Human selection bias drives the linear nature of the more ground truth effect in explainable deep learning optical coherence tomography image segmentation.

Supervised deep learning (DL) algorithms are highly dependent on training data for which human graders are assigned, for example, for optical coherence tomography (OCT) image annotation. Despite the tremendous success of DL, due to human judgment, these ground truth labels can be inaccurate and/or ambiguous and cause a human selection bias. We therefore investigated the impact of the size of the ground truth and variable numbers of graders on the predictive performance of the same DL architecture and repeated each experiment three times. The largest training dataset delivered a prediction performance close to that of human experts. All DL systems utilized were highly consistent. Nevertheless, the DL under-performers could not achieve any further autonomous improvement even after repeated training. Furthermore, a quantifiable linear relationship between ground truth ambiguity and the beneficial effect of having a larger amount of ground truth data was detected and marked as the more-ground-truth effect.

Cynomolgus monkey's retina volume reference database based on hybrid deep learning optical coherence tomography segmentation.

Cynomolgus monkeys (Macaca fascicularis) are commonly used in pre-clinical ocular studies. However, studies that report the morphological features of the macaque retina are based only on minimal sample sizes; therefore, little is known about the normal distribution and background variation. This study was conducted using optical coherence tomography (OCT) imaging to investigate the variations in retinal volumes of healthy cynomolgus monkeys and the effects of sex, origin, and eye side on the retinal volumes to establish a comprehensive reference database. A machine-learning algorithm was employed to segment the retina within the OCT data (i.e., generated pixel-wise labels). Furthermore, a classical computer vision algorithm has identified the deepest point in a foveolar depression. The retinal volumes were determined and analyzed based on this reference point and segmented retinal compartments. Notably, the overall foveolar mean volume in zone 1, which is the region of the sharpest vision, was 0.205 mm3 (range 0.154-0.268 mm3), with a relatively low coefficient of variation of 7.9%. Generally, retinal volumes exhibit a relatively low degree of variation. However, significant differences in the retinal volumes due to the monkey's origin were identified. Additionally, sex had a significant impact on the paracentral retinal volume. Therefore, the origin and sex of cynomolgus monkeys should be considered when evaluating the macaque retinal volumes based on this dataset.

Volumetric subfield analysis of cynomolgus monkey's choroid derived from hybrid machine learning optical coherence tomography segmentation.

This study aimed to provide volumetric choroidal readings regarding sex, origin, and eye side from healthy cynomolgus monkey eyes as a reference database using optical coherence tomography (OCT) imaging. A machine learning (ML) algorithm was used to extract the choroid from the volumetric OCT data. Classical computer vision methods were then applied to automatically identify the deepest location in the foveolar depression. The choroidal thickness was determined from this reference point. A total of 374 eyes of 203 cynomolgus macaques from Asian and Mauritius origin were included in the analysis. The overall subfoveolar mean choroidal volume in zone 1, in the region of the central bouquet, was 0.156 mm3 (range, 0.131-0.193 mm3). For the central choroid volume, the coefficient of variation (CV) was found of 6.3%, indicating relatively little variation. Our results show, based on analyses of variance, that monkey origin (Asian or Mauritius) does not influence choroid volumes. Sex had a significant influence on choroidal volumes in the superior-inferior axis (p ≤ 0.01), but not in the fovea centralis. A homogeneous foveolar choroidal architecture was also observed.

Cynomolgus monkey's choroid reference database derived from hybrid deep learning optical coherence tomography segmentation.

Cynomolgus monkeys exhibit human-like features, such as a fovea, so they are often used in non-clinical research. Nevertheless, little is known about the natural variation of the choroidal thickness in relation to origin and sex. A combination of deep learning and a deterministic computer vision algorithm was applied for automatic segmentation of foveolar optical coherence tomography images in cynomolgus monkeys. The main evaluation parameters were choroidal thickness and surface area directed from the deepest point on OCT images within the fovea, marked as the nulla with regard to sex and origin. Reference choroid landmarks were set underneath the nulla and at 500 µm intervals laterally up to a distance of 2000 µm nasally and temporally, complemented by a sub-analysis of the central bouquet of cones. 203 animals contributed 374 eyes for a reference choroid database. The overall average central choroidal thickness was 193 µm with a coefficient of variation of 7.8%, and the overall mean surface area of the central bouquet temporally was 19,335 µm2 and nasally was 19,283 µm2. The choroidal thickness of the fovea appears relatively homogeneous between the sexes and the studied origins. However, considerable natural variation has been observed, which needs to be appreciated.

