小鼠单纯疱疹病毒性角膜炎中基质金属蛋白酶的表达及活性研究

目的 探讨角膜感染Ⅰ型单纯疱疹病毒(HSV-1)后基质金属蛋白酶(MMPs)及其组织抑制剂(TIMPs)在角膜中的分布及酶活性表达。方法 BALB／c小鼠眼角膜接种HSV-1(KOS株)以诱发单纯疱疹病毒性角膜炎(HSK)。分别收集正常眼球及感染后第2、7、14及28天的感染眼球。应用免疫组织化学法和Western blot方法检测MMP-2、-8、-9及TIMP-1、-2在角膜组织中的表达,并应用酶谱(Zymography)技术检测MMPs的酶活性。结果 感染后第2天,感染眼的MMP-2、-9及TIMP-1、-2表达比未感染眼增加且表达主要位于浅表基质层及上皮下的炎性细胞中。感染后第14和28天可见坏死性角膜炎及角膜溃疡形成,同时角膜基质和浸润的炎性细胞中尤其溃疡处,可见MMP-2、-9及TIMP-1、-2表达显著增加。溃疡区域有大量MMP-8阳性染色的中性粒细胞。角膜感染HSV-1后,明胶酶(MMP-2、-9)活性和胶原酶(MMP-8)活性均增强。结论 HSV-1角膜感染后,由角膜细胞和浸润的炎性细胞分泌产生的MMPs可能对上皮性角膜炎与溃疡形成过程起重要的促进作用。MMPs与TIMPs的相互作用可能对HSK的坏死性病变起重要调节作用。（中华眼科杂志,2004,40：395-399)     

MMP-9及TIMP-1在碱烧伤小鼠角膜中的表达

目的探讨角膜烧伤后基质金属蛋白酶9(MMP9)及其组织型抑制剂(tissueinhibitorofmetallopoteinase1,TIMP1)在小鼠角膜中的表达及意义。方法采用1mol·L-1氢氧化钠溶液烧伤昆明小鼠角膜,建立角膜碱烧伤动物模型;用免疫组织化学染色方法和计算机图像分析系统检测小鼠角膜烧伤后不同时间点MMP9及TIMP1在角膜中的分布及其积分吸光度(A)值。结果小鼠角膜碱烧伤后第2d炎性细胞增多,第7d炎症达到高峰,21d后基本消失。小鼠角膜上皮层、基底膜、以及基质层中大量炎性细胞和新生血管内皮细胞均有MMP及TIMP1表达。角膜中MMP9在第2d出现表达,第7d达高峰,以后逐渐降低;TIMP1开始表达不明显,第7d出现表达,14d达高峰,以后逐渐降低。结论小鼠碱烧伤模型炎症早期MMP9活性增高,继而TIMP1分泌增多,MMP9活性受抑,炎症减轻。提示MMP9可能是参与碱烧伤角膜溃疡形成、角膜融解及纤维化的重要调控因子,而TIMP1则在炎症的抑制过程中发挥了重要作用。     

