The architecture of corneal stromal striae on optical coherence tomography and histology in an animal model and in humans.

The purpose of this study was to use a portable optical coherence tomography (OCT) for characterization of corneal stromal striae (CSS) in an ovine animal model and human corneas with histological correlation, in order to evaluate their architectural pattern by image analysis. Forty-six eyes from female adult sheep (older than 2 years), and 12 human corneas, were included in our study. The eyes were examined in situ by a portable OCT, without enucleation. All OCT scans were performed immediately after death, and then the eyes were delivered to a qualified histology laboratory. In the ovine animal model, CSS were detected with OCT in 89.1% (41/46) of individual scans and in 93.4% (43/46) of histological slices. In human corneas, CSS were found in 58.3% (7/12) of cases. In both corneal types, CSS appeared as "V"- or "X"-shaped structures, with very similar angle values of 70.8° ± 4° on OCT images and 71° ± 4° on histological slices (p ≤ 0.01). Data analysis demonstrated an excellent degree of reproducibility and inter-rater reliability of measurements (p < 0.001). The present study demonstrated that by using a portable OCT device, CSS can be visualized in ovine and human corneas. This finding suggests their generalized presence in various mammals. The frequent observation, close to 60%, of such collagen texture in the corneal stroma, similar to a 'truss bridge' design, permits to presume that it plays an important structural role, aimed to distribute tensile and compressive forces in various directions, conferring resilience properties to the cornea.

Collagen XII Is a Regulator of Corneal Stroma Structure and Function.

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.

Mapping of Corneal Layer Thicknesses With Polarization-Sensitive Optical Coherence Tomography Using a Conical Scan Pattern.

Purpose: We demonstrate segmentation and mapping of corneal layers (epithelium, Bowman's layer, and stroma) across the entire cornea (limbus to limbus), using additional contrast provided by polarization-sensitive optical coherence tomography (PS-OCT) and analyze the reproducibility of the procedure. Methods: A custom built PS-OCT system operating at 1045 nm central wavelength with conical scanning was used for image acquisition. Conical scanning allows for almost perpendicular beam incidence on the corneal surface and provides good signal quality over the entire field of view. Epithelium, Bowman's layer, and stroma were segmented using the additional contrast provided by PS-OCT. Thickness maps were computed and analyzed in sectors. Both eyes of 20 healthy volunteers were imaged at least three times to test this method and to quantify reproducibility. Results: Thickness maps of the epithelium show significant (P < 0.001) superior thinning and an inferior thickening. Bowman's layer appears homogeneous within the central 7 to 8 mm diameter of the cornea and gets thinner toward the periphery until this layer disappears between 4 and 5.5 mm eccentricity from the center. Intersubject variations of the measured thicknesses of epithelium (coefficient of variation [CV] ∼8%), Bowman's layer (CV∼25%), and stroma (CV∼10%) were observed. Very good reproducibility of thickness measurements of epithelium (CV < 3%), Bowman's layer (CV < 5%), and stroma (CV < 2%) was found. Furthermore, a significant correlation (P < 0.001) between layer thicknesses of the right and left eyes of the same subject was found. Conclusions: PS-OCT with conical scanning is a feasible approach for determining thickness maps of corneal layers on a large field of view with high reproducibility.

A comparative study of the elastic fibre system within the mouse and human cornea.

The cornea relies on its organised extracellular matrix for maintaining transparency and biomechanical strength. Studies have identified an elastic fibre system within the human posterior cornea, thought to allow for slight deformations in response to internal pressure fluctuations within the eye. However, the type of elastic fibres that exist within the cornea and their roles remain elusive. The aim of this study was to compare the distribution and organisation of the elastic fibres within the posterior peripheral mouse and human cornea, and elucidate how these fibres integrate with the trabecular meshwork, whilst characterising the distribution of their main likely components (fibrillin-1, elastin and type VI collagen) in different parts of the cornea and adjacent sclera. We identified key differences in the elastic fibre system between the human and mouse cornea. True elastic fibres (containing elastin) were identified within the human posterior peripheral cornea. Elastic fibres appeared to present as an extensive network throughout the mouse corneal stroma, but as fibrillin-rich microfibril bundles rather than true elastic fibres. However, tropoelastin staining indicated the possibility that true elastic fibres had yet to develop in the young mice studied. Differences were also apparent within the anatomy of the trabecular meshwork. The human trabecular meshwork appeared to insert between the corneal stroma and Descemet's membrane, with elastic fibres continuing into the stroma from the trabecular meshwork anterior to Descemet's membrane. Within the mouse cornea, no clear insertion point of the trabecular meshwork was seen, instead the elastic fibres within the trabecular meshwork continued into Descemet's membrane, with the trabecular meshwork joining posterior to Descemet's membrane.

