Control Corneas Research Articles

Meganuclease targeting HSV‐1 limits viral endothelitis in vivo

AbstractPurpose The aim of this study is to assess the antiviral property of a meganuclease targeting HSV‐1 in the prevention of HSV endothelitis in a newly develloped rabbit in vivo model.Methods NZW received intracamerular injection of recombinant adeno‐associated virus equipped with a constitutive expression cassette containing either the meganuclease gene or a non‐coding sequence. After treatment with intravitreal triamcinolone acetonide, HSV‐1 F(1) virus recombinant for LacZ reporter was injected in the inner chamber of the eye,for an analysis 2 days later. Corneal edema, keratic precipitates and the number of contamination plaques were evaluated.Results In 4 tests with 24 experimental or control corneas, the meganuclease‐treated groups systematically showed a decrease in their average number of contamination plaques while compared to controls (from 15 to 71%). Presence of protein and inflammatory cells in the aqueous humor or at the endothelium surface, as well as the extension of focal edema, was also limited by the endonuclease.Conclusion Meganuclease gene therapy targeting HSV‐1 DNA restricts degradation of the endothelium initiated by HSV‐1 injection.

Spatial arrangement of collagen fibrils in normal and keratoconus human cornea studied by low‐frequency dielectric spectroscopy

AbstractPurpose Spatial structure of collagen lamellae of the human cornea is thought to be an important determinant of corneal rigidity. Analysis of the low‐frequency (1–107 Hz) dielectric spectra of the normal and keratoconus human cornea in the temperature range of 140‐300 K and for different hydration states were examined.Methods For the measurements, the 100 – 200 μm specimens containing the middle layers of corneal stroma were used. The 9 specimens of healthy, control corneas from Eye Bank were obtained with second microkeratom section during Ultra Thin‐DSAEK. The 12 specimens of keratoconus corneas were manually prepared from corneas removed during penetrating keratoplasty.Results Experimental results were interpreted in terms of ionic diffusion and space charge polarization according to the Sawada’s theory. The new presentation of the dielectric spectra, i.e.: (delta eps’/delta ln f)*f, was used. The presented method and the Sawada’s expression were applied to the analysis of the changes in spatial molecular structure of collagen fibril network in human cornea.Conclusion Fitting procedure of the theoretical function to the experimental data allowed us to determine two diffusive relaxation regions with two structural distance parameters ds, describing spatial arrangement of collagen fibrils in cornea.

The Endothelial Safety of Using a Gentian Violet Dry-Ink “S” Stamp for Precut Corneal Tissue

To determine the endothelium damage resulting from the application of "dry" gentian violet (GV) stromal markings on corneas precut for Descemet stripping automated endothelial keratoplasty (DSAEK) using a Moria "S" Stamp. Five precut corneas had the stromal graft bed marked with GV using the Moria "S" stamp and 6 precut corneas were left unmarked as controls. After applying the ink to the stamp, care was taken to allow the alcohol carrier to dry for 10 seconds before applying the dry dye to the stromal surface. Tissue was then trephinated and stained with calcein AM to assess endothelial viability. Grafts were photographed and digital pixel planometry, using an established analysis technique, was used to compare the damage between the control and experimental groups. The mean percent cell damage of corneas treated with GV "S" stamp (n = 5) was 8.6% (range 4.4-12.9), and it was 8.1% (range 3.9-15.1) in the DSAEK control set (n = 6). Median percent cell damage was 6.7% among GV-treated corneas and 7.4% among control corneas. The distributions were not significantly different between groups (Mann-Whitney U test = 15.0, two-tailed P = 1.0). Moreover, no "S" pattern of damage was seen in any study eye. There were no significant differences in endothelial damage between the 2 groups. GV stromal markings may be applied without undue damage to the endothelium using the dry-ink technique described.

