Thymosin-beta4 inhibits corneal epithelial cell apoptosis after ethanol exposure in vitro

PURPOSE: The purpose of this study was to determine the effect of thymosin beta 4 (Tbeta(4)) treatment on human corneal epithelial cells exposed to ethanol in vitro. The efficacy of Tbeta(4) in preventing mitochondrial disruption and in inhibiting caspase-mediated apoptosis was examined. METHODS: Nontransformed human corneal epithelial cells (HCECs) at passage 4 were untreated or treated with ethanol (20% for 20 seconds) or a combination of ethanol and Tbeta(4). The cells were allowed to recover from ethanol treatment for 24 hours. Mitochondrial membrane integrity and the release of cytochrome c to the cytoplasm were assessed using microscopy, Western blot, and ELISA. Bcl-2 expression and cell proliferation were measured using ELISA. Colorimetric activity assays were completed for caspase-2, -3, -8, and -9. RESULTS: Tbeta(4) treatment decreased deleterious mitochondrial alterations, significantly decreased cytochrome c release from mitochondria, and increased Bcl-2 expression in ethanol-exposed human corneal epithelial cells. In ethanol-exposed corneal epithelium Tbeta(4) treatment inhibited caspase-2, -3, -8, and -9 activity, with caspase-8 showing the most significant inhibition. Tbeta(4) treatment resulted in no significant effect on the proliferation of human corneal epithelial cells after ethanol exposure. CONCLUSIONS: Tbeta(4) plays an antiapoptotic role under conditions of epithelial cell challenge with an external stress such as exposure to ethanol. Tbeta(4) may function as an antiapoptotic agent by inhibiting the release of cytochrome c from mitochondria and by suppressing the activation of caspases.     

Effect of metalloproteinase inhibitor on corneal cytokine expression after alkali injury.

PURPOSE: Interleukin (IL)-1alpha and IL-6 levels in the cornea are greatly elevated during the early stages after an alkali burn in mice. The authors investigated the effect of synthetic inhibitor of matrix metalloproteinase (SIMP) on the expression of inflammatory cytokines in alkali-burned murine corneas and evaluated the clinical appearance of the eyes. METHODS: After 0.5N NaOH-alkali burns to 400 corneas of ICR mice, 200 received 400 microg/ml of SIMP topically 4 times a day while 200 corneas were similarly treated with vehicle only. At days 4, 7 and 14 after injury, each cornea was assigned a clinical score for corneal opacity, corneal epithelial defect, hyphema and cataract. Extracts of injured corneas in each group were then assayed for cytokine production using ELISA systems for IL-1alpha, IL-1beta, IL-6 and tumor necrosis factor-alpha (TNF-alpha). RESULTS: The levels of IL-1alpha, IL-1beta and IL-6 were significantly lower in the SIMP-treated group than in the vehicle-treated group 7 days after the burn. However, levels of these cytokines were similar in the SIMP and non-SIMP groups at days 4 and 14. Levels of TNF-alpha did not differ between both groups at any postinjury time. In the SIMP-treated corneas, there was less opacification and hyphema formation and epithelial regeneration was faster. CONCLUSIONS: Topical application of SIMP in alkali-burned murine corneas reduced the expression of IL-1alpha, IL-1beta, and IL-6 and lessened the severity of the injury.     

碱烧伤后角膜新生淋巴管与炎症反应指数间的关联

目的 探讨角膜碱烧伤后的角膜新生淋巴管与炎症反应指数间的关联.方法 实验研究.制备大鼠角膜碱烧伤模型.采用5'核苷酸酶-碱性磷酸酶(5'-NA-ALP)双重酶组织化学染色及全角膜免疫荧光法分别检测碱烧伤后1、3 d,1、2、3、4、5、6、7及8周的角膜新生淋巴管和血管的动态变化,并进行淋巴管计数(LVC)和血管计数(BVC).同时,于裂隙灯显微镜下观察角膜炎症反应的变化,记录炎症反应指数(IF),并比较LVC和IF之间的关联.11例人角膜取自碱烧伤后行角膜移植的11例患者.淋巴管内皮细胞受体(LYVE-1)免疫组织化学染色法标记人角膜中的新生淋巴管,LVC和IF之间的关联运用Pearson's相关分析,采用配对t检验比较角膜中存在淋巴管和不存在淋巴管的患者之间IF、炎性细胞计数、碱烧伤病史、年龄的差异.结果 碱烧伤后,角膜基质层存在着新生淋巴管.碱烧伤后3 d时出现角膜新生淋巴管,2周末达到高峰,5周末消退.新生淋巴管的出现滞后于炎症反应,但先于炎症反应和新生血管而消退.LVC与IF之间呈正相关(r=0.572,P＜0.01).11例患者中3例存在着角膜新生淋巴管.与另8例角膜中无新生淋巴管的患者相比,前者IF显著性升高(t=3.28,P＜0.05)、炎性细胞计数显著性增加(t=2.42,P＜0.05),年龄显著性下降(t=2.62,P＜0.05),而碱烧伤病史无显著性差异(t=1.28,P＞0.05).结论 角膜碱烧伤后有淋巴管生成,角膜新生淋巴管和炎症反应指数之间存在着密切的关联.     

