An appreciation of the respective structural roles of the three stromal matrix KSPGs in the cornea is of fundamental importance for a fuller understanding of corneal ultrastructure and transparency. X-ray fiber diffraction studies of the corneas of lumican-null mice and keratocan-null mice have disclosed clear matrix changes, though to different extents.
14 17 In the current investigation, we used similar methodologies to probe the fine structure of the cornea in mice lacking the other main corneal KSPG, mimecan. The results showed that (1) the average collagen interfibrillar spacing in mimecan null corneas was not significantly different from that in wild-type corneas, (2) the level of local order in the fibrillar array was similar in the two tissues, and (3) the mean collagen fibrillar diameter in the corneas of mimecan-null mice was essentially unchanged. The data we report were obtained from unfixed corneas maintained at close to physiologic hydration. Moreover, all collagen fibrils throughout the whole of the tissue volume through which the x-ray beam passes contribute to the diffraction pattern, and in these experiments this is a volume measuring 1.5 mm
2 at the cornea’s surface extended throughout the whole of the cornea’s thickness. Thus, we sampled an extensive number of the collagen fibrils in the mouse cornea and generated highly representative, quantitative measurements of fibrillar architecture.
Previous work has indicated that, in vitro, mimecan has the ability to regulate the fibrillogenesis of type I collagen.
23 In the mimecan-null cornea, manual measurements from electron micrographs of glutaraldehyde-fixed tissue have reported the existence of larger than normal collagen fibrils, although it was not ascertained quantitatively how widespread these changes were.
18 The large sampling achieved by x-ray diffraction provided for an excellent evaluation of the overall structural dimensions of the stromal matrix, and whereas our analysis did not entirely rule out the existence of some larger than normal fibrils in the corneas of mimecan-null mice, it clearly indicated that, throughout the whole tissue thickness, the collagen fibrillar diameter was, on average, essentially unchanged. This is not the case in keratocan-null corneas where x-ray scattering investigations have shown a small but consistent increase in average fibril diameter throughout the tissue.
17 Nor is it the case in lumican-null corneas where the marked variation in collagen fibril diameter seen on electron microscopy
12 was so extensive that it precluded the formation of a measurable x-ray reflection that would have allowed us to calculate average collagen fibril diameter.
14 Recent work has indicated that the severity of phenotype in lumican-null corneas might be because this molecule, as well as serving as a structural regulator in its own right, also modulates keratocan gene expression.
24
Light-scattering from the extracellular stromal matrix of cornea, like x-ray scattering, is based on the combined scattering from all fibrils in the path of the radiation. Theories of corneal transparency
2 state that the fraction of light transmitted through a cornea,
F(λ), falls off exponentially with theproduct of the total scattering cross-section (σ), the collagen fibril number density (ρ), and the thickness of the tissue (
t):
\[F({\lambda})\ {=}\ e^{{-}{\sigma}{\rho}t}\]
Detailed calculations of corneal transparency are not trivial, particularly because σ is itself a complex function of the wavelength of light, the diameters of the collagen fibrils, their mode of packing, and the ratio of the refractive index of the hydrated fibrils to the refractive index of the extrafibrillar matrix.
2 Nevertheless, when considering the transparency of lumican-deficient corneas, one could reasonably argue that the increased average interfibrillar spacing seen by x-ray diffraction
14 is indicative of a lower ρ. The lumican-null cornea is considerably thinner than normal, and so
t is also reduced.
12 Both of these changes point to an increase in
F(λ), the corollary being that the alterations in matrix structure must increase the value of σ sufficiently to bring about the three-fold increase in backscattered light from the corneal stroma that is seen in the lumican-null mouse.
12 Keratocan-null corneas are also thinner than normal, with a wider average interfibrillar spacing (and therefore lower
t and ρ), but no detectable reduction in corneal clarity.
16 17 This can be explained if we accept that stromal matrix alterations in this mutant are less extensive than in the lumican-null animal, leading to less pronounced changes in σ. Normal corneal thickness (
t) has been reported in mimecan-null mice.
18 Because the current investigation discloses no significant alterations in matrix architecture in mimecan-null corneas we can reasonably surmise that any changes in σ and ρ are small. Thus, it is not surprising that these corneas show no detectable loss of corneal clarity.
18
Mouse cornea, unlike the corneas of most other species that have been investigated, contains KS that is predominantly undersulfated.
25 26 Nevertheless, these PGs are considered to be instrumental in the formation and maintenance of a structurally normal corneal stroma.
11 12 16 18 Based on the findings of this and previous
14 17 x-ray scattering experiments on all three KSPG-null mouse corneas, there appears to be a hierarchy within the KSPG population in terms of their relative influence as structural regulatory molecules: lumican, keratocan, and mimecan in decreasing order of importance.
The authors thank Gunter Grossmann for help with data collection at the SRS, and Craig Boote for help with data handling.