Donald J. Brown

Nonlinear Optical Macroscopic Assessment of 3-D Corneal Collagen Organization and Axial Biomechanics

PURPOSE To characterize and quantify the collagen fiber (lamellar) organization of human corneas in three dimensions by using nonlinear optical high-resolution macroscopy (NLO-HRMac) and to correlate these findings with mechanical data obtained by indentation testing of corneal flaps. METHODS Twelve corneas from 10 donors were studied. Vibratome sections, 200 μm thick, from five donor eyes were cut along the vertical meridian from limbus to limbus (arc length, 12 mm). Backscattered second harmonic-generated (SHG) NLO signals from these sections were collected as a series of overlapping 3-D images, which were concatenated to form a single 3-D mosaic (pixel resolution: 0.44 μm lateral, 2 μm axial). Collagen fiber intertwining was quantified by determining branching point density as a function of stromal depth. Mechanical testing was performed on corneal flaps from seven additional eyes. Corneas were cut into three layers (anterior, middle, and posterior) using a femtosecond surgical laser system and underwent indentation testing to determine the elastic modulus for each layer. RESULTS The 3-D reconstructions revealed complex collagen fiber branching patterns in the anterior cornea, with fibers extending from the anterior limiting lamina (ALL, Bowmans layer), intertwining with deeper fibers and reinserting back to the ALL, forming bow spring-like structures. Measured branching-point density was four times higher in the anterior third of the cornea than in the posterior third and decreased logarithmically with increasing distance from the ALL. Indentation testing showed an eightfold increase in elastic modulus in the anterior stroma. CONCLUSIONS The axial gradient in lamellar intertwining appears to be associated with an axial gradient in the effective elastic modulus of the cornea, suggesting that collagen fiber intertwining and formation of bow spring-like structures provide structural support similar to cross-beams in bridges and large-scale structures. Future studies are necessary to determine the role of radial and axial structural-mechanical heterogeneity in controlling corneal shape and in the development of keratoconus, astigmatism, and other refractive errors.

Overexpression of matrix metalloproteinase-10 and matrix metalloproteinase-3 in human diabetic corneas: A possible mechanism of basement membrane and integrin alterations

We have previously described decreased immunostaining of nidogen-1/entactin; laminin chains alpha1, alpha5, beta1,gamma1; and epithelial integrin alpha3beta1 in human diabetic retinopathy (DR) corneas. Here, using 142 human corneas, we tested whether these alterations might be caused by decreased gene expression levels or increased degradation. By semiquantitative reverse transcription-polymerase chain reaction, gene expression levels of the alpha1, alpha5, and beta1 laminin chains; nidogen-1/entactin; integrin alpha3 and beta1 chains in diabetic and DR corneal epithelium were similar to normal. Thus, the observed basement membrane and integrin changes were unlikely to occur because of a decreased synthesis. mRNA levels of matrix metalloproteinase-10 (MMP-10/stromelysin-2) were significantly elevated in DR corneal epithelium and stroma, and of MMP-3/stromelysin-1, in DR corneal stroma. No such elevation was seen in keratoconus corneas. These data were confirmed by immunostaining, zymography, and Western blotting. mRNA levels of five other proteinases and of three tissue inhibitors of MMPs were similar to normal in diabetic and DR corneal epithelium and stroma. The data suggest that alterations of laminins, nidogen-1/entactin, and epithelial integrin in DR corneas may occur because of an increased proteolytic degradation. MMP-10 overexpressed in the diabetic corneal epithelium seems to be the major contributor to the observed changes in DR corneas. Such alterations may bring about epithelial adhesive abnormalities clinically seen in diabetic corneas.

Increased gelatinolytic activity in keratoconus keratocyte cultures. A correlation to an altered matrix metalloproteinase-2/tissue inhibitor of metalloproteinase ratio.

