No abstract

Mutations in the the glaucoma gene GCL1A, also known as trabecular meshwork glucocorticoid response (TIGR) or myocilin (Myoc), have been shown to be associated with juvenile-onset primary open-angle glaucoma. Very little is known about the pattern of expression of the TIGR gene in human ocular tissues. In-situ hybridization experiments demonstrated the localization of TIGR mRNA in cells throughout the iris, ciliary muscle, and the filtering portion of the trabecular meshwork of normal eye donors. The expression of TIGR protein was investigated by Western blot using an epitope-directed antibody to the carboxy terminus region of TIGR. This antibody was able to distinguish a recombinant TIGR fusion protein from a truncated TIGR form containing the naturally occurring Gln(368)-->stop mutation. In tissue extracts from the iris, ciliary body, and trabecular meshwork, the antibody recognized a major protein band of 57-kDa molecular mass. Deglycosylation treatment with PNGase F, NANase II, and O-glycosidase indicated that the 57-kDa protein in these tissues was unglycosylated. In agreement with this observation, in coupled in-vitro transcription/translation systems, the 57-kDa TIGR protein was unaffected by the presence of the processing and glycosylation activities of canine pancreatic microsomal membranes. These findings support the view that the expression of TIGR mRNA in cells of the iris, ciliary body, and trabecular meshwork correlates with that of TIGR protein, and that the 57-kDa TIGR protein was unglycosylated. These results, which are in contrast with earlier reports, raise the possibility that the TIGR protein might be processed into distinct forms in a tissue-specific manner.

Aims: The problem of automatic estimation of endothelial cell density from microscopy images in donor corneas was addressed. Methods: The spatial frequencies contained in digital endothelium images are extracted with a two dimension discrete Fourier transform (DFT) technique. A circular band in the DFT of the images is shown to contain the frequency information related to the cell density. An algorithm for reliably recovering this spatial frequency information and for extracting from it an estimate of endothelial cell density has been developed and implemented in a computer program. An evaluation was performed on a data set containing 100 donor corneas, by comparing automatic values with manual counts performed by three eye bank experts on two images for each cornea. Results: The mean difference of automatic densities v manual ones was 14 cells/mm 2 (0.9%), with a standard deviation of 119 cells/mm 2 (5.1%) and mean absolute difference of 92 cells/mm 2 (3.9%). The ANOVA based overall inter-rater reliability was 0.935. The algorithm was also capable of identifying all non-processable images. Running times were in the order of 1-2 seconds per image. Conclusion: A new algorithm was developed for the fully automatic estimation of endothelial cell density. The results of a clinical evaluation on 100 corneas suggest that it is capable of reliably estimating endothelium cell density in donor corneas.

Online OCP was suitable for continuous measurements of corneal changes caused by dehydration. These dehydration effects should be particularly considered in refractive corneal surgery.