Spatial and temporal alterations in the collagen fibrillar array during the onset of transparency in the avian cornea

In the latter stages of development, the embryonic avian cornea undergoes significant changes in structure, composition and transparency. The rearrangement of stromal collagen fibrils at this time is important because it is believed to play a key role in the acquisition of corneal transparency. Here, we investigate spatial alterations in the internal fine structure of the avian cornea during development. Chicken corneas at developmental days 14, 16 and 18 were examined by transmission electron microscopy and quantitative image analysis. For anterior and posterior regions we determined fibril number densities, two-dimensional distribution functions, and, where appropriate, radial distribution functions. Stromal collagen fibrils became more closely spaced over the developmental range studied here. Changes in fibril number density indicated that fibrils became compacted first in the anterior stroma, and later (i.e. after day 16) in the posterior stroma. By day 18 collagen fibril number densities were essentially the same in superficial and deep tissue regions. At day 14, two-dimensional distribution functions of collagen fibrils in the posterior stroma pointed to a fibrillar array that was unlike that in the anterior stroma because there was no clear radial symmetry. Rather, in the deep stroma at day 14 there was evidence of different nearest neighbour spacings in two orthogonal directions. By day 18, fibril distributions in the anterior and posterior stroma were spatially homogeneous and radially symmetric, with radial distribution functions typical of those ordinarily found in mature cornea. Corneal transparency requires the stromal matrix to have some degree of regularity in the arrangement of its uniformly thin collagen fibrils. The chicken cornea becomes progressively transparent between days 14 and 18 of development as the stroma dehydrates and thins. We show that over this time period collagen fibrils in the anterior stroma become configured in advance of fibrils in deeper stromal regions, leading to questions over the potential roles of sulphated proteoglycans in different regions of the corneal stroma during morphogenesis.     

The organization of collagen in the corneal stroma

The cornea has evolved to fulfil the dual functions of enclosing and protecting the inner contents of the eye, and focussing light onto the retina with minimum scatter and optical degradation. It does this by means of the arrangement of the constituent collagen fibrils, an arrangement that is unique in connective tissues. This article reviews our current knowledge about the detailed organization of collagen in the corneal stroma, and presents new data suggesting that a significant proportion of collagen fibrils running across the cornea, change direction near the limbus and fuse with the circumferential limbal collagen.     

Characterization of corneal keratocyte morphology and mechanical activity within 3-D collagen matrices

The purpose of this study was to assess quantitatively the differences in morphology, cytoskeletal organization and mechanical behavior between quiescent corneal keratocytes and activated fibroblasts in a 3-D culture model. Primary cultures of rabbit corneal keratocytes and fibroblasts were plated inside type I collagen matrices in serum-free media or 10% FBS, and allowed to spread for 1-5 days. Following F-actin labeling using phalloidin, and immunolabeling of tubulin, α-smooth muscle actin or connexin 43, fluorescent and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. In other experiments, dynamic imaging was performed using differential interference contrast microscopy, and finite element modeling was used to map ECM deformations. Corneal keratocytes developed a stellate morphology with numerous cell processes that ran a tortuous path between and along collagen fibrils without any apparent impact on their alignment. Fibroblasts on the other hand, had a more bipolar morphology with pseudopodial processes (P ≤ 0.001). Time-lapse imaging of keratocytes revealed occasional extension and retraction of dendritic processes with only transient displacements of collagen fibrils, whereas fibroblasts exerted stronger myosin II-dependent contractile forces (P < 0.01), causing increased compaction and alignment of collagen at the ends of the pseudopodia (P < 0.001). At high cell density, both keratocytes and fibroblasts appeared to form a 3-D network connected via gap junctions. Overall, this experimental model provides a unique platform for quantitative investigation of the morphological, cytoskeletal and contractile behavior of corneal keratocytes (i.e. their mechanical phenotype) in a 3-D microenvironment.     

