Corneal opacity in lumican-null mice: defects in collagen fibril structure and packing in the posterior stroma

PURPOSE: Gene targeted lumican-null mutants (lum(tm1sc)/lum(tm1sc)) have cloudy corneas with abnormally thick collagen fibrils. The purpose of the present study was to analyze the loss of transparency quantitatively and to define the associated corneal collagen fibril and stromal defects. METHODS: Backscattering of light, a function of corneal haze and opacification, was determined regionally using in vivo confocal microscopy in lumican-deficient and wild-type control mice. Fibril organization and structure were analyzed using transmission electron microscopy. Biochemical approaches were used to quantify glycosaminoglycan contents. Lumican distribution in the cornea was elucidated immunohistochemically. RESULTS; Compared with control stromas, lumican-deficient stromas displayed a threefold increase in backscattered light with maximal increase confined to the posterior stroma. Confocal microscopy through-focusing (CMTF) measurement profiles also indicated a 40% reduction in stromal thickness in the lumican-null mice. Transmission electron microscopy indicated significant collagen fibril abnormalities in the posterior stroma, with the anterior stroma remaining relatively unremarkable. The lumican-deficient posterior stroma displayed a pronounced increase in fibril diameter, large fibril aggregates, altered fibril packing, and poor lamellar organization. Immunostaining of wild-type corneas demonstrated high concentrations of lumican in the posterior stroma. Biochemical assessment of keratan sulfate (KS) content of whole eyes revealed a 25% reduction in KS content in the lumican-deficient mice. CONCLUSIONS: The structural defects and maximum backscattering of light clearly localized to the posterior stroma of lumican-deficient mice. In normal mice, an enrichment of lumican was observed in the posterior stroma compared with that in the anterior stroma. Taken together, these observations indicate a key role for lumican in the posterior stroma in maintaining normal fibril architecture, most likely by regulating fibril assembly and maintaining optimal KS content required for transparency.     

An x-ray diffraction study of corneal structure in mimecan-deficient mice

PURPOSE: Keratan sulfate proteoglycans (KSPGs) in the corneal stroma are believed to influence collagen fibrillar arrangement. This study was performed to investigate the fibrillar architecture of the corneal stroma in mice homozygous for a null mutation in the corneal KSPG, mimecan. METHODS: Wild-type (n = 9) and mimecan-deficient (n = 10) mouse corneas were investigated by low-angle synchrotron x-ray diffraction to establish the average collagen fibrillar spacing, average collagen fibril diameter, and level of fibrillar organization in the stromal array. RESULTS: The mean collagen fibril diameter in the corneas of mimecan-null mice, as an average throughout the whole thickness of the tissue, was not appreciably different from normal (35.6 +/- 1.1 nm vs. 35.9 +/- 1.0 nm). Average center-to-center collagen fibrillar spacing in the mutant corneas measured 52.6 +/- 2.6 nm, similar to the 53.3 +/- 4.0 nm found in wild-type mice. The degree of local order in the collagen fibrillar array, as indicated by the height-width (H:W) ratio of the background-subtracted interfibrillar x-ray reflection, was also not significantly changed in mimecan-null corneas (23.4 +/- 5.6), when compared with the corneas of wild-types (28.2 +/- 4.8). CONCLUSIONS: On average, throughout the whole depth of the corneal stroma, collagen fibrils in mimecan-null mice, unlike collagen fibrils in lumican-null mice and keratocan-null mice, are of a normal diameter and are normally spaced and arranged. This indicates that, compared with lumican and keratocan, mimecan has a lesser role in the control of stromal architecture in mouse cornea.     

Neonatal development of the corneal stroma in wild-type and lumican-null mice

PURPOSE: Between days 8 and 14 of neonatal development, the corneal stroma of the mouse undergoes critical changes in tissue thickness, cell density, and light scattering. The authors investigate the stromal matrix structure in wild-type and lumican-deficient corneas in this developmental phase. METHODS: Wild-type (n = 44) and lumican-deficient (n = 42) mouse corneas at neonatal days 8, 10, 12, and 14 were investigated by synchrotron x-ray diffraction to establish the average collagen fibril spacing, average collagen fibril diameter, and level of fibrillar organization in the stromal matrix. RESULTS: Collagen interfibrillar spacing in the normal mouse cornea became more closely packed between days 8 and 14, though not significantly so. In lumican-null mice, interfibrillar spacing was significantly elevated at days 8, 10, and 12, but not day 14, compared with that in wild-type mice. At all stages investigated, collagen fibrils were, on average, marginally thinner than normal in lumican-null mutants, and the spatial distribution of the fibrils was less well organized. CONCLUSIONS: Transient thickening of the corneal stroma of the normal mouse at eye opening is probably not caused by widespread, homogeneous rearrangement of collagen fibrils but more likely by a temporary increase in cell or stromal "lake" volume. Lumican, structurally influential in adult mouse corneas, is also a key molecule in the neonatal development of the stromal matrix.     

