Ultrastructural morphology and expression of proteoglycans, ig-h3, tenascin-C, fibrillin-1, and fibronectin in bullous keratopathy

AIMS To investigate the ultrastructural localisation of proteoglycans (PG), ig-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 ig-h3, TN-C, fibrillin, and fibronectin were immunolocalised with rabbit anti-ig-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 ig-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 ig-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 ig-h3, throughout stroma. The modified expression of the proteins studied in these cases of BK may be regarded as part of an injury response

Correlation of the appearance of the keratoconic cornea in vivo by confocal microscopy and in vitro by light microscopy

OBJECTIVE: To compare morphologic features of keratoconus as observed in vivo with a slit scanning confocal microscope and in vitro using light microscopy. METHODS: Slit scanning confocal microscopy (CM) was used to evaluate the central cornea of 29 keratoconic subjects (mean age, 31 +/- 10 years; range, 16-49). Light microscopy (LM) examination was performed on 2 of the keratoconic corneas post-keratoplasty. RESULTS: With CM, the epithelium appeared more abnormal with increasing severity of keratoconus. In severe disease, the epithelium displayed the following characteristics: superficial cells were elongated and spindle shaped, wing cell nuclei were larger and more irregularly spaced, and basal cells were flattened. These findings were confirmed by LM. Images obtained using CM revealed disruption to Bowman's layer and the occasional presence of epithelial cells and stromal keratocytes. This was shown with LM to be due to breaks in Bowman's layer. Stromal haze and hyperreflectivity observed with CM corresponded with apical scarring seen on slit-lamp biomicroscopy. Hyperreflective keratocyte nuclei observed with CM are thought to indicate the presence of fibroblastic cells seen with LM. Increasing levels of haze detected with CM were found with LM to be due to fibroblast accumulation and irregular collagen fibers. Descemet's membrane appeared normal with both CM and LM. Evidence of endothelial cell elongation was apparent in 1 subject with CM. CONCLUSIONS: The current study confirms the application of CM for assessing morphologic alterations to the epithelium, Bowman's layer, and stroma in keratoconus. Many of the tissue changes observed with CM could be reconciled with observations made using LM. This work provides a framework against which tissue changes in keratoconus can be studied in a clinical context in vivo using CM

Deposits and proteoglycan changes in primary and recurrent granular dystrophy of the cornea

Objective: To investigate the origin and distribution of granular deposits in the corneas of 3 patients with granular dystrophy, 1 of whom had previously received a lamellar keratoplasty in which the granular dystrophy had recurred. Method: Corneal tissue from 2 patients with primary granular dystrophy (patients 1 and 2) and from a patient with recurrent granular dystrophy (patient 3) was examined. Corneal graft tissue was fixed in (1) 3% glutaraldehyde in sodium cacodylate buffer, (2) 2.5.% glutaraldehyde in sodium acetate buffer containing cuprolinic blue, and (3) 4% paraformaldehyde in phosphate-buffered saline. Results: In patient 1 (aged 48 years), electron-dense granular structures were observed in epithelium, Bowman layer, and throughout the stroma. Bowman layer was absent in several places. Patient 2 (aged 78 years) showed similar features except with more deposits in the stroma. In patient 3 (aged 48 years), granular structures were heavily deposited in the epithelium; there were also some deposits in the posterior (host) stroma, some of which were associated with partially degenerated keratocytes. Bowman layer appeared normal. In all 3 patients, the intracellular or extracellular granular structures were surrounded by fine fibrillar material and abnormal proteoglycans. Electron-lucent spaces within the corneal stroma contained large quantities of abnormal proteoglycan filaments that were attached in part to collagen fibrils. Conclusions: Results from patient 3 support an epithelial origin for the deposits, presumably from keratoepithelin, aggregated with other proteins. The role of keratocytes is less clear, although the presence of deposits in the stroma of all 3 patients, some associated with keratocytes, suggests that these cells might produce granular material in addition to abnormal proteoglycans