圆锥角膜各期的共焦显微镜表现

目的评价共焦显微镜在圆锥角膜临床研究中的应用价值。方法应用共焦显微镜观察圆锥角膜患者32例（48眼）及正常对照组17例（28眼），分别比较早、中、晚期圆锥角膜与正常对照组的图像特点。结果早期圆锥角膜出现激活状态的角膜细胞、浅基质层的细小皱褶、深基质层的暗纹、部分内皮细胞异形性明显，中、晚期圆锥角膜出现角膜上皮细胞拉伸、细胞核皱缩；基质细胞排列紊乱、基质层暗纹；而对照组未发现上述表现。各期圆锥角膜的角膜基质层厚度、不同深度角膜基质细胞密度、内皮细胞密度与对照组之间差异有统计学意义（P〈0．05）。结论共焦显微镜对早期圆锥角膜的发现以及圆锥角膜病理发展的研究具有重要的临床价值。     

角膜移植术后角膜在共焦显微镜下的形态学改变

目的：研究角膜移植术后角膜组织在共焦显微镜(Confocal microscopy)下的形态学改变。方法：应用Conoscan2．0共焦显微镜对板层角膜移植术后3～7d患者12例(12只眼)，术后1a患者8例(8只眼)，穿透性角膜移植术后3～7d患者10例(10只眼)，术后1a患者11例(11只眼)进行扫描检查，记录各层角膜图像。结果：板层角膜移植术后3～7d，植片中基质细胞较小，可见裂隙状暗纹和细小神经，层间为大面积高反光区，有点状颗粒沉积，植床水肿，细胞成像不清。移植术后1a，植片中未见神经，层间反光明显减弱，但仍有点状高反光颗粒沉积，植床中出现粗大裂隙状暗纹，内皮细胞密度正常。穿透角膜移植术后3～7d，植片中基质细胞“激活”，可见神经和后基质层的粗大暗纹，内皮细胞密度正常，细胞间可镶嵌有高反光点。术后1a，植片中基质细胞仍较小，未见神经，后基质层仍有裂隙状暗纹，内皮细胞体积增大，密度减少，高反光点消失。结论：Confoscan 2．0共焦显微镜可活体检查角膜移植术后角膜组织结构和细胞的病理改变，这对评估手术效果和临床观察以及跟踪随访具有重大意义。     

共焦显微镜在角膜营养不良诊断中的应用

目的 探讨共焦显微镜在角膜营养不良诊断中的应用价值。设计 病例系列研究。研究对象 6例角膜营养不良，包括4例Reis-Bueckleas角膜营养不良、1例角膜斑点状营养不良、1例。Fuchs角膜内皮营养不良。方法 患者双眼行裂隙灯显微镜及共焦显微镜检查，选择病变在不同角膜层次的共焦显微镜图像，对角膜沉淀进行形态学评价，并与裂隙灯检查比较。主要指标角膜病变的裂隙灯显微镜及共焦显微镜图像。结果 共焦显微镜显示Reis-Bficklers角膜营养不良病变主要累及前部基质，包括角膜上皮、基底细胞及前弹力层；斑点状角膜营养不良病变仅累及基质层，而角膜上皮层及内皮层正常；在Fuchs角膜内皮营养不良中，可直接观察角膜小滴及角膜内皮情况。结论 共焦显微镜检查提供了一种评价角膜病变的方法，较裂隙灯显微镜的分辨率高。     

角膜内皮炎共焦显微镜形态学分析

目的:观察角膜内皮炎的共焦显微镜下形态学特征。方法:应用Confoscan 4.0共焦显微镜对24例24眼角膜内皮炎患者的角膜进行扫描检查,记录并分析各层角膜图像。结果:所有患者前部基质混浊,角膜深基质层可见基质细胞排列紊乱及条索状高反光结构,深基质层中还可见低反光带为后弹力层皱褶,角膜内皮细胞前可见斑片状大小不等的高反光结构,病变区角膜内皮细胞水肿、变性呈无结构暗区,内皮细胞呈多形性改变。4眼角膜上皮细胞边界不清,排列疏松,细胞较大,细胞核呈高反光结构,其中可见泡状暗区。结论:共焦显微镜可活体检查角膜内皮炎患者角膜组织各层结构,起到类似病理组织切片的作用;角膜内皮炎以深基质层及内皮细胞层损害为特征;共焦显微镜检查对角膜内皮炎具有一定的参考价值。     

