25 research outputs found

    IMAGING AND BIOLOGICAL MARKERS IN RETINAL DISORDERS TO ASSESS GENE THERAPY SAFETY AND INVESTIGATE VASCULAR DISEASE MECHANISMS

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    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. -- 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

    OCT Angiography (OCTA) in Retinal Diagnostics

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    Optical coherence tomography angiography (OCTA) is an imaging modality which can be applied in ophthalmology to provide detailed visualization of the perfusion of vascular networks in the eye. Compared to previous state of the art dye-based imaging, such as fluorescein angiography, OCTA is non-invasive, time-efficient, and it allows for the examination of retinal vasculature in 3D. These advantages of the technique combined with the good usability in commercial devices led to a quick adoption of the new modality in the clinical routine. However, the interpretation of OCTA data is not without problems: Commonly observed image artifacts and the quite involved algorithmic details of OCTA signal construction can make the clinical assessment of OCTA exams challenging. In this article we describe the technical background of OCTA and discuss the data acquisition process, common image visualization techniques, as well as limitations and sources of artifacts of the modality. Examples of clinical cases underline the increasing importance of the OCTA technology in ophthalmology and its relation to dye-based angiography

    Clinical outcomes of ranibizumab treatment in diabetic eye disease

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    Background: The vascular endothelial growth factor (VEGF) inhibitor ranibizumab is emerging as an efficacious treatment for diabetic macular oedema. Large clinical trials have shown improvements in visual acuity and reduced central retinal thickness. Details of its effect on other retinal functional parameters are lacking. There is a concern that repeated ranibizumab treatment could exacerbate macular ischaemia or lead to global retinal dysfunction by inhibiting physiological isoforms of VEGF. Outcomes of surgery for advanced proliferative retinopathy remain variable and post-operative complications including recurrent haemorrhage can limit visual recovery. VEGF is strongly implicated in the pathogenesis of advanced retinopathy, so VEGF inhibition prior to surgery may improve outcomes. Trials have failed to demonstrate a clear benefit for bevacizumab, so investigation of the licensed intraocular agent ranibizumab represents a logical next step. Aims: To investigate the effects of ranibizumab and laser treatment in diabetic macular oedema on the following parameters: visual acuity, protan and tritan colour contrast sensitivity, 4° and 12° macular sensitivity by microperimetry, electrophysiological indices from pattern and full field electroretinograms. To report structural retinal changes following ranibizumab and laser treatment in terms of qualitative and quantitative optical coherence tomography outcomes, and to quantify macular ischaemia by fluorescein angiography. To investigate the effect on visual acuity at three months post-surgery of ranibizumab pre-treatment in patients undergoing vitrectomy for advanced proliferative diabetic retinopathy. Methods: Randomised clinical trial of intravitreal ranibizumab vs. laser in 36 subjects with centre-involving diabetic macular oedema (The LUCIDATE study). Randomised clinical trial of pre-operative intravitreal ranibizumab vs. subconjunctival saline injection in 30 subjects undergoing vitrectomy-delamination for advanced proliferative diabetic retinopathy (The RaDiVit study). Results: Thirty six subjects with diabetic macular oedema were recruited and 33 completed the trial. Ranibizumab treated subjects gained a mean of 6 letters compared with 0.9 letter loss for laser at 48 weeks. Retinal sensitivity improved in the central macular 4° and 12° in both groups but to a greater extent with ranibizumab. There was no evidence of worsening global retinal dysfunction by electroretinograms in either group. Retinal thickness decreased in both groups: there was a 132 µm reduction in central macular thickness with ranibizumab compared with 103 µm for laser. Fluorescein angiography showed no evidence of significantly increased macular ischaemia in either group. Thirty subjects with advanced proliferative diabetic retinopathy were recruited, underwent surgery, and completed the study. At three months post-surgery, visual acuity in the ranibizumab group was 53 letters compared with 47 letters in the control group. Conclusion: In diabetic macular oedema, there is evidence that ranibizumab leads to greater improvements in visual acuity and retinal sensitivity than laser, with a corresponding greater reduction in retinal thickness. There is no evidence that it worsens macular ischaemia or indices of global retinal electrophysiological function, but larger trials designed to address each of the outcomes investigated here would be required to confirm these findings. In proliferative diabetic retinopathy, there is evidence from this small pilot study that ranibizumab treatment leads to better visual acuity at 3 months post-surgery. An appropriately powered trial would be required to confirm this