Altered corneal stromal matrix organization is associated with mucopolysaccharidosis I, III and VI.

The presence of cloudy corneas is a prominent feature of mucopolysaccharidosis (MPS) types I and VI, but not MPS IIIA or IIIB. The cause of corneal cloudiness in MPS I and VI is speculative. Transparency of the cornea is dependent on the uniform diameter and the regular spacing and arrangement of the collagen fibrils within the stroma. Alterations in the spacing of collagen fibrils in a variety of conditions including corneal edema, scars, and macular corneal dystrophy is clinically manifested as corneal opacity. The purpose of this study was to compare the structural organization of the stromal extracellular matrix of normal corneas with that of MPS corneas. The size and arrangement of collagen fibrils in cloudy corneas from patients with MPS I were examined. The alterations observed were an increased mean fibril diameter with a broader distribution in the MPS corneas. The MPS I corneas also had altered fibril spacing and more irregular packing compared with normal control corneas. The clear corneas of patients with MPS IIIA and IIIB also showed increases in mean fibril diameter and fibril spacing. However, there was less variation indicating more regularity than seen in MPS I. In addition, corneas from cat models of certain MPS were compared to the human corneas. Cats with MPS I and VI, as well as normal control cats, were examined. Structural alterations comparable to those seen in human MPS corneas were seen in MPS I and VI cats relative to normal clear corneas. The findings suggest that cloudy corneas in MPS I and VI are in part a consequence of structural alterations in the corneal stroma, including abnormal spacing, size, and arrangement of collagen fibrils.     

Annulus of collagen fibrils in mouse cornea and structural matrix alterations in a murine-specific keratopathy

PURPOSE: Mouse corneas were investigated to see whether a limbal annulus of corneal collagen exists as in humans. Mice with corneas predisposed to topographical changes (the SKC strain) were also examined, to establish the size and spacing of stromal collagen fibrils and the integrity of the annulus. METHODS: X-ray diffraction was used to measure collagen fibril spacing and diameter in normal (the BALB/c strain; four male, two female) and SKC (six male and six female) corneas and to identify the degree of preferred collagen orientation at 200- microm intervals across two BALB/c and four SKC corneas. RESULTS: The average collagen fibril diameter measured 35.5 nm in 3-month-old BALB/c corneas, and 36.9 nm and 37.0 nm, respectively, in corneas of age-matched male and female SKC mice. In male and female SKC corneas, average collagen interfibrillar Bragg spacing was significantly higher (64.5 and 59.9 nm, respectively) than in corneas of BALB/c mice (49.7 nm). Circumferentially aligned collagen, indicative of a limbal annulus of fibrillar collagen 2.2 mm in diameter, was identified in mouse cornea. On occasion, this was disturbed in the SKC phenotype. CONCLUSIONS: Collagen fibrils are marginally larger in the corneas of SKC mice than in the corneas of BALB/c mice and are considerably more widely spaced. An annulus of fibrillar collagen probably exists near the limbus of the normal mouse cornea that may help promote biomechanical stability and maintain corneal shape. A loss of structural integrity in the annulus of some SKC mice may predispose the corneas to biomechanical instability and shape changes.     

Structural abnormalities of the cornea and lid resulting from collagen V mutations

