Pump function of the human corneal endothelium. Effects of age and cornea guttata

The specific binding of tritiated ouabain to endothelial Na/K ATPase was used to quantitate the density of pump sites in the human corneal endothelium. Donor eyes, unsuitable for use in keratoplasty, were obtained from the Wisconsin Lions Eye Bank. The endothelium of each donor eye was examined using wide-field specular microscopy, and the specular micrographs were traced and digitized for the determination of cell density. Ouabain binding was measured in matched pairs of isolated endothelial sheets. A total of 26 pairs of donor eyes, ranging in age from 11 through 91 years, were studied. Twenty pairs, determined to have normal endothelia, were found to have a constant pump site density which was independent of donor age. Six donor pairs had moderate guttata; in this group pump site density was significantly increased. These results indicate that, although pump site density is normally constant in the human corneal endothelium, conditions which increase endothelial permeability, such as guttata, can cause a compensatory increase in pump site density and presumably pump function.     

Confocal microscopy of the human cornea in vivo

In vivo, scanning-slit, confocal microscopy offers improved resolution and has resulted in new discoveries of corneal pathology at the cellular level.The ability to provide high resolution, real-time images of the full thickness of theliving human cornea gives the clinician and the researcher an important new tool.     

Imaging posterior polymorphous corneal dystrophy by in vivo confocal microscopy

To identify features of posterior polymorphous dystrophy (PPMD) by in vivo confocal microscopy, the corneas of a female patient with PPMD were examined using slit‐lamp biomicroscopy and slit‐scanning in vivo confocal microscopy. Characteristic endothelial vesicular and band lesions were seen clinically and easily identified using in vivo confocal microscopy. However, endothelial pleomorphism, an increased density and reflectance of posterior stromal keratocytes, and prominence of corneal nerves were also delineated. In vivo confocal microscopy enhances clinicopathological diagnosis and follow up of corneal dystrophies with subtle clinical presentations, such as PPMD.     

In vivo confocal microscopic evaluation of keratic precipitates and endothelial morphology in Fuchs' uveitis syndrome

Purpose

To evaluate the endothelial cell layer in patients with Fuchs'' uveitis syndrome (FUS) with respect to the type and distribution of keratic precipitates (KP), endothelial cell morphology, and endothelial cell density (ECD), using in vivoconfocal microscopy (IVCM).

Methods

Forty eyes of 40 patients (mean age of 32.2±12.5 years) with the clinical diagnosis of FUS were evaluated with IVCM (Confoscan 3.0, Vigonza, Italy). KP were classified as type I (small, round), type II (stippled), type III (dendritiform), and type IV (globular). When >1 KP type was present, differentiation between the predominant and less frequent KP was made as ‘primary'' and ‘secondary''. ECD was measured and compared with age-matched 60 control subjects. Endothelial blebs were classified as small (3–10 μm) or large (>10 μm).

Results

In 36 (90.0%) cases with FUS, more than one KP type was observed with IVCM. Type III (dendritiform) KP was the most frequently observed primary KP type (85.0%), followed by type II (stippled) KP (15.0%). Secondary KP included type II (58.3%), type IV (globular) (27.8%), and type III (13.9%). The mean endothelial cell density of eyes with FUS (2588±396 cells/mm²) was significantly lower than that of control subjects (2930±364 cells/mm²) (t-test; P<0.001). Eyes with FUS had lower proportion of hexagonal cells and higher percentage of polymegethism compared with the uninvolved contralateral eyes. Endothelial blebs (21 small, 16 large blebs) were observed in 37 (92.5%) eyes.

Conclusions

FUS is characterized by dendritiform KP and is associated with decreased ECD and altered endothelial cell morphology.     

