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1 From the Biophysics Group, Department of Optometry and Vision Sciences, Cardiff University, United Kingdom; and the 2 Departments of Medicine, Genetics and Ophthalmology, Case Western Reserve University, Cleveland, Ohio. 3 Present affiliation: Departments of Medicine, Cell Biology and Ophthalmology, Johns Hopkins University, Baltimore, Maryland.
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Abstract |
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METHODS. X-ray diffraction patterns were obtained from the corneas of 6-month-old and 2-month-old lumican-null and wild-type mice. Measured in each cornea were the average collagen fibril diameter, average collagen fibril spacing, and the level of order in the collagen array.
RESULTS. The x-ray reflection arising from regularly packed collagen was well-defined on all x-ray patterns from 6-month-old wild-type corneas. Patterns from 6-month-old lumican-deficient corneas, however, contained interfibrillar reflections that were measurably more diffuse, a fact that points to a widespread alteration in the way the collagen fibrils are configured. The same distinction between mutant and wild-type corneas was also noted at 2-months of age. Average collagen fibril spacing was marginally higher in corneas of 6-month-old lumican-null mice than in corneas of normal animals. Unlike x-ray patterns from wild-type corneas, patterns from lumican-deficient corneas of both ages registered no measurable subsidiary x-ray reflection, evidence of a wider than normal range of fibril diameters.
CONCLUSIONS. The spatial arrangement of stromal collagen in the corneas of lumican-deficient mice is in disarray. There is also a considerable variation in the diameter of the hydrated collagen fibrils. These abnormalities, seen at 2 months as well as 6 months of age, probably contribute to the reduced transparency.
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Introduction |
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In the cornea, three types of small proteoglycan have been identified that contain keratan sulfate side chains: lumican,11 keratocan,12 13 and mimecan (or osteoglycin).14 Recently, work at the genetic level has directly demonstrated the importance of lumican in corneal transparency when it was shown that mice homozygous for a null mutation in this molecule have opaque corneas.15 16 In these animals, a bilateral corneal opacity is usually detectable by slit lamp examination anywhere from 6 weeks to 5 months and is generally diffuse and confined to the central cornea with a peripheral clear zone. In vivo confocal microscopy has disclosed increased backscattering (an indication of loss of transparency) in the corneas of the null mice as early as 3 to 4 weeks of age.17 We have not detected progression of these opacities in follow-up studies.
It is believed that keratan sulfate in the cornea helps (along with other corneal glycosaminoglycans) to maintain collagen fibrils in a specific spatial conformation.18 This arrangement of fibrils, in turn, is understood to be a significant contributory factor to the cornea’s light transmission properties.19 20 21 22 The corollary is that a breakdown of the normal stromal architecture, if it is severe enough, leads to increased light scattering and hence to corneal cloudiness. Indeed, an electron microscopic inspection of the corneas of lumican-deficient mice has indicated the presence of structural collagen abnormalities in localized regions of the tissue that might well be to the detriment of tissue transparency.17
To investigate the ultrastructure of the corneas of lumican-null mice, we undertook a series of experiments using synchrotron x-ray diffraction, a technique that directs a focused beam of monochromatic x-rays through the whole thickness of the cornea, and in doing so provides average values for regularly occurring spatial elements in the whole volume of the tissue through which the beam, ordinarily a few millimeters in size, passes.23 In recent years, a substantial amount of new data has been gained by x-ray diffraction experiments in the cornea.24 25 26 27 28 29 This is because, as well as providing structural information from an immense number of scattering elements (i.e., collagen fibrils), the approach has the added advantage that corneas can be studied at close to physiologic hydration. Thus, dehydration- and fixation-induced artifacts, unavoidable in electron microscopy,30 are not a major issue.
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Methods |
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Synchrotron X-ray Diffraction
Corneas, secured in airtight specimen holders between two sheets
of polyester film, were analyzed at the Synchrotron Radiation Source
(SRS), Daresbury Laboratory (Cheshire, UK). Each specimen was placed in
the path of a focused (1.5 x 1.0 mm), monochromatic (
=
0.154 nm) x-ray beam on SRS Station 2.1 and the shutters opened to
expose the cornea for 10 minutes. In this manner we were able to record
low-angle x-ray diffraction patterns for all specimens on a multiwire,
gas proportional-area detector situated directly behind the cornea,
8.5 m away. An evacuated tube with polyester film windows
separated the specimen from the detector, and the window nearest the
detector contained a lead backstop positioned to stop the x-ray beam
that passed through the cornea undeviated.