PURPOSE: Type V collagen forms heterotypic fibrils with type I collagen and accounts for 10% to 20% of corneal collagen. The purpose of this study was to define the ocular phenotype resulting from mutations in the type V collagen genes COL5A1 and COL5A2 and to study the pathogenesis of anomalies in a Col5a1-deficient mouse. METHODS: Seven patients with classic Ehlers-Danlos syndrome (EDS) due to COL5A1 haploinsufficiency and one with an exon-skipping mutation in COL5A2 underwent an ocular examination, corneal topography, pachymetry, and specular microscopy. A Col5a1-haploinsufficient mouse model of classic EDS was used for biochemical and immunochemical analyses of corneas. Light and electron microscopy were used to quantify stromal thickness, fibril density, fibril structure, and diameter. RESULTS: Five males and three females (mean age, 26 +/- 13.57 years; range, 11-52) were studied. All patients had "floppy eyelids." The corneas of all eyes were thinner (mean corneal thickness: 435.75 +/- 12.51 microm) when compared with control corneas (568.89 +/- 28.46 microm; P < 0.0001). In the Col5a1+/- mouse cornea, type V collagen content was reduced by approximately 49%, and stromal thickness was reduced by approximately 26%. Total collagen deposition in Col5a1(+/-) corneas also was reduced. Collagen fibril diameters were increased, but fibril density was decreased throughout the stroma at all developmental stages. CONCLUSIONS: In the eye, COL5A1 and COL5A2 mutations manifest as abnormally thin and steep corneas with floppy eyelids. Mechanisms involved in producing the latter anomalies probably involve altered regulation of collagen fibrillogenesis due to abnormalities in heterotypic type I/V collagen interactions similar to those observed in the Col5a1+/- mouse cornea.     

Spatial and temporal alterations in the collagen fibrillar array during the onset of transparency in the avian cornea

In the latter stages of development, the embryonic avian cornea undergoes significant changes in structure, composition and transparency. The rearrangement of stromal collagen fibrils at this time is important because it is believed to play a key role in the acquisition of corneal transparency. Here, we investigate spatial alterations in the internal fine structure of the avian cornea during development. Chicken corneas at developmental days 14, 16 and 18 were examined by transmission electron microscopy and quantitative image analysis. For anterior and posterior regions we determined fibril number densities, two-dimensional distribution functions, and, where appropriate, radial distribution functions. Stromal collagen fibrils became more closely spaced over the developmental range studied here. Changes in fibril number density indicated that fibrils became compacted first in the anterior stroma, and later (i.e. after day 16) in the posterior stroma. By day 18 collagen fibril number densities were essentially the same in superficial and deep tissue regions. At day 14, two-dimensional distribution functions of collagen fibrils in the posterior stroma pointed to a fibrillar array that was unlike that in the anterior stroma because there was no clear radial symmetry. Rather, in the deep stroma at day 14 there was evidence of different nearest neighbour spacings in two orthogonal directions. By day 18, fibril distributions in the anterior and posterior stroma were spatially homogeneous and radially symmetric, with radial distribution functions typical of those ordinarily found in mature cornea. Corneal transparency requires the stromal matrix to have some degree of regularity in the arrangement of its uniformly thin collagen fibrils. The chicken cornea becomes progressively transparent between days 14 and 18 of development as the stroma dehydrates and thins. We show that over this time period collagen fibrils in the anterior stroma become configured in advance of fibrils in deeper stromal regions, leading to questions over the potential roles of sulphated proteoglycans in different regions of the corneal stroma during morphogenesis.     

Hyaluronidase treatment, collagen fibril packing, and normal transparency in rabbit corneas

PURPOSE: Hyaluronidase treatment is the initial step of corneaplasty, a treatment under development that induces stromal softening and involves the application of a custom designed forming lens to achieve modification of refractive error. The purpose of this investigation was to examine changes in the arrangement of stromal collagen fibrils after hyaluronidase treatment. METHODS: Rabbit corneas were evaluated by slit-lamp microscopy at 0, 2 and 7 days after treatment and haze was assessed by subjective observation. Molecular and interfibrillar Bragg spacing of corneal collagen were measured from synchrotron x-ray scattering patterns. Transmission electron microscopy and digital image analysis were used to calculate radial distribution functions from the positions of collagen fibrils. The calculated fibril sizes and positions were also used to predict the transmission of visible light through these corneas. RESULTS: Hyaluronidase-treated corneas were shown to have a decreased interfibrillar Bragg spacing of 15% to 21%. Fibril hydration did not change. Transparency of these corneas remained unaltered. CONCLUSIONS: Hyaluronidase reduced the hydration of the corneal stroma, which led to a more compacted collagen fibril arrangement. This compression was predicted to cause a small reduction in the transmission of visible light through the cornea but not to a point likely to cause visual impairment.     

