Architectural and biochemical expressions of mustard gas keratopathy: preclinical indicators and pathogenic mechanisms

A subset of victims of ocular sulfur mustard (SM) exposure develops an irreversible, idiotypic keratitis with associated secondary pathologies, collectively referred to as mustard gas keratopathy (MGK). MGK involves a progressive corneal degeneration resulting in chronic ocular discomfort and impaired vision for which clinical interventions have typically had poor outcomes. Using a rabbit corneal vapor exposure model, we previously demonstrated a clinical progression with acute and chronic sequelae similar to that observed in human casualties. However, a better understanding of the temporal changes that occur during the biphasic SM injury is crucial to mechanistic understanding and therapeutic development. Here we evaluate the histopathologic, biochemical and ultrastructural expressions of pathogenesis of the chronic SM injury over eight weeks. We confirm that MGK onset exhibits a biphasic trajectory involving corneal surface regeneration over the first two weeks, followed by the rapid development and progressive degeneration of corneal structure. Preclinical markers of corneal dysfunction were identified, including destabilization of the basal corneal epithelium, basement membrane zone abnormalities and stromal deformation. Clinical sequelae of MGK appeared abruptly three weeks after exposure, and included profound anterior edema, recurring corneal erosions, basement membrane disorganization, basal cell necrosis and stromal degeneration. Unlike resolved corneas, MGK corneas exhibited frustrated corneal wound repair, with significantly elevated histopathology scores. Increased lacrimation, disruption of the basement membrane and accumulation of pro-inflammatory mediators in the aqueous humor provide several mechanisms for corneal degeneration. These data suggest that the chronic injury is fundamentally distinct from the acute lesion, involving injury mechanisms that operate on different time scales and in different corneal tissues. Corneal edema appears to be the principal pathology of MGK, in part resulting from persistent necrosis of the basal corneal epithelium and deterioration of the basement membrane. The findings also provide a potential explanation as to why administration of anti-inflammatories transiently delays, but does not prevent, the development of MGK sequelae.     

Stimulation of ion transport by ascorbic acid through inhibition of 3′:5′-cyclic-AMP phosphodiesterase in the corneal epithelium and other tissues

Ascorbic acid stimulates active transport of Cl^? by the isolated intact corneas. The effect is not present in corneas previously stimulated by theophylline, an inhibitor of 3′: 5′-cyclic-AMP phosphodiesterase (EC 3.1.4.17), and vice versa, theophylline has no action after stimulation with ascorbic acid. This indicated inhibition of 3′: 5′-cyclic-AMP phosphodiesterase by ascorbic acid. Assay of phosphodiesterase using ³H-labeled cyclid AMP of frog and rabbit corneal epithelial homogenates showed an inhibitory effect of ascorbic acid. Concentration of 5 mM produced 16% inhibition with 20 mM producing 46 %. This compares with 58 % inhibition by theophylline at 5 mM. Phosphodiesterase activity is mostly soluble in frog corneal epithelium but in rabbit 45 % is particulate. Soluble and particulate fractions are inhibited by ascorbate, but in rabbits greater inhibition (50 %) was observed in the particulate fraction than in the soluble fraction. Other tissues showed inhibition also: frog retina 12 %, rat brain (caudate nucleus) 48 %, rabbit brain 14 %, rabbit liver 16 %. It is concluded that ascorbate produces an increase in cyclic AMP content of corneal epithelium and other tissues by inhibition of 3′: 5′-cyclic-AMP phosphodiesterase. This action may be one of the main functions of the high ascorbic acid content of ocular tissues and explain some of the effects of high dosis of ascorbate in other systems.     

Quantitative changes and ultrastructural alterations of the cornea in response to ultraviolet light II. Effects on amphibia; elucidation of desmosomal structure and basement membrane synthesis

The cornea of the urodele amphibian Triturus c. cristatus was studied ultrastructurally in order to provide the basis for a comparison among corneas throughout the vertebrate phylum. The cornea of this salamander consists of relatively thick epithelium and basement membrane and thin Descemet's membrane, unlike the mammalian corneas. The outermost epithelial cells contain Ruthenium Red stainable extracellular filaments and intracellular vesicles which are thought to play a role in the process of lubricating the corneal surface. Occluding junctions have been observed in the apical region of the superficial epithelial cells and are considered as barriers to the intercellular passage of material. A thin substantia propria (stroma) consists of about 40 collagenous highly organized lamellae. The thicknesses of the basement membrane, Descemet's membrane and the epithelium are believed to represent the primitive situation in the process of corneal evolution.     