X-ray patterns (512 x 512 pixels) were analyzed with purpose-written, Unix-based software followed by a graphics and statistics package (Statistica; Statsoft, Tulsa, OK). First, x-ray patterns were normalized using ion chamber counts to account for beam intensity decay. A detector response from a 2.5-hour exposure to a radioactive source (Fe55) was then subtracted from each x-ray pattern to correct for any nonlinearities in the detector. Next, a vertical scan, 28 pixels wide, of x-ray intensity (I) versus radial position (R) was taken across the center of the pattern. From this, the intensity profile of the first-order equatorial x-ray reflection was readily visible. The intensity profile was then summed about its center, and a plot of R versus I · R generated to correct for the fact that the scan across the circular x-ray pattern was linear. A background representing diffuse x-ray scatter was then fitted to the intensity scans (a power law in the form y = a + b · xc was found to be suitable) and subtracted from the pattern to leave peaks corresponding to the low-angle x-ray reflections from the cornea.
As is described in detail elsewhere,23
the equatorial
portion of the low-angle x-ray scattering pattern from the cornea is
given by
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As well as providing a measure of collagen fibril separation, the interfibrillar reflection also provides an estimate of the degree of local order in the arrangement of the collagen matrix. The sharper the reflection (i.e., the narrower the peak in the x-ray intensity scan), the narrower the range of nearest-neighbor spacings and vice versa. The quantity used in this study to represent this is the ratio of peak height to peak width at half height.
In cornea, the scattered amplitude (F) is commonly
called the fibril transform, and gives a measure of the average size of
the collagen fibrils that contribute to the diffraction pattern. If,
like Worthington and Inouye,31
we assume that the
interference function is essentially constant after the innermost
maximum and that subsidiary x-ray intensity maxima arise solely from
the fibril transform, we can use the reciprocal space positions
(Qn) of the subsidiary maxima to calculate
the average collagen fibril diameter (D) from
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Results |
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X-ray scattering patterns from the mutant and wild-type corneas that had been stored frozen contained better quality data than those that had been stored in fixative. Patterns from the cryopreserved corneas were therefore further analyzed to provide average measurements of corneal ultrastructure. When these x-ray intensity profiles were summed around their centers (Fig. 2) , it was recognized that the interfibrillar reflections were invariably much broader in patterns from 6-month-old lumtm1sc/lumtm1sc corneas than from 6-month-old wild-type corneas. As previously,34 we quantified the sharpness of the reflection by measuring the ratio of peak height to peak width at half height (after the subtraction of background scatter). Higher ratios are evidence of more local order in the collagen matrix, and it was discovered that the ratio for the 6-month-old wild-type corneas was at least double that for the 6-month-old mutant corneas (Table 1) .
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To determine whether the structural aberrations in the corneas of 6-month-old lumican-deficient mice might be a function of age, we similarly examined the corneas of 2-month-old animals. The data from the corneas that had been stored frozen (the tissues from which the better-quality x-ray data were obtainable) are shown in Figure 3 , and reveal that structural differences in the corneas of the younger mice tended to mirror those of their older counterparts. The interfibrillar spacings in the corneas of 2-month-old lumican-deficient mice were virtually identical with those of the older mutants, as was the interfibrillar spacing in one of the two wild-type corneas (Table 2) . Of particular importance, as in the 6-month-old corneas, the ratio of peak height to peak width at half height of the interfibrillar reflection for the 2-month-old lumtm1sc/lumtm1sc corneas was at least double that for the 2-month-old lum+/lum+ corneas, pointing to more fibrillar disorder in the mutant tissue. Also, the first subsidiary maximum of the fibril transform—a reflection that showed the average collagen fibril diameter in the corneas of the 2-month-old wild-type mice, similar to that of the 6-month-old wild-type mice, to be approximately 35 nm (Table 2) —was greatly diminished in x-ray patterns from 2-month-old mutant corneas. Again, this is consistent with the fact that, on average, throughout the whole thickness of the tissue, the diameter of the corneal collagen fibrils in mice was less uniform when lumican was not present.
