Relationship between Corneal Sensation, Blinking, and Tear Film Quality.

PURPOSE: To examine the possible role of corneal sensitivity and tear film quality in triggering a blink by investigating the relationship between blink rate, central corneal sensitivity threshold (CST), ocular surface temperature (OST), tear meniscus height (TMH), tear film quality (noninvasive tear break-up time [NIBUT]), and tear film lipid pattern under normal conditions. METHODS: Forty-two volunteers (average age, 27.76 ± 5.36 years; 11 males) with good ocular health (Ocular Surface Disease Index, <15.0) were recruited for this cross-sectional cohort study. Blink rate, CST (noncontact corneal air gas aesthesiometry, NCCA), minimum and maximum OST in the central and inferior cornea between blinks (thermal infrared camera), TMH, NIBUT, and lipid pattern of the tear film (Keeler Tearscope Plus) were recorded on the right eye only. RESULTS: Median blink rate was 11 blinks/min (interquartile range [IR], 6.95 to 17.05), CST was 0.35 mbars (IR, 0.30 to 0.40), minimum OST in the central cornea was 35.15°C (IR, 34.58 to 35.50), and NIBUT was 34.55 s (IR, 12.45 to 53.80). Moderate but statistically significant correlations were observed between CST and NIBUT (r = 0.535, p < 0.001), CST and blink rate (r = -0.398, p < 0.001), lipid pattern and OST (r = 0.556, p < 0.001), and between CST and OST (r = 0.371, p = 0.008). The correlations between blink rate and NIBUT (r = -0.696, p < 0.001) and between OST and NIBUT (r = 0.639, p < 0.001; Spearman test) achieved higher significance; this was highlighted by the linear regression model where NIBUT and minimum central and inferior OST were identified as significant predictor variables. CONCLUSIONS: There is strong evidence for significant interactions between corneal sensitivity, NIBUT, OST, and blink frequency, emphasizing that ocular surface conditions represent a possible important trigger for the initiation of a blink. However, the mechanisms involved in the initiation of a blink are complex, with local ocular sensory input as only one trigger, along with other external influences and internal factors under cortical control.

Measuring the Refractive Index of Bovine Corneal Stromal Cells Using Quantitative Phase Imaging.

The cornea is the primary refractive lens in the eye and transmits >90% of incident visible light. It has been suggested that the development of postoperative corneal haze could be due to an increase in light scattering from activated corneal stromal cells. Quiescent keratocytes are thought to produce crystallins that match the refractive index of their cytoplasm to the surrounding extracellular material, reducing the amount of light scattering. To test this, we measured the refractive index (RI) of bovine corneal stromal cells, using quantitative phase imaging of live cells in vitro, together with confocal microscopy. The RI of quiescent keratocytes (RI = 1.381 ± 0.004) matched the surrounding matrix, thus supporting the hypothesis that keratocyte cytoplasm does not scatter light in the normal cornea. We also observed that the RI drops after keratocyte activation (RI = 1.365 ± 0.003), leading to a mismatch with the surrounding intercellular matrix. Theoretical scattering models showed that this mismatch would reduce light transmission in the cornea. We conclude that corneal transparency depends on the matching of refractive indices between quiescent keratocytes and the surrounding tissue, and that after surgery or wounding, the resulting RI mismatch between the activated cells and their surrounds significantly contributes to light scattering.

Quantifying the microstructural and biomechanical changes in the porcine ventricles during growth and remodelling.