Morphological evaluation of Schnyder's crystalline corneal dystrophy by laser scanning confocal microscopy and Fourier-domain optical coherence tomography

A 29-year-old Chinese woman with Schnyder's crystalline corneal dystrophy was referred to our clinic. Ocular examination revealed abnormal deposits of cholesterol and lipid within the corneal stroma, appearing as crystalline needle-shaped deposits, in both eyes. In vivo laser scanning confocal microscopy revealed pathological alterations of the normal corneal anatomy in the patient, such as highly reflective crystalline structures in the anterior-and mid-stroma. The images acquired from Fourier-domain optical coherence tomography indicated that the main presence of crystalline deposits was localized within the anterior stroma, extending from the basal epithelium layer to a depth of 80 to 150 µm. To the best of the author's knowledge, it is consistent with the results of the previous histological study.     

应用共焦显微镜观察Fuch角膜内皮营养不良患者的病变形态学特征

目的探讨应用共焦显微镜观察我国Fuch角膜内皮营养不良患者角膜各层的活体形态学特征。方法对19例(38只眼)Fuch角膜内皮营养不良患者的中央部角膜进行活体共焦显微镜检查,分为有症状组(19只眼)和无症状组(19只眼),并选取30只眼作为正常对照组,应用NAVIS软件测量、分析角膜各层组织细胞形态和密度,以及滴状赘疣和角膜神经的直径。结果 (1)有症状组:19只眼的角膜内皮层均见到滴状赘疣,直径20-60 μm,内皮细胞密度与正常对照组比较差异有显著意义(t=18.74,P<0.01);9只眼后弹力膜增厚;14只眼角膜后基质层有长条形暗区结构;19只眼角膜基质反光普遍增强;17只眼Bowman膜有局灶性高反光区域;19只眼基底上皮细胞形态大致正常;10只眼显示正常的角膜神经结构;后、前基质细胞密度,与正常对照组比较差异无显著意义(t=0.854、1.173,P=0.38、0.24)。(2)无症状组:19只眼的角膜内皮层均见到滴状赘疣,数目较有症状组者少,直径15-40μm;内皮细胞密度,与正常对照组比较,差异无显著意义(t=1.998,P=0.053);角膜其余各层未见异常。有症状组与无症状组的内皮细胞密度计数比较,差异有非常显著意义(t=8.352,P<0.01)。结论活体共焦显微镜检查有助于Fuch角膜内皮营养不良患者的诊断,特别适用于角膜水肿、角膜内皮镜无法成像的患者。(     

Characterization of the Descemet's membrane/posterior collagenous layer isolated from Fuchs' endothelial dystrophy corneas

The combined Descemet's membrane (DM) and posterior collagenous layer (PCL) of Fuchs' endothelial dystrophy corneas were isolated and characterized by biochemical and immunofluorescence methods. The amino acid composition of the Fuchs' DM-PCL was similar to age-matched normal Descemet's membranes (DM). As determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ¹²⁵I two-dimensional peptide mapping, normal DM and Fuchs' DM-PCL contained the same collagen types [type IV and endothelial cell (EC) collagen], but a slight discrepancy was seen in the electrophoretic mobility of some collagen chains. Immunofluorescence staining localized fibrinogen/fibrin to Fuchs' DM-PCL but not to normal DM. These data suggest (1) that the appearance of 110 nm banded material in sheets and fusiform bundles characteristic of Fuchs' PCL is not due to the presence of a new (abnormal) collagen type but may represent altered assembly of collagen molecules, and (2) that the fibrinolytic system may play a role in the degenerative process of Fuchs' endothelial dystrophy.     

活体共聚焦显微镜检查在角膜营养不良诊断中的应用

活体共聚焦显微镜检查(in vivo confocal microscopy,IVCM)无创、快速,可活体观察眼表组织及细胞结构的生理、病理变化,在感染性角膜炎诊断及治疗评价、干眼病眼表评估、眼表手术治疗效果评估及糖尿病早期筛查等方面均有广泛应用。利用IVCM观察角膜营养不良有助于诊断、监测疾病进展、评价治疗效果及了解其病理生理变化等。     

