Academic literature on the topic 'Retina – Pathophysiology'

Diabetes, being a metabolic disease dysregulates a large number of metabolites and factors. However, among those altered metabolites, hyperglycemia is considered as the major factor to cause an increase in oxidative stress that initiates the pathophysiology of retinal damage leading to diabetic retinopathy. Diabetes-induced oxidative stress in the diabetic retina and its damaging effects are well known, but still, the exact source and the mechanism of hyperglycemia-induced reactive oxygen species (ROS) generation especially through mitochondria remains uncertain. In this study, we analyzed precisely the generation of ROS and the antioxidant capacity of enzymes in a real-time situation under ex vivo and in vivo conditions in the control and streptozotocin-induced diabetic rat retinas. We also measured the rate of flux through the citric acid cycle by determining the oxidation of glucose to CO2 and glutamate, under ex vivo conditions in the control and diabetic retinas. Measurements of H2O2 clearance from the ex vivo control and diabetic retinas indicated that activities of mitochondrial antioxidant enzymes are intact in the diabetic retina. Short-term hyperglycemia seems to influence a decrease in ROS generation in the diabetic retina compared to controls, which is also correlated with a decreased oxidation rate of glucose in the diabetic retina. However, an increase in the formation of ROS was observed in the diabetic retinas compared to controls under in vivo conditions. Thus, our results suggest of diabetes/hyperglycemia-induced non-mitochondrial sources may serve as major sources of ROS generation in the diabetic retina as opposed to widely believed hyperglycemia-induced mitochondrial sources of excess ROS. Therefore, hyperglycemia per se may not cause an increase in oxidative stress, especially through mitochondria to damage the retina as in the case of diabetic retinopathy.

Ischemia/reperfusion (I/R) events are involved in the pathophysiology of numerous ocular diseases. The purpose of this study was to test the hypothesis that betulinic acid protects from I/R injury in the mouse retina. Ocular ischemia was induced in mice by increasing intraocular pressure (IOP) to 110 mm Hg for 45 min, while the fellow eye served as a control. One group of mice received betulinic acid (50 mg/kg/day p.o. once daily) and the other group received the vehicle solution only. Eight days after the I/R event, the animals were killed and the retinal wholemounts and optic nerve cross-sections were prepared and stained with cresyl blue or toluidine blue, respectively, to count cells in the ganglion cell layer (GCL) of the retina and axons in the optic nerve. Retinal arteriole responses were measured in isolated retinas by video microscopy. The levels of reactive oxygen species (ROS) were assessed in retinal cryosections and redox gene expression was determined in isolated retinas by quantitative PCR. I/R markedly reduced cell number in the GCL and axon number in the optic nerve of the vehicle-treated mice. In contrast, only a negligible reduction in cell and axon number was observed following I/R in the betulinic acid-treated mice. Endothelial function was markedly reduced and ROS levels were increased in retinal arterioles of vehicle-exposed eyes following I/R, whereas betulinic acid partially prevented vascular endothelial dysfunction and ROS formation. Moreover, betulinic acid boosted mRNA expression for the antioxidant enzymes SOD3 and HO-1 following I/R. Our data provide evidence that betulinic acid protects from I/R injury in the mouse retina. Improvement of vascular endothelial function and the reduction in ROS levels appear to contribute to the neuroprotective effect.

Glaucoma, the leading cause of irreversible blindness, is a heterogeneous group of diseases characterized by progressive loss of retinal ganglion cells (RGCs) and their axons and leads to visual loss and blindness. Risk factors for the onset and progression of glaucoma include systemic and ocular factors such as older age, lower ocular perfusion pressure, and intraocular pressure (IOP). Early signs of RGC damage comprise impairment of axonal transport, downregulation of specific genes and metabolic changes. The brain is often cited to be the highest energy-demanding tissue of the human body. The retina is estimated to have equally high demands. RGCs are particularly active in metabolism and vulnerable to energy insufficiency. Understanding the energy metabolism of the inner retina, especially of the RGCs, is pivotal for understanding glaucoma’s pathophysiology. Here we review the key contributors to the high energy demands in the retina and the distinguishing features of energy metabolism of the inner retina. The major features of glaucoma include progressive cell death of retinal ganglions and optic nerve damage. Therefore, this review focuses on the energetic budget of the retinal ganglion cells, optic nerve and the relevant cells that surround them.

Platelets play a critical role in the pathophysiology of amaurosis fugax. Emboli to retinal vessels apparently produce amaurosis but, in addition, we propose that augmented vasoconstrictor responses and vasospasm may contribute to amaurosis. In this study we tested the hypothesis that constrictor responses of retinal vessels to serotonin, which is released when platelets aggregate, are potentiated in experimental atherosclerosis. Blood flow to the retina was measured in normal and atherosclerotic cynomolgus monkeys. In normal monkeys, infusion of serotonin did not alter flow to the retina. In atherosclerotic monkeys, infusion of serotonin reduced retinal blood flow (in milliliters per minute per 100 g) from 66 ± 7 (mean ± SE) to 5 ±2. Infusion of serotonin in atherosclerotic monkeys abolished the retinal response to light. Thus, atherosclerosis greatly potentiates constrictor responses to serotonin in the retinal circulation and produces a profound but reversible impairment of retinal function. We propose that altered responses to vasoactive substances that are released by platelets may contribute to the pathogenesis of amaurosis fugax.