小鼠角膜碱烧伤行羊膜移植对MMP-9、TIMP-1在新生血管形成中的影响

目的探讨小鼠角膜碱烧伤行羊膜移植对基质金属蛋白酶9(matrixmetalloproteinase9,MMP9)、金属蛋白酶组织抑制剂1(tissueinhibitorofmetalloproteinase1,TIMP1)在新生血管形成中的影响以及MMP9及TIMP1在角膜新生血管形成中的作用。方法昆明小鼠40只,随机分为实验组和对照组,每只鼠均取右眼为实验眼。采用1mol·L-1NaOH溶液烧伤小鼠角膜,建立炎症性角膜碱烧伤动物模型。对实验组小鼠右眼角膜行羊膜移植,对照组不行羊膜移植。分别在羊膜移植后的0d、2d、7d、14d处死2组小鼠,摘除右眼球,行免疫组织化学染色并用计算机图像分析系统检测2组小鼠MMP9及TIMP1在角膜中的分布及其平均吸光度值的变化。结果免疫组化结果:对照组碱烧伤角膜后第2d,可见MMP9出现表达,第7dMMP9的染色较第2d增强,基质层可见大量新生血管,且血管内皮细胞可见表达,14d达高峰;TIMP1开始表达不明显,第7d出现表达,14d达高峰。羊膜移植组各时间点MMP9表达低于对照组,而TIMP1表达高于对照组。结论碱烧伤后小鼠角膜中MMP9表达增加,在新生血管形成的过程中起一定作用。羊膜可能具有抑制MMP9在碱烧伤小鼠角膜中表达的作用,而且羊膜同时促使TIMP1在后期表达水平升高,从而抑制MMP9的活性,抑制和减缓碱烧伤后角膜新生血管的发生和发展。     

大鼠角膜新生血管模型中基质金属蛋白酶-2及其抑制物的表达

目的 探讨大鼠角膜碱烧伤后基质金属蛋白酶－2（MMP－2）及其组织型抑制剂（TIMP－2）在角膜中的表达和意义。方法 采用碱烧伤大鼠角膜建立角膜新生血管动物模型；应用免疫组织化学技术检测MMP－2及TIMP－2在角膜新生血管模型不同阶段的表达和变化。结果 实验鼠于伤后3－天开始形成大量新生血管，可见炎性细胞浸润，伤后14－21天新生血管和炎性细胞均明显减少。免疫组化显示MMP－2于伤后1天表达开始升高，3天达最高，以后逐渐下降。而TIMP－2则于早期变化不明显，7天表达开始升高，14天达高峰。结论 大鼠角膜新生血管形成早期MMP－2活性增高，继而TIMP－2分泌增多，MMP－2活性受抑，基底膜降解受阻，新生血管延伸停滞。     

碱烧伤小鼠行羊膜移植后MMP及TIMP表达的变化

目的:通过检测羊膜移植对小鼠角膜碱烧伤后不同时间点基质金属蛋白酶(metalloproteinases,MMP-2,8,9)及金属蛋白酶组织抑制剂(tissue inhibitor of metalloproteinases,TIMP-1,2)的表达,探讨羊膜移植在碱烧伤病程中所起的作用。方法:将40只Balb/c小鼠随机分为实验组和对照组,采用1mol/L氢氧化钠溶液烧伤小鼠角膜;实验组小鼠右眼行羊膜移植加睑裂缝合术,对照组仅行睑裂缝合术。分别在羊膜移植后的第0,2,7,14d处死小鼠,应用Western blot检测不同时间点MMP-2,8,9及TIMP-1,2的表达,增强化学发光法(enhanced chemiluminescent,ECL)对结果进行分析。结果:对照组角膜中MMP-2,8,9在第2d出现表达,第14d达到峰值,且表达主要位于基质层及上皮下的炎性细胞中。碱烧伤后第2d TIMP-1仅微弱表达,第7d可见表达增加,第14d到达峰值。TIMP-2第2d即可见表达增加,后持续增强。实验组各时间点MMP-2,8,9表达均低于对照组(P<0.01),TIMP-1,2的表达均高于对照组(P<0.01)。结论:行羊膜移植可通过抑制MMP的表达,促进TIMP表达,从而抑制和延迟碱烧伤后角膜炎性浸润及溃疡的发生和发展,对碱烧伤后角膜的重塑起着重要作用。     