Keratoconus is a noninflammatory corneal disorder characterized by gradual stromal thinning and astigmatism. Altered degradation of corneal extracellular matrix is a suggested etiology for this disorder. In the present study we established keratocyte cultures from normal and keratoconus corneas and investigated the roles that matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMP, TIMP-2) may play. After chemical modification (reduction and alkylation) to remove the inhibitor and activation of enzyme with p-aminophenylmercuric acetate (APMA), keratoconus-conditioned media displayed a significant increase (p < 0.05) in the total potential gelatinolytic activity when compared with normal culture media treated in a similar manner. Basal levels of gelatinolytic activity in keratoconus culture media (no reduction, alkylation, or APMA treatment), determined by two different assay methods, tended to be about twice that of normal cell cultures. By zymography, both keratoconus and normal cultures snowed identical enzyme patterns, which represented MMP-2 (72 kDa) in its proform and, depending on the treatment of the media, varying amounts of activated MMP-2 (65 kDa). This suggests that the increased gelatinolytic activity in keratoconus was not correlated with an increased appearance of either the 65-kDa-activated form of MMP-2 or a new MMP species. In addition, no differences in the amount of MMP-2 were detected that could account for the increased activities in keratoconus cultures. However, a relative decline in the detectable TIMP levels in keratoconus cultures resulted in an apparent three-fold increase in the ratio of MMP-2/TIMP. Northern blots showed no significant changes in mRNA levels for MMP-1, MMP-2, MMP-3, TIMP, or TIMP-2. These data suggest that a possible alteration in the interaction between MMP-2 and TIMP may play a role in the increased gelatinolytic activity seen in keratoconus tissues.

Application of second harmonic imaging microscopy to assess structural changes in optic nerve head structure ex vivo

Glaucoma represents the second leading cause of blindness worldwide. While both age and intraocular pressure (IOP) are well-recognized risk factors for this disease, the underlying pathologic process involves the accelerated death of retinal ganglion cells (RGCs) that is associated with progressive loss of vision. The loss of RGCs has been postulated to occur primarily by injury to axons in the optic nerve head (ONH) due to its anatomic features and the mechanical vulnerability of the lamina cribrosa, the specialized ONH zone comprised of collagen beams that define the channels or pores through which axon bundles exit the eye. Recent advances in multiphoton microscopy using femtosecond lasers that generate second harmonic (SH) signals from collagen allows for direct optical imaging of the lamina cribrosa. We assess the application of SH generated microscopy (SHG) to the study of the ONH, and test the general hypothesis that increasing intraocular pressure in the same eye results in the movement of ONH collagen beams leading to distortion of the lamina cribrosa channels and compression of the axon bundles.

Characterization of an endogenous metalloproteinase in human vitreous

The vitreous is a gel-like connective tissue that undergoes liquefaction during aging and pathological processes. We isolated and characterized a degradative enzyme from the vitreous of different species and identified it to be matrix metalloproteinase-2 (MMP-2). This enzyme was found in a latent form and may be associated with endogenous inhibitors. Vitreous isolated from both non-diabetic and diabetic patients contained MMP-2 in the same concentrations. However, the diabetic samples had an additional gelatinase activity at 92 kDa which may be associated with a compromised vasculature. These results suggest that the normal human vitreous contains an endogenous MMP and the appearance of an additional activity is associated with pathologic conditions.

Keratoconus corneas: Increased gelatinolytic activity appears after modification of inhibitors

We examined the metalloproteinase activity from normal and keratoconus corneal extracts. No differences were detected in the total amount of the metalloproteinase or its physical form of activation. However, there was a significant elevation in enzymatic activity in the keratoconus extracts after chemical modification of inhibitory elements. This suggests either a difference in the enzymatic capabilities of keratoconus corneas or, as suggested previously, a decrease in the amount of TIMP (tissue inhibitor of metalloproteinase) present in the tissue.

Characterization of the major matrix degrading metalloproteinase of human corneal stroma. Evidence for an enzyme/inhibitor complex

The human cornea is an avascular, highly organized tissue with the unique property of transparency. While the extracellular matrices of this tissue are composed of a variety of collagen types, proteoglycans and glycoproteins, little is known of the normal degradation and remodeling of these components. We examined the capacity of organ cultured human ocular tissues to produce and secrete metalloproteinases, a family of related enzymes capable of digesting a variety of extracellular matrices. We demonstrated that while enzymatic activities similar to type I collagenase and stromelysin are produced, the predominant activities of the corneal stroma and keratocyte cultures are a 68-kDa gelatinase. In our hands, this enzyme does not appear to be induced significantly by phorbol esters in vitro. In addition, this enzyme appears to be secreted as a complex with a 21-kDa protein that functions as an enzymatic inhibitor. Moreover, the keratocytes also produce a 28-kDa inhibitor which has similar properties to tissue inhibitor of metalloproteinase (TIMP).