Increasing evidence suggests that the conditions of retinal microvessels are indicators to a variety of cerebrovascular, neurodegenerative, psychiatric, and developmental diseases. Thus noninvasive visualization of the human retinal microcirculation offers an exceptional opportunity for the investigation of not only the retinal but also cerebral microvasculature. In this review, we show how the conditions of the retinal microvessels could be used to assess the conditions of brain microvessels because the microvascular network of the retina and brain share, in many aspects, standard features in development, morphology, function, and pathophysiology. Recent techniques and imaging modalities, such as optical coherence tomography (OCT), allow more precise visualization of various layers of the retina and its microcirculation, providing a “microscope” to brain microvessels. We also review the potential role of retinal microvessels in the risk identification of cerebrovascular and neurodegenerative diseases. The association between vision problems and cerebrovascular and neurodegenerative diseases, as well as the possible role of retinal microvascular imaging biomarkers in cerebrovascular and neurodegenerative screening, their potentials, and limitations, are also discussed.

Spreading depression is a neurohumoral phenomenon that has been related to the pathophysiology of migraine. The recently introduced 5HTID agonist anti-migraine compound sumatriptan blocks neurogenic extravasation and induces cerebral vasoconstriction, but the actual mechanism of action against migraine remains obscure. Retinal spreading depression (RSD) velocity has been measured in isolated chick retinas in the presence of 0.05-2.00:nM sumatriptan. This drug reversibly blocks RSD in a concentration-dependent manner. Since the preparation is blood-vessel free, this effect must be related to the nervous tissue.

Diet-based xanthophylls (zeaxanthin and lutein) are conditionally essential polar carotenoids preferentially accreted in high concentrations (1 mM) to the central retina, where they have the capacity to impart unique physiologically significant biophysical biochemical properties implicated in cell function, rescue, and survival. Macular xanthophylls interact with membrane-bound proteins and lipids to absorb/attenuate light energy, modulate oxidative stress and redox balance, and influence signal transduction cascades implicated in the pathophysiology of age-related macular degeneration. There is exclusive transport, sequestration, and appreciable bioamplification of macular xanthophylls from the circulating carotenoid pool to the retina and within the retina to regions required for high-resolution sensory processing. The distribution of diet-based macular xanthophylls and the lutein metabolite meso-zeaxanthin varies considerably by retinal eccentricity. Zeaxanthin concentrations are 2.5-fold higher than lutein in the cone-dense central fovea. This is an ~20-fold increase in the molar ratio relative to eccentric retinal regions with biochemically detectable macular xanthophylls. In this review, we discuss how the differences in the specific properties of lutein and zeaxanthin could help explain the preferential accumulation of zeaxanthin in the most vulnerable region of the macula.

Emerging evidence underscores an association between age-related macular degeneration (AMD) and periodontal disease (PD), yet the biological basis of this linkage and the specific role of oral dysbiosis caused by PD in AMD pathophysiology remains unclear. Furthermore, a simple reproducible model that emulates characteristics of both AMD and PD has been lacking. Hence, we established a novel AMD+PD murine model to decipher the potential role of oral infection (ligature-enhanced) with the keystone periodontal pathogen Porphyromonas gingivalis, in the progression of neovasculogenesis in a laser-induced choroidal-neovascularization (Li-CNV) mouse retina. By a combination of fundus photography, optical coherence tomography, and fluorescein angiography, we documented inflammatory drusen-like lesions, reduced retinal thickness, and increased vascular leakage in AMD+PD mice retinae. H&E further confirmed a significant reduction of retinal thickness and subretinal drusen-like deposits. Immunofluorescence microscopy revealed significant induction of choroidal/retinal vasculogenesis in AMD+PD mice. qPCR identified increased expression of oxidative-stress, angiogenesis, pro-inflammatory mediators, whereas antioxidants and anti-inflammatory genes in AMD+PD mice retinae were notably decreased. Through qPCR, we detected Pg and its fimbrial 16s-RrNA gene expression in the AMD+PD mice retinae. To sum-up, this is the first in vivo study signifying a role of periodontal infection in augmentation of AMD phenotype, with the aid of a pioneering AMD+PD murine model established in our laboratory.

Diabetic Retinopathy (DR) is one of the leading causes of decreased vision and blindness in developed countries. Diabetes-induced metabolic disorder is believed to increase oxidative stress in the retina. This results in deleterious change through dysregulation of cellular physiology that damages both neuronal and vascular cells. In this review, we first highlight the evidence of potential metabolic sources and pathways which increase oxidative stress that contribute to retinal pathology in diabetes. As oxidative stress is a central factor in the pathophysiology of DR, antioxidants therapy would be beneficial towards preventing the retinal damage. A number of experimental studies by our group and others showed that dietary flavonoids cause reduction in increased oxidative stress and other beneficial effects in diabetic retina. We then discuss the beneficial effects of the six major flavonoid families, such as flavanones, flavanols, flavonols, isoflavones, flavones and anthocyanins, which have been studied to improve retinal damage. Flavanoids, being known antioxidants, may ameliorate the retinal degenerative factors including apoptosis, inflammation and neurodegeneration in diabetes. Therefore, intake of potential dietary flavonoids would limit oxidative stress and thereby prevent the retinal damage, and subsequently the development of DR.