羊膜移植对实验性HSK中基质金属蛋白酶表达的影响

目的研究羊膜移植（AMT）对单纯疱疹性角膜炎（HSK）中基质金属蛋白酶（MMP-2，-9）表达的影响。方法40只BALB／c小鼠角膜感染Ⅰ型单纯疱疹病毒（HSV-1），实验组角膜行AMT。术后第0、2、7、14d取出角膜。常规病理切片、免疫组化染色和计算机图像分析检测角膜中MMP-2及-9的表达及平均光度值的变化。结果对照组20只鼠眼中17只发生HSK；AMT组仅有9只眼发生，差异有显著统计学意义（P〈0．01）。AMT组角膜上皮、基质病变程度及新生血管发生率明显低于对照组（P〈0．05）。免疫组化及图像分析显示对照组角膜细胞和浸润炎性细胞中表达的MMP-2及-9在第2d增加，14d时达高峰。AMT组各时间点MMP-2及-9表达低于对照组，差异有显著统计学意义（P〈0．05）。结论羊膜移植可能通过抑制角膜细胞及浸润的炎症细胞产生MMPs，从而抑制HSK的发生和发展。     

不同浓度重组Canstatin蛋白对碱烧伤后角膜MMP-2及TIMP-2表达的影响

目的：探讨不同浓度重组Canstatin蛋白对碱烧伤后小鼠角膜基质金属蛋白酶－2（ matrix metalloproteinase－2,MMP－2）及其组织抑制剂－2（ tissue inhibitor of metalloproteinase－2, TIMP－2）表达的影响及其调节作用。方法：BALB／c小鼠60只随机分为实验组A、实验B及对照组C,每组20只。采用1mol／L氢氧化钠溶液烧伤小鼠右眼角膜,建立炎症性角膜碱烧伤动物模型,分别予以A组、B 组重组 Canstatin 蛋白3μg／mL、5μg／mL 点右眼,4次／d,对照组C组予以生理眼水点右眼。在碱烧伤后第1、3、7、14d以形态学分析评价角膜上皮损伤面积及新生血管生长的情况,并于碱烧伤后第1、3、7、14 d 应用Western－blot检测角膜MMP－2和TIMP－2的表达,增强化学发光法（ ECL）对结果进行分析。结果：形态学分析显示,A组和B组小鼠在碱烧伤后第3d起各时间点角膜上皮缺损面积均小于对照组（P＜0．01）,角膜新生血管均得到抑制,CNV面积明显小于对照组（ P＜0．01）。 Western－blot结果显示,碱烧伤后各时间点MMP－2的表达,A组和B组均明显低于对照组（P＜0．01）,TIMP－2的表达高于对照组（P＜0．01）,且A组和B组间MMP－2的表达在第14d比较差异有统计学意义（P＜0．05）,TIMP－2的表达在第7d及第14d比较差异有统计学意义（P＜0．05）。结论：重组Canstatin蛋白可通过抑制角膜细胞及浸润的炎性细胞产生MMP－2,促进TIMP－2表达,从而抑制和延迟碱烧伤后角膜融解的发生和发展,对碱烧伤后角膜的重塑起着重要作用。     

兔棘阿米巴角膜炎的组织病理学研究

目的 观察兔棘阿米巴角膜炎角膜组织感染的病理变化及其特点,探讨棘阿米巴角膜炎的发病机制.方法 新西兰白兔20只,其中16只角膜基质内注射纯化培养的棘阿米巴原虫悬液建立兔棘阿米巴角膜炎模型,观察兔角膜组织的病理学及免疫学的相应变化以及与组织降解和修复相关的细胞因子基质金属蛋白酶13(MMP13)及碱性成纤维细胞生长因子2(bFGF2)的表达;4只为对照.结果 兔棘阿米巴角膜炎感染,初期以中性粒细胞浸润为主,21d后以巨噬细胞为主,并伴有成纤维细胞的增生,7d和14d时 CD4+CD8+细胞比值异常;感染初期,组织内开始出现MMP13阳性表达,从14d开始MMP13合成减少,FGF2的表达逐渐增强,28d时达峰值,其后减弱.结论 兔棘阿米巴角膜炎早期的自然病程以感染性炎症为主,后期主要特征为角膜组织修复.MMP13和FGF2的表达变化与临床表现有一定相关.     

