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.     

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.     

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.     

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.     

The role of dermatopontin in the stromal organization of the cornea

PURPOSE: Dermatopontin (DPT) is an abundant component of the stromal extracellular matrix; however, its function in the cornea is poorly understood. This study was conducted to determine whether DPT has a direct role in corneal matrix organization by investigating the ultrastructure of Dpt-null (Dpt(-/-)) mouse corneas. METHODS: Conventional light microscopy was used to compare the corneal thickness of Dpt(-/-) mice with that of the wild type. Collagen fibril distribution was studied using transmission electron microscopy and the datasets analyzed using image analysis software to determine fibrillar volume, fibril diameter, and spacing. RESULTS: Light microscopy demonstrated that Dpt(-/-) corneas in 2-month-old mice showed a 24% reduction in average stromal thickness compared with wild type (P < 0.001). The epithelium and Descemet's membrane appeared normal. Examination of Dpt(-/-) stroma by transmission electron microscopy indicated significant disruption of fibril spacing within the posterior lamellae, whereas the mid and anterior regions appeared largely unaffected compared with wild type. The collagen fibrils in Dpt(-/-) stroma showed a lower fibril volume fraction and a pronounced change in posterior fibrillar organization. There was no apparent difference in fibril diameter between Dpt(-/-) and wild-type mice. CONCLUSIONS: Collectively, these data suggest that DPT plays a key role in collagen fibril organization. The defects in collagen organization in Dpt(-/-) cornea appear to be most severe in the posterior stroma. It is possible that DPT interacts with corneal proteoglycans and that this interaction is involved in the maintenance of stromal architecture.     

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.     

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.     

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.     

Immunohistochemical study of localization of extracellular matrix after holmium YAG laser irradiation in rat cornea

PURPOSE: To better understand the corneal responses to holmium YAG (Ho:YAG) laser irradiation, we used immunofluorescent microscopy to examine changes in the localization of the extracellular matrix components, which play important roles in the maintenance of corneal morphology and functions. METHODS: Rats were irradiated with a Ho:YAG laser. On days 1, 3, and 7 after irradiation, the eyes were enucleated and frozen. Cryosections were made with a cryostat and were stained with antibodies against type I collagen, fibronectin, type IV collagen, or laminin for immunohistochemical study. RESULTS: One day after Ho:YAG laser irradiation, contraction of the stromal collagen fibrils was observed. Keratocytes could not be observed at the irradiated stromal region on day 1 after irradiation. One week later, however, keratocytes returned to the irradiated area. Although the stromal collagen fibrils had contracted, they were stained by an antibody against type I collagen. Dense fluorescence for fibronectin was observed at the margin of the stromal acellular zone. Both laminin and type IV collagen were observed at the basement membrane under the corneal epithelium, regardless of whether or not the corneas had been irradiated. CONCLUSION: These results suggest that Ho:YAG laser irradiation might be useful for the collagen contraction of stroma, without causing serious damage to the corneal epithelium and the basement membrane.     

Localization of extracellular matrix proteins after holmium YAG laser radiation in rat cornea]

PURPOSE: To understand corneal responses to holmium YAG (Ho: YAG) laser radiation, we used immunofluorescent microscopy to examine changes in the localization of extracellular matrix proteins. METHODS: Rats were radiated with an Ho: YAG laser. On days 1, 3, and 7 after radiation, the eyes were enucleated and frozen. The cryosections were stained by immunofluorescent microscopy using antibodies against type I collagen, fibronectin, type IV collagen, and laminin. RESULTS: One day after Ho: YAG laser radiation, contraction of the stromal collagen fibrils was observed. Keratocytes could not be observed at the radiated stromal region on day 1 after radiation. One week after radiation, keratocytes returned to the radiated area. Although the stromal collagen fibrils were contracted, they were stained by an antibody against type I collagen. Dense fluorescence for fibronectin was observed at the margin of the stromal acellular zone. Both laminin and type IV collagen were observed at the basement membrane under the corneal epithelium regardless of whether the corneas were radiated or not. CONCLUSIONS: These results suggest that Ho: YAG laser radiation might be useful for collagen contraction of the stroma, without causing serious damage to the corneal epithelium or the basement membrane.     

