To investigate choriocapillaris flow voids (FV) with a new optical coherence tomography angiography (OCTA) image processing strategy that can eliminate artifacts caused by vitreous opacities, ...sub-retinal pigment epithelium fluid and deposits, and subretinal fluid (SRF) by thresholding the en-face OCT image of the outer retina.
We retrospectively reviewed medical records of patients with drusen and patients with active central serous chorioretinopathy (CSC). FV number (FVn), average area (FVav), and maximum area (FVmax) and the percentage of nonperfused choriocapillaris area (PNPCA) obtained using the proposed strategy were compared with those obtained by removing only artifacts caused by the superficial capillary plexus (SCP).
The SRF group included 21 eyes with active CSC and the drusen group included 29 eyes with nonexudative age-related macular degeneration. FVav, FVmax, FVn, and PNPCA obtained using the algorithm were significantly lower than those obtained by removing only SCP-related artefacts in both groups (all p<0.05). The algorithm was also able to remove 96.9% of artifacts secondary to vitreous opacities and all artifacts secondary to serous pigment epithelial detachments.
Choriocapillaris nonperfusion areas on OCTA images may be overestimated in eyes with RPE abnormalities and SRF due to artifacts. These artifact areas on choriocapillaris OCTA images can be removed using thresholded images of the outer retina en-face OCT scans. Our new artifact-removal strategy is useful in the assessment of choriocapillaris FV in eyes with SRF, drusen, drusen-like deposits, and pigment epithelial detachment.
Purpose
Keratoconus is a progressive disease which results thinning of the cornea and increase in the forward curvature of the cornea until corneal rupture. The first choice in clinical management of ...keratoconus is Ultraviolet‐A(UV‐A) crosslinking procedure with riboflavin. This procedure is painful, prolonged and has many side effects on the patient. Eosin‐y is well known molecule and it has been used as crosslinker with visible light on hydrogels with various cells.
Methods
Firstly, we have examined effects of sequential concentrations of eosin‐y on proliferation and apoptosis of human corneal epithelial cells (HCECs). We have used MTT cytotoxicity assay, Xcelligence real‐time cell analysis, Ki67 and Caspase‐3 immunofluorescence staining. After these tests, visible light (525 nm) induced crosslinking has performed with Eosin‐Y in fresh bovine corneas at selected doses. Corneas which applied eosin‐y or riboflavin, or saline are observed under scanning electron microscopy for topographic, cross‐sectional and elemental analysis. Their young moduli measured with hybrid rheometer. Lastly, resistance to osmotic stress and chemical digestion of the corneas compared with water swelling test and enzymatic digestion test, respectively.
Results
We have demonstrated non‐toxic doses of eosin‐y with cytotoxicity and immunofluorescence experiments on HCECs. We determined LD50 concentration of eosin‐y for HCECs. After that, we have showed significant crosslinking with eosin‐y in fresh bovine corneas. Eosin‐y and visible light procedure has created faster and stronger corneal crosslinking reaction on bovine corneas compared to riboflavin and UV‐A procedure.
Conclusions
We have observed, eosin‐y initiate faster and stronger crosslinking in corneal stroma at subtoxic doses than standard procedure. It could be new photo‐initiator agent for corneal crosslinking procedure in clinics. For next step, we will examine its toxicity on corneal stromal cells and corneal endothelial cells.
Purpose: The purpose of this study is to explore the effects of dihydrotestosterone (DHT) on lipopolysaccharide (LPS)-induced proinflammatory cytokine release in human ocular surface epithelial cells ...exposed to LPS and LPS-binding protein (LBP).
Methods: Immortalized human corneal, conjunctival, and meibomian gland epithelial cells were cultured in keratinocyte-free medium. After confluency, they were exposed to a stratification medium Dulbecco's modified Eagle medium (DMEM)/F12 in the presence of fetal bovine serum and were exposed to vehicle, LPS + LBP, or DHT. Culture media were processed for multiplex-bead analysis of specific proinflammatory cytokines including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-4, IL-8, IL-6, IL-10, IL-1β, vascular endothelial growth factor (VEGF)-A. Cytokine concentrations were compared by analysis of variance with Tukey post hoc testing. p < 0.05 was considered statistically significant.