Evolution of the vertebrate corneal stroma.

Although the cornea is the major refractive element of the eye, the mechanisms controlling corneal shape and hence visual acuity remain unknown. To begin to address this question we have used multiphoton, non-linear optical microscopy to image second harmonic generated signals (SHG) from collagen to characterize the evolutionary and structural changes that occur in the collagen architecture of the corneal stroma. Our studies show that there is a progression in complexity of the stromal collagen organization from lower (fish and amphibians) to higher (birds and mammals) vertebrates, leading to increasing tissue stiffness that may control shape. In boney and cartilaginous fish, the cornea is composed of orthogonally arranged, rotating collagen sheets that extend from limbus to limbus with little or no interaction between adjacent sheets, a structural paradigm analogous to 'plywood'. In amphibians and reptiles, these sheets are broken down into broader lamellae that begin to show branching and anastomosing with adjacent lamellae, albeit maintaining their orthogonal, rotational organization. This paradigm is most complex in birds, which show the highest degree of lamellar branching and anastomosing, forming a 'chicken wire' like pattern most prominent in the midstroma. Mammals, on the other hand, diverged from the orthogonal, rotational organization and developed a random lamellar pattern with branching and anastomosing appearing highest in the anterior stroma, associated with higher mechanical stiffness compared to the posterior stroma.

Corneal thickness of eyes of healthy goats, sheep, and alpacas manually measured by use of a portable spectral-domain optical coherence tomography device.

OBJECTIVE To determine corneal thickness of eyes of healthy goats, sheep, and alpacas by use of a portable spectral-domain optical coherence tomography (SD-OCT) device and evaluate intraoperator reliability for measurements. ANIMALS 11 female goats, 10 female sheep, and 11 (4 males and 7 females) alpacas. PROCEDURES Each animal was sedated, and gentle manual restraint was used to ensure proper positioning of the head and globe. Corneal pachymetry was performed (in triplicate) with a portable SD-OCT device on both eyes of each animal. All corneal measurements were obtained manually by use of the integrated caliper function. Corneal epithelial thickness (CET), corneal stromal thickness (CST), Descemet membrane thickness (DMT), and total corneal thickness (TCT) were measured twice on each image, and a mean value was calculated. RESULTS Mean ± SD values for CET, CST, DMT, and TCT were 96.1 ± 5.0 µm, 486.0 ± 10.3 µm, 36.8 ± 4.8 µm, and 616.9 ± 7.1 µm, respectively, for the goats; 111.6 ± 5.7 µm, 599.8 ± 10.0 µm, 31.0 ± 4.5 µm, and 741.1 ± 9.9 µm, respectively, for the sheep; and 147.4 ± 5.7 µm, 446.1 ± 7.4 µm, 44.5 ± 5.0 µm, and 634.8 ± 6.2 µm, respectively, for the alpacas. Intraclass correlations ranged from 0.49 to 0.83 for CET, CST, and TCT and from 0.13 to 0.36 for DMT. CONCLUSIONS AND CLINICAL RELEVANCE SD-OCT provided manual measurement of corneal thickness (CET, CST, and TCT) with clinically acceptable intraoperator reliability for eyes of healthy goats, sheep, and alpacas.

Ophthalmic variables in rehabilitated juvenile Kemp's ridley sea turtles (Lepidochelys kempii).