In Vitro Quantification of the Stiffening Effect of Corneal Cross-linking in the Human Cornea Using Radial Shearing Speckle Pattern Interferometry

To quantify the magnitude of the stiffening effect of corneal cross-linking (CXL) by studying intact human corneas exposed to physiological pressure transients. Nine organ-cultured human corneas mounted in artificial anterior chambers were studied. A radial shearing speckle pattern interferometer was used to measure changes in corneal strain following an increase in artificial anterior chamber pressure from 15.0 to 15.5 mmHg before and after treatment. Measurements were taken from all corneas with their epithelium intact before all underwent epithelial debridement. Three specimens were used as controls and did not receive any further treatment; three underwent riboflavin/ultraviolet A (UVA) CXL using 30 minutes of 370-nm irradiation at 3 mW/cm2 following epithelial removal and saturation with 0.1% riboflavin; and three were fixed with the cross-linking agent 2.5% glutaraldehyde. Strain measurements were repeated after these treatments. Young's moduli of individual corneas were calculated by mathematical analysis. Mean donor age was 81.7 years. Before treatment, the mean Young's moduli of the control, riboflavin/UVA CXL, and glutaraldehyde-fixed corneas did not differ significantly: 0.46±0.03, 0.48±0.03, and 0.47±0.03 MPa, respectively. Following treatment these values changed to 0.46±0.2, 2.06±0.22, and 3.48±0.41 MPa, respectively. In proportional terms, this was equivalent to an increase in corneal Young's modulus by a factor of 4.3 (P<.05) following riboflavin/UVA CXL and 7.3 (P<.05) after glutaraldehyde fixation. Riboflavin-UVA CXL increases the stiffness of organ-cultured corneas by a factor of more than four. This finding quantifies the efficacy of CXL in a physiologic configuration.

Analysis of the Role of ZEB1 in the Pathogenesis of Posterior Polymorphous Corneal Dystrophy

To determine how nonsense mutations in the transcription factor ZEB1 lead to the development of posterior polymorphous corneal dystrophy type 3 (PPCD3). Whole-cell extracts were obtained from cultured human corneal epithelial cells (HCEpCs) as a source of ZEB1 protein. DNA-binding assays were performed using the whole-cell extract and oligonucleotide probes consisting of the two conserved E2-box motifs and surrounding nucleotides upstream of COL4A3. ZEB1 and COL4A3 mRNA expression in primary human corneal endothelial cells (HCEnCs) was assayed in both PPCD3 and control corneas by RT-PCR. Immunohistochemistry was used to localize ZEB1 and COL4A3 expression in normal human cornea. Electromobility shift assays (EMSAs) and competition EMSAs demonstrated binding of protein(s) in the cultured HCEpCs to the E2-box motifs in the probes. The supershift EMSA confirmed that ZEB1, demonstrated to be present in the whole-cell extracts, binds to both the proximal and distal E2-box motifs in the COL4A3 promoter region. Both COL4A3 and ZEB1 are expressed in normal HCEnCs, although in PPCD3, ZEB1 expression is decreased and COL4A3 expression is increased compared with levels of both genes in healthy control corneas. Inversely related HCEnC expression levels of ZEB1 and COL4A3 in PPCD3 indicate that ZEB1-mediated alterations in COL4A3 expression are most likely associated with the pathogenesis of this corneal endothelial dystrophy. However, the demonstration of COL4A3 expression in healthy adult primary HCEnCs suggests that PPCD3 is more likely to involve an alteration in the timing and/or degree of COL4A3 expression than to result from the dichotomous change implied by the previously proposed ectopic expression model.

Interleukin-1 receptor role in the viability of corneal myofibroblasts

The purpose of this study was to investigate the role of interleukin-1 (IL-1) in modulating myofibroblast viability in mouse corneas with stromal opacity. Twenty-four female B6; 129S1-Il1r1tm1Roml/J homozygous IL-1RI knockout mice and 24 control B6129SF2/J mice were included in this study. Each mouse had opacity-generating irregular phototherapeutic keratectomy (PTK) performed with an excimer laser in one eye. Groups of 8 mice from each group were euthanized at one month, three months and six months after surgery and the eyes cryo-preserved. The contralateral eye served as unwounded control. Immunohistochemistry was performed for α-smooth muscle actin (SMA) in central sections of all corneas. The TUNEL assay for apoptosis was performed on 8 sections of four eyes from each group. No SMA+ cells were detected in the stroma of unwounded control or knockout corneas. SMA+ myofibroblast density was significantly higher (p < 0.001) in the IL-1RI knockout group than in the control group at one month, three and six months after irregular PTK. Mean TUNEL+ stromal cells in the anterior 50 μm of stroma was significantly lower in the IL-1RI knockout group compared to the control group at six months after irregular PTK (p = 0.04). These results corroborate the findings of recent in vitro work that demonstrated an antagonistic effect of TGFβ and IL-1 on myofibroblast viability, and found that IL-1-triggered myofibroblast apoptosis was suppressed by TGFβ. Thus, IL-1 is an important modulator of myofibroblast viability during corneal wound healing.

