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Purpose: Patients with cystic fibrosis (CF) are at risk to develop CF related diabetes (CFRD) and subsequently even diabetic neuro- and/or vasculopathy. We sought to determine if there are typical signs of diabetes-related retinal alterations present in CF patients with preserved and impaired glycemic control., Methods: During routine annual examination CF patients were offered an additional 7-day period of real time continuous glucose monitoring (rtCGM) and an ophthalmological examination including retinal optical coherence tomography (OCT). Patients were categorized according to the glycemic control, i.e. the results of an oral glucose tolerance test (OGTT) and rtCGM were taken into consideration. OCT data was analyzed by our previously published visual analysis software generating dedicated and spatially resolved deviation maps for visualization and quantification of differences in total retinal thickness and thickness of retinal nerve fiber layer (RNFL) as well as ganglion cell layer (GCL) in comparison to age-matched healthy controls and patients with either type 1 or type 2 diabetes mellitus., Results: Results of the rtCGM and/or OGTT enabled discrimination between patients with normal glycemic control (CFNG; n  = 6), with abnormal glycemic control (CFAG; n  = 6) and overt CFRD ( n  = 4). OCT data indicates gradually increasing retinal thinning in all 3 groups, depending on the degree of glucose metabolism disorder compared to healthy controls. At the foveal region total retinal thickness and GCL thickness were significantly thinner in CFRD patients compared to CFNG patients (total retinal thickness: 260.4 µm (239.3-270.8) vs. 275.4 µm (254.3-289.5); GCL: 11.82 µm (11.16-15.25) vs. 17.30 µm (13.95-19.82); each p  < 0.05)., Conclusion: Although we investigated a rather small number of patients, we obtained evidence that intraretinal neurodegenerative changes occur in each of our subgroups (CFNG, CFAG, CFRD). Beyond this, our results favor the detrimental role of additional diabetes, as the deviations from healthy controls were most pronounced in the CFRD group and are similar to those seen in patients suffering from type 1 or type 2 diabetes.

Magnetic resonance imaging of the eye and orbit (MReye) is a cross-domain research field, combining (bio)physics, (bio)engineering, physiology, data sciences and ophthalmology. A growing number of reports document technical innovations of MReye and promote their application in preclinical research and clinical science. Realizing the progress and promises, this review outlines current trends in MReye. Examples of MReye strategies and their clinical relevance are demonstrated. Frontier applications in ocular oncology, refractive surgery, ocular muscle disorders and orbital inflammation are presented and their implications for explorations into ophthalmic diseases are provided. Substantial progress in anatomically detailed, high-spatial resolution MReye of the eye, orbit and optic nerve is demonstrated. Recent developments in MReye of ocular tumors are explored, and its value for personalized eye models derived from machine learning in the treatment planning of uveal melanoma and evaluation of retinoblastoma is highlighted. The potential of MReye for monitoring drug distribution and for improving treatment management and the assessment of individual responses is discussed. To open a window into the eye and into (patho)physiological processes that in the past have been largely inaccessible, advances in MReye at ultrahigh magnetic field strengths are discussed. A concluding section ventures a glance beyond the horizon and explores future directions of MReye across multiple scales, including in vivo electrolyte mapping of sodium and other nuclei. This review underscores the need for the (bio)medical imaging and ophthalmic communities to expand efforts to find solutions to the remaining unsolved problems and technical obstacles of MReye, with the objective to transfer methodological advancements driven by MR physics into genuine clinical value.

The cornea is a transparent outermost structure of the eye anterior segment comprising the highest density of innervated tissue. In the process of corneal innervation, trigeminal ganglion originated corneal nerves diligently traverse different corneal cell types in different corneal layers including the corneal stroma and epithelium. While crossing the stromal and epithelial cell layers during innervation, due to the existing physical contacts, close interactions occur between stromal keratocytes, epithelial cells, resident immune cells and corneal nerves. Furthermore, by producing various trophic and growth factors corneal cells assist in maintaining the growth and function of corneal nerves. Similarly, corneal nerve generated growth factors critically modify the corneal cell function in all the corneal layers. Due to their close association and contacts, on-going cross-communication between these cell types and corneal nerves play a vital role in the modulation of corneal nerve function, regeneration during wound healing. The present review highlights the influence of different corneal cell types and growth factors released from these cells on corneal nerve regeneration and function.

