Histopathology in a dissecting conjunctival filtering bleb

CASE REPORT: Four years after trabeculectomy, a patient developed unilateral tearing and presented with an ipsilateral conjunctival filtering bleb that had dissected into the cornea. The corneal portion of the bleb was excised for symptomatic relief. Histopathological examination disclosed dissection of the conjunctival filtering bleb into the cornea between Bowman's layer and the corneal epithelium. The internal portion of the bleb consisted of loose stromal tissue lacking any internal epithelial lining. COMMENTS: A conjunctival bleb dissecting into the cornea is a well-described late complication of trabeculectomy; however, its pathophysiology remains controversial. The subepithelial dissection plane in this specimen supports the concept that the conjunctival filtering bleb may dissect into, as well as "overhang", the limbal cornea.     

In vivo confocal microscopy of the inflamed anterior segment: A review of clinical and research applications

In vivo confocal microscopy (IVCM) allows non‐invasive imaging of the living human cornea, specifically enabling the detection of immune cells in the healthy and diseased ocular anterior segment. Studies using IVCM have provided insight into the effects of contact lens wear on corneal Langerhans cell density and morphology, and the effects of eye drops on conjunctiva‐associated lymphoid tissue. IVCM has also been shown to be a useful adjunctive diagnostic tool in distinguishing infective and non‐infective uveitis and in diagnosing atypical infective keratitis. In the research setting, this technology has enhanced our understanding of the role of inflammatory cells in corneal neuropathy and angiogenesis. In vivo‐ex vivo correlation using animal models has helped overcome some of the difficulties in identifying cell type on IVCM images. As highlighted in this review, currently there are multiple established, and emerging, clinical and research applications for IVCM in the inflamed anterior segment.     

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.     

Suppression of avascular bleb formation by a thin biodegradable film in a rabbit filtration surgery with mitomycin C

Background

Avascularity of the bleb is regarded as a risk of bleb infection, which is the most serious complication after filtration surgery with mitomycin C (MMC). There is no perfect way to prevent avascular bleb formation. We hypothesized that keeping the conjunctiva away from direct exposure to aqueous filtration may suppress avascular bleb formation after filtration surgery with MMC. In order to prove our hypothesis, we investigated whether a thin biodegradable, honeycomb-patterned film (HPF) can reduce bleb avascularity in a rabbit model of filtration surgery with mitomycin C (MMC).

Methods

A fornix-based full-thickness filtration surgery was performed in one eye of each of five rabbits (control group). The same surgery with intraoperative MMC treatment was done in both eyes of six rabbits, with one eye receiving a 14-μm-thick HPF made from poly(L-lactide-co-ε-caprolactone), which was placed subconjunctivally over the filtration site with the honeycomb surface facing up. Intraocular pressure (IOP) measurements, bleb evaluations using ultrasound biomicroscopy (UBM), and in vivo confocal microscopy (IVCM) were performed periodically for 4?weeks postoperatively followed by histological examination.

Results

The postoperative IOP decrease and bleb survival were significantly greater in MMC-treated eyes than in control eyes, and were similar between MMC-only and MMC + HPF eyes. The avascular area in the bleb persisted for 4?weeks in MMC-only eyes. Postoperative IVCM showed morphological changes of the conjunctival epithelial cells (i.e., enlarged and variable in size and shape) and epithelial defects in MMC-only eyes, and significantly lower density of connective tissue and vascularity in the subepithelial space in MMC-only eyes compared to the control eyes. These IVCM findings agreed with those by UBM and histology. Bleb avascularity shown by clinical bleb appearance, IVCM and histology, conjunctival epithelial damage shown by IVCM and histology, and loose subepithelial connective tissue shown by UBM, IVCM, and histology were all reduced in MMC + HPF eyes compared to MMC-only eyes.

Conclusions

IVCM successfully showed the characteristic conjunctival damage in MMC-treated blebs. The concomitant use of a thin biodegradable HPF reduced avascularity and conjunctival damage in blebs, without compromising filtration in rabbits following filtration surgery with MMC.     

Confocal Microscopy of Corneal Dystrophies

In vivo confocal microscopy (IVCM) of the cornea is becoming an indispensable tool in the cellular study of corneal physiology and disease. This technique offers non-invasive imaging of the living cornea with images comparable to that of ex vivo histology. The ability to provide high-resolution images of all layers in the living cornea has resulted in new discoveries of corneal pathology at the cellular level. The IVCM analysis of corneal dystrophies is of importance to clinicians, as current methods of diagnosis involve slit-lamp characteristics, genetic analysis, and invasive biopsy. IVCM is helpful in evaluating the morphological characteristics of corneal dystrophies at the histological level and may be helpful in diagnosis, determination of progression, and understanding the pathophysiology of disease. The purpose of this review is to describe the principles, applications, and clinical correlation of IVCM in the study of corneal dystrophies.     

