Effect of procyanidolic oligomers on corneal collagen of rabbits treated by excimer laser photoablation.

Procyanidolic oligomers (PCO) are mainly used for their therapeutic effect on the vascular wall. We could show that the mechanism of this effect involves interactions with mesenchymal cells and extracellular matrix (ECM). Recently we demonstrated in vitro that they also act on cornea, a tissue rich in ECM. For instance they stimulate the corneal biosynthesis of type VI collagen and proteoglycans. A potent antiprotease effect also could be demonstrated on corneas. In our present work we examined in vivo action of PCO on corneas. A group of rabbits received during 1 week on their right eye a treatment by procinaidolic oligomers, the left eyes were kept as controls. After 1 week both eyes underwent excimer laser photobalation. Another group of rabbits also received the PCO treatment on the right eye, not on the left, but was not treated by photoablation. One week after the surgical intervention corneas were collected and biochemical and morphological observations were carried out. Ocular administration of PCO was well tolerated and no toxic or inflammatory side-effects could be seen. In the control group photoablation was followed by a decrease of the content of corneas in type I collagen and a strong increase in type III collagen. On the corneas treated by PCO these alterations of the composition were not observed. These results indicate that PCO treatment before excimer laser photoablation maintains within normal limits the biochemical composition of the operated corneas.

Classical LCAT deficiency resulting from a novel homozygous dinucleotide deletion in exon 4 of the human lecithin: cholesterol acyltransferase gene causing a frameshift and stop codon at residue 144.

Lecithin: cholesterolacyltransferase (LCAT) transacylates the fatty acid at the sn-2 position of lecithin to the 3beta-OH group of cholesterol forming lysolecithin and the majority of cholesteryl ester found in plasma. LCAT participates in the reverse cholesterol transport pathway in man where it esterifies tissue-derived cholesterol following efflux from peripheral cells into HDL. Only 38 unique mutations in the human LCAT gene have been reported worldwide. Our French female proband presented with corneal opacity and no detectable plasma LCAT activity using either endogenous or exogenous assays. Her total plasma cholesterol and HDL cholesterol were low (2.34 mmol/l and 0.184 mmol/l, respectively) with a very high cholesterol/cholesteryl ester molar ratio (10.9:1). Plasma triglycerides were 0.470 mmol/l with low apo B (40.5 mg/dl), apo A-I (14.7 mg/dl), apo A-II (6.8 mg/dl) and apo E (2.1 mg/dl) levels. Plasma lipoprotein analysis by ultracentrifugation showed very low HDL concentrations and a characteristic shift of the lipoprotein profile towards larger, less dense particles. No proteinuria, renal dysfunction or signs of atherosclerosis were noted at age 45. Sequence analysis of her LCAT gene showed a novel homozygous TG-deletion at residues 138-139 that resulted in a frameshift causing the generation of a stop codon and premature termination of the LCAT protein at amino acid residue 144. Western blotting of the patient's plasma using a polyclonal IgY primary antibody against human LCAT failed to demonstrate the presence of a truncated LCAT protein. A 53 bp mismatched PCR primer was designed to generate an Fsp 1 restriction site in the wild type sequence of exon 4 where the mutation occurred. The 155 bp PCR product from the wild type allele produced a 103 bp and 52 bp fragment with Fsp 1 and no cleavage products with the mutant allele thus permitting rapid screening for this novel mutation.

Retinal TUNEL-positive cells and high glutamate levels in vitreous humor of mutant quail with a glaucoma-like disorder.

PURPOSE: To investigate whether retinal cell death observed in an avian glaucoma-like disorder occurs by apoptosis and whether an increase in excitotoxic amino acid concentration in the vitreous humor is associated temporally with cell death in the retina. METHODS: Presumptive retinal apoptotic nuclei were identified by histochemical detection of DNA fragmentation (by TdT-dUTP terminal nick-end labeling [TUNEL]), and vitreal concentrations of glutamate and several other amino acids were determined by high-pressure liquid chromatography with fluorometric detection in the al mutant quail (Coturnix coturnix japonica) in which a glaucoma-like disorder develops spontaneously. RESULTS: TUNEL-labeled nuclei were located mostly in the ganglion cell layer (GCL) in the retina of mutant quails 3 months after hatching. However, labeled nuclei were also observed in the inner and outer nuclear layers. At 7 months, most TUNEL-positive nuclei were detected in the inner nuclear layer, whereas labeled cells in the GCL were reduced in number. No TUNEL-labeled nuclei were detected in the retina of control quails at any age. Vitreal concentrations of glutamate and aspartate were significantly increased in 1-month-old mutant quails compared with control animals. Concentrations decreased at 3 months, and no significant differences were observed between strains at 7 months. CONCLUSIONS: Presumptive apoptotic cell death is detected from 3 months after hatching in mutant quails and is not restricted to retinal ganglion cells. Cell death appears just after a significant increase in excitotoxic amino acid concentrations in the vitreous humor, suggesting a correlation between both events.

