Michèle Savoldelli

Mineralocorticoid receptor is involved in rat and human ocular chorioretinopathy

Central serous chorioretinopathy (CSCR) is a vision-threatening eye disease with no validated treatment and unknown pathogeny. In CSCR, dilation and leakage of choroid vessels underneath the retina cause subretinal fluid accumulation and retinal detachment. Because glucocorticoids induce and aggravate CSCR and are known to bind to the mineralocorticoid receptor (MR), CSCR may be related to inappropriate MR activation. Our aim was to assess the effect of MR activation on rat choroidal vasculature and translate the results to CSCR patients. Intravitreous injection of the glucocorticoid corticosterone in rat eyes induced choroidal enlargement. Aldosterone, a specific MR activator, elicited the same effect, producing choroid vessel dilation -and leakage. We identified an underlying mechanism of this effect: aldosterone upregulated the endothelial vasodilatory K channel KCa2.3. Its blockade prevented aldosterone-induced thickening. To translate these findings, we treated 2 patients with chronic nonresolved CSCR with oral eplerenone, a specific MR antagonist, for 5 weeks, and observed impressive and rapid resolution of retinal detachment and choroidal vasodilation as well as improved visual acuity. The benefit was maintained 5 months after eplerenone withdrawal. Our results identify MR signaling as a pathway controlling choroidal vascular bed relaxation and provide a pathogenic link with human CSCR, which suggests that blockade of MR could be used therapeutically to reverse choroid vasculopathy.

Microglia/Macrophages Migrate through Retinal Epithelium Barrier by a Transcellular Route in Diabetic Retinopathy: Role of PKCζ in the Goto Kakizaki Rat Model

Diabetic retinopathy is associated with ocular inflammation, leading to retinal barrier breakdown, macular edema, and visual cell loss. We investigated the molecular mechanisms involved in microglia/macrophages trafficking in the retina and the role of protein kinase Cζ (PKCζ) in this process. Goto Kakizaki (GK) rats, a model for spontaneous type 2 diabetes were studied until 12 months of hyperglycemia. Up to 5 months, sparse microglia/macrophages were detected in the subretinal space, together with numerous pores in retinal pigment epithelial (RPE) cells, allowing inflammatory cell traffic between the retina and choroid. Intercellular adhesion molecule-1 (ICAM-1), caveolin-1 (CAV-1), and PKCζ were identified at the pore border. At 12 months of hyperglycemia, the significant reduction of pores density in RPE cell layer was associated with microglia/macrophages accumulation in the subretinal space together with vacuolization of RPE cells and disorganization of photoreceptors outer segments. The intraocular injection of a PKCζ inhibitor at 12 months reduced iNOS expression in microglia/macrophages and inhibited their migration through the retina, preventing their subretinal accumulation. We show here that a physiological transcellular pathway takes place through RPE cells and contributes to microglia/macrophages retinal trafficking. Chronic hyperglycemia causes alteration of this pathway and subsequent subretinal accumulation of activated microglia/macrophages.

The neuroretina is a novel mineralocorticoid target: aldosterone up-regulates ion and water channels in Müller glial cells

