[Advantages and limits of multiple grafts (third keratoplasty) under local cyclosporin 2%]. / Intérêt et limite de la greffe multiple (troisième kératoplastie) sous ciclosporine locale 2 %

INTRODUCTION: iterative penetrating keratoplasty procedures (PKPs) increase the risk of graft rejection. Surgeons are reluctant to perform a third procedure following two previous graft failures. We evaluated the outcomes of patients who had three successive PKPs treated with a local combination of cyclosporin 2% A and dexamethasone 0.1% eye drops. PATIENTS AND METHODS: this is a retrospective study of a series of cases including 15 eyes of 15 patients treated and followed in the ophthalmology unit at the Hôtel-Dieu Hospital in Paris (2006-2009). RESULTS: after an average follow-up of 20.5±2 months, the risk of transplant rejection was 13.33% and the duration of follow-up corresponded in all patients to the duration of treatment with cyclosporin eye drops. The treatment was not interrupted by any patient because of intolerance. CONCLUSION: in this study, we observed a success rate justifying the indication of the third keratoplasty under local cyclosporin 2% classically considered debatable.

Expression of extracellular matrix proteins fibulin-1 and fibulin-2 by human corneal fibroblasts.

PURPOSE: The fibulins are a family of extracellular matrix (ECM) molecules that regulate the organ shape along with other growth factors and stromal cells. We report here the in vitro expression of ECM proteins fibulin-1 and fibulin-2 by human corneal fibroblasts. The ability of fibulin-1 to modulate cell motility was investigated. METHODS: Fibulin-1 and fibulin-2 mRNA and proteins expression were analyzed in primary and immortalized human corneal fibroblasts (CHN) respectively by gene array, RT-PCR, and immunocytochemistry. The motility and adhesion of the cells transfected with fibulin-1 siRNA were analyzed on tissue culture polystyrene coated with Matrigel or ECM secreted by those fibroblasts. RESULTS: (1) The microarray analysis shows the expression of fibulin-1, fibulin-2, and their binding partners (i.e., fibronectin, nidogen-1, aggrecan, fibrilin-1, endostatin, and laminin alpha-2 chain). Interestingly, a matrix metalloprotease, ADAMTS-1, for which fibulin-1 acts as a cofactor, was also detected in CHN. (2) The synthesis by CHN of fibulin-1 and 2 mRNA and proteins was confirmed respectively by RT-PCR and immunocytochemistry. (3) Transfection of CHN by fibulin-1 siRNA has no effect on cell adhesion but increases cell migration compared with that of the control cells. This observation suggests an important role of fibulin-1 on cell motility. CONCLUSIONS: The expression of fibulins and that of their binding partners by human corneal fibroblasts indicate the important role of these proteins in the organization of supramolecular ECM structures of cornea. The variation of their expression and the structural changes of fibulins remain to be determined in corneal pathology.

[Visual outcome in keratoconus patients 1 and 2 years after penetrating keratoplasty]. / Acuité visuelle un et deux ans après kératoplastie transfixiante pour kératocône.

PURPOSE: To quantify the visual outcome obtained in patients operated on for penetrating keratoplasty for keratoconus (KC) after 1 and 2 years. PATIENTS: and methods: The files from consecutive keratoconus patients operated on for PKP after more than 2 years of follow-up were retrospectively studied. The demographic charts, the KC stage, and the surgical features were noted. The best corrected visual acuities (BSCVA) were analyzed at the initial examination and after 1 and 2 years of follow-up. RESULTS: One hundred and two eyes from 98 patients were analyzed, including 83 grade 4 (81%) and 19 grade 3 (18%) cases of KC. The mean preoperative BSCVA was 0.08 on the decimal chart, which increased to 0.5 and 0.6 1 and 2 years after penetrating keratoplasty (p<0.001, respectively). The mean keratometric astigmatism was 3.80+/-2.9 D and 3.94+/-2.3 D for the grade 3 and 4 KC, respectively, after 1 year and 2.52+/-1.9 D and 3.34+/-2.2 D after 2 years (NS). At this time point, 81% of the eyes had less than 5 D astigmatism. CONCLUSION: One and 2 years after penetrating keratoplasty, a mean BSCVA of 0.5 and 0.6 could reasonably be expected in keratoconus patients and three-quarters of patients can be expected to see better than 0.5 after 2 years.

