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Purpose: The aim of this study was to determine the roles of collagen XII in the regulation of stromal hierarchical organization, keratocyte organization, and corneal mechanics., Methods: The temporal and spatial expression of collagen XII at postnatal days 4, 10, 30, 90, and 150 were evaluated in wild-type (WT) mice. The role of collagen XII in hierarchical organization was analyzed by measuring fibril diameter and density, as well as stromal lamellar structure, within ultrastructural micrographs obtained from WT and collagen XII-deficient mice (Col12a1-/-). Keratocyte morphology and networks were assessed using actin staining with phalloidin and in vivo confocal microscopy. The effects of collagen XII on corneal biomechanics were evaluated with atomic force microscopy., Results: Collagen XII was localized homogeneously in the stroma from postnatal day 4 to day 150, and protein accumulation was shown to increase during this period using semiquantitative immunoblots. Higher fibril density (P < 0.001) and disruption of lamellar organization were found in the collagen XII null mice stroma when compared to WT mice. Keratocyte networks and organization were altered in the absence of collagen XII, as demonstrated using fluorescent microscopy after phalloidin staining and in vivo confocal microscopy. Corneal stiffness was increased in the absence of collagen XII. Young's modulus was 16.2 ± 5.6 kPa in WT and 32.8 ± 6.4 kPa in Col12a1-/- corneas. The difference between these two groups was significant (P < 0.001, t-test)., Conclusions: Collagen XII plays a major role in establishing and maintaining stromal structure and function. In the absence of collagen XII, the corneal stroma showed significant abnormalities, including decreased interfibrillar space, disrupted lamellar organization, abnormal keratocyte organization, and increased corneal stiffness.

Purpose: To investigate the depth-dependent mechanical anisotropy of the human corneal stroma at the tissue (stroma) and molecular (collagen) level by using atomic force microscopy (AFM)., Methods: Eleven human donor corneas were dissected at different stromal depths by using a microkeratome. Mechanical measurements were performed in 15% dextran on the surface of the exposed stroma of each sample by using a custom-built AFM in force spectroscopy mode using both microspherical (38-μm diameter) and nanoconical (10-nm radius of curvature) indenters at 2-μm/s and 15-μm/s indentation rates. Young's modulus was determined by fitting force curve data using the Hertz and Hertz-Sneddon models for a spherical and a conical indenter, respectively. The depth-dependent anisotropy of stromal elasticity was correlated with images of the corneal stroma acquired by two-photon microscopy., Results: The force curves were obtained at stromal depths ranging from 59 to 218 μm. At the tissue level, Young's modulus (ES) showed a steep decrease at approximately 140-μm stromal depth (from 0.8 MPa to 0.3 MPa; P = 0.03) and then was stable in the posterior stroma. At the molecular level, Young's modulus (EC) was significantly greater than at the tissue level; EC decreased nonlinearly with increasing stromal depth from 3.9 to 2.6 MPa (P = 0.04). The variation of microstructure through the thickness correlated highly with a nonconstant profile of the mechanical properties in the stroma., Conclusions: The corneal stroma exhibits unique anisotropic elastic behavior at the tissue and molecular levels. This knowledge may benefit modeling of corneal behavior and help in the development of biomimetic materials.

