Control Corneas Research Articles

Comparison of Corneal Biomechanical Efficacy Between Rose Bengal-Green Light and Riboflavin-UVA Crosslinking

Purpose To investigate corneal biomechanical changes after corneal cross-linking (CXL) treatments with rose bengal–green light (RB-CXL) and riboflavin-UVA (RF-CXL). Methods A total of 60 freshly enucleated lamb eyes were obtained for this experimental study. Fifteen eyes were treated with RB-CXL using 0.1% RB solution (Group 1), 15 eyes were treated with RB-CXL using 0.2% RB solution (Group 2), 15 eyes were treated with RF-CXL using 0.1% RF solution (Group 3), and 15 eyes were used as controls (Group 4). The same treatment protocol (10-minute irradiation using a total of 5.4 J/cm2 energy) was applied to all treatment groups. To evaluate corneal biomechanical changes, the stress-strain test was used for both the treated and control corneas. The elastic modulus was calculated using the tension strain curves obtained during the test. Results The average elastic modulus values were calculated to be 18.9, 23.5, 22.3, and 14.1 MPa in Groups 1, 2, 3, and 4, respectively. Statistically significant differences were found between the groups (p < 0.001 for Group 1 vs. 2; p < 0.001 for Group 1 vs. 3; p < 0.001 for Group 1 vs. 4; p = 0.002 for Group 2 vs. 3; p < 0.001 for Group 2 vs. 4; and p < 0.001 for Group 3 vs. 4) Conclusions In this study, the efficacy of RB-CXL treatment applied using different concentrations of RB solutions at a total energy of 5.4 J/cm2 was investigated, and 0.2% RB solution was found to have at least as much and even more effective than the RF-CXL (0.1% RF) on the corneal elasticity module. These results are encouraging for the treatment of ectatic corneas particularly below 400 μm. It is considered that the findings obtained from this study will guide future experimental and clinical studies.

Evidence for persistent UV-induced DNA damage and altered DNA damage response in xeroderma pigmentosa patient corneas

Xeroderma pigmentosum (XP) is a rare genetic disorder characterized by injury to the ocular surface due to exposure to ultraviolet (UV) radiation. UV-induced damage in the cells leads to the formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts that are repaired by the NER (Nucleotide Excision Repair) pathway. Mutations in the genes coding for NER proteins, as reported in XP patients, would lead to sub-optimal damage repair resulting in clinical signs varying from photo-keratitis to cancerous lesions on the ocular surface. Here, we aimed to provide evidence for the accumulation of DNA damage and activation of DNA repair pathway proteins in the corneal cells of patients with XP. Corneal buttons of patients who underwent penetrating keratoplasty were stained to quantify DNA damage and the presence of activated DNA damage response proteins (DDR) using specific antibodies. Positive staining for pH2A.X and thymidine dimers confirmed the presence of DNA damage in the corneal cells. Positive cells were found in both control corneas and XP samples however, unlike normal tissues, positive cells were found in all cell layers of XP samples indicating that these cells were sensitive to very low levels of UV. pH2A.X-positive cells were significantly more in XP corneas (p < 0.05) indicating the presence of double strand breaks in these tissues. A positive expression of phosphorylated-forms of DDR proteins was noted in XP corneas (unlike controls) such as ataxia telangiectasia mutated/Rad-3 related proteins (ATM/ATR), breast cancer-1 and checkpoint kinases-1 and -2. Nuclear localization of XPA was noted in XP samples which co-localized (calculated using Pearson's correlation) with pATM (0.9 ± 0.007) and pATR (0.6 ± 0.053). The increased presence of these in the nucleus confirms that unresolved DNA damage was accumulating in these cells thereby leading to prolonged activation of the damage response proteins. An increase in pp53 and TUNEL positive cells in the XP corneas indicated cell death likely driven by the p53 pathway. For comparison, cultured normal corneal epithelial cells were exposed to UV-radiation and stained for DDR proteins at 3, 6 and 24 h after irradiation to quantify the time taken by cells with intact DDR pathway to repair damage. These cells, when exposed to UV showed nuclear translocation of DDR proteins at 3 and 6 h which reduced significantly by 24 h confirming that the damaged DNA was being actively repaired leading to cell survival. The persistent presence of the DDR proteins in XP corneas indicates that damage is being actively recognized and DNA replication is stalled, thereby causing accumulation of damaged DNA leading to cell death, which would explain the cancer incidence and cell loss reported in these patients.

