibody increased the cell number in the aqueous humor. These observations indicate that T lymphocytes, especially CD4+ T lymphocytes, have an extremely important role in the development of EIU in mice.     

Effects of experimental ocular inflammation on ocular immune privilege.

PURPOSE: To determine whether the inflammation of endotoxin-induced uveitis (EIU) and experimental autoimmune uveoretinitis (EAU) alters key in vivo and in vitro parameters of ocular immune privilege. METHODS: For EIU induction, C3H/HeN mice received 200 microg lipopolysaccharide (LPS). For EAU induction, B10.A mice were immunized with 50 microg interphotoreceptor retinoid-binding protein (IRBP) mixed with complete Freund's adjuvant. Aqueous humor (AqH) was collected at periodic intervals and assayed for leukocyte content and the ability to suppress or enhance T-cell proliferation. Eyes with EAU were assessed for the capacity to support anterior chamber (AC)-associated immune deviation (ACAID) induction after injection of ovalbumin (OVA). RESULTS: Inflammation within the anterior segment in EIU peaked at 12 to 24 hours and was detected from 10 days onward in EAU. In AqH of EIU, protein content rose within 4 hours, followed by infiltrating leukocytes. EIU AqH promptly lost its capacity to suppress T-cell proliferation and became mitogenic for T cells. In AqH of EAU, protein and leukocyte content rose at 11 days and continued to remain elevated thereafter. Whereas 11-day EAU AqH failed to suppress T-cell proliferation, AqH at later time points reacquired immunosuppressive properties. Injection of OVA into the AC of eyes of mice with EAU failed to induce ACAID. CONCLUSIONS: The intraocular inflammation of EIU and EAU disrupted important parameters of immune privilege, ranging from breakdown of the blood- ocular barrier, to loss of an immunosuppressive microenvironment, to abrogation of ACAID. Because AqH from inflamed EAU reacquired the ability to suppress T-cell proliferation, the authors conclude that the capacity to regulate immune expression and inflammation can be a property even of inflamed eyes.     

内毒素诱导的葡萄膜炎中虹膜固有组织细胞上MyD88与TLR-4的共表达

目的观察小鼠内毒素诱导的葡萄膜炎(endotoxin-induced uveitis,EIU)模型中Toll样受体-4(Toll-like receptor-4,TLR-4)、髓样分化因子88(myeloid differentiation factor 88,MyD88)、CD163的共表达。方法 6～8周龄雄性C3H/HeN(野生型)小鼠25只,C3H/HeJ(TLR-4基因缺陷型)小鼠5只,分别腹腔注射霍乱弧菌内毒素细胞壁脂多糖(lipopolysaccharide,LPS)诱导急性前葡萄膜炎动物模型,处死小鼠后虹膜、睫状体铺片,用免疫荧光三标记的方法检测虹膜、睫状体组织内不同时间CD163、TLR-4、MyD88的表达,并对虹膜内CD163、TLR-4和MyD88阳性细胞进行计数分析。结果 C3H/HeN小鼠LPS注射后12h结膜囊内出现炎症反应,24～48h达到高峰,72h炎症逐渐消退,而C3H/HeJ小鼠LPS注射后没有发现炎症反应。在内毒素诱导的C3H/HeJ小鼠虹膜中未检测到CD163、TLR-4和MyD88阳性细胞。C3H/HeN小鼠腹腔注射LPS后在虹膜铺片内0h未见CD163、TLR-4与MyD88阳性表达,12h后可见阳性细胞(40.3±8.9、45.2±6.3、42.5±4.1),24h阳性细胞数(121.0±39.5、138.6±28.3、125.5±36.1)较12h明显增多,差异均有统计学意义(均为P<0.05);48h阳性细胞数(132.3±54.5、129.9±36.2、122.1±29.3)与24h相比,差异均无统计学意义(均为P>0.05);72h阳性细胞数(12.8±3.2、10.4±5.6、9.3±5.2)较48h减少,差异均有显著统计学意义(均为P<0.01)。结论小鼠EIU模型中,LPS激活了CD163标记的巨噬细胞膜表面的TLR-4,TLR-4与MyD88途径可能是EIU主要的信号传导途径。     

IL-6 antagonizes TGF-beta and abolishes immune privilege in eyes with endotoxin-induced uveitis