共焦显微镜观察虹膜角膜内皮综合征及其继发性青光眼的手术治疗

目的应用共焦显微镜检查探讨虹膜角膜内皮综合征的临床特征及其继发性青光眼的手术治疗。方法 8例（8眼）虹膜角膜内皮综合征行共焦显微镜下双眼检查;描述角膜内皮的病变结构特点,分析双眼角膜内皮细胞密度、内皮细胞平均面积、六角形细胞比例及中央角膜厚度,并对6例继发性青光眼中的5例施行手术治疗。结果患眼角膜内皮细胞呈风筝样或上皮细胞样改变;细胞排列紊乱,大小不均;细胞内可见高反光的细胞核,部分可见双核。患眼和对侧眼的角膜内皮细胞密度、内皮细胞平均面积、六角形细胞比例分别为：789.7±75.8个/mm^2、2223.7±80.6个/mm^2;1106.9±89.4μm^2、379.8±20.6μm^2;17.2±1.2%、56.3±1.7%,下降明显。二者的中央角膜厚度相当。4例继发性青光眼手术后眼压控制正常,另1例手术无效。结论共焦显微镜能够发现ICE患者的角膜内皮细胞的特征性改变;对该病的诊断具有很高的临床意义。并发继发性青光眼要及时手术治疗控制眼压。     

Corneal dystrophies. II. Endothelial dystrophies

In general, endothelial dystrophies present three types of clinical manifestations: 1) production of collagenous tissue posterior to Descemet's membrane which appears as cornea guttata, polymorphic excrescences or gray sheets; 2) a disrupted endothelial mosaic in specular reflection; and 3) corneal edema as a reflection of decreased endothelial barrier and pump functions. In this review, the authors discuss three endothelial dystrophies — Fuchs', posterior polymorphous and congenital hereditary. They describe the clinical, histopathologic and biochemical features, and illustrate each dystrophy with a composite drawing. Dystrophies of the epithelium, Bowman's layer, and stroma were reviewed separately in the September–October 1978 issue of this journal.     

In vivo confocal microscopy of human cornea covered with human amniotic membrane

Purpose  Amniotic membrane transplantation has been widely performed to reconstruct the surface of the eye and treat chemical burns or epithelial defects. However, we have difficulty observing the cornea through the opaque transplanted amniotic membrane by slit-lamp biomicroscopy. We investigated the use of confocal microscopy for observation of human corneas covered with amniotic membrane. Methods  Human amniotic membrane was placed onto the normal corneas of five volunteers aged 22–24 years. Then, all layers of the covered corneas were observed by in vivo confocal microscopy. Results  Confocal microscopy displayed the epithelium, basement membrane, and stroma of the amniotic membrane. It also displayed the corneal epithelium. Furthermore, corneal stromal keratocytes and the corneal endothelium were clearly observed through the amniotic membrane by confocal microscopy. Conclusions  We demonstrated that in vivo confocal microscopy enabled us to observe all layers of corneas covered with amniotic membrane in normal human eyes. Our findings suggest that confocal microscopy may have advantages for clinical examination of the ocular surface, including all layers of the cornea.     

Contemporary in vivo confocal microscopy of the living human cornea using white light and laser scanning techniques: a major review

In vivo confocal imaging of the cornea has evolved exponentially over the last few decades and it has increasingly emerged from the laboratory to be used in the clinical setting in relation to inherited corneal diseases, corneal infections, contact lens wear and the effects of corneal surgery. This evolution has led to significant enhancement of our knowledge of the living cornea in both its physiological and pathological states. A number of in vivo confocal microscope devices using white, and more recently coherent, light sources have been developed to provide non-invasive assessment of the corneal microstructure at a lateral resolution of 1-2 microm. The fundamental principles of in vivo confocal microscopy and the key differences between these devices are highlighted in this review. By providing a systematic review of the extensive literature on the human cornea, this perspective paper aims to provide an overview of how in vivo confocal microscopy has contributed to our greater understanding of the human cornea in health, in disease, and following surgery, with a particular emphasis on quantitative data. The utility and limitations of available data are highlighted as are possibilities for the future development of this innovative technology.     