Retina is a unique site in the human body where the microcirculation can be imaged directly and non-invasively, allowing us to study in vivo the structure and pathology of the human microcirculation. Retinal images can be quantitatively assessed with computerized imaging techniques, enabling us to measure several different quantitative traits derived from the retinal vasculature. Arterial and venular calibres are the most extensively studied traits of the retinal microvasculature and numerous epidemiological studies demonstrated promising associations with systemic and ocular diseases as well as with disease markers. However, there has been a lack of research into pathophysiological processes leading to retinal vascular signs, and how they link retinal microcirculation with coronary and cerebral microvasculature change. Information about genetic determinants underlying retinal vascular structure is therefore important for understanding the processes leading to microvascular pathophysiology. Two genome wide association studies have been published so far revealing four loci associated with retinal venular calibre and one locus with arteriolar calibre. Here the results from the genome-wide association analysis of 10 different retinal vessel traits in two population based cohorts are presented. Retinal images were measured in non-mydriatic fundus images from 808 subjects in the Orkney Complex Disease Study (ORCADES) and 390 subjects from the Croatian island of Korcula, using the semi-automated retinal vasculature measurement programme SIVA and VAMPIRE. Using pairwise estimates of kinship based on genomic sharing, heritability was calculated for each trait. Estimates of tortuosity measure and fractal dimensions present first published reports of heritability estimates for those traits. In addition correlation analysis with systemic risk factor was also completed, confirming already published results as well as revealing some new associations. A genome wide association analysis of retinal arteriolar width revealed a genome wide significant hit (1.8x10-7) in a region of chromosome 2q32 (within TTN gene). Replication was sought in a further independent Scottish population (LBC) and additional 400 retinal images were graded. The result did not replicate, however the direction of the effect was consistent and a larger sample size is required. Analysis of the remaining traits did not yield genome wide significant result,s and will also require larger sample sizes. Genetic analysis of a binary retinal trait was also explored in a case control study of retinal detachment, which is an important cause of vision loss. A two-stage genetic association discovery phase followed by a replication phase in a combined total of 2,833 RRD cases and 7,871 controls was carried out. None of the SNPs tested in the discovery phase reached the threshold for association. Further testing was carried out in independent case-control series from London (846 cases) and Croatia (120 cases). The combined meta-analysis identified one association reaching genome-wide significance for rs267738 (OR=1.29, p=2.11x10-8), a missense coding SNP and eQTL for CERS2 encoding the protein ceramide synthase 2. Additional genetic risk score, pathway analysis and genetic liability analysis were also carried out.

Electrical stimulation of retinal neurons is known to elicit visual sensations. When applied to the retina in a spatial pattern, electrical stimulation may be capable of providing rudimentary patterned vision that may be of benefit to sufferers of degenerative retinal disorders. No such device has yet been devised to provide for chronic study of the psychophysical perceptions elicited from a prosthesis for retinal stimulation. In this study, steps towards achieving this goal have been successfully carried out. Foregoing research was reviewed such that appropriate stimulation parameters were incorporated in the design of a 100 stimulation channel, complimentary metal oxide semiconductor (CMOS) integrated circuit, small enough in size so as to be capable of being implanted within the ocular anatomy or surrounding orbit. The device, and its associated external hardware and software were designed, modeled, fabricated, and interfaced with stimulating electrodes in acute testing in a highorder mammal (Ovis aries) so as to assess the capabilities of the device to elicit cortical potentials as a direct result of stimulation of the neural retina. Testing was performed under conditions similar to those anticipated in chronic in-situ configurations wherein radio-frequency telemetry was used to deliver power and configuration parameters to the device thus avoiding the passage of wires through tissue in order to communicate to the implant circuit. The results of the testing indicate that the circuit is indeed capable of eliciting physiological responses in the animal and evidence is present that these responses could be elicited in patterned form. Further work undertaken includes the development of surgical methods for implantation, and application of the prosthesis circuit in functional electronic stimulation.

Background. Lacunar strokes account for 25% of all ischaemic stroke but the exact nature of the causative cerebral small vessel abnormality remains unknown. Pathological studies are technically difficult and brain imaging cannot adequately characterise the cerebral small vessels. The retinal blood vessels are of similar size and physiology to the cerebral small vessels and may act as a surrogate marker for these cerebral small vessels. We therefore investigated retinal microvascular abnormalities in lacunar stroke. Methods. We performed a systematic review of retinal microvascular abnormalities in lacunar stroke to clarify associations and identify where further research was required. We then established a cohort of patients presenting with lacunar stroke with cortical stroke controls to investigate differences in retinal microvascular abnormalities between stroke subtypes. All patients had MRI brain at presentation and digital retinal photography of both eyes. We investigated the prevalence of retinopathy (hard and soft exudates or haemorrhages/microaneurysms), focal arteriolar narrowing and arteriovenous nicking . We developed, validated and used novel semi-automated techniques for measuring retinal arteriolar and venular widths, retinal arteriolar geometry (branching co-efficients (change in arteriolar cross sectional area across a bifurcation) and branching angles) and fractal dimensions (reflecting branching complexity) of the vasculature. We also assessed MRI parameters in lacunar stroke. We used multivariable analysis to correct for baseline imbalances in vascular risk factors. Results. From the systematic review we demonstrated that retinal microvascular abnormalities are associated with incident and prevalent stroke but that in general, strokes were inadequately characterised and there were no data regarding retinal microvascular abnormalities in ischaemic stroke subtypes. We recruited 253 patients, 129 lacunar strokes and 124 cortical strokes, mean age 68 years. We found no difference in the prevalence of retinopathy, arteriovenous nicking, focal arteriolar narrowing or arteriolar widths between lacunar and cortical stroke subtypes. We found that venules were wider in lacunar stroke. We found no differences in arteriolar branching co-efficients or arteriolar branching angles between lacunar and cortical strokes but found that deep white matter white matter hyperintensities on MRI were associated with increased branching co-efficients and periventricular white matter hyperintensities associated with decreased branching co-efficients. We found that the fractal dimension of the vascular tree was decreased in lacunar stroke. Furthermore we found that enlarged perivascular spaces on MRI are associated with lacunar stroke and white matter disease. Conclusions. We have clearly demonstrated that retinal microvascular abnormalities differ between lacunar and cortical stroke suggesting that a distinct small vessel vasculopathy may cause lacunar stroke. We have also identified MR markers of lacunar stroke. These results suggest that venular disease (a hitherto underresearched area) may play a role in the pathophysiology of lacunar stroke. Retinal microvascular abnormalities can act as markers for cerebral small vessel disease. We plan collaborative analyses with colleagues who have performed similar studies to further assess retinal abnormalities in lacunar stroke.