Reduction of corneal edema in endotoxin-induced uveitis after application of L-NAME as nitric oxide synthase inhibitor in rats by iontophoresis.

PURPOSE: To investigate the involvement of the cornea during endotoxin-induced uveitis (EIU) in the rat and the effect of Ngamma-nitro-L-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor, administered by iontophoresis. METHODS: EIU was induced in Lewis rats that were killed at 8 and 16 hours after lipopolysaccharide (LPS) injection. The severity of uveitis was evaluated clinically at 16 hours, and nitrite levels were evaluated in the aqueous humor at 8 hours. Corneal thickness was measured, 16 hours after LPS injection, on histologic sections using an image analyzer. Transmission electron microscopy (TEM) was used for fine analysis of the cornea. Transcorneoscleral iontophoresis of L-NAME (100 mM) was performed either at LPS injection or at 1 and 2 hours after LPS injection. RESULTS: At 16 hours after LPS injection, mean corneal thickness was 153.7+/-5.58 microm in the group of rats injected with LPS (n=8) compared with 126.89+/-11.11 microm in the saline-injected rats (n=8) (P < 0.01). TEM showed stromal edema and signs of damage in the endothelial and epithelial layers. In the group of rats treated by three successive iontophoreses of L-NAME (n=8), corneal thickness was 125.24+/-10.36 microm compared with 146.76+/-7.52 microm in the group of rats treated with iontophoresis of saline (n=8), (P=0.015). TEM observation showed a reduction of stromal edema and a normal endothelium. Nitrite levels in the aqueous humor were significantly reduced at 8 hours by L-NAME treatment (P=0.03). No effect on corneal edema was observed after a single iontophoresis of L-NAME at LPS injection (P=0.19). Iontophoresis of saline by itself induced no change in corneal thickness nor in TEM structure analysis compared with normal rats. CONCLUSIONS: Corneal edema is observed during EIU. This edema is significantly reduced by three successive iontophoreses of L-NAME, which partially inhibited the inflammation. A role of nitric oxide in the corneal endothelium functions may explain the antiedematous effect of L-NAME.

EIU in the rat promotes the potential of syngeneic retinal cells injected into the vitreous cavity to induce PVR.

PURPOSE: To determine whether syngeneic retinal cells injected in the vitreous cavity of the rat are able to initiate a proliferative process and whether the ocular inflammation induced in rats by lipopolysaccharide (LPS) promotes this proliferative vitreoretinopathy (PVR). METHODS: Primary cultured differentiated retinal Müller glial (RMG) and retinal pigmented epithelial (RPE) cells isolated from 8 to 12 postnatal Lewis rats were injected into the vitreous cavity of 8- to 10-week-old Lewis rats (10(5) cells/eye in 2 microlieter sterile saline), with or without the systemic injection of 150 microgram LPS to cause endotoxin-induced uveitis (EIU). Control groups received an intravitreal injection of 2 microliter saline. At 5, 15, and 28 days after cell injections, PVR was clinically quantified, and immunohistochemistry for OX42, ED1, vimentin (VIM), glial fibrillary acidic protein (GFAP), and cytokeratin was performed. RESULTS: The injection of RMG cells, alone or in combination with RPE cells, induced the preretinal proliferation of a GFAP-positive tissue, that was enhanced by the systemic injection of LPS. Indeed, when EIU was induced at the time of RMG cell injection into the vitreous cavity, the proliferation led to retinal folds and localized tractional detachments. In contrast, PVR enhanced the infiltration of inflammatory cells in the anterior segment of the eye. CONCLUSIONS: In the rat, syngeneic retinal cells of glial origin induce PVR that is enhanced by the coinduction of EIU. In return, vitreoretinal glial proliferation enhanced the intensity and duration of EIU.

Corneal wound healing in monkeys after repeated excimer laser photorefractive keratectomy.