Trehalose protects the corneal epithelium in alcohol delamination: a structural and ultrastructural study

During laser subepithelial keratomilieusis (LASEK) the corneal epithelium undergoes to peculiar morphological changes owing to the dilute alcohol used to facilitate its mechanical separation from the stroma. As it was shown that trehalose, a non-reducing disaccharide of glucose, protects corneal epithelial cells from drying [1] and is effective in the treatment of experimental [2] and of human dry eye [3], aim of the present work was to evaluate the advantages of a pretreatment with trehalose to improve the structural and ultrastructural features of the corneal epithelium. Twelve patients undergoing LASEK were consecutively included in the study and treated as follows: topical anesthesia with oxybuprocaine hydrochloride 0.4 %, 20% ethanol in distilled water for 25 seconds followed by Merocel wetting, treatment with hypotonic BSS in distilled water, lifting of the epithelial flap with a beaver blade, excimer laser treatment (0.8 mm flying spot), reposition of the epithelial flap, BSS wash of the entire surface, application of a contact lens for 5-7 days. The right eyes of each patient were pretreated, together with the anesthetic, with 3% trehalose eye drops, whilst the left eyes were used as controls. Small parts of the epithelium were collected with a forceps at the end of the epithelial reposition and were processed for light and transmission electron microscopy. From the micrographs obtained with both techniques a morphometric analysis was also performed. In the controls, the corneal epithelium showed flat superficial cells with well-preserved apical microfolds, wing cells with intracellular vesicles and slightly dilated intercellular spaces, and irregularly shaped basal cells filled with vesicle, separated by wide spaces. In the trehalose-treated epithelium superficial cells showed normal shape, regular apical microfolds and well-preserved intercellular borders; wing cells had a well evident cytoskeleton, sometimes apparently double nuclei and normal intercellular borders, glued by desmosomes. The basal cells demonstrated polygonal shape, round nuclei and evident intracytoplasmic vesicles. The morphometric analysis carried out on the height and on the number of the layers of the corneal epithelium demonstrated in the trehalose-treated group values significantly lower than the control group. Similarly, basal hemidesmosomes were more numerous in the trehalose-treated group when compared to the control group. The morphological changes of the corneal epithelium during LASEK represent a simple and reproducible experimental model to evaluate the antagonists of an acute stress, such as the alcohol delamination. Our results demonstrate that trehalose administration before LASEK is able to maintain better morphological and morphometric features when compared to the control cornea.

Transparency in a Fibrin and Fibrin–Agarose Corneal Stroma Substitute Generated by Tissue Engineering

To examine the transparency characteristics at different times of development in the culture of 2 different types of human corneal stroma substitutes generated by tissue engineering using human fibrin or human fibrin and 0.1% agarose, with human keratocytes entrapped within. The transparency of these artificial corneal stromas was analyzed after 1, 7, 14, 21, and 28 days of development in culture by determining the scattering and absorption coefficients from the spectral reflectance data of each sample using the Kubelka-Munk equations. The scattering coefficient of both types of bioengineered tissues tended to increase with culture time and wavelength until 550 nm, whereby a slight decrease was observed for longer wavelengths. In general, the spectral distribution of the Kubelka-Munk scattering coefficient of the fibrin-agarose corneal constructs was more stable than that of the fibrin constructs. The absorption coefficient of the human fibrin and fibrin-agarose corneal substitutes tended to decrease with increasing wavelength, and their absolute values were higher for fibrin-agarose than for fibrin scaffolds, especially for short wavelengths. In addition, the spectral transmittance behavior of both types of tissue analyzed was similar to the ones of the theoretical and control corneas, with absolute values above 90% for all wavelengths at 28 days of development. The transparency, scattering, and absorption of both fibrin and fibrin-agarose corneal stroma substitutes indicate that these new biomaterials could be adequate for clinical use.