颗粒状角膜营养不良活体共焦显微镜形态学研究

目的研究颗粒状角膜营养不良角膜各层组织的共焦显微镜形态改变。方法应用Confoscan2.0共焦显微镜对13例(26眼)颗粒状角膜营养不良患者的角膜进行扫描检查,记录与分析各层角膜图像。结果所有患眼前基质细胞及16/26眼后基质细胞结构不清,排列紊乱,并可见短棒状多形性强反光;6/26眼前弹力层不规则并增厚,神经纤维密度明显下降;6/26眼角膜上皮基底细胞层可见不定型的强反光;2/26眼角膜上皮细胞边界不清,排列呈疏松的蜂窝状,并出现不透明的强反光;所有患者角膜内皮细胞形态基本正常。视力0.3以下的患眼角膜上皮细胞层、上皮基底细胞层、前弹力层、后基质层发生形态异常的比例高于0.3以上的患眼(P<0.05)。结论1.共焦显微镜可活体检查颗粒状角膜营养不良角膜组织各层结构,起到类似病理组织切片的作用。2.前基质层形态异常可能是颗粒状角膜营养不良最基本的共焦显微镜形态特征,病情越重,前基质层以外的其它层次发生形态异常的可能性越大,但内皮细胞层一般不受累。3.共焦显微镜检查对颗粒状角膜营养不良手术方式的选择具有一定的参考价值。     

圆锥角膜对侧无明显改变眼的共焦显微镜所见

目的　了解圆锥角膜患者对侧无明显临床改变眼的共焦显微镜的影像特点 ,探索共焦显微镜在早期圆锥角膜的临床检查中的意义和诊断价值。方法　前瞻性的应用共焦显微镜研究评价37例圆锥角膜患者对侧眼无明显临床改变者、 6 2例中高度近视和 4 3例中高度近视角膜接触镜佩戴者作对照 ,比较各组之间的图像、角膜基质、内皮细胞的密度等。结果　圆锥角膜对侧眼无明显临床改变者组和中高度近视组、中高度近视接触镜组比较 ,其年龄、近视度数、散光度数均无统计学差异(均P >0 .0 5)。共焦显微镜检查中发现圆锥角膜患者对侧眼无明显临床改变者组中有 8例出现前或后基质暗纹 ,对照组角膜没有发现类似的改变。角膜内皮细胞密度和角膜前、中、后基质细胞密度在早期圆锥角膜与对照组之间无明显统计学差异 (均P >0 .0 5)。各组间上皮下神经的形态结构没有明显差别。结论　共焦显微镜可活体检查圆锥角膜组织结构和细胞的改变 ,是一种新的临床检测手段 ,它为早期圆锥角膜的诊断提供了一种新的思路 ,具有一定的诊断价值。     

板层角膜移植术后全层角膜组织改变的共焦显微镜观察

目的研究活体扳层角膜移植术后角膜各层组织的共焦显微镜(confocal microscopy)形态学改变，方法应用Confoscan 2.0共焦显微镜对板层角膜移植术后3月～3年，角膜植片稳定的患者共17例17眼，进行扫描检查，记录与分析各层角膜图像。结果共焦显微镜上能够清楚地观察到稳定角膜的上皮细胞、基底层细胞、前弹力层，它们同正常角膜的相应细胞在图像上没有显著差别。但植片与植床的基质细胞在共焦显微镜图像上有着明显的差异。板层角膜移植术后稳定角膜植片的基质细胞，细胞核较小呈中等反光，排列较植床的基质细胞紊乱。而角膜植床的基质细胞，细胞核较大也呈中等反光，排列较植片的基质细胞稍整齐，结论1、Confoscan 2.0共焦显微镜可活体检查，板层角膜移植术后角膜组织结构和细胞的病理改变。2、板层角膜移植术后可成功的重建眼表。3、活体板层角膜移植术后稳定角膜上有自体角膜基质细胞与异体的角膜基质细胞同时共存，角膜内发细胞的形态结构正常。4、角膜板层间的不平滑与植片基质细胞结构紊乱可能是导致板层角膜移植术后视力不佳的重要原因。     