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.

[Acanthamoeba stromal keratitis: epidemiology and prognosis factors]. / Kératites stromales amibiennes: épidémiologie et facteurs pronostiques.

PURPOSE: Early treatment of amebic stromal keratitis can change patients' final visual and esthetic prognosis. The aim of this study was to review all proven Acanthamoeba stromal keratitis cases presenting at our ophthalmology emergency department over the past 3 years, in order to delineate the incidence and the factors still leading to this serious complication. PATIENTS: and methods: All cases of Acanthamoeba stromal keratitis confirmed with a parasitology examination and treated between January 2000 and December 2002 were recorded. During this period of time, eight patients were identified with this condition. Their charts, as well as biomicroscopic, therapeutic, and outcome data were analyzed. RESULTS: All patients were referred in a second or third intention. Only one patient did not usually wear contact lenses. Six patients had an associated bacterial abscess. The typical amebic annular infiltrate was only observed in one patient. Three patients received an effective antiamebic treatment in the 15 days following the first symptoms and five patients 1 month or more after. An emergency keratoplasty was carried out in one patient. Finally, visual acuity was lower than 5/10 in all cases. The patients treated in the first 15 days had the best final visual acuity. DISCUSSION: Soft contact lenses are still responsible for the majority of serious amebic corneal complications. Late diagnosis remains the main cause of Acanthamoeba stromal keratitis development, particularly in the absence of contact lens history, because of the delay in treatment. CONCLUSION: The catastrophic consequences of this complication justify a systematic treatment against ameba in each case of suspected diagnosis. An early and effective treatment of the stromal amebic keratitis can increase the final visual prognosis.

[Femtosecond laser: a micromachining system for corneal surgery]. / Laser femtoseconde: système de micro-usinage pour chirurgie de la cornée.

INTRODUCTION: The authors present the diode-pumped, all-solid state, neodymium:glass femtosecond laser from the Laboratory of Ocular Biotechnology, Hotel-Dieu Hospital. MATERIALS AND METHODS: We worked with a 1,065-nm wavelength infrared laser. This laser is composed of an oscillator and amplification glass matrix mixed with neodymium. Its stretching and compression system is capable of producing pulses lasting a few hundred femtoseconds. The repetition rate is adjustable, ranging from 1 to 10 kHz, and can reach energies up to 60 microJ. The delivery system was set up on an optical table, with human corneal samples fixed to an anterior chamber system, which can be moved over the X-Y-Z axis by a computer-guided translation motor with micrometric precision. We analyzed the biological effects of laser impacts in human corneal tissue, obtained from the French Eye Bank. RESULTS: The femtosecond laser provides automated corneal cutting with a high level of precision, which can be verified on the corneal surface regularity by scanning electron microscopy analysis. Silicon samples can also be cut and can be used for calibration testing of the laser. CONCLUSION: The set-up composed of the femtosecond laser and the described delivery system enable precise corneal cutting and offer the opportunity to study its characteristics.