Cardiac tissue growth and remodelling (G & R) occur in response to the changing physiological demands of the heart after birth. The early shift to pulmonary circulation produces an immediate increase in ventricular workload, causing microstructural and biomechanical changes that serve to maintain overall physiological homoeostasis. Such cardiac G & R continues throughout life. Quantifying the tissue's mechanical and microstructural changes because of G & R is of increasing interest, dovetailing with the emerging fields of personalised and precision solutions. This study aimed to determine equibiaxial, and non-equibiaxial extension, stress-relaxation, and the underlying microstructure of the passive porcine ventricles tissue at four time points spanning from neonatal to adulthood. The three-dimensional microstructure was investigated via two-photon excited fluorescence and second-harmonic generation microscopy on optically cleared tissues, describing the 3D orientation, rotation and dispersion of the cardiomyocytes and collagen fibrils. The results revealed that during biomechanical testing, myocardial ventricular tissue possessed non-linear, anisotropic, and viscoelastic behaviour. An increase in stiffness and viscoelasticity was noted for the left and right ventricular free walls from neonatal to adulthood. Microstructural analyses revealed concomitant increases in cardiomyocyte rotation and dispersion. This study provides baseline data, describing the biomechanical and microstructural changes in the left and right ventricular myocardial tissue during G & R, which should prove valuable to researchers in developing age-specific, constitutive models for more accurate computational simulations. STATEMENT OF SIGNIFICANCE: There is a dearth of experimental data describing the growth and remodelling of left and right ventricular tissue. The published literature is fragmented, with data reported via different experimental techniques using tissues harvested from a variety of animals, with different gender and ages. This prevents developing a continuum of data spanning birth to death, so limiting the potential that can be leveraged to aid computational modelling and simulations. In this study, equibiaxial, non-equibiaxial, and stress-relaxation data are presented, describing directional-dependent material responses. The biomechanical data is consolidated with equivalent microstructural data, an important element for the development of future material models. Combined, these data describe microstructural and biomechanical changes in the ventricles, spanning G &R from neonatal to adulthood.

The ultrastructural development and 3D reconstruction of the transparent carapace of the ostracod Skogsbergia lerneri.

The Skogsbergia lerneri is a marine ostracod which possesses a carapace that is both protective and transparent. Since development of this carapace and how it is maintained in the adult is not known, the aim of this investigation was to carry out an in-depth ultrastructural study of the ostracod carapace at different developmental stages. Standard transmission electron microscopy and novel serial block face scanning electron microscopy (SBF-SEM) were undertaken to discern carapace ultrastructure in both two and three dimensions. Analysis revealed a carapace consisting of the same basic layer structure as other myodocopid ostracods, namely an epicuticle, exocuticle, endocuticle and membranous layer, but with a thinner adult carapace of mean thickness of 19.2 ± 1.78 µm, n = 5. The carapace layers, except for instar 1 ostracods, had similar relative proportions throughout development. The endocuticle and membranous layer thickened through advancing developmental stages due to an increase in calcified crystalline polyhedrons and a greater number of chitinous lamellae in the membranous layer. Crystalline polyhedron dimensions were significantly smaller near the boundary with the membranous layer. The borders between the carapace layers were indistinct; SBF-SEM revealed an abundance of epicuticle projections into the exocuticle and apparent gradual merging at the boundary of the exocuticle and the endocuticle. Here, we discuss how the S. lerneri carapace layer structure has evolved to serve a specific mechanical function, allowing surface protection and rigidity. In addition, we suggest that the lack of pigment and graduated layer boundaries contribute to the transparency of the carapace. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00227-021-04006-7.

Characterisation of carapace composition in developing and adult ostracods (Skogsbergia lerneri) and its potential for biomaterials.