白内障激光乳化术后角膜组织的共焦显微镜观察

目的研究人白内障激光乳化手术后角膜各层组织的共焦显微镜下形态学改变。方法应用con-foscan 3.0共焦显微镜对Er:YAG激光乳化手术后1周~1月、无严重手术并发症的患者共10例(10只眼),进行扫描检查,记录和分析各层角膜组织图像。结果confoscan 3.0能够清楚地显示角膜的上皮细胞、基底层细胞、前弹力层、基质层细胞以及内皮细胞。激光乳化术后稳定的角膜同正常角膜的相应细胞在图像上没有显著差异,但有水肿的角膜在共焦显微镜下各层细胞均有明显改变。结论(1)confoscan 3.0共焦显微镜可以活体检查白内障激光乳化术后角膜组织结构和细胞的病理改变;(2)激光乳化作为一种新的白内障手术方式对角膜组织的损伤轻微;(3)活体共焦显微镜可以作为评价白内障手术后角膜组织改变的最佳检查。     

Chandler综合征的共焦显微镜研究

目的 应用角膜激光共焦显微镜观察Chandler综合征患者活体角膜各层组织的形态特点.方法 对21例(21只眼)的Chandler综合征患者,采用角膜激光共焦显微镜观察其活体角膜的各层结构,总结其图像特点.结果 共焦显微镜下Chandler综合征患者角膜有如下特点:(1)角膜上皮细胞及角膜基质细胞在共焦显微镜下可有不同程度的水肿.(2)角膜营养不良是Chandler综合征患者的特征性改变之一,在共焦显微镜下角膜内皮细胞出现不同程度的疣锥状突起,病变的内皮细胞彻底丧失六边形结构,细胞变圆、变大,在中高反光的细胞体外有一圈由于细胞突起引起的暗反光圈结构,严重的患者可以形成山脊状隆起.(3)部分患者的虹膜在共焦显微镜下可出现粗细不同的支架状结构,这些虹膜支架结构在共焦显微镜下呈中-高度反光,部分呈树枝状排列,部分呈树干状排列,其周围可散在大小不同的色素颗粒附着.结论 共焦显微镜在临床诊断Chandler综合征中有重要作用,角膜营养不良是Chandler综合征患者的特征性改变之一,在共焦显微镜下角膜内皮细胞出现不同程度的疣锥状突起,这种内皮细胞是Chandler综合征与其他类型ICE综合征鉴别的特点之一.     

正常中国汉族儿童角膜的共焦显微镜研究

目的采用角膜激光共焦显微镜对正常中国汉族儿童活体角膜各层组织结构进行观察,分析正常中国汉族儿童角膜各层细胞的活体细胞形态学特征及密度。方法 49例6～13岁正常中国汉族儿童中央部角膜进行活体共焦显微镜检查,研究角膜各层结构的图象特点,并分析角膜各层细胞的密度,与成年人进行对比。结果中国汉族儿童正常角膜上皮表层细胞排列疏松,边界清楚,胞体较大,核反光较强,又称翼状细胞;基底层细胞排列紧密,呈规则的蜂巢状,胞质反光弱,正常情况下未见细胞核,细胞平均密度为5947.58±769.3个/mm2。前弹力膜为无细胞结构的膜状物,可见串珠状的神经纤维走行。基质层中可见在相对较暗的背景下明亮的角膜基质细胞核。角膜基质内有神经纤维分布。前基质层角膜细胞平均密度为1621.12±123.26个/mm2,后基质层角膜细胞平均密度为984.71±113.17个/mm2,前后基质层细胞密度有显著性差别（P〈0.05）。后弹力层中无细胞结构,为均一无结构组织。内皮细胞层表现为规则的六边形结构,细胞结构清晰,细胞平均密度为3682.38±251.87个/mm2。左右眼及男女之间角膜各层细胞密度的差异无统计学意义（P=0.341～0.611和P=0.194～0.855）。各层角膜细胞的面积和密度与性别和眼别无差异。中国汉族儿童角膜各层细胞的密度较正常成人高。结论角膜激光共焦显微镜能够在实时、活体和三维空间从细胞水平清晰观察中国汉族儿童活体角膜各层细胞的形态结构,可以对角膜各层结构进行定性和定量分析,在儿童角膜病的研究和临床诊断方面是十分有用的工具。     

