Synchrotron x-ray diffraction studies of the cornea, with implications for stromal hydration

Abstract

The intermolecular and interfibrillar spacings of collagen in bovine corneal stroma have been measured as a function of tissue hydration. Data were recorded from low- and high-angle x-ray diffraction patterns obtained using a high intensity synchrotron source. The most frequently occurring interfibrillar spacing varied from 34 nm in dry corneas to 76 nm at H = 5 (the hydration, H, is defined as the ratio of the weight of water to the dry weight). The most frequently occurring intermolecular Bragg spacing increased from 1.15 nm (dry) to approximately 1.60 nm at normal hydration (H approximately 3.2) and continued to increase only slowly above normal hydration. Most of the increase in the intermolecular spacing occurred between H = O and H = 1. Over this hydration range the interfibrillar and intermolecular spacings moved in tandem, which suggests that the initial water goes equally within and between the fibrils. Above H = 1 water goes preferentially between the fibrils. The results suggest that, even at normal hydration, water does not fill the interfibrillar space uniformly, and a proportion is located in another space or compartment. In dried-then-rehydrated corneas, a larger proportion of the water goes into this other compartment. In both cases, it is possible to postulate a second set or population of fibrils that are more widely and irregularly separated and therefore do not contribute significantly to the diffraction pattern.

Transparency, swelling and scarring in the corneal stroma

“…The corneas were obtained from the Eye Bank in culture medium, and were dehydrated to close to physiological hydration using polyethylene glycol. 21 It should be noted that the value for the fibril number density is lower than previously reported, partly owing to the scaling procedure.…”

“…By extrapolating this line to H ¼ 0, it is possible to plot a theoretical graph of the expected interfibril spacing on the assumption that all the water entering the stroma has gone towards separating the constituent fibrils. 5,21 This theoretical plot is shown in Figure 5 (line b). The shading around the theoretical plot is the uncertainty owing to the uncertainty in determining the spacing at H ¼ 0 from the experimental data.…”

“…However, as the cornea swells, the extra water will change the refractive indices of the interfibrillar matrix and also, possibly, of the collagen fibrils themselves. However, we know from previous studies 21 that collagen fibrils do not swell appreciably above physiological hydration, so their refractive index is independent of tissue hydration and stays constant at 1.416. As water or electrolyte enters the interfibrillar matrix, it dilutes it and the refractive index falls.…”

“…The amount by which it falls can be estimated by measuring the change in the refractive index of the stroma as a function of tissue thickness or hydration ( Figure 8) and then applying Gladstone and Dales's law of refractive indices to the system. 2 Since the imbibed fluid does not enter the fibrils themselves, 21 this fall in the refractive index as the stroma swells leads to an increase in the ratio of the refractive index of the fibrils to that of the interfibrillar matrix and to a corresponding increase in light scattering. We found that between physiological hydration and H ¼ 3.8, there was a 0.15% reduction in the refractive index of the matrix and a corresponding 0.1% increase in the ratio of the refractive indices of the fibrils and the matrix (S Khan, S Dennis, and K Meek, unpublished results).…”

“…The hydration of the stroma was altered by dialysing the denuded stroma against phosphate buffer pH 7.1 36 to which different amounts of the osmotic agent polyethylene glycol were added. 21 The continuous line is a leastsquares best fit to the data points. Transparency, swelling, scarring in corneal stroma KM Meek et al 933…”