    Clinical Phenotypes and Cellular Mediators in Diabetic Retinopathy

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    The aim of this work was to establish meaningful clinical end-points and surrogate markers for diabetic macular ischaemia, a condition for which there is no treatment. The relationship between diabetic eye disease and circulating cellular mediators of angiogenesis and inflammation was further explored with a view to developing therapy in the longer term. Visual loss in diabetic macular ischaemia was observed to occur only in moderate to severe disease, progresses at a rate of 5-10% increase in area per year and associated with thinning of the retinal nerve fibre layer. Direct visualisation of cells in the vitreous was achieved using optical coherence tomography. Novel methods for this were further developed in inflammatory eye disease, with a view for application in diabetic eye disease. A method for in vivo labelling of cells using ICG to enhance visualisation was described. In the field of regenerative medicine, this technique may allow direct visualisation of cell-mediated inflammation regardless of the type of cell or tissue transplanted. EPC and monocyte profiles were analysed in the context of diabetic eye disease. Elevated levels of EPCs as defined by CD34+ CD309+ were observed in diabetes, but no associations were observed with progression. There were no initial associations between monocyte subsets and diabetic eye disease severity at the outset but differences were observed in the context of progression. Observations from this work support the notion that inflammation plays an important role in diabetic eye disease and will inform development of new treatments in this field

    Clinical Phenotypes and Cellular Mediators in Diabetic Retinopathy

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    The aim of this work was to establish meaningful clinical end-points and surrogate markers for diabetic macular ischaemia, a condition for which there is no treatment. The relationship between diabetic eye disease and circulating cellular mediators of angiogenesis and inflammation was further explored with a view to developing therapy in the longer term. Visual loss in diabetic macular ischaemia was observed to occur only in moderate to severe disease, progresses at a rate of 5-10% increase in area per year and associated with thinning of the retinal nerve fibre layer. Direct visualisation of cells in the vitreous was achieved using optical coherence tomography. Novel methods for this were further developed in inflammatory eye disease, with a view for application in diabetic eye disease. A method for in vivo labelling of cells using ICG to enhance visualisation was described. In the field of regenerative medicine, this technique may allow direct visualisation of cell-mediated inflammation regardless of the type of cell or tissue transplanted. EPC and monocyte profiles were analysed in the context of diabetic eye disease. Elevated levels of EPCs as defined by CD34+ CD309+ were observed in diabetes, but no associations were observed with progression. There were no initial associations between monocyte subsets and diabetic eye disease severity at the outset but differences were observed in the context of progression. Observations from this work support the notion that inflammation plays an important role in diabetic eye disease and will inform development of new treatments in this field

    Central serous chorioretinopathy: An evidence-based treatment guideline.

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    Central serous chorioretinopathy (CSC) is a relatively common disease that causes vision loss due to macular subretinal fluid leakage and it is often associated with reduced vision-related quality of life. In CSC, the leakage of subretinal fluid through defects in the retinal pigment epithelial layer's outer blood-retina barrier appears to occur secondary to choroidal abnormalities and dysfunction. The treatment of CSC is currently the subject of controversy, although recent data obtained from several large randomized controlled trials provide a wealth of new information that can be used to establish a treatment algorithm. Here, we provide a comprehensive overview of our current understanding regarding the pathogenesis of CSC, current therapeutic strategies, and an evidence-based treatment guideline for CSC. In acute CSC, treatment can often be deferred for up to 3-4 months after diagnosis; however, early treatment with either half-dose or half-fluence photodynamic therapy (PDT) with the photosensitive dye verteporfin may be beneficial in selected cases. In chronic CSC, half-dose or half-fluence PDT, which targets the abnormal choroid, should be considered the preferred treatment. If PDT is unavailable, chronic CSC with focal, non-central leakage on angiography may be treated using conventional laser photocoagulation. CSC with concurrent macular neovascularization should be treated with half-dose/half-fluence PDT and/or intravitreal injections of an anti-vascular endothelial growth factor compound. Given the current shortage of verteporfin and the paucity of evidence supporting the efficacy of other treatment options, future studies-ideally, well-designed randomized controlled trials-are needed in order to evaluate new treatment options for CSC

    Clinical Perspectives and Trends: Microperimetry as a trial endpoint in retinal disease

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    Endpoint development trials are underway across the spectrum of retinal disease. New validated endpoints are urgently required for the assessment of emerging gene therapies and in preparation for the arrival of novel therapeutics targeting early stages of common sight-threatening conditions such as age-related macular degeneration. Visual function measures are likely to be key candidates in this search. Over the last two decades, microperimetry has been used extensively to characterize functional vision in a wide range of retinal conditions, detecting subtle defects in retinal sensitivity that precede visual acuity loss and tracking disease progression over relatively short periods. Given these appealing features, microperimetry has already been adopted as an endpoint in interventional studies, including multicenter trials, on a modest scale. A review of its use to date shows a concurrent lack of consensus in test strategy and a wealth of innovative disease and treatment-specific metrics which may show promise as clinical trial endpoints. There are practical issues to consider, but these have not held back its popularity and it remains a widely used psychophysical test in research. Endpoint development trials will undoubtedly be key in understanding the validity of microperimetry as a clinical trial endpoint, but existing signs are promising