共焦显微镜——开启干眼诊疗的细胞水平时代

活体共焦显微镜（IVCM）能活体无创观察角膜上皮、基质细胞、免疫炎症细胞、神经,以及结膜、睫毛毛囊、睑板腺等组织结构,并进行客观量化分析,从而帮助从细胞水平对干眼进行早期诊断、细化分型、明确病因、治疗指导、预后判断等,为干眼诊疗开启了一个全新的时代。     

Do injections of 5-fluorouracil after trabeculectomy have toxic effects on the anterior segment?

OBJECTIVE: To discourage fibrosis of the filtering bleb, 5 fluorouracil (5-FU) may be injected after trabeculectomy. 5-FU is an antimetabolite that also can damage extraocular tissues at concentrations as low as 0.5%. This study ascertained whether repeated injection of 5-FU has toxic effects on intraocular structures. METHODS: After unilateral trabeculectomy in anesthetized New Zealand rabbits, 5-FU (5.0 mg/0.1 mL) was injected at the trabeculectomy site every 5 days for 15 days. Evaluation included slit-lamp examination, confocal microscopy, and intraocular pressure (IOP). After sacrifice, aqueous humor (AH) was drawn and eyes excised for scanning electron microscopy (SEM) and light microscopy. RESULTS: The 5-FU injection not decrease IOP beyond trabeculectomy alone. Bleb height remained constant, thickness increased, and vascularity decreased. No changes in cornea or anterior segment were observed. No inflammation was observed in the bleb or surrounding tissues by slit-lamp or histologic examination. Protein in AH increased from 0.6 +/- 0.5 microg/mL at baseline to 19.8 +/- 4.4 microg/mL after trabeculectomy but only to 0.9 +/- 0.6 microg/mL after trabeculectomy plus 5-FU. Both in vivo confocal microscopy and SEM revealed deleterious effects on corneal epithelial and endothelial cells with a minor shift toward smaller cells. CONCLUSIONS: In this study 5-FU did not provoke an intraocular inflammatory response and had minimal effect on extraocular structures. Changes in corneal epithelium and endothelium detectable by confocal microscopy suggest a small toxic effect. These in vivo measurements by confocal microscopy were confirmed by SEM. Repeated administration did not cause additional cumulative toxic effects in the anterior segment. Therefore, multiple injections of 5- FU into the filtering bleb pose minimal risk to intraocular structures.     

Dystrophia Helsinglandica – corneal morphology,topography and sensitivity in a hereditary corneal disease with recurrent erosive episodes

Purpose: The aim of this study was to describe the morphology, corneal topography and sensitivity in individuals with Dystrophia Helsinglandica. This autosomal dominant corneal disease is characterized by recurrent corneal erosive episodes and progressive subepithelial fibrosis not significantly affecting visual acuity. Methods: The corneas of nine affected and nine unaffected individuals were examined using slit‐lamp biomicroscopy, in vivo confocal microscopy (IVCM) and videokeratography. Corneal mechanical sensitivity was also measured using a non‐contact esthesiometer. Results: Slit‐lamp biomicroscopy revealed that the affected individuals represented different stages of corneal changes, from a nearly normal cornea to subepithelial fibrosis of the central cornea. Corneal changes in affected individuals did not significantly decrease the best spectacle‐corrected visual acuity. In vivo confocal microscopy detected morphological changes in the epithelium and stroma. Subepithelial opacity formation including altered keratocytes could be found in the anterior stroma in all affected eyes. With the exception of two eyes (one affected and one unaffected), all videokeratographies showed irregular astigmatism. Corneal sensitivity was significantly lower in affected individuals (p = 0.01). Age and corneal sensitivity showed no correlation. Conclusion: The main morphological findings in affected individuals were discrete and progressive subepithelial fibrosis, in the in vivo confocal microscope corresponding to optically dense extracellular matrix and activated keratocytes. Subbasal nerve morphology was changed in the affected family members who also showed a decreased corneal sensitivity. The findings are per se not specific to the disease. The changes probably reflect a healing response to erosive events on the corneal surface influenced by the genotype.     