Glucocorticoids reduce diabetic macular edema, but the mechanisms underlying glucocorticoid effects are imperfectly elucidated. Glucocorticoids may bind to glucocorticoid (GR) and mineralocorticoid (MR) receptors. We hypothesize that MR activation may influence retinal hydration. The effect of the MR agonist aldosterone (24 h) on ion/water channel expression (real‐time PCR, Western blot, immunofluorescence) was investigated on cultured retinal Müller glial cells (RMGs, which contribute to fluid homeostasis in the retina), in Lewis rat retinal explants, and in retinas from aldosterone‐injected eyes. We evidenced cell‐specific expression of MR, GR, and 11‐β‐hydroxysteroid dehydrogenase type II. Aldosterone significantly enhances expression of sodium and potassium channels ENaC‐α (6.5‐fold) and Kir4.1 (1.9‐fold) through MR and GR occupancy, whereas aquaporin 4 (AQP4, 2.9‐fold) up‐regulation is MR‐selective. Aldosterone intravitreous injection induces retinal swelling (24% increase compared to sham‐injected eyes) and activation of RMGs. It promotes additional localization of Kir4.1 and AQP4 toward apical microvilli of RMGs. Our results highlight the mineralocorticoid‐sensitivity of the neuroretina and show that aldosterone controls hydration of the healthy retina through regulation of ion/water channels expression in RMGs. These results provide a rationale for future investigations of abnormal MR signaling in the pathological retina.—Zhao, M., Valamanesh, F., Celerier, I., Savoldelli, M., Jonet, L., Jeanny, J.‐C., Jaisser, F., Farman, N., Behar‐Cohen, F. The neuroretina is a novel mineralocorticoid target: aldosterone up‐regulates ion and water channels in Müller glial cells. FASEB J. 24, 3405–3415 (2010). www.fasebj.org

Corneal Wound Healing in Monkeys 18 Months After Excimer Laser Photorefractive Keratectomy

Excimer laser keratomileusis (photorefractive keratectomy, direct corneal ablation) for myopic corrections of 2.00 diopters (n = 1), 4.00 D (n = 4), and 8.00 D (n = 3) was performed on eight corneas of four Rhesus monkeys. All animals were followed for 18 months. The ablations healed normally and no epithelial erosions occurred. Serial slit-lamp microscope examinations revealed that a variable amount of corneal haze developed in all animals; this haze progressively faded during the follow-up period. Histopathology revealed an epithelium of normal thickness, basement membrane abnormalities, increased number and activity of stromal keratocytes, and a variable amount of newly secreted extracellular matrix in the anterior stroma. These findings suggest that excimer laser keratomileusis induces a mild wound healing response in the anterior cornea which displays considerable individual variability and persists up to 18 months.

The outer limiting membrane (OLM) revisited: clinical implications

Purpose The outer limiting membrane (OLM) is considered to play a role in maintaining the structure of the retina through mechanical strength. However, the observation of junction proteins located at the OLM and its barrier permeability properties may suggest that the OLM may be part of the retinal barrier. Material and methods Normal and diabetic rat, monkey, and human retinas were used to analyze junction proteins at the OLM. Proteome analyses were performed using immunohistochemistry on sections and flat-mounted retinas and western blotting on protein extracts obtained from laser microdissection of the photoreceptor layers. Semi-thin and ultrastructure analyses were also reported. Results In the rat retina, in the subapical region zonula occludens-1 (ZO-1), junction adhesion molecule (JAM), an atypical protein kinase C, is present and the OLM shows dense labeling of occludin, JAM, and ZO-1. The presence of occludin has been confirmed using western blot analysis of the microdissected OLM region. In diabetic rats, occludin expression is decreased and glial cells junctions are dissociated. In the monkey retina, occludin, JAM, and ZO-1 are also found in the OLM. Junction proteins have a specific distribution around cone photoreceptors and Müller glia. Ultrastructural analyses suggest that structures like tight junctions may exist between retinal glial Müller cells and photoreceptors. Conclusions In the OLM, heterotypic junctions contain proteins from both adherent and tight junctions. Their structure suggests that tight junctions may exist in the OLM. Occludin is present in the OLM of the rat and monkey retina and it is decreased in diabetes. The OLM should be considered as part of the retinal barrier that can be disrupted in pathological conditions contributing to fluid accumulation in the macula.

A new mutation (A546T) of the βig-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.