Purpose: To characterize the depth-dependent shear modulus of the central and peripheral human cornea along the superior-inferior and nasal-temporal directions with a high spatial resolution., Methods: Cylindrical explants from the central and peripheral corneas of 10 human donors were subjected to a 5% shear strain along the superior-inferior and nasal-temporal directions using a microscope-mounted mechanical testing device. Depth-dependent shear strain and shear modulus were computed through force measurements and displacement tracking., Results: The shear modulus G of the human cornea varied continuously with depth, with a maximum occurring roughly 25% of the way from the anterior surface to the posterior surface. G also varied with direction in the superior region and (at some depths) was significantly higher for superior-inferior shear loading. In the anterior half of the cornea, the shear modulus along the nasal-temporal direction (GNT) did not vary with location; however, the superior region had significantly higher GNT in posterior cornea. In contrast, the shear modulus along the superior-inferior direction (GSI) was independent of location at all depths., Conclusions: This study demonstrates that the peak shear modulus of the human cornea occurs at a substantial distance within the corneal stroma. Depth-dependent differences between central and peripheral cornea possibly reflect the location-dependent mechanical environment of the cornea. Moreover, the cornea is not a transverse isotropic material, and must be characterized by more than a single shear modulus due to its dependence on loading direction. The material properties measured in this study are critical for developing accurate mechanical models to predict the vision-threatening morphological changes that can occur in the cornea., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: To determine the efficacy of intratissue refractive index shaping (IRIS) using 400-nm femtosecond laser pulses (blue light) for writing refractive structures directly into live cat corneas in vivo, and to assess the longevity of these structures in the eyes of living cats., Methods: Four eyes from two adult cats underwent Blue-IRIS. Light at 400 nm with 100-femtosecond (fs) pulses were tightly focused into the corneal stroma of each eye at an 80-MHz repetition rate. These pulses locally increased the refractive index of the corneal stroma via an endogenous, two-photon absorption process and were used to inscribe three-layered, gradient index patterns into the cat corneas. The optical effects of the patterns were then tracked using optical coherence tomography (OCT) and Shack-Hartmann wavefront sensing., Results: Blue-IRIS patterns locally changed ocular cylinder by -1.4 ± 0.3 diopters (D), defocus by -2.0 ± 0.5 D, and higher-order root mean square (HORMS) by 0.31 ± 0.04 μm at 1 month post-IRIS, without significant changes in corneal thickness or curvature. Refractive changes were maintained for the duration they were tracked, 12 months post-IRIS in one eye, and just more than 3 months in the remaining three eyes., Conclusions: Blue-IRIS can be used to inscribe refractive structures into live cat cornea in vivo that are stable for at least 12 months, and are not associated with significant alterations in corneal thicknesses or radii of curvature. This result is a critical step toward establishing Blue-IRIS as a promising technique for noninvasive vision correction., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: We evaluated the efficacy and safety of photochemical corneal stiffening by palladium bacteriochlorin 13'-(2-sulfoethyl)amide dipotassium salt (WST11) and near infrared (NIR) illumination, using ex vivo and in vivo rabbit eye models., Methods: Corneas of post mortem rabbits and living rabbits were pretreated topically with 2.5 mg/mL WST11 in saline or in 20% dextran T-500 (WST-D), washed and illuminated with an NIR diode laser (755 nm, 10 mW/cm(2). Studies with corneas of untreated fellow eyes served as controls. Tensile strength measurements, histopathology, electron spin resonance, and optical spectroscopy and fluorescence microscopy were used to assess treatment effects. Comparative studies were performed with standard riboflavin/ultraviolet-A light (UVA) treatment., Results: WST11/NIR treatment significantly increased corneal stiffness following ex vivo or in vivo treatment, compared to untreated contralateral eyes. The incremental ultimate stress and Young's modulus of treated corneas increased by 45, 113, 115%, and 10, 79, and 174% following 10, 20, and 30 minutes of incubation with WST11, respectively. WST-D/NIR had a similar stiffening effect, but markedly reduced post-treatment edema and shorter time of epithelial healing. WST11/NIR and WST-D/NIR generate hydroxyl and superoxide radicals, but no singlet oxygen in the cornea. Histology demonstrated a reduction in the keratocyte population in the anterior half of the corneal stroma, without damage to the endothelium., Conclusions: Treatment of rabbit corneas, with either WST11/NIR or WST-D/NIR, increases their biomechanical strength through a mechanism that does not involve singlet oxygen. The WST-D/NIR treatment showed less adverse effects, demonstrating a new potential for clinical use in keratoconus and corneal ectasia after refractive surgery.

Purpose: To compare different methods of glycerol-preserved corneas intended for deep anterior lamellar keratoplasty (DALK)., Methods: We analyzed transparency, transmittance, thickness, biomechanics, morphology, and antigenicity of donor corneas preserved by four different glycerol-based methods (n = 6 per group) for 3 months, as follows: tissues in anhydrous glycerol without aluminosilicate molecular sieves at room temperature (GRT); tissues in anhydrous glycerol with aluminosilicate molecular sieves at room temperature (SRT); tissues in anhydrous glycerol without aluminosilicate molecular sieves at -78°C (G78); and tissues in anhydrous glycerol without aluminosilicate molecular sieves at -20°C (G20)., Results: Slit lamp images and transmittance curves obtained by spectrophotometer show that the G78 cornea was the most transparent tissue. Stress-strain behavior indicated that corneas in the G78 group were the most pliable, and SRT corneas were the stiffest. Electron microscopy analysis indicated that corneal cytoarchitecture and keratocyte integrity was destroyed in all glycerol-preserved corneas. Disorganized stromal collagen fibers were evident in groups stored at RT. Especially in SRT corneas, parallelism was lost, fibrils were extremely tortuous and discontinuous, and widespread fibril degeneration could be found. Antigenicity of tissue, assessed via immunohistochemistry for CD45-positive cells, HLA-ABC and HLA-DR, was lowered after glycerol preservation relative to fresh cornea tissues, and immunoreactivity was located mainly on corneal epithelium and limbus rather than stroma., Conclusions: Anhydrous glycerol preservation without molecular sieves in a -78°C freezer was the best method to obtain DALK-eligible tissues that were both transparent and pliable.