PURPOSE: To determine the immunosuppressive status of aqueous humor (AqH) from mouse eyes afflicted with endotoxin-induced uveitis (EIU) and to identify the relevant cytokines responsible for immunomodulatory activity within EIU AqH. METHODS: Bacterial lipopolysaccharide (LPS) was injected into hind footpads of C3H/HeN mice; and AqH, collected at 6, 12, 24, and 48 hours, was evaluated for content of transforming growth factor (TGF)-beta, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and interferon (IFN)-gamma and capacity to suppress anti-CD3-driven T-cell proliferation. Cytokine mRNA expression in iris-ciliary body (I/CB) was analyzed by RNase protection assays. RESULTS: During 6 to 24 hours after LPS injection, total TGF-beta levels in AqH increased even though the fluid lost its capacity to suppress T-cell activation. At this time, AqH contained high levels of IL-6, and I/CB contained high levels of IL-6 mRNA. When IL-6 was neutralized with specific antibodies, inflamed AqH reacquired its capacity to suppress T-cell activation, which correlated with high levels of TGF-beta. Coinjection of IL-6 plus antigen into the anterior chamber of the eye of normal mice prevented antigen-specific anterior chamber-associated immune deviation (ACAID). CONCLUSIONS: LPS-induced intraocular inflammation is associated with local production of IL-6, which robs AqH of its immunosuppressive activity, perhaps by antagonizing TGF-beta. The fact that IL-6 antagonized ACAID induction in normal eyes suggests that strategies to suppress the intraocular synthesis of IL-6 may reduce inflammation and restore ocular immune privilege.     

Interleukin 10 inhibits inflammatory cells infiltration in endotoxin-induced uveitis

Background: Endotoxin-induced uveitis (EIU) is a model for acute anterior uveitis associated with a variety of pro-inflammatory cytokines and nitric oxide production. Interleukin 10 (IL-10) down-regulates these inflammatory mediators. We report a study of the effect of systemic administration of IL-10 on the inflammatory parameters of EIU. Methods: Uveitis was induced in C3H/HeN mice by subcutaneous injection of 200 g lipopolysaccharide (LPS) per mouse. Intraocular inflammation was assessed by leukocyte count and measurement of the protein concentration in the aqueous humor (AH). Mouse recombinant IL-10 at 1000 U or its vehicle alone were administered by three intravenous injections given 4.0 h and 0.5 h before and 8.0 h after LPS injection. Results: The inflammatory cell infiltration in the eyes was significantly reduced in four of five experiments from 40% to 64% in the groups treated with IL-10 compared to the control groups (P<0.05). In contrast, the level of protein exudation in the anterior chamber (AC) was not significantly affected by IL-10 treatment. Conclusion: IL-10 reduces the cellular infiltration in the ocular inflammation produced by endotoxin. This result suggests potential usefulness for IL-10 in the treatment of severe anterior uveitis with a strong cellular component.     

Intraocular production of a cytokine (CINC) responsible for neutrophil infiltration in endotoxin induced uveitis.

AIMS/BACKGROUND: The subcutaneous injection of bacterial endotoxin in Lewis rats produces an acute intraocular inflammation evolving over a 24 hour period. This endotoxin induced uveitis (EIU) is characterised by a biphasic protein exudation and a cellular infiltrate composed of macrophages and polymorphonuclear neutrophils (PMNs). This model was used to study the mechanism of cellular infiltration in ocular inflammation. METHODS: EIU was induced by a subcutaneous injection of lipopolysaccharide (LPS) (S typhimurium) at 350 micrograms/kg. The levels of cytokine induced neutrophil chemoattractant (CINC) were measured every 2 hours in the serum and in the aqueous humour by ELISA. The intraocular inflammation was quantified by protein measurement and leucocyte counting. RESULTS: The kinetics of CINC production in the systemic circulation showed a rapid rise, peaking 2 hours after LPS injection, followed by a progressive decline over the next 8 hours. In the eye, the CINC levels increased above the serum levels 10 hours after EIU induction corresponding to the time of cellular infiltration. When leucocyte entry in the eye was inhibited by 56% and 64% with an antiadhesion molecule antibody, there was only a slight reduction in the aqueous humour CINC levels of 9% and 16%, respectively, indicating that CINC was produced by ocular tissue cells. The specific effect of CINC in the eye was confirmed when a direct intraocular injection of 250 ng of purified CINC was followed by significant PMN infiltration, in the absence of protein exudation. CONCLUSION: The data indicate that the production of the CINC chemotactic factor by ocular tissue participates in the inflammatory reaction in EIU.     