Uncovering of intraspecies macular heterogeneity in cynomolgus monkeys using hybrid machine learning optical coherence tomography image segmentation.

The fovea is a depression in the center of the macula and is the site of the highest visual acuity. Optical coherence tomography (OCT) has contributed considerably in elucidating the pathologic changes in the fovea and is now being considered as an accompanying imaging method in drug development, such as antivascular endothelial growth factor and its safety profiling. Because animal numbers are limited in preclinical studies and automatized image evaluation tools have not yet been routinely employed, essential reference data describing the morphologic variations in macular thickness in laboratory cynomolgus monkeys are sparse to nonexistent. A hybrid machine learning algorithm was applied for automated OCT image processing and measurements of central retina thickness and surface area values. Morphological variations and the effects of sex and geographical origin were determined. Based on our findings, the fovea parameters are specific to the geographic origin. Despite morphological similarities among cynomolgus monkeys, considerable variations in the foveolar contour, even within the same species but from different geographic origins, were found. The results of the reference database show that not only the entire retinal thickness, but also the macular subfields, should be considered when designing preclinical studies and in the interpretation of foveal data.

Unraveling the deep learning gearbox in optical coherence tomography image segmentation towards explainable artificial intelligence.

Machine learning has greatly facilitated the analysis of medical data, while the internal operations usually remain intransparent. To better comprehend these opaque procedures, a convolutional neural network for optical coherence tomography image segmentation was enhanced with a Traceable Relevance Explainability (T-REX) technique. The proposed application was based on three components: ground truth generation by multiple graders, calculation of Hamming distances among graders and the machine learning algorithm, as well as a smart data visualization ('neural recording'). An overall average variability of 1.75% between the human graders and the algorithm was found, slightly minor to 2.02% among human graders. The ambiguity in ground truth had noteworthy impact on machine learning results, which could be visualized. The convolutional neural network balanced between graders and allowed for modifiable predictions dependent on the compartment. Using the proposed T-REX setup, machine learning processes could be rendered more transparent and understandable, possibly leading to optimized applications.

Risdiplam treatment has not led to retinal toxicity in patients with spinal muscular atrophy.

OBJECTIVE: Evaluation of ophthalmologic safety with focus on retinal safety in patients with spinal muscular atrophy (SMA) treated with risdiplam (EVRYSDI®), a survival of motor neuron 2 splicing modifier associated with retinal toxicity in monkeys. Risdiplam was approved recently for the treatment of patients with SMA, aged ≥ 2 months in the United States, and is currently under Health Authority review in the EU. METHODS: Subjects included patients with SMA aged 2 months-60 years enrolled in the FIREFISH, SUNFISH, and JEWELFISH clinical trials for risdiplam. Ophthalmologic assessments, including functional assessments (age-appropriate visual acuity and visual field) and imaging (spectral domain optical coherence tomography [SD-OCT], fundus photography, and fundus autofluorescence [FAF]), were conducted at baseline and every 2-6 months depending on study and assessment. SD-OCT, FAF, fundus photography, and threshold perimetry were evaluated by an independent, masked reading center. Adverse events (AEs) were reported throughout the study. RESULTS: A total of 245 patients receiving risdiplam were assessed. Comprehensive, high-quality, ophthalmologic monitoring assessing retinal structure and visual function showed no retinal structural or functional changes. In the youngest patients, SD-OCT findings of normal retinal maturation were observed. AEs involving eye disorders were not suggestive of risdiplam-induced toxicity and resolved with ongoing treatment. INTERPRETATION: Extensive ophthalmologic monitoring conducted in studies in patients with SMA confirmed that risdiplam does not induce ophthalmologic toxicity in pediatric or adult patients with SMA at the therapeutic dose. These results suggest that safety ophthalmologic monitoring is not needed in patients receiving risdiplam, as also reflected in the United States Prescribing Information for risdiplam.

A 3D model to evaluate retinal nerve fiber layer thickness deviations caused by the displacement of optical coherence tomography circular scans in cynomolgus monkeys (Macaca fascicularis).

The main objective of the study was to analyze deviations in retinal nerve fiber layer (RNFL) thickness measurements caused by the displacement of circular optic disc optical coherence tomography scans. High-density radial scans of the optic nerve heads of cynomolgus monkeys were acquired. The retinal nerve fiber layer was manually segmented, and a surface plot of the discrete coordinates was generated. From this plot, the RNFL thicknesses were calculated and compared between accurately centered and intentionally displaced circle scans. Circle scan displacement caused circumpapillary retinal nerve fiber layer thickness deviations of increasing magnitude with increasing center offset. As opposed to the human eye, horizontal displacement resulted in larger RNFL thickness deviations than vertical displacement in cynomolgus monkeys. Acquisition of high-density radial scans allowed for the mathematical reconstruction and modelling of the nerve fiber layer and extrapolation of its thickness. Accurate and strictly repeatable circle scan placement is critical to obtain reproducible values, which is essential for longitudinal studies.