阿米巴角膜炎感染的初步病理观察

目的 观察阿米巴角膜炎患者角膜组织病理变化及相关细胞因子基质金属蛋白酶13(matrix metalloproteinase 13,MMP-13)及成纤维细胞生长因子2(fibroblast growth factor2,FGF2)的表达.方法 收集8例(8眼)阿米巴角膜炎患者接受角膜移植手术后切除的角膜组织,采用常规HE染色和抗MMP-13、抗FGF2免疫组织化学染色,观察其病理变化及MMP-13和FGF2的表达.结果 病程在1个月之内,角膜基质坏死,可见中性粒细胞为主的炎性细胞浸润,组织内可见大量包囊及滋养体.同时,MMP-13呈强阳性表达,平均光密度值为0.308 5±0.078 1;FGF2表达较弱,平均光密度值为0.205 7±0.038 7;病程1个月至半年内,患者角膜处于修复状态,仅有少量中性粒细胞浸润,仍可见到阿米巴存在,组织内MMP-13呈弱阳性表达,平均光密度值为0.187 5±0.029 9,FGF2表达增强,平均光密度值为0.373 5±0.056 2;病程大于半年以上,角膜基质层可见大量新生血管,MMP-13及FGF2均呈微弱表达,平均光密度值分别为0.157 5±0.056 8、0.036 3±0.078 1,未见阿米巴包囊.结论 阿米巴角膜炎感染早期药物治疗不理想,应及时手术清除病灶,可以有助于控制病情,利于组织修复.     

小鸡形觉剥夺性近视眼后极部巩膜MMP-2与TIMP-2 mRNA表达的动态变化

目的　探讨小鸡形觉剥夺性近视眼 (form deprivationmyopia,FDM )后极部巩膜基质金属蛋白酶 2 (matrixmetalloproteinase 2 ,MMP 2 )及其特异性组织抑制剂 (tissuein hibitorofmatrixmetalloproteinase 2 ,TIMP 2 )mRNA表达的时间动态性变化。方法　 5 0只 1d龄来亨雏鸡以半透明眼罩分别遮盖右眼 4、7、14、2 1、30d制备FDM动物模型 ,每组10只 ,未遮盖眼为自身对照眼 ,并随机选取同数目同龄小鸡作为正常对照眼。实验前及预定实验时间进行视网膜检影验光和眼轴长度测量。摘除眼球 ,提取后极部巩膜总RNA ,采用一步法逆转录 聚合酶链反应检测每组小鸡后极部巩膜MMP 2、TIMP 2mRNA表达水平。结果　与正常组、自身对照组相比 ,FDM后极部巩膜MMP 2mRNA显著增高 ,TIMP 2mRNA表达明显降低 ,组间差异非常显著 (P <0 .0 1)。随遮盖时间延长 ,MMP 2mRNA表达逐渐上调 ,7～ 2 1d达最高峰 ,以后轻度下降 ,但仍维持于一较高水平 ,不同遮盖时间组间差异显著 (P <0 .0 1) ,而TIMP 2mRNA表达与之相反。自身对照组MMP 2mRNA表达较同龄正常对照组有轻度上调趋势 ,TIMP 2有下调趋势 ,但组间均无统计学差异 (P >0 0 5 )。结论　MMP 2 /TIMP 2之间动态平衡失调极可能是启动小鸡FDM巩膜细胞外基质早期主动重塑的关键     

Matrix metalloproteinases (MMP-2 and 9) and tissue inhibitors of matrix metalloproteinases (TIMP-1 and 2) during the course of experimental necrotizing herpetic keratitis