The protective carapace of Skogsbergia lerneri, a marine ostracod, is scratch-resistant and transparent. The compositional and structural organisation of the carapace that underlies these properties is unknown. In this study, we aimed to quantify and determine the distribution of chemical elements and chitin within the carapace of adult ostracods, as well as at different stages of ostracod development, to gain insight into its composition. Elemental analyses included X-ray absorption near-edge structure, X-ray fluorescence and X-ray diffraction. Nonlinear microscopy and spectral imaging were performed to determine chitin distribution within the carapace. High levels of calcium (20.3%) and substantial levels of magnesium (1.89%) were identified throughout development. Amorphous calcium carbonate (ACC) was detected in carapaces of all developmental stages, with the polymorph, aragonite, identified in A-1 and adult carapaces. Novel chitin-derived second harmonic generation signals (430/5 nm) were detected. Quantification of relative chitin content within the developing and adult carapaces identified negligible differences in chitin content between developmental stages and adult carapaces, except for the lower chitin contribution in A-2 (66.8 ± 7.6%) compared to A-5 (85.5 ± 10%) (p = 0.03). Skogsbergia lerneri carapace calcium carbonate composition was distinct to other myodocopid ostracods. These calcium polymorphs and ACC are described in other biological transparent materials, and with the consistent chitin distribution throughout S. lerneri development, may imply a biological adaptation to preserve carapace physical properties. Realisation of S. lerneri carapace synthesis and structural organisation will enable exploitation to manufacture biomaterials and biomimetics with huge potential in industrial and military applications.

Involvement of the CD200 receptor complex in microglia activation in experimental glaucoma.

The interaction of the myeloid restricted molecule CD200R with its widely expressed ligand CD200 is involved in the down-regulation of microglia activation. In the present study, we examined the involvement of CD200R in microglia activation in experimental ocular hypertension to determine the role of microglia activation in retinal ganglion cell (RGC) death, the key pathological event in glaucoma. Experimental glaucoma was induced in adult Brown Norway rats by sclerosis of the episcleral veins with the injection of hypertonic saline. Immunohistochemical methods were used to determine the involvement of microglia using GFAP, CD45, OX42 and OX41 and the involvement of CD200 and CD200R in the optic nerve head. Our data demonstrate the increased presence of microglia within the optic nerve head during ocular hypertension, identified by positive staining with OX42 and OX41. The peak of microglia correlates with peak in RGC death at days 20-27 (T3) post OHT induction. In addition, CD200 and CD200R positive cells were increased in ocular hypertensive eyes. Increased expression of CD200 was detected in the early phase (days 1-7; T1) of OHT and decreased over time, whilst the expression of CD200R was detected in the middle phase (days 20-27; T3) of OHT, correlating with the increase in microglia markers. Changes in the expression of CD200R/CD200 occur early in experimental glaucoma and precede the peak in microglia infiltration and RGC death, suggesting that CD200R-positive microglia play an important role in the initiation of RGC death during OHT, indicating a potential area for therapeutic intervention in treating glaucoma.

A role for Notch signaling in corneal wound healing.

To identify the role of the Notch signaling pathway in corneal wound healing, rat corneas receiving either epithelial or stromal wounds were placed in organ culture for up to 3 and 14 days, respectively. Localization of Notch receptors--Notch1, Notch2, and their ligands--Delta1, Jagged1 was determined by immunofluorescence. Wounds were treated with a γ-secretase inhibitor to suppress Notch signaling or recombinant Jagged1 to enhance Notch signaling and morphological changes in the epithelium and stroma were recorded. The expressions of markers of cell proliferation (Ki67) and epithelial differentiation (cytokeratin 3) were assessed by immunohistology. Notch1 and Notch2 were localized to suprabasal epithelial cells in normal corneas. During corneal wound healing, both Notch receptors were detected in suprabasal and superficial epithelial layers. Delta1 and Jagged1 were observed throughout all corneal epithelial cell layers and occasional keratocytes of the stroma in normal and wounded corneas. γ-secretase inhibition of Notch resulted in increased epithelial cell layers, with recombinant Jagged1 activation of Notch leading to a reduction in epithelial cell layers during corneal wound healing. Correspondingly, the activation of Notch resulted in a decreased cytokeratin 3 expression in the corneal epithelium, with no effect on cellular expression of Ki67. Notch signaling pathway suppressed corneal epithelial differentiation during corneal wound healing, but had no effect on epithelial cell proliferation.

Biomimetic Transparent Eye Protection Inspired by the Carapace of an Ostracod (Crustacea).

