To detect and identify, in situ, the lipid composition of drusen, diffuse Bruch's membrane deposits, and sclera in aging human eyes using hot-stage polarizing microscopy (HSPM), a method that allows ...qualitative determination of lipid subtypes within histologic sections based on morphology and melting temperatures of liquid crystals as monitored by birefringence during heating and cooling.
Full-thickness buttons of the central macula and the periphery of human eyes from 17 patients were fixed in 5% calcium-buffered formalin. Frozen sections were stained with oil red O or Sudan black or were analyzed by HSPM.
Birefringent anisotropic droplets ("maltese crosses") with melting characteristics of cholesterol esters were identified within diffuse Bruch's membrane deposits, drusen, and sclera. Deposits that melted from crystal to oil without any maltese cross formation when cooled were present in the sclera and are consistent with triglyceride-rich deposits. Deposits with optical properties consistent with phospholipids were identified in a single aged eye. Eyes from young donors did not show these changes.
HSPM is a valuable technique for evaluating the nature of lipid deposits in aging eyes. Further studies are warranted to determine whether similar changes are also present in eyes with age-related macular degeneration.
To document the cytokeratin expression patterns in the normal human conjunctival epithelium obtained directly from patients using impression cytology.
Impression cytology specimens were obtained from ...normal volunteers using pure nitrocellulose membranes rather than cellulose acetate. The 31 volunteers of both sexes ranged in age from 18 to 79 years. Impression cytology specimens were analyzed for individual cytokeratins by either immunocytochemistry or electrophoresis with immunoblotting using a defined panel of monoclonal antibodies.
Using the corroborative methods of immunocytochemistry and electrophoresis with immunoblotting, cytokeratins characteristic of nonkeratinized, stratified (K4 and K13), simple (K8 and K19), and glandular epithelia (K7) were present in the superficial layer (s) of normal human conjunctiva. Cytokeratins typical of keratinized epithelia (K1, K2, and K10) and the keratinization-related proteins filaggrin and involucrin were not expressed in normal conjunctival epithelium.
The normal human conjunctiva demonstrates a unique cytokeratin expression pattern containing cytokeratins characteristic of nonkeratinized, stratified epithelia, as well as others more typical of a simple differentiation pattern, a glandular differentiation pattern, or both. These findings provide a foundation for examining changes in the cytokeratin expression pattern in diseased human conjunctival epithelium using impression cytology.
To determine if changes in the structure and complexity of junctions between endothelial cells lining Schlemm's canal (SC) occur in normal human eyes with changes in perfusion pressure.
Twelve normal ...human eyes were either perfusion-fixed (at 15 or 45 mm Hg) or immersion-fixed (0 mm Hg) in modified Karnovsky's fluid. 'Outflow facility was measured continually during the perfusion fixation. The intercellular junctions of the endothelial cells of SC were ultrastructurally examined in thin sections, including serial sections and freeze-fracture replicas. Morphometric data on the number of junctional strands per total length of tight junction were documented and categorized by the number of strands (one, two, or three or more). The length of endothelial cell overlap was measured on thin sections.
In freeze-fracture replicas, perfusion-fixed eyes demonstrated less complex junctions. At 15 mm Hg, 18.06% of the total junctional length was represented by three or more strands; at 45 mm Hg, this percentage decreased to 8.59%. In immersion-fixed eyes, 24.17% of the total junctional length was represented by three or more strands. These differences were statistically significant (P < 0.0012). In sections, the amount of endothelial cell overlap, and thus the length of paracellular pathway, was reduced in perfusion-fixed versus immersion-fixed eyes (P < 0.02). Extensive serial sectioning demonstrated that giant vacuoles were formed, either by individual endothelial cells or by two or more adjacent endothelial cells.
When compared with specimens fixed at zero pressure, overlap between endothelial cells of SC is reduced significantly when this cell layer is under conditions of flow similar to those encountered in vivo. The tight junctions between cells of the inner wall of SC become less complex with increasing pressure. Our data suggest that the paracellular pathway into SC in the normal eye is sensitive to modulation within a range of physiologically relevant pressures.
