High Levels of C-Reactive Protein with Low Levels of Pentraxin 3 as Biomarkers for Central Serous Chorioretinopathy.

Purpose: To investigate the association between the 2 acute phase proteins, C-reactive protein (CRP) and pentraxin 3 (PTX3) with central serous chorioretinopathy (CSCR), as PTX3 is a glucocorticoid-induced protein. Design: Cross-sectional multicenter study. Participants: Patients with CSCR compared with age- and sex-matched healthy participants. Methods: Patients with CSCR from 3 centers in Europe were included in the study. The clinical form of CSCR was recorded. Blood samples from patients with CSCR and healthy participants were sampled, and high-sensitivity CRP and PTX3 levels were measured in the serum. Main Outcome Measures: C-reactive protein and PTX3 serum level comparison between patients with CSCR with age- and sex-matched healthy participants. Results: Although CRP levels were higher in patients with CSCR (n = 216) than in age- and sex-matched controls (n = 130) (2.2 ± 3.2 mg/l vs. 1.5 mg/l ± 1.4, respectively, P = 0.037), PTX3 levels were lower in patients with CSCR (10.5 ± 19.9 pg/ml vs. 87.4 ± 73.2 pg/ml, respectively, P < 0.001). There was no significant difference in CRP or PTX3 levels between patients with acute/recurrent and chronic CSCR. Conclusions: In patients with CSCR, high CRP and low PTX3 levels suggest a form of low-grade systemic inflammation together with a lack of glucocorticoid pathway activation, raising new hypotheses on the pathophysiology of CSCR. Financial Disclosures: The author(s) have no proprietary or commercial interest in any materials discussed in this article.

In Vivo Retinal Pigment Epithelium Imaging using Transscleral Optical Imaging in Healthy Eyes.

Objective: To image healthy retinal pigment epithelial (RPE) cells in vivo using Transscleral OPtical Imaging (TOPI) and to analyze statistics of RPE cell features as a function of age, axial length (AL), and eccentricity. Design: Single-center, exploratory, prospective, and descriptive clinical study. Participants: Forty-nine eyes (AL: 24.03 ± 0.93 mm; range: 21.9-26.7 mm) from 29 participants aged 21 to 70 years (37.1 ± 13.3 years; 19 men, 10 women). Methods: Retinal images, including fundus photography and spectral-domain OCT, AL, and refractive error measurements were collected at baseline. For each eye, 6 high-resolution RPE images were acquired using TOPI at different locations, one of them being imaged 5 times to evaluate the repeatability of the method. Follow-up ophthalmic examination was repeated 1 to 3 weeks after TOPI to assess safety. Retinal pigment epithelial images were analyzed with a custom automated software to extract cell parameters. Statistical analysis of the selected high-contrast images included calculation of coefficient of variation (CoV) for each feature at each repetition and Spearman and Mann-Whitney tests to investigate the relationship between cell features and eye and subject characteristics. Main Outcome Measures: Retinal pigment epithelial cell features: density, area, center-to-center spacing, number of neighbors, circularity, elongation, solidity, and border distance CoV. Results: Macular RPE cell features were extracted from TOPI images at an eccentricity of 1.6° to 16.3° from the fovea. For each feature, the mean CoV was < 4%. Spearman test showed correlation within RPE cell features. In the perifovea, the region in which images were selected for all participants, longer AL significantly correlated with decreased RPE cell density (R Spearman, Rs = -0.746; P < 0.0001) and increased cell area (Rs = 0.668; P < 0.0001), without morphologic changes. Aging was also significantly correlated with decreased RPE density (Rs = -0.391; P = 0.036) and increased cell area (Rs = 0.454; P = 0.013). Lower circular, less symmetric, more elongated, and larger cells were observed in those > 50 years. Conclusions: The TOPI technology imaged RPE cells in vivo with a repeatability of < 4% for the CoV and was used to analyze the influence of physiologic factors on RPE cell morphometry in the perifovea of healthy volunteers. Financial Disclosures: Proprietary or commercial disclosure may be found after the references.