Glycosaminoglycans and collagen fibril distribution at various depths of the corneal stroma of normal and CXL treated rats

Corneal collagen cross-linking (CXL) is widely used to treat keratoconus and ecstatic corneal disorders. The present studies were carried out to investigate the distribution of glycosaminoglycans (GAGs) and collagen fibril (CF) at different depths of the normal and CXL treated corneal stroma of four week old rats 7 days after standard CXL application. Ten Wistar rats’ corneas were used for the study. The epithelium of the cornea from the left eye of each rat was removed and treated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm2 for 30 min). The cornea from the right eye was used as the control cornea. The cornea was removed from the eye and processed for transmission electron microscopy. A bottom mounted Quemesa camera was used to capture digital images and these images were analysed using iTEM software. In the control cornea, the GAGs area size was not significantly different in the anterior, middle, and posterior stroma. In the CXL treated rats the GAGs area size gradually increased from the anterior to the posterior stroma whereas the spacing between the GAGs gradually decreased. There were very large GAGs present in the posterior stroma of the CXL treated rats. When comparing the control and CXL cornea, the GAGs area in the CXL cornea was significantly higher and inter-GAGs-spacing was smaller than in the control cornea. In the control cornea, the collagen fibrils diameter was higher in the anterior stroma and lowest in the posterior stroma. In the CXL treated cornea, the CF diameter and the interfibrillar spacing gradually decreased from the anterior to the posterior stroma. On comparison between the control and the CXL treated cornea, the interfibrillar spacing was significantly smaller in the CXL treated cornea than the control cornea in the anterior, middle, and posterior stroma but there was no difference in the diameter. The CXL treatment significantly increased the GAGs area and decreased the inter-GAGs-spacing, and inter–CF–spacing. This could be due to the gradual decline in the availability of riboflavin, UVA, and oxygen in the middle and posterior stroma. Further studies are required to investigate the role of keratan sulphate and chondroitin sulphate by using monoclonal antibodies with immunogold technique.

Intraoperative collagen imaging of sutured cornea: A way towards managing post-penetrating keratoplasty astigmatism

The birefringent nature of the human cornea plays an important role in comprehending its structural behavior in both diseased and surgical conditions. During corneal transplantation, irregular astigmatism is a common post-surgical complication that depends on the characteristics of suturing. Four human cadaver corneas are subjected to an in-vitro model of a typical full-thickness penetrating keratoplasty (PK) procedure using 16 simple interrupted 10–0 vicyrl sutures. The birefringence of these four corneas is analyzed using digital photoelasticity and compared with the control cornea (without PK). It is found that the sutures and their mutual interaction influence the morphology of the peripheral birefringence of the cornea. The findings of the present investigation are pertinent to intraoperative suture management during PK. Results suggest conserving the typical diamond-shaped morphology of peripheral birefringence would ensure uniform distribution of sutures. Therefore, birefringence imaging could be useful in suture management to ensure proper apposition of the graft-host junction, thus minimizing the risk of irregular astigmatism.

Birefringence analysis of collagen supraorganization in cat corneas with tropical keratopathy.

To evaluate the birefringent properties of the cornea and examine the supraorganizational aspects of collagen fibers in cats with tropical keratopathy. In this study, 10-micrometer-thick sections of corneal tissue from cats with tropical keratopathy were examined, both in the opaque and transparent areas of the anterior stroma. Control samples were obtained from healthy cat corneas. Polarized light microscopy was employed to evaluate the birefringent properties using two distinct methods. The first method involved measuring the optical retardation associated with corneal birefringence, while the second method assessed the alignment/waviness of the birefringent collagen fibers. Differences were significant when p < .05. Tropical keratopathy resulted in a significant rise (p < .05) in optical retardation in both opaque and transparent regions of the cat cornea. In the anterior stroma, both the opaque zones and transparent tissue exhibited a higher degree of collagen fiber packing than the control corneas. However, no significant differences (p > .05) in alignment were observed between the transparent tissue of the diseased cornea and the healthy corneas. Supraorganizational changes in collagen fiber packing are not restricted to lesion zones in cat corneas affected by tropical keratopathy. Such alterations also occur in the corneal tissue of the anterior stroma adjoining the lesions. Therefore, it is plausible that the transparent tissue of the anterior stroma in corneas affected by the disease may have functional abnormalities, despite its macroscopic healthy appearance. Additional investigations are required to clarify the implications of these potential defects and their conceivable contribution to tropical keratopathy.

