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Tea tree oil (TTO) is used in ophthalmology to maintain healthy eyelid skin and to combat parasitic eyelid infections. Keratocytes belong to the structure of the corneal stoma and enable to maintain corneal homeostasis. TTO that reaches the surface of the eye in too high concentration may disturb the functions of these cells. The aim of the study was to test what concentration of TTO is safe for corneal keratocytes in vitro without causing a toxic effect. A normal human keratocytes (HK) cell line was used in the study. Morphology was visualized by light and fluorescence microscopy, cytometric analysis of the cell cycle and cytometric and spectrophotometric viability evaluation were performed. The level of nitric oxide was tested by Griess spectrophotometric method. TTO concentrations exceeding 0.01% significantly reduced cell viability. The IC50 values were on average 0.057%. Increasing TTO concentrations stimulated HK cells to release NOx. The observed values did not exceed 1 μM. The lowest TTO concentration increased the number of HK cells in the G1 phase of the cell cycle. Increasing TTO concentrations (≥0.1%) increased the number of cells in late apoptosis. TTO at concentrations ranging from 0.1% to 0.5% significantly changed cell morphology. Fluorescence analyzes confirmed that TTO at concentrations ≥0.1% induced apoptotic cell death. TTO exerts strong effect on ocular keratocytes depending on applied concentration. Concentrations exceeding 0.1% have a toxic effect on keratocytes, which die mainly by apoptosis. The ocular surface should be protected from excessive exposure to TTO, which may damage corneal stroma cells., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Ocular autonomic control is mediated by sympathetic and parasympathetic nerve fibres. Their interactions are complemented by primary afferent nerve fibers of and intrinsic choroidal neurons (ICN). As the vasodilatative neuropeptide, vasoactive intestinal peptide (VIP), is expressed in extrinsic and intrinsic ocular neurons, it is of special interest in ophthalmic research. Since circadian changes of ocular blood flow are known in humans and birds, this study aimed at investigating VIP expression at different daytimes in chicken choroid, the preferred model species in ICN research., Methods: 12 eyes of 12 chickens were retrieved, slaughtered at 8.00-9.30 a.m. (n = 6) and 8.00 p.m. (n = 6), respectively, and choroidal wholemounts were prepared for immunofluorescence of VIP. VIP-positive ICN of both groups were quantified and density of VIP-positive axons assessed semi-quantitatively. In 28 additional eyes retrieved in the morning (n = 14) and evening (n = 14), choroidal VIP content was determined by ELISA. Morning and evening data were analyzed statistically. NADPH-diaphorase (NADPH-d, ICN cell marker) was done at additional 12 whole mount choroids of 12 chicken, retrieved in the morning (n = 6) and evening (n = 6)., Results: (1) Numbers of VIP positive neurons differed significantly between morning: (239.17 ± 113.9) and evening: (550.83 ± 245.7; p = 0.018). (2) Numbers of VIP-positive perikarya were significantly more accumulated in the temporal part of the choroid in the evening than in the morning (p = 0.026). (3) VIP positive axon density was found to be similar throughout the choroid in the morning and evening. (4) Number of NADPH-d positive neurons was not significantly different between morning (848.8 ± 399.5) and evening (945.8 ± 622.1, p > 0.05). (5) ELISA demonstrated a significant difference of VIP content (p = 0.012) in tissues harvested in the morning (145.41 ± 43.3 pg/ml) compared to evening (221.44 ± 106.3 pg/ml)., Conclusions: As VIP positive axon density was similar in the morning and the evening throughout the choroid, PPG and ICN seemed to contribute equally to the axon network. Yet, changes in the total choroidal VIP content, the numbers of VIP positive perikarya, reflecting the intracellular VIP content, and their topographical distribution at two different days-times argue for a different status of activation of both neuronal sources in contrast to the equal amount of NADPHD-d positive neurons. The higher VIP content in the evening, compared to the morning, correlates with a known circadian rhythm of a lower IOP and a higher choroidal thickness at night. Thus, these changes may argue for a potential role of ICN in the regulation of ocular homeostasis and integrity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Purpose: To comparatively investigate the effects of TGF-beta(1) and TGF-beta(2) on extracellular matrix production, proliferation, migration, and collagen contraction of cultured human Tenon's capsule fibroblasts derived from patients with pseudoexfoliation (PEX) syndrome, PEX glaucoma, primary open-angle glaucoma (POAG), and cataract., Methods: Tenon's capsule fibroblasts obtained from four groups of patients were cultured and stimulated with different concentrations (0.1-10 ng ml(-1)) of TGF-beta(1) or TGF-beta(2) for up to 14 days. Cell proliferation was determined with the WST-1 colorimetric assay, cell migration by using the Transwell assay system, and collagen contraction by computerised analysis of three-dimensional collagen lattices and immunohistochemistry for alpha-smooth muscle actin expression. Expression and synthesis of extracellular matrix components (fibronectin, collagen types I and III) was assessed by enzyme-linked immunosorbent assays, by real-time RT-PCR, and by transmission electron microscopy., Results: Both TGF-beta(1) and TGF-beta(2) in pathophysiological concentrations of 0.1-5 ng ml(-1) stimulated cell proliferation, migration, collagen contraction, alpha-smooth muscle actin expression as well as mRNA expression and secretion of fibronectin, collagen type I, and collagen type III by Tenon's fibroblasts derived from all groups of patients. TGF-beta stimulation occurred in a concentration-dependent manner with different peak activities associated with different fibroblast functions. There was some variability among the different groups of patients with an increased response of cells derived from PEX and POAG patients as compared to cataract patients. Although no statistically significant differences were found between both TGF-beta isoforms, TGF-beta(1) had a more pronounced stimulatory effect on expression and synthesis of extracellular matrix components including the production of elastic microfibrils, particularly in cells derived from patients with PEX syndrome/glaucoma., Conclusions: These findings suggest a significant contribution of TGF-beta(1) in addition to TGF-beta(2) to the conjunctival scarring process following glaucoma filtration surgery. Due to its pronounced fibrogenic potential, TGF-beta(1) may become another focus for targeting drug therapy, particularly in patients with PEX glaucoma.