[Truncated abstract] Developmentally, the rodent retinocollicular projection is often thought of as a homogenous projection of retinal ganglion cell (RGC) axons, however the extensive period of RGC neurogenesis and sequential arrival of their axons into central targets such as the superior colliulus (SC) suggests otherwise. RGC axons are already present in the developing SC at embryonic (E) day 16.5-17. RGCs born on E15 have innervated the SC by birth, whereas axons derived from RGCs that are born last (E19) do not grow into the SC until postnatal (P) days 4-6 (Dallimore et al., 2002). These observations may go someway to explaining why, after SC lesions in rats at P2, there is greater growth distal to the lesion site compared to lesions made at P6 (Tan and Harvey, 1997b). It may be that the post lesion growth is simply de novo growth of axons from late-born RGCs rather than regeneration of pre-existing, injured axons. Early and late cohorts of growing RGC axons presumably encounter different developmental terrains as they grow from retina to central targets, possibly resulting in differences in developmental milestones and growth potentials. There may also be differences in guidance cues, further suggesting that gene expression in early vs late born RGCs may differ. To examine differences between early (E15) and late (E19) born RGCs during development, the time-course and extent of programmed RGC death in normal rat pups, and RGC death following the removal of target-derived trophic factors, was assessed. ... On the other hand, LCM captured GCL analysed for gene expression at P0 and P7 revealed decreases in AKT, Math5, Notch1, c-jun, DCC, Arginase-1 mRNA levels and a considerable decrease in GAP-43 expression. It is not surprising to see differences in gene expression between whole eye and the more specific GCL samples, as the cells in all layers of the retina have very different functions and different developmental profiles. It is important to note decreases in mRNA expression in the GCL for a number of the genes analysed at P0 and P7, reflecting cessation of RGC death and completion of axonal growth into central visual targets. I also examined at the protein level expression of DCC, Arginase1, c-Jun and Bcl-2 at birth (P0) in BrdU labeled RGCs born on E15 or E19. When comparing the percentage of double labelled cells compared to the total number of cells expressing each protein, Bcl-2, c-Jun and Arg1 were expressed more in E15 RGCs (22.90%, 72.71%, and 16.44% respectively in E15 RGCs, compared with 0.52%, 13.17% and 3.59% in E19 RGCs). In contrast, DCC was expressed more at birth in E19 RGCs (18.05% in E19 RGCs compared with 9.23% in E15 RGCs). This shows there is clearly a difference in the expression of proteins in the two cohorts of RGCs, which is consistent with PCR data and with their growth state as their axons encounter the changes in the newborn brain. The overall findings of this research suggest that seemingly homogenous populations of neurons are quite different in their developmental profile and in their response to injury. This work may provide new ways of determining better strategies for CNS repair and the most effective way of targeting cells for regeneration and survival.