Porcine Cornea Storage ExVivo Model as an Alternative to Human Donor Tissues for Investigations of Endothelial Layer Preservation.

Due to the growing shortage of human corneas for research, we developed a porcine cornea storage model with qualitative features comparable to human tissues. We established a decontamination procedure for porcine eye bulbs to ensure corneal storage at 31°C to 35°C for up to 28 days without contamination. We compared human and porcine corneas under hypothermic (2-8°C) or culture (31-35°C) conditions for central corneal thickness (CCT), corneal transparency, endothelial morphology, endothelial cell density (ECD), and a novel method to quantify whole endothelial mortality. We also examined portions of lamellar tissues consisting of Descemet's membrane and endothelial cells under the microscope after Alizarin red staining. Our decontamination procedure reduced corneal contamination from 94% (control corneas without decontamination) to 18% after 28 days of storage at 31°C to 35°C. ECD, CCT, transparency, and morphology were significantly higher in porcine corneas than in human corneas at day 0. Nevertheless, the qualitative parameters of porcine and human corneas showed comparable trends under both investigated storage conditions for up to 14 days. The presented corneal storage model provides a reliable alternative to human tissues for preliminary corneal investigations. The porcine cornea storage model can be used to investigate the efficacy and safety of new media, substances, or storage conditions. Furthermore, the method developed to assess the percentage of endothelial mortality is tissue conservative and can be used in eye banks to monitor endothelial mortality during storage of tissues intended for transplantation.

Characterization of Descemet membrane of Fuchs endothelial corneal dystrophy by chromatic confocal microscopy

AbstractPurpose: Fuchs corneal endothelial dystrophy (FECD) causes chronic corneal edema and vision loss, and accounts for more than 50% of keratoplasty indications in occidental countries. The Descemet membrane (DM) gradually thickens because of extracellular matrix deposition and round excrescences named guttae accumulate. Chromatic confocal microscopy (CCM) is on optical device that analyses the nanotopography of surfaces.Aim: to study the feasibility of CCM technology to characterize guttae in FECD.Methods: Descemetorhexis of approximately 8 mm in diameter were obtained during endothelial grafts in 11 patients with FECD and control corneas were obtained from 3 donor corneas. The DM were fixed in 0.5% paraformaldehyde immediately after dissection. The CCM analysis was performed at room temperature on surfaces of 400 x 400 μm to characterize the DM with and without guttae. We measured positive and negative heights (peaks and valleys) relative to the mean altitude, as well as their distribution by asymmetry coefficient.Results: CCM resolution allowed to map nanotopography of the DM of FECD patients and controls. The surface of DM control was flat and homogeneous with maximum positive and negative heights of 1.07 ± 0.09 μm and 1.05 ± 0.07 μm, respectively. The height distribution was uniform with an asymmetry coefficient of 0.002 ± 0.23 μm, reflecting a Gaussian distribution (npeaks = nvalleys). The surface of FECD membranes was completely altered with a maximum peak of 6.05 ± 1.49 μm, up to 5.7 times higher than the control. The height distribution was disturbed with asymmetry value of 1.61 ± 0.79 μm (npeaks &gt; nvalleys) and a maximum valley height (3.09 ± 0.79 μm) that was half that of the peak one.Conclusions: CCM is a promising technology to quantitatively characterize the 3D organization of the DM surface. CCM can be used to classify the different types of surface lesions of the FECD corneas and to follow evolution of DM alteration at different stages of the disease.

Expression Pattern of Tenascin-C, Matrilin-2, and Aggrecan in Diseases Affecting the Corneal Endothelium.