Screening of Donated Whole Globes for Photorefractive Keratectomy

To introduce a screening method for diagnosis of photorefractive keratectomy (PRK) in donated whole globes. Donated whole globes with either no or an unknown history of refractive surgery on family interview were evaluated in the Central Eye Bank of Iran. After disinfection of globes by immersion in 3% povidone iodine for 3 minutes, followed by irrigation with 0.9% normal saline, gross inspection and slit-lamp biomicroscopic examination of the corneas were performed. Corneas with a disciform, round-shaped hazy area within the central 8 mm of the cornea, suggestive of previous PRK (case), and corneas without this criterion (control) were excised and sent for histopathology. Then, sensitivity and specificity of the screening method were calculated by analyzing the histopathology results of case and control corneas. Twelve thousand five hundred fifty-four donated whole globes from 6832 donors were examined. Twenty case corneas from 10 donors and 54 controls from 27 donors were evaluated by histopathology. Age and sex of the donor population in both case and control groups were comparable with the entire donor population. PRK was confirmed by histopathology in all 20 case corneas but not in any of the control corneas. Both the sensitivity and specificity of this screening method were 100%. Gross inspection of donated whole globes after exposure to povidone iodine followed by slit-lamp biomicroscopic examination, with particular attention to finding a midperipheral-to-midperipheral disciform hazy area within the central 8 mm of cornea, is a safe, simple, and inexpensive screening method, with high sensitivity and specificity for diagnosing PRK.

Targeted Decorin Gene Therapy Delivered with Adeno-Associated Virus Effectively Retards Corneal Neovascularization In Vivo

Decorin, small leucine-rich proteoglycan, has been shown to modulate angiogenesis in nonocular tissues. This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the rabbit stroma with adeno-associated virus serotype 5 (AAV5) impedes corneal neovascularization (CNV) in vivo without significant side effects. An established rabbit CNV model was used. Targeted decorin gene therapy in the rabbit stroma was delivered with a single topical AAV5 titer (100 µl; 5×1012 vg/ml) application onto the stroma for two minutes after removing corneal epithelium. The levels of CNV were examined with stereomicroscopy, H&E staining, lectin, collagen type IV, CD31 immunocytochemistry and CD31 immunoblotting. Real-time PCR quantified mRNA expression of pro- and anti-angiogenic genes. Corneal health in live animals was monitored with clinical, slit-lamp and optical coherence tomography biomicroscopic examinations. Selective decorin delivery into stroma showed significant 52% (p<0.05), 66% (p<0.001), and 63% (p<0.01) reduction at early (day 5), mid (day 10), and late (day 14) stages of CNV in decorin-delivered rabbit corneas compared to control (no decorin delivered) corneas in morphometric analysis. The H&E staining, lectin, collagen type IV, CD31 immunostaining (57–65, p<0.5), and CD31 immunoblotting (62–67%, p<0.05) supported morphometric findings. Quantitative PCR studies demonstrated decorin gene therapy down-regulated expression of VEGF, MCP1 and angiopoietin (pro-angiogenic) and up-regulated PEDF (anti-angiogenic) genes. The clinical, biomicroscopy and transmission electron microscopy studies revealed that AAV5–mediated decorin gene therapy is safe for the cornea. Tissue-targeted AAV5-mediated decorin gene therapy decreases CNV with no major side effects, and could potentially be used for treating patients.