In this study we mimic the unique, transparent protective carapace (shell) of myodocopid ostracods, through which their compound eyes see, to demonstrate that the carapace ultrastructure also provides functions of strength and protection for a relatively thin structure. The bulk ultrastructure of the transparent window in the carapace of the relatively large, pelagic cypridinid (Myodocopida) Macrocypridina castanea was mimicked using the thin film deposition of dielectric materials to create a transparent, 15 bi-layer material. This biomimetic material was subjected to the natural forces withstood by the ostracod carapace in situ, including scratching by captured prey and strikes by water-borne particles. The biomimetic material was then tested in terms of its extrinsic (hardness value) and intrinsic (elastic modulus) response to indentation along with its scratch resistance. The performance of the biomimetic material was compared with that of a commonly used, anti-scratch resistant lens and polycarbonate that is typically used in the field of transparent armoury. The biomimetic material showed the best scratch resistant performance, and significantly greater hardness and elastic modulus values. The ability of biomimetic material to revert back to its original form (post loading), along with its scratch resistant qualities, offers potential for biomimetic eye protection coating that could enhance material currently in use.

Cellular inhibitor of apoptosis (cIAP1) is down-regulated during retinal ganglion cell (RGC) maturation.

Apoptosis, is the main type of cell death that occurs in ageing and neurodegenerative disease, such as glaucoma. This study therefore characterises the expression profile of caspases (pro-apoptosis) and inhibitors of apoptosis (IAPs; anti-apoptosis) during maturation of the Brown Norway rat retina between 6 weeks and >24 weeks and also examines concomitant changes in expression of tumor necrosis factor receptor associated factor 2 (TRAF2). The expression profiles of caspases (initiator caspases 8, 9 and effector caspases 6, 7, 3) and inhibitors of apoptosis (IAPs) (Neuronal IAP), cellular IAP1 and 2 (cIAP1/2), X-chromosome linked IAP (XIAP), Survivin, Bruce and Livin) were examined in retinae from 6 weeks and >24 weeks old BN rats using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, Western blotting and immunofluoroscence analysis. Caspase expression was not altered significantly during the study interval. IAP expression showed a general reduction during maturation of BN retina, which was statistically significant for cIAP1. cIAP1 reduction was confirmed by Western blotting and immunoflouroscence and was restricted to cells in the retinal ganglion cell layer (RGCL). Accumulation of TRAF2 was observed in the RGCL accompanying the down-regulation of cIAP1 observed. Our results suggest that cells in the mature RGCL may have a greater susceptibility to cell death compared to their younger counterparts and this may be due in part to a reduction in activation of survival pathways involving IAPs and TRAFs.

Retinal ganglion cell death postponed: giving apoptosis a break?

Glaucoma is characterised by the preferential death of retinal ganglion cells (RGCs). However, mammalian models indicate that neurons pass through a period in which they manifest signs of neuronal damage, but have yet to fully commit to death. Mounting evidence suggests that one of the clearest indications of this process is the reduction in RGC dendritic arborisation, resulting in functional compromise. The extent to which this may be reversible is unclear, since the molecular events that precede changes in dendritic structure have received little attention. Furthermore, there are likely to be many factors involved in this process potentially acting in different individual cells at different times. Recent work in Drosophila shows that dendritic reorganisation/remodelling involves local activation and tight regulation of caspase activity. Here, we propose a model in which the balance between caspases and inhibitors of apoptosis (IAPs) contributes towards the regulation of dendritic remodelling. Thus, RGC dendrite reorganisation and cell death represent opposite ends of a spectrum of events regulated by apoptosis signalling pathways. We summarise relevant events in apoptosis, focusing on caspases and IAPs. We also discuss mechanisms of dendrite development, structure and reorganisation and the implications for early diagnosis and treatment of glaucoma and neurodegenerative disease.

Establishment of long-term ostracod epidermal culture.