The 7‐year cumulative incidence of cornea guttata and morphological changes in the corneal endothelium in the Reykjavik Eye Study

Purpose: To examine the corneal endothelium and establish the 7‐year cumulative incidence of cornea guttata (CG). Methods: Population‐based prospective cohort study with 573 participants (third wave of the Reykjavik Eye Study (RES) in 2008). Four hundred and thirty‐seven subjects had either right or left eyes available for analysis after excluding confounding eye conditions. The baseline for eyes at risk for developing CG is the second wave of the RES in 2001. Participants underwent specular microscopy and a standardized eye examination. Results: The cumulative 7‐year incidence of CG in either eye was estimated as a 95% confidence interval for the expected value for both genders combined (15–23%), for males (8–18%) and for females (19–29%). In right eye only, the 7‐year cumulative incidence for both genders combined was estimated to be 6–11%. For genders combined and for males only, the data indicated no correlation between 7‐year cumulated incidence and age at baseline. In women, however, the change of 7‐year incidence for CG in at least one eye appeared to be correlated to age at baseline. Reduction of endothelial cell density for corneas with CG at baseline was found [CI (0.95)?132 ± 94]. Conclusion: The cumulative 7‐year incidence of primary central CG for a middle‐aged and older Caucasian population without history of potentially confounding eye disease has been established. Women tend to have higher incidence if onset occurs at middle age. If CG is present, the cell density and the cell size variation decrease within a 7‐year period.     

Granular dystrophy of the cornea

Background : Granular corneal dystrophy is the commonest of the dystrophies and usually results in visual disability in the fourth or fifth decades. Case history : A patient with granular corneal dystrophy is reported and the clinical characteristics described. Discussion : The classical clinical features and the pathology and management of granular dystrophy are reviewed. Two unusual variations of the corneal dystrophy, namely, juvenile granular dystrophy and Avellino dystrophy, which is a concurrence of the features of granular and lattice dystrophies, are also described.     

Avellino角膜营养不良在共焦显微镜下的形态学特征

目的 探讨Avellino角膜营养不良（ACD）患者的共焦显微镜图像特点。方法对经基因型检测明确为ACD患者（R124H突变）的10例20眼角膜进行共焦显微镜检查，其中1例2眼为穿透角膜移植（PK）术后眼，一眼病变复发，另一眼无复发表现。并取颗粒状角膜营养不良I型（GCD I，R555W突变）患者9例18眼作为对照。结果ACD患者未手术眼共焦显微镜图像特点：基底上皮、Bowman膜及整个角膜基质均可见不同程度的异常反光改变；PK术后复发眼Bowman膜及紧邻其下的前基质可见由细小沉积物组成的不规则混浊；PK术后无复发表现眼角膜各层未见明显异常。GCD I型最显著的共焦显微镜图像特点为角膜基质内以大型沉积物为主。结论首次对经基因检测确诊的Avellino角膜营养不良患者的角膜进行活体共焦显微镜检查。ACD患者基底上皮、Bowman膜及整个角膜基质均可见不同程度的异常反光改变，但较GCD I型患者的沉积物为小。     

In vivo confocal microscopy in recurrent granular dystrophy in corneal graft after penetrating keratoplasty

Two case reports of recurrent granular dystrophy in corneal grafts after penetrating keratoplasty are presented. Slit-lamp examination and confocal microscopy (HRT II) were performed in two patients with recurrent granular dystrophy. All confocal microscopic findings of granular dystrophy were evaluated in the graft. Dystrophic lesions of the donor cornea presented the same confocal microscopic aspects in both eyes, and were similar to granular dystrophy lesions. Confocal microscopy is an imaging method that may provide new information on corneal microanatomy in dystrophies. It may be particularly useful in improving the early diagnosis of dystrophic lesions in corneal grafts.     