Pathogenic Effects of Mineralocorticoid Pathway Activation in Retinal Pigment Epithelium.

Glucocorticoids are amongst the most used drugs to treat retinal diseases of various origins. Yet, the transcriptional regulations induced by glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) activation in retinal pigment epithelium cells (RPE) that form the outer blood-retina barrier are unknown. Levels of endogenous corticoids, ligands for MR and GR, were measured in human ocular media. Human RPE cells derived from induced pluripotent stem cells (iRPE) were used to analyze the pan-transcriptional regulations induced by aldosterone-an MR-specific agonist, or cortisol or cortisol + RU486-a GR antagonist. The retinal phenotype of transgenic mice that overexpress the human MR (P1.hMR) was analyzed. In the human eye, the main ligand for GR and MR is cortisol. The iRPE cells express functional GR and MR. The subset of genes regulated by aldosterone and by cortisol + RU-486, and not by cortisol alone, mimics an imbalance toward MR activation. They are involved in extracellular matrix remodeling (CNN1, MGP, AMTN), epithelial-mesenchymal transition, RPE cell proliferation and migration (ITGB3, PLAUR and FOSL1) and immune balance (TNFSF18 and PTX3). The P1.hMR mice showed choroidal vasodilation, focal alteration of the RPE/choroid interface and migration of RPE cells together with RPE barrier function alteration, similar to human retinal diseases within the pachychoroid spectrum. RPE is a corticosteroid-sensitive epithelium. MR pathway activation in the RPE regulates genes involved in barrier function, extracellular matrix, neural regulation and epithelial differentiation, which could contribute to retinal pathology.

Polymeric micelle mediated follicular delivery of spironolactone: Targeting the mineralocorticoid receptor to prevent glucocorticoid-induced activation and delayed cutaneous wound healing.

Impaired wound healing in patients receiving glucocorticoid therapy is a serious clinical concern: mineralocorticoid receptor (MR) antagonists can counter glucocorticoid-induced off-target activation of MR receptors. The aim of this study was to investigate the cutaneous delivery of the potent MR antagonist, spironolactone (SPL), from polymeric micelle nanocarriers, prepared using a biodegradable copolymer, methoxy-poly(ethylene glycol)-di-hexyl-substituted-poly(lactic acid). Immunofluorescent labelling of the MR showed that it was principally located in the pilosebaceous unit (PSU), justifying the study rationale since polymeric micelles accumulate preferentially in appendageal structures. Cutaneous biodistribution studies under infinite and finite dose conditions, demonstrated delivery of pharmacologically relevant amounts of SPL to the epidermis and upper dermis. Preferential PSU targeting was confirmed by comparing amounts of SPL in PSU-containing and PSU-free skin biopsies: SPL nanomicelles showed 5-fold higher delivery of SPL in the PSU-containing biopsies, 0.54 ± 0.18 ng/mm2vs. 0.10 ± 0.03 ng/mm2, after application of a hydrogel in finite conditions. Canrenone, an active metabolite of SPL, was also quantified in skin samples. In addition to being used for the treatment of delayed cutaneous wound healing by site-specific antagonism of the MR, the formulation might also be used to treat pilosebaceous androgen-related skin diseases, e.g. acne vulgaris, since SPL is a potent androgen receptor antagonist.

Oral Ursodeoxycholic Acid Crosses the Blood Retinal Barrier in Patients with Retinal Detachment and Protects Against Retinal Degeneration in an Ex Vivo Model.

Rhegmatogenous retinal detachment (RD) is a threatening visual condition and a human disease model for retinal degenerations. Despite successful reattachment surgery, vision does not fully recover, due to subretinal fluid accumulation and subsequent photoreceptor cell death, through mechanisms that recapitulate those of retinal degenerative diseases. Hydrophilic bile acids are neuroprotective in animal models, but whether they can be used orally for retinal diseases is unknown. Ursodeoxycholic acid (UDCA) being approved for clinical use (e.g., in cholestasis), we have evaluated the ocular bioavailability of oral UDCA, administered to patients before RD surgery. The level of UDCA in ocular media correlated with the extent of blood retinal barrier disruption, evaluated by the extent of detachment and the albumin concentration in subretinal fluid. UDCA, at levels measured in ocular media, protected photoreceptors from apoptosis and necrosis in rat retinal explants, an ex vivo model of RD. The subretinal fluid from UDCA-treated patients, collected during surgery, significantly protected rat retinal explants from cell death, when compared to subretinal fluid from control patients. Pan-transcriptomic analysis of the retina showed that UDCA upregulated anti-apoptotic, anti-oxidant, and anti-inflammatory genes. Oral UDCA is a potential neuroprotective adjuvant therapy in RD and other retinal degenerative diseases and should be further evaluated in a clinical trial.