Stimulation of ion transport by ascorbic acid through inhibition of 3':5'-cyclic-AMP phosphodiesterase in the corneal epithelium and other tissues.

Ascorbic acid stimulates active transport of Cl-minus by the isolated intact cornea. The effect is not present in corneas previously stimulated by the theophylline, an inhibitor of 3':5"-cyclic-AMP phosphodiesterase (EC 3.1.4.17), and vice versa, theophylline has no action after stimulation with ascorbic acid. This indicated inhibition of 3':5'-cyclic-AMP phosphodiesterase by ascorbic acid. Assay of phosphodiesterase using 3-H-labeled cyclid AMP of frog and rabbit corneal epithelial homogenates showed an inhibitory effect of ascorbic acid. Concentration of 5 mM produced 16% inhibition with 20 mM producing 46%. This compares with 58% inhibition by theophylline at 5 mM. Phosphodiesterase activity is mostly soluble in frog corneal epithelium but in rabbit 45% is particulate. Soluble and particulate fractions are inhibited by ascorbate, but in rabbits greater inhibition (50%) was observed in the particulate fraction than in the soluble fraction. Other tissues showed inhibition also: frog retina 12%, rat brain (caudate nucleus) 48%, rabbit brain 14%, rabbit liver 16%. It is concluded that ascorbate produces an increase in cyclic AMP content of corneal epithelium and other tissues by inhibition of 3':5'-cyclic-AMP phosphodiesterase. This action may be one of the main functions of the high ascorbic acid content of ocular tissues and explain some of the effects of high dosis of ascorbate in other systems.     

Cell adhesion molecules in stromal corneal dystrophies

The aim of the present study was to investigate the expression pattern of different cell adhesion molecules in corneal stromal dystrophies. Fifteen corneal buttons from patients diagnosed with three different types of stromal corneal dystrophies and healthy corneas were investigated. Paraffin embedded sections were stained immunohistochemically with monoclonal antibodies against human intercellular adhesion molecule-1 (ICAM-1), endothelial selectin (E-selectin) and endothelial cadherin (E-cadherin) using the avidin-biotin-peroxidase-complex technique. The sections were compared to normal eye bank controls. In corneas from granular dystrophy patients ICAM-1 was expressed focally in epithelial cells and in keratocytes, and expressed diffusely in endothelial cells. In corneas from macular dystrophy patients diffuse epithelial staining was observed and the stromal and endothelial expression was found to be similar to that of granular dystrophy. In lattice dystrophy, only the epithelial cells and endothelium were intensively positive for ICAM-1. E-selectin was not present on any layer of the corneal specimens. E-cadherin was observed only in the epithelium of all three types of corneal dystrophies. Normal corneas did not express any of the investigated adhesion molecules. We found different expression patterns of adhesion molecules in corneas from stromal dystrophies. Our results suggest that adhesion molecules may be involved in the pathogenesis of corneal stromal dystrophies.     

Stromal assemblies containing collagen types IV and VI and fibronectin in the developing embryonic avian cornea

The morphogenesis of type IV collagen-containing structures in the stromal matrix of the developing avian cornea was investigated using immunofluorescence and immunoelectron microscopic histochemistry. Two forms of type IV collagen-containing structures were seen; these differed in their probable origin, structure, molecular composition, and developmental fate. The major form of stromal type IV collagen-containing material, termed "strings," was observed only after swelling of the primary stroma and the onset of mesenchymal invasion. These strings are presumed to be products of the stromal cells. In immunofluorescence histochemistry they appeared as linear segments of type IV collagen-specific immunoreactivity. In immunoelectron microscopy, they appeared initially as electron-dense sausages of variable length and orientation. They frequently were associated with cell surfaces and, in fortuitous sections, appeared to connect adjacent cells. The strings also contained type VI collagen and fibronectin, but very little, if any, of the basement membrane components laminin and heparin sulfate proteoglycan (HSPG). As the stroma continued to expand in thickness, more of these structures were observed in a radial orientation, becoming quite long and less tortuous. Later in development, as stromal condensation proceeded, they disappeared. We suggest that the strings function to stabilize the stromal matrix, and perhaps to limit the rate and/or extent of stromal expansion, during a phase of rapid swelling and matrix deposition. The other form of type IV collagen-containing stromal material appeared as irregularly shaped plaques of basement membrane-like material identical to those previously described in mature corneas. These are likely derived from the corneal endothelial cells. They contained other basement membrane-associated components (laminin, HSPG) and fibronectin, but not type VI collagen. This material persists in mature corneas as sparse irregular stromal plaques and as matrix in the interface between Descemet's membrane and the corneal stroma.     