Effect of a metalloproteinase inhibitor on established corneal ulcers after an alkali burn.

Proteinase inhibitors have been shown to prevent corneal ulceration and perforation when used immediately after an experimental alkali burn injury. To evaluate the clinical efficacy of a synthetic metalloproteinase inhibitor, HSCH2CH[CH2CH(CH3)2]CO-Phe-Ala-NH2(SIMP), treatment with inhibitor was withheld until corneal ulceration ensued after a standard alkali injury to the rabbit eye. When topical therapy with a 1 mmol/l solution of SIMP was initiated after corneal ulceration had progressed to a mid-stromal level (clinical score of 2), there was no significant difference in the progression of corneal ulceration between the treated vs. control group after 6 d of therapy. In the second study in which treatment was initiated earlier at the onset of superficial ulceration (clinical score of 1), there was a significant difference in clinical scores between the two groups after 1 day of treatment until termination of the experiment at 21 d (P less than 0.005). In the inhibitor-treated group, 88.9% of the corneas showed a reversal or cessation of progression of the ulceration process. Eighty-seven-and-a-half percent of the control corneas progressed to descemetocele formation or perforation by day 14 of treatment. This study suggests that SIMP may be used for effective treatment of corneal ulcers resulting from an alkali burn injury in the human eye. It also shows that early and aggressive initiation of therapy is critical.     

角膜碱烧伤中PMNs与MMP-9的关系

目的：研究角膜碱烧伤后基质损伤修复病程中多形核中性白细胞(PMNs)的浸润和基质金属蛋白酶-9(MMP-9)的表达的关系。

方法：建立25只家兔角膜碱烧伤模型,分别于伤后3、7、14、21、28d随机处死5只兔,裂隙灯下观察角膜病理修复情况,摘除角膜做病理切片,测定PMNs的浸润量值。免疫组化法测定MMP-9的表达。

结果：PMNs和MMP-9在角膜碱烧伤后的3d开始升高,14d上升达到最大峰值,之后逐渐降低,碱烧伤后角膜基质在第14d溃疡面积及深度最为严重。

结论：角膜碱烧伤病灶中PMNs和MMP-9的量值呈正相关,且角膜的病理损伤与PMNs的浸润和MMP-9的表达密切相关。     

SPARC基因在角膜碱烧伤中的表达

目的　动态检测酸性富含胱氨酸分泌型蛋白 (SPA RC)在角膜碱烧伤中的表达 ,为探讨 SPA RC基因在角膜创伤中的作用提供依据。方法　首先建立角膜碱烧伤模型 ,并设角膜物理损伤组为对照组 ,在不同时间段提取各组角膜上皮细胞总RN A。用基因引物进行逆转录 -聚合酶链反应 (RT - PCR )扩增 ,半定量 SPA RC m RN A的表达水平。然后对 PCR扩增结果用限制性内切酶酶切验证。在取角膜上皮之前 ,对受试动物进行裂隙灯检查 ,以判断角膜碱烧伤程度与基因表达的关系。结果　(1)正常角膜上皮细胞可见 SPA RC基因的低表达 ;(2 ) SPA RC在角膜碱烧伤及物理损伤后均见较高水平表达 ,且与正常细胞表达相比较有显著的统计学差异 (P<0 .0 5) ;角膜创伤越严重 ,基因表达水平越高 ;(3 )所检测基因表达结果与显微镜观察角膜水肿、混浊、角膜新生血管等损伤结果呈同步趋势。结论　 SPARC在角膜碱烧伤和创伤过程中具有高水平表达。是角膜创伤修复过程中的重要因素。     

