Effect of porcine chondrocyte-derived extracellular matrix on the pterygium in mouse model

Purpose

To investigate the effect of porcine chondrocyte-derived extracellular matrix (PCDECM) on an experimental mouse model of human pterygial epithelial cells.

Methods

Cultured human pterygial epithelial cells (hPECs) were stained with pan-cytokeratin (CK), CK3/2p, vimentin, and CK13 antibodies to characterize the cells. A pterygium mouse model was developed by injecting 1X10⁴ hPECs into the nasal subconjunctival space in athymic nude mice. PCDECM (25 mg/mL, 10 μL) was injected into the nasal subconjunctival space in the right eye 7, 10 and 14 days after the epithelial cell injection (PCDECM group). Image analysis was performed using ImageJ® to compare the lesion size. A histopathological analysis of the cornea was conducted to evaluate the state of the epithelium and the expression of pterygial epithelial cell markers.

Results

The isolated pterygial cells were positive for pan-CK, CK3/2p and vimentin, and they were negative for CK13 under immunofluorescence microscopy. On day 17 after epithelial cell injection, the size of the lesion compared to the entire cornea was increased to 37.1 % in the control group. However, in the PCDECM group, the lesion covered only 26.3 % of the entire cornea. The corneas of the pterygium mice showed an epithelium of irregular thickness, proliferation of the stroma, extracellular matrix breakdown and overexpression of pterygium-positive markers. However, these changes were significantly suppressed by the application of PCDEDM.

Conclusions

Our findings suggest that PCDECM seems to suppress pterygial epithelial cell growth and it could be used as a promising biomaterial for the noninvasive treatment of pterygium.     

准分子激光屈光性角膜切削术后兔眼角膜组织细胞外基质成分的变化

目的 动态观察准分子激光屈光性角膜切削术（photorefractive keratectomy,PRK) 后角膜组织细胞外基质（extracellular matrix,ECM)成分的变化,并探讨其与角膜雾状混浊（haze）的关系。方法 24只兔按术后即刻、24h、1周、2周、1个月、3个月、6个月及12个月不同处死时间分为8个实验组,每组3只,按设计矫正度数－10．00D行右眼PRK术,术后裂隙灯显微镜检查各组角膜的haze分级,并取角膜组织以及免疫组织化学方法检测ECM成分,包括Ⅰ型胶原蛋白、Ⅲ型胶原蛋白、Ⅳ型胶原蛋白、细胞纤维连接蛋白（cellular fibronectin,cFN)、细胞黏合素（tenascin,TN)以层黏连蛋白（laminin,LN)。结果 PRK术后haze的发生率为100％;角膜基质浅层内沉积新合成的Ⅰ、Ⅲ、Ⅳ型胶原蛋白及cFN、TN、LN等ECM成分,其表达量变化与haze分级变化基本一致。结论 PRK术后切削区角膜基质浅层ECM成分的沉积与角膜haze的出现密切相关。     

VEGF siRNA对兔眼碱烧伤后角膜新生血管的抑制作用

目的:研究血管内皮生长因子小干扰RNA(VEGF small in-terfering RNA,VEGF siRNA)对兔眼碱烧伤后角膜新生血管的抑制作用。方法:普通家兔25只以碱烧伤法(1mol/LNaOH溶液)诱导角膜新生血管(CNV)生成。碱烧伤后立即以脂质体(LF2000)为载体,右眼球结膜下注射VEGF siRNA重组质粒,左眼球结膜下注射pSilencer 2.1-U6 hygro空白质粒作为阴性对照。碱烧伤后1,3,5,7,14d,形态学分析评价角膜新生血管的生长情况。结果:与对照眼相比,碱烧伤后球结膜下注射VEGF siRNA重组质粒,实验眼在各时间段(3,5,7,14d),CNV长度明显变短,面积明显变小,差异有统计学意义(P<0.05)。结论:VEGF siRNA能有效地抑制碱烧伤后CNV的形成。     