Fully automated detection, segmentation, and analysis of in vivo RPE single cells.

OBJECTIVE: To develop a fully automated method of retinal pigmented epithelium (RPE) cells detection, segmentation and analysis based on in vivo cellular resolution images obtained with the transscleral optical phase imaging method (TOPI). METHODS: Fourteen TOPI-RPE images from 11 healthy individuals were analysed. The developed image processing method encompassed image filtering and normalisation, detection and removal of blood vessels, cell detection and cell membrane segmentation. The produced measures were cellular density of RPE layer, cell area, number of neighbouring cells, eccentricity, circularity and solidity. In addition, we proposed coefficient of variation (CV) of RPE cellular membrane (CMDCV) and the solidity of the RPE cell membrane-shape as new metrics for the assessment of RPE single cells. RESULTS: The observed median cellular density of the RPE layer was 3743 cells/µm2 (interquartile rate (IQR) 1687), with a median observed RPE cell area of 193 µm2 (IQR 141). The mean number of neighbouring cells was 5.22 (standard deviation (SD) 0.05) per RPE cell. The mean RPE cell eccentricity was 0.67 (SD 0.02), median circularity 0.83 (IQR 0.01), and median solidity 0.92 (IQR 0.00). The median CMDCV was 0.19 (IQR 0.02). The method is characterised by a median image processing and analysis time of 48 sec (IQR 12) per image. CONCLUSIONS: The present study provides the first fully automated quantitative assessment of human RPE single cells in vivo. The method provides a baseline for future research in the field of clinical ophthalmology, enabling characterisation and diagnostics of retinal diseases at the single-cell level.

[Anti-TNF-α in the treatment of non-infectious uveitis]. / Les anti-TNF-α pour le traitement des uvéites non infectieuses.

Non-infectious uveitis is a heterogenous group of potentially blinding ocular autoimmune diseases that may represent a manifestation of a systemic condition or may affect the eyes only. A systemically administered anti-TNF has recently been approved for the treatment of non-infectious uveitis, broadening the therapeutic arsenal available to control intraocular inflammation and reduce uveitis complications that can lead to vision loss. When uveitis affects only the eyes, a local anti-TNF-α administration strategy could optimize the ocular therapeutic effect and reduce undesirable systemic side-effects. A new ocular method of non-viral gene therapy, currently in development, may broaden the indications for ocular anti-TNF-α agents, not only for uveitis but also for other diseases in which TNF-α-mediated neuro-inflammation has been demonstrated.

TITLE: Les anti-TNF-α pour le traitement des uvéites non infectieuses. ABSTRACT: Les molécules anti-TNF-α administrés par voie générale ont été approuvés récemment pour le traitement des uvéites non inflammatoires, élargissant l'arsenal thérapeutique dans le traitement de ces pathologies responsables de cécité évitable si l'inflammation est contrôlée. Quand seul l'œil est atteint, des stratégies d'administration locale permettraient d'optimiser les effets intraoculaires des molécules anti-TNF-α et d'en réduire les effets indésirables. Une nouvelle méthode de thérapie génique non virale, actuellement en développement, pourrait élargir les indications des molécules anti-TNF-α oculaires, non seulement pour les uvéites, mais également pour d'autres maladies dans lesquelles une neuro-inflammation impliquant le TNF-α a été démontrée.

Mechanisms of FH Protection Against Neovascular AMD.