Retinal Features in Cynomolgus Macaques (Macaca fascicularis) Assessed by Using Scanning Laser Ophthalmoscopy and Spectral Domain Optical Coherence Tomography.

Cynomolgus macaques are an important and commonly used species in preclinical toxicology studies, but structural reports of in vivo retinal findings are rare in this species. The purpose of this study was to diminish this gap and document optical coherence tomography and scanning laser ophthalmoscopy imaging data in the healthy posterior pole of cynomolgus monkeys' eyes at predose examinations. The current study is a retrospective assessment of baseline spectral domain OCT data obtained from the 768 eyes of 384 cynomolgus monkeys (192 males and 192 females) of Mauritian origin. The data set was obtained from studies conducted over a 4-y period in the context of ocular safety evaluations of various compounds under preclinical development. The most prevalent findings were the presence of Bergmeister papilla and intravitreal hyperreflective spots. Less common findings included disorganization of retinal zones, abnormalities of the retinal vasculature, partial posterior vitreous detachment, and abnormally shaped foveal pits. Thoughtful consideration of these physiologic findings will aid in distinguishing normal features from toxic outcomes in future preclinical ophthalmic studies.

Challenging a Myth and Misconception: Red-Light Vision in Rats.

Due to the lack of L-cones in the rodent retina, it is generally assumed that red light is invisible to rodents. Thus, red lights and red filter foils are widely used in rodent husbandry and experimentation allowing researchers to observe animals in an environment that is thought to appear dark to the animals. To better understand red-light vision in rodents, we assessed retinal sensitivity of pigmented and albino rats to far-red light by electroretinogram. We examined the sensitivity to red light not only on the light- but also dark-adapted retina, as red observation lights in husbandry are used during the dark phase of the light cycle. Intriguingly, both rods and cones of pigmented as well as albino rats show a retinal response to red light, with a high sensitivity of the dark-adapted retina and large electroretinogram responses in the mesopic range. Our results challenge the misconception of rodents being red-light blind. Researchers and housing facilities should rethink the use of red observation lights at night.

Macular thickness measurements of healthy, naïve cynomolgus monkeys assessed with spectral-domain optical coherence tomography (SD-OCT).

The purpose of this study was to measure central macular thickness in an unprecedented number of cynomolgus monkeys. Macular thickness was measured with Heidelberg spectral-domain OCT in 320 eyes of healthy and treatment-naïve cynomolgus monkeys (80 males and 80 females). The macula was successfully measured in all 320 eyes. Macular thickness was not significantly different between the sexes. The mean central macular thickness was 244 µm (+/- 21 µm). Macular thicknesses in the quadrants were 327 +/-17 µm (temporal inner), 339 +/- 17 µm (inferior inner), 341 +/- 14 µm (superior inner), 341 +/-18 µm (nasal inner), and 299 +/- 20 µm (temporal outer), 320 +/- 16 µm (superior outer), 332 +/-23 µm (inferior outer), and 337 +/-18 µm (nasal outer). Highly significant differences between the nasal and temporal quadrants were detected. This study successfully demonstrated the feasibility of retinal thickness measurements in healthy cynomolgus monkeys. The present findings indicate that the macula is thicker in cynomolgus monkeys than in humans and provide important normative data for future studies.

Indocyanine green molecular angiography of choroidal neovascularization.

Retinal diseases such as proliferative diabetic retinopathy and neovascular AMD are characterized by the formation of new blood vessels. Current imaging techniques such as fluorescein and ICG angiography help to identify areas of vascular leakage but are limited in their applicability due to their nonspecific nature. However, as new treatment paradigms emerge in an effort to have patient specific treatments, the development of new imaging techniques that are capable of identifying patient specific biomarkers will become crucial for the success of these approaches. In this study, we create and characterize an endoglin (CD105) targeted imaging probe that can be used for indocyanine green (ICG) molecular angiography. This anti-endoglin-ICG bioconjugate has a self-quenching "off-on" capacity to enable high contrast imaging of proliferative blood vessels at a molecular level in vivo. Using the laser CNV mouse model we demonstrate an approximate 3-fold increase in lesion visualization compared to non-targeting controls.