OBJECTIVE: To determine central corneal thickness (total corneal thickness [TCT], epithelial thickness [ET], and stromal thickness [ST]), anterior chamber depth (ACD), and intraocular pressure (IOP) in Kemp's ridley sea turtles (Lepidochelys kempii). DESIGN: Prospective cross-sectional study. ANIMALS: 25 healthy rehabilitated juvenile Kemp's ridley sea turtles. PROCEDURES; Body weight and straight-line standard carapace length (SCL) were recorded. All turtles underwent a complete anterior segment ophthalmic examination. Central TCT, ET, ST, and ACD were determined by use of a spectral-domain optical coherence tomography device. Intraocular pressure was determined with a rebound tonometer; the horse setting was used to measure IOP in all 25 turtles, and the undefined setting was also used to measure IOP in 20 turtles. For each variable, 3 measurements were obtained bilaterally. The mean was calculated for each eye and used for analysis purposes. RESULTS: The mean ± SD body weight and SCL were 3.85 ± 1.05 kg (8.47 ± 2.31 lb) and 29 ± 3 cm, respectively. The mean ± SD TCT, ET, ST, and ACD were 288 ± 23 µm, 100 ± 6 µm, 190 ± 19 µm, and 581 ± 128 µm, respectively. Mean ± SD IOP was 6.5 ± 1.0 mm Hg when measured with the horse setting and 3.8 ± 1.1 mm Hg when measured with the undefined setting. CONCLUSIONS AND CLINICAL RELEVANCE: Results provided preliminary reference ranges for objective assessment of ophthalmic variables in healthy juvenile Kemp's ridley sea turtles.

Polarimetric Interferometry for Assessment of Corneal Stromal Lamellae Orientation.

PURPOSE: To analyze corneal stromal lamellae orientation in normal subjects and patients with various corneal conditions using a polarimetric interferometer. METHODS: Thirty-two healthy control subjects (59 eyes) and 16 patients (22 eyes) with corneal conditions or postkeratoplasty were included in the study. All patients were imaged using the interferometer and slit lamp photography. The interferometer produces an orthogonal cross-like image of stromal lamellae by illuminating the cornea in phase-light polarization angle. Corneal haze was graded on a scale from 0 to 4. Interferometer cross-like images were graded on a scale from 0 to 5 by a masked observer. Keratometry, corneal central pachymetry, and optical densitometry were obtained with Scheimpflug corneal tomography. RESULTS: The cross-like image was observed in 31 of 32 healthy control subjects (58 of 59 eyes) and in 13 of 16 patients (19 of 22 eyes). The image was not detectable in 3 patients, 1 with total corneal neovascularization and scarring, 1 with central leukoma, and 1 with failed deep lamellar keratoplasty. Corneal haze was the main factor obscuring the cross-like image (P < 0.05). Clarity of the images was influenced by older age, steeper keratometry, higher pachymetry, and optical density (P < 0.05). There was no correlation between the orientation of the keratometric axes and the orientation of the 2 arms of the cross-like image (P > 0.05). CONCLUSIONS: Corneal stromal lamellae orientation can be efficiently assessed and displayed as a cross-like image by the polarimetric interferometer. Stromal lamellae orientation imaging may have potential implications in corneal diagnostics and surgery.

Deep Laser-Assisted Lamellar Anterior Keratoplasty With Microkeratome-Cut Grafts.

PURPOSE: The goals of this laboratory study were to evaluate the interface quality in laser-assisted lamellar anterior keratoplasty (LALAK) with microkeratome-cut grafts and achieve good graft-host apposition. METHODS: Simulated LALAK surgeries were performed on 6 pairs of eye-bank corneoscleral discs. Anterior lamellar grafts were precut with microkeratomes. Deep femtosecond (FS) laser cuts were performed on host corneas followed by excimer laser smoothing. Different parameters of FS laser cuts and excimer laser smoothing were tested. Optical coherence tomography was used to measure corneal pachymetry and evaluate graft-host apposition. The interface quality was quantified in a masked fashion using a 5-point scale based on scanning electron microscopy images. RESULTS: Deep FS laser cuts at 226 to 380 µm resulted in visible ridges on the host bed. Excimer laser smoothing with a central ablation depth of 29 µm and saline as a smoothing agent did not adequately reduce ridges (score = 4.0). Deeper excimer laser ablation of 58 µm and Optisol-GS as a smoothing agent smoothed ridges to an acceptable level (score = 2.1). Same sizing of the graft and host cut diameters with an approximately 50-µm deeper host side cut relative to the central graft thickness provided the best graft-host fit. CONCLUSIONS: Deep excimer laser ablation with a viscous smoothing agent was needed to remove ridges after deep FS lamellar cuts. The host side cut should be deep enough to accommodate thicker graft peripheral thickness compared with the center. This LALAK design provides smooth lamellar interfaces, moderately thick grafts, and good graft-host fits.