Impact of organ culturing on metabolic profile of human corneas: preliminary results

It is suggested that the quality of corneal graft may depend on modifications that appear in the tissue during culturing. The aim of this study was to investigate the differences in the metabolic profile between cultured and noncultured human corneas. Corneas from 12 donors were obtained post-mortem and cultured for 6-20 days. Control corneas were obtained from four patients with malignant melanoma of the chorioidea and were kept frozen at -80 °C until analysed. The metabolic profiles of the samples were investigated using high-resolution, magic angle spinning (1) H nuclear magnetic resonance spectroscopy and special software for: (i) analysis of complex mixtures, (ii) principal component analysis and (iii) specialized statistical analysis. Twenty metabolites were detected and assigned in the corneas. Significant differences in metabolic profiles between cultured and noncultured corneas were revealed. It was also shown in samples kept in culture for 9-14 days that the levels of (i) alanine, formate, lactate and (ii) acetate, alanine, arginine, lactate were elevated in comparison with the samples kept for <9 and more than 14 days, respectively. Corneal culturing affects the metabolic profile of the tissue. The increases in the levels of some metabolites within the second week of culturing likely result from variations in tissue metabolic or enzymatic activity caused by changed (organ culture) environment. As the mechanisms responsible for these changes are not clear, further research is indicated.

Pan-Corneal Endothelial Viability Assessment: Application to Endothelial Grafts Predissected by Eye Banks

To present an experimental method for determining the viable cell pool of corneal endothelia and its application to assessing predissected endothelial grafts. The endothelial cell density (ECD) of five pairs of human organ cultured corneas was determined using a standard counting method with a calibrated image analysis system. A thin posterior graft (30-50 μm) was manually predissected from a cornea chosen at random. Predissected and control corneas were shipped to the remote center, where standard ECD determination was repeated and was immediately followed by a triple Hoechst/ethidium/calcein labeling coupled with image analysis of the whole graft surface. Numeration of nuclei (H+), dead cells (E+), and total area covered by viable cells (C+) allowed the calculation of viable ECD corresponding to the cell density that the cornea may have after redistribution of viable cells over the whole Descemet surface. The median (range) viable ECD was lower than the standard ECD determined immediately earlier in predissected and control corneas: 1628 (1138-2379) and 2065 (1492-2876) cells/mm(2) (P = 0.043), corresponding to -20% (-1%-38%) and -12% (-3%-26%), respectively (P = 0.08). Standard counting by eye banks overestimates the actual pool of viable endothelial cells. This may be the main explanation for the initially rapid decrease in ECD universally described in patients after all types of keratoplasty. Early low postoperative ECD may indicate that surgeons graft fewer living cells than the eye banks' ECD let suppose, rather than a massive pre- and postoperative cell death. The novel concept of viable ECD can be useful for assessing all types of corneal processing.

Multiphoton Microscopy of Ex Vivo Corneas after Collagen Cross-Linking

To investigate changes in the morphology of the corneal stroma after collagen cross-linking (CXL) treatment in bovine and porcine eyes using a nonlinear microscope providing both two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) corneal images. Freshly enucleated eyes were imaged using a tomographic nonlinear imaging method that was highly suitable to track temporal changes in corneal structures. CXL (riboflavin instillation plus UV irradiation) was applied on the enucleated eyes using similar protocols as in the clinic. A set of eyes without treatment were measured to be used as control. In control corneas, SHG images showed a regular distribution of lamellae across the stroma that appeared stable for at least 6 hours postmortem. CXL changed the collagen distribution pattern showing some abnormal structures. TPEF revealed a large reduction in corneal thickness in CXL corneas immediately after treatment. The changes in the distribution of collagen bundles appeared also in corneas treated with riboflavin only, but not followed by UV irradiation. SHG tomography also revealed a partial recovery of the corneal thickness with time. Nonlinear microscopy (in both tomographic and regular XY imaging configurations) was used to study spatial and temporal changes in the cornea during and after CXL on intact ocular globes. SHG imaging showed changes in the morphology of anterior corneal stroma after CXL. Regular collagen patterns turned into random distributed structures with thicker bundles at some localized areas. This might be a consequence of the corneal thickness decrease as a result of riboflavin-dextran instillation.