兔角膜碱烧伤的病理和纤维连接蛋白变化

目的 角膜碱烧伤同理组织和纤维结合蛋白的变化,以探讨其在临床上的作用。方法 在家兔角膜碱烧伤模型中,用病理和免疫组化方法观察其变化。结果 １０只眼在５～１０ｄ发生角膜溃疡,５只眼在７～１２ｄ发生角膜穿孔。烧伤后角膜纤维连接蛋白（ｈｂｒｏｎｅｃｔｉｎ,ＦＮ）增多,与炎症反应相一致。结论 ＦＮ参与角膜碱烧伤全过程,并担钡修复和治疗作用。     

Deposition of extracellular matrix on silicone intraocular lens implants in rabbits

Purpose: To examine the deposition of extracellullar matrix on silicone intraocular lenses (IOLs) implanted experimentally into rabbit eyes by electron microscopy and to determine the immunolocalization of extracellular matrix components, including collagen types and cellular fibronectin, on these IOLs. Methods: We performed phacoemulsification and aspiration of the crystalline lens and implanted a foldable silicone IOL in the capsular bag of one eye of each of 26 adult albino rabbits under general anesthesia. After 8 weeks the animals were killed and the eyes were enucleated. The silicone IOLs were processed for electron microscopy and for immunohistochemical detection of collagen types I, III, and IV and cellular fibronectin. Results: Electron microscopy revealed deposition of a presumed cell matrix complex on the optic portion of all silicone IOLs, as well as the adhesion of presumed macrophages and foreign-body giant cells. Cellular deposits showed immunoreactivity for cellular fibronectin. Fibrous or membranous deposits exhibited immunoreactivity for cellular fibronectin and collagen types I and III. A few type IV collagen-immunoreactive deposits were also seen. Conclusion: Deposits of extracellular matrix components were observed on silicone IOLs. These deposits may form the scaffolding for the adhesion and proliferation of cells. These matrix components appeared to be the products of cells adhering to the surfaces of IOLs, including lens epithelial cells, macrophages and foreign-body giant cells, indicating that the process of granulation was incomplete.     

Advances in corneal stem-cell transplantation in rabbits with severe ocular alkali burns

PURPOSE: To evaluate the efficacy of autologous corneal epithelial sheet implantation in restoring transparency of rabbit corneas severely injured by alkaline and the effect of photocoagulation in arresting corneal neovessel ingrowth. SETTING: Ophthalmology Department, School of Biomedical Sciences, Universidad Austral, Buenos Aires, Argentina. METHODS: Limbal stem-cell deficiency (LSCD) was induced in 14 rabbits by alkali burns. A limbal cell biopsy was done in the contralateral eye, and the cells were cultured on a fibroblast feeder layer grown on autologous clotted platelet-poor plasma or commercial fibrin for 21 days. Anterior keratectomy was followed by suturing corneal cell sheets over the stroma. If regrowth of vessels occurred, argon laser photocoagulation was applied to them. Rabbits were killed at 30, 60, 90, 180, and 360 days and the corneas processed for histopathology and inmunohistochemistry. RESULTS: A small (2.5 mm(2)) limbal biopsy achieved stem-cell replication in vitro. Corneal clarity and epithelial defects evolved with a trend toward improvement. There was a significant reduction in corneal neovascularization. Histology showed a multilayered stratified epithelium including several epithelial-like cells with clear cytoplasm in the deepest part. There were no signs of intraepithelial mucin cells on the implanted corneas. Immunohistochemical results showed expression of cytokeratins 3 and 12 in the central corneal epithelium and an absence of cytokeratin 19. CONCLUSIONS: Autologous limbal epithelial cell transplantation improved the corneal surface in eyes with LSCD. Photocoagulation of neovessel ingrowth was effective over the 1-year follow-up. Results may facilitate the application of this technique in patients.     