The alterations in corneal structure at III/IV stage of keratoconus by means of confocal microscopy and ultrasound biomicroscopy before penetrating keratoplasty

PURPOSE: The aim of the study was in-real time observation and morphological evaluation of the human corneas at III/IV stage of keratoconus, using the scanning slit confocal microscope Confoscan P4 and ultrasound biomicroscopy--UBM. MATERIAL AND METHODS: The patients with keratoconus were examined according to the Amsler scale. The material consisted of 12 corneas of 11 patients (8 men, 3 women), where assessment of the corneal structure was performed with the confocal microscope ConfoScan P4 (Tomey) and ultrasound biomicroscopy--UBM Model 840 (Humphrey Instruments). The comparison of different corneal regions (central and peripheral) was evaluated. RESULTS: The confocal microscopy and UBM revealed thinning of the layers of the corneal structure and pathological changes in the central area, especially at IV stage of keratoconus. The desquamating superficial cells were elongated, arranged around the apex of the cornea. Below the Bowman's membrane a considerable disarrangement of collagen fibers reflected by bright background illumination was observed. In the posterior part of the stroma the folds were detected. The examination of the cornea showed thickening in the peripheral part, central detachment of the Descemet's membrane and the endothelium from the posterior surface of the cornea. The thickness of the cornea varied from 0.201 to 0.384 mm in the central part and 0.675 to 0.740 mm in the peripheral area. CONCLUSION: Confocal scanning microscopy combined with ultrasound biomicroscopy enables the cornea to be examined in vivo. It can be used to localize pathological changes in individual corneal layers and to assess their extent.     

Observations of banding patterns (Vogt striae) in keratoconus: a confocal microscopy study

PURPOSE: To investigate Vogt striae in keratoconus using confocal microscopy. METHODS: The central cornea of 51 eyes of 29 subjects with keratoconus was observed using a slit-lamp biomicroscope, slit-scanning confocal microscope (TOMEY Confoscan 1), and corneal topographer (EyeSys 2000). RESULTS: Alternating dark and light bands were seen in the stromal images of 23 eyes examined. The bands corresponded with the appearance of Vogt striae on slit-lamp biomicroscopy examination. Bands were found most commonly in the posterior stroma. Posterior bands varied in width, ran mainly in a nearly vertical direction, and appeared to run a straight course through individual image frames. Keratocyte nuclei were located in between the bands. Posterior keratocyte density was unaffected by the presence of bands. Nerve fibers appeared to run a straight course through the bands. When present, bands in the anterior stroma showed greater variability in width and direction within a single frame. Bands were only present in the anterior stroma in more severe levels of keratoconus. The difference in banding pattern noted between the anterior and posterior stroma parallels the known collagen fiber arrangement in the anterior and posterior stroma. CONCLUSIONS: The bands apparent on confocal microscopy of the stroma of the keratoconic cornea correspond with Vogt striae on slit-lamp microscopy. It appears that these bands (and hence Vogt striae) represent collagen lamellae under stress. The stress pattern appears to radiate from the center of the cone and is consistent with the direction of striae when viewed with the confocal microscope.     

Histologic findings of recipient corneas obtained via deep anterior lamellar keratoplasty

Purpose

We sought to investigate the histologic findings of recipient corneas obtained via deep anterior lamellar keratoplasty (DALK).