[Cyclosporine 2% eye drops preparation]. / Préparation d'un collyre de ciclosporine à 2%

PURPOSE: Cyclosporine administration is very effective in the case of immunological diseases of the cornea, conjunctive or uvea. Moreover, it is widely used in the case of high-risk rejection corneal transplantation. We present a preparation of cyclosporine 2% eye drops. METHODS: Cyclosporine 2% eye drops are prepared following a particular formulation including one part commercially available cyclosporine oral solution (Sandimmun) diluted in four parts of sterile castor oil. Manufacturing procedures maintain the sterile state of the preparation with a laminar airflow hood placed in a particulate controlled room, with pharmacists, technicians and clerical personnel wearing sterile clothes. Physical and chemical monitoring during and after manufacture for each batch guarantees the process and minimizes the risk of batch rejection. Chemical analysis of cyclosporine is conducted using a validated stability-indicating high-performance liquid chromatographic assay (reverse-phase). Blood dosages taken after the first administration at the 24th hour (after administration of the 6th drop) check for systemic integration. RESULTS: Cyclosporine 2% eye drops are fairly stable: 12 months after manufacturing, concentrations result in levels not statistically different from concentrations measured the day of preparation. After a daily regimen of six drops in the eye, cyclosporine 2% eye drops have a very low systemic bioavailability, because the blood concentrations only reach the detection limit of the fluorescence polarization immunoassay used for cyclosporine drug monitoring. This explains the absence of systemic toxicity. CONCLUSION: Cyclosporine 2% eye drops can be available in the hospital pharmacy. The eye drops are stable at room temperature and can be delivered up to 12 months after manufacture. No local adverse effects have been noted, probably in relation with the very low concentration of ethanol in the ocular preparation.

[Corneal edema in pseudophakia. A case report]. / Oedème cornéen du pseudophake. A propos d'une observation anatomo-clinique.

Pseudophakic bullous keratopathy is one of the most common complications of intraocular lenses, which explains the high rate of this condition in series of patients experiencing corneal grafts. It is caused by irreversible damage to the endothelial layer of the implanted eye involving the artificial lens, and occurs either during cataract extraction or after the operation. Intraoperative endothelial protection and the choice of an appropriate lens may contribute to decreasing this secondary disease, which can have severe consequences for many patients whose corneal graft may not restore their visual potential as well as expected. We report a clinicopathological case of a 59-year-old male, previously operated on for cataract in both eyes with an anterior chamber lens. A bilateral corneal edema occurred a few years after the operation, with a bilateral decrease in vision as a consequence. A corneal graft was performed in 2000 on the left eye in order to improve the patient's visual autonomy. The best treatment of this condition remains prevention, avoiding the use of this type of intraocular lens, whose main complication is corneal edema.

[Corneal burns and matrix metalloproteinases (MMP-2 and -9): the effects of human amniotic membrane transplantation]. / Brûlures cornéennes et métalloprotéases: influence des greffes de membranes amniotiques.

PURPOSE: Human amniotic membranes have recently been used in ophthalmology to restore deleted ocular surface after burns. Matrix metalloproteinases-2 and -9 have been implicated in the development of neovascularization. In this study, MMP-2 and MMP-9 expression was analyzed by in situ zymography on rabbit corneal chemical burns with and without human amniotic membrane graft. METHOD: Corneal neovascularization was induced in 10 Fauve de Bourgogne rabbits by means of a heptanol chemical burn on controlled deep keratotomy using a Chiron ALK-E corneal shaper. Half of rabbits received acute amniotic membrane transplantation 30mn after chemical burn; the remaining five rabbits received medical treatment. In situ zymography is a recent nondestructive technique which preserved the fine morphological details of the cornea and showed the active enzyme location in different corneal layers. The MMP-2 and -9 substrate was gelatin, which was detected by fluorescent microscopy. RESULTS: There was an overexpression of MMP-2 and -9 in corneal burns versus control corneas. Expression of MMP-2 and -9 was low in corneal burn without amniotic membrane graft. Following amniotic membrane transplantation, MMP-2 and -9 were strongly expressed and clinical neovascularization and inflammation decreased. Active enzymes were located in epithelium layers in the uncovered group. In the covered group, the active enzymes were located in the anterior and posterior stromal layers. CONCLUSION: The results support a role for MMP-2 and MMP-9 in corneal burn neovascularization. Amniotic membrane transplantation can play a protective role by up-regulation of their biological expression.