The Graft of Autologous Adipose-Derived Stem Cells in the Corneal Stromal after Mechanic Damage

This study was designed to explore the feasibility of using autologous rabbit adipose derived stem cells (rASCs) as seed cells and polylactic-co-glycolic acid (PLGA) as a scaffold for repairing corneal stromal defects. rASCs isolated from rabbit nape adipose tissue were expanded and seeded on a PLGA scaffold to fabricate cell-scaffold constructs. After 1 week of cultivation in vitro, the cell-scaffold complexes were transplanted into corneal stromal defects in rabbits. In vivo, the autologous rASCs-PLGA constructed corneal stroma gradually became transparent without corneal neovascularization after 12 weeks. Hematoxylin and eosin staining and transmission electron microscopy examination revealed that their histological structure and collagen fibril distribution at 24 weeks after implantation were similar to native counterparts. As to the defect treated with PLGA alone, the stromal defects remained. And scar tissue was observed in the untreated-group. The implanted autologous ASCs survived up to 24 weeks post-transplantation and differentiated into functional keratocytes, as assessed by the expression of aldehyde-3-dehydrogenase1A1 (ALDH1A1) and cornea-specific proteoglycan keratocan. Our results revealed that autologous rASCs could be one of the cell sources for corneal stromal restoration in diseased corneas or for tissue engineering of a corneal equivalent.     

Monoclonal antibody analysis of ocular basement membranes during development

As a first step in a study of the role(s) of basement membranes in ocular morphogenesis, we have produced a variety of monoclonal antibodies against native lens capsule from adult chicks, and have used these reagents to stain histological sections of ocular tissues from 4 1/2- to 18-day-old chicken embryos. Four different patterns of immunofluorescence were observed in sections of corneas of 18-day-old chicken embryos stained with these antibodies. The antibodies in group 1 stained the basement membranes of both the corneal epithelium and the endothelium (as well as Descemet's membrane). Those in groups 2 and 3 stained only the epithelial or endothelial basement membranes, respectively. The group 4 antibody stained the corneal stroma as well as Bowman's membrane and Descemet's membrane. The antibodies in group 1 could be further subdivided into groups 1a and 1b on the basis of temporal differences in the onset of staining in corneas from 4 1/2- to 7-day-old embryos. Thus, this series of monoclonal antibodies appears to recognize at least five different antigenic determinants. When these antibodies were used to stain sections of eyes at different stages of development, we found that the characteristic differential staining of some basement membranes was maintained throughout development, while the staining properties of others changed. This indicates that many of the ocular basement membranes may differ from one another in composition or conformation, and that at least some of them may undergo developmental changes. We also noticed a similarity in the pattern of fluorescence associated with the basement membranes of the limbal blood vessels and the corneal endothelium that is consistent with the hypothesis that the corneal endothelium is derived from the early periocular vascular endothelium. Our observations of developing corneas also revealed that the antigen recognized by the group 4 antibody may be produced by both the corneal epithelium and the stromal fibroblasts. The suitability of monoclonal antibodies for probing basement membrane heterogeneity is discussed.     

