An amplified-target loss approach for photoreceptor layer segmentation in pathological OCT scans

Segmenting anatomical structures such as the photoreceptor layer in retinal optical coherence tomography (OCT) scans is challenging in pathological scenarios. Supervised deep learning models trained with standard loss functions are usually able to characterize only the most common disease appeareance from a training set, resulting in suboptimal performance and poor generalization when dealing with unseen lesions. In this paper we propose to overcome this limitation by means of an augmented target loss function framework. We introduce a novel amplified-target loss that explicitly penalizes errors within the central area of the input images, based on the observation that most of the challenging disease appeareance is usually located in this area. We experimentally validated our approach using a data set with OCT scans of patients with macular diseases. We observe increased performance compared to the models that use only the standard losses. Our proposed loss function strongly supports the segmentation model to better distinguish photoreceptors in highly pathological scenarios.Comment: Accepted for publication at MICCAI-OMIA 201

Toward genome editing in X-linked RP-development of a mouse model with specific treatment relevant features

Genome editing represents a powerful tool to treat inherited disorders. Highly specific endonucleases induce a DNA double strand break near the mutant site, which is subsequently repaired by cellular DNA repair mechanisms that involve the presence of a wild type template DNA. In vivo applications of this strategy are still rare, in part due to the absence of appropriate animal models carrying human disease mutations and knowledge of the efficient targeting of endonucleases. Here we report the generation and characterization of a new mouse model for X-linked retinitis pigmentosa (XLRP) carrying a point mutation in the mutational hotspot exon ORF15 of the RPGR gene as well as a recognition site for the homing endonuclease I-SceI. Presence of the genomic modifications was verified at the RNA and protein levels. The mutant protein was observed at low levels. Optical coherence tomography studies revealed a slowly progressive retinal degeneration with photoreceptor loss starting at 9 months of age, paralleling the onset of functional deficits as seen in the electroretinogram. Early changes to the outer retinal bands can be used as biomarker during treatment applications. We further show for the first time efficient targeting using the I-SceI enzyme at the genomic locus in a proof of concept in photoreceptors following adeno-associated virus mediated gene transfer in vivo. Taken together, our studies not only provide a human-XLRP disease model but also act as a platform to design genome editing technology for retinal degenerative diseases using the currently available endonucleases

Photobiomodulation in Inherited Retinal Degeneration

The retinal degenerative disease, retinitis pigmentosa (RP), is the most common cause of inherited blindness in the developed world and is caused by the progressive degeneration of rod photoreceptor cells preceding cone degeneration. Mitochondrial dysfunction and oxidative stress have been shown to play a significant role in the pathogenesis of RP and other retinal degenerative diseases. A growing body of evidence indicates that exposure of tissue to low energy photon irradiation in the far-red to near-infrared (NIR) range of the spectrum, (photobiomodulation or PBM) acts on mitochondria-mediated signaling pathways to attenuate oxidative stress and prevent cell death. These studies tested the hypothesis that PBM acts in the retina to promote mitochondrial integrity and function, prevent photoreceptor cell death and preserve retinal function in an established rodent model of retinitis pigmentosa, the P23H rhodopsin transgenic rat. Retinal function, structural integrity, surviving photoreceptors and the mitochondrial redox state were assessed using electroretinography, spectral domain optical coherence tomography, histomorphometry and cryofluorescence redox imaging. PBM did not alter the structural and functional characteristics of retina in a non-dystrophic animal strongly supporting the safety of PBM. Establishing the safety of PBM is essential to advance the therapy to clinical use. 830 nm PBM exerted a robust retinoprotective effect compared to 670 nm PBM in the P23H transgenic rat model. 830 nm PBM during the critical period of photoreceptor degeneration in P23H transgenic rat profoundly attenuated retinal degeneration resulting in the preservation of retinal function; retinal morphology and retinal metabolic state in comparison to the sham-treated group. An in vivo longitudinal study corroborated the structural preservation observed in the cross-sectional study. These findings provide evidence supporting the therapeutic utility of PBM in the treatment of retinal degenerative disease. They also further our understanding of the mechanism of action of PBM by showing that it improves mitochondrial function in the retinae of RP animals. By exploiting, the cells own mechanism of self-repair, PBM has the potential for translating into clinical practice as an innovative, non-invasive stand-alone or adjunct therapy for the prevention and treatment of retinal diseases