阿柏西普对碱烧伤大鼠角膜新生血管的抑制作用

目的探讨阿柏西普对大鼠角膜碱烧伤后新生血管的抑制作用。方法制备SD大鼠角膜碱烧伤动物模型,随机分成4组,每组各9只,A组、B组与C组分别给予20 g·L^-1、25 g·L^-1、30 g·L^-1的阿柏西普滴眼液,D组为生理盐水组(给予生理盐水),每组每天2次滴眼治疗,共14 d。分别于碱烧伤后第3天、7天、14天,观察角膜新生血管(corneal neovascularization,CNV)情况并计算CNV面积。碱烧伤后第14天,处死全部小鼠,进行组织学和免疫组织化学检测各组CD34、VEGF蛋白表达情况。结果碱烧伤后第3天、7天和14天,A组、B组与C组的CNV面积均小于D组,差异均有统计学意义(均为P<0.05);B组与C组在各时间点上CNV面积差异均无统计学意义(均为P>0.05);但B组和C组与A组相比,差异均有统计学意义(均为P<0.05)。HE染色及免疫组织化学染色结果显示:角膜碱烧伤后第14天,各组角膜CD34和VEGF蛋白表达增强,与A组、B组、C组比较,D组CNV旺盛致密,CD34和V...     

Analysis of corneal surface evolution after moderate alkaline burns by using impression cytology

PURPOSE: To compare corneal surface evolution after moderate alkaline burns by impression cytology in patients treated with medical therapy or with amniotic membrane transplantation (AMT). METHODS: A prospective study of 24 eyes from 18 patients (13 men and 5 women) with moderate alkaline burns was performed. All patients were divided according to the clinical ocular severity and the therapy used. Twelve eyes were treated surgically with AMT and the other 12 eyes received only medical therapy. Corneal cytology was obtained immediately after the burns, and 1, 2, 5, and 9 months later. We differentiated between samples obtained from affected areas and areas not affected by the burns. Cellular size, nuclear size, and nuclear-cytoplasmic (N:C) ratio were examined in corneal epithelial cells, as was the presence of goblet cells in corneal epithelium. RESULTS: Nuclear size, cellular size, and N:C ratio in non-burn-affected corneal areas had no significant alterations in comparison with normal eyes. In contrast, in burn-affected corneal areas, these parameters were significantly worse, and the presence of goblet cells in corneal epithelium was frequent 1 month after severe burns. Cellular size, nuclear size, N:C ratio, and corneal conjunctivalization improved during the study in all patients, but corneal reepithelialization occurred earlier in patients treated with AMT than in patients with only medical therapy. CONCLUSION: Morphologic and morphometric analysis of corneal cells by impression cytology after ocular burns permits the establishment of cellular reepithelialization patterns in relation with limbal deficiency level and with clinical ocular severity. AMT improves corneal reepithelialization earlier than medical therapy in moderate alkaline burns.     

TGF-β1短期眼部应用对兔眼角膜碱烧伤后整合素β1表达的作用

目的：研究TGF—β1短期眼部应用对兔角膜碱烧伤后整合素β1表达和角膜上皮愈合的影响，探求其对角膜碱烧伤的治疗作用。方法：制备大耳自家兔角膜碱烧伤模型，一组给予TGF—β1（浓度为200ng／m1）局部滴眼，每日3次，连续7日；另一组给予PBS溶液代替，处理相同。于角膜碱烧伤后每13观察角膜上皮愈合面积，并于烧伤后6h、1d、3d、7d和14d5个时间点应用免疫组化方法检测TGF—β1实验组与PBS组角膜整合素β1表达情况。结果：烧伤后4d、10d、11d、12d和14d实验组和对照组上皮愈合率比较，差异有统计学意义（P〈0．05），两组随着上皮修复过程的进行，整合素B1的表达均逐渐增加，烧伤后7d、14d两个时间点实验组和对照组整合素B1平均灰度值比较，差异有显著性（P〈0．05）。结论：TGF—β1在活体实验中能促进整合素β1的表达，而后者的增加可以促进角膜上皮细胞向损伤区域的移行和粘附，从而减少碱烧伤愈合过程中上皮再次脱落现象，有利于创伤愈合。     

Changes in extracellular matrix proteins and actin during corneal endothelial growth