Evaluation of Total Corneal Thickness and Corneal Layers With Spectral-Domain Optical Coherence Tomography.

PURPOSE: To evaluate total corneal thickness and corneal layers in healthy young adults using spectral-domain optical coherence tomography and to describe its repeatability and reproducibility. METHODS: Eighty-six eyes from 86 healthy volunteers were prospectively and consecutively enrolled. Manual measurements of central corneal thickness (CCT) and central thickness of epithelium, Bowman's layer, stroma, and the Descemet-endothelium complex were performed using Spectralis OCT (Heidelberg Engineering, Heidelberg, Germany). To assess the reliability of the repeated measurements, intraclass correlation coefficients and coefficients of variation were used. RESULTS: Mean CCT, epithelium, Bowman's layer, stroma, and Descemet-endothelium values were 555.50 ± 29.64, 54.60 ± 4.25, 16.70 ± 1.73, 467.51 ± 28.91, and 16.74 ± 1.66 µm, respectively. The intraclass correlation coefficients ranged from 0.746 (Bowman's layer) to 0.999 (CCT and stroma) and from 0.483 (Bowman's layer) to 0.995 (CCT) and 0.998 (stroma) for intraobserver repeatability and interobserver reproducibility, respectively. The measurements showed coefficients of variation lower than 11% in all cases. CONCLUSIONS: This study establishes a normal database for corneal thickness and all its layers in healthy young adults with spectral-domain optical coherence tomography. This device exhibited a high degree of intraobserver repeatability and interobserver reproducibility for all regions except Bowman's layer.

Modified big-bubble technique compared to manual dissection deep anterior lamellar keratoplasty in the treatment of keratoconus.

PURPOSE: To evaluate the clinical findings and results of manual dissection deep anterior lamellar keratoplasty (DALK) compared to a modified big-bubble DALK technique in eyes affected by keratoconus. METHODS: Sixty eyes of 60 patients with keratoconus were treated with one of the two surgical techniques manual DALK (n = 30); big-bubble DALK (n = 30). The main outcomes measured were visual acuity, corneal topographic parameters, thickness of residual stroma and endothelial cell density (ECD). Patients were examined postoperatively at 1 month, 6 months, 1 year and 1 month after suture removal. RESULTS: Final best spectacle-corrected visual acuity (BSCVA) measured 1 month after suture removal was 0.11 ± 0.08 LogMAR in the big-bubble group compared to 0.13 ± 0.08 in the manual DALK group (p = 0.227). In patients treated with the big-bubble technique without complications (Descemet's membrane completely bared), the stromal residue was not measureable. Mean stromal residual thickness in the manual DALK group was 30.50 ± 27.60 µm. Data analysis of the manual DALK group demonstrated a significant correlation between BSCVA and residual stromal thickness; lower residual stromal thickness correlated with better BSCVA values (Spearman ρ = 0.509, p = 0.018). Postoperative ECD was similar in both groups at all intervals, with no statistically significant differences. In both groups, ECD loss was only significant during the 1- to 6-month interval (p = 0.001 and p < 0.001 in the big-bubble DALK and manual DALK groups, respectively). CONCLUSION: Manual DALK provides comparable results to big-bubble DALK. Big-bubble DALK permits faster visual recovery and is a surgical technique, which can be easily converted to manual DALK in cases of unsuccessful 'big-bubble' formation.

Biocompatibility of Ferrara intracorneal ring segment with and without chondroitin sulfate coating. Clinical and histopathological evaluation in rabbits.