A common allele (402H) of the complement factor H (FH) gene is the major risk factor for age-related macular degeneration (AMD), the leading cause of blindness in the elderly population. Development and progression of AMD involves vascular and inflammatory components partly by deregulation of the alternative pathway of the complement system (AP). The loss of central vision results from atrophy and/or from abnormal neovascularization arising from the choroid. The functional link between FH, the main inhibitor of AP, and choroidal neovascularization (CNV) in AMD remains unclear. In a murine model of CNV used as a model for neovascular AMD (nAMD), intraocular human recombinant FH (recFH) reduced CNV as efficiently as currently used anti-VEGF (vascular endothelial growth factor) antibody, decreasing deposition of C3 cleavage fragments, membrane attack complex (MAC), and microglia/macrophage recruitment markers in the CNV lesion site. In sharp contrast, recFH carrying the H402 risk variant had no effect on CNV indicating a causal link to disease etiology. Only the recFH NTal region (recFH1-7), containing the CCPs1-4 C3-convertase inhibition domains and the CCP7 binding domain, exerted all differential biological effects. The CTal region (recFH7-20) containing the CCP7 and CCPs19-20 binding domains was antiangiogenic but did not reduce the microglia/macrophage recruitment. The antiangiogenic effect of both recFH1-20 and recFH-CCP7-20 resulted from thrombospondin-1 (TSP-1) upregulation independently of the C3 cleavage fragments generation. This study provides insight on the mechanistic role of FH in nAMD and invites to reconsider its therapeutic potential.

Glial cells of the human fovea.

Purpose: The exact cellular types that form the human fovea remain a subject of debate, and few studies have been conducted on human macula to solve this question. The purpose of this study was to perform immunohistochemistry on fresh human samples to characterize the glial cells that form the human fovea. Methods: Immunohistochemistry was performed using antibodies against proteins expressed in astrocytes or in retinal Müller glial cells or both types of cells on six human macula obtained from eyes enucleated for peripheral intraocular tumors and on two postmortem eyes from healthy donors. The posterior poles of the enucleated eyes were cryosectioned and stained with antibodies against the glial proteins GFAP, vimentin, CRALBP, glutamine synthetase, and connexin 43. Results: A population of cells positive for GFAP and negative for glutamine synthetase and CRALBP that express connexin 43 were identified at the roof of the foveal pit. These cells are distinct from the Müller cone cells described by Yamada and Gass, suggesting that another type of foveal glial cells, most likely astrocytes, are present in the human fovea. Conclusions: This study showed that in humans, astrocytic glial cells cover the foveal pit. Their roles in macula homeostasis and mechanisms of macular diseases disease remain to be determined.

CHANGES IN VISUAL ACUITY AND PHOTORECEPTOR DENSITY USING ADAPTIVE OPTICS AFTER RETINAL DETACHMENT REPAIR.

PURPOSE: To quantify changes in photoreceptor density using adaptive optics fundus camera in patients after retinal detachment (RD) and to correlate them with macular involvement and best-corrected visual acuity. METHODS: At 1 and 3 months (M1 and M3) after vitrectomy, 194 patients underwent adaptive optics imagery in both eyes, at 5 locations, that we matched between time points using anatomical landmarks. Twenty-two patients (10 fovea-OFF [OFF] and 12 fovea-ON [ON]) had matched and analyzable adaptive optics images. We used analysis of variance for repeated measures. RESULTS: Best-corrected visual acuity (logarithm of the minimum angle of resolution and Snellen equivalent [SE]) was significantly different between OFF and ON RDs at baseline: 2.0 (2.3-0.95) (SE: 20/2000) versus 0 (0.1-0) (SE: 20/20); at M1: 0.35 (0.5-0.1) (SE: 20/40) versus 0.05 (0-0.1) (SE: 20/25); and at M3: 0.25 (0.3-0.1) (SE: 20/32) versus 0 (0-0) (SE: 20/20). We observed that cone density was stable in fellow eyes between M1 and M3 (P = 0.67); decreased in treated eyes than in fellow eyes (P < 0.05); and increased postoperatively in the ON group (P = 0.02) but not in the OFF group (P = 0.97). Visual acuity and RD type were independently correlated with cone density (P = 0.004, P = 0.000). CONCLUSION: Postoperative cone density was reduced in OFF RD, but also in the ON group, although the drop recovered during the 3-month follow-up. Cone density was significantly correlated with both visual acuity and type of RD at both time points.