Purpose: The aim of this study was to examine the expression pattern of tenascin-C, matrilin-2, and aggrecan in irreversible corneal endothelial pathology such as pseudophakic bullous keratopathy (PBK) and Fuchs’ endothelial corneal dystrophy (FECD), which most frequently require corneal transplantation. Materials and methods: Histological specimens of corneal buttons removed during keratoplasty were investigated in PBK (n = 20) and FECD (n = 9) and compared to healthy control corneas (n = 10). The sections were studied by chromogenic immunohistochemistry (CHR-IHC) and submitted for evaluation by two investigators. Semiquantitative scoring (0 to 3+) was applied according to standardized methods at high magnification (400x). Each layer of the cornea was investigated; in addition, the stroma was subdivided into anterior, middle, and posterior parts for more precise analysis. In case of non-parametric distribution Mann–Whitney test was applied to compare two groups. Kruskal–Wallis and Dunn’s multiple comparisons tests have been applied for comparison of the chromogenic IHC signal intensity among corneal layers within the control and patient groups. Differences of p < 0.05 were considered as significant. Results: Significantly elevated tenascin-C immunopositivity was present in the epithelium and every layer of the stroma in both pathologic conditions as compared to normal controls. In addition, also significantly stronger matrilin-2 positivity was detected in the epithelium; however, weaker reaction was present in the endothelium in PBK cases. Minimal, but significantly elevated immunopositivity could be observed in the anterior and posterior stroma in the FECD group. Additionally, minimally, but significantly higher aggrecan immunoreaction was present in the anterior stroma in PBK and in the posterior stroma in both endothelial disorders. All three antibodies disclosed the strongest reaction in the posterior stroma either in PBK or in FECD cases. Conclusions: These extracellular matrix molecules disclosed up to moderate immunopositivity in the corneal layers in varying extents. Through their networking, bridging, and adhesive abilities these proteins are involved in corneal regeneration and tissue reorganization in endothelial dysfunction.

Enzymatic Digestion of Fresh-Frozen Human Cornea After Riboflavin/Ultraviolet-A Collagen Crosslinking

Objective: Crosslinking (CXL) of the cornea by using riboflavin (RBF)/ultraviolet-A (UVA) has been developed for stiffening the collagenous matrix of the cornea. Collagenases and other metalloproteinases are known to play an important role in the pathogenesis of corneal ulceration. Our aim in this study was to show and compare the morphological and histopathological changes of the CXL human corneas against enzymatic digestion by collagenase, trypsin and pepsin solutions. Materials and Methods: Eighteen corneas from nine fresh frozen cadavers were included in this study. The crosslinked group (n=12) was treated with the combined riboflavin (RBF) and ultraviolet-A (UVA) and the control group (n=6) was only treated with RBF. After treatment corneas were exposed to collagenase, trypsin and pepsin enzymes. For each enzyme group one cornea was evaluated morphologically and the other one was evaluated microscopically. Results: The collagenase treated control group, digestion of the cornea was noted in the first days and completed on the 5th day. The control cornea treated with pepsin was fully digested after 14 days. In the trypsin treated CXL cornea complete digestion was noted after 18 days. In both groups, enzyme activity was observed to be parallel to each other morphologically, but CXL cornea was evaluated to be more resistant to enzymatic digestion. Conclusion: In conclusion, CXL increases resistance of the cornea against enzymatic digestion. In addition to the biomechanical support, this study was also showing the histopathological changes of CXL procedure and the results of enzymatic digestion, supporting new treatment options in the corneal diseases.

Longitudinal assessment of the effect of alkali burns on corneal biomechanical properties using optical coherence elastography.

Eye injury due to alkali burn is a severe ocular trauma that can profoundly affect corneal structure and function, including its biomechanical properties. Here, we assess the changes in the mechanical behavior of mouse corneas in response to alkali-induced injury by conducting longitudinal measurements using optical coherence elastography (OCE). A non-contact air-coupled ultrasound transducer was used to induce elastic waves in control and alkali-injured mouse corneas in vivo, which were imaged with phase-sensitive optical coherence tomography. Corneal mechanical properties were estimated using a modified Rayleigh-Lamb wave model, and results show that Young's modulus of alkali-burned corneas were significantly greater than that of their healthy counterparts on days 7 (p=0.029) and 14 (p=0.026) after injury. These findings, together with the changes in the shear viscosity coefficient postburn, indicate that the mechanical properties of the alkali-burned cornea are significantly modulated during the wound healing process.

Biomechanical Evaluation of Topographically and Tomographically Normal Fellow Eyes of Patients With Keratoconus.

