Specular microscopy in clinical practice

Specular microscopy is a noninvasive diagnostic tool that allows for in vivo evaluation of corneal endothelium in health and various diseased states. Endothelial imaging helps in the diagnosis and management of several endothelial disorders. The review focuses on the principles of specular microscopy, limitations of endothelial imaging, and its interpretation in common conditions seen in the clinical practice. A thorough PubMed search was done using the keywords specular microscopy, corneal endothelium, and endothelial imaging.     

Anterior segment optical coherence tomography

Optical coherence tomography (OCT) provides non-contact, rapid in vivo imaging of ocular structures, and has become a key part of evaluating the anterior segment of the eye. Over the years, improvements to technology have increased the speed of capture and resolution of images, leading to the increasing impact of anterior segment OCT imaging on clinical practice. In this review, we summarize the historical development of anterior segment OCT, and provide an update on the research and clinical applications of imaging the ocular surface, cornea, anterior chamber structures, aqueous outflow system, and most recently anterior segment vessels. We also describe advancements in anterior segment OCT technology that have improved understanding with greater detail, such as tear film in dry eye disease evaluation, intra-operative real-time imaging for anterior segment surgery, and aqueous outflow with angle assessment for glaucoma. Improvements to image processing and software have also improved the ease and utility of interpreting anterior segment OCT images in everyday clinical practice. Future developments include refinement of assessing vascular networks for the anterior segment, in vivo ultra-high resolution anterior segment optical coherence tomography with histology-like detail, en-face image with 3-dimensional reconstruction as well as functional extensions of the technique.     

Capillaries in the epithelium of pterygium

AIM—To present new morphological observations of intraepithelial capillaries in pterygium and to provide some explanations for this phenomenon.
METHODS—The ultrastructural features of pterygia from 26 patients were examined. Surgically excised tissue was processed for conventional light and transmission electron microscopy.
RESULTS—Individual capillaries within the epithelium of the anterior half towards the head of pterygia were identified in 11 specimens out of 26 pterygia examined (42.3%). The perivascular connective tissue of the intraepithelial capillaries contained fibroblasts, collagen fibrils, and elastin-like material. Epithelial cells surrounding these capillaries showed defects in the basal lamina in contrast with the continuous basal lamina of the endothelium. In the intercellular space of the epithelium an amorphous substance, occasional fibroblast processes, and collagen fibrils were frequently observed.
CONCLUSION—Capillaries in the epithelium of pterygia are rare, but not exceptional. The ingrowth of these vessels from the stroma into the epithelium can be interpreted as a reaction to hypoxia or deficiency of any other substance transported via the bloodstream. Apparently, the perivascular connective tissue can be used by ingrowing fibroblasts as a migration pathway. The migrating fibroblasts appear to use the defects of the epithelial basal lamina (whether partially or complete) in order to reach the intercellular space. It is possible that collagen fibrils in the epithelial intercellular space have been laid down by fibroblasts which contribute to the pathological dedifferentiation of the conjunctival epithelium.

Keywords: pterygium; ultrastructure; epithelium; blood vessels     

眼底血管病理改变特征提取方法研究进展

眼底血管是活体能唯一观察到的人体终末血管,许多疾病在眼底血管的病理改变可通过多种手段进行观察,如检眼镜、眼底血管照相、激光共聚焦扫描眼底成像、荧光素眼底血管造影、光学相干断层扫描、多普眼底血管成像等,如何将观察到的病理改变准确提取,一直是学术界的难题,本文总结了眼底血管病理学改变的主要观察手段及病理改变的多种提取方法,包括眼底血管追踪法、像素分类法、匹配滤波法、阈值分割法等,并对眼底血管提取后的图像处理方法进行了总结,希望可以促进眼底血管病理特征提取方法的进一步发展,以利于临床疾病的诊断。     

A single cell atlas of human cornea that defines its development,limbal progenitor cells and their interactions with the immune cells

PurposeSingle cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood.MethodsFour human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs).ResultsscRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of LPCs, which predate the formation of limbal niche by a few weeks.ConclusionsOur scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of ocular surface disorders.     