后部多形性角膜营养不良49例共聚焦显微镜影像学观察

目的:探讨49例后部多形性角膜营养不良(PPCD)患者在共聚焦显微镜(IVCM)下的影像学特征。方法:回顾性病例研究。收集我院2013-01/2021-01诊断为PPCD患者49例86眼,包括男32例,女17例,平均年龄42.5±22.9岁。所有患者均进行IVCM检查,分析角膜内皮细胞密度及不同类型病变的镜下特点。结果:所有患者病灶区内皮细胞数量均较外周细胞数量低。IVCM下病变呈1型囊泡型的有44眼(51%),表现为角膜内皮层的圆形或不规则弹坑样病灶,单个或多个出现;2型条带型16眼(19%),表现为边缘弧形凸起,部分可见似赘疣样影像散在或条索状分布;3型弥漫型26眼(30%),表现为内皮细胞大面积缺失,大部分区域内皮成像不清,呈匍匐样、条索样延展的凸起,成像较清晰处部分同条带型病灶。观察病例中有2例患者随访4～5a, IVCM下病灶均较前有变化,包括中央角膜内皮数量的减少和上皮层铁质沉着等。结论:IVCM能够显示PPCD患者各期内皮细胞和Descemet膜水平的特征性显微结构改变,通过观察其影像学特点可以为疾病提供有效的诊断价值。     

Epithelial dendritic cell distribution in normal and inflamed human cornea: in vivo confocal microscopy study

PURPOSE: To evaluate dendritic cell (DCs) density, distribution, and morphology in central corneal and limbal epithelium in normal subjects and patients with immune-mediated corneal inflammation using in vivo confocal microscopy (IVCM). DESIGN: Comparative case-controlled, observational confocal microscopy study. METHODS: A total of 135 eyes of 135 patients were investigated. Group 1 (normal eyes) included 45 eyes of 45 healthy volunteers, group 2 photorefractive keratectomy (PRK-treated eyes) included 45 myopic eyes of 45 patients treated with PRK, and group 3 (inflamed eyes) comprised 45 eyes of 45 patients affected by immune-mediated corneal inflammation. The central cornea and limbus were examined for epithelial dendritic-shaped cells using laser scanning IVCM. DCs density was calculated using image analysis software. RESULTS: Cells with a branching dendritic morphology were visualized in the basal epithelial layer, basal lamina, and subbasal nerve plexus, in the central cornea, and in the basal layer and basal membrane of the limbal epithelium. The limbal epithelium demonstrated DCs in 93.3%, 89%, and 97.7% of eyes in group 1, 2, and 3, respectively (P = ns). Central epithelial DCs were observed in 20.0% and 13.3% of eyes in group 1 and 2 (P = ns), while in 93.3% of eyes in group 3 (P < .001). DCs were found to be significantly higher at the limbus compared with central cornea in each group (P < .001). Cell densities observed in group 3 were significantly greater than groups 1 and 2, at both locations (P < .05). CONCLUSIONS: Laser scanning IVCM is a useful method for evaluating epithelial DCs distribution at the limbus and central cornea in both healthy and diseased eyes.     

Distribution of Antigen Presenting Cells in the Human Cornea: Correlation of In Vivo Confocal Microscopy and Immunohistochemistry in Different Pathologic Entities

Purpose: The purpose of this study was to determine the quantity and distribution of antigen presenting cells (APC) in various inflammatory and non-inflammatory corneal diseases, comparing in vivo confocal microscopy (IVCM) and immunohistochemistry. Material and methods: Corneae of 41 eyes, composed of group 1 (status post herpes-keratitis), group 2 (keratoconus) and group 3 (graft rejection after keratoplasty) were investigated. IVCM was used preoperatively to assess the distribution and density of dendritic cells in the corneal center versus the paracentral area. Afterwards, all patients underwent penetrating keratoplasty. The host corneas were analyzed by immunohistochemistry for antigen presenting cell distribution, density and characterization by using specific markers for CD207/Langerin, CD209/DC-SIGN and HLA-DR. The IVCM findings were compared with immunohistochemistry results in the corneal epithelium. Results: Cells with branching dendritic morphology were visualized by IVCM mainly in the basal epithelial layer and subepithelial nerve plexus of the central and paracentral cornea. The density of APC in IVCM decreased in all groups towards the central part of the cornea. The highest gradient was observed in group 2, followed by groups 1 and 3. The corneal paracenter showed similiar distribution of APC in group 1 and 2 (76.7 cells/mm(2) and 74.4 cells/mm(2)). The highest density of central APC was observed in group 1 (53.76 cells/mm(2)), followed by group 3 (27.0 cells/mm(2)) and group 2 (24.2 cells/mm(2)). In immunohistochemistry positive stained, APC were distributed similarly to IVCM but with a higher density (p < 0.05). Conclusion: Distribution, density and stage of maturation of corneal epithelial APCs can be evaluated on morphological basis by IVCM. However, the corneal APCs density was about three-fold lower compared to immunohistochemistry findings.     