Le Syndrome de Wolfram (SW; OMIM #222300, prévalence 1-9 / 1000 000) est une maladie neurodégénérative, qui se présente avec un début juvénile, intégrant le diabète insipide, diabète sucré, l’atrophie optique (AO), et la surdité. AO est généralement son premier symptôme neurologique, commençant à l’âge de 11 ans et se terminant par la cécité 8 ans plus tard. Malheureusement, un modèle murin du SW approprié aux symptômes ophtalmologiques n'a pas encore été trouvé, donc la recherche de la thérapie pour sauver la vision en est à ces débuts. Dans cette thèse j’ai étudié l’atteinte visuelle de deux modèles de souris mutantes pour le SW et succès d’une approche de thérapie génique (TG) avec le gène humain WFS1.Premièrement, les souris Wfs1exon8del sont été examinées à 3 et 6 mois pour l’acuité visuelle (AV) et la sensibilité aux contrastes (SC) via changements dans le reflexe optomoteur (ROM), la fonction rétinienne neurale par électrorétinogramme (ERG), ainsi que la physiologie de l’œil par la fondoscopie et tomographie par cohérence optique (TCO). De plus, la proportion des cellules ganglionnaire de la rétine (CGRs) et la perte axonale dans le nerf optique (NO) à 7 mois ont été examinés avec marquage anti-Brn3a et microscopie électronique, respectivement. Il y avait une perte progressive de l’AV et la SC chez les souris KO à partir du 1 mois. Elle était accompagnée d'une pâleur du disque optique (DO), d'amincissement de la rétine ainsi que des lésions axonales. Par contre, il n’avait pas de perte des CGRs ni stress du réticulum endoplasmique dans la rétine. Brièvement, les souris KO présentent un phénotype ophtalmique du SW significatif et peuvent servir comme modèle.Deuxièmement, à la recherche d'un autre modèle du SW, les fonctions visuelles de la lignée Wfs1E864K de la souris ont été étudiées. Déjà à 1 mois, les souris Wfs1E864K/E864K avait une perte drastique de la fonction des CGRs, mais en gardant le nombre de cellules à un niveau normal. Ceci a été accompagné par un amincissement de la rétine et d’un sévère dommage du NO, comme montré par le TCO et la fondoscopie, respectivement. En conséquence, les souris Wfs1E864K/E864K, avec leur fort phénotype ophtalmique, pourraient servir comme modèle du SW classique.Enfin, pour enquêter sur les futures options de traitement contre le SW, les souris de la lignée Wfs1exon8del à 1 mois ont subi une TG intravitréenne avec AAV-2/2-CMV-WFS1. Les examens à 3 et 6 mois ont montré une amélioration de l’AV, ainsi que le sauvetage de la pâleur du DO et réduction des lésions axonales chez les souris KO. En outre, aucun effet indésirable lié à des injections TG n’ont été noté. Suivant cette idée, les souris Wfs1E864K/E864K ont également été soumis à la TG intravitréenne, délivrée à P14, mais sans succès.En conclusion, la lignée Wfs1exon8del de la souris est un modèle fiable du SW, y compris les aspects visuels. Je propose le modèle Wfs1E864K/E864K comme une alternative, en particulier pour enquêter sur la fonction de Wfs1 dans l'œil. Enfin, la GT intravitréenne avec WFS1 a un potentiel pour sauver partiellement le phénotype ophtalmique, ouvrant la voie vers le traitement pour les patients du SW
Wolfram Syndrome (WS; OMIM #222300, prevalence 1-9 / 1 000 000) has a juvenile onset and incorporates diabetes insipidus, diabetes mellitus, optic atrophy (OA), and deafness; leading to death in middle age. OA is its first neurological symptom, starting in adolescence and ending with blindness within 8 years. Unfortunately, a suitable WS mouse model comprising ophthalmologic symptoms has not yet been found, therefore the search for its treatment is delayed. In this thesis, I studied visual impairment in two WS mouse models along with a success of a gene therapy (GT) approach with the human WFS1 gene.Firstly, 3 and 6 months old Wfs1exon8del mice were examined for the visual acuity (VA) and contrast sensitivity via changes in the opto-motor reflex (OMR), the neural retinal function via electroretinogram (ERG), as well as the eye physiology via fundoscopy and optic coher-ence tomography (OCT). Also, the proportion of retinal ganglion cells (RGC) and the axonal loss at the age of 7 months were determined with anti-Brn3a immuno-labeling of retinal sections and electron microscopy of optic nerve (ON) sections, respectively. There was a progressive loss of VA and contrast sensitivity in Wfs1exon8del-/- mice, starting already at 1 month of age. It was accompanied by optic disc pallor, retinal thinning as well as axonal damage. However, there was no RGC loss and the endoplasmic reticulum (ER) stress in the retina was at a normal level. It suggested a presence of another cause for the reported degeneration in KO mice; in opposition to what was proposed in the literature. I brief, KO mice exhibit significant WS ophthalmic phenotype.Secondly, in search for another model, visual functions of Wfs1E864K mouse line were investigated. This line was originally a model of Wolfram-like Syndrome, characterized by dominant mutations in WFS1 leading to congenital progressive hearing impairment, diabetes mellitus and OA. Only homozygous mutants, however, showed expected visual impairment. Already at 1 month of age, Wfs1E864K/E864K mice had drastic loss of RGC function, albeit keeping the cell number at a normal level. This was accompanied by retinal thinning and a severe ON damage, as shown with OCT and fundoscopy, respectively. In contrast, the RGC function in Wfs1E864K/+ mice dropped slightly only at the age of 7 and 12 months, showing that the pathology of the E864K mutation-driven disease in mice is different than in humans. Therefore, Wfs1E864K/E864K mice, with their strong ophthalmic phenotype, could potentially serve as a model of the classical WS.Finally, to investigate future treatment options, 1 month old Wfs1exon8del+/+ (WT) and Wfs1exon8del-/- (KO) mice underwent a uni- and bi-lateral intravitreal gene therapy (GT) with AAV-2/2-CMV-WFS1. Exams at 3 and 6 months of age showed improved VA, as well as optic pallor and axonal damage rescue in KO mice. Also, no adverse effects related to either GT or sham injections were noted. Following this idea, the Wfs1E864K/E864K mice were also subjected to intravitreal GT, delivered at P14, but without success.In conclusion, Wfs1exon8del mouse line is a reliable model of WS, including the visual aspects. I propose the Wfs1E864K/E864K model as an alternative, especially to investigate Wfs1 function in the eye. Finally, the intravitreal AAV-driven GT with WFS1 has a potential to partially rescue the ophthalmic phenotype, paving the wave towards the treatment for WS patients

Relatively new technology called optical coherence tomography allows direct and non invasive in vivo imaging of retinal anatomy in human subjects. There are several interesting applications of this technique, including testing models relating retinal anatomy (structural measures) to behavioral thresholds of light sensitivity (functional measures). In addition to potentially improving our understanding of this relationship and how it changes during the course of neurodegenerative diseases of the eye such as glaucoma, analyses of these data may allow for early identification of glaucomatous neural damage in the retina, which has considerable clinical relevance. Here, the underlying assumptions and generalization of a previously developed model of the structure function relationship in glaucoma was tested by applying this model to a novel dataset. This model has been influential in the literature because it purports to accurately estimate the number of retinal ganglion cells; however, it was found to have several questionable assumptions and did not generalize well. Next, a new method of estimating the number of retinal ganglion cells from optical coherence tomography was developed. This method uses fewer and more defensible assumptions and demonstrated good agreement with independent histological estimates. Finally, a new method, using computer simulations, was developed for analyzing data from optical coherence tomography in order to distinguish early signs of glaucomatous changes in retinal anatomy from variability in structure among healthy retinas, and this method performed better than previously published techniques.