Imaging the Corneal Endothelium in Fuchs Corneal Endothelial Dystrophy

ABSTRACT

Fuchs endothelial corneal dystrophy (FECD) is characterized by the progressive degeneration of the corneal endothelium (CE). The purpose of this article is to review the diagnostic tools available to image and assess the CE in FECD. Slit-lamp biomicroscopy with specular reflection and retroillumination are important techniques to assess the CE. Objective diagnostic tests, such as retroillumination photographic analysis, specular microscopy, in vivo confocal microscopy (IVCM), and anterior segment optical coherence tomography, are valuable tools to evaluate the CE in FECD. Specular microscopy can be performed rapidly without touching the eye but requires a clear cornea with a smooth CE. In contrast, IVCM can image all layers of the cornea, even in advanced FECD. However, IVCM is contact-based and more technically challenging. It is important to select the appropriate objective diagnostic test to image and assess the CE in managing patients with FECD.     

The role of Tenonplasty in the management of limbal and scleral ischemia due to acute ocular chemical burns

Of the various manifestations of ocular chemical burns (OCBs), ischemia of the limbus and the peri-limbal sclera indicates poor prognosis and in severe cases threaten the integrity of the globe. Tenonplasty is a surgical procedure which involves advancing the Tenon’s capsule over the ischemic areas to provide a vascular supply and to enable migration of the conjunctival epithelium. This review aims to provide an overview of the diagnosis of limbal ischemia and its management with Tenonplasty. A literature review was conducted using the keywords “Tenonplasty,” “Tenon’s capsule,” “ocular chemical injury,” “ocular thermal injury,” “Tenon advancement,” “scleral ischemia,” and “limbal ischemia,” and outcomes were studied from seven selected articles. In addition to clinical evaluation, in vivo imaging techniques such as anterior segment optical coherence tomography angiography can provide an objective method of measuring and monitoring the ischemia and re-perfusion of the peri-limbal vasculature. Tenonplasty can be performed in eyes with acute OCBs with scleral or limbal ischemia by dissecting the Tenon’s layer from the orbit and securing it to the limbus. The indications, mechanism of action, peri-operative considerations, surgical technique, and post-operative care of Tenonplasty are discussed in detail. The average time for post-operative re-epithelization ranges from 1 to 6 months with the formation of a symblepharon being the most common complication. In conclusion, Tenonplasty is a globe-salvaging procedure in cases with severe limbal and scleral ischemia because of OCBs and has good anatomical outcomes priming the globe for subsequent re-constructive and vision-restoring surgeries.     

The clinical value of in vivo confocal microscopy for diagnosis of ocular surface squamous neoplasia

Purpose

To determine the reliability and efficiency of in vivo confocal microscopy for the diagnosis of ocular surface squamous neoplasia (OSSN).

Methods

A case series with five consecutive cases of OSSN were investigated retrospectively, of which the characteristics and subspecial types had been estimated by in vivo confocal microscopy before surgery. The structure and cellular features of OSSN were analyzed with other examinations, such as anterior-segment optical coherence tomography (AS-OCT), and confirmed by histopathological biopsy.

Results

The tumors revealed red gelatinous surfaces with vascular dilatation on the ocular surface of the conjunctival and corneal epithelium in anterior segment photography. Involvement of only corneal epithelium was observed by AS-OCT in three cases, whereas the Bowman''s layer and anterior stroma were also invaded in the other two cases. In vivo confocal microscopy showed cellular anisocytosis and enlarged nuclei with high nuclear to cytoplasmic ratio in three cases diagnosed as conjunctival intraepithelial neoplasia; moreover, nests were partially formed by isolated keratinized, binucleated, and actively mitotic dysmorphic epithelial cells in the other two cases diagnosed as carcinoma in situ and ocular surface squamous carcinoma (OSSC). The characteristics assessed from histopathological biopsy were similar to that revealed by in vivo confocal microscopy in all five cases.

Conclusion

In vivo confocal microscopy analysis of cytological characteristics of OSSN is a safe, relatively noninvasive, and effective diagnostic tool in detecting characteristics of OSSN before surgical resection. Although in vivo confocal microscopy cannot replace excisional biopsy for definitive diagnosis, it can be valuable for initial diagnosis and management of patients with OSSN.     