Analysis of interleukin-6 in endotoxin-induced uveitis

The mechanisms underlying the induction of intraocular inflammation in the rat model of endotoxin-induced uveitis (EIU) and the subsequent development of tolerance after repeated endotoxin injections are poorly understood. Interleukin-6 (IL-6) was measured in the aqueous humor and serum of Lewis rats after single and repeated injections of endotoxin into the footpad. After a single injection, a rise in serum and aqueous-humor levels of IL-6 was seen after 2 and 16 hr, respectively. The highest aqueous-humor level of IL-6 was seen 20 hr postinjection and was tenfold that seen in the serum sample taken at the same time, suggesting intraocular synthesis of this cytokine. Four hours later the most active uveitis and the highest total aqueous-humor protein level were observed. Repeated injection of endotoxin still resulted in a moderate but significant systemic release of IL-6 but no detectable IL-6 in the aqueous humor and the absence of uveitis. Intravitreal injection of endotoxin-free human recombinant IL-6 (10-10(5) U) in rats resulted in uveitis, resembling the ocular response to endotoxin. There appeared to be a prozone effect regarding the total aqueous-humor protein concentration. The largest amount of aqueous-humor protein was seen in the eyes injected with 10(2) U of IL-6, but increasing concentrations of intravitreal IL-6 showed a corresponding decrease in protein levels. In the fellow saline-injected eyes, a clear consensual response was observed with regard to the extravasation of protein, although the uveitic grade in these eyes was low or zero. Repeated intravitreal injection of IL-6 resulted in ocular unresponsiveness in nine of 11 rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endotoxin-induced uveitis is partially inhibited by anti-IL-8 antibody treatment.

PURPOSE: To examine the potential therapeutic effect of a neutralizing anti-IL-8 monoclonal antibody in endotoxin-induced uveitis (EIU) in the rabbit. METHODS: An anti-IL-8 antibody (WS-4) was injected intravitreal 2 hours before, simultaneously with, or 6 hours after endotoxin challenge in rabbits. Eyes were examined for clinical signs of inflammation, and aqueous humor (AH) was sampled to study cellular infiltration and protein content. Leukocyte subset analysis was performed on Giemsa-stained AH cytospins. Histologic grading of inflammation was performed on hematoxylin-eosin-stained sagittal sections of enucleated eyes. In separate experiments, animals received the anti-IL-8 antibody simultaneously with the endotoxin challenge, before repeated anterior chamber paracentesis was performed (at 6, 12, 24, 48, and 72 hours after injection) to estimate the kinetics and durability of changes in total cell count and protein concentration in AH. RESULTS: Anti-IL-8 therapy caused a decrease in the clinical and histologic grade of inflammation in EIU. The mean cell count in the AH at the peak of inflammation (24 hours) in eyes receiving endotoxin only was 6419+/-1165/microl (mean +/- SE) compared to 2546+/-573/microl in rabbits treated simultaneously with 250 microg of anti-IL-8 antibody (P < 0.05). The protein concentration in the AH was not significantly altered by anti-IL-8 treatment. Kinetic analysis of the leukocyte count in the AH demonstrated persistent inhibition of leukocyte accumulation (range, 60%-91% compared to control eyes) by the anti-IL-8 antibody administered simultaneously with endotoxin. This inhibition was sustained for up to 72 hours after injection. CONCLUSIONS: Anti-IL-8 antibody treatment partially blocks EIU in rabbits. A consistent decrease in the recruitment of polymorphonuclear leukocytes into the anterior chamber was obtained when neutralizing antibody was injected simultaneously with endotoxin. These findings suggest that IL-8 contributes to the chemotactic signal for the recruitment of leukocytes in EIU.     