血小板源性生长因子与角膜损伤修复

血小板源性生长因子(PDGF)在机体伤口愈合过程中具有重要的调节作用。由于角膜的特殊结构和功能,其伤口愈合的调节显得至关重要。本文简述PDGF的结构、受体、生物学活性及其在角膜伤口愈合中的作用。     

ECM,MMP与角膜结构和角膜损伤修复

细胞外基质是角膜的主要结构成分,赋予角膜特殊的生理功能。基质金属蛋白酶是调节细胞外基质降解代谢的主要酶类。本文概述了成熟角膜细胞外基质的组成及基质金属蛋白酶在角膜损伤修复过程中的作用。     

rhEGF基因治疗角膜碱烧伤的实验研究

目的探讨重组人表皮生长因子（rhEGF）基因缓释型滴眼液对免角膜碱烧伤的基因治疗。方法新西兰白兔75只，分为A（rhEGF-pcDNA3．1-脂质体基因滴眼液）、B（rhEGF衍生物滴眼液）、c（pcDNA3.1空质粒-脂质体对照组）三组分别点眼治疗免角膜碱烧伤。每天观察模型眼眼表、荧光素染色并照相。分别测定hEGFmRNA、hEGF和EGFR在角膜组织中的表达。结果A组眼表结膜充血、分泌物、角膜水肿和角膜混浊程度均比B组和C组轻。角膜荧光素染色显示A组角膜上皮的溃疡面积较B组和C组范围小，21d转为阴性。A组基因在行滴眼24h后在全层角膜细胞内可见hEGFmRNA和蛋白质表达，第3d达高峰，细胞阳性率高达95％，随后逐渐降低，21d仍有弱表达。结论rhEGF-PcDNA3．1-脂质体基因滴眼液能够降低角膜的炎症反应，促进角膜上皮细胞的增生，加速角膜碱烧伤的损伤修复过程。     

表皮生长因子对家兔角膜内皮损伤修复作用的实验研究

目的 研究表皮生长因子对家兔角膜内皮损伤的修复作用。方法 将溶于透明持酸钠的ＥＧＦ注入角膜内皮已损伤的兔眼前房,对照眼只注入ＮａＨＡ。结果 １０μｇ和５μｇ的ＥＧＦ均可使角膜内皮缺损面积显著缩小,并可使再生的角膜中央内皮细胞密度,六边形细胞出现率显著上升及细胞面积变异系数明显减小。     

飞秒激光制瓣LASIK手术后的角膜伤口愈合反应

任何屈光手术都会对角膜组织造成损伤,发生伤口愈合过程。飞秒激光作为新兴技术逐渐成为角膜屈光手术的主流方法,其伤口愈合过程直接影响术后早期炎症刺激及远期临床疗效。此前国内外对准分子激光引起的愈合反应已有详细的研究,但飞秒激光造成的角膜损伤及愈合过程报道较少。飞秒激光制瓣LASIK手术后早期炎症反应较机械刀制瓣LASIK术后重,并有其特点。（国际跟科纵览,2012,36：323—327）     

A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma

The purpose of the present study was to re-evaluate the three-dimensional organization of collagen fibrils and proteoglycans (PGs) in the human corneal stroma using an improved ultrastructural approach. After a short aldehyde prefixation, one half of seven fresh corneal buttons was stained for PGs with Quinolinic Phtalocyanin (QP) or Cupromeronic Blue (CB). Strips of 1 mm width were cut, subsequently treated with aqueous phosphotungstic acid (PTA) and further processed for light and electron microscopy. The other half of the corneas served as control and was routinely processed with OsO4. Embedding was as such that ultrathin sections could be cut precisely parallel (frontal sections) or perpendicular (cross sections) to the corneal surface. The mutual connections between collagen fibrils and PGs were studied and the length of PGs and their mutual distance were measured manually at a calibrated final magnification of 70,000 x. Prefixed fresh corneal tissue treated with QP and CB shows no signs of swelling and exhibits well contrasted PGs. In cross sections PGs form a repeating network of ring-like structures (approximately 45 nm) around the collagen fibrils. In frontal sections PGs are aligned orthogonal to the collagen fibrils, are equidistant (approximately 42 nm) attached to the collagen fibrils along their full length and have a thickness of approximately 11 nm and a length of approximately 54 nm. The observed maximal length of the PGs and the occurrence of ring-like structures enwrapping the collagen fibrils urged us to revisit the prevailing model of maurice (1962) on the organization of the corneal stroma. In the new model hexagonal arranged collagen fibrils are interconnected at regular distances with their next-nearest neighbours by groups of six PGs, attached orthogonal to the circumference of the fibrils. In this way a regular meshwork of ring-like structures enwrapping the collagen fibrils is formed. It is discussed that this new model more convincingly explains corneal resistance to compression and stretching and further rationalizes corneal transparency because of the low refractive index difference between the regularly arranged collagen fibrils and their inter-space filled with PGs.     