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Discussion |
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What, then, are the implications for corneal transparency of the loss
of order in the collagen array in lumican-null mice? Theory has
it35
that the fraction of light transmitted undeviated
through the cornea F() is a function of the total
scattering cross-section per fibril per unit length (
), the number
density of fibrils in the stroma (
), and the thickness of the tissue
(t) and takes the form
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) 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.35
More extensive structural
information from x-ray diffraction and electron microscopic
investigations is required to fully model corneal transparency in
lumican-deficient mice; however, we can predict that the altered fibril
packing will modify and and thus influence corneal
transparency. Indeed, recent work with confocal microscopy through
focusing (CMTF) has disclosed higher levels of backscatter in the
corneas of
lumtm1sc/lumtm1sc
mice and has shown that that increased backscatter is a feature of
younger (1–2-month-old) as well as older (4–5-month-old)
animals.17
Our results concur with this, in that they
indicate that collagen disorganization is present early (2 months) as
well as later (6 months) in life. Although the collagen fibrils in the lumican-null corneas of both ages studied were more disordered than normal, we were able to obtain a value for the mean center-to-center collagen fibril spacings in the specimens that had been stored frozen (Tables 1 2) . Apart from one of the two 2-month-old wild-type corneas, the results were fairly consistent and indicated that the corneas of lumican-null mice, on average, had collagen fibrils that were a little more widely spaced than normal. It must be remembered, however, that we are quoting average values and when compared with the wild-type corneas, the mutant corneas, as indicated by the relative diffuseness of the interfibrillar reflections they generate, possessed rather more collagen fibrils that lay away from the quoted average. Also bear in mind that the values quoted are the interfibrillar Bragg spacings, values that do not take into account the mode of packing of the fibrils. If, as has often been done, a liquidlike packing of corneal collagen fibrils is envisaged, then the interfibrillar Bragg spacings should be multiplied by the factor 1.12 to give the actual center-to-center collagen fibril spacing.31
Previous x-ray diffraction studies of pathologic human corneas have shown collagen fibril spacing to be normal in keratoconus,36 but compacted (in a nonuniform manner) in macular corneal dystrophy.37 It is thus reasoned that keratoconic corneas are likely to be thin because the stroma contains less collagen than normal. Corneal thinness in macular corneal dystrophy, on the other hand, is judged to be caused mainly by the heterogeneous close packing of normal-diameter collagen fibrils. In the present study, the average interfibrillar spacings in the lumtm1sc/lumtm1sc and lum+/lum+ corneas that had been stored frozen were not markedly different (Tables 1 2) , but the corneas of lumican-deficient mice are invariably in the region of 40% thinner than normal.17 In view of this, we can reasonably conclude that the thinness of the mutant corneas is unlikely to be caused by the compaction of collagen fibrils. The most persuasive explanation is that there are simply fewer collagen fibrils in the mutant corneas. With this in mind, we point out the recent suggestion that lumican-null corneas are thin because of restricted collagen biosynthesis or because of a smaller pool of keratocytes.17 This remains to be seen, although, if true, it would fit with our reasoning.
A prevailing theme of prior investigations into lumtm1sc/lumtm1sc corneas has been that it is the posterior stroma that is chiefly affected. Specifically, relatively more backscattered light is detected in deeper regions of the stroma, more lamellar disruption is reported there, collagen fibrils as a whole are a few percentage points larger than normal there, and more large and irregular fibrils abound.17 Most of this information is acquired from electron microscopy. The x-ray reflections described herein were formed by a contribution to scattering from all collagen fibrils in a volume of the cornea through which the x-ray beam passes—in this case a volume that measured 1.5 x 1.0 mm x corneal thickness. This is an immense number of fibrils (of the order of 108), and our data thus provide highly representative average values for various stromal structures. The x-ray data also give a measure of these structures in a hydrated state. However, precisely because the x-ray beam passes through the whole cornea in these experiments, we were unable to identify the location with respect to depth of the structural alterations that we found. That said, the x-ray diffraction data are readily reconcilable with the results of the electron microscopic study of the lumican-null corneas, in that the interfibrillar reflection is "smeared out" because of fibrillar disorganization in the stromal matrix—a disorganization that electron microscopy has identified to be mostly in the posterior stroma.17