To determine the effectiveness of parameters and indices based on biomechanical measures at discriminating fellow eyes with topographically and tomographically normal corneas in patients with keratoconus from normal control corneas. The study included 47 keratoconus suspect eyes, defined as the topographically and tomographically normal fellow eyes of patients with frank keratoconus in the other eye. Eyes were imaged using the Pentacam HR and Corvis ST (both Oculus Optikgeräte GmbH). Fellow eyes were then categorized as topographically/tomographically normal fellow eyes (TNF) and topographically/tomographically borderline fellow eyes (TBF). The ability of each of the Corvis Biomechanical Index (CBI), Tomographic and Biomechanical Index (TBI), stiffness parameter at applanation 1 (SP-A1), and stress-strain index (SSI) at discriminating between normal controls and keratoconus suspects was assessed. The TBI had the best discriminative ability with the greatest area under the receiver operating characteristic (AUROC) curve value of 0.946 for normal controls versus TBF eyes, and 0.824 for normal controls versus TNF eyes. Compared to the TBI AUROC curves, SP-A1 and CBI had AUROC curve values of 0.833 (P = .09) and 0.822 (P = .01) for normal controls versus TBF eyes, respectively, and AUROC curve values of 0.822 (P = .96) and 0.550 (P = .0002) for normal controls versus TNF eyes, respectively. The TBI had the best positive predictive value for TNF and TBF eyes, followed by CBI and SP-A1. The TBI and the purely biomechanical parameter SP-A1 were of moderate utility in distinguishing between normal and keratoconus suspect eyes. In the absence of topographic/tomographic evidence of keratectasia, an independently abnormal biomechanical parameter may suggest an increased risk of ectasia. [J Refract Surg. 2022;38(5):318-325.].

Effect of Ultraviolet-A and Riboflavin treatment on the architecture of the center and periphery of normal rat cornea: 7 days post treatment

Corneal collagen cross-linking (CXL) is a treatment that is widely applied to halt the progression of ectatic diseases such as keratoconus by creating biomechanical strength in the cornea. Most of the studies assessed the effect of the CXL on the cornea without any differentiation of its effect between periphery and the center of the untreated control cornea especially after the 7 days of CXL application. We investigate the ultrastructural changes in the architecture of the center and periphery of rat corneas, 7 days after standard CXL application. Five Wistar rats (10 corneas) were used in the present study. The left eye corneas (5 mm area) were de-epithelialized and irradiated with standard CXL application using riboflavin and Ultraviolet-A (UVA) (3 mW/cm2 for 30 min). The right eye corneas were used as a control. The sclera-cornea button was removed and processed for electron microscopy. Digital images were captured with a bottom mounted Quemesa camera and analyzed using the iTEM software. The ultrastructure of epithelium, hemi-desmosomes, Bowman's layer and stroma were organized in both untreated control and CXL rat cornea in both untreated control and CXL rat cornea. Within the same CXL cornea, both the collagen fibril (CF) diameter and interfibrillar spacing at the center were significantly smaller compared to the peripheral diameter and spacing of the cornea. When comparing the untreated control and CXL cornea, the central interfibrillar spacing of the CXL cornea was significantly smaller than the central spacing the untreated control cornea. In the CXL cornea the peripheral spacing was significantly higher compared to the peripheral interfibrillar spacing of the untreated control cornea. Within the CXL cornea, the proteoglycans (PGs) area and density of the periphery was significantly higher compared to the area and density of the center of the cornea. It suggests that CXL was more effective at the periphery of the cornea. This could be due to the higher amount of leucine rich PG lumican and higher diffusion of oxygen and riboflavin at the periphery cornea.

Correlation of the Corvis Biomechanical Factor with tomographic parameters in keratoconus.

To investigate the relationship between corneal biomechanics and keratoconus (KC) severity as described by tomographic parameters. University-based German ophthalmology department. Retrospective cross-sectional study. A total of 448 KC corneas of the Homburg Keratoconus Center and 112 healthy corneas (448+112 patients) were examined by Pentacam high-resolution and Corneal Visualization Scheimpflug Technology (Pentacam HR and Corvis ST). The KC population included a wide spectrum of disease severity based on Belin's ABCD classification. Linear regression analysis was performed between the linear term of the Corvis Biomechanical Index (CBI) (CBI beta) and the tomographic values anterior radius of curvature (ARC), posterior radius of curvature (PRC), and thinnest corneal thickness (TCT). A linear transformation of the CBI beta was performed to provide an intuitive scaling, which was referred to as the Corvis Biomechanical Factor (CBiF = -0.24294226 × CBI beta + 6.02). This scaling adjusted the CBI beta to the same scale as posterior corneal curvature (PRC). There was a high correlation of the CBI beta and its modification, the CBiF, with TCT (Pearson, r = -0.775), ARC (r = -0.835), and PRC (r = -0.839) in the KC population (P < .001). In the control corneas, the correlation between the CBI beta and ARC was weak (r = -0.216, P = .022), not significant (PRC, r = -0.146, P = .125), or moderate (TCT, r = -0.628, P < .001). The linear term of the CBI was highly associated with KC severity as defined by corneal tomography. The CBiF represents a new scale based on biomechanical characteristics in KC, which could serve as a basis for a biomechanical KC classification in the future.