Previous studies of the hydraulic conductivity of connective tissues have failed to show a correspondence between ultrastructure and specific hydraulic conductivity. We used the technique of ...quick-freeze/deep-etch to examine the ultrastructure of the corneal stroma and then utilized morphometric studies to compute the specific hydraulic conductivity of the corneal stroma. Our studies demonstrated ultrastructural elements of the extracellular matrix of the corneal stroma that are not seen using conventional electron microscopic techniques. Furthermore, we found that these structures may be responsible for generating the high flow resistance characteristic of connective tissues. From analysis of micrographs corrected for depth-of-field effects, we used Carmen-Kozeny theory to bound a morphometrically determined specific hydraulic conductivity of the corneal stroma between 0.46 x 10(-14) and 10.3 x 10(-14) cm2. These bounds encompass experimentally measured values in the literature of 0.5 x 10(-14) to 2 x 10(-14) cm2. The largest source of uncertainty was due to the depth-of-field estimates that ranged from 15 to 51 nm; a better estimate would substantially reduce the uncertainty of these morphometrically determined values.
To investigate the relationship between outflow facility and separation between the inner wall of the aqueous plexus and the juxtacanalicular connective tissue (JCT) during washout in the bovine eye.
...Facility was recorded during 3 hours of anterior chamber perfusion at 15 mm Hg in eight pairs of bovine eyes. One eye of each pair was then lowered to 0 mm Hg for 1 hour, whereas the fellow eye was kept at 15 mm Hg. After a brief perfusion at 15 mm Hg, both eyes were perfusion fixed and processed for electron microscopy. Micrographs of the inner wall were analyzed for separation from the JCT. To study the role of cellular adhesion between the inner wall and JCT, 12 additional pairs were perfused with integrin-binding peptide (RGD: Arg-Gly-Asp) or sham control peptide (RGE: Arg-Gly-Glu) at 2 micro M to 2 mM, before IOP was reduced.
During the first 3 hours, facility increased in both eyes because of "washout." However, after 1 hour of 0 mm Hg, facility decreased by 13% (P < 0.006), whereas facility increased by 20% (P < 0.001) in the fellow eyes maintained at 15 mm Hg. Two types of separation were observed between the inner wall and JCT: cell-matrix separation between the endothelial cell and basal lamina and matrix-matrix separation between the basal lamina and JCT. A significant positive correlation (P = 0.042) was found between the degree of matrix-matrix separation and the change in outflow facility after 1 hour of 0 mm Hg. Compared with RGE control, RGD had no apparent effect on outflow facility (P > 0.35) or on the change in outflow facility after 1 hour at 0 mm Hg (P > 0.15).
The increase in outflow facility that occurs during washout in the bovine eye is reversible and correlates with the degree of separation between the basal lamina of the inner wall endothelium and the JCT. Therefore, adhesions tethering the inner wall to the JCT may be important ultrastructural features involved in the regulation of aqueous humor outflow resistance.
The aim of this study was to determine the structure and complexity of the intercellular junctions between trabecular endothelial cells and Schlemm's canal endothelial (SCE) cells as they exist in ...the normal human eye. Despite the probable relevance of these junctions to aqueous outflow, examination of these junctions has been limited to monkey eyes.
Human eyes (< 24 hours after death) were fixed by immersion in modified Karnovsky's fluid. Radially oriented tissue-chopper sections (190 microns) were trimmed to contain only the trabecular meshwork, Schlemm's canal, and a narrow strip of the sclera. Specimens were processed for conventional electron microscopy and freeze-fracture. Replicas were produced in a freeze-fracture apparatus operated at -115 degrees C and 10(-7) torr. Thin sections were stained with uranyl acetate and lead citrate. Micrographs were taken on a transmission electron microscope.
The trabecular endothelial cells were joined by gap junctions and short discontinuous junctional strands that partitioned predominantly with P-face. The strand pattern varied from short and isolated undulating strands to radiating arrays of short junctional strands. No continuous zonulae occludentes were observed. The SCE cells were joined by continuous tight junctions (tj) composed of discontinuous strands that rarely branched or anastomosed. The tj strands fractured preferentially with the E-face, where they were positioned at the bases of shallow valleys. On the P-face, a complementary system of shallow ridges was observed with few particles at their crests. Often, only a single continuous strand was present for long distances, with occasional short discontinuous strands running parallel to the single strand. Less often, short lengths of remarkably complex junctions, which occasionally branched or anastomosed, were also encountered. When more than one strand was present, numerous free endings and transjunctional pathways, described in monkey eyes as "slit-pores," were evident.
The overall structure of the junctions between endothelial cells of Schlemm's canal in the human eye was more complex, and thus less permeable, than that reported in the monkey eye. The role of slit-pores and other junctions of SCE and trabecular endothelial cells will require further investigation under conditions of flow and in glaucoma to determine if these junctions change in a manner that might influence outflow resistance.