Basement membranes influence growth, shape and differentiation of cells and tissues. However, the role and influence of Descemet's membrane during corneal development is not understood. To address this question, the relationships between cell growth and fibronectin, laminin and actin distribution in the developing rat corneal endothelium in vivo has been examined. During fetal development, rat corneal endothelial cells undergo DNA synthesis and mitosis. However, at day 14 of gestation both processes begin to decline and neither can be detected in endothelium of 1-month-old animals. By this time cell number has increased to approximately 100,000 and tissue area has increased 25-fold. However, as the tissue area increased, cell density decreased, indicating that cell spreading occurred in order to maintain tissue integrity. Changes in endothelial growth were accompanied by changes in the distribution of laminin, fibronectin and actin. Laminin and fibronectin were diffusely localized within endothelial cells in newborn animals. By 4 weeks of age, no proliferation was demonstrated and both extracellular matrix proteins were localized in pericellular patterns. Actin, on the other hand, which appeared diffuse at 16 days in utero, was distributed at or near the cell membrane by 19 days in utero. Thus, the reorganization of extracellular matrix glycoproteins and actin may indicate important roles for these components in regulating the growth and formation of the corneal endothelium in vivo.     

Changes of matrix metalloproteinases in the stroma after corneal cross-linking in rabbits

AIM:To observe changes in the content of matrix metalloproteinases(MMPs)in the corneal stroma after corneal cross-linking(CXL)in rabbits,and further explore the corneal pathophysiological process after CXL.METHODS:Forty-two rabbits(42 eyes)were randomly divided into seven groups.One group served as the control group,while the other six groups were treated with CXL.The concentrations of MMPs in corneal stroma were evaluated through parallel reaction monitoring at baseline and 3,7,15,30,90,and 180 d after treatment.RESULTS:The levels of MMP-2 in the corneal stroma of rabbits were 0.76±0.07,2.78±1.39,4.12±0.69,2.00±0.29,2.00±0.30,1.22±0.18,and 1.35±0.18(10^-9mol/g)at baseline and 3,7,15,30,90,and 180 d after treatment,respectively.The contents of tissue inhibitor of metalloproteinase-1(TIMP-1)were 1.83±0.26,7.94±0.58,6.95±2.64,3.81±0.48,3.07±0.92,1.72±0.19,and 1.69±0.74(10^-9mol/g),respectively.The ratios of MMP-2/TIMP-1 were 0.42±0.33,0.36±0.20,0.62±0.10,0.54±0.15,0.68±0.13,0.71±0.10,and 0.68±0.09,respectively.After CXL,the expression of MMP-2 and TIMP-1 in the rabbit corneal stroma was initially increased and subsequently decreased.The levels of MMP-2 remained higher than those recorded at baseline 180 d after treatment,but it was not statistically significant.The levels of TIMP-1 returned to baseline levels at 90 d after treatment.The ratio of MMP-2/TIMP-1 started to rise from 7 d after CXL.It was significantly higher than that calculated at baseline 30-180 d after CXL.The results for MMP-1,-3,-7,-9,-13,and TIMP-2 were negative.CONCLUSION:CXL can lead to changes in the content of MMP-2 and TIMP-1 in the rabbit corneal stroma.The ratio of MMP-2/TIMP-1 remains higher versus baseline,indicating that MMP-2 is involved in the corneal pathophysiological process after CXL.     

The effect of topical parasympathomimetics on corneal epithelial healing in rabbits