Primary crustacean cell culture was introduced in the 1960s, but to date limited cell lines have been established. Skogsbergia lerneri is a myodocopid ostracod, which has a body enclosed within a thin, durable, transparent bivalved carapace, through which the eye can see. The epidermal layer lines the inner surface of the carapace and is responsible for carapace synthesis. The purpose of the present study was to develop an in vitro epidermal tissue and cell culture method for S. lerneri. First, an optimal environment for the viability of this epidermal tissue was ascertained, while maintaining its cell proliferative capacity. Next, a microdissection technique to remove the epidermal layer for explant culture was established and finally, a cell dissociation method for epidermal cell culture was determined. Maintenance of sterility, cell viability and proliferation were key throughout these processes. This novel approach for viable S. lerneri epidermal tissue and cell culture augments our understanding of crustacean cell biology and the complex biosynthesis of the ostracod carapace. In addition, these techniques have great potential in the fields of biomaterial manufacture, the military and fisheries, for example, in vitro toxicity testing.

Collagen ultrastructural changes during stromal wound healing in organ cultured bovine corneas.

Corneal collagen ultrastructural changes occur during the healing process. The present study was designed to compare collagen ultrastructural changes after trephine wounding or flap creation. Bovine corneas were injured and maintained in organ culture for up to 4 weeks. Samples were removed from culture at 0, 1, 2, 3 and 4 weeks and snap frozen in liquid N(2). X-ray scattering was used to measure changes in collagen interfibrillar spacing, intermolecular spacing and fibrillar diameter. Some samples were fixed in 10% Neutral Buffered Formalin solution and wax embedded for immunohistochemistry to monitor myofibroblast differentiation in corneal flaps. Swelling effects (i.e. changes in interfibrillar spacing) were more severe in trephined corneas than in those with stromal flaps. Collagen fibrillar diameter remained normal in the periphery of injured corneas, but increased significantly in areas within and around the wound in trephined samples and in the epithelial incision site in corneal flaps. Intermolecular spacing was unchanged in all samples. In the flaps, alphaSMA expression was only detected in an area adjacent to the epithelial plug, and cell numbers gradually increased during the culture. We conclude that stromal swelling is more rapid for trephine-wounded corneas than in stromal flaps, indicating that the intensity of the corneal healing response depends on the type of injury.

Does air gas aesthesiometry generate a true mechanical stimulus for corneal sensitivity measurement?

BACKGROUND: Belmonte Ocular Pain Meter (OPM) air jet aesthesiometry overcomes some of the limitations of the Cochet-Bonnet aesthesiometer. However, for true mechanical corneal sensitivity measurement, the airflow stimulus temperature of the aesthesiometer must equal ocular surface temperature (OST), to avoid additional response from temperature-sensitive nerves. The aim of this study was to determine: (A) the stimulus temperature inducing no or least change in OST; and (B) to evaluate if OST remains unchanged with different stimulus durations and airflow rates. METHODS: A total of 14 subjects (mean age 25.14 ± 2.18 years; seven women) participated in this clinical cohort study: (A) OST was recorded using an infrared camera (FLIR A310) during the presentation of airflow stimuli, at five temperatures, ambient temperature (AT) +5°C, +10°C, +15°C, +20°C and +30°C, using the OPM aesthesiometer (duration three seconds; over a four millimetre distance; airflow rate 60 ml/min); and (B) OST measurements were repeated with two stimulus temperatures (AT +10°C and +15°C) while varying stimulus durations (three seconds and five seconds) and airflow rates (30, 60, 80 and 100 ml/min). Inclusion criteria were age <40 years, no contact lens wear, absence of ocular disease including dry eye, and no use of artificial tears. Repeated measures (analysis of variance) and appropriate post-hoc t-tests were applied. RESULTS: (A) Stimulus temperatures of AT +10°C and +15°C induced the least changes in OST (-0.20 ± 0.13°C and 0.08 ± 0.05°C). (B) OST changes were statistically significant with both stimulus temperatures and increased with increasing airflow rates (p < 0.001), and were more marked with stimulus temperature AT +10°C. CONCLUSION: A true mechanical threshold for corneal sensitivity cannot be established with the air stimulus of the Belmonte OPM because its air jet stimulus with mechanical setting is likely to have a thermal component. Appropriate stimulus selection for an air jet aesthesiometer must incorporate stimulus temperature control that can vary with stimulus duration and airflow rate.