Purpose: To investigate the biomechanics of the anterior human corneal stroma using atomic force microscopy (AFM)., Methods: AFM measurements were performed in liquid on the anterior stroma of human corneas, after gently removing the epithelium, using an atomic force microscope in the force spectroscopy mode. Rectangular silicon cantilevers with tip radius of 10 nm and spring elastic constants of 25- and 33-N/m were used. Each specimen was subjected to increasing loads up to a maximum of 2.7 μN with scan speeds ranging between 3- and 95-μm/s. The anterior stromal hysteresis during the extension-retraction cycle was quantified as a function of the application load and scan rate. The elastic modulus of the anterior stroma was determined by fitting force curve data to the Sneddon model., Results: The anterior stroma exhibited significant viscoelasticity at micrometric level: asymmetry in the curve loading-unloading response with considerable hysteresis dependent both on the application load and scan rate (P < 0.01). The mean elastic modulus ranged between 1.14 and 2.63 MPa and was constant over the range of indentation depths between 1.0 and 2.7 μm in the stroma., Conclusions: At microscale level, the mechanical response of the most anterior stroma is complex and nonlinear. The microstructure (fibers' packing, number of cross-links, water content) and the combination of elastic (collagen fibers) and viscous (matrix) components of the tissue influence the type of viscoelastic response. Efforts in modeling the biomechanics of human corneal tissue at micrometric level are needed.

Purpose: To measure the transverse shear modulus of the human corneal stroma and its profile through the depth by mechanical testing, and to assess the validity of the hypothesis that the shear modulus will be greater in the anterior third due to increased interweaving of lamellae., Methods: Torsional rheometry was used to measure the transverse shear properties of 6 mm diameter buttons of matched human cadaver cornea pairs. One cornea from each pair was cut into thirds through the thickness with a femtosecond laser and each stromal third was tested individually. The remaining intact corneas were tested to measure full stroma shear modulus. The shear modulus from a 1% shear strain oscillatory test was measured at various levels of axial compression for all samples., Results: After controlling for axial compression, the transverse shear moduli of isolated anterior layers were significantly higher than central and posterior layers. Mean modulus values at 0% axial strain were 7.71 ± 6.34 kPa in the anterior, 1.99 ± 0.45 kPa in the center, 1.31 ± 1.01 kPa in the posterior, and 9.48 ± 2.92 kPa for full thickness samples. A mean equilibrium compressive modulus of 38.7 ± 8.6 kPa at 0% axial strain was calculated from axial compression measured during the shear tests., Conclusions: Transverse shear moduli are two to three orders of magnitude lower than tensile moduli reported in the literature. The profile of shear moduli through the depth displayed a significant increase from posterior to anterior. This gradient supports the hypothesis and corresponds to the gradient of interwoven lamellae seen in imaging of stromal cross-sections.

Purpose: The mechanical properties of corneal tissue are linked to prevalent ocular diseases and therapeutic procedures. Brillouin microscopy is a novel optical technology that enables three-dimensional mechanical imaging. In this study, the feasibility of this noncontact technique was tested for in situ quantitative assessment of the biomechanical properties of the cornea., Methods: Brillouin light-scattering involves a spectral shift proportional to the longitudinal modulus of elasticity of the tissue. A 532-nm single-frequency laser and a custom-developed ultrahigh-resolution spectrometer were used to measure the Brillouin frequency. Confocal scanning was used to perform Brillouin elasticity imaging of the corneas of whole bovine eyes. The longitudinal modulus of the bovine corneas was compared before and after riboflavin corneal collagen photo-cross-linking. The Brillouin measurements were then compared with conventional stress-strain mechanical test results., Results: High-resolution Brillouin images of the cornea were obtained, revealing a striking depth-dependent variation of the elastic modulus across the cornea. Along the central axis, the Brillouin frequency shift varied gradually from 8.2 GHz in the epithelium to 7.5 GHz near the endothelium. The coefficients of the down slope were measured to be approximately 1.09, 0.32, and 2.94 GHz/mm in the anterior, posterior, and innermost stroma, respectively. On riboflavin collagen cross-linking, marked changes in the axial Brillouin profiles (P < 0.001) were noted before and after cross-linking., Conclusions: Brillouin imaging can assess the biomechanical properties of cornea in situ with high spatial resolution. This novel technique has the potential for use in clinical diagnostics and treatment monitoring.

Purpose: A novel nanoindentation technique was used to biomechanically characterize each of three main layers of the cornea by using Hertzian viscoelastic formulation of creep, the deformation resulting from sustained-force application., Methods: The nanoindentation method known as mechanical interferometry imaging (MII) with <1-nm displacement precision was used to observe indentation of bovine corneal epithelium, endothelium, and stroma by a spherical ferrous probe in a calibrated magnetic field. For each specimen, creep testing was performed using two different forces for 200 seconds. Measurements for single force were used to build a quantitative Hertzian model that was then used to predict creep behavior for another imposed force., Results: For all three layers, displacement measurements were highly repeatable and were well predicted by Hertzian models. Although short- and long-term stiffnesses of the endothelium were highest of the three layers at 339.2 and 20.2 kPa, respectively, both stromal stiffnesses were lowest at 100.4 and 3.6 kPa, respectively. Stiffnesses for the epithelium were intermediate at 264.6 and 12.2 kPa, respectively., Conclusions: Precise, repeatable measurements of corneal creep behavior can be conveniently obtained using MII at mechanical scale as small as one cell thickness. When interpreted in analytical context of Hertzian viscoelasticity, MII technique proved to be a powerful tool for biomechanical characterization of time-dependent biomechanics of corneal regions.