Corneal wound healing is an important process that involves interaction between the different corneal cell layers, growth factors, and environmental conditions. More powerful therapies for the treatment of delayed epithelial wound healing are still being proposed. The objective of this study is to investigate the effects of the direct-acting parasympathomimetic agents on the healing process of corneal epithelium in rabbits. The corneal epithelial defects, 10 mm in diameter, were created in 32 eyes of 16 island rabbits by combination of chemical debridement using n-heptanol and mechanical scraping. Animals were randomly divided into four groups. Groups 1, 2 and 3 were treatment groups; each group consisted of four rabbits (8 eyes). The animals in these groups were treated with topical 1% acetylcholine (ACh), 2% pilocarpine, and 0.75% carbachol drops respectively. In group 4, four rabbits (8 eyes) were used as control group and left for spontaneous healing. The length and area of the defect were measured at days 3,6,9,12,15,18 and 22 after wounding. Areas of the photographically documented fluorescein-stained defects were measured by planimetry. All eyes in the treatment groups reepithelialized completely. The duration for reepithelialization in Groups 1 and 2 was 12 days, and 18 days for Group 3. In the control group reepithelialization occurred within 22 days. The healing rates of corneal epithelium were statistically significantly faster in all treatment groups as compared with the control group at all times (p=0.0001 to 0.0279). Although the rates of wound healing varied, all of the parasympathomimetics used in the present study were found to facilitate wound healing. Our results indicate that direct-acting cholinergic agents, especially ACh and pilocarpine, may have an important therapeutic role in the treatment of severe corneal epithelial injury.     

The effect of liquid silicone on the corneal endothelium in rabbits

Specular microscopic and histopathologic findings in the corneal endothelium were compared after the injection of liquid silicone into the anterior chamber of both eyes in 15 rabbits. The findings demonstrated serious damage to the endothelium in the area of contact of liquid silicone with the posterior surface of the cornea and disclosed some problems in the interpretation of specular microscopic findings. An abnormal endothelial mosaic can be probably found even in a zone of preserved intercellular borders or in a zone with a relief of the bases of extinct endothelial cells.     

Differential tyrosine phosphorylation of paxillin in human corneal epithelial cells on extracellular matrix proteins

Purpose: To understand the interaction of corneal epithelial cells with laminin, fibronectin, and collagen type IV, major components of basement membrane, we investigated whether tyrosine phosphorylation of paxillin was increased during attachment of these cells to matrix proteins. Paxillin is one of the focal adhesion proteins and it is tyrosine phosphorylated during cell adhesion.Methods: SV 40-transformed human corneal epithelial (HCE) cells were plated on these extracellular matrix proteins and incubated. The cellular lysates were submitted to immunoprecipitation and Western blotting to determine tyrosine phosphorylation of paxillin.Results: When the cells were plated on laminin matrix, the stained band indicating tyrosine phosphorylated paxillin increased in proportion to cultivation periods. The increase was significant at 6 to 24 hours of cultivation. When HCE cells were cultured on various concentrations of laminin, paxillin was up-regulated in phosphorylation in a dose-dependent fashion. On a fibronectin matrix, tyrosine phosphorylation of paxillin increased in a time-dependent fashion and peak time point was 6 hours of cultivation. Paxillin was up-regulated in tyrosine phosphorylation in direct relation with the fibronectin concentration. On a type IV collagen matrix, tyrosine phosphorylation of paxillin increased in relation to time, but not so rapidly as cultures on a laminin or fibronectin matrix. Conclusion: Differential tyrosine phosphorylation of paxillin may have been caused by different extracellular matrix proteins.     

羊膜移植抑制兔角膜碱烧伤后新生血管增殖的研究

目的　比较采用保存羊膜和新鲜羊膜移植抑制角膜碱烧伤后新生血管增殖的效果 ;探讨应用保存人羊膜移植防治角膜碱烧伤后新生血管的手术时机。方法　制备角膜碱烧伤后新生血管增殖的动物模型 ;2 2只家兔 ( 2 2眼 )随机分为4组 :A组 ( 4眼 )作为对照组 ;B组 ( 6眼 )在碱烧伤的急性期行新鲜羊膜移植 ;C组 ( 6眼 )在急性期行保存人羊膜移植 ;D组 ( 6眼 )在瘢痕期行保存人羊膜移植。应用计算机彩色图像处理系统测定角膜新生血管面积。结果　 3个移植组和对照组比较 ,角膜新生血管面积的差异均有显著性意义 (P <0 0 1) ;B组与C组比较无显著性差异 (P >0 0 5 ) ;C组的新生血管面积明显少于D组 (P <0 0 1)。结论　保存羊膜和新鲜羊膜移植均能有效地抑制角膜碱烧伤后新生血管的增殖 ,治疗效果无显著性差异 ;在角膜碱烧伤的急性期施行羊膜移植防治新生血管增殖的效果要优于在瘢痕期手术。     

