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.     

Second-harmonic imaging microscopy of normal human and keratoconus cornea

PURPOSE: The purpose of this study was to evaluate the ability of second-harmonic imaging to identify differences in corneal stromal collagen organization between normal human and keratoconus corneas. METHODS: Six normal corneas from eye bank donors and 13 corneas of patients with keratoconus, obtained after penetrating keratoplasty were examined. A femtosecond titanium-sapphire laser with 800-nm output was used to generate second-harmonic signals collected at 400 nm from central and paracentral corneal tissue blocks. Three-dimensional (3-D) data sets were collected and reconstructed to evaluate the location and orientation of stromal collagen lamellae. RESULTS: Imaging of second-harmonic signals combined with 3-D reconstruction of the normal cornea identified a high degree of lamellar interweaving, particularly in the anterior cornea. Of note was the detection of lamellae that inserted into Bowman's layer and were oriented transverse to the corneal surface, penetrating posteriorly approximately 120 mum. In keratoconus corneas, imaging second-harmonic signals identified less lamellar interweaving and a marked reduction or loss of lamellae inserting into Bowman's layer in 12 of 13 cases, particularly in regions associated with cone development without breaks in Bowman's layer or scarring. CONCLUSIONS: Compared with normal adult corneas, marked abnormalities were detected in the organization of the anterior corneal collagen lamellae of keratoconus corneas by second harmonic imaging. These structural abnormalities are consistent with the known changes in collagen organization and biomechanical strength of keratoconus.     

Distribution of specific collagen types and fibronectin in normal and keratoconus corneas

The distribution of five types of collagen and fibronectin in 6 normal and 9 keratoconus corneas was examined, using immunofluorescent staining and the enzyme-labeled antibody method. Types I, III and V collagens were detected in the corneal stroma. There was essentially no difference between normal and keratoconus corneas in their distribution. Type IV collagen and fibronectin were detected in the basement membrane of the normal corneal epithelium, while in the keratoconus corneas the disruption of the basement membrane as well as the excrescence of basement membrane materials was observed. The abnormal distribution of the type IV collagen and fibronectin was also observed in the anterior stromal area of keratoconus corneas.     

Changes in collagen orientation and distribution in keratoconus corneas

PURPOSE: To map the collagen orientation and relative distribution of collagen fibrillar mass in keratoconus corneal buttons. METHODS: Structural analysis was performed by obtaining synchrotron x-ray scattering patterns across the samples at 0.25-mm intervals. The patterns were analyzed to produce two-dimensional maps of the orientation of the lamellae and of the distribution of total and preferentially aligned lamellae. RESULTS: Compared with normal corneas, in keratoconus the gross organization of the stromal lamellae was dramatically changed, and the collagen fibrillar mass was unevenly distributed, particularly around the presumed apex of the cone. CONCLUSIONS: The development of keratoconus involves a high degree of inter- and probably intralamellar displacement and slippage that leads to thinning of the central cornea and associated changes in corneal curvature. This slippage may be promoted by a loss of cohesive forces and mechanical failure in regions where lamellae bifurcate.     

Interlamellar adhesive strength in human eyebank corneas

The interlamellar biomechanical properties of stromal collagen are relatively unknown, yet may be highly significant with respect to wound healing and the efficacy of certain keratorefractive surgical procedures. Interlamellar adhesive strength was measured as the tearing force required to separate corneal lamellae at a 50% stromal depth in 16 human eyebank corneas. The mean value for the central cornea was found to be 14.2 (+/- 0.5 SEM) g-wt/mm of tissue width. Histology showed a smooth separation between the lamellae along the tearing plane in the central cornea. We believe that the adhesive strength measured in the central cornea may be primarily the force needed to break interlamellar proteoglycan bonds between collagen lamellae, because no torn lamellae were found in this region. The mean adhesive strength and the SEM increased toward the periphery in a symmetrical fashion. The mean adhesive strength in the far periphery was 31.6 (+/- 3.7 SEM) g-wt/mm at 5 mm nasally, and 28.4 (+/- 3.2 SEM) g-wt/mm at 5 mm temporally, and was approximately twice the mean central value. The rising value of the mean adhesive strength with increasing distance from the central cornea was believed to be due to a more highly disorganized collagen network in which greater numbers of lamellae passed obliquely in depth through the tearing plane. These lamellae would contribute their tensile strength to the adhesive strength measurement along the tearing plane. Histology from the peripheral cornea confirmed the existence of depth-varying collagen lamellae and the torn ends of lamellae that passed across the tearing plane.     