Effects of a new dexamethasone-delivery system (Surodex) on experimental intraocular inflammation models

Background To investigate the effects of a new biodegradable dexamethasone drug delivery system, Surodex, in two experimental intraocular inflammation models; endotoxin-induced uveitis (EIU) and experimental autoimmune uveoretinitis (EAU).Methods Surodex was inserted into the right anterior chambers (ACs) of rats. In the EIU experiment, protein concentration, cell infiltration, and myeloperoxidase (MPO) activity in the aqueous humor were measured 24 h after injection. Eyes were evaluated histopathologically. In the EAU experiment, firstly, Surodex was administered at various days after immunization. Then, Surodex was administered on day 9 and eyes were evaluated histopathologically. Intraocular cytokine levels (IFN- and IL-4) were investigated.Results In the EIU experiments, eyes with Surodex exhibited significantly reduced inflammation compared with contralateral controls. Protein concentrations, cell infiltrations, as well as MPO activity were reduced. In the EAU experiments, all rats with Surodex given on days 0 or 7 showed no or significantly reduced inflammation in both eyes. Rats treated on day 12 developed reduced inflammation only in the treated eyes. IFN- levels were significantly lower in the eyes with Surodex, whereas IL-4 was not detectable.Conclusions This new, biodegradable corticosteroid drug-delivery system is highly effective in suppressing intraocular inflammation, and should be a useful tool to manage uveitis in humans.     

Systemic interferon-gamma suppresses the development of endotoxin-induced uveitis in mice

PURPOSE: It has been shown that interferon (IFN)-gamma is involved in the development of endotoxin-induced uveitis (EIU), but its exact role is unclear. We aimed to elucidate the role that endogenous systemic IFN-gamma plays in EIU pathogenesis. METHODS: EIU was induced in wild-type (WT) or IFN-gamma knockout (GKO) mice on the C57BL/6 background by injecting Salmonella typhimurium endotoxin into a hind footpad. Twenty-four hours later, the eyes were harvested for histological analysis, and the serum was collected for cytokine ELISAs. WT and GKO mice were also intraperitoneally injected with 1 microg of recombinant murine IFN-gamma (rmIFN-gamma) just after and 6 h after EIU induction, and their eyes and sera were evaluated 24 h after EIU induction, as above. RESULTS: The GKO mice had significantly more severe EIU as determined by the number of ocular infiltrating cells and lower serum IL-6 levels after EIU induction compared to WT mice. The injection of rmIFN-gamma suppressed the severity of EIU and increased the serum IL-6 levels in both the WT and GKO mice. CONCLUSIONS: Endogenous IFN-gamma suppresses EIU pathogenesis. In addition, the systemic administration of IFN-gamma suppresses EIU. The suppressive mechanism involved is unclear but may relate to the production of IL-6.     

内毒素诱导葡萄膜炎中炎症细胞凋亡与TRAIL表达的研究

目的:在内毒素诱导的葡萄膜炎(endotoxin induced uveitis,EIU)模型中,观察葡萄膜炎病变中炎症细胞凋亡的发生,研究大鼠虹膜组织中炎症细胞肿瘤坏死因子相关凋亡诱导配体(TRAIL)的表达与炎症细胞凋亡的关系,探讨TRAIL凋亡途径可能参与炎症细胞的凋亡。方法:以1mg/kg内毒素注射于大鼠后足垫建立EIU模型,分别于注射后不同时间点观察大鼠眼内的炎症反应。吸取房水观察细胞数。摘取眼球,行HE(hematoxylin eosin)染色观察大鼠虹膜组织的病理改变;TUNEL(terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling)法检测炎症细胞凋亡情况;同时运用SABC法检测在内毒素诱导后的不同时间TRAIL在炎症细胞上的表达,并行图像分析。结果:内毒素注射6h开始出现炎症,以后炎症加重,于18～24h达到炎症高峰,48h炎症明显消退。房水中的细胞数也在24h组达到最多。HE染色显示:内毒素注射后6h,即出现炎症细胞,细胞数目在24h组最多,多数为多形核中性粒细胞,少数为单核细胞和淋巴细胞,48h组炎症细胞几乎消失。TUNEL染色显示:在内毒素注射后6h组即有炎症细胞出现阳性着色,24h组凋亡数达到最多。免疫组化显示:TRAIL蛋白在大鼠的虹膜色素上皮层呈弱阳性着色;TRAIL在炎症细胞上呈阳性着色,24h组在炎症细胞中的着色数量及着色强度达到最高。结论:以1mg/kg的内毒素注射于SD大鼠后足垫可诱导出葡萄膜炎反应,炎症反应在24h组最为强烈。炎症细胞凋亡可能是EIU炎症迅速消退的原因之一。TRAIL可能参与了炎症细胞的凋亡。     