Methods

The histology of three recipient corneas obtained from patients during DALK was investigated. In all cases, the Descemet membrane was successfully exposed without any perforation during surgery. The isolated corneal tissue was stained with hematoxylin-eosin and periodic acid-Schiff. In two cases, the tissues were examined using a transmission electron microscope.

Results

In one cornea obtained via DALK, only the corneal stroma was observed, and the Descemet membrane was not confirmed. In another case, the recipient cornea was detached within the Descemet membrane. In the third cornea, the banded layer membrane was partially confirmed.

Conclusions

These findings suggest that the recipient corneas separated at different layers during the DALK procedure. With our surgical technique, the detachment of the Descemet membrane may occur at a mechanically weak segment. This separation site may not be between the Descemet membrane and the corneal stroma.     

The expression of tenascin and fibronectin in keratoconus, scarred and normal human cornea

Background: The etiology and pathogenesis of keratoconus remain unclear, and therefore we decided to study the distribution of different isoforms of tenascin (Tn) and fibronectin (Fn) in normal human corneas and in those obtained from penetrating keratoplasty for keratoconus and corneal scarring. Methods: Frozen sections of human cornea and conjunctiva were stained by immunohistochemical methods with a panel of monoclonal antibodies (MAbs) against different isoforms of Tn and Fn. Results: In the normal human eye, Tn was found in the limbal and conjunctival basement membrane region, in the conjunctival blood vessels and at the junction of the cornea and sclera, but no immunoreaction was seen in the normal cornea. In the corneas from the keratoconus patients, a clear immunoreaction for Tn was seen in the defects of Bowman's membrane as well as in the distorted stroma beneath the defects. In some of the keratoconus corneas, basement membrane adjacent to the defects also showed reactivity for Tn, and in clinically and histologically scarred keratoconus corneas the scars expressed Tn. In the scarred corneas, only blood vessels in the posterior portion of the cornea showed immunoreactivity for Tn, while no Tn was noted in the scar area or in Bowman's membrane. No major differencies were noticed in the reactivity of different MAbs against Tn isoforms. Fn, extradomain A Fn (EDA-Fn) and oncofetal Fn (onc-Fn) were found in the basement membrane of the central cornea of the normal eye. In keratoconus corneas, the defects and clinical and histological scars bound MAbs against Fn, EDA-Fn and onc-Fn, but in the scarred corneas no enhancement in the expression of Fns was noted. Extradomain B cellular Fn (EDB-Fn) was not expressed in any of the eyes studied. Conclusions: The results suggest that the anterior portion of the cornea is involved in the pathogenesis of keratoconus. Furthermore, it seems that the expression of Tn and Fns in the clinically scarred keratoconus corneas is due to a process in which both repairing and scar-forming mechanisms operate at the same time. However, the origin of the defects in Bowman's membrane seen in keratoconus still remains unclear. They may be minor scars due to the disease or primary defects in the process leading to keratoconus.     

Structural analysis of normal corneas and diseased corneas by applying second harmonic generation

We have established a second harmonic generation (SHG) microscopy system for imaging of the human cornea with a mode-locked femtosecond laser and a laser confocal microscope. This SHG microscopy system has allowed us to scan corneal tissue noninvasively ex vivo and to obtain three-dimensional images of corneal collagen lamellae. Such three-dimensional imaging of the normal anterior cornea revealed that collagen lamellae at the anterior stroma are inter-woven and adhere to Bowman membrane with these adherent lamellae being designated "sutural lamellae." Sutural lamellae adhere to Bowman membrane at an angle of approximately 19 degrees, whereas the angle of lamellae in the mid-stroma relative to Bowman membrane is smaller. We hypothesize that the structural unit consisting of both Bowman membrane and the sutural lamellae contributes to the rigidity and anterior curvature of the cornea. SHG imaging of keratoconic corneas revealed an either abnormal or a total lack of structure of the sutural lamellae, suggesting that this abnormality might be related to that of the corneal anterior curvature in such corneas. Furthermore, SHG imaging of corneas affected by stromal edema showed that the structure of the sutural lamellae was maintained, although abnormal collagen signals both above and below Bowman membrane were detected in corneas affected by clinical stromal edema for more than 12 months. SHG imaging of the structure of collagen lamellae in normal and diseased corneas thus has the potential to provide insight both into the mechanism for maintenance of corneal curvature as well as into the pathophysiology of corneal diseases.     