PURPOSE: To investigate and compare the biocompatibility of two types of Ferrara intracorneal ring segment: with and without chondroitin sulfate coating by clinical and histopathological evaluation. METHODS: A randomized experimental study was carried out on thirty right-eye corneas from 30 Norfolk albino rabbits allocated into two experimental groups: Group G1 - implanted with Ferrara intracorneal ring segment without coating (FICRS) and Group G2 - implanted with Ferrara intracorneal ring segment with chondroitin sulfate coating (FICRS-CS). Left eyes formed the control group. Clinical parameters analyzed were: presence of edema, vascularization, infection and ring extrusion one, 30, and 60 days after surgery. Histopathological parameters analyzed were: number of corneal epithelial layers over and adjacent to the ring, presence of spongiosis, hydropic degeneration, basement membrane thinning, inflammatory cells, neovascularization and pseudocapsule formation. RESULTS: At clinical examination 60 days after implant, edema, vascularization and extrusion were observed respectively in 20%, 26.7%, 6.7% of FICRS corneas and in 6.7%, 6.7%, and 0% of FICRS-CS corneas. Histopathological evaluation showed epithelial-layer reduction from 5 (5;6) to 3 (3;3) with FICRS and from 5 (5;5) to 4 (3;5) with FICRS-CS in the region over the ring. Epithelial spongiosis, hydropic degeneration, and basement membrane thinning were present in 69.2%, 53.8%, and 69.2% of FICRS and in 73.3%, 73.3%, and 46.7% with FICRS-CS, respectively. Vascularization was present in 38.5% of FICRS and 13.3% with FICRS-CS, inflammatory cells in 75% of FICRS and 33.3% with FICRS-CS, and pseudocapsule in 66.7% of FICRS and 93.3% with FICRS-CS. Giant cells occurred only in the FICRS-CS group (20%). CONCLUSION: Ferrara intracorneal rings coated with chondroitin sulfate (FICRS-CS) caused lower frequency of clinical and histopathological alterations than Ferrara intracorneal rings without the coating (FICRS), demonstrating higher biocompatibility of the FICRS-CS.

Biocompatibility of Ferrara intracorneal ring segment with and without chondroitin sulfate coating: clinical and histopathological evaluation in rabbits

PURPOSE: To investigate and compare the biocompatibility of two types of Ferrara intracorneal ring segment: with and without chondroitin sulfate coating by clinical and histopathological evaluation. METHODS: A randomized experimental study was carried out on thirty right-eye corneas from 30 Norfolk albino rabbits allocated into two experimental groups: Group G1 - implanted with Ferrara intracorneal ring segment without coating (FICRS) and Group G2 - implanted with Ferrara intracorneal ring segment with chondroitin sulfate coating (FICRS-CS). Left eyes formed the control group. Clinical parameters analyzed were: presence of edema, vascularization, infection and ring extrusion one, 30, and 60 days after surgery. Histopathological parameters analyzed were: number of corneal epithelial layers over and adjacent to the ring, presence of spongiosis, hydropic degeneration, basement membrane thinning, inflammatory cells, neovascularization and pseudocapsule formation. RESULTS: At clinical examination 60 days after implant, edema, vascularization and extrusion were observed respectively in 20%, 26.7%, 6.7% of FICRS corneas and in 6.7%, 6.7%, and 0% of FICRS-CS corneas. Histopathological evaluation showed epithelial-layer reduction from 5 (5;6) to 3 (3;3) with FICRS and from 5 (5;5) to 4 (3;5) with FICRS-CS in the region over the ring. Epithelial spongiosis, hydropic degeneration, and basement membrane thinning were present in 69.2%, 53.8%, and 69.2% of FICRS and in 73.3%, 73.3%, and 46.7% with FICRS-CS, respectively. Vascularization was present in 38.5% of FICRS and 13.3% with FICRS-CS, inflammatory cells in 75% of FICRS and 33.3% with FICRS-CS, and pseudocapsule in 66.7% of FICRS and 93.3% with FICRS-CS. Giant cells occurred only in the FICRS-CS group (20%). CONCLUSION: Ferrara intracorneal rings coated with chondroitin sulfate (FICRS-CS) caused lower frequency of clinical and histopathological alterations than Ferrara intracorneal rings without the coating (FICRS), demonstrating higher biocompatibility of the FICRS-CS.