Tese no âmbito do Programa de Doutoramento em Ciências da Saúde – ramo de Medicina, apresentada à Faculdade de Medicina da Universidade de Coimbra
Background: Recent studies have reported that the choroidal thickness may be a prognostic factor for diabetic retinopathy and diabetic macular edema (DME), but there are conflicting results in the literature. Moreover, diabetic choroidopathy and the nature of diabetic retinopathy, including macular edema, have been recognized as complex traits, with an inflammatory component. Alterations in the choroid, such as vascular remodelling and capillary depletion of the choriocapillaris, and in the retina, as glial cell reactivity and migration, have been described in diabetic rats. However, there is no consensus about the role of baseline choroidal thickness as a prognostic factor in DME under treatment. Likewise, it is unknown how the choroidal thickness changes in animal models of diabetes, as well as the cellular and molecular alterations occurring simultaneously in the choroid and retina in diabetes. Purpose: To determine the prognostic value of choroidal thickness and to search other prognostic factors in patients with DME. To evaluate the choroidal thickness and changes in cellular and molecular signatures in the choroid and retina in the course of diabetes, in animal models of Type 1 and Type 2 diabetes. Methods: In a prospective study, 126 eyes of 126 patients with DME were enrolled to assess the anatomical (central retinal thickness, CRT, decrease ≥ 10% from baseline) and functional (best corrected visual acuity, BCVA, gain ≥ 5 ETDRS letters from baseline) prognostic value of baseline subfoveal choroidal thickness (SFCT) on anti-vascular endothelial growth factor (anti-VEGF), ranibizumab or aflibercept, treatment response after 3 (early outcome) and 6 months (late outcome). A comparison was made between SFCT and other choroidal thicknesses collected at different locations from the fovea to establish the value of SFCT as a surrogate of the choroidal thickness. In addition, 122 eyes of 122 patients were prospectively enrolled to search for anatomical (CRT) and functional (BCVA) baseline prognostic factors, other than SFCT, for recent onset DME under anti-VEGF agents’ treatment. Furthermore, two rat models of diabetes, streptozotocin (STZ)-induced Type 1 diabetes in Wistar rats (8 weeks-old; with further 8 weeks of diabetes duration) and Goto-Kakizaki (GK) Type 2 diabetes rats (1 year old) were used. In vivo choroidal thickness was evaluated in both models by optical coherence tomography (OCT). Vascular density of the choriocapillaris and middle/outer choroid was quantified in sclerochoroidal whole mounts of eyes perfused by 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate (DiI). The immunoreactivity of vascular endothelial growth factor (VEGF), VEGF-receptor 2 (VEGFR2), as well as the immunoreactivity of vimentin (marker of macroglial cells), Iba1 and MHC II (markers of non-activated and activated microglial cells/macrophages, respectively) and NG2 (marker of pericytes and perivascular mural cells), were assessed by immunohistochemistry, in the choroid and retina, in eye cryosections and in sclerochoroidal whole mounts. Images were acquired by fluorescence and confocal microscopy and the immunofluorescence was quantified by ImageJ. Moreover, Iba1, MHC II and NG2 positive cells were counted. Results: In diabetic patients, treatment of DME with anti-VEGF agents, ranibizumab and aflibercept, decreased the choroidal thickness. However, the SFCT was not a predictor of the anatomical or functional outcomes. SFCT was an excellent surrogate of the choroidal thickness, showing an excellent correlation with the other choroidal thickness parameters evaluated. The subretinal fluid was a predictor of the anatomical outcome, whereas the ellipsoid zone status and a good metabolic control were predictors of functional outcome, regardless of being early or late outcomes. In experimental models, significant differences between STZ (serious metabolic-imbalance) and GK (longer lasting diabetes and light metabolic-imbalance) rats were found. In vivo choroidal thickness increased in GK rats only and the choriocapillaris vascular density decreased in GK rats only, as well. Moreover, VEGFR2 immunoreactivity was upregulated in the retina of GK rats, being downregulated in the retina of STZ rats. The number of Iba1+ cells increased in the outer retina of both animal models. However, in the choroid, the number of Iba1+ cells and MHC II+ cells increased in STZ rats only. The aforementioned results for Iba1+ and MHC II+ cells indicate that the degree of such increase may depend on metabolic status and/or disease duration. Signs of pericyte depletion at the choriocapillaris were present in both models, being more evident in GK rats. Conclusions: Although there were alterations in the SFCT in DME under anti-VEGF treatment, the baseline SFCT was not a useful prognostic tool for DME. It was an indicator of time-dependent anti-VEGF’s subsiding effect on the choroid instead, and a good surrogate of the choroidal thickness as such. Good metabolic control and an intact ellipsoid zone were predictors of functional outcome while subfoveal neuroretinal detachment was a predictor of anatomic outcome only. The number of Iba1+ cells and MHC II+ cells increased in the choroid and retina in diabetic rats but the magnitude of such increase changed considerably when the metabolic status was seriously imbalanced. VEGFR2 immunoreactivity increased in the retina in longer diabetes duration and slighter metabolic imbalance. Conversely, VEGFR2 immunoreactivity decreased when there was a serious metabolic imbalance. Vascular remodelling or vascular depletion at the choriocapillaris was also a trait of the long lasting disease.
Introdução: Estudos recentes indicam que a espessura da coroide pode ser considerada fator de prognóstico na retinopatia diabética (RD) e no edema macular diabético (EMD), embora os resultados sejam contraditórios. A coroidopatia diabética e a natureza da RD, incluindo do EMD, têm características complexas, que incluem um componente inflamatório. Alterações na coroide, como a renovação vascular e a depleção capilar a nível da coriocapilar, e alterações na retina, tais como a ativação e migração de células da glia, foram descritas em ratos diabéticos. No entanto, o papel da espessura basal da coroide como fator de prognóstico no EMD não é consensual. Em modelos animais de diabetes, desconhece-se como varia a espessura da coroide e se existem alterações celulares e moleculares que ocorrem simultaneamente na coroide e na retina. Objetivos: Determinar o valor prognóstico da espessura basal da coroide e pesquisar outros fatores de prognóstico em doentes com EMD. Avaliar a espessura da coroide e alterações celulares e moleculares na coroide e na retina em modelos animais de diabetes tipo 1 (T1D) e tipo 2 (T2D). Métodos: Cento e vinte e seis olhos de 126 doentes com EMD foram incluídos num estudo prospetivo, para avaliar o valor prognóstico da espessura coroideia subfoveal (ECSF) inicial, definido como anatómico (baixa da espessura basal central da retina ≥ 10%,) e como funcional (ganho na melhor acuidade visual corrigida, MAVC, basal ≥ 5 letras ETDRS), na resposta ao tratamento com ranibizumab ou aflibercept, ao final de 3 e 6 meses. Para determinar o valor da ECSF como indicador de espessura coroideia comparou-se a ECSF com espessuras da coroide à volta da fovea. Adicionalmente, 122 olhos de 122 doentes com EMD foram prospetivamente incluídos, para determinar outros fatores de prognóstico no EMD recente sob tratamento com anti-angiogénicos. Relativamente à diabetes experimental, utilizaram-se dois modelos de ratos diabéticos. Ratos Wistar em que foi induzida T1D através de uma injeção de estreptozotocina (STZ, às 8 semanas de idade, com mais 8 semanas de duração da diabetes) e ratos Goto-Kakizaki (GK) com um ano de idade, como modelo de T2D. A espessura da coroide foi avaliada in vivo por tomografia de coerência ótica (OCT) em ambos os modelos animais. A densidade vascular da coriocapilar e da coroide vascular, média e externa, foi quantificada em explantes esclero-coroideus de olhos perfundidos por perclorato de 1,1’-dioctadecyl-3,3,3’,3’-tetramethilindocarbocianina (DiI). As imunorreatividades do fator de crescimento do endotélio vascular (VEGF) e do seu recetor 2 (VEGFR2), assim como a da vimentina (marcador das células da macroglia), de Iba1 e MHC II (marcadores da microglia/macrófagos não ativados e ativados, respetivamente), e de NG2 (marcador de pericitos e células murais peri-vasculares), foram determinadas por imuno-histoquímica na coroide e na retina, em criosecções e em explantes esclero-coroideus. As imagens foram adquiridas por microscopia de fluorescência ou confocal e a imunofluorescência foi quantificada pelo ImageJ. Procedeu-se também à contagem de células positivas para Iba1, MHC II e NG2. Resultados: A espessura coroideia subfoveal diminuiu com o tratamento do EMD, mas não revelou possuir valor prognóstico, quer anatómico quer funcional, precoce ou tardio. A ECSF revelou-se um bom marcador da espessura coroideia, possuindo uma boa correlação com os outros parâmetros de espessura coroideia. A existência basal de fluido sub-retiniano revelou-se fator de bom prognóstico anatómico, enquanto que uma zona elipsoide íntegra e um bom equilíbrio metabólico se revelaram fatores de bom prognóstico funcional, quer precoces quer tardios. Nos modelos experimentais, observaram-se diferenças significativas entre os ratos com diabetes induzida pela STZ (desequilíbrio metabólico acentuado) e os ratos GK (maior duração de diabetes com desequilíbrio metabólico ligeiro/moderado). Observou-se um aumento da espessura da coroide e uma diminuição da densidade vascular da coriocapilar, in vivo, apenas em ratos GK. A imunorreactividade para o VEGFR2 aumentou na retina dos ratos GK e diminuiu na retina dos ratos STZ. O número de células Iba1+ aumentou na retina externa em ambos os modelos animais, embora apenas nos ratos STZ se encontrasse aumentado no estroma da coroide. O número de células MHC II+ também aumentou apenas na coroide de ratos STZ. Estes resultados indicam que o incremento das células inflamatórias na coroide depende do estado metabólico e da duração da doença. Além disso, também se observaram sinais de rarefação de pericitos a nível da coriocapilar em ambos os modelos, embora essa alteração fosse mais evidente em ratos GK. Conclusões: Embora a ECSF diminua no EMD sob tratamento, não se revelou um fator de prognóstico para o EMD. Revelou-se apenas um indicador de duração de ação do anti-angiogénico e um bom índice de espessura coroideia em geral. Um bom controlo metabólico e uma zona elipsoide íntegra revelaram-se fatores de bom prognóstico funcional, enquanto que o fluido subretiniano se revelou fator de bom prognóstico anatómico. A espessura coroideia aumentou e a densidade vascular da coroide diminuiu apenas no modelo animal de T2D. O número de células Iba1+ e MHC II+ encontrava-se aumentado na coroide e na retina dos ratos T1D e T2D, mas esse aumento variou com o desequilíbrio metabólico e com a duração da doença. A imunorreactividade do VEGFR2 encontrava-se aumentada quando a duração da diabetes era mais prolongada e quando existia apenas um desequilíbrio metabólico ligeiro/moderado. Pelo contrário, a imunorreactividade do VEGFR2 revelou-se diminuída quando o desequilíbrio metabólico era acentuado. A rarefação e o aumento da renovação vasculares a nível da coriocapilar era uma característica que se acentuava numa situação de doença prolongada.
Novartis Portugal financiou a contribuição de João Martins.