Ocular Biodistribution of Spironolactone after a Single Intravitreal Injection of a Biodegradable Sustained-Release Polymer in Rats.

Sustained-release formulations for ocular delivery are of increasing interest given their potential to significantly improve treatment efficacy and patient adherence. The objectives of this study were (i) to develop a sustained-release formulation of spironolactone (SPL) using a biodegradable and injectable polymer, hexyl-substituted poly-lactic acid (hexPLA) and (ii) to investigate the ocular biodistribution and tolerability of SPL and its metabolites in rats in vivo over 1 month following a single intravitreal injection (IVT inj). The concentrations of SPL and its two principal active metabolites, 7α-thiomethylspironolactone and canrenone (CAN), in the different ocular compartments were determined at different time points (3, 7, and 31 days after IVT inj) using a validated ultra-high-performance liquid chromatography-mass spectrometry method. Systemic exposure following a single IVT inj of 5% SPL-hexPLA formulation was evaluated by quantifying SPL and its metabolites in the plasma. Ocular tolerability of the formulation was evaluated using in vivo retinal imaging and histology. In vitro release studies revealed a sustained release of SPL from 5% SPL-hexPLA for up to 65 days. In vivo studies showed that SPL and its metabolites were detected in all ocular tissues at 3 and 7 days post-IVT inj. At 31 days post-IVT inj, SPL and CAN were mainly detected in the retina. These results also highlighted the clearance pathway of SPL and its metabolite involving the anterior and posterior routes in the first week (days 3 and 7), then mainly the posterior segment in the last week (day 31). This study showed that a single IVT inj of 5% SPL-hexPLA in rats enabled sustained delivery of therapeutic amounts of SPL for up to 1 month to the retina without systemic exposure. This formulation may be of interest for the local treatment of diseases involving overactivation of the mineralocorticoid receptor in the chorioretina such as chronic central serous chorioretinopathy.

Iron is neurotoxic in retinal detachment and transferrin confers neuroprotection.

In retinal detachment (RD), photoreceptor death and permanent vision loss are caused by neurosensory retina separating from the retinal pigment epithelium because of subretinal fluid (SRF), and successful surgical reattachment is not predictive of total visual recovery. As retinal iron overload exacerbates cell death in retinal diseases, we assessed iron as a predictive marker and therapeutic target for RD. In the vitreous and SRF from patients with RD, we measured increased iron and transferrin (TF) saturation that is correlated with poor visual recovery. In ex vivo and in vivo RD models, iron induces immediate necrosis and delayed apoptosis. We demonstrate that TF decreases both apoptosis and necroptosis induced by RD, and using RNA sequencing, pathways mediating the neuroprotective effects of TF are identified. Since toxic iron accumulates in RD, we propose TF supplementation as an adjunctive therapy to surgery for improving the visual outcomes of patients with RD.

Proteome and Metabolome of Subretinal Fluid in Central Serous Chorioretinopathy and Rhegmatogenous Retinal Detachment: A Pilot Case Study.