Results: The results are LPS + LBP-induced the secretion of IFN-γ, IL-6, IL-10, IL-1β, VEGF-A cytokines in corneal epithelial cells; TNF-α, IL-2, IL-8, IL-6, IL-1β, VEGF-A cytokines in conjunctival epithelial cells; and IL-8, IL-6, IL-1β, VEGF-A cytokines in meibomian gland epithelial cells. When these LPS + LBP-stimulated cells were exposed to DHT for 2 days, it was found that DHT suppressed the secretion of IL-6, IL-10, IL-1β, VEGF-A cytokines in corneal epithelial cells; TNF-α, IL-6, IL-1β, VEGF-A cytokines in conjunctival epithelial cells; and IL-6, IL-1β, VEGF-A cytokines in meibomian gland epithelial cells.
Conclusion: LPS + LBP is shown to induce the secretion of certain proinflammatory cytokines from ocular surface and adnexal epithelial cells. DHT showed anti-inflammatory activity by suppressing some of those cytokines in these cell lines.
ObjectivesThe purpose of the study was to determine interobserver and intraobserver agreement, repeatability, and intrasubject variation of the detection of Demodex infestation in eyelids of ...blepharitis patients using in vivo confocal microscopy (IVCM). MethodsEighty-three eyes of 42 blepharitis patients were included in the study. All eyelids were evaluated from temporal to nasal with IVCM using section mode and 10 lashes with their follicles were imagined, and every image with suspicion of Demodex infestation was recorded. Two experienced and two inexperienced ophthalmologists were masked for the diagnosis and interpreted the IVCM images regarding the presence of Demodex infestation with a 3-week interval. Interobserver and intraobserver agreements were calculated with Cohen's kappa and its variant statistics between and within experienced observers and between inexperienced observers. ResultsWhile average sensitivity for the diagnosis of demodicosis in IVCM images was 83.35% for experienced and 51.35% for inexperienced observers, the average positive predictive value was 88.6% for experienced observers and 91.05% for inexperienced ones. Interobserver agreement between experienced observers was moderate (κ = 0.529) and intraobserver agreements within experienced observers were perfect (κ = 0.918 for observer-1; κ = 0.958 for observer-2). Interobserver agreement between inexperienced observers was poor (κ = 0.162) and intraobserver agreements within inexperienced observers were fair (κ = 0.427 for observer-3; κ = 0.475 for observer-4). ConclusionThe sensitivity, interobserver and intraobserver agreements in IVCM image analysis for the detection of Demodex infestation were highly associated with the clinical experience on IVCM imaging. In the hands of an experienced clinician, IVCM could be reliable for the diagnosis of ocular demodicosis.
In vivo confocal microscopy (IVCM) is a noninvasive, reproducible, and inexpensive diagnostic tool for corneal diseases. However, widespread and effortless image acquisition in IVCM creates serious ...image analysis workloads on ophthalmologists, and neural networks could solve this problem quickly. We have produced a novel deep learning algorithm based on generative adversarial networks (GANs), and we compare its accuracy for automatic segmentation of subbasal nerves in IVCM images with a fully convolutional neural network (U-Net) based method.
We have collected IVCM images from 85 subjects. U-Net and GAN-based image segmentation methods were trained and tested under the supervision of three clinicians for the segmentation of corneal subbasal nerves. Nerve segmentation results for GAN and U-Net-based methods were compared with the clinicians by using Pearson's R correlation, Bland-Altman analysis, and receiver operating characteristics (ROC) statistics. Additionally, different noises were applied on IVCM images to evaluate the performances of the algorithms with noises of biomedical imaging.
The GAN-based algorithm demonstrated similar correlation and Bland-Altman analysis results with U-Net. The GAN-based method showed significantly higher accuracy compared to U-Net in ROC curves. Additionally, the performance of the U-Net deteriorated significantly with different noises, especially in speckle noise, compared to GAN.
