27 research outputs found
IMAGING AND BIOLOGICAL MARKERS IN RETINAL DISORDERS TO ASSESS GENE THERAPY SAFETY AND INVESTIGATE VASCULAR DISEASE MECHANISMS
The retina is the neurosensory tissue responsible for the acquisition of visual stimuli. It is biologically separated from the systemic circulation by blood-retinal barriers, limiting the possibility for circulating markers to reflect retinal changes due to disease or therapeutic intervention. However, due to the transparency of ocular media, the retina is highly accessible to high-resolution imaging, and image processing provides access to physiological parameters quantitatively. In addition, the analysis of ocular media sampled during surgical procedures provides access to biological data regarding disease processes. In this work, imaging and biological markers were developed for several experimental and clinical situations: a gene therapy preclinical safety study (Project 1); the analysis of disease mechanisms in the choroical choroidal vascular disorder central serous chorioretinopathy (CSCR) (Project 2), and a similar translational approach in retinal vascular telangiectatic disorders (Project 3). Specific image processing algorithms were designed.
In Project 1, the preclinical safety of a lentiviral subretinal gene therapy for RPE65 replacement in Leber congenital amaurosis, LV-RPE65, was assessed on healthy non-human primates by conventional methods (in vivo electrophysiology, ex vivo immunohistochemistry, systemic biodistribution study) combined to in vivo analysis of the retinal structure by optical coherence tomography (OCT) at different timepoints, including early follow-up within 7 days. Imaging techniques revealed a transient and pronounced inflammatory process linked to LV-RPE65 injection that delayed retinal reattachment. Partial and transient photoreceptor loss was observed in the macular region, that was to a lesser extent also observed in control eyes injected with the vehicle. This work highlights the need to improve the surgical procedure for subretinal gene therapy delivery, and to consider using anti-inflammatory agents to prevent damaging processes occurring rapidly after subretinal injection.
In Project 2, mechanisms of CSCR, a retinal disease caused by choroidal vessel dilation leading to subretinal fluid accumulation, were explored. We analyzed predictive multimodal imaging factors of episode duration (2a) and recurrence (2b), evidencing in particular choroidal thickness as prognosis factor. Non-invasive OCT angiography images of the choriocapillaris, the innermost layer of the choroid beneath the retinal pigment epithelium, were processed to detect flow voids and investigate their distribution (2c). Finally, the molecular composition of subretinal fluid from a unique case of CSCR requiring subretinal surgery, was explored using a multi-omics approach (2d).
In Project 3, mechanisms of retinal vasculopathy were investigated in two pure phenotypes represented by telangiectatic disorders: type 1 macular telangiectasia (Mactel 1) (3a and 3b), and radiation maculopathy (3c). For the investigation of Mactel 1, image processing tools were used to compute global and local capillary density on OCT angiography images, showing that non-perfusion is a critical feature in Mactel 1, related to visual outcome and telangiectasia formation. This approach was combined to the biological investigation of aqueous humor from Mactel 1 cases. Intraocular levels of angiogenic factors demonstrated the involvement of placental growth factor in the pathophysiology of MacTel 1, that was correlated with multimodal imaging findings (3b). Finally, an image processing algorithm was designed and applied to radiation maculopathy, to compute automatically the fractal dimension of OCT angiography images. This parameter was relevant in assessing capillary network disruption, and demonstrated that alterations of the deep plexus influence independently visual function.
The strategies developed throughout these three projects demonstrate the interest of quantitative image analysis for the investigation of retinal disorders, and the possibility of coupling imaging and biological data. This approach contributed to identify potential imaging or biological markers for diagnosis, prognosis, therapeutic response and toxicity in several biomedical situations.
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La rétine est un tissu neurosensoriel responsable de l'acquisition des signaux visuels. Elle se trouve biologiquement séparée de la circulation systémique par les barrières hémato-rétiniennes, limitant la possibilité pour des marqueurs circulants de refléter des altérations du tissu rétinien, dus à des maladies ou secondaires à des interventions thérapeutiques. Cependant, en raison de la transparence des milieux oculaires, la rétine est accessible à l'imagerie haute résolution, et l’analyse d'images permet d’extraire des paramètres physiologiques quantitatifs. En outre, l'analyse des milieux oculaires prélevés au cours d’interventions chirurgicales permet d'accéder à des données biologiques concernant les processus physiopathologiques. Dans ce travail, des marqueurs biologiques et d'imagerie ont été développés pour plusieurs situations expérimentales et cliniques : une étude de sécurité préclinique en thérapie génique (Projet 1), l'analyse de mécanismes pathologiques dans la choriorétinopathie séreuse centrale (CRSC) (Projet 2), et dans les pathologies télangiectatiques vasculaires rétiniennes (Projet 3). Des algorithmes de traitement d'image spécifiques ont été conçus.