Collagen fibrils appear more closely packed in the prepupillary cornea: optical and biomechanical implications

PURPOSE: The size and organization of stromal collagen fibrils influence the biomechanical and optical properties of the cornea and hence its function. How fibrillar structure varies with position across the cornea has not been fully characterized. The present study was designed to quantify the collagen fibril spacing and diameter across the normal human cornea and to relate this to the properties of the tissue. METHODS: Small-angle x-ray diffraction was used to map in detail the variation in fibril spacing and fibril diameter along orthogonal medial-lateral and inferior-superior meridians of five normal human corneoscleral discs. RESULTS: Mean fibril diameters remained constant across all corneas up to the limbus, whereupon a sharp increase was observed. However, mean fibril spacing across the central 4 x 3 mm (prepupillary) cornea measured 5% to 7% lower than in the peripheral cornea. CONCLUSIONS: Collagen fibrils in the prepupillary cornea appear to be more closely packed than in the peripheral cornea. Anisotropy in fibril packing across the cornea has potential implications for the transparency and refractive index of the tissue. Biomechanically, it is possible that the higher packing density of stress-bearing collagen fibrils in the prepupillary cornea is necessary for maintaining corneal strength, and hence curvature, in a region of reduced tissue thickness. By inference, these results could have important implications for the development of corneal models for refractive surgery.     

Collagen fibril characteristics at the corneo‐scleral boundary and rabbit corneal stromal swelling

Background: The aim of this investigation was to reassess the impact of the scleral rim on the swelling of the mammalian corneal stroma and to investigate the ultrastructural features of the scleral rim and corneal stromal tissues. Methods: The epithelium and endothelium were removed from corneas excised from three‐month‐old female rabbits. The resulting preparations consisted of the corneal stroma plus a surrounding scleral rim, excised corneal stroma or a nine‐millimetre button of central corneal stroma. These preparations were immersed in a 35 mM bicarbonatebuffered mixed salt solution (equilibrated with five per cent CO₂‐air, pH 7.54 at 37 degrees Centigrade for nine hours. Some sclero‐corneal preparations were fixed for light or transmission electron microscopy. Results: The initial rate of swelling of corneal stromal buttons was greatest at 127 ± 8 per cent per hour, less for complete stromal preparations (118 ± 9 per cent per hour) and least for sclerostromal preparations (76 ± 12 per cent per hour). The swelling continued over three to nine hours but sclera preparations swelled up to 40 per cent with no further swelling. Light microscopy demonstrated that the sclero‐corneal rim tissue limited the swelling of the posterior corneal stroma. TEM sections of the episclera and sclera indicate that most fibril bundles show a radial orientation to the cornea. There are marked anterio‐posterior differences in the collagen fibrils of the scleral surround that are distinctly different from previous reports. Average fibril diameters were 62.8 ± 7.9 nm in the episclera, 122.4 ± 18.9 nm, 133.5 ± 51.9 nm and 56.5 ± 11.2 nm in the anterior, mid‐ and posterior scleral stroma, compared to an average fibril diameter of 33.5 ± 3.5 nm for the posterior corneal stroma. Conclusions: When there is a scleral rim in place, the swelling of the corneal stroma is substantially less than for isolated corneal stroma. The effect can be attributed to the a b sence of a cut‐edge effect for the sclero‐corneal stromal preparation but the unique and largely radial arrangement of the collagen fibrils in the scleral rim plays a part in limiting the swelling of the adjacent corneal stroma. The heterogeneous nature of this sclero‐corneal interface requires further investigation to define the mechanism of the effect.     

Ultrastructural analysis of collagen fibrils and proteoglycans in keratoconus

Purpose: To investigate ultrastructural alterations in the distribution of collagen fibrils (CFs) and proteoglycans (PGs) in the keratoconus cornea. Methods: Four normal corneas (donor age 24–75 years) and four severe and one mild keratoconus corneas (donor age 24–47 years) were fixed in 2.5% glutaraldehyde containing 0.05% cuprolinic blue dye for electron microscopy. Analyses were carried out on approximately 39 000 CF and 66 000 PG filaments in the anterior, middle and posterior stroma, using analySIS^® soft imaging software. Results: In severe keratoconus, stromal lamellae were seen to undulate in most regions, whereas in mild keratoconus only the middle and posterior lamellae were affected. In keratoconus corneas the mean diameter and interfibrillar spacing of CFs was reduced in all zones (p < 0.0001) and the CF and PG number density and area fractions were significantly increased (p < 0.0001) compared with in normal corneas and were higher (p < 0.0001) in the corneas with severe keratoconus than in that with mild keratoconus. The lamellae contained microfibrils (8–9 nm wide) and, in addition, PGs embedded within CFs. Degenerate keratocytes containing PGs were found in all keratoconus corneas. Conclusions: These studies suggest that as keratoconus progresses, the PG content of the stroma increases, whereas fibril diameter is reduced. The altered stromal content of PGs may influence CF diameters and their organization in keratoconus, weakening lateral cohesion and resulting in significant disorder of CF packing.     