Purpose : To analyze the use of deviation maps (DevMs) to understand thickness changes of inner retinal layers in optical coherence tomography (OCT) data. To test a new visual analytics (VA) method with reduced complexity of OCT data analysis by comparing the layer thickness of children with type 1 diabetes mellitus (T1DM) to matched controls. Methods : OCT was performed on unilateral eyes of 26 children with T1DM without diabetic retinopathy and 29 healthy children to obtain macular volume scans. Subsequently, segmented inner retinal layers were analyzed using VA. Deviation maps were generated to readily visualize thickness differences between both groups and to investigate thickness changes of individual patients in relation to the control group. Results : In DevMs of the patient group, the total retina (TR) demonstrated localized, irregular areas of thinning (mean ± standard deviation) involving foveal center, inner macula, and inferior-nasal outer macula (-9.31 ± 1.73 µm; p < 0.05). Similarly, retinal nerve fiber layer showed continuous and localized areas of thinning in both inner and outer macula, extending nasally (-5.45 ± 4.31 µm; p < 0.05). In DevMs of individual patients, the TR and inner retinal layers revealed remarkable changes in thickness that were present between patients at both late and early stages of diabetes. Conclusion : The VA method simplifies the in-depth analysis of OCT volume data from different groups and is effective in detecting retinal thickness changes in children with diabetes. It can be easily adopted in a clinical set-up and intuitively used in complex multidisciplinary studies.

Objective: Children with chronic kidney diseases (CKD) are at risk for neurological diseases at early adulthood. Spectral-domain optical coherence tomography (SD-OCT) of the retina is especially suitable for determination of intraretinal layer thickness. We wonder whether retinal thinning is already present in pediatric patients with mild-to-moderate CKD., Patients and Methods: Children (n = 15; 14.9 ± 2.4 years) with mild-to-moderate CKD (median eGFR of 95ml/min/1.73m 2 ; range: 28-187ml/min/1.73m 2 ) due to glomerulopathy, congenital anomalies of kidney and urinary tract (CAKUT), or haemolytic uremic syndrome (HUS) underwent a detailed ophthalmologic examination including high-resolution SD-OCT. Three OCT scans were obtained from the right eyes of all patients. Within each scan, retinal layers were separated and the mean thickness was determined at the foveal, parafoveal, and perifoveal area. The results were compared to those we obtained previously from healthy children., Results: At the parafoveal area, thickness (median, range) of the total retina (ALL), ganglion cell layer (GCL), and inner plexiform layer (IPL) were reduced compared to healthy volunteers (339µm, (288-361µm) vs. 348µm, (320-385µm); 49.8µm (30.5-56.6µm) vs. 53.5μm (49.5-60.5µm) and 41.0µm (29.4-43.7µm) vs. 43.46µm (39.5-46.3μm); each p < 0.05). The intraretinal thickness measurements at the foveal and perifoveal areas revealed no statistically significant differences between patients and controls., Conclusion: Distinct changes within the parafoveal area of the total retina, GCL, and IPL are present in children with mild-to-moderate CKD. Prospective studies are required to assess the clinical significance of our findings.

Purpose/Aim of the study: A recently proposed technique enables the generation of continuously increasing mosaic images of the corneal sub-basal nerve plexus (SNP) using in vivo corneal confocal microscopy (CCM). The aim of the present study was to investigate the progression of the corneal nerve fiber length (CNFL) measured in the growing mosaic images with regard to their increasing area., Materials and Methods: Five large datasets from three healthy volunteers were examined using the proposed CCM technique. Intermediate mosaic images were created and assessed for CNFL., Results: The measured CNFL progression shows both over- and underestimation of the CNFL for small observed areas. Increasing the mosaic image area stabilizes the CNFL values and reduces the moving variance in all five datasets. The relative deviation of means from values of first and second examination of two of the subjects shows high differences for an observed area of <1.5 mm 2 ., Conclusions: The present examination provides two measures to quantify different area-dependent aspects of the CNFL measured in an expanding mosaic image. The moving variance measures how stable the CNFL can be considered at a certain mosaic size. The relative deviation of means from two repeated CCM examinations on the other hand gives some indication on the level of reliability that can be expected from the measured CNFL. The progression of CNFL in the examined datasets manifests a potentially very high variability for mosaic sizes of less than about 1.5 mm 2 . Above that size, CNFL progression and the intra-patient relative deviations both stabilize significantly in all five datasets. The results of the present examination suggest a recommendation for a minimum sampled area of the central SNP of 1.5 mm 2 for reliable and meaningful measurement of CNFL.