Inflammatory reaction following cataract surgery and implantation of acrylic intraocular lens in rabbits with endotoxin-induced uveitis

PURPOSE: To investigate whether inflammatory responses are more severe in uveitic eyes than nonuveitic eyes when acrylic intraocular lens (IOL) is implanted after cataract surgery. METHODS: Clear lens removal (phacoemulsification and aspiration) was conducted and the hydrophobic acrylic IOL (AR40e, AMO) was implanted in adult albino rabbits. Just after the operation, rabbits were divided into two groups. One group (nine rabbits) received intravitreal injection of lipopolysaccharide (LPS, 200 ng/10 microl) into both eyes to induce endotoxin-induced uveitis (EIU) and the other group (nine rabbits) received intravitreal injection of phosphate-buffered saline (PBS, 10 microl) into both eyes as the control. Aqueous humour (AH) and IOLs were harvested 1, 3 , and 7 days after the intravitreal injection. The infiltrating cell number in AH was counted and the protein concentration of AH was measured. IOLs were evaluated morphologically. RESULTS: At 1 day after intravitreal injection, both the infiltrating cell number in AH and protein concentration of AH were significantly higher in the LPS-injected group than in the PBS-injected group. Similarly, more inflammatory cells attached to the surfaces of the IOLs in the LPS-injected group. However, 7 days later, inflammatory reactions subsided and no clear differences in any of the parameters examined were observed between the two groups. CONCLUSIONS: At 7 days after the operation, inflammatory reactions in eyes implanted with the hydrophobic acrylic IOLs were similar in uveitic eyes and nonuveitic eyes. The data suggest that the hydrophobic acrylic IOLs may be suitable for patients with uveitis.     

Murine endotoxin-induced uveitis, but not immune complex-induced uveitis, is dependent on the IL-8 receptor homolog.

PURPOSE: To determine the roles of the murine interleukin-8 receptor homolog (mIL-8Rh, neutrophil chemokine CXC receptor 2) and macrophage inflammatory protein-1alpha (MIP-1alpha, a CC chemokine) in two eye inflammation models: endotoxin-induced uveitis (EIU) and immune complex-induced uveitis (reverse passive Arthus reaction (RPAR) uveitis). METHODS: For the EIU model, 250 ng E.coli endotoxin was injected into the vitreous of mIL-8Rh-/- mice or heterozygous littermate mIL-8Rh+/- controls and into MIP-1alpha-/- mice or congenic MIP-1alpha+/+ controls. Eyes were harvested after 24 h for histologic characterization of infiltrating cells and IL-6 bioassays. For the RPAR model, mouse antiserum against human serum albumin (HSA) was injected into the vitreous of mIL-8Rh-/-, mIL-8Rh+/-, MIP-1alpha-/-, and MIP-1alpha+/+ mice. Twenty-four hours later, animals were challenged with intravenous HSA. Eyes were harvested after 4 h for analysis. RESULTS: RPAR resulted in the deposition of immune complexes at the ciliary area and iris with the subsequent development of uveitis. Genetic deficiency of mIL-8Rh reduced the median number of infiltrating cells in EIU by 63% (p < 0.01) but had no effect on RPAR-induced inflammation. In the EIU model, macrophages comprised a much higher percentage (45%) of infiltrating cells in mice lacking mIL-8Rh than in controls (17%). Loss of the MIP-1alpha gene had no apparent effect on RPAR uveitis and a 39% reduction of infiltrating cells in EIU that was not statistically significant. IL-6 activity in aqueous humor was much less in mice with RPAR uveitis than in those with EIU. Neither gene deletion had a significant impact on IL-6 levels in either disease model. CONCLUSIONS: Chemokines acting via mIL-8Rh have a significant role in the induction of neutrophil infiltration during EIU but not during RPAR uveitis. MIP-1alpha is not critical for either EIU or RPAR-induced uveitis. The differential dependence on IL-8-like chemokines is in accord with the two forms ofuveitis having different etiologies and, therefore, potentially different optimal therapies.     