Purpose: Decellularization of animal corneas is a promising method for the development of artificial human corneas by tissue engineering. In this study, two different decellularization protocols were evaluated to determine which one is able to best preserve the histologic structure, composition, and optical behavior of decellularized porcine corneas. Then, these corneas were recellularized with human keratocytes to obtain a partial human corneal substitute., Methods: Two different decellularization protocols were applied, using NaCl and SDS, to determine which one is able to best preserve the histologic structure, composition, and optical behavior of the decellularized corneas. Then, those decellularized corneas that showed the most appropriate results were recellularized with human keratocytes and evaluated at the histologic, biochemical, and optical levels for use in regenerative medicine., Results: The results showed that 1.5 M NaCl treatment of porcine corneas is able to generate an acellular corneal stroma with adequate histologic and optical properties and that human keratocytes are able to penetrate and spread within this scaffold with proper levels of cell differentiation. In contrast, 0.1% SDS treatment of porcine corneas resulted in high levels of fibril disorganization and poor optical behavior of these corneas., Conclusions: In conclusion, the authors suggest that the decellularization of animal corneas with 1.5 M NaCl represents a useful method for the development of human bioengineered corneas with therapeutic potential.

Purpose: To determine whether changes in the expression of type IV alpha1, alpha2, or alpha3 collagen isoforms are stringently associated with corneal stromal cell activation., Methods: Keratocytes isolated from rabbit corneal stroma by collagenase digestion were plated in serum-free or insulin-, bFGF/heparin sulfate (HS)-, TGF-beta1-, or fetal bovine serum (FBS)-supplemented DMEM/F12 medium. Expression of type IV collagen isoforms and keratan sulfate proteoglycans (KSPGs) was evaluated by immunocytochemical analysis, Western blot analysis, or both. Concentrations of mRNAs were estimated by quantitative RT-PCR using SYBR Green RT-PCR reagents., Results: Immunohistochemical analysis indicated that type IV alpha1, alpha2, and alpha3 collagens were expressed in normal rabbit corneal stroma and in keratocytes cultured in serum-free and insulin-supplemented media. However, alpha3(IV) collagen was not detectable in the regenerating stroma after photorefractive keratectomy (PRK) in rabbit or in corneal stromal cells cultured in media supplemented with FBS, bFGF/HS, or TGF-beta1. alpha3(IV) collagen mRNA levels were also diminished in the stromal cells cultured in these growth factor-supplemented media. KSPGs (lumican and keratocan) were expressed and secreted in serum-free medium. Although the expression of KSPGs was promoted by insulin, the expression and intracellular levels of lumican and keratocan mRNAs were downregulated by TGF-beta1 and FBS. bFGF/HS promoted the downregulation of intracellular keratocan but not lumican mRNA levels., Conclusions: The loss in the expression of alpha3(IV) collagen is a stringent phenotypic change associated with activation of keratocytes in vivo and in vitro. This phenotypic change in activated corneal stromal cells is induced by bFGF/HS and by TGF-beta1, and it accompanies the downregulation of keratocan expression.

Purpose: To elucidate biological processes underlying the keratocyte, fibroblast, and myofibroblast phenotypes of corneal stromal cells, the gene expression patterns of these primary cultures from mouse cornea were compared with those of the adult and 10-day postnatal mouse cornea., Methods: Murine Genome&#95;U74Av2 arrays (Affymetrix Inc., Santa Clara, CA) were used to elucidate gene expression patterns of adult and postnatal day-10 corneal stroma, keratocytes, fibroblasts, and myofibroblasts., Results: Mobilization of stromal cells by culturing led to a wound-healing cascade in which specific extracellular matrix and cornea-transparency-related genes were turned off, and a repertoire of macrophage genes were switched on. Thus, novel transparency-related crystallins detected in the corneal gene expression patterns were downregulated in culture, whereas macrophage genes, mannose receptor type-1, Cd68, serum amyloid-A3, chemokine ligands (Ccl2, Ccl7, Ccl9), lipocalin-2, and matrix metalloproteinase-3 and -12 of innate immunity were induced in primary keratocyte cultures. Fibroblasts expressed the growth-related genes lymphocyte antigen 6 complex locus-A and preprokephalin-1, and myofibroblasts expressed annexin-A8, WNT1-inducible signaling pathway protein-1, arginosuccinate synthetase-1, and procollagen XI of late-stage wound healing., Conclusions: The emergent biological process suggests a dual role for resident stromal keratocytes in the avascular cornea: one of cornea maintenance, which involves synthesis of proteins related to the extracellular matrix and corneal transparency, and a second of barrier protection macrophage functions, which are switched on during corneal infection and injury.