Purpose: The aim of this study is to determine age-related morphological changes in the corneal subbasal nerve plexus (SNP) in two inbred mouse strains., Materials and Methods: The corneal SNP was investigated by in vivo confocal laser scanning microscopy (CLSM) in 0.5-, 1-, 1.5-, and 2-year-old C57BL/6J mice and in 0.5- and 1-year-old BALB/c mice (n = 4 per age category and strain; 10 images per mouse). Fixed corneal samples from C57BL/6J mice were also analyzed after PGP9.5 staining. Nerve fiber density (NFD) was determined using the semi-automated NeuronJ program. In addition, a new custom-designed, fully automated computerized technique based on oriented multiscale matched filtering was tested to objectify and accelerate image analysis., Results: C57BL/6J mice showed low NFD (11.7 ± 0.5 mm/mm 2 ). Aging from 0.5 to 1, 1.5, and 2 years resulted in significant reductions in subbasal NFD by 34%, 49%, and 66%, respectively. The decline in nerve fibers revealed by in vivo CLSM together with NeuronJ quantification was confirmed by ex vivo immunohistochemical analyses. Subbasal NFD in BALB/c mice (30.0 ± 1.4 mm/mm 2 ) was 3-fold higher than in C57BL/6J mice. Aging from 0.5 to 1 year resulted in a significant 17% reduction in NFD. With the automated approach, NFD of 22.6 ± 2.9 mm/mm 2 and a 45% reduction during aging was determined from the same images., Conclusions: An age-related reduction in subbasal corneal nerve fibers was observed. The differing extent of reduction in the two mouse strains may be accounted for by genetic factors. Automated NFD quantification of corneal nerve fibers in mice appears to be a useful, reliable, objective, and time-saving tool.

Purpose: The corneal subbasal nerve plexus (SNP) offers high potential for early diagnosis of diabetic peripheral neuropathy. Changes in subbasal nerve fibers can be assessed in vivo by confocal laser scanning microscopy (CLSM) and quantified using specific parameters. While current study results agree regarding parameter tendency, there are considerable differences in terms of absolute values. The present study set out to identify factors that might account for this high parameter variability., Materials and Methods: In three healthy subjects, we used a novel method of software-based large-scale reconstruction that provided SNP images of the central cornea, decomposed the image areas into all possible image sections corresponding to the size of a single conventional CLSM image (0.16 mm2), and calculated a set of parameters for each image section. In order to carry out a large number of virtual examinations within the reconstructed image areas, an extensive simulation procedure (10,000 runs per image) was implemented., Results: The three analyzed images ranged in size from 3.75 mm2 to 4.27 mm2. The spatial configuration of the subbasal nerve fiber networks varied greatly across the cornea and thus caused heavily location-dependent results as well as wide value ranges for the parameters assessed. Distributions of SNP parameter values varied greatly between the three images and showed significant differences between all images for every parameter calculated (p < 0.001 in each case)., Conclusions: The relatively small size of the conventionally evaluated SNP area is a contributory factor in high SNP parameter variability. Averaging of parameter values based on multiple CLSM frames does not necessarily result in good approximations of the respective reference values of the whole image area. This illustrates the potential for examiner bias when selecting SNP images in the central corneal area.

Keratocytes are specialized, neural crest-derived mesenchymal cells occupying approximately 3% of the corneal stromal volume. They reside between the collagen lamellae and are responsible for the secretion of extracellular matrix macromolecules, thus contributing to the corneal transparency and integrity. During the regeneration process after infection, traumata and refractive surgery, the keratocytes undergo transition into divergent phenotypes, which are referred to as "activated keratocytes". Quite shortly after injury, the keratocytes lose their quiescence, enter into the cell cycle and migrate toward the site of injury. In certain types of injury, which affect the integrity of basement membrane, activated keratocytes also participate in wound closure by production of α-smooth muscle actin (α-SMA). Since the activated keratocytes are the major cell type contributing to tissue repair during corneal wound healing, their morphological and biochemical properties have been studied in details in experimental studies using light and electron microscopy. More recently, emerging of in vivo microscopy techniques has opened new possibilities to investigate cornea in vivo. The non-invasive nature of this imaging modality enables repeated examination of the same tissue over time and is an ideal tool to rapidly and accurately investigate corneal wound healing. However, the in vivo data on activated keratocytes are not as uniform as data from experimental ex vivo studies. There is still inconsistency in the literature findings on activated phenotypes, and often the described morphologies cannot be appreciated in in vivo images. In this article, a literature review was performed in order to interpret the morphology of different activated phenotypes, based on biological processes underlying the morphological alterations.