Localization of extracellular matrix proteins after holmium YAG laser radiation in rat cornea

Purpose: To understand corneal responses to holmium YAG (Ho:YAG) laser radiation, we used immunofluorescent microscopy to examine changes in the localization of extracellular matrix proteins. Methods: Rats were radiated with an Ho:YAG laser. On days 1, 3, and 7 after radiation, the eyes were enucleated and frozen. The cryosections were stained by immunofluorescent microscopy using antibodies against type I collagen, fibronectin, type IV collagen, and laminin.Results: One day after Ho:YAG laser radiation, contraction of the stromal collagen fibrils was observed. Keratocytes could not be observed at the radiated stromal region on day 1 after radiation. One week after radiation, keratocytes returned to the radiated area. Although the stromal collagen fibrils were contracted, they were stained by an antibody against type I collagen. Dense fluorescence for fibronectin was observed at the margin of the stromal acellular zone. Both laminin and type IV collagen were observed at the basement membrane under the corneal epithelium regardless of whether the corneas were radiated or not. Conclusions: These results suggest that Ho:YAG laser radiation might be useful for collagen contraction of the stroma, without causing serious damage to the corneal epithelium or the basement membrane.     

Use of a corneal stroma perfusion technique and transmission electron microscopy to assess ultrastructural changes associated with exposure to slightly acidic pH 5.75 solutions

PURPOSE: To quantitatively assess the effects of pH on the corneal stroma under an applied hydrostatic pressure. METHODS: Sclera-stroma preparations from adult sheep eyes were perfused with isotonic mixed salts solutions at 37 degrees C for 9 hr under an applied hydrostatic pressure and then fixed for electron microscopy. RESULTS: Exposure to pH 7.25 solutions resulted in slight swelling, retention of lamellae, and the keratocytes were intact. The collagen fibril diameter was 40.0+/-3.5 nm, which was the same as non-perfused samples (40.9+/-3.7 nm). Perfusion at pH 5.75 resulted in slight compaction, some disorganization of the lamellae, and almost total destruction of the keratocytes. The collagen fibril profiles in cross section were irregular and reduced to a mean of 30.2+/-3.5 nm (p<0.001). CONCLUSIONS: Exposure of the corneal stroma to slightly acid pH at the so-called critical electrolyte concentration pH for proteoglycan-ionic interactions can cause substantial structural changes.     

Effect of galardin on collagen degradation by Pseudomonas aeruginosa

The authors examined the effect of a synthetic peptidyl hydroxamate inhibitor of matrix metalloproteinase, Galardin, on collagen degradation by Pseudomonas aeruginosa (P. aeruginosa) in the presence or absence of keratocytes. Type I collagen gels, with or without suspended keratocytes, were incubated under medium containing sterile P. aeruginosa culture broth and/or Galardin for 24 hr. Degradation of collagen fibrils during culture was measured by the release of hydroxyproline. The conditioned media were also subjected to gelatin zymography and Western blotting to analyse the activation, by P. aeruginosa factor(s), of matrix metalloproteinases (MMPs) released by keratocytes. The effects of protease inhibitors, aprotinin, leupeptin and pepstatin, on collagen degradation by P. aeruginosa were also examined. P. aeruginosa broth by itself induced collegen gel degradation. When keratocytes were present, P. aeruginosa broth increased the amount of degraded collagen even further. Galardin significantly reduced the amounts of collagen degraded by P. aeruginosa culture broth, whether keratocytes were present or absent in the gel. However, the protease inhibitors had no inhibitory effects on collagen degradation. Gelatin zymography and Western blotting revealed that inactive proMMP-1, -2 and -3, released by keratocytes, were converted to active forms in the presence of P. aeruginosa broth. Galardin decreased the amounts of active MMPs and increased those of inactive proMMPs, suggesting that Galardin inhibited the activation of proMMPs by P. aeruginosa. The present results suggest that Galardin inhibits the keratocyte-mediated collagen degradation by P. aeruginosa culture broth, resulting from preventing the conversion of proMMPs to active MMPs.     