Purpose: To measure the refractive index (RI) of the mammalian corneal stroma in relation to hydration in vitro and human corneal stroma before and after LASIK., Methods: RI at the anterior stromal surface of bovine corneal buttons was measured after the epithelium was scraped away. Samples were weighed and oven dried to calculate hydration. RI of the stromal bed surface was measured with a modified hand-held Abbé refractometer immediately before and after excimer laser photoablation. Thirty-one untreated persons (group 1: 44 corneas; age range, 19-65 years) and eight re-treated patients (group 2: 10 corneas) were examined., Results: RI of bovine stromal surface was significantly associated with hydration (H) (RI = 1.4067 - 0.00599H, r = -0.9079, P < 0.001). Photoablation significantly increased the RI of the midstroma (group 1: 1.3721 +/- 0.0041 to 1.3839 +/- 0.0050; group 2: 1.3717 +/- 0.0038 to 1.3819 +/- 0.0039). Differences between groups were not significant. In group 1 (n = 31), change in RI (DeltaRI) was significantly related to preoperative RI (DeltaRI = 1.155 - 0.833RI, r = 0.595, P < 0.001) and RI was significantly related to age (x) (RI = 1.3634 + 0.00026x, r = 0.603, P < 0.001)., Conclusions: Mammalian corneal stromal RI correlates with hydration. LASIK significantly increases the refractive index of the treated stromal bed, and this equates to an average change in hydration from 4.3 to 2.9. For individual cases, change in RI is associated with the pre-op RI. The lack of any difference between untreated and re-treated corneas suggests that with time hydration returns back to normal levels. The RI in the older corneal stroma is slightly higher relative to the RI in the younger corneal stroma.

Purpose: The adult rabbit limbal basal epithelium contains corneal epithelial stem cells, which have been characterized by a negative expression of keratin-3 (K3) and a lower expression of connexin 43 (Cx43). This study was conducted to determine whether the limbal stroma dictates the plasticity of limbal and corneal epithelial differentiation., Methods: Viable epithelial sheets of the central cornea and the pigmented limbus were isolated from Dutch belted rabbits by incubation of 50 mg/mL of dispase II in supplemental hormonal epithelial medium (SHEM) for 18 hours at 4 degrees C. The cleavage plane was studied by immunostaining with antibodies against K3, Cx43, integrin beta1, and collagen IV. Viability of single cells derived from these sheets was assessed by a live-dead assay. Such limbal (L) and corneal (K) epithelial sheets were recombined with either limbal (Ls) or corneal (Ks) stroma, and cultured in SHEM for 10 days before lifting to the air-fluid interface for 1 week. The resultant epithelial phenotype was determined by histology and immunostaining to K3 and Cx43, and apoptosis was investigated by Hoechst and TUNEL nuclear staining., Results: Viability of isolated limbal and corneal epithelial sheets was determined to be 91.1% +/- 2.9%. The basal epithelium of isolated limbal epithelial sheets was positive for integrin beta1, negative for K3, but weakly positive for Cx43, and still retained patches of collagen IV. All recombinants showed stratified epithelia, with intraepithelial cysts with desquamated debris noted only in K/Ks, and epithelial outgrowth onto the insert filter from L/Ls. As expected, expression of K3 was negative in the basal layer of L/Ls, but positive in that of K/Ks. Expression of K3 was sporadically positive in the basal layer of L/Ks but largely negative in that of K/Ls. Expression of Cx43 was uniformly expressed in the basal layer of the K/Ks, but weak in that of L/Ls, K/Ls, and L/Ks. A higher apoptosis index was only noted in intraepithelial cysts of K/Ks., Conclusions: These results strongly indicate that the limbal stroma modulates epithelial differentiation, proliferation and apoptosis in the direction favoring stemness, whereas the corneal stroma promotes differentiation. Further investigation into elements constituting such a niche should help unveil the secrecy whereby stemness is controlled.

Purpose: To investigate the migratory and contractile behavior of isolated human corneal fibroblasts in fibrillar collagen matrices., Methods: A telomerase-infected, extended-lifespan human corneal fibroblast cell line (HTK) was transfected by using a vector for enhanced green fluorescent protein (GFP)-alpha-actinin. Cells were plated at low density on top of or within 100-microm-thick fibrillar collagen lattices. After 18 hours to 7 days, time-lapse imaging was performed. At each 1- to 3-minute interval, GFP and Nomarski differential interference contrast (DIC) images were acquired in rapid succession. Serum-containing (S+) medium was used initially for perfusion. After 2 hours, perfusion was switched to either serum-free (S-) or S+ medium containing the Rho-kinase inhibitor Y-27632 for 1 to 2 hours. Finally, perfusion was changed back to S+ medium for 1 hour., Results: Two to 4 days after plating, many cells underwent spontaneous contraction and/or relaxation in S+ medium. A decrease in the distance between consecutive alpha-actinin-dense bodies along stress fibers was measured during contraction, and focal adhesion and matrix displacements correlated significantly. Removal of serum or inhibition of Rho-kinase induced cell body elongation and relaxation of matrix stress, as confirmed using finite element modeling. Rapid formation and extension of pseudopodia and filopodia were also observed, and transient tractional forces were generated by these extending processes., Conclusions: Cultured human corneal fibroblasts can undergo rapid changes in the subcellular pattern of force generation that are mediated, in part, by Rho-kinase. Sarcomeric shortening of stress fibers in contracting corneal fibroblasts is also demonstrated for the first time.