Significant Inhibition of Corneal Scarring In Vivo with Tissue-Selective, Targeted AAV5 Decorin Gene Therapy

This study tested a hypothesis that tissue-selective targeted decorin gene therapy delivered to the stroma with adeno-associated virus serotype 5 (AAV5) inhibits corneal fibrosis in vivo without significant side effects. An in vivo rabbit model of corneal fibrosis was used. Targeted decorin gene therapy was delivered to the rabbit cornea by a single topical application of AAV5 (100 μL; 6.5 × 10(12) μg/mL) onto the bare stroma for 2 minutes. The levels of corneal fibrosis were determined with stereomicroscopy, slit lamp biomicroscopy, α-smooth muscle actin (αSMA), fibronectin, and F-actin immunocytochemistry, and/or immunoblotting. CD11b, F4/80 immunocytochemistry, and TUNEL assay were used to examine immunogenicity and cytotoxicity of AAV5 to the cornea. Transmission electron microscopy (TEM) was used to investigate ultrastructural features. Slot-blot-quantified the copy number of AAV5-delivered decorin genes. Selective decorin delivery into the stroma showed a significant (P < 0.01) decrease in corneal haze (1.3 ± 0.3) compared with the no-decorin-delivered control rabbit corneas (3 ± 0.4) quantified using slit lamp biomicroscopy. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA, F-actin, and fibronectin proteins (59%-73%; P < 0.001 or <0.01) in decorin-delivered rabbit corneas compared with the no-decorin-delivered controls. The visual clinical eye examination, slit lamp clinical studies, TUNEL, CD11b, and F4/80 assays revealed that AAV5-mediated decorin gene therapy is nonimmunogenic and nontoxic for the cornea. TEM studies suggested that decorin gene delivery does not jeopardize collagen fibrillogenesis as no significant differences in collagen fibril diameter and arrangement were observed in decorin-delivered and no-decorin-delivered control corneas. Tissue-targeted AAV5-mediated decorin gene therapy is effective and safe for treating corneal fibrosis in vivo.

Clinical and histopathologic analysis of superficial tissue proliferation following the implantation of keratoprosthesis

To investigate the clinical and histopathologic features of the superficial tissue proliferation (STP) following the implantation of MICOF keratoprosthesis, and to analyze the formation and treatment of STP. Retrospective study. Eighty-five patients (85 eyes) received MICOF keratoprosthesis surgery from January 2000 through December 2009 in General Hospital of PLA, which included 72 males and 13 females. The mean age of the patients was (45 ± 15) years. Preoperative diagnoses were ocular burn (56 eyes), end-stage of autoimmune dry eye (14 eyes), severe ocular trauma (10 eyes) and repeated graft failure (5 eyes). Postoperatively, STPs of Kpro were observed and treated. The membranes anterior to the optical cylinder were removed and investigated by histological and immunohistochemical methods, and anterior segment specimens from normal eyes were taken as control. Twenty-two (26%) patients presented STP during the follow-up, and proliferations occurred ranging from 2 to 63 months (median, 7 months). The incident rates of STP were 34% (19/56 eyes) in burned eyes, 14% (2/14 eyes) in end-stage dry eye, 10% (1/10 eyes) in severe mechanical ocular trauma, and none in repeated grafts failure. Difference among four groups did not arrive significance statistically (χ(2) = 5.93, P = 0.11). The epithelial proliferations were observed in 11 patients, which were removed easily. To prevent from recurrence, the height of the cylinder was adjusted. Other 4 patients underwent ultra-high frequency ocular surface plastic operation and 7 patients received membranectomy. Histologically, the superficial proliferative membrane was composed of proliferative epithelium and fibrovascular tissue incorporating inflammatory cells. The immunohistochemical staining demonstrated the expression of PCNA increased in the epithelium, compared with control cornea and conjunctiva. Many vimentin-positive fibroblasts and a few α-SMA-positive myofibroblasts presented in the interstitial tissue, and the numbers of CD45RO-positive T cells, CD11c-positive dendritic cells, and CD68-positive macrophages were increased in proliferative membranes. The tissue proliferation around optical cylinder results in membrane formation anterior to the Kpro. The excessive inflammation at the prosthesis-corneal junction and the unsuited height of the optical cylinder might have been the main reasons of STP.

Evaluation of Irradiated Corneas Using Scatterometry and Light and Electron Microscopy.