Three-dimensional supramolecular organization of the extracellular matrix in human and rabbit corneal stroma, as revealed by ultrarapid-freezing and deep-etching methods

The present work was carried out to clarify the three-dimensional fine structure of extracellular matrix in the cornea, using ultrarapid-freezing and deep-etching methods for electron microscopy. Fresh and glutaraldehyde-fixed samples of human and rabbit posterior corneas were ultrarapidly-frozen onto a copper block cooled by liquid helium or liquid nitrogen, freeze-fractured, deeply etched for 8-10 min and rotary replicated with platinum-carbon. Replicas were examined in a transmission electron microscope equipped with a tilting device. Only structures with repeatedly observed, similar architectural profiles free from ice crystal damage, were taken into account. The very recognizable major collagen fibrils revealed 8-10 nm subfibrils running helically along the fibril long axis. The other extracellular matrix components consisted of: (1) 8-12 nm interfibrillar bridging filaments, frequently ornamented with globular domains, joining neighbouring collagen fibrils like steps of a ladder; (2) 10-20 nm filaments with relatively large globular domains, running on the surface of collagen fibrils along their long axes, and projecting finger-like structures into interfibrillar spaces sometimes attaching to adjacent collagen fibrils; (3) 10-15 nm beaded filaments with a periodicity of 75-110 nm, forming extended networks, especially at the interlamellar interfaces; and (4) 8-14 nm straight or sinuous strands consisting of 4-6 nm repeating subunits or modules, forming extended sheets by lateral association at the Descemet's membrane/stroma interface. In the light of the information available from studies on the localization of extracellular matrix components in the cornea, and by reference to the structural models of extracellular matrix molecules and macromolecular assemblies, we have related the deep-etched extracellular matrix structures described above to: (1) proteoglycans; (2) fibril-associated collagens with interrupted triple helices or FACIT collagens; (3) type VI collagen; and (4) fibronectin, respectively.     

An x-ray diffraction investigation of corneal structure in lumican-deficient mice.

PURPOSE. The corneas of mice homozygous for a null mutation in lumican, a keratan sulfate-containing proteoglycan, are not as clear as normal. In the present study, mutant corneas were examined by synchrotron x-ray diffraction to see what structural changes might lie behind the loss of transparency. 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.     

Characterization of corneal keratocyte morphology and mechanical activity within 3-D collagen matrices

The purpose of this study was to assess quantitatively the differences in morphology, cytoskeletal organization and mechanical behavior between quiescent corneal keratocytes and activated fibroblasts in a 3-D culture model. Primary cultures of rabbit corneal keratocytes and fibroblasts were plated inside type I collagen matrices in serum-free media or 10% FBS, and allowed to spread for 1-5 days. Following F-actin labeling using phalloidin, and immunolabeling of tubulin, α-smooth muscle actin or connexin 43, fluorescent and reflected light (for collagen fibrils) 3-D optical section images were acquired using laser confocal microscopy. In other experiments, dynamic imaging was performed using differential interference contrast microscopy, and finite element modeling was used to map ECM deformations. Corneal keratocytes developed a stellate morphology with numerous cell processes that ran a tortuous path between and along collagen fibrils without any apparent impact on their alignment. Fibroblasts on the other hand, had a more bipolar morphology with pseudopodial processes (P ≤ 0.001). Time-lapse imaging of keratocytes revealed occasional extension and retraction of dendritic processes with only transient displacements of collagen fibrils, whereas fibroblasts exerted stronger myosin II-dependent contractile forces (P < 0.01), causing increased compaction and alignment of collagen at the ends of the pseudopodia (P < 0.001). At high cell density, both keratocytes and fibroblasts appeared to form a 3-D network connected via gap junctions. Overall, this experimental model provides a unique platform for quantitative investigation of the morphological, cytoskeletal and contractile behavior of corneal keratocytes (i.e. their mechanical phenotype) in a 3-D microenvironment.     