The measurement of corneal thickness from center to limbus in vivo in C57BL/6 and BALB/c mice using two-photon imaging.

The mouse corneal thickness is very important for research into the fields of eye disease. However, the in vivo corneal thickness for the entire cornea from the pupil to the limbus was not determined. We measured in vivo corneal layer thicknesses in different corneal areas, from the central cornea to the limbus, in the widely used inbred C57BL/6 and BALB/c mouse strains using two-photon (2 PH) imaging. Eight corneas of the C57BL/6 or BALB/c were scanned using a 2 PH laser scanning fluorescence microscopy system. A total of 14 thicknesses of the different corneal layers, from different corneal regions, were measured using image processing software. In both C57BL/6 and BALB/c mice, the thickness of the corneal layers was inhomogeneous in different areas of the cornea, and all of the layers had their minimum thickness at the limbus. In C57BL/6 mice, the thickness of the corneal layers gradually increased from the central to the paracentral cornea, peaked at the fifth measurement point in the paracentral area, and decreased from this point to the limbus. In BALB/c mice, the thickness of the entire cornea and corneal epithelium had its maximum at the central cornea and gradually decreased from the central cornea to the peripheral cornea and to the limbus. The thickness of the corneal stroma and endothelium had its maximum at the fourth measurement point in the paracentral cornea and gradually decreased from the paracentral cornea to the limbus. The ratio of epithelial thickness to the total corneal thickness gradually decreased from the central cornea to the limbus in both C57BL/6 and BALB/c mice. The minimum ratio was observed at the fourteenth measurement point in both C57BL/6 and BALB/c mice. The ratio of stromal and endothelial to the total corneal thickness gradually increased from the central cornea to the limbus in both C57BL/6 and BALB/c mice. The maximum ratio was observed at the fourteenth measurement point in C57BL/6 mice. The ratio at the first eight measurement points was significantly lower in BALB/c than in C57BL/6 mice (P < 0.05). Our study demonstrated that the thickness of the entire cornea, the corneal epithelium, the corneal stroma and the endothelium was inhomogeneous in different areas of the cornea. Moreover, all of the layers exhibited a minimum thickness at the limbus in both C57BL/6 and BALB/c mice. Furthermore, the corneal thickness in different areas varied between C57BL/6 and BALB/c mice, and the variation in thickness with respect to corneal location for these strains was dissimilar. When using the mouse as an animal model to examine the cornea, it is important to note the differences between humans and mice.

Bulbar conjunctival thickness measurements with optical coherence tomography in healthy chinese subjects.

PURPOSE: To conduct in vivo measurements of the bulbar conjunctiva epithelial, stromal, and total thicknesses in healthy Chinese subjects using optical coherence tomography (OCT) and to compare the conjunctival thickness differences between sex and age. METHODS: From January to July 2012, healthy Chinese subjects were recruited to undergo a Cirrus HD-OCT examination using the Anterior Segment 5 Line Raster scanning protocol. Ophthalmoscopy and slit-lamp examinations were performed on all eyes, along with the determination of visual acuity. The bulbar conjunctiva epithelial thickness, stromal thickness, and full thickness were measured on cross-sectional conjunctiva images. The average values of each parameter were compared between males and females and among eight age groups (<20, 21-30, 31-40, 41-50, 51-60, 61-70, 71-80, and >80 years). RESULTS: A total of 711 eyes in 711 subjects (average age 46.5 years) were included. The average conjunctiva epithelial, stromal, and full thickness measurements were 42.4 ± 7.4 µm, 197.7 ± 32.5 µm, and 240.1 ± 29.8 µm, respectively. No significant difference was found for any of the three parameters between sexes. The conjunctiva epithelial thickness values decreased significantly after age 20 and were then maintained at relatively low levels, followed by a sharp increase at 60 years of age. The conjunctiva stromal thickness decreased throughout the entire lifetime, and the full thickness decreased from the teenage to the elderly periods. CONCLUSIONS: None of the Chinese conjunctiva epithelial, stromal, and full thickness was sex-related, but all of them were age-related.