Wound healing in rabbit corneas after photorefractive keratectomy and laser in situ keratomileusis

PURPOSE: To compare the wound-healing process in the rabbit cornea after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) with the same refractive correction. SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: Adult albino rabbits (N = 24) were used. One eye of each animal had PRK or LASIK with the same refractive correction. Each animal was killed after an interval of up to 6 months. The expression pattern of corneal stromal injury-related molecules with the 2 treatments were compared. Paraffin sections of the cornea were processed immunohistochemically for alpha-smooth muscle actin (alpha-SMA), collagen type IV [alpha1(IV)](2),alpha2(IV), and heat shock protein (HSP) 47 as well as other HSPs. Sections were also examined after hematoxylin and eosin or periodic acid-Schiff staining. RESULTS: Hematoxylin and eosin staining showed the central epithelium to be thick in PRK-treated corneas. The thick epithelium was restricted to the area around the corneal flap edge adhesion in LASIK-treated corneas at 3 months. Periodic acid-Schiff staining showed an absence of or interruption in the epithelial basement membrane in PRK-treated corneas for up to 6 months. Heat shock protein 47 was detected in keratocytes on day 3 but not after that in PRK-treated corneas. There was no difference in the expression of other HSPs. Alpha-smooth muscle actin was expressed in keratocytes repopulated in the central anterior cornea of PRK-treated corneas at 28 days. Keratocytes with immunoreactivity for these 2 proteins were not seen in LASIK-treated corneas. Collagen IV [alpha1(IV)](2),alpha2(IV) was not detected in either group of corneas. The central epithelium became transiently thicker in PRK-treated corneas. CONCLUSION: Keratocyte responses to laser stromal ablation were more marked in corneas treated with PRK than in those treated with LASIK.     

Rabbit cornea microstructure response to changes in intraocular pressure visualized by using nonlinear optical microscopy

PURPOSE: To characterize the microstructural response of the rabbit cornea to changes in intraocular pressure (IOP) by using nonlinear optical microscopy (NLOM). METHODS: Isolated rabbit corneas were mounted on an artificial anterior chamber in series with a manometer and were hydrostatically pressurized by a reservoir. The chamber was mounted on an upright microscope stage of a custom-built NLOM system for corneal imaging without using exogenous stains or dyes. Second harmonic generation in collagen was used to image through the full thickness of the central corneal stroma at IOPs between 5 and 20 mm Hg. Microstructural morphology changes as a function of IOP were used to characterize the depth-dependent response of the central cornea. RESULTS: Regional collagen lamellae architecture through the full thickness of the stroma was specifically imaged as a function of IOP. Hypotensive corneas showed gaps between lamellar structures that decreased in size with increasing IOP. These morphologic features appear to result from interwoven lamellae oriented along the anterior-posterior axis and parallel to the cornea surface. They appear throughout the full thickness and disappear with tension in the anterior but persist in the posterior central cornea, even at hypertensive IOP. CONCLUSIONS: NLOM reveals interwoven collagen lamellae sheets through the full thickness of the rabbit central cornea oriented along the anterior-posterior axis and parallel to the surface. The nondestructive nature of NLOM allows 3-dimensional imaging of stromal architecture as a function of IOP in situ. Collagen morphologic features were used as an indirect measure of depth-dependent mechanical response to changes in IOP.     