Long Noncoding RNA 3632454L22Rik Contributes to Corneal Epithelial Wound Healing by Sponging miR-181a-5p in Diabetic Mice.

PurposeThis work explores the abnormal expression of long noncoding RNAs (lncRNAs), microRNAs (miRNAs) and messenger RNAs (mRNAs) in diabetic corneal epithelial cells (CECs) and constructs an associated competitive endogenous RNA (ceRNA) network. Moreover, we revealed that Rik may exert advantageous effects on diabetic corneal epithelial wound closure by sponging miR-181a-5p.MethodsWe obtained the profiles of differentially expressed lncRNAs (DELs) of CECs of type 1 diabetic versus control corneas by microarray and summarized the differentially expressed miRNAs (DEmiRs) and differentially expressed genes (DEGs) data by published literature. Subsequently, the ceRNA network was constructed using bioinformatics analyses. The levels of lncRNA ENSMUST00000153610/3632454L22Rik (Rik) and miR-181a-5p were verified. The localization of Rik was identified with fluorescence in situ hybridization (FISH), and dual-luciferase assays proved the targeted relationship between Rik and miR-181a-5p. Furthermore, we validated the functional impact of Rik in vitro.ResultsOverall, 111 upregulated and 117 downregulated DELs were detected in diabetic versus control CECs. The level of Rik located in both the cytoplasm and the nucleus was clearly downregulated, whereas miR-181a-5p was upregulated in vitro and in vivo in the diabetic group versus the control group. Rik can act as a ceRNA to bind to miR-181a-5p, thus promoting diabetic corneal epithelial wound healing in vitro.ConclusionsThis work investigated the expression profile of DELs and constructed ceRNA networks of diabetic CECs for the first time. Furthermore, we revealed that Rik may positively impact diabetic corneal epithelial wound healing by sponging miR-181a-5p, providing a novel potential therapeutic target of diabetic keratopathy (DK).

Corneal biomechanical properties following corneal cross-linking: Does age have an effect?

PurposeTo explore the effect of age on corneal biomechanical properties following corneal cross-linking (CXL). MethodsA total of 12 pairs of human eye-banked corneas (24 corneas, from 14 females and 10 males) were used in the study. The mean donor age was 48.5 years (ranging from 26 to 71 years). Corneas were divided into three age groups: A (26–41 years), B (42–57 years) and C (58–71 years), with four pairs in each group. For each pair, the right corneas were cross-linked using accelerated CXL with UVA (10 mW/cm2) and riboflavin, while the left corneas served as controls and were not exposed to either UVA irradiation or riboflavin. The corneal elastic modulus of the anterior, mid and posterior corneal stroma was measured using nanoindentation. ResultsThe difference in the corneal elastic modulus following CXL was significant in the anterior (p = 0.00002) and mid stroma (p = 0.001); however, the difference was not significant in the posterior stroma (p = 0.27) when compared to control corneas. The corneal elastic modulus of the anterior stroma increased by 178.44% in Group A, 119.7% in Group B and 50.73% in Group C compared to control corneas. For the mid stroma, the elastic modulus increased by 47.35% in Group A, 25% in Group B and 24.56% in Group C. No differences were observed in the posterior stroma between age groups. ConclusionsCorneal elasticity showed a greater response to CXL in the younger group compared to older groups. CXL treatment showed effectiveness in enhancing stromal strength, and the effect was concentrated in the anterior and mid stroma with minimal impact on the posterior stroma in all age groups.

Cytokeratin 13 Is a New Biomarker for the Diagnosis of Limbal Stem Cell Deficiency.