PURPOSE: To investigate the molecular composition of subretinal fluid (SRF) in central serous chorioretinopathy (CSCR) and rhegmatogenous retinal detachment (RRD) using proteomics and metabolomics. METHODS: SRF was obtained from one patient with severe nonresolving bullous CSCR requiring surgical subretinal fibrin removal, and two patients with long-standing RRD. Proteins were trypsin-digested, labeled with Tandem-Mass-Tag and fractionated according to their isoelectric point for identification and quantification by tandem mass spectrometry. Independently, metabolites were extracted on cold methanol/ethanol, and identified by untargeted ultra-high performance liquid chromatography and high-resolution mass spectrometry. Bioinformatics analyses were conducted. RESULTS: In total, 291 proteins and 651 metabolites were identified in SRF samples. Compared with RRD, 128 proteins (77 downregulated; 51 upregulated) and 76 metabolites (43 downregulated; 33 upregulated) differed in the SRF from CSCR. Protein and metabolites notably deregulated in CSCR were related to glycolysis/gluconeogenesis, inflammation (including serum amyloid P component, versican), alternative complement pathway (complement factor H and complement factor H-related protein), cellular adhesion, biliary acid metabolism (farnesoid X receptor/retinoid X receptor), and gluco- and mineralocorticoid systems (aldosterone, angiotensin, and corticosteroid-binding globulin). CONCLUSIONS: Proteomics and metabolomics can be performed on SRF. A unique SRF sample from CSCR exhibited a distinct molecular profile compared with RRD. TRANSLATIONAL RELEVANCE: This first comparative multiomics analysis of SRF improved the understanding of CSCR and RRD pathophysiology. It identified pathways potentially involved in the better photoreceptor preservation in CSCR, suggesting neuroprotective targets that will require additional confirmation.

Mechanisms of macular edema: Beyond the surface.

Macular edema consists of intra- or subretinal fluid accumulation in the macular region. It occurs during the course of numerous retinal disorders and can cause severe impairment of central vision. Major causes of macular edema include diabetes, branch and central retinal vein occlusion, choroidal neovascularization, posterior uveitis, postoperative inflammation and central serous chorioretinopathy. The healthy retina is maintained in a relatively dehydrated, transparent state compatible with optimal light transmission by multiple active and passive systems. Fluid accumulation results from an imbalance between processes governing fluid entry and exit, and is driven by Starling equation when inner or outer blood-retinal barriers are disrupted. The multiple and intricate mechanisms involved in retinal hydro-ionic homeostasis, their molecular and cellular basis, and how their deregulation lead to retinal edema, are addressed in this review. Analyzing the distribution of junction proteins and water channels in the human macula, several hypotheses are raised to explain why edema forms specifically in the macular region. "Pure" clinical phenotypes of macular edema, that result presumably from a single causative mechanism, are detailed. Finally, diabetic macular edema is investigated, as a complex multifactorial pathogenic example. This comprehensive review on the current understanding of macular edema and its mechanisms opens perspectives to identify new preventive and therapeutic strategies for this sight-threatening condition.

Tolerance of high and low amounts of PLGA microspheres loaded with mineralocorticoid receptor antagonist in retinal target site.

Mineralocorticoid receptor (MR) contributes to retinal/choroidal homeostasis. Excess MR activation has been shown to be involved in pathogenesis of central serous chorioretinopathy (CSCR). Systemic administration of MR antagonist (MRA) reduces subretinal fluid and choroidal vasodilation, and improves the visual acuity in CSCR patients. To achieve long term beneficial effects in the eye while avoiding systemic side-effects, we propose the use of biodegradable spironolactone-loaded poly-lactic-co-glycolic acid (PLGA) microspheres (MSs). In this work we have evaluated the ocular tolerance of MSs containing spironolactone in rat' eyes. As previous step, we have also studied the tolerance of the commercial solution of canrenoate salt, active metabolite of spironolactone. PLGA MSs allowed in vitro sustained release of spironolactone for 30days. Rat eyes injected with high intravitreous concentration of PLGA MSs (10mg/mL) unloaded and loaded with spironolactone maintained intact retinal lamination at 1month. However enhanced glial fibrillary acidic protein immunostaining and activated microglia/macrophages witness retinal stress were observed. ERG also showed impaired photoreceptor function. Intravitreous PLGA MSs concentration of 2mg/mL unloaded and loaded with spironolactone resulted well tolerated. We observed reduced microglial/macrophage activation in rat retina compared to high concentration of MSs with normal retinal function according to ERG. Spironolactone released from low concentration of MSs was active in the rat retina. Low concentration of spironolactone-loaded PLGA MSs could be a safe therapeutic choice for chorioretinal disorders in which illicit MR activation could be pathogenic.

Nephropathy in Pparg-null mice highlights PPARγ systemic activities in metabolism and in the immune system.