A role for notch signaling in human corneal epithelial cell differentiation and proliferation.

PURPOSE: To identify the role of Notch signaling in the human corneal epithelium. METHODS: Localization of Notch1, Notch2, Delta1, and Jagged1 in the human corneal epithelium was observed with the use of indirect immunofluorescence microscopy. Gene and protein expression of Notch receptors and ligands in human corneal epithelial cells was determined by RT-PCR and Western blot analysis, respectively. The effects of Notch inhibition (by gamma-secretase inhibition) and activation (by recombinant Jagged1) on epithelial cell proliferation (Ki67) and differentiation (CK3) were analyzed after Western blotting and immunocytochemistry. RESULTS: Immunofluorescent labeling localized Notch1 and Notch2 to suprabasal epithelial cell layers, whereas Delta1 and Jagged1 were observed throughout the corneal epithelium. Notch1, Notch2, Delta1, and Jagged1 genes and proteins were expressed in human corneal epithelial cells. gamma-Secretase inhibition resulted in decreased Notch1 and Notch2 expression, with an accompanying decrease in Ki67 and increased CK3 expression. The activation of Notch by Jagged1 resulted in the upregulation of active forms of Notch1 and 2 proteins (P < 0.05), with a concurrent increase in Ki67 (P < 0.05) and a decrease in CK3 (P < 0.05) expression. Interestingly, gamma-secretase inhibition in a three-dimensional, stratified corneal epithelium equivalent had no effect on Ki67 or CK3 expression. In contrast, Jagged1 activation resulted in decreased CK3 expression (P < 0.05), though neither Notch activation nor inhibition affected cell proliferation in the 3D tissue equivalent. CONCLUSIONS: Notch family members and ligands are expressed in the human corneal epithelium and appear to play pivotal roles in corneal epithelial cell differentiation.

Connective tissue structure of the tree shrew optic nerve and associated ageing changes.

PURPOSE: To identify the structure and composition of the tree shrew optic nerve to determine its potential as a model for glaucoma. METHODS: Tree shrew optic nerves, aged 4 weeks to 5 years, were wax or cryoembedded for analysis of overall morphology and cellular (glial fibrillary acidic protein [GFAP]) and extracellular matrix (collagen types I, III, IV, V, VI; fibronectin; and elastin) immunolocalization studies. In addition, transmission and scanning electron microscopy were performed. In vivo optic disc imaging was performed by HRT2 and fundus camera photography. RESULTS: The optic nerve of the tree shrew comprised regions comparable to the human prelaminar and lamina cribrosa (LC) in the optic nerve head and the retrolaminar region, immediately posterior. The multilayered connective tissue plates of tree shrew LC stretched across the optic nerve canal at the level of the sclera and consisted of collagen types I, III, IV, V, and VI; elastin; and fibronectin. Significant age-related alterations in connective tissue components were indicated. Connective tissue was present in the central retinal vessel sheaths and was identified as longitudinally oriented collagen fibrils in the retrolaminar optic nerve. GFAP immunofluorescence indicated a high concentration of astrocytic processes in the LC. Myelination of axons was evident in the retrolaminar optic nerve. Ultrastructural studies supported the structural organization and spatial distribution of connective tissue. CONCLUSIONS: In contrast to many rodent models of glaucoma, since the tree shrew optic nerve resembles that in humans, especially at the LC, the tree shrew offers an ideal opportunity to investigate glaucoma pathophysiology in a subprimate model.

Deformation of the Lamina Cribrosa and Optic Nerve Due to Changes in Cerebrospinal Fluid Pressure.