Imaging of vascular abnormalities in ocular surface disease

The vascular system of the ocular surface plays a central role in infectious, autoimmune, inflammatory, traumatic and neoplastic diseases. The development, application, and monitoring of treatments for vascular abnormalities depends on the in vivo analysis of the ocular surface vasculature. Until recently, ocular surface vascular imaging was confined to biomicroscopic and color photographic assessment, both limited by poor reproducibility and the inability to image lymphatic vasculature in vivo. The evolvement and clinical implementation of innovative imaging modalities including confocal microscopy, intravenous, and optical coherence tomography–based angiography now allows standardized quantitative and functional vascular assessment with potential applicability to automated analysis algorithms and diagnostics.     

Idiopathic bilateral lipid keratopathy.

A 52-year-old Mexican man presented with asymptomatic, bilaterally symmetrical lipid infiltrates of the cornea and adjacent limbus. No evidence of previous ocular disease or systemic disorder of lipid metabolism could be detected. Penetrating keratoplasty of the right eye was required. The cornea was rigid and thick, with posterior bulging into the anterior chamber. Light microscopy revealed deep corneal lipid granules, foamy histiocytes, vascularisation, and chronic non-granulomatous inflammation. Transmission electron microscopy showed extracellular lipid spaces and numerous intracytoplasmic lipid vacuoles in histiocytes, keratocytes, conjunctival epithelium, and the endothelium of blood vessels in the corneal stroma and adjacent limbal conjunctiva. Histochemical analysis revealed the presence of neutral fats, free fatty acids, cholesterol, and phospholipids.     

角膜缘干细胞培养上清液对血管内皮细胞增殖的影响

目的：观察角膜缘干细胞培养液对血管内皮细胞增殖的影响。

方法：分别培养角膜缘干细胞及球结膜上皮细胞,并通过免疫组化检测AE-5蛋白鉴别角膜缘干细胞。搜集两组培养细胞的上清液加入培养的人脐静脉血管内皮细胞中,并设立对照组。培养24h后通过MTT法检测细胞增殖情况并进行统计学分析。

结果：角膜缘干细胞AE-5染色呈阴性,而球结膜上皮细胞为阳性。加入角膜缘干细球结膜上皮细胞和对照组培养液的血管内皮细胞MTT值分别为2.097±0.079,1.981±0.034和1.990±0.044。三组间有显著性差异(F=9.169,P=0.000)。加入角膜缘干细胞培养上清液的血管内皮细胞增殖活性明显高于其他两组(P=0.005和P=0.007)。

结论：角膜缘干细胞培养液能够促进血管内皮细胞的增殖,这可能是角膜缘干细胞的特征。本研究从功能学角度为角膜缘干细胞理论提供更多的依据。     

Limbal stem cell disease: Treatment and advances in technology

Anterior segment stem cell technology, due to its already well-defined corneal limbal stem cells with greater ease of evaluation, has been at the forefront of ophthalmic stem cell treatment and technology since 1997. This paper provides an overview of the current standard of care for treatment of limbal stem-cell deficient conditions and reviews recent treatment technologies using ex vivo expansion of cultivated limbal stem cells of the cornea.     

Differential diagnosis of corneal oedema assisted by in vivo confocal microscopy

The purpose of this study was to demonstrate microstructural differences between clinically similar, but aetiologically different, cases of corneal oedema in four subjects. In vivo confocal microscopy highlighted oedema of the basal epithelium, prominent nerve–keratocyte interactions, and typical ‘epithelialization’ of the endothelium in a case of iridocorneal endothelial syndrome; however, a similar microstructural appearance was observed in a case of presumed herpetic disciform keratitis. The latter diagnosis was subsequently revised on this basis. Confocal examination of Fuchs’ endothelial dystrophy demonstrated oedema of the basal epithelium, prominent wing cells, anterior stromal alterations, fibrosis of Descemet’s membrane and a typical ‘strawberry’ appearance of the endothelium. In contrast, in vivo microstructural examination of bilateral keratoconus with hydrops confirmed oedema mainly involving the epithelium and anterior stroma. In vivo confocal microscopy allows the clinician to observe the living cornea at a microstructural level and to better diagnose and differentiate borderline or unusual cases of corneal oedema.     