The first subsidiary x-ray reflection denoted by the elevated x-ray intensity around position Q = 0.045 nm-1 in Figures 2 and 3 is referred to as the fibril transform, and its position enables measurement of the average diameter of collagen fibrils throughout the whole tissue thickness. We did this in the 2-month-old and 6-month-old wild-type corneas that had been stored frozen (Tables 1 2) and found that the value of approximately 35 nm was in the predicted range for hydrated mammalian corneas.33 In stark contrast to the x-ray diffraction patterns from the corneas of wild-type mice, those from the corneas of lumican-null mutants did not possess a clear and consistently measurable fibril transform (Figs. 2 3) . This means that whereas collagen fibrils in normal corneas tend to be of a uniform diameter, those in the corneas of young and old lumican-deficient mice exhibit a clear variation is size. The electron microscopy concurs with this and further locates most large-diameter collagen fibrils in localized regions, in the deeper stromal layers.17
Regarding the possible mechanisms involved in the formation of larger than normal collagen fibrils, it is notable that, in vitro, lumican retards collagen fibrillogenesis and results in the formation of smaller fibrils.38 In mice, we point out that higher levels of lumican are usually found in the posterior stroma,17 and thus its absence is perhaps most strongly felt there, resulting in more large fibrils in that region of the tissue.17 The picture regarding the influence of small leucine-rich proteoglycans in relation to the size of collagen fibrils in the corneal stroma is not yet fully resolved, however. For example, decorin, like lumican, retards collagen fibrillogenesis in vitro and results in the formation of smaller fibrils.38 Nevertheless, initial reports show that gene-targeted decorin-null mice exhibit no apparent corneal abnormalities on electron microscopy.39
Work with transgenic models such as this can shed light on the pathologic mechanisms at play in human corneal disease. For example, in the inherited condition, macular corneal dystrophy, corneal clouding, and collagen ultrastructural abnormalities possibly caused by keratan sulfate proteoglycan deficiencies, are present, just as they are in the lumican-null mice.9 10 15 16 17 However, there are key clinical, ultrastructural, and biochemical differences between the known types of macular corneal dystrophy and the lumtm1sc/lumtm1sc mouse investigated in this study. For example, unlike the lumican-null corneal phenotype in which corneal opacification is predominantly central with a peripheral clear zone, corneal clouding in macular corneal dystrophy extends to the limbal area. Also, in vivo CMTF has indicated increased backscattering (opacity) that is restricted to the posterior stroma in the lumican-deficient mouse, whereas in macular corneal dystrophy the opacification is superficial initially and later extends throughout the stroma. At the ultrastructural level, lumican-null corneas possess thicker than normal collagen fibrils and show a general lamellar disorganization in the posterior stroma that coincides well with the zone of increased backscattering.17 These ultrastructural abnormalities are similar in some respects to those seen in macular corneal dystrophy, where an irregular pattern of collagen fibril packing has been reported,40 along with some larger than normal collagen fibril diameters.41 42
Unlike the lumican-null cornea, however, a previous x-ray diffraction study on macular corneal dystrophy provided evidence for a heterogeneous, closer than normal interfibrillar spacing that was considered to be the underlying cause for the thin cornea.37 Another difference between macular corneal dystrophy and the lumican deficient model in the present study is the reason for the absence of sulfated lumican. In the mutant mouse the lumican core protein, the major keratan sulfate–bearing core protein, is absent, and an assessment of total keratan sulfate content of whole eyes has indicated a 25% reduction in this molecule. In macular corneal dystrophy, on the other hand, mutations in a new carbohydrate sulfotransferase gene (CHST6)43 44 and reduced enzyme activity45 have been identified as the underlying cause of the poorly sulfated keratan sulfate proteoglycans.
Currently, experiments using transgenic technology aligned with a number of structural and biochemical tools are beginning to further enhance our understanding of corneal structure and function with regard to proteoglycans—for example, one form of the inherited condition cornea plana is now known to be caused by mutations in the gene that encodes keratocan, a small, leucine-rich proteoglycan that, similar to lumican, bears keratan sulfate side chains.46 For a fuller understanding of the pathogenesis of macular corneal dystrophy we await the development of a transgenic model of this disease.
It is widely accepted that if the physical requirements for corneal transparency are to be met, then the collagen fibrils that make up the corneal stroma must be of approximately the same diameter and configured in a fairly regular array. The spatial arrangement of stromal collagen in the corneas of lumican-deficient mice was in disarray, and there was a significant variation in the diameter of the hydrated collagen fibrils that is not ordinarily seen in normal corneas. These abnormalities, which are present from an early age, probably contribute to the reduced transparency in the corneas of lumican-null mice.
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Acknowledgements |
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Footnotes |
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Supported by Grant EY11654-03 from the National Institutes of Health, the BBSRC, Daresbury Lab CLRC, and a programme grant from the Medical Research Council, United Kingdom.
Submitted for publication December 13, 2000; revised March 2, 2001; accepted March 21, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: Andrew J. Quantock, Biophysics Group, Department of Optometry and Vision Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, Cathays Park, Cardiff CF10 3NB, UK. quantockaj{at}cf.ac.uk
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