Dans le Projet 1, la tolérance préclinique d'une thérapie génique sous-rétinienne lentivirale pour remplacement du gène RPE65 dans l'amaurose congénitale de Leber, LV-RPE65, a été évaluée sur des primates non humains sains par des méthodes conventionnelles (électrophysiologie in vivo, immunohistochimie ex vivo, étude de biodistribution systémique), et par analyse in vivo de la structure rétinienne par tomographie par cohérence optique (OCT) à différents points, y compris un suivi précoce dans les 7 jours. Les techniques d'imagerie ont révélé un processus inflammatoire transitoire lié à l'injection de LV-RPE65 qui a retardé le réattachement rétinien. Une perte partielle et transitoire des photorécepteurs a été observée dans la région maculaire, détécté également, et dans une moindre mesure dans les yeux témoins, injectés avec la solution véhicule. Ce travail souligne la nécessité d'améliorer la procédure chirurgicale pour l’administration de thérapies géniques sous- rétiniennes, et d'envisager l’usage d’agents anti-inflammatoires pour limiter ces altérations.
Dans le projet 2, les mécanismes de la CRSC, une maladie rétinienne causée par la dilatation des vaisseaux choroïdiens menant à l'accumulation de liquide sous-rétinien, ont été explorés. Nous avons analysé les facteurs d'imagerie multimodaux prédictifs de la durée des épisodes (2a) et de récurrence (2b), mettant en évidence notamment l'épaisseur choroïdienne comme facteur pronostic. Des images en OCT angiographie, non invasive, de la choriocapillaire, la couche la plus interne de la choroïde sous l'épithélium pigmentaire rétinien, ont été traitées pour détecter des lacunes dans le flux sanguin, et étudier leur distribution (2c). Enfin, la composition moléculaire du liquide sous-rétinien d'un cas rare de CRSC nécessitant une chirurgie sous-rétinienne, a été explorée en utilisant une approche multi- omique collaborative (2d).
Dans le projet 3, les mécanismes de vasculopathie rétinienne ont été étudiés dans deux phénotypes purs représentés par les troubles télangiectasiques : télangiectasie maculaire de type 1 (Mactel 1) (3a et 3b), et maculopathie radique (3c). Pour l'étude de Mactel 1, des outils de traitement d'images ont été utilisés pour calculer la densité capillaire globale et locale sur des images d’OCT angiographie, montrant que la non-perfusion est un paramètre critique dans les Mactel 1, corrélé à la fonction visuelle et à la formation des télangiectasies. Cette approche a été combinée à l'étude biologique de l'humeur aqueuse dans des cas de Mactel 1. Des niveaux intraoculaires de facteurs angiogéniques ont démontré l'implication du facteur de croissance placentaire dans la physiopathologie de MacTel1. De plus, ce facteur était corrélé avec la densité capillaire en OCT angiographie (3b). Enfin, un algorithme de traitement d'images a été conçu et appliqué à la maculopathie radique pour calculer automatiquement la dimension fractale des images d’OCT angiographie. Ce paramètre était pertinent dans l'évaluation de la perturbation du réseau capillaire, et a démontré que les altérations du plexus profond influencent indépendamment la fonction visuelle.
Les stratégies développées dans ce travail démontrent l'intérêt de l'analyse d'image quantitative pour l'étude des pathologies rétiniennes, et la possibilité de coupler l'imagerie et les données biologiques. Cette approche a permis d'identifier des marqueurs biologiques ou d'imagerie potentiels pour le diagnostic, le pronostic, la réponse thérapeutique et la toxicité dans les différentes situations étudiées
En Face OCT Imaging for the Diagnosis of Outer Retinal Tubulations in Age-Related Macular Degeneration
Purpose. “En face” is an emerging imaging technique derived from spectral domain optical coherence tomography (OCT). It produces frontal sections of retinal layers, also called “C-scan OCT.” Outer retinal tubulations (ORTs) in age-related macular degeneration (AMD) are a recent finding evidenced by spectral-domain OCT. The aim of this study is to characterize the morphology of ORT according to the form of AMD, using “en-face” spectral domain OCT. Methods. “En face” OCT imaging was prospectively performed in 26 consecutive eyes with AMD that also had ORT. Results. There were 15 neovascular, 8 atrophic, and 3 eyes with a mixed (fibrotic and atrophic) form of AMD. Among the neovascular group, the most frequent tubulation pattern on “en-face” OCT was a branching network emanating from a fibrovascular scar; we term this pattern as “pseudodendritic.” It did not require treatment when observed as an isolated finding. In all cases of atrophic AMD, the tubular network was located at the edge of the geographic atrophy area, and formed a “perilesional” pattern. Six atrophic cases showed tubular invaginations inside this area. Conclusion. “En face” OCT is a valuable technique in the diagnosis and followup of macular disease. It revealed the main characteristic patterns of ORT associated with neovascular and atrophic AMD