Five rhesus monkey eyes underwent repeated argon fluoride (193 nm) excimer laser myopic photorefractive keratectomy 3 months following an initial ablation that had produced mild subepithelial haze. At 3 months all eyes had development of a dense subepithelial opacity and a thickened epithelium (12 cells, 80 microns) with vacuolization of basal cells, fragmented basement membrane, and a layer of subepithelial fibrosis containing activated fibroblasts. By 6 months the opacity was clearing; epithelium was thinner (50 microns); subepithelial fibrosis was more lamellar. By 15 months only mild haze persisted clinically; epithelium was 30 microns thick, with persistent basal vacuolization and focal basement membrane disruption; subepithelial fibrous tissue was more organized. Early repeated excimer laser ablation of the monkey cornea apparently induces vigorous stromal wound healing. Use of shallower ablations, corticosteroids, or a longer delay between ablations may be necessary for repeated laser surgery to be practical clinically.

Keratoprosthesis with biocolonizable microporous fluorocarbon haptic. Preliminary results in a 24-patient study.

BACKGROUND: Most complications of a keratoprosthesis occur at the tissue-to-implant interface. The ideal prosthesis would eliminate this interface by having the tissue actually grow into the supporting material. We present a prospective clinical human study of a novel biocolonizable keratoprosthesis in 24 eyes of 24 patients. DESIGN: To promote implant stability, the 9-mm-diameter haptic was fashioned using a custom-made microporous fluorocarbon with a 4-mm-diameter, 2.67-mm-long, central optic made of medical grade polymethylmethacrylate, giving a global visual field of 110 degrees to 130 degrees. Only bilaterally blind patients with untreatable corneal diseases were included in the study. The haptic was inserted into a lamellar pocket delaminated in the stroma, and the optic was positioned through a hole trephined in the central cornea. RESULTS: The average follow-up was 15.7 months (range, 4 to 28 months). The host corneal fibroblasts penetrated and proliferated into the peripheral microporous fluorocarbon and provided anchorage between the cornea and prosthesis. Seventeen patients (70.8%) had visual acuity improvements. Mean corrected final visual acuity was 20/100 (range, 20/30 to 20/400). Five anatomic failures occurred in the first 6 months (three extrusions, one dislocation of the optic, and one endophthalmitis). We had one case (4.1%) of treatable glaucoma. We successfully removed four of five retroprosthetic membranes that had occurred. No retinal detachment occurred. CONCLUSION: The biocompatible inert microporous polymer did not eliminate all mechanical complications associated with a keratoprosthesis.

Wound healing after excimer laser keratomileusis (photorefractive keratectomy) in monkeys.

Laser myopic keratomileusis (photorefractive keratectomy) was performed on 29 rhesus monkey corneas with an argon fluoride (193-nm) excimer laser and a computer-controlled, moving slit delivery system. The 4-mm-diameter central ablation zone ranged in depth from 11 microns (-2 diopters effect) to 46 microns (-8 diopters effect). Corneas were studied for the 9 months postoperatively by clinical slit-lamp microscopy, and periodically with light and transmission electron microscopy. By 6 weeks, mild to moderate subepithelial haze was apparent in 93% of the corneas, with considerable variability in density. Progressive clearing occurred so that by 6 to 9 months 12 of 13 surviving corneas (92%) were either completely clear (4 corneas) or trace hazy (8 corneas). The epithelium was thickened at 21 days after ablation and returned to normal thickness by 3 months. At 3 weeks, subepithelial fibroblasts were three times the density of normal keratocytes and returned to nearly normal numbers by 9 months. We concluded that the anterior monkey cornea demonstrated a mild, typical wound healing response after excimer laser keratomileusis.

Corneal stromal wound healing in rabbits after 193-nm excimer laser surface ablation.

An argon fluoride excimer laser (193 nm) with a moving slit delivery system was used to perform anterior myopic keratomileusis in both eyes of 24 New Zealand white rabbits. Rabbits were killed immediately after ablation and at intervals up to 100 days. By slit-lamp microscopy, four rabbits at day 100 exhibited four clear corneas and four corneas had central, spotty, subepithelial haze. Light and electron microscopy documented corneal healing. In the early stages a transient acellular zone in the anterior stroma appeared over a period of three weeks, followed by an increased number of fibrocytes. In the corneas with opacification, focal areas of 20-microns-thick subepithelial scarring were present. An unexpected finding was transient damage to posterior stromal keratocytes and endothelial cells. The endothelium produced a layer of granular material that migrated anteriorly across Descemet's membrane. Immunochemistry at day 6 showed a marked staining for collagen IV, proteoglycans, fibronectin, and laminin.