SEM localization of laminin on the basement membrane of the chick corneal epithelium with immunolatex microspheres

Embryonic chick corneal explants were soaked in mild detergent and the anterior corneal epithelium was peeled from its basement membrane, leaving the lamina lucida surface exposed and supported on the subjacent primary stroma. Explants were treated with rabbit anti-laminin IgG, followed by sheep anti-rabbit IgG linked microspheres, and processed for SEM. The lucida surface was heavily decorated with microspheres, whereas controls treated with preimmune rabbit IgG were essentially beadless. Laminin distribution was not regular, appearing denser in some regions than others. However, the connective tissue surface of the basement membrane was never laminin-positive, even after treatment with hyaluronidase. These results suggest the basal lamina of the corneal epithelium is asymmetric, with preferential location of laminin to the lucida surface of the basement membrane.     

Quantitative changes and ultrastructural alterations of the cornea in response to ultraviolet light. II. Effects of amphibia; elucidation of desmosomal structure and basement membrane synthesis.

The effects of far ultraviolet light irradiation upon an amphibian cornea were studied to compare the effects observed both quantitatively and ultrastructurally with data obtained after UV irradiation of mammalian corneas. The ultimate goal of this series of investigations is the elucidation of the alterations and the regeneration mechanisms, which might reflect existing morphological diversities among the species, observed in vertebrate corneas following exposure to UV light. It was found that while the epithelial cells undergo oedema after low dose exposures and are gradually damaged after high doses of UV light, 2-4 days leter a new epithelium has been formed. Intercellular permeability is increased by low dose exposure as was detected by the penetration of Ruthenium Red into the intercellular clefts. Under these conditions desmosomal structure revealed a 21-laminar configuration. The basement membrane of the amphibian, unlike that of the mammal, does not dissolve away upon exposure but shows localized disruptions which are thought to accommodate the passage of leucocytes from stroma to epithelium. That a new basement membrane is subsequently formed is evident by the existence of extracellular and intracellular secretion granules. In comparison to irradiated rabbit corneas, this stroma remains remarkably at the same thickness following a high dose exposure although a noticeable disorganization of collagen arrangement is apparent. Finally, as in the case of the rabbit corneas, a secondary degeneration of endothelium was observed 4 days after a moderate dose exposure.     

Histochemistry of some proteases in the normal rabbit,pig and ox corneas

Summary The distribution of activities of membrane aminopeptides (aminopeptidases M (APM), aminopeptidase A (APA), dipeptidyl peptidase IV (DPP IV), -gluamyltransferase (GGT) and lysosomal exopeptidases (dipeptidyl peptidase I (DPP I), dipeptidyl peptidase II (DPP II) was investigated in rabbit, ox and pig corneas. Cryostat sections of snap-frozen corneas treated with chloroform-acetone (4°C) were used for the demonstration of membrane-bound enzymes and sections of corneas fixed in 4% paraformaldehyde (4°C) for the demonstration of lysosomal enzymes.In activities of proteases species differences were found. The rabbit cornea was most active, followed by ox and pig corneas. Individual corneal layers reacted differently. Of membrane proteases a high APM activity was found in keratocytes, whereas epithelium and endothelium were negative. On the other hand APA and GGT were active in the epithelium and endothelium. Their activities in keratocytes were less pronounced. DPP IV activity was demonstrated in some keratocytes beneath the epithelium only. Lysosomal enzymes DPP I and DPP II were active in all corneal layers. The epithelium displayed the highest activity.Differences in activities in the centro-peripheral and epithelio-endothelial directions were found. DPP I, DPP II, and APM were most active in the limbal region in all corneal layers.     

Reactive oxygen species (ROS)-generating oxidases in the normal rabbit cornea and their involvement in the corneal damage evoked by UVB rays