Discovery of Risdiplam, a Selective Survival of Motor Neuron-2 ( SMN2) Gene Splicing Modifier for the Treatment of Spinal Muscular Atrophy (SMA).

SMA is an inherited disease that leads to loss of motor function and ambulation and a reduced life expectancy. We have been working to develop orally administrated, systemically distributed small molecules to increase levels of functional SMN protein. Compound 2 was the first SMN2 splicing modifier tested in clinical trials in healthy volunteers and SMA patients. It was safe and well tolerated and increased SMN protein levels up to 2-fold in patients. Nevertheless, its development was stopped as a precautionary measure because retinal toxicity was observed in cynomolgus monkeys after chronic daily oral dosing (39 weeks) at exposures in excess of those investigated in patients. Herein, we describe the discovery of 1 (risdiplam, RG7916, RO7034067) that focused on thorough pharmacology, DMPK and safety characterization and optimization. This compound is undergoing pivotal clinical trials and is a promising medicine for the treatment of patients in all ages and stages with SMA.

Preputial diverticulum dilation in a Goettingen minipig.

Preputial dilation is an infrequently reported condition in pigs. The pathophysiology and etiology is unclear. Causes for diverticulum dilation are proposed to be chronic preputial diverticulitis with subsequent fibrosis of the preputial cavity, phimosis of the preputial orifice or the preputial diverticulum, but the large majority of cases are reportedly idiopathic in nature. Surgical interventions include ablative procedures, but many cases are not treated because of an assumed lack of clinical relevance in pigs not used for breeding. We report a case of progressive preputial dilation that recurred after surgical intervention. Histopathological examination revealed no primary inflammatory condition, contrary to literature suggesting a role for inflammatory mediators in pathogenesis. Phimosis of the preputial orifice was noted post mortem and might be a contributing factor. These findings partially contradict the current assumptions in regards to pathophysiology and treatment choices in the literature and warrant further investigation into alternative therapeutic interventions for this condition.

The role of diet and housing-temperature in the production of genetically modified mouse embryos and their developmental capacity after cryopreservation.

Mutant mouse lines are unique models with an enormous scientific potential. Cryopreservation of preimplantation embryos or of spermatozoa is a common approach to save those lines. The breeding of a line can be discontinued if sufficient specimens have been cryopreserved. Prerequisites to economically cryopreserve embryos are high yields of embryos prepared from donors and a high recovery rate after revitalization. Diets for laboratory animals are often produced from phytoestrogen-containing soy; the present study shows that feeding the donor animals with a phytoestrogen-poor diet is more efficient compared to a phytoestrogen-containing, soy-based diet. Additionally, a uterotrophic bioassay indicating the estrogenic role of compounds showed a significant increase of the relative uterus size of females fed with a phytoestrogen-rich diet. The role of the housing-temperature was investigated, too, showing that a housing-temperature of 24 °C results in the best embryo yields. The production of two-cell embryos is more economic than the production of eight-cell embryos. Investigating the recovery rate of frozen/thawed embryos, a very high recovery rate was determined when both, two- and eight-cell embryos were thawed. However, the capacity to develop to the next embryonic stage in vitro was dramatically reduced when two-cell embryos were compared to eight-cell embryos. After embryo transfer, the sex ratio became uneven and more males were delivered. This effect might be due to the procedures to which animals and embryos were subjected. These data show that many parameters can influence the production of animals when using (frozen/thawed) embryos. These parameters need continuous surveillance.

Development of a murine ocular posterior segment explant culture for the study of intravitreous vector delivery.

The objective of this study was to develop a murine retinal/choroidal/scleral explant culture system to facilitate the intravitreous delivery of vectors. Posterior segment explants from adult mice of 2 different age groups (4 wk and 15 wk) were cultured in serum-free medium for variable time periods. Tissue viability was assessed by gross morphology, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. To model ocular gene therapy, explants were exposed to varying transducing units of a lentiviral vector expressing the gene for green fluorescent protein for 48 h. Explant retinal cells remained viable for approximately 1 wk, although the ganglion cell layer developed apoptosis between 4 and 7 d. Following vector infusion into the posterior segment cups, viral transduction was noted in multiple retinal layers in both age groups. An age of donor mouse influence was noted and older mice did not transduce as well as younger mice. This explant offers an easily managed posterior segment ocular culture with minimum disturbance of the tissue, and may be useful for investigating methods of enhancing retinal gene therapy under controlled conditions.