Decreased fibronectin levels in aqueous humor after corneal injury

Fibronectin plays an important role in wound healing and cell differentiation. The present study measures fibronectin levels in aqueous humor from injured corneas. In response to transcorneal freezing treatments, a thickened scar-like matrix is deposited on the posterior surface of rabbit corneas. This posterior collagenous layer (PCL) is visible grossly and has a uniform diameter. Aqueous humor from eyes with established posterior corneal scarring has significantly less fibronectin compared with control eye aqueous humor. Tissue culture experiments show that corneal endothelial cells from control eyes and PCL-associated cells from injured eyes produce similar amounts of fibronectin. The marked decline in fibronectin levels in aqueous humor in scarred corneas supports the hypothesis that fibronectin plays a role in the pathological events which result from corneal injury.     

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。     

Corneal infiltration after recurrent corneal epithelial erosion

AIMS—To describe the clinical features of patients with a history of recurrent corneal epithelial erosion who develop acute corneal infiltration.
METHODS—The records were reviewed of patients who had previously been examined and treated for recurrent corneal epithelial erosion and who presented again with signs suggestive of a microbial keratitis.
RESULTS—11 patients were described; one patient presented with similar signs on two occasions. There was typically a paracentral epithelial defect >2 mm in diameter with an associated stromal infiltrate and an intense anterior uveitis. Three patients had a hypopyon, and four developed a subepithelial ring infiltrate. Samples were taken for microscopy and bacterial culture, with a positive isolate from two of 12 episodes (16%). Treatment with topical antibiotics and topical corticosteroid resulted in rapid re-epithelialisation and a reduction of inflammation. There was good visual outcome for all eyes, with a recurrence or symptoms of epithelial erosion in only one eye after a mean follow up period of 18 months.
CONCLUSIONS—Corneal infiltrates are an uncommon complication of recurrent corneal epithelial erosion. Despite the intensity of the infiltration the majority are culture negative using established techniques. There is typically rapid resolution and a good visual outcome, with a tendency for the episode to mark the end of further symptoms of epithelial erosion.

     

人羊膜匀浆提取液对角膜碱烧伤后新生血管PEDF和VEGF表达及角膜新生血管的影响

目的:探讨人羊膜匀浆提取液对角膜碱烧伤后新生血管形成过程中色素上皮衍生因子(PEDF)和血管内皮生长因子(VEGF)表达及角膜新生血管的影响.方法:选取2015-06/2016-06在佛山爱尔眼科医院治疗的角膜碱烧伤患者32例37眼,随机分为A、B两组.其中A组17例19眼,采用40mg/L人羊膜匀浆提取液治疗,B组15例18眼,采用3g/L泼尼松龙滴眼液治疗.在治疗不同的时间点(1、4、7、14、21、28d)观察角膜新生血管的生长,同时检测新生血管形成过程中PEDF及VEGF的表达水平.结果:A组患者在使用人羊膜匀浆提取液治疗后,PEDF表达水平显著高于B组,差异有统计学意义(P=0.001),在治疗28d后,PEDF表达水平达到了0.721依0.314,而B组患者PEDF表达水平仅有0.538依0.253,两组患者PEDF表达水平间的差异具有统计学意义( P<0.05 );A组VEGF表达水平在不同的时间点检测时均低于B组,在治疗28 d后,A组患者VEGF 表达水平为0.152依0.020,B 组患者VEGF表达水平为0.302依0.031,两组患者VEGF表达水平间的差异具有统计学意义(P<0.05);A组患者角膜新生血管数量显著低于B组患者,差异有统计学意义( P<0郾05 ).结论:人羊膜匀浆提取液可以促进患者角膜碱烧伤后新生血管形成过程中PEDF表达,抑制VEGF的表达和角膜新生血管的增殖.     

Adenosine phosphate and glutathione levels in the regenerated corneal epithelium after abrasion and mild alkali burns

After scraping away the corneal epithelium or after mild alkali burn, the regeneration of the cellular layers on the stromal surface was observed clinically. The average time of regeneration was 7 days after abrasion and 12 days after mild alkali burn. When the stromal surface was just covered by the newly regrown epithelium, the metabolite levels were determined. In the regenerated corneal epithelium after abrasion the ATP/ADP ratios were much higher than in the normal epithelium and much lower after the alcali burns.The GSH levels were diminished in both groups. The GSSG levels were elevated significantly only after the alkali burns resulting in a decreased redox ratio of the glutathione. The results seemed to be in accordance with morphological and enzymatical data of the regenerating corneal epithelium. Changes of the glutathione in the corneal endothelium were cautiously interpreted.Presented at the Annual General Meeting of the Association for Research in Vision and Ophthalmology. Sarasota, Florida 1979.     