Purpose: Cerebrospinal fluid pressure (CSFp) changes are involved or implicated in various ocular conditions including glaucoma, idiopathic intracranial hypertension, and visual impairment and intracranial pressure syndrome. However, little is known about the effects of CSFp on lamina cribrosa and retrolaminar neural tissue (RLNT) biomechanics, potentially important in these conditions. Our goal was to use an experimental approach to visualize and quantify the deformation of these tissues as CSFp increased. Methods: The posterior eye and RLNT of porcine eyes (n = 3) were imaged using synchrotron radiation phase-contrast micro-computed tomography (PC µCT) at an intraocular pressure of 15 mm Hg and CSFps of 4, 10, 20, and 30 mm Hg. Scans of each tissue region were acquired at each CSFp step and analyzed using digital volume correlation to determine 3-dimensional tissue deformations. Results: Elevating CSFp increased the strain in the lamina cribrosa and RLNT of all three specimens, with the largest strains occurring in the RLNT. Relative to the baseline CSFp of 4 mm Hg, at 30 mm Hg, the lamina cribrosa experienced a mean first and third principal strain of 4.4% and -3.5%, respectively. The corresponding values for the RLNT were 9.5% and -9.1%. Conclusions: CSFp has a significant impact on the strain distributions within the lamina cribrosa and, more prominently, within the RLNT. Elevations in CSFp were positively correlated with increasing deformations in each region and may play a role in ocular pathologies linked to changes in CSFp.

Differential regulation of key stages in early corneal wound healing by TGF-beta isoforms and their inhibitors.

PURPOSE: Inhibition of TGF-beta reduces myofibroblast differentiation and fibrosis in the cornea. Determining the actions of distinct TGF-beta isoforms and their inhibitors during early corneal wound healing is an essential step in guiding therapeutic intervention. METHODS: Bovine serum-free corneal cell and wounded organ cultures were challenged with a range of concentrations of TGF-beta1, -beta2, and -beta3; IL-10; and neutralizing human monoclonal antibodies (mAbs) against TGF-beta1 (CAT-192) or -beta2, (CAT-152). Cultures were assessed for re-epithelialization, proliferation (cell counts and cresyl violet assay), morphology (histologic examination), repopulation of the area under the wound, and myofibroblast transformation (alpha-smooth muscle actin) between 0 and 5 days. RESULTS: TGF-beta1 delayed re-epithelialization, increased repopulation of the stroma, increased keratocyte proliferation and was the only isoform to promote myofibroblast differentiation. The anti-TGF-beta1 mAb, CAT-192 promoted re-epithelialization and reduced repopulation of the stroma. Exogenous TGF-beta3 had little effect on re-epithelialization but reduced repopulation of the stroma. IL-10 promoted corneal re-epithelialization at low doses but inhibited this response at high doses. Stromal repopulation was prevented by all doses of IL-10. TGF-beta2 or the anti-TGF-beta2 mAb, CAT-152 had little effect on any repair parameter. CONCLUSIONS: The results confirm TGF-beta1 as the principal isoform in corneal wound healing and suggest that inhibition of the action of TGF-beta1 can promote corneal wound healing. Treatment with the anti-TGF-beta1 mAb CAT-192 accelerates corneal re-epithelialization but reduces cell repopulation of the stroma. The cytokines TGF-beta3 and IL-10 have opposing actions to that of TGF-beta1.

The contribution of DNA repair and antioxidants in determining cell type-specific resistance to oxidative stress.