To determine the distribution and activities of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during the course of experimental herpes simplex virus (HSV) type-1 keratitis, BALB/c mice were corneally infected with 10(5) plaque-forming units (PFU) of HSV-1 (KOS strain) and then observed for the clinical signs of keratitis. Corneas were harvested at days 0, 2, 7 and 14 post-infection (p.i.). MMP-2, MMP-9, MMP-8, TIMP-1 and TIMP-2 were detected by immunohistochemistry and the Western blot technique. The enzymatic activities were analyzed by zymography. Epithelial HSV keratitis was present at day 2 after corneal infection and healed by day 5 p.i. While the expression and activity of MMP-2, MMP-8 and MMP-9 increased in the corneas at day 2 p.i., it was reduced at day 7 p.i. TIMP-1 and -2 were expressed in the corneas before and seven days after infection. Necrotizing stromal keratitis with corneal ulceration and dense polymorphonuclear leukocyte (PMN) infiltration was present at day 14 p.i. This correlated with increased expression of MMP-2, MMP-8 and MMP-9 in the corneas. MMP-8, MMP-9 and MMP-2 staining was particularly intense in the proximity of the ulcers and in areas of PMN infiltration. At day 14 p.i., MMP-2, -8 and -9 activities were upregulated, and TIMP-2 was expressed. These data suggest that MMPs produced by resident corneal cells and PMNs may possibly play a role in early epithelial keratitis and in the ulcerative process in the late phase after corneal HSV-1 infection. The ratio of MMPs to TIMPs may be important for the course of necrotizing HSV keratitis. TIMPs might participate in the repair process.     

Induction of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs correlates with outcome of acute experimental pseudomonal keratitis

This study aimed to investigate expressions and sources of matrix metalloproteinases (MMP)-2 and MMP-9, and of tissue inhibitors of MMP (TIMP)-1 and TIMP-2 in experimental Pseudomonas aeruginosa keratitis in rabbits. Pseudomonal keratitis was induced in New Zealand white rabbits, and macroscopic and microscopic examinations were performed at appropriate time points (3, 9, 12, 18, 24, 72 h). Expressions and sources of MMP-2, 9, and TIMP-1, 2 were determined using immunohistochemistry, gelatin zymography, ELISA, and RT-PCR. A typical corneal ulcer with a ring abscess was observed 12-72 h post-infection (p.i.) with P. aeruginosa. In microscopic examinations, massive inflammatory cell (mostly polymorphonuclear leukocytes, PMNs) infiltration and liquefactive necrosis were characteristic features. MMP-2 was constitutively expressed in keratocytes, and its expression was not apparently enhanced after pseudomonal infection as evidenced by zymography, immunostaining, and RT-PCR. However, MMP-9 and its activated form were induced, and were significantly enhanced 12-24 h p.i. MMP-9 appeared to derive from PMNs rather than from resident corneal cells. TIMP-1 was expressed in PMNs, macrophages, and keratocytes, and its expression was enhanced 72 h p.i. Although TIMP-2 was constitutively expressed as seen by immunostaining and RT-PCR, its concentration was below detection limits during the experiments. We demonstrated that MMP-9 was one of the important factors for corneal tissue destruction, because it was induced and significantly expressed in keratocytes and inflammatory cells after pseudomonal infection. Although TIMP-1 was expressed in later stages of infection, enhancement and activation of MMP-9 were much faster and stronger than those of TIMP-1, thereby facilitating tissue destruction leading to corneal ulceration.     

Matrix metalloproteinases (MMP-8, MMP-9) and the tissue inhibitors of metalloproteinases (TIMP-1, TIMP-2) in patients with fungal keratitis