The corneas of albino rabbits were irradiated (5 min exposure once a day) with UVB rays (312 nm) for 4 days (shorter procedure) or 8 days (longer procedure). The eyes were examined microbiologically and only the corneas of sterile eyes or eyes with non-pathogenic microbes were employed. Histochemically, the activities of reactive oxygen species (ROS)-generating oxidases (xanthine oxidase, D-amino acid oxidase and alpha-hydroxy acid oxidase) were examined in cryostat sections of the whole corneas. Biochemically, the activity of xanthine oxidoreductase/xanthine oxidase was investigated in the scraped corneal epithelium. UVB rays significantly changed enzyme activities in the corneas. In comparison to the normal cornea, where of ROS-generating oxidases only xanthine oxidase showed significant activity in the corneal epithelium and endothelium, D-amino acid oxidase was very low and alpha-hydroxy acid oxidase could not be detected at all, in the cornea repeatedly irradiated with UVB rays, increased activities of xanthine oxidase and D-amino acid oxidase were observed in all corneal layers. Only after the longer procedure the xanthine oxidase and D-amino acid oxidase activities were decreased in the thinned epithelium in parallel with its morphological disturbances. Further results show that the xanthine oxidase/xanthine oxidoreductase ratio increased in the epithelium together with the repeated irradiation with UVB rays. This might suggest that xanthine dehydrogenase is converted to xanthine oxidase. However, in comparison to the normal corneal epithelium, the total amount of xanthine oxidoredutase was decreased in the irradiated epithelium. It is presumed that xanthine oxidoreductase might be released extracellularly (into tears) or the enzyme molecules were denatured due to UVB rays (particulary after the longer procedure). Comparative histochemical and biochemical findings suggest that reactive oxygen species-generating oxidases (xanthine oxidase, D-amino acid oxidase) contribute to the corneal damage evoked by UVB rays.     

Lanthanum-induced damage to microvilli in cells of the posterior epithelium of the cornea and its trigger mechanism

Destructions in filament-membranous connections of microvilli of cells of the rabbit corneal posterior epithelium have been investigated after the effect of an ionized form of lanthanum. Rare-earth ions are used as a specific antagonist and as a substituent for calcium on the surface of biopolymeres. Twelve corneas from 6 rabbits of Shinshilla strain at the age of 2 months are used. Lanthanum ions are stated to have selective connections with the luminal part of the plasmolemma and with the membrane of the microvilli in the cells of the corneal posterior epithelium. The changes in the microvilli are accompanied with destruction of the filament-membranous connections producing protrusions and vesiculation of their plasmolemma, disaggregation of microfilaments adjoining the luminal part of the cellular plasmolemma. A suggestion is made on an impostant role of calcium and C++-binding biopolymeres in the mechanism regulating the filament-membranous connections.     

束状刺盘孢角膜炎的实验研究

目的初步研究束状刺盘孢感染兔角膜的临床表现和组织病理学特征。方法选择15只新西兰白兔,应用基质内直接注射法建立模型。肉眼观察角膜变化,并在接种后第5 d、14 d、21 d处死家兔,对角膜行HE染色和PAS染色,观察其组织病理学改变。结果兔束状刺盘孢角膜炎动物模型成功建立,肉眼可以见到典型的真菌性角膜炎的表现。组织病理学检查,在感染早期即可看到大量真菌病原体生长,菌丝垂直于角膜基质,后期出现新生血管和瘢痕。结论角膜基质内直接注射法复制真菌性角膜炎动物模型成功率高、稳定。兔束状刺盘孢角膜炎有典型的真菌性角膜炎的表现,组织学中菌丝垂直于角膜基质生长。     

Non‐invasive optical method for real‐time assessment of intracorneal riboflavin concentration and efficacy of corneal cross‐linking

Keratoconus is the primary cause of corneal transplantation in young adults worldwide. Riboflavin/UV‐A corneal cross‐linking may effectively halt the progression of keratoconus if an adequate amount of riboflavin enriches the corneal stroma and is photo‐oxidated by UV‐A light for generating additional cross‐linking bonds between stromal proteins and strengthening the biomechanics of the weakened cornea. Here we reported an UV‐A theranostic prototype device for performing corneal cross‐linking with the ability to assess corneal intrastromal concentration of riboflavin and to estimate treatment efficacy in real time. Seventeen human donor corneas were treated according to the conventional riboflavin/UV‐A corneal cross‐linking protocol. Ten of these tissues were probed with atomic force microscopy in order to correlate the intrastromal riboflavin concentration recorded during treatment with the increase in elastic modulus of the anterior corneal stroma. The intrastromal riboflavin concentration and its consumption during UV‐A irradiation of the cornea were highly significantly correlated (R = 0.79; P = .03) with the treatment‐induced stromal stiffening effect. The present study showed an ophthalmic device that provided an innovative, non‐invasive, real‐time monitoring solution for estimating corneal cross‐linking treatment efficacy on a personalized basis.      