Atypical composition and ultrastructure of proteoglycans in the mouse corneal stroma

PURPOSE: Recently, gene-targeted strains of mice with null mutations for specific proteoglycans (PGs) have been used for investigations of the functional role of these molecules. In the present study, the corneal stroma of the mouse was examined to provide some baseline PG morphologies in this species. METHODS: Monoclonal antibodies to specific glycosaminoglycan (GAG) chain sulfation patterns were used to characterize PG composition in corneal extracts by SDS-PAGE and Western blot analysis and to identify their tissue distribution by immunofluorescence microscopy. PGs were also visualized by transmission electron microscopy after contrast enhancement with cationic dye fixation. RESULTS: Western blot analysis of pooled corneal extracts and immunofluorescence of tissue sections identified 4-sulfated, but not 6-sulfated, chondroitin sulfate/dermatan sulfate (CS/DS). Keratan sulfate (KS) was present only as a low-sulfated moiety. Electron microscopic histochemistry disclosed a complex array of corneal PGs present as (1) fine filaments radiating from collagen fibrils, and (2) elongate, straplike structures, running either along the fibril axis or weaving across the primary fibril orientation. These large structures were digested by chondroitinase ABC, but not by keratanase. CONCLUSIONS: KS in the mouse is predominantly undersulfated and generates an immunostaining pattern that differs from that observed in corneas of other mammalian species thus far investigated. The mouse cornea resembles other mammalian corneas in the presence of filamentous arrays of small, collagen-associated stromal PGs visualized by cationic dye staining. However, large dye-positive structures with a CS/DS component are also present and appear to be unique to the cornea of this species.     

Collagen organization in the secondary chick cornea during development

PURPOSE: The latter stages of morphogenesis in the embryonic chick cornea are instrumental in the establishment of a properly formed corneal stroma. This study was designed to provide better appreciation of collagen reorganization in the avian corneal stroma during the latter stages of embryogenesis. METHODS: High-angle synchrotron x-ray diffraction patterns were obtained from 47 developing chick corneas daily at developmental days 13 through 18 (n = 7 or 8 at each time point) and analyzed to establish collagen molecular spacing and fibril orientation. RESULTS: Collagen intermolecular x-ray reflections were of approximately constant intensity between days 13 and 15 of development, but thereafter became progressively more intense, suggesting that extra collagen is deposited in embryonic chick corneas after day 16 of development. At all times, the mean collagen intermolecular spacing measured approximately 1.43 nm. X-ray intensity was not uniform around the intermolecular x-ray reflections at earlier time points. Rather, a fourfold symmetry was evident, indicative of an orthogonal array of collagen fibrils. An index of this symmetry was essentially unchanged between developmental days 13 and 15, but thereafter diminished considerably. CONCLUSIONS: The lateral spacing of fibril-forming collagen molecules does not change as the chick cornea develops between days 13 and 18. An orthogonal array of collagen fibrils is present in the corneas of developmental day-13 to -18 chicks, but starting at developmental day 16, additional collagen is deposited in a less well-oriented manner and thus acts to obscure the overall orthogonality, with implications for the biomechanical strength and shape of the cornea.     

Organization of collagen types I and V in the embryonic chicken cornea

The distribution and organization of type I and type V collagens were studied in the embryonic chicken cornea using anti-collagen, type specific, monoclonal antibodies and immunoelectron microscopy. These studies were performed on lathyritic 17-day corneas treated at 4 degrees C or 37 degrees C. At the lower temperature, collagen fibril structure is disrupted; at the higher temperature, normal fibril structure is maintained. Corneas from non-lathyritic 17-day chick embryos, reacted at the two different temperatures, were studied for comparison. In Bowman's membrane, the thin (20 nm) fibrils were labelled by antibodies against both type I and type V collagen under all conditions studied. In the corneal stroma, the striated collagen fibrils (25 nm) were labelled with the antibodies against type I collagen in all cases, and by antibodies against type V collagen under conditions where fibril structure was disrupted. These results are consistent with the concept of heteropolymeric fibrils consisting of both type I and type V collagen molecules assembled such that the epitopes on the type V molecule are unavailable to antibody unless the fibrillar structure is disrupted. We suggest that the interaction of type V collagen with type I collagen may be responsible for the small diameter fibrils and the rigid control of fibril structure found in the cornea.     

