Pierre Gastaud

Histopathological effects of topical ophthalmic preservatives on rat corneoconjunctival surface

PURPOSE Long term use of topical drugs has clearly been shown to induce toxic immunopathological changes in the ocular surface. However, little is known concerning the respective roles of active compounds and preservatives. Benzalkonium chloride (BAC) is the most used preservative and its cytotoxicity is well known, but other preservatives have not yet been clearly evaluated. We thus performed a comparative study to investigate toxic side effects induced in the rat ocular surface by applications of various preservatives, with special attention to inflammatory infiltrates. METHODS A total of 35 brown Norway rats were divided into seven groups of five each. They received, for one month, in both eyes, either 0.01% cetrimonium chloride, 0.01% benzalkonium chloride, 0.01% benzododecinium bromide, 0.004% thiomersal, 0.05% methyl parahydroxybenzoate or phosphate-buffered saline (PBS), the last group remaining untreated. Then, animals were sacrificed and eyes were processed for histological and immunological procedures with monoclonal antibodies to rat immunocompetent cells. RESULTS When compared to controls, all preservative-treated eyes consistently showed corneal and conjunctival damage, including epithelial alterations, various degrees of keratinization and inflammatory infiltrates at the limbus and within the conjunctival stroma and epithelium. No difference was found between the five tested drugs. CONCLUSIONS This study confirms that most preservatives used in ophthalmic eyedrops may similarly induce strong histopathological and inflammatory changes in the ocular surface after short term use. Although obtained in animal model, these results confirm strong toxic side effects in patients with preexisting ocular surface disorders and/or receiving topical drugs for long periods.

Results of proton therapy of uveal melanomas treated in Nice

PURPOSE To present the first results of uveal melanomas treated with the Medicyc Cyclotron 65 MeV proton beam facility in Nice, analyzing the factors that affect the cause-specific survival (CSS), metastatic rate, and reporting the visual outcome. METHODS AND MATERIALS This study concerns 538 patients referred by French institutions between June 1991 and December 1996. The eye and tumor parameters were measured using ultrasonography and angiography. Since 1994, CT scans were performed in most patients to help determine the axial length and the shape of the ocular globe. Tantalum clips were inserted around the tumor by the referring ophthalmologist. There were 349 posterior pole tumors (64.9%), 130 equatorial tumors (24.1%), and 59 ciliary body tumors (11%). Two hundred four patients (37.9%) had T1 or T2 tumors, and 334 patients (62.1%) had T3 or T4 tumors. The median tumor diameter was 14.6 mm, and the median tumor height was 5.1 mm. All patients received 52 Gy (57.20 Gy Co-equivalent dose) on 4 consecutive days. The data were analyzed by December 1997. RESULTS The CSS was 77.4% at 78 months, the overall survival was 73.8% and the local control was 89.0%. The CSS was not influenced by the patient age or the site of the tumor. It was 81.5% for T1 and T2 tumors, versus 75% for T3 and T4 tumors (P = 0.035). It was found that the tumor diameter, rather than the height, was the most important parameter affecting outcome. The metastatic rate was 8%. It depended on the T stage, tumor diameter and thickness, but not the tumor site. Thirty-eight enucleations were performed, most of them due to tumor progression and/or glaucoma. One-third of the patients in whom visual acuity was adequately scored before and after treatment had a stable, if not improved vision, and half the patients retained useful vision after treatment. CONCLUSION The outcome of patients suffering from uveal melanoma and treated with high-energy protons compares favorably with other techniques of treatment. The tumor dimensions affected CSS and metastatic rate. Even though two-thirds of patients had posterior pole tumors, half of them retained useful vision.

Expression of Inflammatory Membrane Markers by Conjunctival Cells in Chronically Treated Patients with Glaucoma

PURPOSE Recent histologic studies of conjunctival tissues in patients who have had long-term treatment for glaucoma have shown in situ an abnormal infiltration by inflammatory cells. In this study, conjunctival inflammatory antigens were investigated in impression cytology specimens from patients who have been and those who have not been treated for glaucoma. METHODS This study included 107 eyes from 55 patients with primary open-angle glaucoma. Of these, 48 had received prolonged topical treatments, all containing benzalkonium chloride as a preservative. Seven glaucomatous eyes could be examined before any treatment. In addition, the authors examined 11 patients (21 eyes) receiving anticataract eye drops preserved with chlorhexidine and 15 normal untreated subjects (30 eyes). In all patients, immunocytochemistry was performed in impression cytology specimens, using two monoclonal antibodies against HLA-DR antigens and receptor to IgE CD23. RESULTS None of the untreated eyes showed reactivity for either monoclonal antibody. In contrast, HLA-DR expression by conjunctival cells was found in 43 of 88 treated eyes (mean percentage of reactive cells, 70% +/- 28%) and positive staining for receptor to IgE in 26 of 68 eyes (52% +/- 28% of conjunctival cells). Results were not related to a specific treatment or combination of anti-glaucoma drugs. However, the proportion of positive specimens (3/14 for both antigens) in the group receiving chlorhexidine-containing eye drops was significantly lower than that found in the patients with glaucoma. CONCLUSION This study showed abnormal expression of inflammatory markers without clinical inflammation at the level of conjunctival cells in repetitive contact with various anti-glaucomatous treatments and their common preservative, benzalkonium chloride. Failure in filtering glaucoma surgery was found to be related to prolonged medical treatment; therefore, a topical sensitization to preservatives and/or anti-glaucoma drugs has been hypothesized. An immunocytologic test thus could be useful for qualitative and quantitative investigation of drug-induced conjunctival inflammation and predict high-risk patients.