Effect of hyaluronan on MMP expression and activation.

Matrix metalloproteases (MMPs) play a crucial role in tissue remodelling in a variety of physiological and pathological processes. Hyaluronan is also involved in the same processes. Several cytokines and growth factors are involved in the regulation of the biosynthesis of hyaluronan and also of MMPs. The activity of MMPs has been shown to be regulated at the level of transcription and activation of the zymogen form. In order to explore the possible relationship between matrix components and especially hyaluronan, we studied the effect of hyaluronan on MMP expression (biosynthesis and activation) in the culture of human skin fibroblasts and corneal keratocytes (explant cultures and cell cultures). These cells were shown to exhibit distinct phenotypes as far as matrix biosynthesis is concerned. Using a synthetic substrate N-Suc(ala)3pNA, we measured elastase-type endopeptidase activity produced by fibroblasts and keratocytes and characterized the MMPs by zymography. Hyaluronan added to fibroblast cultures stimulated the membrane-bound elastase-type endopeptidase activity in a dose dependent fashion. Similar results were obtained with keratocyte cultures. In the presence of 1 mg/ml hyaluronan there was an increase in MMP expression and also an activation of latent MMPs both by fibroblasts and keratocytes.

Nineteen years of penetrating keratoplasty in the Hotel-Dieu Hospital in Paris.

PURPOSE: To identify changing trends in penetrating keratoplasties (PKs) performed at the Hotel-Dieu Hospital in Paris between January 1980 and December 1999 and to explain the reasons for the changes. METHODS: We retrospectively reviewed 3,736 of the 3,836 PKs performed between January 1, 1980, and December 31, 1999, and classified them into diagnostic categories. RESULTS: The most common indications for PK were keratoconus (28.8%), herpetic infections (10.9%), graft failures (9.9%), aphakic and pseudophakic corneal edema (9.9%), Fuchs' endothelial dystrophy (9.4%), and nonherpetic leucoma (7.7%). Other indications represented 23.4% of the cases. The incidence of aphakic and pseudophakic corneal edema progressively increased between 1980 and 1991, became the most frequent indication in 1991 (21.4%), and then progressively decreased. The annual number of PKs increased between 1980 and 1986, decreased between 1987 and 1997, and increased again after September 1997. The decrease was caused by both a shortage of corneal buttons, and, in 1987, the fear of transmitting diseases through corneal transplantation, particularly human immunodeficiency virus. Beginning in 1992, decreases were also associated with stringent governmental regulations of eye bank tissue. CONCLUSION: Changes in the incidence and management of corneal disorders were the primary factors leading to modifications of grafting until 1987. After 1987, corneal button shortage probably corresponded to the acquired immune deficiency syndrome epidemic. Governmental regulations of eye banking led to a severe corneal button shortage between 1992 and 1997. Despite an increase in the number of PKs performed after 1997, corneal buttons are still preferentially allocated to patients in whom there is a high probability of graft success.

[Histologic evaluation in rabbits of the biocompatibility of episcleral implant material: S-PTFEe (silicone core covered with expanded polytetrafluoroethylene)]. / Evaluation histologique chez le lapin de la biocompatibilité d'un matériel d'indentation épiscléral: le S-PTFEe (noyau en silicone recouvert de polytétrafluoro-éthylène expansé).

PURPOSE: To study the tolerance of a silicone band wrapped with expanded polytetrafluoroethylene (e-PTFE) in "fauve de Bourgogne" rabbits. METHODS: A 5.7mmx3.2mm band of S-PTFE (silicone-polytetrafluoroethylene, France Chirurgie Instruments) was used. A tight seal was produced between the two materials by silicone gluing. Eight eyes were implanted episclerally and fixed with Mersilene 5-0. One silicone band was used on a control eye. RESULTS: Three eyes were removed at 4 weeks, one at 8 weeks and four at 12 weeks. No extrusion was observed except for the silicone band rejected 2 weeks after implantation. On histopathologic examination, PTFE colonisation was present in all eyes with fibroblast, inflammatory cells, and neovascularization. CONCLUSION: The S-PTFE implant was colonized and well tolerated in rabbit eyes. Advantages of silicone band (quality, stability of indentation) are combined to the tolerance of PTFE.