Preconditioned donor corneal thickness for microthin endothelial keratoplasty.

PURPOSE: To describe a novel surgical technique to produce thin endothelial grafts for Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: Thirteen human cadaveric corneas in organ culture were randomized into conventional (n = 7) and microthin (n = 6) DSAEK groups. Grafts in the conventional DSAEK group were prepared using the conventional DSAEK technique of a single microkeratome pass with a 350-µm cutting head. Corneas in the microthin group were preconditioned to achieve a target central thickness of 530 µm before graft dissection with a 350-µm microkeratome head. Preconditioning involved stromal dehydration under pachymetric control using sterile airflow for 15-second increments. Donor and graft thicknesses were assessed with optical coherence tomography, and endothelial viability with trypan blue and alizarin red staining. RESULTS: Mean endothelial graft thickness obtained using the microthin DSAEK technique was 106 µm (SD, 32 µm) compared with 177 µm (SD, 33 µm) obtained using conventional DSAEK technique (P = 0.0024). Donor preconditioning yielded a predicted reduction of 100 µm in graft stromal thickness at a rate of 1.5 µm/s and mean duration of 72 seconds. The average anterior lamella thickness (cut depth) obtained in microthin and conventional DSAEK groups were 424 and 431 µm, respectively (P = 0.84). There was no difference in endothelial viability between the 2 groups. There were no corneal perforations during graft preparation in this study. CONCLUSIONS: Donor preconditioning by pachymetry-controlled stromal dehydration achieved significantly thinner endothelial grafts compared with the conventional DSAEK technique without compromise to endothelial viability or graft wastage.

Variation in central corneal thickness during open- or closed-eye riboflavin instillation for corneal collagen cross-linking.

PURPOSE: To assess the effect of having an open or closed eye on the variation in central corneal thickness during riboflavin instillation for corneal collagen cross-linking (CXL). METHODS: Thirty eyes of 15 New Zealand White rabbits underwent an in vivo anterior segment optical coherence tomography (OCT) examination at 0, 10, 20, and 30 min after riboflavin instillation on the de-epithelialized corneal surface. Each eye of every rabbit was randomly placed into one of two different treatment groups (open-eye or closed-eye) during the instillation; the examinations were performed one after the other. After instillation for 30 min, the changes in the corneal stroma and anterior chamber were observed by slit lamp. RESULTS: A significant decrease in the central corneal thickness (CCT) was demonstrated during riboflavin instillation; the variations were smaller in the measurements performed with the eye closed than with the eye open (81.36 +/- 15.13 microm and 129.20 +/- 12.05 microm respectively). Both methods turned the corneal stroma and anterior chamber yellow. CONCLUSION: Keeping the eye closed during riboflavin instillation reduced the decrease in the CCT. The same yellow change in the corneal stroma and anterior chamber occurred, but the exposure time of the ocular surface was shorter. Therefore, keeping the eye closed was a more effective and safer method than keeping the eye open.

How normal is the transparent cornea? Effects of aging on corneal morphology.