The aims of this study were; (i) to elucidate the mechanisms involved in determining cell type-specific responses to oxidative stress and (ii) to test the hypothesis that cell types which are subjected to high oxidative burdens in vivo, have greater oxidative stress resistance. Cultures of the retinal pigment epithelium (RPE), corneal fibroblasts, alveolar type II epithelium and skin epidermal cells were studied. Cellular sensitivity to H2O2 was determined by the MTT assay. Cellular antioxidant status (CuZnSOD, MnSOD, GPX, CAT) was analyzed with enzymatic assays and the susceptibility and repair capacities of nuclear and mitochondrial genomes were assessed by QPCR. Cell type-specific responses to H2O2 were observed. The RPE had the greatest resistance to oxidative stress (P>0.05; compared to all other cell types) followed by the corneal fibroblasts (P < 0.05; compared to skin and lung cells). The oxidative tolerance of the RPE coincided with greater CuZnSOD, GPX and CAT enzymatic activity (P < 0.05; compared to other cells). The RPE and corneal fibroblasts both had up-regulated nDNA repair post-treatment (P < 0.05; compared to all other cells). In summary, variations in the synergistic interplay between enzymatic antioxidants and nDNA repair have important roles in influencing cell type-specific vulnerability to oxidative stress. Furthermore, cells located in highly oxidizing microenvironments appear to have more efficient oxidative defence and repair mechanisms.

Effects of Peripapillary Scleral Stiffening on the Deformation of the Lamina Cribrosa.

PURPOSE: Scleral stiffening has been proposed as a treatment for glaucoma to protect the lamina cribrosa (LC) from excessive intraocular pressure-induced deformation. Here we experimentally evaluated the effects of moderate stiffening of the peripapillary sclera on the deformation of the LC. METHODS: An annular sponge, saturated with 1.25% glutaraldehyde, was applied to the external surface of the peripapillary sclera for 5 minutes to stiffen the sclera. Tissue deformation was quantified in two groups of porcine eyes, using digital image correlation (DIC) or computed tomography imaging and digital volume correlation (DVC). In group A (n = 14), eyes were subjected to inflation testing before and after scleral stiffening. Digital image correlation was used to measure scleral deformation and quantify the magnitude of scleral stiffening. In group B (n = 5), the optic nerve head region was imaged using synchrotron radiation phase-contrast microcomputed tomography (PC µCT) at an isotropic spatial resolution of 3.2 µm. Digital volume correlation was used to compute the full-field three-dimensional deformation within the LC and evaluate the effects of peripapillary scleral cross-linking on LC biomechanics. RESULTS: On average, scleral treatment with glutaraldehyde caused a 34 ± 14% stiffening of the peripapillary sclera measured at 17 mm Hg and a 47 ± 12% decrease in the maximum tensile strain in the LC measured at 15 mm Hg. The reduction in LC strains was not due to cross-linking of the LC. CONCLUSIONS: Peripapillary scleral stiffening is effective at reducing the magnitude of biomechanical strains within the LC. Its potential and future utilization in glaucoma axonal neuroprotection requires further investigation.

Phase-Contrast Micro-Computed Tomography Measurements of the Intraocular Pressure-Induced Deformation of the Porcine Lamina Cribrosa.

The lamina cribrosa (LC) is a complex mesh-like tissue in the posterior eye. Its biomechanical environment is thought to play a major role in glaucoma, the second most common cause of blindness. Due to its small size and relative inaccessibility, high-resolution measurements of LC deformation, important in characterizing LC biomechanics, are challenging. Here we present a novel noninvasive imaging method, which enables measurement of the three-dimensional deformation of the LC caused by acute elevation of intraocular pressure (IOP). Posterior segments of porcine eyes were imaged using synchrotron radiation phase contrast micro-computed tomography (PC µCT) at IOPs between 6 and 37 mmHg. The complex trabecular architecture of the LC was reconstructed with an isotropic spatial resolution of 3.2 µm. Scans acquired at different IOPs were analyzed with digital volume correlation (DVC) to compute full-field deformation within the LC. IOP elevation caused substantial tensile, shearing and compressive devformation within the LC, with maximum tensile strains at 30 mmHg averaging 5.5%, and compressive strains reaching 20%. We conclude that PC µCT provides a novel high-resolution method for imaging the LC, and when combined with DVC, allows for full-field 3D measurement of ex vivo LC biomechanics at high spatial resolution.