Immunopathological findings in conjunctival cells using immunofluorescence staining of impression cytology specimens.

The conventional technique of impression cytology provides a non-invasive method for the evaluation of conjunctival epithelium alterations. Using indirect immunofluorescence procedures two inflammatory markers, class II MHC antigens HLA DR and the receptor to IgE (CD23), were sought in impression cytology specimens obtained from 80 patients. In normal subjects conjunctival epithelial cells did not show any reactivity. Only scattered dendritic cells were found to express class II antigens but not the receptor to IgE. In contrast patients with chronic conjunctivitis of various aetiologies, mainly infectious or allergic, had 40-100% of brightly positive conjunctival cells for one or both antigens. In these cases epithelial cells and goblet cells reacted similarly. Twenty four eyes in 12 patients with idiopathic dry eye syndrome disclosed results similar to those from normal conjunctival specimens. However 18 other specimens from patients suffering from idiopathic tear deficiency but undergoing multiple substitutive treatments for dry eye had moderate to strong positivity for HLA DR and/or the receptor to IgE (20-100% of cells). As these results were independent of the degree of squamous metaplasia the expression of these membrane markers may reflect local inflammation in addition to tear deficiency, possibly due to sensitisation to the eye drops used. These immunocytological techniques thus provide useful methods of investigating conjunctival inflammation and allergy. They may constitute valuable aid in the diagnosis and appropriate treatment of ocular surface disorders.

Growth Factors in Vitreous and Subretinal Fluid Cells from Patients with Proliferative Vitreoretinopathy

Mechanisms accounting for the development of proliferative vitreoretinopathy (PVR) in patients with rhegmatogenous retinal detachment remain poorly understood. In a previous study, we found the presence of various growth factors in preretinal membranes that were surgically removed from patients with PVR. The present immunohistological study was undertaken in intravitreal and subretinal fluid cells from patients suffering from PVR in various stages of development, in order to seek the presence of 4 growth-promoting factors for retinal pigment epithelial cells: acidic fibroblast growth factor (FGF), epidermal growth factor (EGF), insulin-like growth factor type I (IGF-I) and transforming growth factor-beta (TGF-beta). Results were quite similar in vitreous and subretinal fluid. Acidic FGF was found in all vitreous and subretinal specimens, in 30-100% of the examined cells. Immunoreactivity for EGF could be found in 53% of intravitreal cells and 69% of subretinal fluid cells. Positive cells were seen in all vitreous specimens and in all but 1 of the subretinal fluid specimens. IGF-I-containing cells were present in 13 of 15 vitreous specimens and in 18 of 20 subretinal fluid samples (mean percentages of reactivity in positive specimens 70% and 78%, respectively). In contrast, TGF-beta 1 reactivity was found in only 8 of 15 vitreous specimens and in 11 of 20 subretinal samples. Mean percentages of reactive cells were 30% and 50%, respectively. These results suggest that several growth factors could be involved in the proliferation and migration of retinal pigment epithelial cells during the course of PVR.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistologic Study of Epiretinal Membranes in Proliferative Vitreoretinopathy

We performed an immunohistologic study on 11 specimens of epiretinal membranes surgically obtained from patients who had rhegmatogenous retinal detachment with proliferative vitreoretinopathy. Immunostaining procedures were used to identify immunoglobulin and complement deposits, to visualize class II antigen expression by proliferating cells, and to determine eventual infiltration by cells of the immune system. Diffuse deposits of IgG, IgA, IgE, C1q, C3c, and C3d were found in epiretinal membranes, whereas numerous cells, including glial or pigmented epithelial cells, expressed HLA-DR and HLA-DQ antigens. Some macrophages and B or T8 lymphocytes were identified. These results suggest activation of the immune system during the course of proliferative vitreoretinopathy. Class II antigen expression could be dependent upon growth-promoting factors and interferon gamma and could play a crucial role in this immune reaction, which resulted in immunoglobulin deposition and activation of complement. However, the eventual role of immune phenomena in the extension of proliferative processes remains to be determined.

Influence of topical anesthesia on tonometric values of intraocular pressure.