Escape of HIV-1 from a Small Molecule CCR5 Inhibitor Is Not Associated with a Fitness Loss
Fitness is a parameter used to quantify how well an organism adapts to its environment; in the present study, fitness is a measure of how well strains of human immunodeficiency virus type 1 (HIV-1) replicate in tissue culture. When HIV-1 develops resistance in vitro or in vivo to antiretroviral drugs such as reverse transcriptase or protease inhibitors, its fitness is often impaired. Here, we have investigated whether the development of resistance in vitro to a small molecule CCR5 inhibitor, AD101, has an associated fitness cost. To do this, we developed a growth-competition assay involving dual infections with molecularly cloned viruses that are essentially isogenic outside the env genes under study. Real-time TaqMan quantitative PCR (QPCR) was used to quantify each competing virus individually via probes specific to different, phenotypically silent target sequences engineered within their vif genes. Head-to-head competition assays of env clones derived from the AD101 escape mutant isolate, the inhibitor-sensitive parental virus, and a passage control virus showed that AD101 resistance was not associated with a fitness loss. This observation is consistent with the retention of the resistant phenotype when the escape mutant was cultured for a total of 20 passages in the absence of the selecting compound. Amino acid substitutions in the V3 region of gp120 that confer complete AD101 resistance cause a fitness loss when introduced into an AD101-sensitive, parental clone; however, in the resistant isolate, changes elsewhere in env that occurred prior to the substitutions within V3 appear to compensate for the adverse effect of the V3 changes on replicative capacity. These in vitro studies may have implications for the development and management of resistance to other CCR5 inhibitors that are being evaluated clinically for the treatment of HIV-1 infection
Sustained-Release Steroids for the Treatment of Diabetic Macular Edema
Glucocorticoids have been used for decades in the treatment of ocular disorders via topical, periocular, and more recently intravitreal routes. However, their exact mechanisms of action on ocular tissues remain imperfectly understood. Fortunately, two recently approved intravitreal sustained-release drug delivery systems have opened new perspectives for these very potent drugs. To date, among other retinal conditions, their label includes diabetic macular edema, for which a long-lasting therapeutic effect has been demonstrated both morphologically and functionally in several randomized clinical trials. The rate of ocular complications of intravitreal sustained-release steroids, mainly cataract formation and intraocular pressure elevation, is higher than with anti-vascular endothelial growth factor agents. Yet, a better understanding of the mechanisms underlying these adverse effects and the search for the minimal efficient dose should help optimize their therapeutic window
Two-year follow-up of mineralocorticoid receptor antagonists for chronic central serous chorioretinopathy
To evaluate the long-term oral mineralocorticoid receptor antagonist (MRa) treatment in chronic central serous chorioretinopathy (CSC).
Patients with chronic non-resolving CSC (defined as foveal subretinal fluid (SRF) lasting >4 months with retinal pigment epithelium (RPE) alterations) treated with MRa only (eplerenone or spironolactone) for at least 6 months were retrospectively included. Clinical and imaging characteristics were recorded during visits at baseline, 6, 12, 18 and 24 months.
Sixteen eyes of 16 patients were included (mean age 53±11 years; 14 men, 2 women). Mean duration of SRF before treatment initiation was 11.2±19.7 months. MRa treatment was administered during 21.0±5.1 months (range, 10-24 months). There was a progressive improvement of visual acuity (p=0.05), a decrease of foveal SRF height (p=0.011), central macular thickness (p=0.004) and subfoveal choroidal thickness (p=0.002) over 24 months. Changes in SRF were correlated with subfoveal choroidal thickness at 24 months (p=0.006, Spearman r=065). The mean time to complete foveal SRF resolution was 10.5±8.0 months after treatment initiation. At 24 months, foveal SRF resolution was achieved in 13 eyes (81%). Minor side effects occurred in five patients (31%) and resolved after switching between MRa.