Anaylsis of birefringence during wound healing and remodeling following alkali burns in rabbit cornea

The use of synthetic inhibitors of metalloproteinases (SIMP) or medroxyprogesterone (MP) can prevent or significantly delay the ulceration of alkali-injured corneas by influencing collagen degradation. We have examined the remodeling of rabbit corneal stroma following alkali injury and have assessed the effect of SIMP and MP treatment. Following a defined alkali injury to the rabbit cornea, animals were divided into three subgroups, one group treated with topical beta-mercaptomethyl tripeptide (SIMP), one treated by subconjunctival injection of MP and one treated with a control solution. The corneal tissue was taken at 3 days, 1, 2, 3, 4, 9 and 26 weeks after alkali injury and prepared for light microscopy and transmission electron microscopy (TEM). A quantitative measurement of birefringence, in terms of the optical path difference (OPD), was made using a modified polarized microscopy technique based on the analysis of interference colours. The results showed that SIMP effectively prevented deep corneal ulceration. MP could delay the ulceration and the corneas treated with MP appeared to have better transparency than the other groups. There was a significant difference of the OPD between the anterior (5.8 +/-0.3 nm) and posterior (7.8 +/-0.4 nm) stroma of the normal cornea (P<0.001). The OPD values from the central corneas from alkali-injured eyes were generally lower than normal during the first 4 weeks and then gradually recovered to the normal level or above, except for the posterior stroma of the MP-treated eyes. We found that the OPD changes were very dependent on the presence of corneal lesions. The stroma near corneal ulceration, scar tissue, calcified stroma and the retro-corneal collagen layer showed a significant reduction of birefringence (lower OPD values). These OPD values remained much lower than normal up to the end of the experiment. TEM showed disrupted corneal stroma in all three groups, with thinner scar tissue in the MP group. The fibril diameters did not change significantly 3 days and 1 week after the alkali burns (27.1+/-2.3 nm in the control group, 27.3+/-2.2 nm in the SIMP group and 27.7+/-2.1 nm in the MP group) and there were no differences compared with 29.7+/-1.7 nm of the normal cornea (P>0.05). After 2 weeks of tissue remodeling, the fibril diameters in alkali-injured corneas showed a large variation (the range was between 11.5 and 80 nm) with a bimodal distribution, especially in the control group. The technique presented here for birefringence evaluation can provide an alternative way to monitor wound healing and tissue remodeling, both visually and quantitatively.     

Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals

PURPOSE: To investigate the feasibility of using femtosecond-pulse lasers to produce second-harmonic generated (SHG) signals to noninvasively assess corneal stromal collagen organization. SETTING: The Eye Institute, University of California, Irvine, California, USA. METHODS: Mouse, rabbit, and human corneas were examined by two-photon confocal microscopy using a variable-wavelength femtosecond lasers to produce SHG signals. Two types were detected: forward scattered and backward scattered. Wavelength dependence of the SHG signal was confirmed by spectral separation using the 510 Meta (Zeiss). To verify the spatial relation between SHG signals and corneal cells, staining of cytoskeletons and nuclei was performed. RESULTS: Second-harmonic-generated signal intensity was strongest with an excitation wavelength of 800 nm for all 3 species. Second-harmonic-generated forward signals showed a distinct fibrillar pattern organized into bands suggesting lamellae, while backscattered SHG signals appeared more diffuse and indistinct. Reconstruction of SHG signals showed two patterns of lamellar organization: highly interwoven in the anterior stroma and orthogonally arranged in the posterior stroma. Unique to the human cornea was the presence of transverse, sutural lamellae that inserted into Bowman's layer, suggesting an anchoring function. CONCLUSIONS: Using two-photon confocal microscopy to generate SHG signals from the corneal collagen provides a powerful new approach to noninvasively study corneal structure. Human corneas had a unique organizational pattern with sutural lamellae to provide important biomechanical support that was not present in mouse or rabbit corneas.     