A new mutation (A546T) of the betaig-h3 gene responsible for a French lattice corneal dystrophy type IIIA.

PURPOSE: To characterize the betaig-h3 gene defect in a French family affected with lattice corneal dystrophy type IIIA (LCDIIIA). METHODS: Histologic examination was performed from corneal buttons of two patients. Genomic DNA was extracted from leukocytes, and exons of the betaig-h3 gene were amplified by polymerase chain reaction to be directly sequenced. RESULTS: Numerous deposits were evident in the stroma and beneath the Bowman membrane, which had all the features of amyloid deposits. Analysis of exon 12 revealed a heterozygous G to A transition on codon 546. CONCLUSION: In contrast to Japanese patients, these French patients affected with LCDIIIA carry a distinct mutation of the betaig-h3 gene (A546T instead of P501T). Therefore, it is unclear whether different mutations could result in the same dystrophy or whether we are dealing with clinical heterogeneity of LCDIIIA.

Endothelial damage caused by uncoated and fluorocarbon-coated poly(methyl methacrylate) intraocular lenses.

PURPOSE: To assess endothelial damage induced by poly(methyl methacrylate) (PMMA) intraocular lenses (IOLs) coated with a fluorocarbon polymer, Teflon AF, to make them highly hydrophobic. SETTING: Department of Ophthalmology. Hôtel-Dieu Hospital, Paris, France. METHODS: Ten Teflon-coated and 10 uncoated PMMA IOLs were used in an in vitro static touch model. The corneal endothelium was placed in direct contact with the IOL for 15 seconds and then stained with trypan blue and alizarin red. The endothelial damage produced by each IOL in the area of contact was assessed semiquantitatively and quantitatively. RESULTS: Teflon-coated IOLs produced significantly less endothelial damage than uncoated PMMA IOLs (P < .0001). Endothelial cells in contact with Teflon-coated IOLs did not usually adhere to the IOL surface. In contrast, the uncoated IOLs produced large areas of endothelial cell loss. CONCLUSION: Teflon-coated PMMA IOLs have an antiadhesive effect that reduced endothelial damage after IOL insertion in an in vitro model.

Keratoconus and normal cornea: a comparative study of the collagenous fibers of the corneal stroma by image analysis.

Using an automatic image analysis technique, we studied the characteristics of the collagenous fibers of the corneal stroma of keratoconus at different stages of development. The clear portions of keratoconus specimens were studied at three different levels: anterior, middle, and posterior. The parameters obtained were compared with those of a normal adult cornea with the purpose of determining which ultrastructural alterations were caused by the appearance and progression of keratoconus.

The effect of gentamicin on the corneal endothelium. An experimental study.

Gentamicin sulfate with preservatives was injected into the anterior chamber of a rabbit eye to evaluate its effect on the corneal endothelium. Endothelial toxicity was evaluated by contact specular microscopy and transmission electron microscopy 48 h after injection. The results suggest that doses ranging from 100 to 400 micrograms of gentamicin are not toxic to the corneal endothelium. Endothelial toxicity occurred when 1,000 micrograms of gentamicin was injected.

Microcomputer software applied to corneal stromal biometry.

Here we report a new method for image analysis of the corneal stroma. To obtain the biometric characteristics of a given area, we perform three different treatments of the same image. These automated predictions closely match each parameter (length, surface area, etc.), as measured manually by a "blind investigator." Furthermore, to obtain quantifiable, average values, we have increased the number of successive image measurements, which has led to the development of a series of programs designed to optimize automated data handling. Finally, the acquired, calculated parameters are summarized in the form of intermediate tables for each series of images, and as a final summary table incorporating t-test values and permitting comparison between two stromas (e.g., normal and pathological). Multivariate analysis and an ascending hierarchical classification demonstrate the main trends in differences of pathological vs. normal stroma.

A fish-eye disease-like familial condition with massive corneal clouding and dyslipoproteinemia. Report of clinical, histologic, electron microscopic, and biochemical features.