The purpose of this study was to evaluate the expression of cytokeratin (K) 13 on the corneal surface and to validate its application in the diagnosis of limbal stem cell deficiency (LSCD). This prospective comparative study included 26 corneal impression cytology (IC) specimens from patients diagnosed with LSCD. Twenty-three IC specimens from normal donors served as controls. K12 and K13 expression were detected on the IC specimens by immunohistochemistry study. The number of K12 + or K13 + cells in all areas of the IC was quantified using ImageJ software. The epithelial cells harvested from IC specimens from control corneas were all K12 + . In eyes with LSCD, K13 + and K12 + /K13 + cells accounted for 93.8% and 2.6%, respectively, in the cornea. In eyes with sectoral LSCD, the median number of K13 + cells in the clinically affected area was higher than that in the unaffected area (810.0 vs. 115.0 cells/mm 2 ; P < 0.001). No significant correlation was found between the LSCD severity and the number of K12 + cells (r = -0.284, P = 0.16) or K13 + cells (r = -0.011, P = 0.95). The presence of at least 16 K13 + cells/mm 2 was suggestive of LSCD. Identification of K13 + cells on IC specimens provides a simple and reliable method to detect conjunctival epithelial cells on the cornea. K13 is a marker for diagnosing LSCD and localizing the involved area in sectoral LSCD.

Topical onion juice mitigates the morphological alterations of the cornea in the aged male rats

Aging is associated with structural and functional changes of the cornea. Fresh onion juice contains phenolic compounds and flavonoids that may provide an anti-aging effect. The aim of this study was to assess the ability of the onion juice to ameliorate these aging changes. Rats were grouped as adult and aged groups. Rats of both groups received eye drops of diluted onion juice in their right eyes every 8 hours for 12 weeks, while the left ones were served as control eyes. The corneas of both eyes underwent histopathological, immunohistochemical and morphometric assessments, in addition to measuring their intraocular pressure (IOP). The aged group exhibited a significantly elevated IOP, decreased tear secretion, degenerated corneal epithelium and endothelium, surface erosions and stromal edema with irregular collagen fibers. Administration of onion juice led to lowering of IOP, significant increase in tearing, restoration of most of epithelial, endothelial and stromal integrity, and increased epithelial, keratocystic and endothelial cell densities. Immunohistochemically, the epithelium and endothelium revealed positive immune reactions for both epidermal growth factor receptor (EGFR) and paired box protein-6 (PAX6) in the control and onion-treated corneas of the adult group, while these immune reactions were negative in the untreated aged ones. Onion drops in aged corneas showed a positive immune reaction for EFGR and PAX6 involving the epithelial and endothelial layers. In conclusion, topical onion juice improves corneal aging changes through its direct effect, and indirectly through lowering IOP and enhancing tear secretion.

High-Fluence Accelerated Epithelium-Off Corneal Cross-Linking Protocol Provides Dresden Protocol-Like Corneal Strengthening.

PurposeTo assess whether optimized technical settings for accelerated epithelium-off corneal cross-linking may lead to increases in biomechanical stiffness similar to the benchmark 30-minute epithelium-off Dresden protocol.MethodsThree-hundred porcine eyes were divided equally into six groups for analysis. All samples underwent epithelial debridement and soaking with 0.1% iso-osmolar riboflavin solution for 20 minutes. Corneal cross-linking (CXL) was performed using epithelium-off protocols varying in acceleration and total fluence (intensity in mW/cm² * time in minutes, total fluence in J/cm²): standard (S)-CXL (3*30, 5.4), accelerated (A)-CXL (9*10, 5.4), A-CXL (9*13′20″, 7.2), A-CXL (18*6′40″, 7.2), and A-CXL (18*9′15″, 10). Control corneas were not irradiated. The elastic modulus of 5-mm wide corneal strips was measured as an indicator of corneal stiffness.ResultsAll irradiated groups had significantly higher elastic modulus than controls (P < 0.05), with a stiffening effect of 133% S-CXL (3*30, 5.4), 122% A-CXL (9*10, 5.4), 120% A-CXL (9*13′20″, 7.2), 114% A-CXL (18*6′40″, 7.2) and 149% A-CXL (18*9′15″, 10). The high-fluence accelerated epithelium-off protocol (18*9′15″, 10) showed the highest stiffening effect. Elastic modulus at 5% strain (1%–5% strain) showed significant differences between A-CXL (18*9′15″, 7.2) and three other accelerated protocols: A-CXL (9*10, 5.4; P = 0.01), A-CXL (9*13′20″, 7.2; P = 0.003), and A-CXL (18*6′40″, 10; P = 0.0001).ConclusionsAn accelerated high-fluence epithelium-off CXL protocol (18 mW/cm² for 9′15″) was identified to provide a significantly greater stiffening effect than any other accelerated protocols and is indistinguishable from the Dresden protocol, with accelerating irradiation times ranging from 30 to 9 minutes; by combining gentle acceleration with higher fluence, such a protocol does not require supplemental oxygen.Translational RelevanceThis A-CXL (18*9′15″, 10) protocol has the potential to become a new standard in epithelium-off CXL, delivering Dresden protocol–like strengthening over a shorter period.