Chemotactic penetration of keratocytes in ePTFE polymer in vitro.

Expanded polytetrafluoroethylene (ePTFE) is used as a support for artificial corneas. Implanted in corneas, most of the time this polymer is colonized by corneal host cells. The absence of colonization often coincides with extrusion of the polymer. Therefore, we decided to introduce keratocytes into ePTFE in vitro before implantation. Because keratocytes do not spontaneously enter ePTFE, we used several chemoattractants, separately and in a mixture, to stimulate the penetration of cultured keratocytes into the polymer. The influence of the passage number on cell penetration was also studied. No significant differences were observed up to the seventh passage, although seventh-passage cells penetrated somewhat more slowly than younger cells. Satisfactory results were obtained with four of the tested chemotactic factors: IL-6, type alpha transforming growth factor (TGF-alpha), platelet derived growth factor isoform BB (PDGF-BB), and fibroblast growth factor-2 (FGF-2). Under our experimental conditions, two to more than six million keratocytes entered the polymer discs with a volume of 706.5 mm(3) in the presence of these four chemoattractants. TGF-alpha was the most efficient and was selected for further in vitro and in vivo studies.

Effect of procyanidolic oligomers on the permeability of the blood-brain barrier.

Blood-brain barrier (BBB) is the site of regulatory mechanisms which control the exchange of substances between the brain and the blood through the wall of 'true' brain capillaries with tight junctions between endothelial cells. In some pathological situations the permeability of the BBB is increased because of a partial proteolytic degradation of some constituents of the capillary basement lamina. In such cases it is important to restore normal permeability. The effect of procyanidolic oligomers (PCO) on the BBB was investigated in vivo with quantitative morphologic procedures. We also investigated the action of this drug on collagen and basement lamina constituents (Matrigel) in vitro. Collagenase injected in lateral brain ventricles was shown to increase BBB permeability. Per os administration of PCO to rats greatly increased the resistance of brain capillaries to bacterial collagenase, as shown by the inhibition of the diffusion of fluorescein-isothiocyanate-marked dextran particles from the blood-stream into the brain tissues. Calf skin collagen pretreated in vitro with PCO became more resistant to the hydrolytic action of collagenase. Similar, even more intense protective effect was seen when basal lamina constituents containing type IV collagen was incubated with PCO before exposure to pronase. These in vitro effects may partly explain the in vivo protective effect of PCO against the alteration of brain capillaries by i.v. injected bacterial collagenase.

Corneal collagens.

Cornea is a highly differentiated tissue rich in extracellular matrix (ECM) specifically distributed in space in order to insure its dual role--transparency and protection of inner eye-tissues. Corneal ECM is especially rich in collagens. Since the characterisation of a number of distinct collagen types it appeared that most of them are present in the cornea. Their synthesis follows a specific program of sequential expression of the different collagen types to be synthesised during the development and maturation of the cornea. The precise regulation of the diameter and orientation of fibers, and of the interfibrillar spaces is partially at least attributed to interactions between glycosaminoglycans and collagens. The 'program' of vectorial collagen synthesis and GAG-collagen interactions changes also with age and in several pathological conditions as corneal dystrophies and wound healing. The Maillard reaction, especially in diabetes, is one of these important factors involved in age-dependent modifications of corneal structure and function. Far from being inert, corneal collagens were shown to have relatively short half-lives. The biosynthesis of corneal collagens was studied also during wound healing. The refibrillation of wounded corneas does not follow the original 'program' of ECM-synthesis as shown by the comparative study of wound healing using biochemical and morphometric methods. This review recapitulates briefly previous and recent studies on corneal collagens in order to present to clinicians and scientists an overview of the state of the art of this important field at the intersection of eye research and matrix biology.