小梁细胞基质金属蛋白酶及其调控因素

基质金属蛋白酶(MMPs)是一组降解细胞外基质(ECM)成分的含锌蛋白水解酶家族,对于维持ECM不断产生与降解的动态平衡及正常房水通路的流畅性具有重要的意义.基质金属蛋白酶抑制剂(TIMPs)是MMPs的内源性特异性的组织抑制剂,可以抑制MMPs对ECM的降解作用.MMPs活性受多种水平调节,分为基因水平、酶原活化调节、活化后调节.因此各种外界因素、MMPs及其组织抑制因子TIMPs共同参与ECM的降解与重建,它们之间的协同作用以及表达的动态平衡保证组织的正常生理功能运转.就小梁细胞MMPs及其多种调控因素进行综述.     

苏拉明对角膜碱烧伤新生血管抑制作用的研究

目的研究苏拉明对碱烧伤角膜新生血管（CNV）及血管内皮生长因子（VEGF）、胰岛素样生长因子-I（IGF—I）的影响。方法新西兰大白兔48只,分为空白组、对照组、苏拉明组3组,每组16只（32只眼）。对照组与治疗组制备角膜碱烧伤模型,术后分别给予氯霉素、8g／L苏拉明滴眼液点眼。分别于术后第1、4、7、14d观察新生血管情况,免疫组织化学法检测VEGF、IGF—I的表达。结果术后第1d各组无新生血管生长。第4、7、14d,空白组未出现新生血管,对照组与治疗组可见CNV,且治疗组比对照组新生血管面积小（P〈0．01）。第1、4、7d,对照组与治疗组VEGF、IGF—I的表达均比空白组升高,差异有统计学意义（P〈0．01）,治疗组与对照组相比,差异亦有统计学意义（P〈0．01）。第14d,3个组间比较差异无统计学意义（P〉0．05）。结论VEGF、IGF—I参与CNV形成,苏拉明通过降低角膜上皮中二者的表达,抑制角膜碱烧伤新生血管形成。     

Effects of topical vitamin E on corneal superoxide dismutase,glutathione peroxidase activities and polymorphonuclear leucocyte infiltration after photorefractive keratectomy

PURPOSE: Photorefractive keratectomy (PRK) induces free radical formation and polymorphonuclear (PMN) cell infiltration in the cornea. Vitamin E is a free radical scavenger and protects the cells from reactive oxygen species. We investigated the effects of topical vitamin E on corneal PMN cell infiltration and corneal antioxidant enzyme activities after PRK. METHODS: We studied four groups, each consisting of seven eyes. Group 1 were control eyes. In group 2 the corneal epithelium was removed by a blunt spatula (epithelial scrape). In group 3, corneal photoablation (59 micro m, 5 dioptres) was performed after epithelial removal (traditional PRK). In group 4 we tested the effects of topical Vitamin E after traditional PRK. Corneal tissues were removed and studied with enzymatic analysis (measurement of corneal superoxide dismutase and glutathione peroxidase activities) and histologically. RESULTS: Stromal PMN leucocyte counts were significantly higher after mechanical epithelial removal and traditional PRK (p < 0.05). Corneal superoxide dismutase and glutathione peroxidase activities decreased significantly after mechanical epithelial removal and traditional PRK (p < 0.05). In group 4, treated with vitamin E, corneal superoxide dismutase activity did not differ significantly from that in the medically non-treated groups, nor did corneal PMN cell infiltration after traditional PRK. The reduction of corneal glutathione peroxidase activity after PRK was reduced significantly after topical vitamin E treatment. CONCLUSIONS: Topical vitamin E treatment may be useful for reducing the harmful effects of reactive oxygen radical after epithelial scraping and PRK in that it increases corneal glutathione peroxidase activity.     