This study is the first application of GAN-based algorithms on IVCM images. The GAN-based algorithms demonstrated higher accuracy than U-Net for automatic corneal nerve segmentation in IVCM images, in patient-acquired images and noise applied images. This GAN-based segmentation method can be used as a facilitating diagnostic tool in ophthalmology clinics.
Generative adversarial networks are emerging deep learning models for medical image processing, which could be important clinical tools for rapid segmentation and analysis of corneal subbasal nerves in IVCM images.
To investigative the effects of sex steroids on hyperosmolar stress-induced proinflammatory cytokine expression of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-8, and IL-6, and on the ...mitogen-activated protein kinase pathway in immortalized human corneal epithelial cells (hCECs).
Immortalized hCECs were cultured with keratinocyte-free medium until reaching 80% confluency with either 10 M dihidrotestosteron (DHT) or 10 M 17-β-estradiol, and then, the medium was changed to hyperosmolar for various time points. After hyperosmolar treatment, a real-time polymerase chain reaction was performed to show the TNF-α, IL-8, and IL-6 gene expression levels in hCECs. In addition, the treated cells were lysed, and Western blot analysis was applied for phosphorylated and nonphosphorylated forms of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 kinase, and c-Jun N-terminal kinase 1/2 (JNK1/2). hCECs viability was measured with Annexin V/propidium iodide.
Pretreatment with 10 M DHT or 17-β-estradiol inhibited the high osmolarity-induced expression of TNF-α, IL-8, and IL-6. The upregulation of p-ERK, p-JNK, and p-p38 with high osmolarity was inhibited partially by DHT, but 17-β-estradiol pretreatment only affected p-p38 for a short time interval. In addition, DHT increased cell viability of hCECs under hyperosmolar conditions.
Our results demonstrated that DHT and 17-β-estradiol decreased the proinflammatory cytokine gene expression levels which were stimulated by high osmolarity in immortalized hCECs. The mitogen-activated protein kinase signaling pathway is partially involved in the regulatory effects of DHT on hCECs. These findings may contribute to the etiologic role and therapeutic implications of sex steroids in certain ocular surface diseases.
Type 2 Diabetes mellitus (DM) is a major health problem and its ocular complications like orbital infections, cataract and diabetic retinopathy cause blindness. Meibomian gland (MG) dysfunction and ...dry eye disease are also important ocular complications of type 2 DM but not enough research has been conducted on these complications. Our hypothesis suggests type 2 DM can alter significant gene expressions of MG. In our study, MGs of leptin-deficient spontaneous diabetic and non-diabetic mice were extracted, and gene expression profiles were analyzed with microarray technology.
Mice were divided into two groups; nine Lep
b/ob
spontaneous diabetic mice as type 2 DM group and nine non-diabetic Balb/c mice as controls. Blood glucose levels, tearfilm break-up time and fluorescein scores were measured in both two groups for 12 weeks. MGs were dissected and RNAs were isolated for microarray gene expression analysis. We filtered probes with standard deviation of more than 0.1 and we used 40452 of 45281 probes for processing. We performed fold change analysis and identified which genes are affected, and we analyzed the impact of genes on proteins, pathways and gene ontologies by using various databases.
We observed 172 up-regulated and 118 down-regulated genes in type 2 diabetic mice when compared to non-diabetic mice. Interestingly, expression of collagen type I, integrin beta-I binding protein-I, pyruvate dehydrogenase kinase, TNF receptor genes up-regulated with DM; on the other hand, IL-33, cholecystokinin, plasminogen activator, IL-1 and serine peptidase inhibitor genes down-regulated significantly. Also, we have seen a significant decrease in WNT signaling and pentose phosphate pathways-related genes.
Our data show these changes in gene expression caused by endocrine and immune mechanisms of type 2 DM which result disrupted homeostasis of epithelial cells of MG. Increased expressions of apoptosis and inflammation-related genes and their effects on related pathways have proven that MGs were negatively affected by type-2 DM.