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.

Purpose: Within the corneal stroma, keratocytes communicate through gap junctions. These plasma membrane channels, which connect the cytoplasm of adjacent cells, are composed of connexins. In a cell culture model, an investigation was conducted to determine whether connexin-based gap junction intercellular communication is present in fibroblasts and myofibroblasts, both of which replace keratocytes after wounding., Methods: Fibroblasts and myofibroblasts were grown according to preestablished methods. Phenotype was determined by immunocytochemistry. A gap junction-permeant dye, Lucifer yellow or Cascade blue, and nonpermeant 10-kDa Texas red-dextran were used. Tracer fluorescent dyes were introduced by scrape-loading or by microinjection, and their diffusion into adjacent cells was recorded photographically. Inhibition of gap junction dye transfer was elicited by treatment with 18-alpha-glycyrrhetinic acid (AGA)., Results: In confluent fibroblast or myofibroblast cultures, the scrape-loaded dextran probe remained within wounded cells, whereas the Lucifer yellow or Cascade blue dye diffused into adjacent intact cells. Similarly, in nonconfluent fibroblast and myofibroblast cultures, microinjected Lucifer yellow rapidly diffused from the microinjected cell to adjacent cells. Treatment with 2 microM AGA, an uncoupling agent, blocked the spread of Lucifer yellow in fibroblast and myofibroblast cultures., Conclusions: Cultured fibroblasts and myofibroblasts have functional gap junctions as has previously been demonstrated for keratocytes in vivo. Thus, fibroblasts and myofibroblasts have the ability to establish and maintain intercellular communication with themselves and with nonactivated keratocytes. This property may be critical in the wound-healing process, especially in the avascular corneal environment.

Purpose: Transparency and biomechanical properties of the cornea depend on the structure and organization of collagen fibrils. The authors determined diameter, axial period, and lateral molecular spacing of collagen fibrils in human corneal stroma as a function of age., Methods: Seventeen normal human corneas were investigated in their native state by means of small-angle and wide-angle x-ray scattering., Results: The mean radius of collagen fibrils, the axial period of collagen fibrils, and the lateral intermolecular Bragg spacing were found to be age dependent. The authors determined fibril radii of 16.1 +/- 0.5 nm in persons older than 65 years of age (n = 10) and 15.4 +/- 0.5 nm (mean +/- SD) in persons younger than 65 years (n = 7) (P < 0.022). The related age-dependent values were 66.4 +/- 0.7 nm (> 65 years) and 65.2 +/- 0.8 nm (< 65 years) for the axial period (P < 0.006) and 1.515 +/- 0.010 nm (> 65 years) and 1.499 +/- 0.013 nm (< 65 years) for the intermolecular Bragg spacing (P < 0.022)., Conclusions: Aging is related to a three-dimensional growth of collagen fibrils in the human corneal stroma. The age-related growth of the fibril diameter was mostly a result of an increased number of collagen molecules and, in addition, to some expansion of the intermolecular Bragg spacing probably resulting from glycation-induced cross-linking. The observed expansion of the fibrils in an axial direction may result from reduction of the molecular tilting angle within collagen fibrils. The observed alterations of the collagen framework may have implications for refractive surgery and ocular tonometry achieved through related changes in the biomechanical properties of the cornea.

Purpose: Development of postoperative corneal haze and regression of refractive effect are unfavorable clinical complications of excimer laser photorefractive keratectomy (PRK). Although exact mechanisms remain to be elucidated, these outcomes have been attributed to post-PRK corneal wound healing. The purpose of this study was to evaluate corneal wound repair quantitatively after PRK in a rabbit model using a newly developed in vivo technique, termed confocal microscopy through focusing (CMTF)., Methods: Twelve rabbit corneas received a monocular, 6-mm diameter, 9.0-diopter PRK myopic correction. Animals were evaluated sequentially up to 6 months after surgery by in vivo CMTF, which uses an image-intensity depth profile to measure epithelial and stromal thickness and uses corneal light reflectivity as an objective estimate of corneal haze. At differing temporal intervals, in vivo morphology was correlated with ex vivo histology using fluorescence microscopy., Results: One week after PRK, an acellular layer of 86 +/- 24 microns was found anteriorly in the remaining stroma, which demonstrated surgically induced keratocyte death. Underlying keratocytes became activated and migrated toward the wound bed; repopulation was completed within 3 weeks. One week after PRK, there was a significant increase (P < 0.001) in light reflections detected from the photoablated stromal surface (1745 +/- 262 U) and from the underlying activated fibroblasts (713 +/- 607 U). Corneal reflectivity peaked at 3 weeks (4648 +/- 1263 U) and decreased linearly to 889 +/- 700 U by 6 months after the PRK; this corresponded to a reflectivity six times greater than the level seen in unoperated corneas. Two weeks after PRK, initial corneal edema had resolved, revealing an actual ablation depth (maximal stromal thinning) of 118 +/- 8 microns. Starting at 2 weeks after surgery, the stroma underwent gradual rethickening that reached 98% of the preoperative thickness at 6 months after PRK; at that time, only 6% of the initial photoablation depth persisted. By contrast, the central corneal epithelium showed no significant postoperative hyperplasia., Conclusions: Rabbit corneas treated by PRK showed a remarkable stromal wound-healing response that ultimately led to the restoration of the original stromal thickness by 6 months after surgery, demonstrating complete regression of the initial photoablative effect. Additionally, corneal wound healing was associated with increased light reflections from both the photoablated stromal surface and the activated wound-healing keratocytes underlying this area. Based on these findings, the authors hypothesize that the development of clinically observed corneal haze in PRK patients may be related, in part, to activation of corneal keratocytes and to putative changes in the extracellular matrix.