Magnetic resonance imaging (MRI) has been used to examine conditions that alter the permeability of the blood–retinal barrier. Our goal was to determine if blood-aqueous barrier permeability could be ...similarly assessed, because MRI offers the theoretical advantage of providing quantitative data directly from inflamed uveal tissues rather than from the aqueous humor into which the inflammatory reaction spills. As an additional challenge, we sought to use MRI to measure differences between the inflamed uveal tissues of corticosteroid-treated and placebo-treated uveitic eyes. Anterior uveitis was induced in one eye of eight rabbits by subcutaneous injection of Mycobacterium tuberculosis, followed after 10 days with intravitreal challenge. One rabbit of each pair was treated with topical 1% prednisolone acetate while control rabbits were treated with artificial tears. Contrast-enhanced MRI studies were performed prior to uveitis induction, one day after induction and then weekly for at least 2 weeks. MR image data were analyzed to determine percent change in peak enhancement of the ciliary body and anterior chamber. The initial rate of change of enhancement of the anterior chamber was also measured. Extensive contrast agent-induced MR image enhancement of both the anterior chamber and the ciliary processes was measured following the induction of uveitis. More rapid improvement was measured for the 1% prednisolone acetate-treated rabbit eyes (P<0.001). MR signal enhancement data obtained from the ciliary processes proved to be the most reliable indicator of disease activity in this rabbit model of uveitis. Such data can only be obtained using MRI.
The purpose of this research was to reassess the effects of topical pilocarpine on the integrity of the blood-aqueous barrier, using high resolution, magnetic resonance imaging, and the standard ...intravenous contrast agent gadolinium dimeglumine. It has long been known that topical pilocarpine gives rise to an increase in protein levels in the anterior chamber of the eye. This protein scatters light and is referred to clinically as ‘flare’. Prior studies concluded that pilocarpine-induced flare resulted from disruption of the blood-aqueous barrier. These studies relied upon indirect methods that precluded direct visualization of the posterior chamber of the eye.
Five normal, human volunteers (age 22–40) received a single drop of 3% pilocarpine in one eye, following baseline measurements of pupil size and anterior chamber ‘flare’. These measurements were repeated every 15
min for 45
min. The subject was then positioned in the magnet and the eye that received pilocarpine was taped closed and covered with a 3
in.-diameter receive-only surface coil. The open contralateral eye focused on a target to maintain fixation of the imaged eye. A baseline image of the eye was obtained and the contrast agent was administered intravenously. A series of additional images was obtained during the following 60
min to track the movement of contrast material from the bloodstream into the tissues and compartments of the eye. Percent enhancement was calculated from selected regions of interest in the images, including the ciliary body, and the anterior and posterior chambers.
Within the 45
min after administration of pilocarpine, pupil size in mm decreased from (mean±
s.d.) 5.7±1.5 to 2.5±0.5 (
p=0.0106). During this period, average flare/
s.d. (photons
msec
−1) increased from 3.7±1.1 to 12.5±4.7 (
p=0.0151). In all cases, MRI images showed rapid enhancement of the ciliary body, followed by a progressive increase in signal in the anterior chamber but not the posterior chamber.
These studies confirm that topical pilocarpine gives rise to ‘flare’ in the anterior chamber. But the lack of enhancement in the posterior chamber strongly suggests that the presence of this added protein in the anterior chamber is not the result of increased permeability of the blood-aqueous barrier of the ciliary body.
These studies also introduce the novel concept that not all clinically observed flare is the result of blood-aqueous barrier compromise.
Previous studies have used conventional electron microscopy and freeze fracture to identify the morphological equivalents of the blood-aqueous barrier in the mammalian eye. These equivalents are the ...tight junctions that form a part of the apicolateral junctional complex between adjacent non-pigmented ciliary epithelial cells and the tight junctions present between endothelial cells of the iris vasculature. Recent investigations have begun to unravel the molecular assembly of the tight junction and some variability has been found. Our goal in the present study was to probe the ciliary epithelium and iris vascular endothelium of the rabbit eye to determine if certain molecular constituents associated with tight junctions in other tissues are also present as parts of the blood-aqueous barrier. The selected constituents were occludin, ZO-1, and a representative, adherens junction-related cadherin. Immunohistochemical and immunoelectron microscopic methods were used. The results showed that occludin was distributed exclusively at known locations of tight junctions. ZO-1 was also expressed at these locations but its distribution extended beyond that of occludin, along the adjacent membranes. Pan-cadherin was expressed ubiquitously within the ciliary epithelium and negligibly in iris vascular endothelium. Our results demonstrate that occludin and ZO-1 are integral components of the blood-aqueous barrier of the normal rabbit eye.