Effects of age and dysfunction on human meibomian glands.

OBJECTIVE To identify age-related changes in human meibomian glands that may be associated with meibomian gland dysfunction (MGD). METHODS Excess eyelid tissue from 36 patients (age range, 18-95 years, 19 female, 17 male) who underwent canthoplasty procedures were used. Dermatologic history, age, and presence of MGD were recorded. Samples were frozen, sectioned, and stained with specific antibodies against peroxisome proliferator-activated receptor γ (PPARγ) to identify meibocyte differentiation, Ki67 nuclear antigen to identify cycling cells, and CD45 to identify inflammatory cell infiltration. RESULTS Staining for PPARγ showed cytoplasmic and nuclear localization in the 2 youngest subjects (ages, 18 and 44 years). Older individuals (>60 years) showed predominantly nuclear staining, with cytoplasmic staining limited to the basal acinar cells in 17 of 31 subjects. The number of Ki67 positively stained basal cells were significantly elevated in the younger compared with older subjects based on linear regression analysis (r(2) = 0.35; P < .001). There was also a significant correlation between MG expression grade and CD45 cell infiltration (r = 0.414; P = .05). CONCLUSIONS These results indicate that aging human meibomian glands show decreased meibocyte differentiation and cell cycling that is associated with the development of MGD. Findings also suggest that altered PPARγ signaling may lead to acinar atrophy and development of an age-related hyposecretory MGD. CLINICAL RELEVANCE Meibomian gland dysfunction and evaporative dry eye are common age-related eyelid disorders. Understanding the underlying mechanism of MGD may lead to the development of novel therapeutic strategies to treat this disease.

Eye-Specific IOP-Induced Displacements and Deformations of Human Lamina Cribrosa

PURPOSE To measure high-resolution eye-specific displacements and deformations induced within the human LC microstructure by an acute increase in IOP. METHODS Six eyes from donors aged 23 to 82 were scanned using second harmonic-generated (SHG) imaging at various levels of IOP from 10 to 50 mm Hg. An image registration technique was developed, tested, and used to find the deformation mapping between maximum intensity projection images acquired at low and elevated IOP. The mappings were analyzed to determine the magnitude and distribution of the IOP-induced displacements and deformations and contralateral similarity. RESULTS Images of the LC were obtained and the registration technique was successful. IOP increases produced substantial, and potentially biologically significant, levels of in-plane LC stretch and compression (reaching 10%-25% medians and 20%-30% 75th percentiles). Deformations were sometimes highly focal and concentrated in regions as small as a few pores. Regions of largest displacement, stretch, compression, and shear did not colocalize. Displacements and strains were not normally distributed. Contralateral eyes did not always have more similar responses to IOP than unrelated eyes. Under elevated IOP, some LC regions were under bi-axial stretch, others under bi-axial compression. CONCLUSIONS We obtained eye-specific measurements of the complex effects of IOP on the LC with unprecedented resolution in uncut and unfixed human eyes. Our technique was robust to electronic and speckle noise. Elevated IOP produced substantial in-plane LC stretch and compression. Further research will explore the effects of IOP on the LC in a three-dimensional framework.

Alu DNA polymorphism in ACE gene is protective for age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. We report an association between an Alu polymorphism in the angiotensin-converting enzyme (ACE) gene with the dry/atrophic form of AMD. Using the polymerase chain reaction (PCR) on genomic DNA isolated from patients with AMD (n=173), and an age-matched control population (n=189), we amplified a region polymorphic for an Alu element insertion in the ACE gene. The Alu(+/+) genotype occurred 4.5 times more frequently in the control population than the dry/atrophic AMD patient population, (p=0.004). The predominance of the Alu(+/+) genotype within the unaffected control group represents a protective insertion with respect to the human ocular disease, dry/atrophic AMD. This is the first demonstration of an Alu element insertion exerting protective effects against a known human disease.