PURPOSE: To study the infiltrating cells and quantify the levels of matrix metalloproteinases (MMP-8, MMP-9) and tissue inhibitor of metalloproteinases (TIMP-1, TIMP-2) in the cornea, tear, and serum of patients with fungal keratitis. METHODS: Experimental study. Infected corneal tissue from 4 patients with fungal keratitis (group 1) scheduled to undergo therapeutic keratoplasty accounted for the histopathologic and cytospin smear analysis. Ten patients with fungal keratitis from group 2 served for the quantification of MMPs and TIMPs. Five patients with keratoconus undergoing penetrating keratoplasty and 5 cadaver corneas were chosen as controls for group 2. Corneal buttons obtained during keratoplasty, 15 to 20 microL of tears collected using the capillary flow method, and 3 mL of blood was obtained from patients with fungal keratitis and patients with keratoconus. Corneal button sections from group 1 were stained with hematoxylin and eosin and Grocott methenamine silver nitrate for the histopathologic studies and Giemsa staining for the cytospin smear analysis. Enzyme-linked immunosorbent assay was used for the quantification of total MMP-8, MMP-9, TIMP-1, and TIMP-2 in the corneal homogenates, tear, and serum samples of group 2. RESULTS: Corneal sections from group 1 revealed dense fungal filaments and a large proportion (91.4% +/- 38%) of polymorphonuclear leukocytes (PMNs). Significant elevation in the levels of MMP-8 and MMP-9 (P < 0.05) in the fungal keratitis corneas was observed in group 2 compared with the cadaver and keratoconus corneas. The ratio of MMP/TIMP was also higher in the fungal keratitis corneas. CONCLUSIONS: Infiltrating PMNs in the cornea of patients with fungal keratitis contributed to the increased activities of MMP-8 and MMP-9, thereby enhancing tissue destruction and derangement.     

The expression of matrix metalloproteinases and their inhibitors in corneal fibroblasts by alarmins from necrotic corneal epithelial cells

Purpose

Sterile ulceration is frequently observed in the cornea following persistent corneal epithelial damage. We examined the effect of alarmins released by necrotic corneal epithelial cells (HCE) on the production of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by corneal fibroblasts.

Methods

IL-1α and high-mobility group box 1 protein (HMGB1) released into the supernatant derived from necrotic HCE cells were measured with enzyme-linked immunosorbent assay (ELISA). MMPs and TIMPs produced by corneal fibroblasts, stimulated with the supernatant from necrotic HCE cells, were analyzed and measured with protein array and ELISA. To investigate dynamic expression of alarmins in the corneal epithelium, we used immunohistochemistry to observe the expression of human IL-1α in the corneal epithelium of human IL-1α Tg mice with or without cryopexy. We also investigated the expression of MMPs in corneal stroma of the mice treated with cryopexy, using RT-PCR.

Results

We detected IL-1α and HMGB-1 in the supernatant of necrotic HCE cells. These supernatants increased the expression of MMP-3 and MMP-1, and decreased that of TIMP-1 and TIMP-2 in human corneal fibroblasts. Almost always these were inhibited by IL-1 receptor antagonist. Recombinant IL-1α increased the production MMP-3 and MMP-1 in corneal fibroblasts. After cryopexy of the epithelium of human IL-1α Tg mice, the expression of human IL-1α was recognized in the cytoplasm but not nucleus of epithelial cells. The level of MMP-3 and MMP-1 mRNAs was elevated in the corneal stroma in mice treated with cryopexy.

Conclusion

Alarmins, especially IL-1α, released from necrotic HCE cells may play an important role in the expression of MMPs and TIMPs by corneal fibroblast, resulting in sterile ulceration.

     

Effect of prostaglandin analogues on tear proteomics and expression of cytokines and matrix metalloproteinases in the conjunctiva and cornea

The purpose of this work was to identify potential tear-film-based proteins and their effect on changes in the conjunctiva and cornea in eyes using prostaglandin (PG) analogues. Recruited subjects were individuals who had used PG for at least 1 year and comparison with eyes of normal controls and timolol using patients were done. Approximately 3-5?μL of tears were sampled from both eyes of each subject using glass microcapillaries. Proteomic analysis was done to compare the pooled tear samples from each group by Bradford assay and cytokine arrays. Impression cytology was used to gather mRNA from conjunctival epithelial cells, and target protein mRNA was quantified by PCR. Rabbits treated with PG were scarified, and changes in the corneal stroma were evaluated by immunohistochemical staining and western blot analysis. There were increased levels of IL-1β, IL-6, MMP-1, MMP-3, and MMP-9 and decreased levels of TIMP-1 and TIMP-2 in the tears of PG-treated patients. The mRNA of IL-1β, MMP-1, MMP-3, and MMP-9 was elevated and mRNA of TIMP-1 and TIMP-2 was decreased in the conjunctival epithelial cells. Rabbits treated with PG showed corneal thinning with decreased collagen type I expression. The protein of MMP-1 and MMP-9 was elevated and protein of TIMP-1 was decreased in the rabbit cornea by western blot analysis. Immunohistochemical staining showed elevated expression of MMP-1 and MMP-9 and the decreased expression of TIMP-1 in the corneal stroma. The topical use of PG analogues results in an altered balance between MMPs and TIMPs, which may be triggered by inflammatory cytokines. This results in an increase of matrix degradation and decrease of stromal collagens in the cornea by PG treatments.     