Inhibition of lens epithelial cell proliferation by substituted PMMA intraocular lenses.

BACKGROUND: Migration and proliferation of lens epithelial cells (LECs) are based on interactions between cellular integrins and extracellular matrix proteins (ECMs). We were able to design polymers substituted with COO- and SO3- groups that counteract LEC proliferation through modification of the integrin/ECM interaction. The objective of this study was to compare the cell proliferation-inhibiting properties of differently substituted copolymers with those of a homopolymer. METHODS: Polymers were synthesized by radical polymerization and characterized by nuclear resonance spectroscopy. Second-passage rabbit LECs were cultured either on PMMA homopolymers (control) or on substituted PMMA copolymers. Cells were counted on days 2, 7, and 17. Cell vitality was evaluated by trypan blue staining. Experiments were run in quadruplicate. Statistical analysis was performed using the Wilcoxon test and the Mann-Whitney U-test. RESULTS: Compared with control, cells cultured on copolymers grew to significantly lower cell numbers. The inhibitory effect became evident at day 2 after seeding and persisted throughout the experiment. Different ratios of COO-/SO3- substitutions yielded different grades of inhibition. No toxic effect was seen on rabbit LECs. CONCLUSIONS: The tested copolymers inhibit cell proliferation and are nontoxic. Further evaluation could lead to the development of new intraocular lens materials that prevent secondary cataract.

[Avellino dystrophy. Current diagnostic criteria]. / La dystrophie d'Avellino. Critères diagnostiques modernes.

We report a French family suffering from an Avellino corneal dystrophy diagnosed by using clinical, histological, ultrastructural and genetics findings. Our results indicate that direct corneal examination and routine histological examinations must always be associated with an assay for BIGH3 gene mutations to establish a modern and unambiguous diagnosis of a corneal dystrophy.

Clinical, histologic, and ultrastructural features of the corneal dystrophy caused by the R124L mutation of the BIGH3 gene.

OBJECTIVE: This study was designed to describe the clinical, histologic, and ultrastructural features of the corneal dystrophy associated with the R124L mutation of the BIGH3 gene. DESIGN: Retrospective clinical and histologic review of a new genetic mutation. PARTICIPANTS: Thirty-four patients from five unrelated French families with corneal dystrophy caused by the R124L mutation of the BIGH3 gene were studied at the clinical, histologic, and ultrastructural levels. Records of patients carrying this mutation were compared with those from three unrelated patients with corneal dystrophy of Bowman's layer (CDB) type 2 (R555Q mutation) and from three unrelated patients with classic corneal granular dystrophy (R555W mutation). INTERVENTION: The mutational genetic status of the BIGH3 gene was determined for each patient, and the histologic and ultrastructural data available after corneal graft were analyzed. MAIN OUTCOMES MEASURES: Genomic DNA was extracted from peripheral blood leukocytes. Exons 4 and 12 of the BIGH3 gene were amplified by the polymerase chain reaction (PCR), and the PCR products were directly sequenced. RESULTS: All 34 patients with the R124L mutation displayed the clinical, histologic, and electron microscopic features of the dystrophy previously described as a superficial variant of corneal granular dystrophy. Combining molecular genetics with clinical and histologic findings established a clear distinction between the R555Q and R555W dystrophies. CONCLUSIONS: The R124L mutation of the BIGH3 gene is associated with specific clinical and morphologic criteria. This indicates that molecular studies are needed for an adequate classification of corneal dystrophies. All criteria are presently available to segregate the dystrophy caused by the R124L mutation (known as CDB1) from the dystrophy caused by the R555Q mutation (known as CDB2).