Self-limiting corneal edema with multiple parallel lines on the endothelium (SCEMPLE)

The purpose of this study was to report the natural course and in vivo confocal microscopy (IVCM) findings of five cases with unilateral self-limiting corneal edema and multiple parallel lines on the endothelium (SCEMPLE). This study is an observational case series. Five patients, who experienced a blurred vision due to SCEMPLE, were studied using slit-lamp examination and white-light IVCM (Confoscan 4; Nidek Technologies, Padova, Italy). IVCM of the linear deposits revealed characteristic hyperreflective material protruding between the endothelial cells. The lines also displayed spot-like holes and polygonal precipitates, which resembled dislodged endothelial cells. Because other signs of corneal inflammation, such as stromal infiltration and ciliary injection, were lacking, the condition was left untreated. After 1 day, slit-lamp examination and IVCM revealed only a small residual of the lines. Spontaneous, complete resolution occurred in all five cases within 1 week, leaving a lower endothelial cell density of the affected eyes as only sequela. SCEMPLE requires no treatment but may result in endothelial cell loss. The self-limiting character and IVCM appearance dispute SCEMPLE being a form of endotheliitis and suggest a different etiology.     

共焦显微镜观察慢性角膜水肿患者角膜各层形态特点

目的：应用活体共焦显微镜(IVCM)观察慢性角膜水肿患者角膜各层形态特点。

方法：使用IVCM观察不同病因的慢性角膜水肿的患者21例21眼,并与5例拟行白内障手术患者的正常角膜进行对照。

结果：IVCM观察到所有慢性角膜水肿患者角膜上皮层均可见大泡,表现为黑色、圆形、边缘清晰。18眼(86%)上皮细胞出现高反射的无细胞结构的片状区域和瘢痕。12眼(57%)患者中央区角膜上皮下未发现神经纤维,9眼(43%)患者中央区角膜上皮下神经平均密度显著降低。所有患者Bowman膜(BZ)表现为明显的异常,除了瘢痕外,BZ呈分支的、细的、黑线状。13眼(62%)患者前基质表现为细颗粒或粗颗粒,且反光不同。所有患者角膜基质细胞密度降低。所有患者角膜内皮表现为正常六边形结构消失,细胞边界不清。对照组角膜正常未见上述改变。

结论：IVCM可以用来观察慢性角膜水肿角膜各层微观结构上的变化,包括上皮瘢痕形成、上皮下神经纤维及基质细胞的减少。随着角膜内皮移植术的日益普及,本研究支持IVCM在定量评估术前、术后角膜水肿的作用。     

Conjunctival covering (Harms) (author's transl)]

The authors report on 7 cases of conjunctival flap cover in the technique of Harms and Mackensen (1963) in cases of leackage of the bleb after filtering operations. The technique consists in excision of the thin conjunctiva of the bleb, undermining of the conjunctiva until the fold, arched incision of the cornea in front of the bleb, incarceration of the conjunctival lip in the corneal incision with fixation by continuous suture with monophile nylon-string (10X0). In 6 of 7 cases the intraocular pressure was spontaneously regulated after the operation with morphological functioning bleb in 4 cases.     

Cryotherapy to close a corneal subepithelial aqueous track after trabeculectomy

After filtering surgery for primary open-angle glaucoma in a 64-year-old man, a persistent wound leak was encountered from the fornix-based conjunctival flap. Cyanoacrylate adhesive was applied but was unsuccessful in sealing the leak. The limbal margin was sutured with 10/0 nylon. One week later a corneal subepithelial fistulous track developed from the edge of the bleb and extended across the cornea towards the inferonasal limbus. The inferior end of the track leaked aqueous. Further surgery was performed to refashion the conjunctival bleb, ablate the fistula at its origin and re-form the anterior chamber. The track recurred 16 days later. The upper cornea was then treated with cryotherapy, which successfully closed the track with maintenance of a functional bleb (ocular pressure 6 mmHg). A faint asymptomatic subepithelial scar persisted.     