Purpose: To monitor the health of the epithelium and the anterior stroma when porous membranes are implanted into the feline cornea and to determine membrane diffusivity characteristics needed to maintain corneal integrity., Methods: Filtration membranes in a range of effective pore sizes of less than 15 nm (groups 1 and 2, n = 11), 25 nm (group 3, n = 8), 50 nm (group 4, n = 16), and 100 nm (group 5, n = 15) were implanted into an interlamellar corneal pocket of the stroma. The implanted membranes ranged in thickness from 6 nm to 15 nm and were between 8 mm and 12 mm in diameter. Animals were monitored for clinical signs of intolerance to the implants., Results: At 1 month, thinning and ulceration had occurred in the epithelium and the anterior stroma of all animals in groups 1 and 2; epithelial changes, anterior stromal thinning, and ulceration had developed in 75% of animals of group 3; 50% of animals showed vascularization and only 7% showed epithelial degeneration in group 4; and local anterior stromal thinning was observed in 7% of animals in group 5, indicating clinical acceptance of the implanted membrane. In the long term (greater than 50 days), 30% and 73% of the group 4 and 5 corneas, respectively, were clinically quiet. Analysis of the failure times indicated an inverse relation between failure rate and pore size: less than 15 nm > 25 nm > 50 nm > 100 nm. The difference between the 100-nm and 50-nm membranes was significant (P = 0.03)., Conclusions: A corneal implant must have a porosity greater than that provided by 50-nm membranes. The 100-nm membranes used in this study establish the porosity needed to satisfy the nutritional requirements of the cornea.

Purpose: To minimize the injury to endothelial cells during cryopreservation of rabbit corneas with dimethyl sulfoxide., Methods: Rabbit corneas were cryopreserved using 20% wt/wt dimethyl sulfoxide (Me2SO), added and removed in stages to maintain the osmotically induced excursions in cell volume to within +/-40% of their isotonic volume. The vehicle solution, cooling rate, and conditions of storage used were those already reported to be optimal for endothelial cell survival after exposure to low temperatures. Survival was assessed by confocal microscopy with vital staining and by the ability of the endothelium to control stromal hydration during 3 hours of normothermic perfusion. The effect of temperature of addition and removal of Me2SO (room temperature [RT] or 2 degrees C) on endothelial viability also was measured., Results: After thawing, all the cryopreserved corneas appeared structurally intact when assessed by vital staining and could limit stromal swelling during subsequent normothermic perfusion. Analysis of the rate of stromal swelling during the first 1.5 hours of normothermic perfusion indicated a substantial benefit when the Me2SO was removed at RT. Adding and removing the Me2SO at RT, which allowed a briefer exposure to Me2SO before cooling, resulted in better structural integrity of the endothelial layer than when the addition of cryoprotectant took place on ice., Conclusions: These results demonstrate the importance of osmotic stresses in the generation of injury to corneal endothelium during cryopreservation and the possibility of eventual successful cryopreservation of this tissue.

Purpose: The authors sought to discover whether there are differences in the degree of spatial order in the fibrillar ultrastructure between anterior and posterior stroma., Methods: Human corneas were obtained from eye bank eyes. Although they had been classified as normal, some swelling remained after 3 hours of deturgescence. Freshly excised, unswollen rabbit corneas also were used. Image analysis methods were applied to transmission electron micrographs of the anterior, middle, and posterior stroma of these corneas to determine the positions and radii of fibrils, the fraction of total area occupied by fibrils, and the fibril number density. Results were used to calculate the interference factor that appears in the direct summation of the fields for light scattering theory and to estimate the total scattering cross-section per fibril. The interference factor is a measure of the spatial order in the positions and sizes of the fibrils., Results: Electron micrographs showed anterior-posterior variations in size and number density of fibrils. The interference factor at wavelengths of visible light was lower in posterior stroma than in anterior stroma for humans and rabbits. In some instances in humans, the anterior interference factor was characteristic of mildly swollen cornea. When averaged for the electron micrographs analyzed, the anterior stroma was predicted to scatter approximately twice as much light per unit depth as the posterior stroma in humans (at any given wavelength) and approximately three times as much in rabbits., Conclusions: Calculations of the interference factor showed that there were differences in the anterior-posterior spatial ordering of fibrils. In human corneas, the differences could have been caused by intrinsic in vivo differences between anterior and posterior stroma; however, possible anterior-posterior variations in swelling between the two regions in vitro also could have affected the results.