Corneal collagen crosslinking (CXL) is a commonly used minimally invasive surgical technique to prevent the progression of corneal ectasias, such as keratoconus. Unfortunately, riboflavin/UV-A ...light-based CXL procedures have not been successfully applied to all patients, and result in frequent complications, such as corneal haze and endothelial damage. We propose a new method for corneal crosslinking by using a Ruthenium (Ru) based water-soluble photoinitiator and visible light (430 nm). Tris(bipyridine)ruthenium(II) (Ru(bpy)32+) and sodium persulfate (SPS) mixture covalently crosslinks free tyrosine, histidine, and lysine groups under visible light (400–450 nm), which prevents UV-A light-induced cytotoxicity in an efficient and time saving collagen crosslinking procedure. In this study, we investigated the effects of the Ru/visible blue light procedure on the viability and toxicity of human corneal epithelium, limbal, and stromal cells. Then bovine corneas crosslinked with ruthenium mixture and visible light were characterized, and their biomechanical properties were compared with the customized riboflavin/UV-A crosslinking approach in the clinics. Crosslinked corneas with a ruthenium-based CXL approach showed significantly higher young's modulus compared to riboflavin/UV-A light-based method applied to corneas. In addition, crosslinked corneas with both methods were characterized to evaluate the hydrodynamic behavior, optical transparency, and enzymatic resistance. In all biomechanical, biochemical, and optical tests used here, corneas that were crosslinked with ruthenium-based approach demonstrated better results than that of corneas crosslinked with riboflavin/ UV-A. This study is promising to be translated into a non-surgical therapy for all ectatic corneal pathologies as a result of mild conditions introduced here with visible light exposure and a nontoxic ruthenium-based photoinitiator to the cornea.
Keratoconus, one of the most frequent corneal diseases, could be treated with riboflavin and ultraviolet light-based photo-crosslinking application to the cornea of the patients. Unfortunately, this method has irreversible side effects and cannot be applied to all keratoconus patients. In this study, we exploited the photoactivation behavior of an organoruthenium compound to achieve corneal crosslinking. Ruthenium-based organic complex under visible light demonstrated significantly better biocompatibility and superior biomechanical results than riboflavin and ultraviolet light application. This study promises to translate into a new fast, efficient non-surgical therapy option for all ectatic corneal pathologies.
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To investigate cytokine profile of cholesteatoma and to collect information about important intercellular signaling pathways by establishing two different cell culture models, to block important ...intercellular signaling pathways in cholesteatoma by applying immune system modifier drugs to develop alternative medical therapy options for cholesteatoma.
To observe the pathogenesis of cholesteatoma and to apply the immunomodulatory drugs, cholesteatoma tissue culture models were constituted with HEKa cells and cholesteatoma keratinocytes, which were obtained from 3 patients who underwent operations for cholesteatoma. Medicines including 5-fluorourasil, imiquimod, cyclosporine, and tacrolimus were applied on both cholesteatoma keratinocytes and HEKa cells. After 48 h of incubation, IL-1, IL-6, IL-8, IL-10, TNF-α, and Ki67 levels were measured to determine cell viability rates.
In the cholesteatoma control group, IL-6 and TNF-α levels were found higher than in the HEKa control group. All repurposed drugs in the study demonstrated anti-inflammatory, anti-proliferative, and cytotoxic effects on cholesteatoma. Imiquimod and tacrolimus in particular are potential treatment prospects for cholesteatoma due to their strong anti-inflammatory and cytotoxic effects.
Medical therapy options for cholesteatoma are still missing and surgery is not the ultimate solution. We have focused on intercellular inflammatory processes, which play significant roles in the pathogenesis of cholesteatoma in our paper. Inflammation and proliferation of cholesteatoma decreased after all repurposed drug applications in our study. Anti-inflammatory and anti-proliferative effects of tacrolimus and imiquimod was more significant than other drugs in the study. For this reason, tacrolimus and imiquimod should be examined in depth with in vivo studies in terms of efficacy and safety for medical treatment of cholesteatoma.
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