Formation of stromal collagen fibrils and proteoglycans in the developing zebrafish cornea

Purpose: Collagen fibrils and proteoglycans are the main components of the corneal extracellular matrix and corneal transparency depends crucially on their proper arrangement. In the present study, we investigated the formation of collagen fibrils and proteoglycans in the developing cornea of the zebrafish, a model organism used to study vertebrate embryonic development and genetic disease. Methods: We employed thin‐section electron microscopy to investigate the ultrastructure of the zebrafish cornea at different developmental stages. Results: The layering of the zebrafish cornea into an epithelium, a Bowman’s layer, stroma and endothelium was observed starting at 72 hr post‐fertilization. At this stage, the stroma contained orthogonally arranged collagen fibrils and small proteoglycans. The density of proteoglycans increased gradually throughout subsequent development of the cornea. In the stroma of 2‐week‐old larvae, the collagen fibrils were organized into thin lamellae and were separated by very large, randomly distributed proteoglycans. At 4 weeks, a regular arrangement of proteoglycans in relation to the collagen fibrils was observed for the first time and the lamellae were also thickened. Conclusion: The present study, for the first time, provides ultrastructural details of collagen fibril and proteoglycan development in the zebrafish cornea. Furthermore, it directly correlates the collagen fibril and proteoglycan composition of the zebrafish cornea with that of the human cornea. The similarities between the two species suggest that the zebrafish could serve as a model for investigating the genetics of human corneal development and diseases.     

Evidence for mitosis in the adult corneal endothelium

Intracellular structures probably representative of mitotic figures were seen by specular microscopy in the endothelium of a corneal graft following a rejection reaction. Serial measurements over an eight month period initially showed grossly enlarged cells and apparent mitotic figures. Subsequently, clusters of cells smaller than any cells previously seen were observed. Measurements of endothelial cell area over this period demonstrated a highly significant (P less than 0.0001) decrease in cell area, or increase in cell density, with time. These observations indicate that at least under some circumstances mitosis occurs in the endothelium of the adult human cornea.     

Aquaporin-1-facilitated keratocyte migration in cell culture and in vivo corneal wound healing models

Aquaporin-1 (AQP1) water channels are expressed in corneal keratocytes, which become activated and migrate following corneal wounding. The purpose of this study was to investigate the role of AQP1 in keratocyte migration. Keratocyte primary cell cultures from wildtype and AQP1-null mice were compared, as well as keratocyte cultures from pig cornea in which AQP1 expression was modulated by RNAi knockdown and adenovirus-mediated overexpression. AQP1 expression was found in a plasma membrane pattern in corneal stromal and cultured keratocytes. Osmotic water permeability, as measured by calcein fluorescence quenching, was AQP1-dependent in cultured keratocytes, as was keratocyte migration following a scratch wound. Keratocyte migration in vivo was compared in wildtype and AQP1 knockout mice by histology and immunofluorescence of corneal sections at different times after partial-thickness corneal stromal debridement. AQP1 expression in keratocytes was increased by 24 h after corneal debridement. Wound healing and keratocyte appearance near the wound margin were significantly reduced in AQP1 knockout mice, and the number of neutrophils was increased. These results implicate AQP1 water permeability as a new determinant of keratocyte migration in cornea.     