Purpose: For developing injectable lenses the retention properties of the capsular bag are important. Therefore the apparent permeability coefficients of sodium fluorescein and fluorescent dextrans of different sizes were determined for the human anterior lens capsule to calculate a molecular weight cutoff from these data. In addition, permeability coefficients of drugs helpful for the suppression of secondary cataract were determined., Materials and Methods: Capsulorhexis specimens were fixed in a specially designed two compartment diffusion chamber to investigate the permeation of sodium fluorescein and fluorescent dextrans of different sizes (10, 40, 70 and 150 kDa) for 24 h (n ≥ 3) and of the antiproliferative drugs actinomycin D and methotrexate for 0.5, 24, 48 and 72 h (n ≥ 3)., Results: The molecular weight cutoff of the anterior lens capsule was found to be 166 ± 82 kDa. After 0.5 h, no passage of actinomycin D and methotrexate was detectable through the lens capsule. The apparent permeability coefficients for actinomycin D and methotrexate were calculated to 0.71 ± 0.02 µm/s and to 0.80 ± 0.13 µm/s, respectively., Conclusions: The capsular bag retains fluorescent dextrans with a molecular weight of >166 kDa. Hence, prepolymers are required to polymerize rapidly to be retained inside of the capsular bag. In addition, low-molecular substances intended as antiproliferative drugs for secondary cataract prevention should be applied within a time frame of five minutes in such a way that cells adjacent to the capsular bag will not be damaged.

Objective: Magnetic resonance imaging (MRI) at 1.5 and 3.0 Tesla with small surface coils is a well-established procedure in the diagnosis of masses of the eye and orbital cavity. Until now histological examination has been required to obtain definitive information on tumor extent or possible infiltration of surrounding structures. With ultra-high-field MRI, however, it is possible to evaluate tumor morphology as well as possible extension into surrounding structures with submillimeter spatial resolution., Materials and Methods: We present a female patient with a uveal melanoma who underwent a preoperative MRI at 1.5 T (spatial resolution = 0.9 x 0.9 x 4 mm/voxel). Postoperatively, the enucleated specimen was examined in a 7.1 Tesla high-field MRI scanner (slice thickness = 500 µm, matrix size = 512 x 512 pixels, spatial resolution = 78 x 78 x 500 µm/voxel, acquisition time = 8:20 min per plane). Finally, the specimen was examined histologically, and the histological and MRI results were correlated., Results: Ultra-high-field MRI at 7.1 Tesla visualized the uveal melanoma and anatomical structures of the bulb with high resolution, enabling definitive assessment of tumor morphology and extent. Subsequent histological examination confirmed the MRI findings regarding origin, internal structure, and extent of the tumor., Conclusion: MR microscopy correlates strongly with histology, suggesting that this new imaging modality has the potential for noninvasively assessing tumor morphology, extent, and infiltration of surrounding structures. The examination was performed ex vivo and demonstrates that diagnostic assessment of malignant masses is feasible using high-resolution MR microscopy.

Purpose: Trehalose has been shown to protect epithelial cells from desiccation damage in cell culture and the murine dry eye model. The present study evaluates the protective role of trehalose in reconstructed human corneal epithelium (3D-HCE) during desiccation., Materials and Methods: The morphology of 3D-HCE was examined using in vivo an ex vivo confocal laser-scanning microscopy (CLSM). The 3D-HCE was desiccated with or without pre-treatment with trehalose. Evaluation of protective role of trehalose was conducted using different in vitro cell viability assays and CLSM. Tissue thickness for each condition was determined by optical coherence tomography (OCT)., Results: 3D-HCE tissue revealed similar features with human cornea at histological level. After desiccation the percentage of living cells was only 32% in 3D-HCE tissue without pre-incubation and 98% in trehalose-pre-incubated tissue, as shown by a cell viability assay. These findings were confirmed by using a Live-Dead assay. Also, the confocal immunofluorescence analysis revealed much better preservation of tight junctions in trehalose-pre-treated tissue., Conclusions: CLSM and an in vitro cell viability assays could be successfully used for the characterization of 3D-HCE tissue. We demonstrated the protective role of trehalose using reconstructed corneal epithelium (3D-HCE), which mimics HCE and has the potential to become a valuable model in ophthalmic research.