The air pulse noncontact tonometer provides a safe and reliable method for measuring intraocular pressure (IOP), and makes it possible to avoid topical anesthesia. Based on previous reports that suggested possible anesthetic-induced IOP variations, this study was undertaken to investigate with this procedure the influence of local anesthetics on IOP and of some topically used drugs that could modify IOP values. In 212 normal or glaucomatous patients who underwent IOP measurement with a noncontact tonometer, IOP was determined before and in the first minutes following instillation of one of four tested drugs, oxybuprocaine and betoxycaine, two topical anesthetics currently used in applanation tonometry, and indomethacin suspension and metipranolol as controls. No significant effect was observed when comparing IOP values successively measured with the air pulse tonometer or 1 min after instillation of indomethacin suspension and metipranolol. In contrast a significant decrease in IOP was observed 1 and 5 min after instillation of one drop of the local anesthetics oxybuprocaine (mean IOP: 15.53 mm Hg before, 14.77 mm Hg at the 1st minute; p < 0.001) and betoxycaine (16.06 mm Hg before, 15.70 mm Hg at the 1st minute; p = 0.023). This effect was observed at least to the 15th minute, and in some patients, the decrease in IOP reached 8 mm Hg. Metipranolol only decreased IOP significantly at the 15th minute as compared to initial values, which differed from IOP variations following topical anesthesia. This phenomenon could not be related to mechanical effects of repetitive IOP measurements or massage by eyelids secondary to corneal irritation by anesthetic eye drops.(ABSTRACT TRUNCATED AT 250 WORDS)

Proton beam radiotherapy for uveal melanomas at nice teaching hospital: 16 years' experience.

PURPOSE To present the results of uveal melanomas treated at Nice Teaching Hospital. METHODS AND MATERIALS This retrospective study included 886 consecutive patients referred to our clinic for the treatment of uveal melanomas by proton beam radiotherapy from June 1991 to December 2007. Survival rates were determined by using Kaplan-Meier estimates, and prognostic factors were evaluated using the log-rank test or Cox model. RESULTS The number (percent total) of subjects staged according to the TNM classification system (6th edition) of malignant tumors included 39 stage T1 (4.4%), 420 stage T2 (47.40%), 409 stage T3 (46.16%), and 18 stage T4 (2.03%) patients. The median follow-up was 63.7 months. The Kaplan-Meier overall survival rate at 5 years according to the sixth edition TNM classification was 92% for T1, 89% for T2, 67% for T3, and 62% for T4; and at 10 years, 86% for T1, 78% for T2, 43% for T3, and 41% for T4. Five factors were found to be associated with an increased death rate: advanced age, tumor thickness, largest tumor basal diameter, tumor volume, and tumor volume-to-eyeball volume ratio. The metastasis-free survival rates were 88.3 % at 5 years and 76.4 % at 10 years. The local control rates were 93.9% at 5 years and 92.1% at 10 years. The ocular conservation rates were 91.1% at 5 years and 87.3% at 10 years. CONCLUSIONS We report the results of a large series of patients treated for uveal melanomas with a very long follow-up. Despite the large tumor volume treated, our results were similar to previously published findings relating to proton beam therapy.

HLA DR and DQ distribution in normal human ocular structures.

HLA DR and DQ distribution was investigated in normal human ocular tissues, together with class I antigens and immunocompetent cell subsets, by immunofluorescence and immunoperoxidase procedures. In the anterior segment, our findings, consistent with those of previous reports, showed the wide distribution of class I antigens, specially in the corneal epithelium, while class II antigens were restricted to very rare cells scattered in the conjunctiva, the peripheral cornea and the stroma of the ciliary processes. Some non pigmented epithelial cells of the ciliary processes were HLA DR and DQ positive. In the posterior segment, class I antigens were abundantly represented in the choroid and the retinal layers. Few HLA DR and DQ positive cells were seen in the choroid, similar to those found in the anterior segment. Normal RPE did not react with any monoclonal antibody, but numerous cells located in the retina were strongly HLA DR and DQ positive, all around the blood vessels, and not at the sites of endothelial cells. The characterization of those cells, which could be hypothetized as pericytes needs further studies but suggests close relationships between neuroretina and the immune system. This study may provide insight in the implication of the immune system in many poorly understood ocular diseases.

Acidic fibroblast growth factor distribution in normal human eye and possible implications in ocular pathogenesis.

Using a rabbit anti-acidic fibroblast growth factor (anti-aFGF) antiserum, we tried to establish a precise mapping of aFGF localization in normal human ocular structures, from samples obtained by autopsies. Cell cultures of retinal pigment epithelium and ciliary pigment epithelium were also established and immunofluorescence studies were performed after 1 month. Corneal and conjunctival epithelia were strongly positive for anti-aFGF antibodies as well as the subcapsular epithelium of the lens. The cortical fibers were weakly reactive and the lens nucleus negative. A strong intracytoplasmic reactivity was observed in the pigmented and nonpigmented epithelial cells of ciliary processes and pars plana, both ex vivo and in vitro. Retina was brightly positive, mostly in the photoreceptor and plexiform layers. The possible involvement of aFGF in normal eye growth and in various ocular diseases was then discussed.