L'objectif de la présente étude était de développer un système murin de culture d'explant de rétine/choroïde/sclérotique afin de faciliter la livraison intra-vitréenne de vecteurs. Des explants de segments postérieurs provenant de souris adultes de deux groupes d'âge différents (4 sem et 15 sem) furent cultivés dans un milieu sans sérum pour des périodes de temps variables. La viabilité tissulaire fut évaluée par morphologie macroscopique, quantification de la survie cellulaire, expression de la caspase-3 activée, et immunohistochimie. Afin d'imiter la thérapie génique oculaire, les explants furent exposés pendant 48 h à des unités de transduction variables d'un vecteur lentiviral exprimant le gène pour la protéine fluorescente verte. Les explants de cellules de la rétine sont demeurés viables pour environ 1 sem, bien que dans la couche de cellules ganglionnaires on nota le développement de l'apoptose entre 4 à 7 j. Suite à l'infusion de vecteur dans le segment postérieur, la transduction virale fut notée dans plusieurs couches rétiniennes des animaux des deux groupes d'âge. Une influence de l'âge de la souris donneuse fut notée et chez les souris plus âgées la transduction ne se faisait pas aussi bien que chez les jeunes souris. Ce modèle d'explant permet la gestion facile de culture de segment oculaire postérieur avec un minimum de dérangement du tissu, et pourrait être utile pour étudier des méthodes visant à augmenter la thérapie génique sous conditions contrôlées.(Traduit par Docteur Serge Messier).

A retrospective study of the clinical, histological, and immunohistochemical manifestations of 5 dogs originally diagnosed histologically as necrotizing scleritis.

PURPOSE: To describe the clinical, histological, and immunohistochemical manifestations of canine necrotizing scleritis. METHODS: A retrospective examination of the clinical records and samples of ocular tissues from five dogs with a histological diagnosis 'necrotizing scleritis' was completed. Archived, formalin-fixed, paraffin-embedded samples and two control globes were stained with hematoxylin and eosin, Gram, periodic acid-Schiff (PAS) and Masson trichrome stains, and they were immunohistochemically labeled for CD3, CD18, and CD20. RESULTS: Of the five cases reviewed, only two could be confirmed as idiopathic necrotizing scleritis. The other three cases were retrospectively diagnosed as unilateral focal, non-necrotizing scleritis, one as episcleritis and the third was scleritis secondary to a proptosed globe based on our retrospective clinical, histological, and immunohistochemical evaluations. In these two cases, idiopathic necrotizing scleritis manifested as a bilateral, progressive, inflammatory disease of the sclera and cornea that induces significant uveitis. Light microscopic examination confirmed collagen degeneration and granulomatous inflammation. There was no evidence for an infectious etiology based on Gram's and PAS stainings. Immunohistochemical labeling revealed a predominance of B cells in idiopathic, bilateral necrotizing scleritis. Tinctorial staining abnormalities with Masson's trichrome stain were present in scleral collagen of the two cases with idiopathic necrotizing scleritis as well as a case of secondary traumatic scleritis. CONCLUSIONS: Based on a limited number of cases, idiopathic canine necrotizing scleritis shares similar histopathological features with non-necrotizing scleritis and episcleritis; however, necrotizing scleritis is B-cell-dominated and bilateral, and significant collagen alterations manifest with Masson's trichrome stain.

The effect of UV-blocking contact lenses as a therapy for canine chronic superficial keratitis.

OBJECTIVE: To evaluate the effect of UV-blocking soft contact lenses in treatment for chronic superficial keratitus (CSK). METHODS: Twenty six dogs with CSK were treated continuously with UV-blocking contact lenses for 6 months. A contact lens was placed on one eye of each dog; the other eye remained without a lens as a control eye. After this primary study, five of the dogs were further treated and they wore then contact lenses in both eyes. Continuously, all patients were concurrently treated topically with cyclosporine. The contact lenses were changed every 4 weeks and an ophthalmic examination performed. Evaluation criteria included corneal alterations as pigmentation, edema, pannus and vascularization. To determine the transmittance characteristics of the contact lenses before and after use, 32 contact lenses were measured with a UV-vis-NIR spectrophotometer. RESULTS: Pigmentation increased in eyes wearing lenses and in control eyes over the evaluation period of 6 months. Corneal edema increased in the eyes wearing lenses, but remained unaffected in the control eyes. A significant difference in the incidence of pannus and the extent of corneal vascularisation could not be evaluated. Adverse effects were noted in six cases (corneal edema and vascularisation, conjunctivitis, blepharospasm). All new lenses studied reduced UV-radiation to a safe level, whereas used lenses did not maintain their transmittance characteristics. CONCLUSIONS: No positive effect of UV-blocking contact lenses could be proven with the study design used.