Evaluation of the potentials for optical coherence tomography (OCT) to detect early signs of retinal neurodegeneration

Among neuroretinal degenerations, glaucoma and age-related macular degeneration (AMD) have become the most frequent reasons for irreversible blindness globally. Among the causes of the elderly and senile dementia, Alzheimer’s disease (AD) has the leading position, the early ocular symptoms of which can potentially be a prognostic factor. The aim of this thesis was the early in vivo ligand-free detection of degenerative changes in the inner and outer retinal layers, which was possible using high-resolution optical coherence tomography (OCT) with the machine learning (ML) algorithms: support vector machine (SVM) and principal component analysis (PCA). Prior to the application of SVM and PCA for the classification of human OCT images, evaluation of the classifiers was performed in the classification of optical phantoms, the accuracy of which was in the range of 82-100%. This was the first attempt to measure the textural properties of various polystyrene and silica beads optical phantoms. To identify optical changes that characterise early apoptosis, OCT imaging of axotomised retinal ganglion cells (RGCs) in ex vivo retinal murine explants was performed. Substantial optical alterations in RGC dendrites in the early stages of apoptosis (up to 2 hours) were detected. ML algorithms correctly classified the retinal texture of the inner plexiform layer (IPL) of transgenic AD mice in all cases, indicating the potential for further investigation in in vivo animal and human studies. Not only the optical signature but also the transparency of the dissected murine retinal explants was investigated. Moreover, ML classification of 3xTg mice IPL layer was studied in terms of optical changes due to the RGD dendritic atrophy. ML classifiers’ accuracy in the detection of early and neovascular AMD was 93-100% for the texture of retinal pigment epithelium, 69-67% for the outer nuclear layer, 70% for the inner segment and 60-90% for the outer segment of photoreceptors. Classification of AMD stages and comparison with the age-matched healthy controls was carried out in the outer retina and RPE. Grey-level co-occurrence, run-length matrices, local binary patterns features were extracted from the IPL of the macula to classify glaucoma OCT images. The accuracy of linear and non-linear SVMs, linear and quadratic discriminant analyses, decision tree and logistic regression was between 55-70%. Based on the classifiers’ precision, recall and F1-score, Gaussian SVM outperformed other ML techniques. In this study, the observation of early glaucomatous subtle optical changes of human IPL was conducted. Also, the significance of various supervised ML algorithms was investigated. Understanding the optical signature of cumulative inherent speckle of OCT scans arising from apoptotic retinal ganglion cells and photoreceptors may provide vital information for the prevention of retinal neurodegeneration