Indiana University-Purdue University Indianapolis (IUPUI)
Reactive gliosis is known to have a beneficial and a degenerative effect following injury to neurons. Although many factors have been implicated in reactive gliosis, their role in regulating this change is still unclear. We investigated the role of bone morphogenetic proteins in reactive gliosis in vivo and in vitro. In vivo, IHC analysis indicated reactive gliosis in the 6 week Ins2Akita mouse and WPK rat retinas. Expression of BMP7 was upregulated in these models, leading to an increase in the phosphorylation of downstream SMAD1. In vitro, treatment of murine retinal astrocyte cells with a strong oxidizing agent such as sodium peroxynitrite regulated RNA levels of various markers, including GFAP, CSPGs, MMPs and TIMPs. BMP7 treatment also regulated RNA levels to a similar extent, suggesting reactive gliosis. Treatment with high glucose DMEM and BMP4, however, did not elicit increase in levels to a similar degree. Increase in SMAD levels and downstream targets of SMAD signaling such as ID1, ID3 and MSX2 was also observed following treatment with sodium peroxynitrite in vitro and in the 6 week Ins2Akita mouse retinas in vivo. These data concur with previously established data which show an increase in BMP7 levels following injury. It also demonstrates a role for BMP7 in gliosis following disease. Further, it suggests SMAD signaling to play a role in initiating reactivity in astrocytes as well as in remodeling the extracellular matrix following injury and in a disease condition.