Transplantation of amniotic membrane in murine herpes stromal keratitis modulates matrix metalloproteinases in the cornea

PURPOSE: To study matrix metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) in the corneas from mice with ulcerative herpes stromal keratitis (HSK) treated with amniotic membrane transplantation (AMT). METHODS: The corneas from BALB/c mice were infected with HSV-1. Mice with ulcerative HSK on postinfection (PI) day 14 were used for the experiments. In one group of mice, the corneas were treated with transplantation of amniotic membrane (AMT) that was secured with a tarsorrhaphy, and a control group underwent tarsorrhaphy alone. After 2 days, the appearance of corneal ulcers and stromal inflammation was judged clinically. Corneal sections were studied by immunohistochemistry for the expression of MMP-2, -8, and -9 and TIMP-1 and -2. MMP activity in the corneas was investigated by zymography, and the expression of the enzymes was measured by the Western blot technique. RESULTS: At day 14 PI, the ulcers stained intensely positive for MMP-2, -8, and -9 and TIMP-1 and -2. Ulceration (P < 0.001), stromal inflammation (P < 0.01) and inflammatory cell infiltration (P < 0.001) markedly improved by day 2 after AMT. This was associated with reduced expression (P < 0.01) and activity of MMP-8, and -9 and increased localization of TIMP-1 (P < 0.01), whereas TIMP-2 was not affected. In contrast, high levels of expression of MMP-8 and -9 remained in the cornea after tarsorrhaphy, and the TIMP-1 expression was only slightly upregulated. CONCLUSIONS: Rapid improvement of HSV-1-induced ulcerative keratitis is noted after amniotic membrane transplantation. This may be caused by reduced expression and activity of MMP-8 and -9, increased expression of TIMP-1, and sustained expression of TIMP-2.     

Expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloproteinase 1 and 2 in inflammation-induced corneal neovascularization.

PURPOSE: Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) have been linked to the angiogenic process in general. In order to understand the potential roles of MMP-2, MMP-9 and TIMPs in the corneal neovascularization process, we examined the expression and activities of MMP-2, MMP-9 and TIMPs during the course of cauterization-induced corneal neovascularization in a rat model. METHODS: Neovascularization of rat corneas was induced by silver nitrate cauterization. The expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 was examined by immunohistochemistry and RT-PCR. The protein activities of MMPs and TIMPs were compared in pre- and postcauterization corneas by gelatin zymography and reverse zymography, respectively. RESULTS: MMP-2, TIMP-1 and TIMP-2 immunoreactivities were expressed in normal corneas, predominantly in the corneal epithelium. After injury, immunoreactivities of both MMPs and TIMPs were increased, notably in the healing corneal epithelium, infiltrating inflammatory cells, stromal fibroblasts and ingrowing vascular endothelial cells. The increase in gross MMP-2 enzymatic activity paralleled the maximal vascular ingrowth on day 4, while the gross MMP-9 enzymatic activity rose immediately on day 1, then decreased steadily, which paralleled the magnitude of inflammatory cell infiltration. The immunoreactivity of MMPs/TIMPs decreased significantly 2 weeks after cauterization. On day 35, MMP-2, TIMP-1 and TIMP-2 staining was seen only in corneal epithelium and vascular endothelial cells. Both the RT-PCR and reverse zymography results revealed a more constant expression of TIMP-2, while the TIMP-1 expression appeared to be more inducible. CONCLUSION: MMPs as well as TIMPs were upregulated in cauterization-induced corneal neovascularization, suggesting that both may participate in extracellular matrix remodeling in the corneal wound healing, inflammation and neovascularization processes.     