Purpose: Previously, human corneal stromal interlamellar cohesive strength in the horizontal meridian was shown to be twice as strong peripherally as centrally (approximately 2.90 x 10(-1) versus 1.40 x 10(-1) N/mm). In the current study, stromal samples excised from the vertical meridian were studied to determine if meridional differences also exist. Precise knowledge about corneal stromal structure is warranted, because anisotropy can influence postoperative healing strength and corneal shape., Methods: Limbus-to-limbus stromal strips with a constant 2.2-mm width were obtained from the vertical meridians of 52 eyebank corneas (8 single, 22 pairs). Cohesive strength was recorded as samples were split at a constant 1.6 mm/s at 50% stromal depth., Results: The mean cohesive strength in the inferior periphery was found to be only two thirds the strength observed in the nasal or temporal periphery (1.96 x 10(-1) N/mm versus approximately 2.94 x 10(-1) N/mm @ 5 mm). The mean cohesive strength in the inferior periphery was also significantly less than the strength of the superior periphery (1.85 x 10(-1) N/mm +/- 8.83 x 10(-3) SEM versus 2.34 x 10(-1) +/- 1.37 x 10(-2) @ 4 mm from the central cornea; P = 0.0027). Unlike the samples from the horizontal meridian, which could be described by a profile symmetric about the central cornea, force profiles for the vertical data could be characterized with distinct, classifiable patterns that were generally asymmetric. Fellow corneas from a single donor tended to have strength profiles belonging to the same class (P = 0.035; chi-square), although some paired eyes exhibited profiles from distinctly different classes., Conclusions: These data strongly support the concept of an anisotropic collagen macrostructure that is more complex than previously believed. This inherent structural anisotropy may become a significant determinant of corneal shape during ectatic disease and some forms of keratorefractive surgery.

The effects of an externally applied direct-current electric field on the movement of cultured rabbit corneal epithelial cells and stromal fibroblasts were studied. After a latency of approximately 20 minutes in an electric field, both epithelial cells and stromal fibroblasts became spindle shaped and underwent galvanotropism by aligning their long axes perpendicular to the applied electric field. The electric field stimulus thresholds for galvanotropic movements in epithelial cells and stromal fibroblasts were 4V/cm and 6 V/cm, respectively. After an additional latency of 30 minutes, both cell types manifested galvanotaxic movements: epithelial cells commenced migration in the cathodal (downfield) direction and stromal fibroblasts in the anodal (upfield) direction. For both types of cells, ruffled membranes and lamellipodia were abundant at the leading edges of migrating cells and cell processes underwent retraction at the trailing edges. At field strengths of above 10 V/cm, evidence of cellular damage (manifested by cellular rounding and detachment), attributable to the electric field treatment, was observed after 4 hours. These preliminary results suggest that galvanotaxic responses could be exploited clinically in the enhancement of corneal wound healing.

This study used a transcorneal freezing technique to produce a 2-mm circular, central wound in the rabbit cornea for investigating corneal nerve regeneration. All the corneal cells, nerves, and associated Schwann cells were dead inside the wound, but the extracellular matrix components remained intact. The destroyed epithelium and endothelium were replaced in 1 and 5-7 days, respectively. The necrotic keratocytes and stromal and subepithelial nerves were removed completely in 1-3 days by invading macrophage-like cells. The wounded stroma was repopulated centripetally by migrating keratocytes between days 1-5. Two types of nerve growth were identified in the stroma. The first type was novel sprouting of straight, long neurites between days 2-21, initially from the undamaged, periwound nerves and later from regenerated stromal nerves inside the wound. These small-caliber neurites proliferated in a random and disorderly pattern both inside and outside the wound and sometimes terminated on stationary, stellate keratocytes. The second type was genuine regrowth of stromal and subepithelial nerves in a centripetal direction between days 3-7. Schwann cells appeared on the newly formed nerves starting on day 4 or 5. A near-normal pattern and size of the nerves were established in the wound as early as day 10. In the epithelium, transient, wound-oriented neurites (days 1-3), single nerves, and semileashes (days 4-10) appeared. A near-normal leash pattern was restored between days 10-21 only at the wound periphery. Thus, in this model, the major groundwork of nerve regeneration occurred between days 3-10, simultaneously, at all three levels of nerve organization. These data suggest that nerve-Schwann cell interaction contributes to the restoration of stromal and subepithelial nerves, whereas a reparative epithelium deficient in trophic activity may account for the incomplete regrowth of epithelial nerves. The cryodamage model offers an efficient and multifaceted system for the experimental study of corneal nerve regeneration.

Oxygen permeability (Dk) of 15 rabbit and 19 human cornea stromal samples was measured by conventional polarographic means at 36 C. Hydration and thickness were also measured. Rabbit stromal permeability was found to be about 26 X 10(-11) ml O2 cm2/sec ml mmHg at normal in vivo hydration; in vivo human cornea stromal permeability is probably less than 29 X 10(-11) ml O2 cm2/sec ml mmHg.