Optical coherence tomography of Descemet membrane separation by the big bubble technique

PURPOSE: To image Descemet membrane separation by the big bubble technique in human corneas by using anterior segment optical coherence tomography (OCT). METHODS: Five human corneoscleral rims were placed on an artificial anterior chamber and partially trephinated. A 23-gauge needle was inserted into the stroma under slit-lamp control and air was injected. The procedure was continuously imaged by anterior segment OCT. RESULTS: In all corneoscleral rims, a big bubble was created. The spread of air seemed to follow the interlamellar spaces without crossing lamellae. It involved mainly the inner layers of the stroma while sparing the outer 212 +/- 41 microm of the cornea (range, 168-271 microm). Intrastromal pressure build-up forced air above the Descemet membrane, creating tiny air bubbles of approximately 355 +/- 111 microm (range, 210-560 microm). When the pressure inside those bubbles reached a certain level, the bubbles spontaneously coalesced to form a big bubble. CONCLUSIONS: OCT is useful in imaging intracorneal air spread. The main obstacle to creating a big bubble is the impermeability to air of the imperforated posterior stromal lamellae.     

Altered lectin binding sites in keratoconus corneas.

We investigated the glycoconjugates in frozen sections of keratoconus corneas, using a panel of 12 biotin- or fluorescein isothiocyanate-labeled lectins. No differences between the lectin binding sites of the epithelium, endothelium and Descemet's membrane of normal and keratoconus corneas could be observed. However, in contrast to normal corneas, intense staining with peanut agglutinin (PNA) could be detected at breaks in Bowman's layer, in scar tissue and in the adjacent stroma. Furthermore, in the majority of cases binding sites for Phaseolus vulgaris erythroagglutinin (PHA-E) and increased staining with Ricinus communis agglutinin I (RCA-I) and Lens culinaris agglutinin (LCA) could also be detected in ruptures in Bowman's layer and in scar tissue. These data suggest that the scarred regions of the anterior stroma in keratoconus corneas may contain oligosaccharides with terminal D-galactose (beta 1-3)-D-N-acetylgalactosamine disaccharides (recognized by PNA), increased amounts of glycoconjugates with terminal beta-galactose residues (recognized by RCA-I), increased amounts of glycoconjugates with glucose/mannose residues (recognized by LCA), and finally, biantennary complex-type glycopeptides containing two outer galactose residues and a residue of N-acetylglucosamine (recognized by PHA-E). Since corneal scars due to causes other than keratoconus revealed lectin binding sites (particularly for PNA and to a lesser extent also for PHA-E, LCA and RCA-I) similar to those seen in scar tissue of keratoconus corneas, we conclude that it is mainly scar formation that may be responsible for the altered lectin binding sites in keratoconus.     

圆锥角膜组织中EGF和bFGF变化免疫组织化学的研究

目的 观察正常人及圆锥角膜中表皮生长因子(EGF)和碱性成纤维细胞生长因子（bFGF)的表达，探讨EGF和hFGF在圆锥角膜中的变化。方法 采用免疫组织化学SABC法检测角膜组织中EGF、bFGF的表达，结果 在圆锥角膜上皮层、Bowman层和内皮层有EGF表达，EGF比正常角膜的表达强；在圆锥角膜的上皮层、实质层和内皮层可见bFGF的阳性表达，圆锥角膜的基质瘢痕区bFGF有较强的阳性表达。结论 圆锥角膜组织中EGF水平增高，促进上皮细胞和基质细胞的DNA合成，从而使组织愈合。bFGF表达增强可以促进角膜上皮、基质细胞和内皮细胞的增殖。