The conjunctiva in corneal epithelial wound healing

BACKGROUND/AIMS—During the healing of corneal epithelial wounds with limbal involvement, conjunctival epithelium often migrates across the denuded limbus to cover the corneal surface. It is believed that, over a period of time, conjunctival epithelium covering the cornea assumes characteristics of corneal epithelium by a process referred to as conjunctival transdifferentiation. The purpose of this study was to examine, clinically, the fate of conjunctival epithelial cells covering the cornea and to assess the healing of corneal epithelial wounds when the conjunctival epithelium was removed or actively prevented from crossing the limbus and extending onto the cornea.
METHODS—10 patients with conjunctivalisation of the cornea were followed for an average of 7.5 months. Five patients in this group had their conjunctival epithelium removed from the corneal surface and allowed to heal from the remaining intact corneal epithelium. In another four patients with corneal epithelial defects, the conjunctival epithelium was actively prevented from crossing the limbus by mechanically scraping it off.
RESULTS—The area of cornea covered by conjunctival epithelium appeared thin, irregular, attracted new vessels and was prone to recurrent erosions. Conjunctivalisation of the visual axis affected vision. Removal of conjunctival epithelium from the cornea allowed cells of corneal epithelial phenotype to cover the denuded area with alleviation of symptoms and improvement of vision. It was also established that migration of conjunctival epithelium onto corneal surface could be anticipated by close monitoring of the healing of corneal epithelial wounds, and prevented by scraping off conjunctival epithelium before it reached the limbus.
CONCLUSION—This study shows that there is little clinical evidence to support the concept that conjunctival transdifferentiation per se, occurs in humans. "Replacement" of conjunctival epithelium by corneal epithelial cells may be an important mechanism by which conjunctival "transdifferentiation" may occur. In patients with partial stem cell deficiency this approach can be a useful and effective alternative to partial limbal transplantation, as is currently practised.

Keywords: corneal epithelium; conjunctiva; stem cells; transdifferentiation     

In Vivo Confocal Microscopy in Dry Eye Disease and Related Conditions

A new era of ocular imaging has recently begun with the advent of in vivo confocal microscopy (IVCM), shedding more light on the pathophysiology, diagnosis, and potential treatment strategies for dry eye disease. IVCM is a noninvasive and powerful tool that allows detection of changes in ocular surface epithelium, immune and inflammatory cells, corneal nerves, keratocytes, and meibomian gland structures on a cellular level. Ocular surface structures in dry eye-related conditions have been assessed and alterations have been quantified using IVCM. IVCM may aid in the assessment of dry eye disease prognosis and treatment, as well as lead to improved understanding of the pathophysiological mechanisms in this complex disease. Further, due to visualization of subclinical findings, IVCM may allow detection of disease at much earlier stages and allow stratification of patients for clinical trials. Finally, by providing an objective methodology to monitor treatment efficacy, image-guided therapy may allow the possibility of tailoring treatment based on cellular changes, rather than on clinical changes alone.     

Effects of gentamicin on healing of transdifferentiating conjunctival epithelium in rabbit eyes

We examined the effects of commercially prepared gentamicin, a wide-spectrum topical antibiotic, on the healing of epithelial defects of the rabbit cornea. Abrasions were created by: (1) removing the corneal epithelium and 3 mm of the conjunctival epithelium (Group 1); and (2) producing the same initial trauma and subsequently removing the central 8 mm of epithelium 28 days after initial healing (Group 2). The complete healing of the large corneal and conjunctival epithelial defects was not delayed when gentamicin solution was used four times a day (Group 1). When the healed epithelium was reinjured while transdifferentiating from conjunctival to corneal epithelium (day 28, Group 2), treatment with the gentamicin solution and its vehicle, both containing benzalkonium chloride, delayed epithelial healing significantly compared with treatment with saline (P less than .01).     

Actin filament localization in developing and pathologic human corneas

Actin is associated with motility, cell morphology, and cell-substrate adhesion. The molecular probe NBD phallacidin, which reacts with filamentous actin, was used to study the distribution of actin filaments in the corneal and conjunctival epithelium, stroma, and endothelium. Frozen sections of human fetal eyes from 8 weeks to 40 weeks of gestation were reacted with NBD phallacidin. Pathologic tissues included keratoplasty specimens from patients with hereditary posterior polymorphous corneal dystrophy (PPMD) and surgically excised tissues removed for treatment of epithelial down-growth. Normal human cornea was used as a control. Immunofluorescent staining disclosed actin filament distribution in corneal epithelium as early as 9-10 weeks of gestation. Staining increased with maturation until term. Adult human corneal epithelium showed more pronounced staining of the surface layers. Stromal staining was more extensive in earlier stages of gestation and decreased in later stages of gestation, after 20-21 weeks. In pathologic corneas with posterior polymorphous dystrophy, there was localization of actin, as well as keratin, in the abnormal epithelial-like layers lining the posterior cornea. In epithelial downgrowth, actin and keratin were demonstrated in multilayered squamous epithelium on the anterior iris surface. Actin appears to be involved in migration of corneal epithelial and endothelial cells.