Biochemical and ultrastructural changes in rabbit sclera after treatment with 7-methylxanthine, theobromine, acetazolamide, or L-ornithine

AIMS: To examine a possible effect of 7-methylxanthine, theobromine, acetazolamide, or L-ornithine on the ultrastructure and biochemical composition of rabbit sclera. METHODS: Groups of pigmented rabbits, six in each group, were dosed during 10 weeks with one of the substances under investigation, and one untreated group was the control. Samples of anterior and posterior sclera were taken for determination of hydroxyproline, hydroxylysine, proline, proteoglycans, uronic acids and dermatan sulphate, chondroitin sulphate, and hyaluronic acid. Sections were examined with electron microscopy, and the diameter of the individual collagen fibrils was measured. RESULTS: Treatment with theobromine produced a significant increase in the contents of hydroxylysine, hydroxyproline, and proline in both anterior and posterior sclera, while 7-methylxanthine increased the contents of hydroxyproline and proline selectively in posterior sclera. Acetazolamide, on the other hand, significantly decreased the contents of hydroxyproline and proline in samples from anterior sclera. Uronic acids in both anterior and posterior sclera were significantly reduced by treatment with 7-methylxanthine, and L-ornithine significantly reduced uronic acids in posterior sclera. An inverse correlation between contents of hydroxyproline and uronic acids was found. The mean diameter of collagen fibrils was significantly higher in the posterior sclera from rabbits treated with 7-methylxanthine or theobromine, and significantly lower in rabbits treated with acetazolamide or L-ornithine compared with controls. In the anterior sclera, fibril diameter was significantly reduced in all treatment groups compared with controls. A positive, significant correlation between fibril diameter and content of hydroxyproline and proline was found in posterior sclera. CONCLUSION: 7-Methylxanthine, a metabolite of caffeine, increases collagen concentration and the diameter of collagen fibrils in the posterior sclera, and may be useful for treatment or prevention of conditions associated with low level and/or inferior quality of scleral collagen, such as axial myopia, chronic open angle glaucoma, and possibly neovascular age related macular degeneration. The apparent loss of collagen induced by chronic treatment with acetazolamide should be taken into consideration as a potentially harmful side effect. These results may indicate that scleral biochemistry and ultrastructure are influenced by the retinal pigment epithelium. One possible explanation is that the scleral fibroblasts which produce the collagen are sensitive to changes in the physiological electric field created by the retinal pigment epithelium.     

绿脓杆菌对角膜基质细胞介导的胶原降解作用的实验研究

目的 探讨绿脓杆菌性角膜炎组织破坏机制,为临床治疗提供理论依据。方法 Ⅰ型胶原凝胶,混悬及不混悬角膜基质细胞,与绿脓杆菌培养液共同培养２４ｈ,超滤去除天然胶原纤维,超滤液经过水解,分光光度计测定其羟脯氨酸含量。同时检测基质金属蛋白酶抑制剂Ｇａｌａｒｏｄｉｎ对胶原降解作用的影响。结果 绿脓杆菌培养液可直接降解Ⅰ型胶原,如同时存在角膜基质细胞此作用增强。无论 角膜基质细胞存在与否,Ｇａｌａｒｄｉｎ可明     

Association of type XII collagen with regions of increased stability and keratocyte density in the cornea