Ultrasound thermal damage to rabbit corneas after simulated phacoemulsification

PURPOSE: To determine rabbit cornea thermal tolerance and evaluate the effects of ultrasound (US) on this tissue after applying defined US heat doses. SETTING: Eye Clinic; Anatomy Histology and Forensic Medicine, University of Florence, Florence, Italy. METHODS: Hyperthermia was induced in rabbit corneas using US, simulating a phacoemulsification procedure. The US power was set at 100% in continuous mode, and temperature values were reached within 10 seconds of the onset of US treatment. Corneal surface temperatures were continuously monitored and recorded by thermographic registration. The eyes of 16 rabbits were examined: 4 controls, 8 treated at 40 degrees C for 10 seconds, 8 treated at 50 degrees C for 10 seconds, and 12 treated at 60 degrees C for 10 seconds. All 32 corneal buttons were removed and prepared for light microscopic evaluation with hematoxylin and eosin staining, trichromic staining, and zinc iodide-osmium tetroxide solution. The 12 corneas treated at 60 degrees C for 10 seconds were also processed for immunohistochemical analysis. RESULTS: Corneas at 40 degrees C for 10 seconds were grossly and histologically normal and were not different from control corneas. Corneas at 50 degrees C for 10 seconds showed initial stromal damage with collagen disorganization, mild stromal edema, and initial signs of keratocyte damage. Half of the corneas at 60 degrees C for 10 seconds were examined at time 0 and the other half after 1 week. At time 0, massive corneal damage with epithelial cell edema, collagen disorganization, severe stromal edema, intrastromal vacuole formation, plump keratocyte nuclei, and endothelial cell detachment were found, as was a severely impaired nerve plexus. At 1-week follow-up, corneas showed persistent stromal and endothelial cell edema with an increase activated keratocytes and mitotic features in the stroma and the epithelial layer. CONCLUSIONS: Rabbit corneas showed a considerable tolerance to US damage up to 50 degrees C. Higher thermal doses produced severe histological damage, even though corneas showed a considerable plasticity due to their regenerative capacity.     

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.     

Congenital corneal opacification in De Barsy syndrome

A newborn male was noted to have bilateral congenital corneal opacification. Findings from examination disclosed a variety of dysmorphic features, including cutis laxa, progeroid aspect, short stature, multiple hyperextensible subluxated joints, muscular hypotonia, and hyperreflexia. Bilateral penetrating keratoplasties were performed; histopathologic examination revealed diffuse epithelial thickening, loss of the Bowman layer, and stromal attenuation with anterior stromal scarring. Special stains showed no deposition of abnormal material in the corneas. Electron microscopy demonstrated absence of Bowman layer differentiation with a paucity of collagen fibers, as well as extensive small elastic fibers in the anterior stroma. The diagnosis of De Barsy syndrome was made, a rare progeroid syndrome associated with characteristic ocular, facial, skeletal, dermatologic, and neurologic abnormalities. De Barsy syndrome should be included in the differential diagnosis of congenital corneal opacification; its distinctive clinical features enable the clinician to easily differentiate it from other causes of congenitally cloudy corneas.     

Differential response of lens crystallins and corneal crystallins in degenerative corneas

Corneal degenerations, occurring either spontaneously or as a complication to other diseases, cause vision problems by endangering corneal transparency. Our past cornea research projects involving mice revealed that some recruited mice presented corneal problems similar to human corneal degeneration. The present study examines the histology of diseased mice corneas, including ultrastructure. Genome-wide microarray and proteomic methods were utilized to screen for molecular changes in the diseased corneas. It was found that abnormalities affected mainly anterior layers of the corneas. The most often observed histological abnormalities included neoplasm or detachment of the epithelial layer, erosion or breakage of Bowman membranes, blood vessel formation, and bleeding in the stroma. Microarray assay showed that among the 46 up-regulated probes in diseased corneas, 13 were for lens crystallins. However, all corneal crystallins genes remained unchanged. αA-crystallin was among the proteins that showed the greatest increase in diseased corneas, as detected by gel electrophoresis. We propose that lens crystallins, rather than corneal crystallins, are involved in the pathological process of corneal degeneration. Further study along these lines would provide insight into the mechanism of corneal transparency.     