Evaluation of the New Photosensitizer Stakel (WST-11) for Photodynamic Choroidal Vessel Occlusion in Rabbit and Rat Eyes

PURPOSE To evaluate the photodynamic potential of a new hydrosoluble photosensitizer (WST-11, Stakel; Steba Biotech, Toussus-Le-Noble, France), for use in occlusion of normal choroidal vessels in the rabbit eye and CNV (choroidal neovascularization) in the rat eye. METHODS Occlusive and nonocclusive parameters of Stakel and verteporfin photodynamic therapy (PDT) were investigated in pigmented rabbits. Eyes were followed by fluorescein angiography (FA) and histology at various intervals after PDT. RESULTS When occlusive parameters (fluence of 50 J/cm(2), 5 mg/kg drug dose and DLI [distance to light illumination] of 1 minute) were used, Stakel PDT was efficient immediately after treatment without associated structural damage of the RPE and retina overlying the treated choroid in the rabbit eye. Two days later, total occlusion of the choriocapillaries was seen in 100% of the treated eyes, along with accompanying histologic structural changes in the overlying retina. When the occlusive parameters (fluence, 100 J/cm2; drug dose, 12 mg/m2; and DLI, 5 minutes) of verteporfin PDT were used, occlusion of the choriocapillaries was observed in 89% of the treated eyes. Histology performed immediately after treatment demonstrated structural damage of the overlying retina and RPE layer. Weaker, nonocclusive Stakel PDT parameters (25 J/cm2, 5 mg/kg, and DLI of 10 minutes) did not induce choriocapillary occlusion or retinal lesions on FA or histology. Weaker, nonocclusive verteporfin PDT parameters (10 J/cm2, 0.2 mg/kg, and DLI of 5 minutes) did not induce choriocapillary occlusion. However, histology of these eyes showed the presence of damage in the retinal and choroidal tissues. Moreover, preliminary results indicate that selective CNV occlusion can be achieved with Stakel PDT in the rat eye. CONCLUSIONS Unlike verteporfin PDT, Stakel PDT does not cause direct damage to the RPE cell layer or retina. These observations indicate that Stakel PDT may have a high potential for beneficial therapeutic outcomes in treatment of AMD.

Ultrashort pulse laser surgery of the cornea and the sclera

The strongly localized interaction process of ultrashort laser pulses with tissue makes femtosecond lasers a powerful tool for eye surgery. These lasers are now routinely used in refractive surgery and other forms of surgery of the anterior segment of the eye. Several clinical laser systems also offer options for corneal grafting and the potential use of ultrashort pulse lasers in glaucoma surgery has been the object of several recent studies which have shown promising results. While devices aimed for interventions in clear tissue may be based on available solid state or fibre laser technology, the development of tools for surgery in more strongly scattering tissue has to account for the compromised tissular transparency and requires the development of optimized laser sources. The present paper focuses on surgery of clear and pathological cornea as well as sclera. It aims to give an overview over typical medical indications for ultrashort pulse laser surgery, the optics of the tissues involved, the available laser technology, the laser–tissue interaction process, and possible future developments.

In situ monitoring of second-harmonic generation in human corneas to compensate for femtosecond laser pulse attenuation in keratoplasty

The application of femtosecond lasers in corneal transplant surgery requires high pulse energies to compensate for the strong optical scattering in pathological corneas. However, excessive energies deteriorate the quality of the incisions. The aim of this study is to demonstrate the dependence of side effects on local radiant exposure, numerical aperture, and tissue properties, to quantify the penetration depth of the laser for individual corneas, and to provide a method for optimizing the energy in the volume of the cornea. We examine histological and ultrastructural sections of clear and edematous corneas with perforating and lamellar incisions performed at different pulse energies. We demonstrate that the augmented energies in edematous corneas may result in unwanted side effects even when using high numerical apertures. The dependence of the laser beam penetration depth on pulse energy is evaluated by histology and an exponential decrease is observed. We show that the penetration length can be determined by evaluating the backscattered second-harmonic emission associated with the nonlinear optical properties of the tissue. This approach represents a noninvasive method for the in situ quantification of the laser beam attenuation, enabling us to adapt the pulse energy accordingly. Experiments using adapted energies show that the side effects are minimized.

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 Bowmans 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).