Role of the endothelium in modulating functional responses of isolated bovine anterior ciliary arteries to vasoconstrictor agonists

BACKGROUND/AIMS—Endothelium dependent vasodilatation is an important regulator of blood flow to the eye but its role has not been investigated in vessels supplying the ciliary body. This study assessed the role of the endothelium in modulating vasoconstrictor responses of the intraocular bovine anterior ciliary artery.
METHODS—Bovine anterior ciliary arteries (n=33) were mounted in a myograph, containing physiological salt solution at 37°C, for isometric force measurement. Cumulative concentration-response curves were obtained to the constrictor agonists 5-hydroxytryptamine (5-HT), noradrenaline, phenylephrine, prostaglandin F_2α, endothelin-1, and KCl in both endothelium intact and denuded arteries.
RESULTS—All vasoconstrictors produced sustained contractile responses which were unaffected by the removal of the endothelium. Responses to 5-HT were also unaffected by inhibition of nitric oxide synthase.
CONCLUSION—These results indicate that neither agonist stimulated nor basal release of nitric oxide from the endothelium modulates responses to vasoconstrictor agonists in the isolated bovine anterior ciliary artery when measured in a no flow isometric system.

Keywords: anterior ciliary artery; constrictor agonists; endothelium; nitric oxide     

Resident plasmacytoid dendritic cells patrol vessels in the naïve limbus and conjunctiva

Plasmacytoid dendritic cells (pDCs) constitute a unique population of bone marrow-derived cells that play a pivotal role in linking innate and adaptive immune responses. While peripheral tissues are typically devoid of pDCs during steady state, few tissues do host resident pDCs. In the current study, we aim to assess presence and distribution of pDCs in naïve murine limbus and bulbar conjunctiva. Immunofluorescence staining followed by confocal microscopy revealed that the naïve bulbar conjunctiva of wild-type mice hosts CD45⁺ CD11c^low PDCA-1⁺ pDCs. Flow cytometry confirmed the presence of resident pDCs in the bulbar conjunctiva through multiple additional markers, and showed that they express maturation markers, the T cell co-inhibitory molecules PD-L1 and B7–H3, and minor to negligible levels of T cell co-stimulatory molecules CD40, CD86, and ICAM-1. Epi-fluorescent microscopy of DPE-GFP×RAG1^−/− transgenic mice with GFP-tagged pDCs indicated lower density of pDCs in the bulbar conjunctiva compared to the limbus. Further, intravital multiphoton microscopy revealed that resident pDCs accompany the limbal vessels and patrol the intravascular space. In vitro multiphoton microscopy showed that pDCs are attracted to human umbilical vein endothelial cells and interact with them during tube formation. In conclusion, our study shows that the limbus and bulbar conjunctiva are endowed with resident pDCs during steady state, which express maturation and classic T cell co-inhibitory molecules, engulf limbal vessels, and patrol intravascular spaces.     

The effect of reactive oxygen species on the myogenic tone of rat ophthalmic arteries with and without endothelium

Background

Reactive oxygen species (ROS) play an important role in the pathogenesis of various ocular diseases. ROS can induce vasodilation or vasoconstriction depending on the species, the tested vessel bed, and the condition of the vessel. This study investigates the effect of different dosages of ROS on the tone of rat ophthalmic arteries.

Methods

Freshly dissected rat ophthalmic arteries were pressurized in a perfusion setup in steps of 10 mmHg to 180 mmHg in three consecutive cycles. The first cycle was run under mostly physiological conditions, the second cycle was run after ROS treatment, and the third cycle as passive dilation after all Ca²⁺ was removed from the solution. ROS-induced dilation or constriction was calculated in relation to the passive dilation. All experiments were performed with or without endothelium.

Results

For vessels with endothelium, dilation in control experiments was 20.0?±?0.1 %; after 5 s of ROS dilation was 74.4?±?0.6 %, and after 20 s 87.4?±?0.3 %. ANOVA revealed significant differences between these groups (P?=?0.048). For vessels without endothelium, a slight dilation was seen in control experiments (14.5?±?0.4 %), which was also present after 5 s of ROS treatment (15.4?±?0.4 %). Treatment with ROS for 20 s led to a constriction of the vessel preparations (?16.6?±?0.5 %; P?=?0.831).

Conclusions

ROS led to a vasodilation in vessels with endothelium that was not seen in vessels without endothelium. Endothelial function seems to determine the effect of ROS on the vessel tone in isolated rat ophthalmic arteries.     