Role of corneal collagen fibrils in corneal disorders and related pathological conditions

The cornea is a soft tissue located at the front of the eye with the principal function of transmitting and refracting light rays to precisely sense visual information. Corneal shape, refraction, and stromal stiffness are to a large part determined by corneal fibrils, the arrangements of which define the corneal cells and their functional behaviour. However, the modality and alignment of native corneal collagen lamellae are altered in various corneal pathological states such as infection, injury, keratoconus, corneal scar formation, and keratoprosthesis. Furthermore, corneal recuperation after corneal pathological change is dependent on the balance of corneal collagen degradation and contraction. A thorough understanding of the characteristics of corneal collagen is thus necessary to develop viable therapies using the outcome of strategies using engineered corneas. In this review, we discuss the composition and distribution of corneal collagens as well as their degradation and contraction, and address the current status of corneal tissue engineering and the progress of corneal cross-linking.     

Removal of the basement membrane enhances corneal wound healing

Recurrent corneal erosions are painful and put patients’ vision at risk. Treatment typically begins with debridement of the area around the erosion site followed by more aggressive treatments. An in vivo mouse model has been developed that reproducibly induces recurrent epithelial erosions in wild-type mice spontaneously within two weeks after a single 1.5 mm corneal debridement wound created using a dulled-blade. This study was conducted to determine whether 1) inhibiting MMP9 function during healing after dulled-blade wounding impacts erosion development and 2) wounds made with a rotating-burr heal without erosions. Oral or topical inhibition of MMPs after dulled-blade wounding does not improve healing. Wounds made by rotating-burr heal with significantly fewer erosions than dulled-blade wounds. The localization of MMP9, β4 integrin and basement membrane proteins (LN332 and type VII collagen), immune cell influx, and reinnervation of the corneal nerves were compared after both wound types. Rotating-burr wounds remove the anterior basement membrane centrally but not at the periphery near the wound margin, induce more apoptosis of corneal stromal cells, and damage more stromal nerve fibers. Despite the fact that rotating-burr wounds do more damage to the cornea, fewer immune cells are recruited and significantly more wounds resolve completely.     

骨形成蛋白-7在大鼠角膜损伤愈合的表达及作用

目的:通过观察大鼠角膜损伤愈合过程中骨形成蛋白-7(bone morphogenetic protein 7,BMP-7)的表达规律来探讨其在大鼠角膜损伤愈合过程中可能发挥的作用。方法:雄性Wistar大鼠42只,随机分为7组,每组6只,其中一组为正常对照组,动物模型组均取右眼制造角膜针刺损伤模型。于损伤后各时间点(6h;1,3,5,7,14d)取角膜标本,HE染色行组织病理学检查,免疫组织化学染色观察BMP-7的表达与分布,计算机图像分析系统对结果进行分析。结果:鼠角膜上皮BMP-7的表达于损伤后1,3d逐渐升高(P<0.05);损伤后5d时表达逐渐降低,至损伤后14d时表达恢复正常。角膜基质层及角膜内皮层中BMP-7的表达未见明显变化。结论:BMP-7在大鼠角膜上皮层均有表达,可能作为负性的生长因子参与角膜损伤愈合过程。     

Heterogeneity of the bovine corneal collagen

Type III collagen [α₁ (III)] was isolated from the pepsin-solubilized bovine corneal collagen either by differential salt precipitation, or by molecular sieve chromatography. It has been estimated that type III collagen represents about 20% of the total collagen, the predominant part of which is type I collagen. The identity of type III collagen was verified by molecular sieve chromatography and polyacrylamide gel electrophoresis of reduced and alkylated γ-chains, and by CNBr cleavage and amino acid analysis of isolated α₁ (III) chains.