Retinal Structure and Function in Typical Children and Young Adults

Purpose. Children with visual impairment may be referred for ERG and OCT testing to aid in diagnosis and monitoring, particularly those with suspected retinal diseases. To establish if a result is abnormal, knowledge of typical development of retinal structure and function is essential to detect, monitor, and understand pathological processes that may affect the pediatric retina. The purpose of the first study was to investigate the development of the ERG waveforms from childhood until adulthood in healthy children of European descent to better understand how retinal function changes with age. Additionally, the study aimed to provide a pediatric normative dataset for the standard ERGs to be used for clinical interpretations for children with suspected retinal diseases. The purpose of the second study was to investigate the maturation of the retinal structure from childhood until adulthood. Also, the study aimed to provide a pediatric normative dataset of retinal layer thickness maps measurements for each of the seven layers that were automatically generated by SD-OCT in the retinal regions defined by the Early Treatment of Diabetic Retinopathy Study (EDTRS) (Heidelberg Spectralis) in the same population (children of European descent). The additional purpose was to provide reference values for each sector of the peripapillary RNFL thickness. Adults were included in both studies for comparison. Methods. For the first cross-sectional study (ERG study), thirty-two participants of European descent with normal ocular and general health were recruited. The sample included 12 children between 7 and 11 years, 10 older children and adolescents between 12 and 15 years and 10 adults between 20 and 33 years. Full-field ERGs were recorded simultaneously in each eye from thread electrodes (DTL® fiber) using the Espion E3 system with fully dilated pupils (0.5% tropicamide). Stimuli were ISCEV standard dark-adapted ERGs (DA 0.01, DA 3, DA 10) as well as a light-adapted ERG series with flash strengths of 0.3, 1.0, 3.0, 10 & 24 cd.s.m2 (which includes the standard LA 3.0). We measured a- and b-wave amplitudes and implicit times using the average of the right and left eye values to compare age groups. For the cross–sectional second study (OCT study), thirty-six participants of European descent with normal ocular and general health were recruited. The sample included 6 children between 4 and 7 years, 9 children between 8 and 11 years and 10 adolescents between 12 and 15 years and 11 adults between 20 and 33 years. SD-OCT scans centered on the fovea were acquired with fully dilated pupils (0.5% tropicamide). Retinal thickness values were measured for the ETDRS regions for each of the seven layers that were automatically generated by SD-OCT (Heidelberg Spectralis, Eye Explorer software version 1.9.10.0). The peripapillary RNFL thickness measurements were calculated using the Spectralis OCT device. The influence of age on the foveal subfield, inner ring, and outer ring of the ETDRS maps, as well as on the peripapillary RNFL thickness was determined using parametric or ranked correlations. Adjusted Bonferroni correction was applied to correct for multiple comparisons. Results. For the first study, both DA a- and- b-wave implicit times were significantly positively correlated with age for all stimuli except for the b-wave of the DA 3.0 ERG i.e., implicit times were shorter for children compared to adults. Rank correlations of a-wave with age were r=0.573, p = 0.001 for the DA 3.0 ERG, r = 0.570, and p < 0.001 for the DA 10 ERG. DA b-wave implicit times were correlated with age for the weak and strong flash stimuli but not for the LA standard 3.0 ERG (DA 0.01 ERG [r = 0.596, p< 0.001] or DA 10 ERG [r = 0.434, p< 0.013]). With respect to the LA ERGs, a-wave implicit times did not correlate with age except for the LA 0.3 ERG (r = 0.548, p = 0.001). LA b-wave implicit times did not correlate with age except for the LA 1.0 ERG time age (r = 0.363, p= 0.041). In contrast, none of the ERG DA and LA amplitudes for both a- and b-waves were significantly correlated with age. For the second study, average global peripapillary RNFL thickness was 104.86 +/- 9.43µm. The peripapillary RNFL thickness was not significantly correlated with age except for the nasal superior sector where it thinned with age (r = -0.379, p = 0.023). Regarding the ETDRS regions, the total retinal thickness was positively correlated with age in the foveal subfield (r = 0.487, p = 0.003) but not in the other ETDRS rings. All the individual inner retinal layers thickened with age in some regions, except for the ganglion cell layer. While the retinal nerve fiber layer was significantly positively correlated with age in the fovea (r = 0.557, p<0.001) and parafovea (0.474, p = 0.004), the inner plexiform layer was only influenced by age in the parafoveal area (0.495, p= 0.002). In contrast, the inner nuclear layer was positively influenced by age only at the fovea (r = 0.452, p= 0.006). The individual outer retinal layers were associated with age in some regions except for the outer nuclear layer. While the outer plexiform layer thinned significantly with age in the parafoveal area (r = -0.394, p = 0.017), the retinal pigment epithelium thickened with age (r = 0.387, p = 0.020) in the foveal area. In Chapter 5 examples of OCTs and ERGs from children with retinopathy due to HARS syndrome are compared qualitatively with the present results. Conclusion. The present study provides evidence that the functional and morphological development of the retina may not be mature for children aged from 4 to 15 years. So that, the implicit times of both DA a- and b-waves and some of the LA ERGs (0.3[a-wave], 1.0 [b-wave]) increase with age to approach the adult values. Similarly, the OCT findings of the present study indicate that both the inner and outer retinal layers were influenced by age except for the ganglion cells and outer nuclear layers. Nevertheless, the peripapillary RNFL thickness measurements were not affected by age except for the nasal superior sector such that the thickness values decrease with age. In this study, we were able to obtain OCT scans using standard instruments for children as young as 4 years, and as young as 7 years for ERG. For the quantitative measurements from these techniques to be most beneficial in detecting and monitoring retinal disorders in pediatric patients, they have to be compared to an age-matched database. Age-norms and ranges were therefore calculated for those measures that were correlated with age, and overall means/medians and 95% ranges for those that were not correlated with age. These normative values can be used as a reference against which to compare for children with suspected retinal diseases

Quantitative Optical Studies of Oxidative Stress in Rodent Models of Eye and Lung Injuries