Assessment of the apparent intra- and inter-sample variability in the collagen fibril diameter in the posterior corneal stroma of rabbits. A transmission electron microscopy study

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.     

Expression of type XII collagen and hemidesmosome-associated proteins in keratoconus corneas

PURPOSE: Keratoconus is a disease characterized by thinning of the central and paracentral cornea and scarring in advanced cases. This study was performed to examine the expression of type XII collagen, proteins associated with hemidesmosomes, and beta1 integrin in keratoconus corneas. METHODS: Corneal buttons were collected from normal subjects and patients with keratoconus and other corneal diseases. Immunofluorescence staining was performed on frozen sections for type XII collagen, bullous pemphigoid antigen (BP180), and integrin subunits alpha6, beta4, and beta1. RESULTS: To varying degrees, all proteins examined were expressed in normal human corneas. The staining intensity of type XII collagen was diminished in keratoconus corneas in the epithelial basement membrane zone and the stromal matrix. No significant variation was found in either the staining patterns or intensities for BP180, or integrins alpha6, beta4, and beta1. CONCLUSIONS: The level of type XII collagen was reduced in the epithelial basement membrane zone and stromal matrices in keratoconus corneas. These alterations may affect critical interactions of the corneal epithelium with the under-lying basement membrane, and cell-matrix interactions and matrix organization in the stroma.     

The molecular basis of corneal transparency

The cornea consists primarily of three layers: an outer layer containing an epithelium, a middle stromal layer consisting of a collagen-rich extracellular matrix (ECM) interspersed with keratocytes and an inner layer of endothelial cells. The stroma consists of dense, regularly packed collagen fibrils arranged as orthogonal layers or lamellae. The corneal stroma is unique in having a homogeneous distribution of small diameter 25-30 nm fibrils that are regularly packed within lamellae and this arrangement minimizes light scattering permitting transparency. The ECM of the corneal stroma consists primarily of collagen type I with lesser amounts of collagen type V and four proteoglycans: three with keratan sufate chains; lumican, keratocan, osteoglycin and one with a chondroitin sulfate chain; decorin. It is the core proteins of these proteoglycans and collagen type V that regulate the growth of collagen fibrils. The overall size of the proteoglycans are small enough to fit in the spaces between the collagen fibrils and regulate their spacing. The stroma is formed during development by neural crest cells that migrate into the space between the corneal epithelium and corneal endothelium and become keratoblasts. The keratoblasts proliferate and synthesize high levels of hyaluronan to form an embryonic corneal stroma ECM. The keratoblasts differentiate into keratocytes which synthesize high levels of collagens and keratan sulfate proteoglycans that replace the hyaluronan/water-rich ECM with the densely packed collagen fibril-type ECM seen in transparent adult corneas. When an incisional wound through the epithelium into stroma occurs the keratocytes become hypercellular myofibroblasts. These can later become wound fibroblasts, which provides continued transparency or become myofibroblasts that produce a disorganized ECM resulting in corneal opacity. The growth factors IGF-I/II are likely responsible for the formation of the well organized ECM associated with transparency produced by keratocytes during development and by the wound fibroblast during repair. In contrast, TGF-β would cause the formation of the myofibroblast that produces corneal scaring. Thus, the growth factor mediated synthesis of several different collagen types and the core proteins of several different leucine-rich type proteoglycans as well as posttranslational modifications of the collagens and the proteoglycans are required to produce collagen fibrils with the size and spacing needed for corneal stromal transparency.     

Report of a new family with dominant congenital heredity stromal dystrophy of the cornea.

PURPOSE: To report a new family with the rare form of congenital and hereditary stromal dystrophy of the cornea. METHODS: A mother and son, showing a bilateral congenital clouding of the cornea, were studied clinically and by biomicroscopy. After corneal transplantation, light microscopy and electron microscopy were performed. RESULTS: The stroma of the cornea was bilaterally and symmetrically thickened with diffuse and homogeneous small opacities. The opacities were present at birth and slowly progressive. Visual acuity was reduced to 2/10. Electron microscopy of the excised corneas showed a thickened stroma owing to cleaving of the lamellae by alternating layers of small-diameter collagen fibrils arranged in a random fashion. The epithelium, Bowman's membrane, the endothelium, and Descemet's membrane were normal. CONCLUSIONS: This family presents with a congenital stromal dystrophy of the cornea not linked to endothelial defects and thus differs from the more common form of congenital hereditary corneal endothelial dystrophy.