Indiana University-Purdue University Indianapolis (IUPUI)
Human induced pluripotent stem (iPS) cells have the unique ability to differentiate into 200 or so somatic cell types that make up the adult human being. The use of human iPS cells to study development and disease is a highly exciting and interdependent field that holds great promise in understanding and elucidating mechanisms behind cellular differentiation with future applications in drug screening and cell replacement studies for complex and currently incurable cellular degenerative disorders. The recent advent of iPS cell technology allows for the generation of patient-specific cell lines that enable us to model the progression of a disease phenotype in a human in vitro model. Differentiation of iPS cells toward the affected cell type provides an unlimited source of diseased cells for examination, and to further study the developmental progression of the disease in vitro, also called the “disease-in-a-dish” model. In this study, efforts were undertaken to recapitulate the differentiation of distinct retinal cell affected in two highly prevalent retinal diseases, Usher syndrome and glaucoma. Using a line of Type III Usher Syndrome patient derived iPS cells efforts were undertaken to develop such an approach as an effective in vitro model for studies of Usher Syndrome, the most commonly inherited disorder affecting both vision and hearing. Using existing lines of iPS cells, studies were also aimed at differentiation and characterization of the more complex retinal cell types, retinal ganglion cells (RGCs) and astrocytes, the cell types affected in glaucoma, a severe neurodegenerative disease of the retina leading to eventual irreversible blindness. Using a previously described protocol, the iPS cells were directed to differentiate toward a retinal fate through a step-wise process that proceeds through all of the major stages of neuroretinal development. The differentiation process was monitored for a period of 70 days for the differentiation of retinal cell types and 150 days for astrocyte development. The different stages of differentiation and the individually derived somatic cell types were characterized by the expression of developmentally associated transcription factors specific to each cell type. Further approaches were undertaken to characterize the morphological differences between RGCs and other neuroretinal cell types derived in the process. The results of this study successfully demonstrated that Usher syndrome patient derived iPS cells differentiated to the affected photoreceptors of Usher syndrome along with other mature retinal cell types, chronologically analogous to the development of the cell types in a mature human retina. This study also established a robust method for the in vitro derivation of RGCs and astrocytes from human iPS cells and provided novel methodologies and evidence to characterize these individual somatic cell types. Overall, this study provides a unique insight into the application of human pluripotent stem cell biology by establishing a novel platform for future studies of in vitro disease modeling of the retinal degenerative diseases: Usher syndrome and glaucoma. In downstream applications of this study, the disease relevant cell types derived from human iPS cells can be used as tools to further study disease progression, drug screening and cell replacement strategies.

Glaucoma is the second leading cause of irreversible blindness in the world. The biological mechanisms of this disease are not well understood, and the factors contributing to its progression are not well characterized. It is generally accepted that the retinal ganglion cell axons are damaged in glaucoma as they pass through the optic nerve head (ONH). The ONH contains the lamina cribrosa (LC), a fenestrated connective tissue network that spans the scleral canal, through which the axons pass as they leave the eye. It has been postulated that intraocular pressure (IOP)-related stress and strain plays a central role in the pathophysiology of glaucoma [1], so the study of LC biomechanics is important in developing an understanding of the disease.

Glaucomatous vision loss can occur at both normal and elevated levels of intraocular pressure (IOP) and the optic nerve head (ONH) is the principle site of damage. The mechanical environment of the ONH is believed to be critical for retinal ganglion cell pathophysiology [1]. Previous computational models have shown that scleral mechanical properties play an important role in affecting the mechanical environment of the ONH [2]. It is thus important to characterize the mechanical behavior of sclera under physiological loadings. Ultrasonic strain mapping has been developed to measure the internal displacement and strain of soft tissue under external loadings [3, 4]. The purpose of this study was to examine the cross-sectional strain maps at the posterior sclera under IOP elevations using non-invasive ultrasound and a speckle tracking algorithm.

Glaucoma is a leading cause of blindness in the world and is due to the loss of retinal ganglion cell axons. These axons deteriorate in a region in the posterior pole of the eye known as the optic nerve head (ONH). The axons pass through the lamina cribrosa (LC) as they exit the eye at the ONH. The LC is characterized by a porous, connective tissue structure composed of laminar beams. The function of the LC is unclear, but is believed to include providing mechanical support to the axons as they transition from inside the pressurized globe to the lower pressure orbital space. Early experimental glaucoma studies have shown that the LC remodels into a thicker, more posterior structure which incorporates more connective tissue after chronic IOP elevation [1,2]. The process by which this occurs is unknown. These structural changes are assumed to play an important role in the pathophysiology of the ocular disease glaucoma, where elevated IOP is known to be the most relevant risk factor.