Role of anterior segment optical coherence tomography angiography in the assessment of acute chemical ocular injury: a pilot animal model study

To examine the use of anterior segment-optical coherence tomography angiography (AS-OCTA) in the assessment of limbal ischemia in an animal model chemical ocular injury. We conducted a prospective study using an established chemical ocular injury model in 6 rabbits (12 eyes), dividing the cornea limbus into 4 quadrants. Chemical injury grade was induced based on extent of limbal injury (0 to 360 degrees) and all eyes underwent serial slit-lamp with AS-OCTA imaging up to one month. Main outcome measure was changes in AS-OCTA vessel density (VD) comparing injured and control cornea limbal quadrants within 24 h and at one month. AS-OCTA was able to detect differences in limbal VD reduction comparing injured (3.3 ± 2.4%) and control quadrants (7.6 ± 2.3%; p < 0.001) within 24 h of ocular chemical injury. We also observed that AS-OCTA VD reduction was highly correlated with the number of quadrants injured (r = − 0.89; p < 0.001; 95% CI − 5.65 to − 1.87). Corneal vascularization was detected by AS-OCTA in injured compared to control quadrants (10.1 ± 4.3% vs 7.0 ± 1.2%; p = 0.025) at 1 month. Our animal pilot study suggests that AS-OCTA was able to detect limbal vessel disruption from various severities of acute chemical insult, and in the future, could potentially serve as an adjunct in providing objective grading of acute ocular chemical injury once validated in a clinical trial.

LOX-1 Regulates Neutrophil Apoptosis and Fungal Load in A. Fumigatus Keratitis

ABSTRACT Purpose To determine whether LOX-1 regulates neutrophil apoptosis and fungal load in A. fumigatus keratitis. Methods Fas, FasL, CASP3, CASP8, CASP9 and BCL2 were tested in normal and infected corneas of C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of LOX-1 neutralizing antibody or inhibitor (Poly I). Clinical score was recored and HE staining was tested. Fungal load in mice corneas was observed by plate counting. Poly morphonuclear neutrophilic leukocytes (PMNs) were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of LOX-1 neutralizing antibody or Poly I. PCR, western blot and immunostaining tested expression of Fas, FasL, CASP3, CASP8, CASP9, BCL2 and cleaved caspase-3. PMNs infiltration and TUNEL-positive cells were assessed by immunofluorescent staining. Flow cytometry assay tested the percentage of apoptosis neutrophils. Results Fas, Fas ligand, caspase-8, caspase-9 and caspase-3 mRNA levels were significantly higher in C57BL/6 mice corneas infected with A. fumigatus than normal corneas. Poly I treatment alleviated the severity and decreased clinical score at 3, 5 and 7 days post infecrion (p.i.). HE staining showed less infiltration in corneal tissue after LOX-1 inhibition. Plate counting experiment showed that number of viable fungus in corneas of Poly I treated group was significantly less than control group. LOX-1 neutralizing antibody or Poly I treatment significantly decreased neutrophil infiltration, the quantity of TUNEL-positive cells, the expression of Fas, Fas ligand, caspase-8, caspase-9, caspase-3, cleaved caspase-3 and the percentage of apoptosis neutrophils compared with control corneas. LOX-1 neutralizing antibody treatment significantly decreased Fas, FasL, CASP3, CASP8, CASP9 and cleaved caspase-3 expression in neutrophils. Conclusion LOX-1 inhibition decrease neutrophil apoptosis and fungal load in A. fumigatus keratitis.