Immune cells in the human choroid.

AIM: To characterise the leucocytes in human macular choroid with and without drusen, and in eyes with advanced age-related macular degeneration (AMD) with fibrovascular scarring (FVS). METHODS: Ten eyes from nine donors (range 55-91 years of age) were obtained from an eye bank within 38 h post mortem. Fixed macular biopsies were sectioned, stained immunochemically and examined for the presence of leucocyte antigens CD45, CD4, CD8, CD14 and CD83. RESULTS: Four eyes without drusen, four eyes with drusen and two eyes with FVS contained 23.9 (SD 6.2)%, 27.5 (7.2)%, and 19.3 (11.3)% CD45-positive cells, respectively. The corresponding percentages for CD4-positive cells were 5.4 (4.3), 8.9 (3.0) and 7.5 (8.1); for CD8-positive cells, 3.8 (0.7), 6.8 (2.2) and 6.3 (2.1); and for CD14-positive cells, 3.7 (3.7), 3.6 (1.6) and 2.6 (3.6), respectively. The authors found CD83-positive cells solely in one of the two FVS eyes examined that had the more severe form of scarring. CONCLUSION: Human choroid contains similar amounts of CD4-positive cells and monocytes irrespective of the presence of drusen, but CD8-positive cells are more abundant in macular choroid with drusen. The presence of haematopoietic cells in the macular choroid provides further evidence for the possible participation of inflammatory cells in pathogenesis of AMD.

Ultrastructural localization of collagen IV, fibronectin, and laminin in the trabecular meshwork of normal and glaucomatous eyes.

PURPOSE: To determine whether differences in the ultrastructural characteristics or composition of the basement membranes of the trabecular lamellae and Schlemm's canal exist in normal eyes and eyes with primary open-angle glaucoma (POAG). Basement membranes play key roles in the attachment of the overlying trabecular cells and Schlemm's canal cells. METHODS: Electron microscopy used in conjunction with immunogold labeling was used to examine the ultrastructure of the basement membranes in the trabecular meshwork and to determine the presence of collagen IV, laminin, and fibronectin in 6 normal eyes and 6 eyes with POAG. To determine which cells in the meshwork synthesized these molecules in situ hybridization was studied in an additional 8 normal eyes. RESULTS: No distinctive ultrastructural changes were found in the basement membranes of glaucomatous eyes, whether early or advanced disease, when compared with normal eyes. Label for all three proteins was present in the basement membranes of the trabecular lamellae, Schlemm's canal, and in scattered patches within the juxtacanalicular tissue. Laminin and fibronectin were most abundant in the periphery of the sheath material surrounding the elastic tendons in the juxtacanalicular tissue. In contrast to previously published light microscopic studies, no increase in fibronectin was found in glaucoma. Regions of the basement membrane of the canal underlying giant vacuoles were similar to regions without giant vacuoles in both appearance and labeling. In situ hybridization revealed that mRNA for all three proteins was present in most trabecular cells throughout the meshwork; no regional differences in cellular labeling within were observed. CONCLUSION: The ultrastructural characteristics and immunogold labeling of basement membranes were similar in normal and glaucomatous eyes; no additional structures were labeled in POAG eyes that were not also labeled in normal eyes. Label of the patches of amorphous fibrogranular material within the juxtacanalicular tissue suggests it is basement membrane in origin, while the sheath material which is known to accumulate in POAG was not heavily labeled and does not appear to be basement membrane in origin.

A comparison of different embedding media on the ultrastructure of the trabecular meshwork.

PURPOSE: To determine whether the preservation of the extracellular matrix and the ultrastructural appearance of the trabecular meshwork are affected by different histologic processing protocols and embedding media. Conventionally used epoxy resins such as Araldite require complete dehydration of tissue, while the acrylic resin LR White requires only partial dehydration, better preserves tissue antigens, and has been reported to preserve more of the extracellular matrix. METHODS: Seven human eyes ranging in age from 2 months to 78 years were dissected and tissue samples from each eye processed and embedded in both Araldite and LR White media. The ultrastructure of the trabecular cells and the extracellular matrix of the meshwork was compared between media. The preservation of the extracellular matrix in the juxtacanalicular region was determined by measuring the amount of material immediately underlying Schlemm(1)s canal. Immunoelectron microscopy was used to determine the composition of this material. RESULTS: Araldite provided better resolution of ultrastructural details than freshly polymerized LR White. After a period of ripening for several months, however, resolution of tissue details in LR White improved. No significant quantitative difference was found in the amount of extracellular matrix underlying Schlemm's canal when comparing the two media. Neither post-mortem time to fixation (up to 31 hr), donor age, nor immersion vs. perfusion fixation technique affected the amount of extracellular material present in the comparison of the two embedding media. Immunogold labeling of the extracellular material within the juxtacanalicular tissue revealed the presence of collagen IV, laminin, and fibronectin in the basement membrane region immediately underlying the inner wall, and also in scattered patches within the juxtacanalicular tissue. CONCLUSIONS: Despite the less rigorous processing required for LR White than epoxy embedding, neither the appearance nor amount of the extracellular material was affected by the different embedding protocols. Prolonged post-mortem time to fixation did not affect the appearance nor amount of extracellular matrix. Immunolabeling revealed that the extracellular matrix of the juxtacanalicular tissue contains components of basement membrane material.

Antigen retrieval of basement membrane proteins from archival eye tissues.

Antigen retrieval (AR) methods can unmask tissue antigens that have been altered by fixation, processing, storage, or resin interactions. This is particularly important in the study of archival tissues, because primary fixatives and storage times may vary among specimens. We performed an electron microscopic study of basement membrane components of the aqueous humor drainage pathways from archival eye tissue. AR (heated citrate buffer, pH 6.0, LR White resin) increased the amount of label of collagen IV and fibronectin in tissue fixed in four different fixatives, including those containing glutaraldehyde. Labeling density was approximately doubled after AR for most fixatives, with the largest increase for tissues fixed in 4% paraformaldehyde/2% glutaraldehyde. Duration of storage time for archival tissues did not affect AR results. AR did not change the components of the extracellular matrix labeled; no "new" components were labeled after AR. We conclude that AR in citrate buffer can be used on selected extracellular matrix antigens to enhance label that would otherwise be lost due to fixation and storage.

Three-dimensional reconstruction of aqueous channels in human trabecular meshwork using light microscopy and confocal microscopy.

Conventional two-dimensional imaging of the trabecular meshwork (TM) provides limited information about the size, shape, and interconnection of the aqueous channels within the meshwork. Understanding the three-dimensional (3-D) relationships of the channels within this tissue may give insight into its normal function and possible changes present in the eye disease glaucoma. The purpose of our study was to compare laser scanning confocal microscopy with standard 1 micron Araldite-embedded histologic sections for 3-D analysis of the trabecular meshwork. In addition, the study was done to determine whether computerized 3-D reconstruction could isolate the fluid spaces of the trabecular meshwork and determine the size of interconnections between the fluid spaces. Confocal microscopy appears comparable to 1 micron Araldite-embedded tissue sections and has the advantage of inherent registration of the serial tissue sections. Three-dimensional reconstruction allowed the isolation of the fluid spaces within the trabecular meshwork and revealed the presence of numerous interconnections between larger fluid spaces. The distribution of these interconnections was randomly arranged, with no predilection for specific regions within the trabecular meshwork. This distribution of constrictions and "expansion chambers" may provide a clue to the mechanism by which subtle histologic changes are associated with increased ocular pressure in glaucoma.