Keratoconus (KC) is a progressive corneal disorder in which vision gradually deteriorates as a result of continuous conical protrusion and the consequent altered corneal curvature. While the majority of the literature focus on assessing the center of this diseased cornea, there is growing evidence of peripheral involvement in the disease process. Thus, we investigated the organization of collagen fibrils (CFs) and proteoglycans (PGs) in the periphery and center of KC corneal stroma. Three-dimensional transmission electron tomography on four KC corneas showed the degeneration of microfibrils within the CFs and disturbance in the attachment of the PGs. Within the KC corneas, the mean CF diameter of the central-anterior stroma was significantly (p ˂ 0.001) larger than the peripheral-anterior stroma. The interfibrillar distance of CF was significantly (p ˂ 0.001) smaller in the central stroma than in the peripheral stroma. PGs area and the density in the central KC stroma were larger than those in the peripheral stroma. Results of the current study revealed that in the pre- Descemet’s membrane stroma of the periphery, the degenerated CFs and PGs constitute biomechanically weak lamellae which are prone to disorganization and this suggests that the peripheral stroma plays an important role in the pathogenicity of the KC cornea.

Ultraviolet radiation is an environmental carcinogenic agent that enhances inflammation and immunological reactions in the exposed human skin cells leading to oxidative photoaging of the epidermal and dermal segment. In the present study, we investigated the protective role of ursolic acid (UA) against ultraviolet B (UVB) radiation- induced photoaging an in vitro model of human skin dermal fibroblasts. UA-pretreated human skin dermal fibroblast (HDF) cells were exposed to UVB radiation to evaluated cell viability, reactive oxygen species (ROS), mitochondrial membrane potential, lipid peroxidation, antioxidant status, DNA damage, proinflammatory response, apoptotic induction, and matrix metalloproteinase (MMP) alteration. The UA pretreatment of HDFs mitigated the UVB irradiation-induced cytotoxicity, ROS generation, and mitochondrial membrane potential alteration and lipid peroxidation, depletion of antioxidant status, DNA damage, and apoptotic induction. UA pretreatment of HDFs also attenuated the UVB-induced expression of inflammatory (TNF-α and NF-κB) and apoptotic (p53, Bax, and caspase-3) and MMPs (MMP-2 and MMP-9) and enhanced the Bcl-2 protein levels in 20 μM UA treatment, when compared to concentrations. Hence, these results revealed that UA has the potential to mitigate UVB-induced extracellular damage by interfering with the ROS-mediated apoptotic induction and photoaging senescence and thus is a potential therapeutic agent to protect the skin against UVB-irradiation induced photooxidative damage.

AIMS To investigate the ultrastructural localisation of proteoglycans (PG), βig-h3 (keratoepithelin), tenascin-C (TN-C)), fibrillin, and fibronectin in bullous keratopathy (BK) corneas. METHODS Five corneas from cases of pseudophakic bullous keratopathy (BK) were examined by electron microscopy. PG were demonstrated using cuprolinic blue, and the proteins βig-h3, TN-C, fibrillin, and fibronectin were immunolocalised with rabbit anti-βig-h3, mouse anti–TN-C (BC10 and TN2), mouse anti-fibrillin-1 (MAB2502), mouse anti-fibrillin (MAB1919), and rabbit anti-fibronectin by using a standard immunogold technique. RESULTS Epithelial cells contained numerous vacuoles. Epithelial folds and large, electron lucent subepithelial bullae were present. Basal lamina was thickened and traversed by disrupted anchoring filaments. In the stroma, interfibrillar collagen spacing was increased and abnormally large PG were present. Descemet's membrane (DM) contained lucent spaces in which there were small filaments. Keratocyte and endothelial cells contained melanin granules. A posterior collagenous layer (PCL) contained numerous microfilaments and wide spacing collagen fibres with a periodicity of 100 nm. Large quantities of abnormal PG were observed at the endothelial face of the PCL. Very strong labelling with βig-h3 antibody was observed in the basement membrane, Bowman's layer, stroma, DM, and PCL, but not in keratocytes and endothelial cells. Strong labelling with BC10 and TN2 was seen below the epithelium, in electron lucent spaces where the hemidesmosomes were absent, in the fibrotic pannus, in parts of Bowman's layer, the stroma, and Descemet's membrane. Labelling with BC10 was stronger and more evenly distributed than with TN2. Fibrillin-1 (MAB2502) and fibrillin (MAB1919) labelling was similar to TN-C labelling. Fibrillin (MAB1919) labelling was stronger than fibrillin-1 (MAB2502) labelling. CONCLUSIONS Immunoelectron microscopy showed precise labelling of proteins at both the cellular and the subcellular level. Expression of proteins βig-h3, TN-C, fibrillin, and fibronectin was highly increased compared with normal cornea. In the oedematous stroma, increased collagen fibril separation may facilitate a wider distribution of some soluble proteins, such as βig-h3, throughout stroma. The modified expression of the proteins studied in these cases of BK may be regarded as part of an injury response.

Comparative ultrastructural study of normal and scoliotic intervertebral discs. To examine the ultrastructural organization and distribution of proteoglycan in relation to collagen and elastic fibers in normal and scoliotic discs. The mechanical properties of intervertebral discs depend on the collagens and proteoglycans in the matrix; proteoglycan content and the organization of collagen fibers are altered in degenerate discs. However, little is known about the structural relations among disc components and alterations to them in scoliosis. Samples of normal and scoliotic disc from individuals aged between 12 and 16 years were fixed in 2.5% glutaraldehyde containing cuprolinic blue and examined by transmission electron microscopy. The sizes and distribution of proteoglycan particles throughout different matrix regions were quantified. In anulus fibrosus of normal disc, collagen and elastic fibers in lamellas were covered with small proteoglycan particles in a regular pattern; proteoglycan particles of similar sizes were evenly distributed throughout the matrix. Anulus fibrosus of scoliotic disc contained degenerate collagen lamellas, few elastic fibers, and a pericellular accumulation of large proteoglycan aggregates; scoliotic nucleus pulposus also contained an accumulation of proteoglycan aggregates. Most cells in both regions of scoliotic discs were degenerate and vacuolated. We provide ultrastructural confirmation of the disruption of the lamellar organization of collagen and elastic fibers in scoliosis. Our observations of altered proteoglycan distribution in scoliotic tissue suggest that impaired proteoglycan turnover plays a key role in the disruption of the structural integrity of the disc in scoliosis.