The visual and anatomical benefit of MRa treatment prolonged for 6 months or more in chronic, non-resolving CSC appeared to be maintained over a 24-month period. These results suggest that MRa can be proposed as an alternative therapy in severe CSC with advanced RPE alterations
Ocular Spiroplasma Ixodetis in Newborns, France
International audienceCataract and uveitis are rare in newborns but potentially blinding. Three newborns with cataract and severe anterior uveitis underwent cataract surgery. Spiroplasma ixodetis was detected in lens aspirates using bacterial 16S-rRNA PCR and transmission electron microscopy. These findings, which suggest maternal-fetal infection, are consistent with previous experimental Spiroplasma-induced cataract and uveitis
Multimodal imaging including semiquantitative short-wavelength and near-infrared autofluorescence in achromatopsia
Abstract Multimodal imaging provides insights into phenotype and disease progression in inherited retinal disorders. Congenital achromatopsia (ACHM), a cone dysfunction syndrome, has been long considered a stable condition, but recent evidence suggests structural progression. With gene replacement strategies under development for ACHM, there is a critical need for imaging biomarkers to define progression patterns and follow therapy. Using semiquantitative plots, near-infrared (NIR-AF) and short-wavelength autofluorescence (SW-AF) were explored and correlated with clinical characteristics and retinal structure on optical coherence tomography (OCT). In sixteen ACHM patients with genetic confirmation (CNGA3, n = 8; CNGB3, n = 7; PDE6C, n = 1), semiquantitative plots allowed the detailed analysis of autofluorescence patterns, even in poorly fixating eyes. Twelve eyes showed perifoveal hyperautofluorescent rings on SW-AF, and 7 eyes had central hypoautofluorescent areas on NIR-AF, without association between these alterations (P = 0.57). Patients with central NIR-AF hypoautofluorescence were older (P = 0.004) and showed more advanced retinal alterations on OCT than those with normal NIR-AF (P = 0.051). NIR-AF hypoautofluorescence diameter was correlated to patient age (r = 0.63, P = 0.009), size of ellipsoid zone defect on OCT (r = 0.67, P = 0.005), but not to the size of SW-AF hyperautofluorescence (P = 0.27). These results demonstrate the interest of NIR-AF as imaging biomarker in ACHM, suggesting a relationship with age and disease progression
Role of inflammation in a rat model of radiation retinopathy
Abstract Radiation retinopathy (RR) is a major side effect of ocular tumor treatment by plaque brachytherapy or proton beam therapy. RR manifests as delayed and progressive microvasculopathy, ischemia and macular edema, ultimately leading to vision loss, neovascular glaucoma, and, in extreme cases, secondary enucleation. Intravitreal anti-VEGF agents, steroids and laser photocoagulation have limited effects on RR. The role of retinal inflammation and its contribution to the microvascular damage occurring in RR remain incompletely understood. To explore cellular and vascular events after irradiation, we analyzed their time course at 1 week, 1 month and 6 months after rat eyes received 45 Gy X-beam photons. Müller glial cells, astrocytes and microglia were rapidly activated, and these markers of retinal inflammation persisted for 6 months after irradiation. This was accompanied by early cell death in the outer retina, which persisted at later time points, leading to retinal thinning. A delayed loss of small retinal capillaries and retinal hypoxia were observed after 6 months, indicating inner blood‒retinal barrier (BRB) alteration but without cell death in the inner retina. Moreover, activated microglial cells invaded the entire retina and surrounded retinal vessels, suggesting the role of inflammation in vascular alteration and in retinal cell death. Radiation also triggered early and persistent invasion of the retinal pigment epithelium by microglia and macrophages, contributing to outer BRB disruption. This study highlights the role of progressive and long-lasting inflammatory mechanisms in RR development and demonstrates the relevance of this rat model to investigate human pathology
Effect of eplerenone on choroidal blood flow changes during isometric exercise in patients with chronic central serous chorioretinopathy
To investigate choroidal blood flow changes after isometric exercise in patients with chronic central serous chorioretinopathy nontreated or treated with mineralocorticoid receptor antagonists (MRA). Foveolar choroidal laser Doppler flowmetry parameters – velocity (ChVel), volume (ChVol) and blood flow (ChBF) – of 22 eyes of 22 treated patients, 16 eyes of 16 untreated patients and 19 healthy controls were measured during a squatting test. Treatment consisted in MRA administration (eplerenone 50 mg/day or spironolactone 50 mg/day). The experiment comprised three successive periods: 30 seconds of rest, 2 min of continuous squatting exercise, and 150 seconds of recovery. Significance levels were calculated using a generalized estimating equation. During the squatting period, nontreated CSCR eyes had a similar change in ChVel (p = 0.8), ChVol (p = 0.8), ChBF (p = 0.5) and resistance to healthy eyes. Treated CSCR eyes exhibited significantly smaller changes in ChVel (−0.1 ± 11%, p = 0.04) than healthy eyes (6 ± 8%). No significant difference was found for ChVol and ChBF between the groups. The increase in ChVol from baseline in the nontreated CSCR group (4.4 ± 9%) was lower than that of treated group (6.7%±11%; p = 0.01). Finally, ChBF and ChVel changes in the CSCR groups were not significantly different. No abnormalities were detected in the changes in ChBF parameters during increased ocular perfusion pressure in nontreated CSCR patients compared with controls. MRA treatment in CSCR patients induced a significant reduction in ChBVel and an increase in ChBVol in response to isometric exercise, suggesting that MRA exerts effects on choroidal vascular changes