Optical imaging techniques have emerged as essential tools for reliable assessment of organ structure, biochemistry, and metabolic function. The recognition of metabolic markers for disease diagnosis has rekindled significant interest in the development of optical methods to measure the metabolism of the organ. The objective of my research was to employ optical imaging tools and to implement signal and image processing techniques capable of quantifying cellular metabolism for the diagnosis of diseases in human organs such as eyes and lungs. To accomplish this goal, three different tools, cryoimager, fluorescent microscope, and optical coherence tomography system were utilized to study the physiological metabolic markers and early structural changes due to injury in vitro, ex vivo, and at cryogenic temperatures. Cryogenic studies of eye injuries in animal models were performed using a fluorescence cryoimager to monitor two endogenous mitochondrial fluorophores, NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide). The mitochondrial redox ratio (NADH/ FAD), which is correlated with oxidative stress level, is an optical biomarker. The spatial distribution of mitochondrial redox ratio in injured eyes with different durations of the disease was delineated. This spatiotemporal information was helpful to investigate the heterogeneity of the ocular oxidative stress in the eyes during diseases and its association with retinopathy. To study the metabolism of the eye tissue, the retinal layer was targeted, which required high resolution imaging of the eye as well as developing a segmentation algorithm to quantitatively monitor and measure the metabolic redox state of the retina. To achieve a high signal to noise ratio in fluorescence image acquisition, the imaging was performed at cryogenic temperatures, which increased the quantum yield of the intrinsic fluorophores. Microscopy studies of cells were accomplished by using an inverted fluorescence microscope. Fixed slides of the retina tissue as well as exogenous fluorophores in live lung cells were imaged using fluorescent and time-lapse microscopy. Image processing techniques were developed to quantify subtle changes in the morphological parameters of the retinal vasculature network for the early detection of the injury. This implemented image cytometry tool was capable of segmenting vascular cells, and calculating vasculature features including: area, caliber, branch points, fractal dimension, and acellular capillaries, and classifying the healthy and injured retinas. Using time-lapse microscopy, the dynamics of cellular ROS (Reactive Oxygen Species) concentration was quantified and modeled in ROS-mediated lung injuries. A new methodology and an experimental protocol were designed to quantify changes of oxidative stress in different stress conditions and to localize the site of ROS in an uncoupled state of pulmonary artery endothelial cells (PAECs). Ex vivo studies of lung were conducted using a spectral-domain optical coherence tomography (SD-OCT) system and 3D scanned images of the lung were acquired. An image segmentation algorithm was developed to study the dynamics of structural changes in the lung alveoli in real time. Quantifying the structural dynamics provided information to diagnose pulmonary diseases and to evaluate the severity of the lung injury. The implemented software was able to quantify and present the changes in alveoli compliance in lung injury models, including edema. In conclusion, optical instrumentation, combined with signal and image processing techniques, provides quantitative physiological and structural information reflecting disease progression due to oxidative stress. This tool provides a unique capability to identify early points of intervention, which play a vital role in the early detection of eye and lung injuries. The future goal of this research is to translate optical imaging to clinical settings, and to transfer the instruments developed for animal models to the bedside for patient diagnosis

