Search

Purpose: To assess the natural variability in the diameter of small collagen fibrils in the lamellae of corneal stroma of healthy young rabbits., Materials and Methods: The corneas of 6 young adult female New Zealand White rabbits (2.1 kg) were prepared for transmission electron microscopy (TEM) by fixation with a cacodylate-buffered 2% glutaraldehyde (pH 7.2-7.4, 320-340 mosm/kg). The corneas were embedded in Epon-Araldite, thin sections prepared from the central region of the posterior corneal stroma and stained with acidic uranyl acetate followed by acidic lead citrate. High magnification (x 33,000) micrographs were taken, and fibril diameters (FDs) measured at a final magnification of x 275,000 to a resolution of 2 nm., Results: Assessment of sampling-related errors indicated that the average diameter of the fibrils within any particular micrograph could be estimated to within 1% or better by measures of 100 fibrils. Assessments of the intra-sample variance (6 micrographs taken from the same cornea) indicated a group mean FD of 32.4 +/- 3.6 nm, whilst the inter-sample variance (6 micrographs taken from 6 different corneas) yielded an average of 33.1 +/- 4.5 nm (n = 100 fibrils/micrograph, +/-SD). However, group-averaged data sets of FDs, while unimodal, were not normally distributed, and cumulative averaging indicated a fixed range of FDs across the data sets., Conclusions: Intra- and inter-sample variability in collagen FDs is very similar, but the analysis indicates that the collagen fibrils are not homogeneous and that closely adjacent lamellae can have subtle differences in average FD., (Copyright 2006 S. Karger AG, Basel.)

The kinetics of imbibition of unbuffered 1% NaCl at 37 degrees C by whole ovine corneal stroma preparations were compared to those for square samples of stromas with side dimensions of 4, 6, 8, 10 and 13 mm. The rates and extent of swelling over a 24-hour period were proportionately greater for samples with smaller dimensions. After 24 h, the relative magnitude of the changes in wet mass ranged from 465% for complete stromas to 1,185% for 4 x 4-mm tissue squares. This size-dependent difference in swelling may, in part, account for differences between previous studies.

The corneal epithelium was scraped from bovine eyes (Hereford or Holstein) within 3 h post mortem. The mitochondriallysosomal fraction of the cells was assayed for hexosaminidase (HEX) and acid phosphatase (AP), and the membrane-soluble fraction for lactate dehydrogenase (LDH) and leucine arylamidase (LAA) activity. Pairs of eyes with clear corneas showed variances (+/-1 SD, n = 10 pairs) of +/-62, +/-57, +/-72 and +/-31% in the four enzyme activities (HEX, AP, LDH, LAA). Selection based on age (corneal diameter) reduced the variance to +/-41, +/-37, +/-39 and +/-30%. Additional selection for corneal surface wettability reduced variances to +/-26, +/-21, +/-22 and +/-26% in the activities. The latency of the lysosomal enzymes (HEX, AP) increased substantially with eye selection.

Corneal stroma-endothelial preparations from 2-kg albino rabbits were incubated in vitro in a bicarbonate-Ringer solution equilibrated with 5% CO2-9% O2 over 4 h in half-chambers at 37 degrees C. An average net fluid pump activity of 6.8 microliters/h was measured in the 4th hour. If such preparations were initially equilibrated with bicarbonate-Ringer for 90 min and then challenged for 20 min with 15 mM sodium lactate or NaCl, an osmotically induced increase in fluid flow occurred. After removal of the challenge and return to bicarbonate-Ringer for 2.5 h, the net fluid pump returned to baseline and then increased again (to an average of 9.7 microliters/h) for the lactate- but not NaCl-challenged endothelia. Scanning electron microscopy showed that lactate-challenged (but not the control or NaCl-challenged) endothelial contained a moderate number of very large pleomorphic cells within an otherwise regular mosaic. It is proposed that the large cells develop as a result of cell fusion (coalescence).

The aqueous humors of 100 freshly enucleated rabbit eyes were assessed within 10 min postmortem by taking samples in the last stage of corneal dissection following the Dikstein-Maurice procedure. Aqueous levels of partial pressure of carbon dioxide (pCO2) averaged 58.2 +/- 8.5 mm Hg to give calculated tCO2 and bicarbonate levels of 34.9 +/- 2.2 mEq/l and 30.0 +/- 3.4 mM/l, respectively. The pH and ionized Ca2+ levels averaged 7.39 and 1.50 mM while partial pressure of oxygen (pO2) values were variable (31.5 +/- 23.6 mm Hg). The pH showed an inverse correlation to pCO2 values, and Ca2+ increased as pCO2 increased. O2 levels tended to decrease as CO2 increased. The results suggest that significant hypercapnia and hypoxia of the eye fluids can occur even within 10 min postmortem for an enucleated globe at room temperature.

Freshly isolated stroma-endothelial preparations were perfused with a glucose, adenosine and glutathione-supplemented Ringer solution at 37 degrees C at a hydrostatic pressure of 20 cm H2O. The composition (tCO2, pH, Ca2+ and osmolality) of the stromal-side bathing solution was analyzed during a 90-min equilibration period during which this solution was covered or deliberately left open to the atmosphere. If exposed to the atmosphere, even for a few minutes, substantial changes in pH, osmolality and ionized calcium occurred such that, when silicone oil was subsequently applied to the stromal surface, significant increases in both the rate and amplitude of deturgescence were observed. The rates and amplitude were proportional to the corneal thickness prior to silicone oil application. The results further indicate that the physicochemical state of the stroma is an important determinant in the corneal deturgescence mechanism.