To determine if sterilization of donor corneas by irradiation alters optical, histologic, or ultrastructural tissue characteristics. Blinded assessment of 10 irradiated and 10 nonirradiated donor corneas by a scatterometer used to objectively measure backscattered corneal light. Light and electron microscopy were performed on samples from both groups. The mean scattering of the nonirradiated (control) corneas was significantly lower (0.0060 ± 0.0034, mean ± SD) than the scattering of the irradiated corneas (0.023 ± 0.0078; P = 0.0001). There was no statistical difference between ages, days in storage, or central corneal thickness of the 2 groups. Light microscopic evaluation revealed attenuation of the irradiated corneal epithelium with superficial stromal clefting. Transmission electron microscopy demonstrated similar fibril diameter in the nonirradiated and irradiated corneas. Although the mean interfibrillar distance was similar between the nonirradiated and irradiated groups, the interfibrillar distance in the superficial stroma was increased in the nonirradiated compared with the irradiated groups (15.4 ± 2.4 vs. 11.8 ± 0.98 nm; P ≤ 0.05). Our data quantitatively support increased light scatter after irradiation, which cannot be attributed to increased corneal thickness. Irradiation alters the histology of the epithelium and the ultrastructure of the superficial corneal stroma. Increased light scatter because of epithelial changes would not be expected to impact postoperative visual function. However, increased light scatter because of stromal changes may affect postoperative visual function if used for optical corneal rehabilitation. Further investigation is warranted to determine whether attenuated epithelium or superficial stromal changes are the cause of increased light scatter in the irradiated corneal tissue.

Reduction in Corneal Volume with Severity of Keratoconus

Purpose: To compare the corneal volume in keratoconic and normal eyes to improve our understanding of the tissue distribution associated with the disease.Materials and Method: The Oculus Pentacam tomographer (Oculus Inc., Wetzlar, Germany) was used to analyze the corneal volume contained within discs with diameters of 3, 5, 7, and 10 mm in 21 patients with keratoconus and 21 matched healthy control subjects.Results: Corneal volume was significantly decreased in the keratoconus group (keratoconus vs. control group: 3.44 ± 0.39 vs. 4.05 ± 0.29 mm3, 10.34 ± 0.95 vs. 11.79 ± 0.84 mm3, 22.80 ± 1.73 vs. 25.26 ± 1.74 mm3, and 57.17 ± 3.94 vs. 61.90 ± 4.12 mm3 for the 3-, 5-, 7-, and 10-mm diameter discs, respectively; p < 0.001). As the corneal disc diameter analyzed increased, fewer differences were found between the control corneas and keratoconic corneas at different stages of the disease. Within the 3-mm and 5-mm diameter discs, significant differences were detected between the control group, moderate keratoconus, and the severe keratoconus groups (p < 0.05). However, within the 10-mm discs, differences were only detected between the control group and the severe keratoconus group (p = 0.005).Conclusions: Corneal volume was significantly decreased in keratoconus, particularly in the central and paracentral area. The decrease in corneal volume in moderate and severe keratoconus as detected by the Pentacam tomographer, may be explained by loss of corneal tissue. In the early stages of the disease, the altered metabolic activity may cause tissue stretching and, as the disease progresses, this stretching is then accompanied by tissue loss.

Efficacious and Safe Tissue-Selective Controlled Gene Therapy Approaches for the Cornea