Corneal Absorption of a New Riboflavin-Nanostructured System for Transepithelial Collagen Cross-Linking

Corneal collagen cross-linking (CXL) has been described as a promising therapy for keratoconus. According to standard CXL protocol, epithelium should be debrided before treatment to allow penetration of riboflavin into the corneal stroma. However, removal of the epithelium can increase procedure risks. In this study we aim to evaluate stromal penetration of a biocompatible riboflavin-based nanoemulsion system (riboflavin-5-phosphate and riboflavin-base) in rabbit corneas with intact epithelium. Two riboflavin nanoemulsions were developed. Transmittance and absorption coefficient were measured on corneas with intact epithelia after 30, 60, 120, 180, and 240 minutes following exposure to either the nanoemulsions or standard 0.1% or 1% riboflavin-dextran solutions. For the nanoemulsions, the epithelium was removed after measurements to assure that the riboflavin had passed through the hydrophobic epithelium and retained within the stroma. Results were compared to de-epithelialized corneas exposed to 0.1% riboflavin solution and to the same riboflavin nanoemulsions for 30 minutes (standard protocol). Mean transmittance and absorption measured in epithelialized corneas receiving the standard 0.1% riboflavin solution did not reach the levels found on the debrided corneas using the standard technique. Neither increasing the time of exposure nor the concentration of the riboflavin solution from 0.1% to 1% improved riboflavin penetration through the epithelium. When using riboflavin-5-phosphate nanoemulsion for 240 minutes, we found no difference between the mean absorption coefficients to the standard cross-linking protocol (p = 0.54). Riboflavin nanoemulsion was able to penetrate the corneal epithelium, achieving, after 240 minutes, greater stromal concentration when compared to debrided corneas with the standard protocol (p = 0.002). The riboflavin-5-phosphate nanoemulsion diffused better into the stroma than the riboflavin-base nanoemulsion.     

Regulation of corneal collagenase production: stimulation of serially passaged stromal cells by blood mononuclear cells.

After three or more passages, stromal cell outgrowths from explants of normal rabbit corneas and from corneas previously burned with alkali did not produce collagenase until they were stimulated by the addition of rabbit blood-derived mononuclear cells or media conditioned by them. Normal stromal cells required stimulation by lymphocytes or their products from alkali-burned animals, whereas those from alkali-burned corneas were stimulated by lymphocytes from either normal or alkali-burned rabbits.     

Spontaneous corneal opacities in laboratory mice

Corneal opacities were observed in numerous strains and stocks of laboratory mice (Mus musculus) from different microbiological environments. The opacities were characterized as acute and chronic inflammatory lesions of the corneal epithelium and anterior corneal stroma, including corneal ulcers and erosions, acute keratitis, stromal neovascularization and mineralization of the basement membrane zone. Some strains and stocks of mice from barrier-reared colonies had a high incidence of corneal opacities [DBA/2 (29.1%), C3H (16.2%), CF1 (16.2%) and BALB/c (10.0%)] while others had a lower incidence [CD-1 (4.3%) and C57BL/6 (4.1%)]. Axenic and gnotobiotic mice had a very low incidence of corneal opacities (1.6%). An experimental study demonstrated that twice weekly cage cleaning would reduce the incidence of corneal opacities to a very low level. A bacterial product, such as ammonia, is proposed as a significant factor in the pathogenesis of spontaneous corneal opacities in laboratory mice.     

Acanthamoeba castellanii: Morphological analysis of the interaction with human cornea

The present study demonstrates that when Acanthamoeba castellanii trophozoites are co-cultivated with isolated human corneas, the amoeba can be invasive and cause damage to the intact corneal epithelium without the requirement of previous corneal abrasion. After adhesion, A. castellanii trophozoites migrate between cells forming bumps on the corneal cell layers and reaching Bowman´s membrane in 3 h, although no evidence of cell damage was observed until the phagocytic process was detected. Likewise, conditioned medium produced damage to the corneal cells that was proportional to the time of incubation, but this cytophatic effect involved only the most superficial layer of the human cornea and was not enough to explain amoebic invasion of Bowman´s membrane. As a result of our observations, we suggest that the mechanical action of the trophozoites and phagocytosis of corneal cells during the process of corneal invasion are more important than previously suggested.