Suppression of ocular inflammation in endotoxin-induced uveitis by blocking the angiotensin II type 1 receptor

PURPOSE: To examine whether the angiotensin II type 1 receptor (AT1-R) signaling plays a role in ocular inflammation in endotoxin-induced uveitis (EIU). METHODS: EIU was induced in C57BL/6 mice by a single intraperitoneal injection of 150 mug lipopolysaccharide (LPS). Tissue localization, mRNA expression, and protein levels of AT1-R in murine retinas were examined by immunohistochemistry, RT-PCR, and Western blot analyses, respectively. Telmisartan, an AT1-R antagonist widely used as an antihypertensive agent, was administered intraperitoneally at a dose of 10 mg/kg daily for 5 days until the injection of LPS. Twenty-four hours after administration, leukocyte adhesion to the retinal vasculature was evaluated with a concanavalin A lectin perfusion-labeling technique. Retinal mRNA and protein levels of intercellular adhesion molecule (ICAM)-1 were examined by RT-PCR and ELISA, respectively. Protein concentration and inflammatory cells in the aqueous humor were also measured. RESULTS: Retinal vessels were positive for AT1-R. In mice with EIU, retinal AT1-R mRNA and protein levels were significantly increased when compared to the normal control. EIU animals also showed significant increases in the number of inflammatory cells infiltrating the anterior chamber and adhering to the retinal vessels and in retinal ICAM-1 levels. Administration of telmisartan to EIU mice resulted in significant suppression of retinal ICAM-1 expression and leukocyte adhesion and infiltration compared with vehicle treatment. Protein concentration in the aqueous humor of telmisartan-treated EIU mice tended to be lower than that of vehicle-treated EIU mice, but the difference was not statistically significant. CONCLUSIONS: AT1-R signaling blockade inhibited retinal ICAM-1 upregulation and leukocyte adhesion and infiltration in the EIU model. These results suggest the potential use of an AT1-R antagonist as a therapeutic agent to reduce ocular inflammation.     

Visualization of cell death in vivo during murine endotoxin-induced uveitis

PURPOSE: To develop a technology to image cell death in the eye of a live mouse and to apply that technology to characterize the role of apoptosis and necrosis in the evolution of endotoxin-induced uveitis (EIU), a standard model of intraocular inflammation. METHODS: To induce EIU, 250 ng Escherichia coli 055:B5 lipopolysaccharide was injected into the vitreous body of BALB/c mice. At 0, 6, 10, 16, 20, 24, 48, 72, and 96 hours and on day 7 after endotoxin injection, annexin V and propidium iodide were injected into the anterior chamber of these mice, and labeled cells were observed by using intravital epifluorescence video microscopy. Iris and corneal wholemounts isolated from the mice were also evaluated with standard and confocal fluorescence microscopy. TUNEL staining was performed on iris wholemounts taken from additional mice similarly injected with endotoxin, to confirm the in vivo results. RESULTS: Uveitis was induced in all the mice that received an endotoxin injection. The percentages of annexin V(+) propidium(-) cells, annexin V(+) propidium(+) cells, and annexin V(-) propidium(+) cells in the iris tissues visible by intravital microscopy were comparable to those observed by TUNEL staining in vitro. In addition, intravital microscopy allowed observation of labeled cells in the aqueous humor and on the surface of the lens. Both the number and the pattern of labeled cells changed dramatically over time. The cells stained with annexin V had a variety of morphologies, including small and round, round with a lobulated or a kidney-shaped nucleus, dendriform, and irregular. CONCLUSIONS: A technique was developed to image cell death in the anterior segment of the eye in vivo and used to demonstrate that the number and proportion of early apoptotic (annexin V(+) propidium(-)) and late apoptotic or necrotic (annexin V(+) propidium(+) and annexin V(-) propidium(+)) cells change over the course of EIU. A variety of inflammatory cells and resident cells undergo apoptosis, or possibly necrosis, which may contribute to the rapid resolution of EIU. This in vivo technique will be a valuable tool for future studies on the resolution of ocular inflammation.     