Regulation of MMPs and TIMPs by IL-1beta during corneal ulceration and infection

PURPOSE: This study was conducted to investigate the role of IL-1beta in the regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in a mouse model of experimental keratitis and corneal injury. METHODS: Mice were injected subconjunctivally with 10 micro g of anti-mouse IL-1beta antibody 2 hours before challenge with Pseudomonas aeruginosa (strain 6294). Control animals received an equal volume and concentration of isotype control antibody at the same time. Eyes were enucleated at 0, 8, 24, and 72 hours, after bacterial challenge and processed for histologic examination. Some eyes were homogenized and used to evaluate production of MMP-2, MMP-9, TIMP-1, and TIMP-2 protein, by zymography and reverse zymography. RESULTS: Injury without bacterial infection resulted in increases in both MMP-2 and -9 and a slight but significant downregulation of TIMP-1. Administration of anti-IL-1beta just before injury and without bacterial infection resulted in a significant reduction in expression of MMP-2 (at 8 hours), MMP-9 (at 8 hours), TIMP-1 (at 8 and 72 hours), and TIMP-2 (at 8 hours). Mice treated with anti-IL-1beta antibody, before bacterial challenge, demonstrated markedly reduced corneal damage compared with the severe corneal injury and massive neutrophil infiltration observed in infected mice treated with control antibody. Administration of the neutralizing anti-IL-1beta antibody resulted in a significant reduction of MMP-9 and a change in the time course of TIMP-1 and -2 expression. The reduction in MMP-9 by anti-IL-1beta during infection was much greater than the reduction without infection. CONCLUSIONS: The results imply that IL-1beta has a central role in corneal destruction during bacterial keratitis and suggests that targeting IL-1beta may be a novel therapeutic strategy for microbial keratitis.     

Early results of circularity and centration of capsulotomy prepared by three different methods

AIM:To compare the difference of capsulotomy produced by precision pulse capsulotomy(PPC),manual(M-CCC),and femtosecond laser assisted capsulotomy(FLAC)in relation to intraocular lens(IOL)centration,circularity and its effect on visual outcomes.METHODS:Prospective,non-randomized comparative study conducted at LV Prasad Eye Institute,Hyderabad,India.Sixty eyes of 52 patients were grouped into 3(FLAC,PPC and M-CCC)based on capsulotomy techniques used.Twenty consecutive eyes with uneventful phacoemulsification and with no comorbidities affecting the capsulotomy or visual outcome were included in each group.The main outcome measure was IOL centration in relation to capsulotomy and pupil.Secondary outcome measures were post-operative visual acuity,manifest refraction and aberration profile between groups.RESULTS:At 5 wk the visual,refractive outcomes and endothelial cell density were comparable between the 3 groups.The median circularity index of FLAC was statistically significantly different to M-CCC or PPC(1-10)groups(P<0.01)but PPC(11-20)was comparable to FLAC.Decentration of IOL center in relation to capsulotomy was seen only between the PPC(1-10)group and FLAC group(P=0.02).The IOL was well centered in relation to the pupil in all the groups(P=0.46).The quality of vision parameters like the higher order aberrations,spherical aberration,coma,trefoil,modular transfer function,and Strehl ratio were comparable between the groups.CONCLUSION:Our study shows that despite differences in the morphology of capsulotomy produced by PPC,M-CCC,FLAC a well-centered IOL can be achieved.The measured capsular morphology parameters do not affect visual outcomes.