应用共聚焦显微镜观察正常老年人角膜形态学的特点

目的:探讨正常老年人共焦显微镜下角膜各层组织的活体细胞形态学特征。方法:正常老年人19例22眼中央角膜应用共聚焦显微镜(confocal microscope through focus,CMTF)进行观察,记录上皮翼状细胞层、上皮基底细胞层、前基质层、后基质层、内皮层的细胞密度,选取每人上皮基底细胞层下神经丛和中后基质层神经最清晰图像,并记录此幅图像中神经纤维总长度、神经纤维直径、神经纤维数目和每100μm神经纤维包含的念珠状结构(beads)数目,同时比较不同眼别及不同性别之间上述计量和计数资料之间的差别;观察上皮翼状细胞层、上皮基底细胞层、前基质层、后基质层、内皮层及上皮基底细胞层下神经丛(subbasal epithelial nerve plexus)和中后基质层神经(stromal nerves)的组织形态。结果:上皮翼状细胞层、上皮基底细胞层、前基质层、后基质层、内皮层的细胞密度分别为2150±315,5270±539,859±137,627±184,2529±654个/mm2,上皮基底细胞层下神经丛每幅图像中神经纤维总长度、神经纤维直径和数目、每100μm神经纤维包含的念珠状结构分别为944±176μm,2.3±0.5μm,9.0±1.3条,4.9±1.4;中后基质层神经每幅图像中神经纤维总长度、神经纤维直径和数目、每100μm神经纤维包含的念珠状结构分别为306±138μm,5.6±1.7μm,1.8±1.5条,0.0±0.0。上述计量和计数资料左、右眼和男、女之间无统计学差异。同时观察到排列疏松、细胞间夹杂有无结构暗区的表层上皮细胞层,过度形态的翼状上皮细胞层,呈"斑马皮样"外观的基底细胞层,在角膜中后基质层有4眼(18%)可见到微皱褶(microfolds),在排列规则、多呈六边型的角膜内皮层发现有2眼(9%)出现类似滴状赘疣(pseudoguttata-like)结构。结论:共焦显微镜可以在实时、活体和三维空间从细胞水平对角膜各层结构进行定量和定性分析。     

Confocal microscopic observations of the human cornea following overnight contact lens wear

Background: Striae and folds are observed with a slidamp biomicroscope in the cornea following overnight contact lens wear. These phenomena are poorly understood. The aim of this study is to employ confocal microscopy to observe and document these and other morphological changes in die human cornea following overnight contact lens wear. Methods: Slitlamp biomicroscopy, slit‐scanning confocal microscopy and ultrasonic pachometry were performed on both eyes of 13 subjects (3M, 10F, age 24 ± 3 years) before and after eight hours overnight wear of a ‐3.00 D Bausch & Lomb one day disposable soft contact lens (Dk/t = 15.1 times 10^‐9 (cm/sec) x {ml O²/ml x mmHg)) in one eye; the other non‐lens‐wearing eye acted as a control. Results: Following sleep, both corneas were swollen (lens‐wearing eye 11.8 ± 3.8 per cent; control eye 2.1 ± 1.9 per cent) and the stroma of both corneas displayed an apparent reduction in keratocyte density (lens‐wearing eye 21 per cent; control eye 10 per cent). Folds were observed with the slidamp biomicroscope and long, straight, dark, orthogonal lines were observed widi die confocal microscope, in the posterior stroma of the oedematous lens‐wearing eyes. Such features were not observed in die control eyes. The keratocytes appeared less distinct with greater levels of corneal oedema. Conclusion: The apparent loss of keratocytes following overnight lens wear is an optical artefact that can be explained in terms of corneal oedema causing volumetric tissue expansion and a loss of optical clarity, which hampers keratocyte detection. These findings place the onus on researchers postulating a loss of stromal keratocytes following clinical interventions, such as contact lens wear, to account for the effects of oedema.     

In vivo confocal microscopy of pre-Descemet's membrane corneal dystrophy

Pre-Descemet's membrane corneal dystrophy is clinically characterized by the presence of numerous tiny pleomorphic opacities located in the deep stroma immediately anterior to Descemet's membrane. A 35-year-old man, clinically diagnosed with pre-Descemet's corneal dystrophy, was examined by in vivo slit scan confocal microscopy. The pleomorphic structures containing dense hyperreflective inclusions in the posterior stroma were revealed in vivo. To the best of the authors' knowledge, it is consistent with the result of the previous histological study, but different from other reports using in vivo confocal microscopy.