Differential tyrosine phosphorylation of paxillin in human corneal epithelial cells on extracellular matrix proteins]

PURPOSE: To understand the interaction of corneal epithelial cells with laminin, fibronectin, and collagen type IV, major components of basement membrane, we investigated whether tyrosine phosphorylation of paxillin was increased during attachment of these cells to matrix proteins. Paxillin is one of the focal adhesion proteins and it is tyrosine phosphorylated during cell adhesion. METHODS: SV 40-transformed human corneal epithelial (HCE) cells were plated on these extracellular matrix proteins and incubated. The cellular lysates were submitted to immunoprecipitation and western blotting to determine tyrosine phosphorylation of paxillin. RESULTS: When the cells were plated on laminin matrix, the stained band indicating tyrosine phosphorylated paxillin increased in proportion to cultivation periods. The increase was significant at 6 to 24 hours of cultivation. When HCE cells were cultured on various concentrations of laminin, paxillin was up-regulated in phosphorylation in a dose-dependent fashion. On a fibronectin matrix, tyrosine phosphorylation of paxillin increased in a time-dependent fashion and peak time point was 6 hours of cultivation. Paxillin was up-regulated in tyrosine phosphorylation in direct relation with the fibronectin concentration. On a type IV collagen matrix, tyrosine phosphorylation of paxillin increased in relation to time, but not so rapidly as cultures on a laminin or fibronectin matrix. CONCLUSION: Differential tyrosine phosphorylation of paxillin may have been caused by different extracellular matrix proteins.     

基质金属蛋白酶抑制剂GM-6001对大鼠角膜碱烧伤后新生血管的抑制作用

目的:研究基质金属蛋白酶抑制剂GM-6001对大鼠角膜碱性烧伤后角膜新生血管的抑制作用。方法:取健康成年Wistar大鼠20只,随机分成实验组和对照组,每组10只。两组均建立大鼠角膜碱性烧伤模型。实验组滴用GM-6001滴眼液、氧氟沙星滴眼液和硫酸软骨素滴眼液;对照组滴用氧氟沙星滴眼液和硫酸软骨素滴眼液;各种滴眼液每日点眼4次,共7d。裂隙灯下观察角膜溃疡和角膜新生血管生长情况。结果:碱烧伤后实验组和对照组出现新生血管的时间无显著差异(P>0.05)。新生血管的生长长度与生长面积,在伤后1d和3d,实验组和对照组无显著差异(P>0.05);在伤后5d和7d,实验组和对照组有显著差异(P<0.05)。结论:基质金属蛋白酶抑制剂GM-6001点眼对角膜新生血管的生长有抑制作用。     

Cornea with Peters' anomaly: perturbed differentiation of corneal cells and abnormal extracellular matrix in the corneal stroma

PURPOSE: We examined histopathologically the anterior ocular segment including the cornea and lens of an eye which had been enucleated in a patient with Peters' anomaly because of untreatable corneal perforation. Special effort was made to differentiate the corneal stromal and endothelial cells, and the stromal extracellular matrix.METHODS: Light microscopy, with hematoxylin and eosin staining, and transmission electron microscopy were employed.RESULTS: Corneal endothelial cells and Descemet's membrane were not detected in the central cornea, where there were immature cells with a fibroblastic configuration. The inner surface of the peripheral cornea was covered with cells containing pigment granules in the cytoplasm. Cell density in the central corneal stroma was relatively high. The diameter of the stromal collagen fibrils was not uniform. A mature collagen fibril-free area was also seen in the central corneal stroma.CONCLUSIONS: Differentiation of neural crest-derived cells in corneal stroma and endothelium might have been perturbed in the cornea of this patient with Peters' anomaly, inducing the defect in the corneal endothelium and the qualitative and quantitative abnormalities of the extracellular matrix.