In vivo confocal microscopy of climatic droplet keratopathy

We describe the corneal microstructural changes in a patient with spheroidal degeneration using in vivo confocal microscopy. Multiple hypo‐ and hyper‐reflective spherical lesions were observed in the anterior corneal stroma and Bowman's layer ranging from 45 to 220 μm in size. The corneal epithelium, posterior stroma and endothelium were otherwise unaffected. In vivo confocal microscopy demonstrates good correlation with excised histological samples in climatic droplet keratopathy. It provides a non‐invasive technique to examine the living cornea for degenerative disease and acts as a bridge between clinical and laboratory observations.     

Efectos corneales de hipotensores oculares que contienen cloruro de benzalconio: análisis in vivo con microscopia confocal

PurposeTo evaluate the effects of anti-glaucoma treatments containing benzalconium chloride (BAC) on the human cornea.MethodsA prospective single masked cohort study was conducted on the 50 eyes of 50 patients. The inclusion criteria were: recently diagnosed glaucoma or ocular hypertension with previous treatment, or ophthalmologist-prescribed anti-glaucoma therapy, and oral consent to participate in the study. The patients were not randomised, as the ophthalmologist decided the best therapy according to clinical criteria. The patients were divided in 2 cohorts: one exposed to BAC (23 patients), and not exposed (27patients). The mean follow-up period was 22 weeks (range 18-30). The change in cell density before and after therapy was measured in: basal layer epithelium, basal layer of limbal epithelium and endothelium. The change in stromal reflectivity and the number of nerve branches in sub-basal nerve plexus was also measured. BAC exposure was blinded to the main researcher.ResultsA greater increase in basal layer epithelium cell density was observed in BAC exposed cohort (P < .05). No significant differences were detected in the endothelium, limbal cell density, stromal reflectivity, or sub-basal nerve plexus. Age, sex, IOP, active ingredient or BAC concentration did not affect the direction or magnitude of the ocular surface alterations found.ConclusionChronic anti-glaucoma therapy induces changes in the corneal epithelium. Preservative free drops showed less disruption of the ocular surface by confocal microscopy analysis. Further studies should be conducted to evaluate the clinical impact of these histological findings.     

The OCT angular sign of Henle fiber layer (HFL) hyperreflectivity (ASHH) and the pathoanatomy of the HFL in macular disease

The Henle fiber layer (HFL) is comprised of bundles of unmyelinated photoreceptor axons intermingled with outer Müller cell processes. The photoreceptor axons extend from the cell bodies located in the outer nuclear layer and radially project toward the outer plexiform layer, the inner third of which includes the synaptic junctional complexes and the outer two-thirds of which includes the HFL. The oblique path of the HFL provides unique structural and reflectance properties and this radial anatomy is highlighted in many macular disorders including those with macular star exudation and HFL hemorrhage. Recent investigations using multimodal imaging techniques, especially cross sectional and en face optical coherence tomography (OCT), have provided new perspectives regarding HFL disruption in retinal diseases. The aim of this review is to highlight the pathoanatomy and multimodal imaging, especially OCT, associated with HFL disruption that is present in various macular diseases. After describing the current knowledge of the embryology, anatomy, and physiology of the HFL, we review the existing imaging modalities that allow in vivo visualization of the HFL in the healthy and diseased retina. Finally, we report the clinical and imaging findings of acute HFL alteration in various macular disorders, including degenerative, inflammatory, and vascular conditions. Also, we propose a novel and signature OCT biomarker indicative of acute photoreceptor disruption involving the HFL, termed the “angular sign of HFL hyperreflectivity” (ASHH) of macular disease, to unify the pathoanatomy common to these various macular disorders and to provide clarity regarding the underlying pathogenesis.     

The preoperative assessment of the corneal endothelium

The ability of the cornea to maintain its transparency after surgery depends primarily upon the integrity of the corneal endothelium and in corneal grafting this is important for both donor and recipient eyes. The corneal endothelium may be significantly affected in various conditions of the anterior segment, especially if these exist over a considerable period of time. These conditions include superficial keratopathy, long-term contact lens wear, cornea guttata and low-grade anterior uveitis, especially chronic cyclitis which may be almost imperceptible clinically. The suitability of such corneas for use as donor material as well as the response of the eyes to anterior segment surgery can be assessed by specular microscopy and this may become even more important in the future as long-term contact lens wearers become more numerous in the community.