    Functional outcome of retinal oedema and its standard treatment

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    Macular oedema is a pathological condition of fluid accumulation in the retinal tissues. It is a nonspecific sign of several retinal diseases that in the long term can lead to permanent vision loss. The clinical aspect of macular oedema treatment and vision recovery is reduction of the amount of fluid accumulated in the retina. Due to its complex pathophysiological mechanism, macular oedema has proven challenging to manage. Many unanswered questions remain in the ophthalmology world on this subject. The development of recent diagnostic tools such as optical coherence tomography allows better understanding of the morphological changes in the retina. Now we are able to detect retinal oedema and characterise it by location, depth, and amount of fluid. Further, clinicians are now able to assess therapeutic response by examining the anatomical structures of the retina. Yet, with techniques offering objective accuracy, emerging reports have shown discrepancies between clinically examined visual acuity, anatomical changes of the retina, and patients’ self-reported visual ability. The presence of such discrepancies is also supported by the fact that results achieved by randomised clinical trials rarely align with results attained in real-world settings. Today, functional vision testing can be performed with several different methods including questionnaires, colour vision tests, reading speed tests, contrast sensitivity tests etc. Nevertheless, none of these methods are widely used in clinical settings, and their predictive capabilities have yet to be explored. Establishing precise methodology for functional vision testing is likely to provide better understanding of patients’ treatment response. This thesis aims to investigate the potential predictive capabilities of functional vision tests and to compare these capabilities with those of well-established, routine ophthalmic examinations such as visual acuity and retinal thickness tests. In the current research, I focused on the following functional examinations: the visual function questionnaire (VFQ- 25), reading speed testing, and testing of the contrast sensitivity of the macula area (examined by microperimetry). These techniques allowed very specific and sensitive testing of the functionality of the retina. In addition, I explored functional vision tests and their association to the routine ophthalmic tests and their ability to detect sub-clinical changes in vision. I believe further research in this area will offer better understanding of the functional vision changes in patients with macular oedema and potentially will help in improving visionrelated quality of life

    Estudio mediante OCT del comportamiento de los puntos hiperreflectivos en el edema macular uveítico no infeccioso

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    186 p.Las uveítis no infecciosas son un grupo heterogéneo de enfermedades inflamatorias intraoculares de origen autoinmune. Puede presentarse en el contexto de una enfermedad autoinmune sistémica o bien aisladamente. Afecta con mayor frecuencia a pacientes jóvenes o de mediana edad y la causa más frecuente de pérdida de visión es el edema macular. La Tomografía de Coherencia Óptica es una técnica diagnóstica de imagen no invasiva que permite evaluar las características del edema macular como el grosor de la mácula y la presencia de puntos hiperreflectivos, que se cree corresponden a células de la microglía. En esta tesis hemos evaluado el comportamiento de dichos puntos a lo largo del tiempo en cuanto a su número y distribución en las capas de la retina tras el tratamiento del edema macular y hemos valorado su utilidad como biomarcadores pronóstico. Hemos hallado que la disminución del grosor macular central se asocia tanto con la redistribución hacia las capas más internas de la retina de los puntos, como con la reducción del número total de los mismos. El número de puntos hiperreflectivos se reduce de forma paralela al desplazamiento de dichos puntos a las capas más internas de la retina en pacientes con reducción del grosor macular central. La agudeza visual sin embargo, no se asoció con el número de puntos hiperreflectivos ni con la distribución de los mismos en la retina

    Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium Transplantation in Advanced Neovascular Age-Related Macular Degeneration

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    Age-related macular degeneration (AMD) remains one of the leading causes of permanent vision impairment worldwide. It is a disorder of the central retina that manifests with irreversible cell loss, primarily affecting the retinal pigment epithelium (RPE) and subsequently the retina and choroid, leading to blindness through atrophy or neovascularization and exudation. Current treatments are only able to suppress the progression of the early and moderate neovascular AMD, mainly by controlling leakage and haemorrhage, while there is no established therapy for the atrophic type or the advanced neovascular type. RPE transplantation strategies have been attempted with promising outcomes; however, their operational complexity combined with the large patients’ volume has underlined the need for more accessible cell sources and a more feasible surgical paradigm. This thesis aims to examine the feasibility, safety and efficacy of transplantation of a human Embryonic Stem Cell (hESC)-derived RPE sheet in patients with severe neovascular (n) AMD. A fully differentiated hESC-RPE monolayer on a coated synthetic basement membrane (BM) has been bioengineered ex vivo and, using a purpose-designed surgical tool, has been implanted in the subretinal space of two patients with nAMD and acute vision decline. Systemic immunosuppression was administered during the peri- operative periods, while only local, intra-ocular steroids were given for the longer term. The patients were followed-up in a prospective study to assess the safety, and the structural and functional outcomes of this strategy for two years post-operatively. Both subjects demonstrated good safety outcome with no signs of local or distal tumorigenicity or uncontrolled proliferation from the implanted cells. Both showed reconstruction of the RPE-BM complex sufficient to support the retinal structure and the rescue and preservation of the photoreceptors, during the study period. Furthermore, both patients showed significant gain in their visual function, in terms of fixation, retinal light sensitivity, visual acuity and reading speed, maintained for two years. Most importantly, in both cases there was a clear co-localisation of the structural support, provided by the transplant, with the areas of functional improvement. The work in this thesis provides proof that the reconstruction of the RPE using hESC on synthetic BM can rescue and preserve the retinal structure and function over the long term, in severe neovascular AMD
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