Characteristics of polymorphonuclear leukocyte infiltration into the alkali burned eye and the influence of sodium citrate

Polymorphonuclear leukocytes (PMNs) are considered to play a central role in the corneal ulceration process subsequent to an alkali burn. We have described the time course of PMN infiltration into the ocular tissues following an alkali burn. In addition, we examined the effect of sodium citrate upon the accumulation of PMNs in the alkali burned cornea. The accumulation of PMNs into the cornea and iris-ciliary body was quantified by measuring myeloperoxidase (MPO), an enzyme marker for these inflammatory cells. Leukocytes in aqueous humor aspirates and corneal washes were counted directly under a microscope. In the alkali burned cornea, we found an initial transient, yet substantial, infiltration of PMNs, peaking at about 12-24 hr and limited to the peripheral cornea; this subsided by about 72 hr. By 14 days, the MPO activity, and hence the number of leukocytes, had risen again, and by 21 days the level had increased by several fold. Qualitatively similar biphasic patterns of leukocyte infiltration were observed in the iris-ciliary body and aqueous humor. Leukocyte numbers in corneal washes only increased between 4-24 hr following the alkali burn. The exceptionally high degree of leukocyte infiltration into the cornea at 21 days corresponded with the presence of ulceration. Topical administration of sodium citrate (10%) inhibited the early and late phase of PMN accumulation in the alkali burned cornea, i.e. at 24 hr (-63%) and 21 days (-92%). The inhibition of PMN infiltration by sodium citrate correlates with the reduced corneal ulceration observed following treatment with this compound.     

羊膜移植对大鼠角膜碱烧伤后基质金属蛋白酶的影响

目的探讨羊膜移植对大鼠角膜中度碱烧伤后角膜上皮细胞和基质成纤维细胞中基质金属蛋白酶(matrix metalloproteinase，MMP)变化的影响。方法将12只SD大鼠随机分成2组，每组6只，分别作为碱烧伤后1d组和14d组。在2组中，所有大鼠角膜建立中度碱烧伤模型，左眼行羊膜移植，右眼作为对照。应用免疫组化法，分别观察碱烧伤后1d和14d实验组与对照组中MMP-9和MMP-2表达的变化。结果碱烧伤后1d，实验组与对照组中，角膜上皮细胞内的MMP-9和MMP-2的表达与正常角膜上皮细胞内的MMP-9和MMP-2的表达相比明显升高，但两组间的表达差异无显著性；而2组成纤维细胞内的MMP-9和MMP-2的表达与正常成纤维细胞内的MMP-9和MMP-2的表达相比明显升高，两组间的表达差异无显著性。碱烧伤后14d，2组中角膜上皮细胞内的MMP-9和MMP-2的表达与碱烧伤后1d情况相似，两组间的表达差异无显著性；2组成纤维细胞内的MMP-9和MMP-2的表达与碱烧伤后1d组相比明显升高，并且实验组中的MMP-9和MMP-2的表达与对照组相比也有明显升高。结论碱烧伤后大鼠角膜上皮细胞和基质成纤维细胞中的MMP-9和MMP-2的表达都有升高。羊膜移植能增强MMP-9和MMP-2在成纤维细胞中的表达，提示羊膜在加快角膜碱烧伤后的重塑中起一定作用。     

HIF在兔角膜碱烧伤后CNV生成中的作用研究

目的研究缺氧诱导因子(hypoxia inducible factor,HIF)以及诱导性一氧化氮合成酶(inducible nitric oxide synthase,iNOS)和血管内皮生长因子(vascular endothelial growth factor,VEGF)在角膜碱烧伤后角膜新生血管(corneal neovascularization,CNV)形成中的作用及机制。方法 建立兔CNV动物模型,将其随机分为对照组和3个实验组,对照组结膜下注射生理盐水,实验组注射不同剂量的三氧化二砷。观察兔CNV的生长情况,并记录CNV的生长面积。用免疫组织化学方法检测兔角膜HIF、iNOS以及VEGF的表达。结果 对照组6 d、12 d、15 d、21 d、28 d CNV的面积分别为:(53.86±20.60)mm2、(87.21±25.80)mm2、(128.31±40.10)mm2、(114.42±29.40)mm2、(78.15±35.13)mm2。各实验组CNV面积均小于对照组,差异均有统计学意义(均为P<0.05)。免疫组织化学方法检测角膜HIF-1α、iNOS及VEGF蛋白的表达:VEGF表达集中在角膜上皮下基质,存在于血管内皮细胞、炎性细胞胞浆内,呈深黄色。在7 d有较高表达,14 d达到高峰,28 d时明显降低,且对照组明显高于实验组,各实验组的表达随三氧化二砷剂量的增加而减少。蛋白的表达与CNV面积呈正相关。结论 角膜碱烧伤后CNV的形成可能与HIF-1α有关。三氧化二砷通过抑制HIF-1α和iNOS、VEGF表达而抑制兔CNV的形成。