Distribution of fibronectin in human and rabbit corneas

In order to study the possible role of fibronectin (FN) in corneal wound healing and the relationship between FN and sensory innervation, FN was demonstrated immunohistochemically in both normal and sensorily denervated rabbit corneas and in normal or tissue-cultured human corneas. The distribution of FN was the same in the groups examined: a thin subepithelial band of FN-like immunoreactivity was seen at the level of epithelial basement membrane and at the stromal side of Descemet's membrane. Epithelial abrasions were also performed in both normal and denervated rabbit corneas. The results were compared with those obtained from organ-cultured human corneas. Following abrasion of the corneal epithelium, FN was detected in the anterior margin of the denuded stroma 18 hr after the operation in the areas where the epithelium had not healed, but not 49 hr after. Sensory denervation did not affect the distribution of FN in normal, denervated or healing rabbit cornea. It is concluded that FN is probably not controlled by sensory innervation.     

Characterization of stroma from Fuchs' endothelial dystrophy corneas

Fuchs' endothelial dystrophy is commonly regarded as an endothelial cell disorder. In the present study we compared glycoconjugates of Fuchs' and normal corneas using FITC conjugated lectins [peanut agglutinin (PNA), castor bean agglutinin (RCA120), soybean agglutinin (SBA), and wheat germ agglutinin (WGA)]. Our results showed increased staining with RCA120 and PNA in the posterior region of the Fuchs' corneas, indicating an accumulation of terminal beta-galactose and B-D-galactose (1-3)-D-N-acetylgalactosamine residues. The stromal and epithelial regions of normal and Fuchs' corneas exhibited similar staining patterns with all lectins tested. Our collagen studies showed an increased extractability and abnormal amino acid analyses of collagen from Fuchs' corneas as compared with normals. The purified collagens did have similar banding patterns by sodium dodecyl sulfate gels. However, further characterization by 125(1) two-dimensional peptide mapping revealed that Fuchs' alpha 1-sized chains contained fingerprints that were distinctly different from normal cornea stromal collagen. These data suggest that in addition to abnormal accumulation of RCA120- and PNA-specific glycoconjugates in the posterior cornea, Fuchs' corneas contained stromal collagens with altered biochemical properties. We postulate that the characteristic deterioration of endothelial function in Fuchs' dystrophy may compromise the microenvironment of the stroma and its keratocytes, and thereby lead to an altered collagenous extracellular matrix.     

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.     

Depth-dependent cohesive tensile strength in human donor corneas: implications for refractive surgery

PURPOSE: To determine the cohesive tensile strength throughout the stroma of normal human donor corneas and evaluate the relevance of these findings within the context of current excimer laser surgical techniques. METHODS: Twenty normal corneoscleral buttons from 11 donors were obtained from the Georgia Eye Bank. The corneas were cut into 3-mm strips, dissected at varying stromal depths, mechanically separated through the dissection plane using a motorized extensometer, and measured for cohesive tensile strength. Central corneal thickness and dissection depth were measured by routine light microscopy and correlated with cohesive tensile strength measurements. RESULTS: A strong negative correlation was noted between stromal depth and cohesive tensile strength (r = -0.93). The anterior corneal stroma directly adjacent to Bowman's layer followed by the underlying anterior 40% of the corneal stroma had the highest cohesive tensile strength. Cohesive tensile strength plateaued from 40% to 90% corneal stromal depth and then declined rapidly from the posterior 10% of the stroma to Descemet's membrane. The anterior 40% of the corneal stroma had significantly higher cohesive tensile strength than the posterior 60% (33.3 g/mm vs 19.6 g/mm, P < .00001). Within the central 40% to 60% depth, a positive correlation was found between increased age and increased tensile strength (r = 0.67), with corneal tensile strength increasing 38% from ages 20 to 78 years. CONCLUSIONS: The anterior 40% of the central corneal stroma is the strongest region of the cornea, whereas the posterior 60% of the stroma is at least 50% weaker. The risk for ectasia may therefore be greater with ablations into the posterior stroma. Increasing age is associated with increased corneal cohesive tensile strength.     