Vliv proteinu Fam84b na homeostázu retiny

Fam84b je jen málo prostudovaný protein, jehož funkce v eukaryotické buňce je stále nejasná. V této práci je představen myší model s vyřazeným genem FAM84B (FAM84B KO) s detailní charakteristikou jeho fenotypu v oční sítnici. Morfologie sítnice byla u FAM84B KO myší studována pomocí optické koherentní tomografie a histologie. Tyto dvě metody ukázaly dynamické změny vycházející z fotoreceptorové a z pigmentové epiteliální vrstvy. Fenotyp se zhoršoval s věkem a patologické oblasti se postupně rozšiřovaly, ale také pronikaly hlouběji do vnitřních retinálních vrstev. Porovnání lokalizace standartních retinálních buněčných markerů ukázalo, že u FAM84B KO myší dochází k postupnému rozrušení vrstev sítnice spolu s deformací retinální makrostruktury. Sítnice mutovaných myší také vykazují nižší reakci na stimulaci světlem, což bylo prokázáno pomocí elektroretinografie. Fam84b je homologní s HRASLS enzymatickou rodinou, která tlumí signalizaci asociovanou s Ras. K ověření, zda má Fam84b podobnou funkci, byly porovnány hodnoty fosforylovaných a aktivovaných downstream Ras efektorů mezi lyzáty sítnice FAM84B KO a kontrolních nemutovaných zvířat. U KO myší byla naměřena redukce pERK1 (pY204), která poukazuje na možnou funkci v rámci downstream Ras signalizace. Předběžné ko-imunoprecipitační pokusy odhalily...Fam84b is a largely unstudied protein, where its function in eukaryotic cells is unclear. This thesis work presents a FAM84B knockout mouse model and characterises the resulting retinal phenotype in detail. FAM84B KO mice were morphologically assessed by optical coherence tomography and histological processing, revealing dynamic changes stemming from the photoreceptor and pigmented epithelial layers. This potent phenotype progresses with age, spreading inwards towards the inner retinal layers, as well as laterally to adjacent retinal regions. Comparative localisation of standard retinal cell markers demonstrates that FAM84B KO retinal layering becomes increasingly disorganised, together with deformation of the retinal macrostructure. Due to this, KO mice experience reducing responses to light, as demonstrated by electroretinography, where overall retinal efficiency falls. Fam84b shows homology to the HRASLS enzyme family, which are capable of attenuating Ras-associated signalling. To investigate whether Fam84b possesses a similar function, the level of phosphorylated and activated downstream Ras effectors were compared between wild type and FAM84B KO mouse retinal lysates. A reduction of pERK1 (pY204) in KO lysates suggests that Fam84b holds some function related to this pathway downstream of Ras....Department of Cell BiologyKatedra buněčné biologiePřírodovědecká fakultaFaculty of Scienc

Enhanced visualization of retinal pathologies with ultrahigh resolution optical coherence tomography

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2005.Page 150 blank.Includes bibliographical references.Current clinical practice calls for the development of techniques to diagnose diseases in its early stages, when treatment is most effective and significant irreversible damage can either be prevented or delayed. Optical coherence tomography (OCT) is an emerging medical diagnostic technology being investigated for applications in a number of medical fields including ophthalmology, cardiology, and gastroenterology. OCT is analogous to ultrasound except that it uses light waves rather than sound waves. OCT can achieve a much higher resolution than ultrasound in measuring the underlying tissue microstructures. Another advantage of OCT is that it can achieve imaging in a non-contact and non-invasive manner. With typical axial resolution of 10 [mu]m, OCT already provides significantly more detailed structural information than any other conventional clinical imaging technique. The development of OCT with even higher resolution would potentially have significant impact in diagnosing diseases in such fields as ophthalmology, cardiology, gastroenterology, and oncology. Ultrahigh resolution OCT systems have been developed for animal research and clinical ophthalmology. Ultrahigh resolution OCT improves the axial resolution of OCT from the standard 10 [mu]m to 1 [mu]m for animal studies and 3 [mu]m for clinical studies. This improved imaging resolution approaches that of histopathology. Therefore, OCT can potentially function as "optical biopsy" since it permits the imaging of tissue microstructure with resolutions approaching that of histopathology except that imaging can be performed in real time, without the need of tissue removal.(cont.) Using ultrahigh resolution OCT systems, animal imaging studies have been performed on mouse and rat models of retinal diseases and clinical imaging studies have been performed on more than 800 patients at the ophthalmology clinic. The results from patient imaging studies on a wide variety of retinal diseases suggest that ultrahigh resolution OCT can improve the diagnosis and management of retinal diseases as well as possibly increase the understanding of ocular disease pathogenesis. Therefore, ultrahigh resolution OCT has the potential to become an important tool in ophthalmology research and clinics.by Tony Hong-Tyng Ko.Ph.D

Characterizing Stargardt disease-causing mutations to identify ABCA4 gene lesions amenable to splice intervention therapeutics

Stargardt disease (STGD1, OMIM: 248200) is an autosomal recessive retinal dystrophy, characterized by bilateral progressive central vision loss and subretinal deposition of lipofuscin-like substances. The wide spectrum of clinical phenotypes, ranging from childhood-onset cone-rod dystrophy to late-onset macular pattern dystrophy-like disease, indicates a more complex genotype-phenotype correlation than previously believed. The association of mutations in the ATP-binding cassette transporter gene, ABCA4, with STGD1 was first reported in two families in 1997. The ABCA4 protein encoded by ABCA4 is predominantly expressed in outer segments of photoreceptors and retinal pigment epithelial (RPE) cells in retina..