Untargeted and uncontrolled gene delivery is a major cause of gene therapy failure. This study aimed to define efficient and safe tissue-selective targeted gene therapy approaches for delivering genes into keratocytes of the cornea in vivo using a normal or diseased rabbit model. New Zealand White rabbits, adeno-associated virus serotype 5 (AAV5), and a minimally invasive hair-dryer based vector-delivery technique were used. Fifty microliters of AAV5 titer (6.5×1012 vg/ml) expressing green fluorescent protein gene (GFP) was topically applied onto normal or diseased (fibrotic or neovascularized) rabbit corneas for 2-minutes with a custom vector-delivery technique. Corneal fibrosis and neovascularization in rabbit eyes were induced with photorefractive keratectomy using excimer laser and VEGF (630 ng) using micropocket assay, respectively. Slit-lamp biomicroscopy and immunocytochemistry were used to confirm fibrosis and neovascularization in rabbit corneas. The levels, location and duration of delivered-GFP gene expression in the rabbit stroma were measured with immunocytochemistry and/or western blotting. Slot-blot measured delivered-GFP gene copy number. Confocal microscopy performed in whole-mounts of cornea and thick corneal sections determined geometric and spatial localization of delivered-GFP in three-dimensional arrangement. AAV5 toxicity and safety were evaluated with clinical eye exam, stereomicroscopy, slit-lamp biomicroscopy, and H&E staining. A single 2-minute AAV5 topical application via custom delivery-technique efficiently and selectively transduced keratocytes in the anterior stroma of normal and diseased rabbit corneas as evident from immunocytochemistry and confocal microscopy. Transgene expression was first detected at day 3, peaked at day 7, and was maintained up to 16 weeks (longest tested time point). Clinical and slit-lamp eye examination in live rabbits and H&E staining did not reveal any significant changes between AAV5-treated and untreated control corneas. These findings suggest that defined gene therapy approaches are safe for delivering genes into keratocytes in vivo and has potential for treating corneal disorders in human patients.

Biosynthetic Corneal Implants for Replacement of Pathologic Corneal Tissue: Performance in a Controlled Rabbit Alkali Burn Model

To evaluate the performance of structurally reinforced, stabilized recombinant human collagen-phosphorylcholine (RHCIII-MPC) hydrogels as corneal substitutes in a rabbit model of severe corneal damage. One eye each of 12 rabbits received a deep corneal alkali wound. Four corneas were implanted with RHCIII-MPC hydrogels. The other eight control corneas were implanted with either allografts or a simple cross-linked RHCIII hydrogel. In all cases, 6.25 mm diameter, 350 μm thick buttons were implanted by anterior lamellar keratoplasty to replace damaged corneal tissue. Implants were followed for nine months by clinical examination and in vivo confocal microscopy, after which implanted corneas were removed and processed for histopathological and ultrastructural examination. Alkali exposure induced extensive central corneal scarring, ocular surface irregularity, and neovascularization in one case. All implants showed complete epithelial coverage by four weeks postoperative, but with accompanying suture-induced vascularization in 6 out of 12 cases. A stable, stratified epithelium with hemidesmosomal adhesion complexes regenerated over all implants, and subbasal nerve regeneration was observed in allograft and RHCIII-MPC implants. Initially acellular biosynthetic implants were populated with host-derived keratocytes as stromal haze subsided and stromal collagen was remodeled. Notably, RHCIII-MPC implants exhibited resistance to vascular ingrowth while supporting endogenous cell and nerve repopulation. Biosynthetic implants based on RHC promoted cell and nerve repopulation in alkali burned rabbit eyes. In RHCIII-MPC implants, evidence of an enhanced resistance to neovascularization was additionally noted.

Anterior Keratocyte Depletion in Fuchs Endothelial Dystrophy

To determine if keratocyte populations are different in corneas with Fuchs dystrophy compared with control corneas. Eleven corneas excised during penetrating keratoplasty for Fuchs dystrophy and 5 control corneas of eyes enucleated for choroidal melanoma were examined using light microscopy. Twenty control corneas age-matched to the corneas with Fuchs dystrophy were examined using confocal microscopy in vivo. The number of keratocytes in a full-thickness column of central stroma with frontal area of 1 mm(2), determined using histologic and confocal methods, was compared between corneas with Fuchs dystrophy and controls. By histology, the mean (SD) number of cells in a full-thickness column of stroma in Fuchs dystrophy (12215[1394] cells) was less than in control corneas (15628[710] cells; P<.001). The mean (SD) number of keratocytes in the anterior 10% of the stroma of corneas with Fuchs dystrophy (682[274] cells) was less than in the control corneas measured using histology (1858[404] cells; P<.001) and confocal microscopy (1481[397] cells; P<.001). Keratocytes are depleted by 54% to 63% in the anterior 10% of the stroma of corneas that require penetrating keratoplasty for Fuchs dystrophy. Keratocyte loss might contribute to anterior stromal changes that persist and degrade vision after endothelial keratoplasty.