内毒素诱导的葡萄膜炎动物模型研究进展

葡萄膜炎是一种常见的眼部炎症,易反复发作且牵涉到眼内多种组织,其具体发病机制目前尚不清楚,因此有必要建立合适的动物模型以进行研究。已有研究表明,内毒素注射可以诱导动物的实验性葡萄膜炎。内毒素诱导的葡萄膜炎（EIU）动物模型已成为人们研究葡萄膜炎重要的动物模型,它对人们了解葡萄膜炎的发病机制及治疗葡萄膜炎做出了重要贡献。就该模型的发现、建立方法、炎症过程以及模型中涉及到的内毒素信号通路相关转录因子、细胞因子和一氧化氮（NO）等进行综述。     

Downregulation of endotoxin-induced uveitis by intravitreal injection of vasoactive intestinal Peptide encapsulated in liposomes

PURPOSE: To reestablish the immunosuppressive microenvironment of the eye, disrupted by ocular inflammation during endotoxin-induced uveitis (EIU), by means of intravitreal injection of vasoactive intestinal peptide (VIP) in saline or encapsulated in liposomes, to increase its bioavailability and efficiency. METHODS: EIU was induced in Lewis rats by subcutaneous injection of lipopolysaccharide (LPS). Simultaneously, animals were intravitreally injected with saline, saline/VIP, VIP-loaded liposomes (VIP-Lip), or unloaded liposomes. EIU severity and cellular infiltration were assessed by clinical examination and specific immunostaining. VIP concentration was determined in ocular fluids by ELISA. Ocular expression of inflammatory cytokine and chemokine mRNAs was detected by semiquantitative RT-PCR. Biodistribution of rhodamine-conjugated liposomes (Rh-Lip) was analyzed by immunohistochemistry in eyes and regional cervical lymph nodes (LNs). RESULTS: Twenty-four hours after intravitreal injection of VIP-Lip, VIP concentration in ocular fluids was 15 times higher than after saline/VIP injection. At that time, EIU clinical severity, ocular infiltrating polymorphonuclear leukocytes (PMNs), and, to a lesser extent, ED1(+) macrophages, as well as inflammatory cytokine and chemokine mRNA expression, were significantly reduced in VIP-Lip-injected rats compared with rats injected with saline/VIP, unloaded liposomes, or saline. Rh-Lip was distributed in vitreous, ciliary body, conjunctiva, retina, and sclera. It was internalized by macrophages and PMNs, and VIP colocalized with liposomes at least up to 14 days after injection. In cervical LNs, resident macrophages internalized VIP-Rh-Lip, and some adjacent lymphocytes showed VIP expression. CONCLUSIONS: VIP was efficient at reducing EIU only when formulated in liposomes, which enhanced its immunosuppressive effect and controlled its delivery to all tissues affected by or involved in ocular inflammation.     

A Novel Therapeutic Target in Inflammatory Uveitis: Transglutaminase 2 Inhibitor

Purpose

Our goal was to investigate the effects of inhibition of transglutaminase 2 (TGase 2) on endotoxin-induced uveitis (EIU)

Methods

EIU was induced in female Lewis rats by single footpad injections of 200 µg of lipopolysaccharide (LPS). TGase 2 inhibitors were administered intraperitoneally 30 minutes before and at the time of LPS administration. Rats were sacrificed 24 hours after injection, and the effects of the TGase 2 inhibitors were evaluated by the number of intraocular inflammatory cells present on histologic sections and by measuring the TGase 2 activity and TGase products in the aqueous humor (AqH). TGase 2 substrates were also assayed in AqH from uveitis patients.

Results

Clinical indications of EIU, the number of cells present on histologic sections, and TGase 2 activity in AqH increased in a time-dependent manner, peaking 24 hours after LPS injection. Inflammation in EIU was significantly reversed by treatment with TGase inhibitors. A 23-kDa cross-linked TGase substrate was identified in the AqH from EIU rats and uveitis patients. MALDI-TOF analysis showed that this substrate in uveitis patients was human Ig kappa chain C region.

Conclusions

TGase 2 activity and its catalytic product were increased in the AqH of EIU rats. TGase 2 inhibition attenuated the degree of inflammation in EIU. Safe and stable TGase inhibitors may have great potential for the treatment of inflammatory uveitis.