Matrix metalloproteinase 2 activation in cultured corneas

PURPOSE: Corneas that are maintained in tissue culture medium shed their epithelial cells and repopulation following graft surgery is an essential facet of the healing process. Failure to do so may be a result of structural damage to the epithelial basement membrane of a donor cornea. The purpose of the present investigation was to ascertain whether MMP-2, the matrix metalloproteinase produced by corneal keratocytes, may be activated during storage and hence cleave the type IV collagen component of the epithelial cell basement membrane. METHODS: Fresh and transplant rejected corneas that had been stored in culture medium for varying time periods and of known donor age were collected. The soluble protein fractions of these corneas were obtained. Their MMP-2 proteins were visualised by zymography on SDS gelatin polyacrylamide gels and assayed for activity against nitrophenyl acetate and denatured [(3)H]type I collagen. RESULTS: The stromal tissue of fresh, normal corneas produced inactive MMP-2 of M(r) 66,000. Although the cultured corneas did not up-regulate MMP-2 production, they contained additional MMP-2 activities of M(r) 62,000 and M(r) 43,000. The appearance of these additional MMP-2 activities correlated with corneal culture time but not donor age. The ability to cleave denatured [(3)H]type I collagen correlated with the appearance of the M(r) 43,000 activity but not the M(r) 62,000 activity. CONCLUSION: Activated MMP-2 is produced in cultured corneas. For this reason the corneas donated for all graft procedures should not be held in culture medium for periods exceeding 4 weeks.     

Quantitative analysis of collagen fiber in keratoconus

In order to identify the direct pathogenic factors involved in the stromal thinning of keratoconus, quantitative analysis of keratocytes, collagen fibers and collagen lamellae in keratoconus cornea was performed histologically by light and electron microscopy. Both normal and keratoconus corneas showed a similar cell density of keratocytes in the central stroma, therefore the total number of keratocytes in keratoconus cornea might be smaller than that of controls, because of the thinning of stroma in the keratoconus. The collagen lamellae in keratoconus corneas showed a significant decrease in number compared with controls. There was a direct relationship between the stromal thickness and number of collagen lamellae. On the other hand, there was no statistical significance between normal and keratoconus corneas in terms of the thickness of collagen lamellae. These results suggest that the thinning of the cornea in keratoconus might occur as the result of a defect of some collagen lamellae due to a disorganization during the process of collagen lamellae formation.     

Histology of rabbit corneas after 10-diopter photorefractive keratectomy for hyperopia.

PURPOSE: To measure histological changes in the optical and transition zones of rabbit corneas treated with excimer laser photorefractive keratectomy (PRK) to correct hyperopia and to estimate the optical effect of these changes on the induced power profile. METHODS: Corrections of +10.00 D were ablated by means of a Questek 2620 excimer laser and rotational masks on 8 rabbit eyes. On frozen sections of 4 stained corneas and historesine sections of 4 other corneas, stromal, new stromal, epithelial, and total corneal thickness were measured on four significant locations. The optical effect of each parameter on the axial power profile was estimated in a computer simulation. RESULTS: Mean stromal regrowth was 50% of the ablated tissue. Deposition was lenticular and could account for -5.00 D of regression. Stromal thickening without regrowth occurred in the optical zone and over the shoulder, causing augmentation instead of regression. The epithelium thickened 20% in the mid-transition zone and was thinner over the shoulder, accounting for 1.00 D of regression and an increase in asphericity in the optical zone. CONCLUSION: In these hyperopic rabbit PRK corneas, stromal regrowth and epithelial hyperplasia were lenticular and contributed to regression of the optical effect. Stromal swelling around interrupted collagen lamellae seemed to occur, augmenting the optical effect. The epithelium filters high spatial frequency stromal irregularities.     

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.