The anterior avian cornea possesses several distinct cellular and extracellular regions including the epithelial basal lamina, Bowman's layer and the interfacial matrix that separates Bowman's layer from the stroma. These unique regions differ biochemically, physically and morphologically but all contain type XII collagen. Previously, the collagen fibrils of several of these interfacial regions were shown to be stable to thermal and enzymatic denaturation. We reasoned that type XII collagen, a fibril-associated collagen, would be a good candidate to confer such stabilizing properties. The studies described herein were performed to localize type XII collagen and to assess its role in the interfacial matrices (IM). Using antibodies that react with both the short and long type XII collagen isoforms and that react specifically with the long isoform, we demonstrate that it is the short isoform that is present in Bowman's layer and the associated interfacial matrix lying between Bowman's and the stroma proper. In situ hybridization analyses demonstrate that both the epithelial and endothelial cells synthesize type XII collagen. In vitro cell culture analyses, however, demonstrate that in addition to epithelial cell synthesis, the stromal fibroblasts are capable of synthesizing type XII collagen as well. Immunofluorescence analyses performed at elevated temperature demonstrate that type XII collagen is thermally stable in Bowman's layer, but not in the anterior interfacial matrix or Descemet's layer. In addition, we observed that the distribution of type XII collagen during the development of the anterior extracellular matrices correlates precisely with an elevated density of keratocytes populating the interfacial matrix just deep to Bowman's layer. We show that this cellular density is developmentally regulated and does not arise from a localized increase in cell proliferation. These data demonstrate that Bowman's layer and the anterior interfacial matrix have unique biochemical and morphologic properties. Type XII collagen is thermally stable in Bowman's layer and, as a surface component of type I collagen fibrils, may contribute to the stability of the fibrils in this region. Neither type XII nor type I collagen is stable in the adjacent interfacial matrix, suggesting that differences in the type I-XII collagen fibril organization may exist between Bowman's layer and IM.     

Structural macromolecules and supramolecular organisation of the vitreous gel

The vitreous gel is a transparent extracellular matrix that fills the cavity behind the lens of the eye and is surrounded by and attached to the retina. This gel liquefies during ageing and in 25-30% of the oppulation the residual gel structure eventually collapses away from the posterior retina in a process called posterior retina in a process called posterior vitreous detachment. This process plays a pivotal role in a number of common blinding conditions including rhegmatogenous retinal detachment, proliferative diabetic retinopathy and macular hole formation. In order to understand the molecular events underlying vitreous liquefaction and posterior vitreous detachment and to develop new therapies it is important to understand the molecular basis of normal vitreous gel structure and how this is altered during ageing. It has previously been established that a dilute dispersion of thin (heterotypic) collagen fibrils is essential to the gel structure and that age-related vitreous liquefaction is intimately related to a process whereby these collagen fibrils aggregate. Collagen fibrils have a natural tendency to aggregate so a key question that has to be addressed is: what normally maintains the spacing of the collagen fibrils? In mammalian vitreous a network of hyaluronan normally fills the spaces between these collagen fibrils. This hyaluronan network can be removed without destroying the gel structure, so the hyaluronan is not essential for maintaining the spacing of the collagen fibrils although it probably does increase the mechanical resilience of the gel. The thin heterotypic collagen fibrils have a coating of non-covalently bound macromolecules which, along with the surface features of the collagen fibrils themselves, probably play a fundamental role in maintaining gel stability. They are likely to both maintain the short-range spacing of vitreous collagen fibrils and to link the fibrils together to form a contiguous network. A collagen fibril-associated macromolecule that may contribute to the maintenance of short-range spacing is opticin, a newly discovered extracellular matrix leucine-rich repeat protein. In addition, surface features of the collagen fibrils such as the chondroitin sulphate glycosaminoglycan chains of type IX collagen proteoglycan may also play an important role in maintaining fibril spacing. Furthering our knowledge of these and other components related to the surface of the heterotypic collagen fibrils will allow us to make important strides in understanding the macromolecular organisation of this unique and fascinating tissue. In addition, it will open up new therapeutic opportunities as it will allow the development of therapeutic reagents that can be used to modulate vitreous gel structure and thus treat a number of common, potentially blinding, ocular conditions.