Purpose: Surprisingly, a significant reduction (18.4%) in epithelial thickness was found after 15 seconds of eye rubbing measured using Holden-Payor optical pachometer reported in the literature. Hence, we aimed at studying the effects of compressive and shearing pressure associated with eye rubbing on total corneal, epithelial and Bowman's membrane thickness using spectral domain OCT (SD-OCT)., Methods: SD-OCT (Spectralis; Heidelberg Engineering) was used to acquire cross-sectional images of the cornea. Central total-corneal thickness (TCT), epithelial thickness (ET) and Bowman's membrane thickness (BMT) was measured on 20 eyes of 10 subjects with normal corneas without any ocular pathology using in-house developed MATLAB (Mathworks, Inc.) program. Two different measurement methods (distance between inflection points and peak to peak distance) were performed on luminance graph to obtain thickness profile of the corneas. Baseline measurements were compared with the measurements obtained immediately after 30 seconds of circular eye rubbing over the closed eye lid with contra lateral eye at a primary gaze position., Results: We have found that the mean difference in TCT, ET and BMT to be 0.44 ± 6.00 µm, 0.28 ± 1.72 µm, and 0.01 ± 0.77 µm with first method of measurement, respectively, and the mean difference in TCT and ET were -0.26 ± 5.75 µm, and 0.37 ± 1.38 µm with second method of measurement respectively before and after eye rubbing. These differences were statistically insignificant (all p > 0.05) using both measuring methods., Conclusion: Thirty seconds of circular pattern rubbing over closed eye lids using index finger produce no significant changes on total corneal, epithelial and Bowman's membrane thickness.

Purpose: The aim of the present study was to use 7.1T magnetic resonance imaging (MRI) to determine the correct anatomical position of a non-metallic glaucoma microstent following implantation in rabbit eyes., Materials and Methods: In an in vitro experiment three microstents (made from silicone, polyurethane, and nickel titanium) were embedded in a standard vial filled with agar. In a second series of experiments a prefabricated polyurethane microstent connecting the anterior chamber with the suprachoroidal space (SCS) was implanted in rabbit eyes (n = 12). High-resolution images were acquired ex vivo (n = 6) and in vivo (n = 6) on an ultra high-field MRI unit using a 2-channel coil with four coil elements and T2-weighted turbo spin-echo sequences. Finally, the eyes were dissected and processed for histology., Results: Artifact-free MR imaging of silicone and polyurethane microstents was achieved in vitro, with slightly higher contrast for silicone stents. Image quality for the nickel titanium stent was greatly reduced due to artifacts. MRI examination of rabbit eyes ex vivo and in vivo following polyurethane microstent implantation presented no problems in terms of the size and placement of the coil. The sequences showed good image quality; the full length of the microstent was visualized in cross-section and longitudinal section, and correct anatomical placement was demonstrated from the anterior chamber into the SCS. These findings were verified by histology., Conclusions: The excellent image quality obtained with both of the non-metallic materials generates detailed information about microstent placement, including the surrounding tissues. High-resolution MRI can be used to monitor the anatomical position of a microstent after glaucoma surgery in animal experiments and is therefore a valuable tool for documenting experimental research findings ex vivo and in vivo.

Purpose: The aim of the present study was to analyze and compare in vivo morphology of healthy cornea of six different laboratory animals., Materials and Methods: One Pomeranian Coarsewool sheep, 5 Beagle dogs, 1 Norwegian and 2 Domestic Short-haired cats, 20 New Zealand White rabbits, 6 Wistar rats, and 10 Balb/c mice were included. The examination was performed bilaterally, using Heidelberg Retina Tomograph equipped with Rostock Cornea Module. The morphology of living corneal layers was visualized and compared between species. The central corneal thickness, density of keratocytes, and endothelial cells were quantified., Results: The epithelial multilayer showed a similarity in morphology between animal types, displaying three clearly distinguishable layers: superficial, intermediate, and basal. Subbasal nerve fibers were displayed as hyperreflective structures underneath basal cells. The subbasal fibers were confirmed in all species, however, the density varied between species. A pronounced Bowman's membrane was visualized in sheep. In all other species, however, a thin acellular layer with overlying nerve fibers could be seen between basal epithelial cells and anterior stroma. The keratocytes nuclei could be demonstrated in all species except for mice, where no nuclei but only reflective structures resembling keratocytes cell bodies were detected. Overall, the density of keratocytes nuclei was significantly higher in the anterior than in the posterior stroma. Besides endothelial cells density, the endothelial cells morphology was very similar among all species, except for sheep. The endothelial cells were displayed as polygonal structures with bright cytoplasm and dark borders. In sheep, the appearance of the endothelium was very poor because of a thick hyperreflective Descemet's membrane., Conclusions: The present study will help researchers consider appropriate models for animal experiments, depending on focus of investigation. In vivo CLSM can be used for the characterization of the living cornea over time, thus, reducing the number of animal experiments.