PURPOSE: To ascertain the effects of aging on corneal morphology and to illustrate the morphologic diversity of the different layers in the normal cornea as seen by in vivo confocal microscopy (IVCM). DESIGN: Observational cross-sectional study. PARTICIPANTS: A total of 150 healthy subjects, evenly distributed over 5 age categories, comprising 75 men and 75 women. METHODS: Both transparent corneas (n = 300) of all subjects were examined in duplicate by white light IVCM (Confoscan 4, NIDEK Technologies, Albignasego, Padova, Italy). After reviewing the IVCM examinations for morphologic variations of the corneal layers, we selected the 8 most common features to illustrate the morphologic diversity. Subsequently, all 600 IVCM examinations were assessed for the presence of these features. We used binary logistic regression analyses to assess the age-relatedness of each feature. MAIN OUTCOME MEASURES: Age distribution of bright superficial epithelial cells, dendriform cells, alterations characteristic of epithelial basement membrane dystrophy (EBMD), tortuous stromal nerves, stromal microdots in the anterior stroma, folds in the posterior stroma, opacification of Descemet's membrane, and corneal guttae. RESULTS: Four features were found characteristic of the aging cornea: stromal microdots in the anterior stroma (P<0.0001), folds in the posterior stroma (P<0.0001), opacification of Descemet's membrane (P<0.0001), and corneal guttae (P<0.0001). Alterations characteristic of EBMD were found in 3% of all eyes and only detected in subjects aged ≥40 years, suggesting age-relatedness (P = 0.09). Other features, such as bright superficial epithelial cells (n = 38, 13%), dendriform cells (n = 42, 14%), and tortuous stromal nerves (n = 115, 38%), were age-independent. We also found a novel phenotype of corneal endothelium in 4 normal eyes of 2 subjects, which we coined "salt and pepper endothelium." We could not establish whether this novel phenotype represented a morphologic variant of normal endothelium, an early stage of a known corneal endothelial disorder, or a completely new disease entity. CONCLUSIONS: Knowledge of the common morphologic variations of the corneal layers and the effects of aging on corneal morphology as seen by IVCM increases our understanding of corneal degenerative disorders and is essential to detect corneal pathology. Our finding of a novel phenotype of corneal endothelium emphasizes the morphologic diversity of this optically transparent tissue.

An explanation for the central to peripheral thickness variation in the mouse cornea.

BACKGROUND: The mouse corneal stroma varies in thickness across its diameter. The purpose of the present study was to explain this variation and to advance our understanding of stromal lamellar architecture in the mammalian cornea. METHODS: Eight C57BL/6 mice were killed, eyes enucleated, immersed in 2% glutaraldehyde fixative, processed and sectioned transversely for light and transmission electron microscopy. Transmission electron micrographs were assembled into montages and printed at 5000× magnification and used for lamellar counts and thickness assessments. RESULTS: The mouse cornea had an average of 49.8±2.4 lamellae centrally averaging 2.1µm in thickness versus 35.5±3.0 lamellae, averaging 1.9µm in thickness peripherally. The central to peripheral decrease in number lamellae and lamellar thickness measured utilizing the transmission electron microscope was statistically significant (P<0.005). CONCLUSIONS: This study demonstrated that the thickness difference between the thicker central and thinner peripheral mouse cornea is explained primarily by the number of lamellae present and that the peripheral lamellar dropout occurred in the anterior 2/3 of stroma. The decreased lamellar count towards the periphery suggested that not all lamellae cross the cornea limbus to limbus. These findings may be relevant to the thickness variation of the human cornea.

Laser in situ keratomileusis flap-thickness predictability with a pendular microkeratome.

PURPOSE: To assess flap-thickness predictability with a pendular microkeratome (130 µm head). SETTING: Eye Institute of Thrace, Democritus University of Thrace, Alexandroupolis, Greece. DESIGN: Clinical trials. METHODS: The study comprised 263 eyes (132 patients). Laser in situ keratomileusis was performed using the 130 µm head of the Carriazo pendular microkeratome; right eyes were treated first. Ultrasound pachymetry and topography were used for central corneal thickness (CCT) and keratometry (K) measurements. Evaluation included flap thickness, flap diameter, and flap shape. RESULTS: The mean flap thickness was 125 µm ± 22 (SD) (range 74 to 187 µm) in right eyes and 112 ± 21 µm (range 61 to 190 µm) in left eyes. Flap thickness was significantly correlated with preoperative CCT (r = 0.271; P<.001) but not with K values or the manifest refraction spherical equivalent (P>.15). Right eyes had thicker flaps than left eyes (P<.001); both were significantly below the 130 µm head thickness (mean flap thickness 119.2 ± 22.8 µm; P<.001). The mean achieved flap diameter was 9.2 mm using the 9.0 ring and 9.8 mm using the 10.0 mm ring. Flap-thickness stabilization and convergence between right eyes and left eyes occurred after 100 consecutive flap cuts. CONCLUSIONS: Flap-thickness predictability was influenced by preoperative CCT only. All cuts were significantly thinner than the head thickness regardless of the suction ring size. Second surgical eyes had thinner flaps, possibly from blade deterioration from the first cut. Approximately 100 flaps were required as a learning curve.