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-dependent transcription factor involved in many aspects of metabolism, immune response, and development. Total-body deletion of the two Pparg alleles provoked generalized lipoatrophy along with severe type 2 diabetes. Herein, we explore the appearance and development of structural and functional alterations of the kidney, comparing Pparg null-mice to their littermate controls (carrying Pparg floxed alleles). We show that renal hypertrophy and functional alterations with increased glucosuria and albuminuria are already present in 3 weeks-old Pparg null-mice. Renal insufficiency with decreased creatinine clearance progress at 7 weeks of age, with the advance of the type 2 diabetes. At 52 weeks of age, these alterations are accompanied by signs of fibrosis and mesangial expansion. More intriguingly, aged Pparg null-mice concomitantly present an anti-phospholipid syndrome (APS), characterized by the late appearance of microthrombi and a mesangioproliferative pattern of glomerular injury, associated with significant plasmatic levels of anti-ß2- glycoprotein1 antibodies and renal deposition of IgG, IgM, and C3. Thus, in line with the role of PPARγ in metabolic homeostasis, Pparg null-mice first represent a potent model for studying the initiation and the development of diabetic nephropathy. Second, and in relation with the important PPARγ activity in inflammation and in immune system, these mice also highlight a new role for PPARγ signaling in the promotion of APS, a syndrome whose pathogenesis is poorly known and whose current treatment is limited to prevention of thrombosis events.

Cell viability and shock wave amplitudes in the endothelium of porcine cornea exposed to ultrashort laser pulses.

PURPOSE: Some forms of keratoplasty assisted by ultrashort-pulse lasers require performing laser cuts close to the endothelium, which requires the knowledge of "safe" values concerning incision depth and pulse energy preserving endothelial cell viability. Our study aims to determine the thresholds for cell death in porcine corneas exposed to ultrashort laser pulses, in terms of laser pulse energy and nearness of the impacts to the endothelium. METHODS: Using a laboratory laser set-up, lamellar cuts were induced while varying pulse energies and distances from the endothelium. A fluorescent staining protocol was used to determine the percentage of surviving endothelial cells. Numerical simulations of the Euler equations for compressible fluids provided pressure level and axial and radial pressure gradient estimates at the endothelium. RESULTS: Ninety percent of the endothelial cells survived when using 16.5 µJ pulses no closer than 200 µm to the endothelium, or pulses not exceeding 2 µJ at a distance of 50 µm. The comparison of the observed percentage of surviving cells with the estimates of the shock wave amplitudes and gradients generated by the laser pulses yielded cell death thresholds at amplitudes in the megapascal range, or gradients of the order of 108 Pa/m. CONCLUSIONS: Our results provide limits in terms of pulse energy and distance of the incision from the endothelium within which endothelial cell viability is preserved. Current forms of corneal laser surgery are compatible with these limits. However, these limits will need to be considered for the development of future laser routines working in close proximity to the endothelium.

EFFICACY OF INTRAVITREAL AFLIBERCEPT IN MACULAR TELANGIECTASIA TYPE 1 IS LINKED TO THE OCULAR ANGIOGENIC PROFILE.

PURPOSE: To evaluate intravitreal aflibercept in macular telangiectasia Type 1 (MacTel 1) patients and measure their ocular angiogenic profile. METHODS: Eight subjects with MacTel 1 refractory to bevacizumab, ranibizumab, or laser therapy and switched to aflibercept were included. Best-corrected visual acuity, central macular thickness, and cystic areas quantified on optical coherence tomography B-scans were assessed during 12 months. Perifoveal capillary densities were measured on optical coherence tomography angiography. Aqueous humor was sampled from six patients and eight control subjects undergoing cataract extraction. Growth factors were quantified using a multiarray immunoassay. RESULTS: Over 12 months, patients received 6.6 ± 1.4 (range, 5-8) intravitreal aflibercept injections. Twelve months after switching to aflibercept, best-corrected visual acuity increased by ≥5 letters in 5 of 8 patients, compared with preaflibercept levels. Mean best-corrected visual acuity improved from 79.6 (∼20/50) to 88.0 (∼20/35) Early Treatment Diabetic Retinopathy Study letters (P = 0.042), and central macular thickness decreased from 434 ± 98 µm to 293 ± 59 µm (P = 0.014). Compared with control subjects, the profile of angiogenic factors in MacTel 1 eyes revealed no difference in vascular endothelial growth factor-A levels but significantly higher levels of placental growth factor (P = 0.029), soluble vascular endothelial growth factor receptor-1 (sFlt-1; P = 0.013), vascular endothelial growth factor-D (P = 0.050), and Tie-2 (P = 0.019). Placental growth factor levels inversely correlated with both superficial and deep capillary plexus densities on optical coherence tomography angiography (P = 0.03). CONCLUSION: The clinical response to aflibercept coupled to the angiogenic profile of MacTel 1 eyes support the implication of the placental growth factor/Flt-1 pathway in MacTel 1.