Fish-eye disease (FED) is a rare familial condition characterized by progressive bilateral corneal clouding and dyslipoproteinemia previously described in one family and an unrelated woman of Swedish descent. Biochemical studies have clearly demonstrated the existence of this entity as a unique dyslipoproteinemia. We present a non-Swedish family of Mediterranean ancestry afflicted with bilateral corneal clouding and lipoprotein analysis consistent with FED-like state. This family's biochemical profile, corneal button histology, and electron microscopy of one member are reviewed. Other dyslipoproteinemias causing corneal changes are considered. Corneal tissue and familial biochemical analyses differed significantly from previous descriptions. On the basis of these findings, explanation of pathologic deposition and disease mechanism is proposed.

Acute corneal edema in pellucid marginal degeneration or acute marginal keratoconus.

This article reports a case of bilateral corneal pellucid marginal degeneration. The right cornea had an acute hydrops. Both eyes underwent penetrating keratoplasty. A histopathological study of the corneal specimens was performed by light and electron microscopy. The histological changes observed on the right cornea showed breaks on Bowman's layer, edema and disorganization of the stromal collagen, and break of Descemet's membrane. The ultrastructural changes were similar to those observed in acute keratoconus, leading to the belief that these two corneal diseases are closely related.

[Epithelial invasion of the anterior chamber : exploration by scanning electron microscopy (author's transl)]. / Invasion épithéliale de la chambre antérieure : aspect au microscope à balayage.

Transfixing keratoplasty was performed in a patient seven years after a lens extraction, following the appearance of corneal edema with hypertony and proliferation of a retrocorneal veil. Scanning electron microscopy examination of the posterior surface of the removed graft demonstrated the presence of a vast cellular veil formed of epithelial cells. The principal characteristics of these cells were their polygonal shape, slightly raised edges, and the presence of numerous surface microvilli. Because of the particular characteristics of this epithelium, scanning electron microscopy can be used to observe mitoses in the deep layer as well as for differentiation of the superficial layers. The epithelial cell appears to be identical, as far as its evolution is concerned, both in the aqueous humor and when in contact with the lacrymal film. When compared with other modern investigational techniques, the scanning electron microscope appears to be an effective method for studying epithelial invasion of the anterior chamber. In fact, optical microscopy is of little value in such cases, and transmitted light electron microscopy too heavy a technique for the results expected. Scanning electron microscopy enables precise definition of epithelial cells and can confirm their corneal or conjunctival origin.

[Acute keratoconus--an ultrastructural study (author's transl)]. / Kératocône aigu. Une étude ultrastructurale.

The corneal oedema of acute keratoconus is related to a break in endothelial-Descemetic continuity. This break was confirmed by optical and electron microscopical study of an anatomical section taken during a perforating keratoplasty; the methods of repair were studied five months after the incident. The corneal endothelium had almost totally recoversed the surfaces of the detached Descemet's membrane and the posterior surface of the bare corneal stroma. Electron microscopic study tended to indicate that the endothelial repair occurred more by cellular extension rather than mitosis (very flat cells) and showed that the inter-cellular connecting systems (focal tight junctions) were not regular, or even absent altogether, which explained the clinical finding of persistant corneal oedema. The endothelial cells secrete a Descemet neo-membrane over all their area which is more or less complete. They revover an original scarred stroma of fibrocytes of the posterior stroma.

[Gelatinous drop-like dystrophy of the cornea (primary amylosis) (author's transl)]. / Dystrophie gélatineuse en goutte de la cornée. Amylose primitive.

Two cases of gelatinous drop-like dystrophy of the cornea in young North-African (Algeria, Tunisia) subjects are reported. Clinical examination revealed characteristic subepithelial gelatinous drops in the cornea. Histology showed the amyloid nature of the subepithelial deposits, their fibrillary from being demonstrated on ultrastructural examination. Lamellar keratoplasty did not prevent recurrences. A pathogenic hypothesis is suggested involving a role of epithelial induction in the fibroblast development.

[Unilateral progressive annular opacity of the cornea. Apropos of a case. Clinical, histological and ultrastructural study]. / Opacité annulaire progressive unilatérale de la cornée. A propos d'un cas. Etude clinique, histologique et ultrastructurale.

A case of unilateral corneal annular opacification with a slowly progressive thickening of the cornea is reported; clinical, optical and electron microscopic studies have made possible to eliminate oedematous pathological process. The central zone of the cornea was histologically within normal aspect. In the peripheral zone intracellular mucopolysaccharides (MPS) and lipids were found in the stroma. Their presence suggest a collagen degeneration, thus likely revealing a forgotten initial stromal pathology. Its consequence, the unilateral annular thickened opacified cornea, seemed to us confusing enough to be presented and discussed.