Anti-Inflammatory Effect of Dexamethasone Controlled Released From Anterior Suprachoroidal Polyurethane Implants on Endotoxin-Induced Uveitis in Rats.

PURPOSE: Targeted drug delivery to the ocular tissues remains a challenge. Biodegradable intraocular implants allow prolonged controlled release of drugs directly into the eye. In this study, we evaluated an anterior suprachoroidal polyurethane implant containing dexamethasone polyurethane dispersions (DX-PUD) as a drug delivery system in the rat model of endotoxin-induced uveitis (EIU). METHODS: In vitro drug release was studied using PUD implants containing 8%, 20%, and 30% (wt/wt) DX. Cytotoxicity of the degradation products of DX-PUD was assessed on human ARPE-19 cells using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) test. Short-term ocular biocompatibility of suprachoroidal DX-PUD implants was evaluated in normal rat eyes. Endotoxin-induced uveitis was then induced in rat eyes preimplanted with DX-PUD. Clinical examination was performed at 24 hours; eyes were used to assess inflammatory cell infiltration and macrophage/microglial activation. Cytokine and chemokine expression in the iris/ciliary body and in the retina was investigated using quantitative PCR. Feasibility of anterior suprachoroidal PUD implantation was also tested using postmortem human eyes. RESULTS: A burst release was followed by a sustained controlled release of DX from PUD implants. By-products of the DX-PUD were not toxic to human ARPE-19 cells or to rat ocular tissues. Dexamethasone-PUD implants prevented EIU in rat eyes, reducing inflammatory cell infiltration and inhibiting macrophage/microglial activation. Dexamethasone-PUD downregulated proinflammatory cytokines/chemokines (IL-1ß, IL-6, cytokine-induced neutrophil chemoattractant [CINC]) and inducible nitric oxide synthase (iNOS) and upregulated IL-10 anti-inflammatory cytokine. Polyurethane dispersion was successfully implanted into postmortem human eyes. CONCLUSIONS: Dexamethasone-PUD implanted in the anterior suprachoroidal space may be of interest in the treatment of intraocular inflammation.

A new CRB1 rat mutation links Müller glial cells to retinal telangiectasia.

We have identified and characterized a spontaneous Brown Norway from Janvier rat strain (BN-J) presenting a progressive retinal degeneration associated with early retinal telangiectasia, neuronal alterations, and loss of retinal Müller glial cells resembling human macular telangiectasia type 2 (MacTel 2), which is a retinal disease of unknown cause. Genetic analyses showed that the BN-J phenotype results from an autosomal recessive indel novel mutation in the Crb1 gene, causing dislocalization of the protein from the retinal Müller glia (RMG)/photoreceptor cell junction. The transcriptomic analyses of primary RMG cultures allowed identification of the dysregulated pathways in BN-J rats compared with wild-type BN rats. Among those pathways, TGF-ß and Kit Receptor Signaling, MAPK Cascade, Growth Factors and Inflammatory Pathways, G-Protein Signaling Pathways, Regulation of Actin Cytoskeleton, and Cardiovascular Signaling were found. Potential molecular targets linking RMG/photoreceptor interaction with the development of retinal telangiectasia are identified. This model can help us to better understand the physiopathologic mechanisms of MacTel 2 and other retinal diseases associated with telangiectasia.