Hydrogel-Induced Cell Membrane Disruptions Enable Direct Cytosolic Delivery of Membrane-Impermeable Cargo.

Intracellular delivery of membrane-impermeable cargo offers unique opportunities for biological research and the development of cell-based therapies. Despite the breadth of available intracellular delivery tools, existing protocols are often suboptimal and alternative approaches that merge delivery efficiency with both biocompatibility, as well as applicability, remain highly sought after. Here, a comprehensive platform is presented that exploits the unique property of cationic hydrogel nanoparticles to transiently disrupt the plasma membrane of cells, allowing direct cytosolic delivery of uncomplexed membrane-impermeable cargo. Using this platform, which is termed Hydrogel-enabled nanoPoration or HyPore, the delivery of fluorescein isothiocyanate (FITC)-dextran macromolecules in various cancer cell lines and primary bovine corneal epithelial cells is convincingly demonstrated. Of note, HyPore demonstrates efficient FITC-dextran delivery in primary human T cells, outperforming state-of-the-art electroporation-mediated delivery. Moreover, the HyPore platform enables cytosolic delivery of functional proteins, including a histone-binding nanobody as well as the enzymes granzyme A and Cre-recombinase. Finally, HyPore-mediated delivery of the MRI contrast agent gadobutrol in primary human T cells significantly improves their T1 -weighted MRI signal intensities compared to electroporation. Taken together, HyPore is proposed as a straightforward, highly versatile, and cost-effective technique for high-throughput, ex vivo manipulation of primary cells and cell lines.

Isorhamnetin Ameliorates Aspergillus fumigatus Keratitis by Reducing Fungal Load, Inhibiting Pattern-Recognition Receptors and Inflammatory Cytokines.

Purpose: Isorhamnetin is a natural flavonoid with both antimicrobial and anti-inflammatory properties, but its effect on fungal keratitis (FK) remains unknown. The current study aims to investigate the antifungal and anti-inflammatory effects of isorhamnetin against mouse Aspergillus fumigatus keratitis. Methods: In vitro, the lowest effective concentration of isorhamnetin was assessed by minimum inhibitory concentration and cytotoxicity tests in human corneal epithelial cells (HCECs) and RAW264.7 cells. The antifungal property was investigated by scanning electron microscopy and propidium iodide uptake test. The anti-inflammatory effect of isorhamnetin in HCECs and RAW264.7 cells was observed by quantitative real-time polymerase chain reaction (qRT-PCR). In the eyes of mice with A. fumigatus keratitis, FK severity was evaluated using clinical score, plate counting, histological staining and periodic acid Schiff staining. In vivo, the anti-inflammatory effect of isorhamnetin was examined by immunofluorescence staining, myeloperoxidase assay, Western blot, enzyme-linked immunosorbent assay, and qRT-PCR. Results: In HCECs and RAW264.7 cells, isorhamnetin significantly inhibited A. fumigatus conidia growth and hyphae viability at 80 µg/mL without affecting cell viability. In vitro, isorhamnetin altered A. fumigatus hyphal morphology and membrane integrity. In A. fumigatus keratitis mouse model, isorhamnetin treatment alleviated the severity of FK by reducing corneal fungal load and inhibiting neutrophil recruitment. In addition, the mRNA and protein expression levels of TLR-2, TLR-4, Dectin-1, IL-1ß, and tumor necrosis factor-α were significantly decreased in isorhamnetin-treated groups in vivo and in vitro. Conclusions: Isorhamnetin improves the prognosis of A. fumigatus keratitis in mice by inhibiting the growth of A. fumigatus, reducing the recruitment of neutrophils and downregulating inflammatory factors.

Neutralization of the eye and skin irritant benzalkonium chloride using UVC radiation.

PURPOSE: Benzalkonium chloride (BAK) is a widely used disinfectant and preservative which is effective against a wide range of viruses (e.g. SARS-CoV and SARS-CoV-2), bacteria and fungi. However, it is toxic to the eye and skin. This study investigated the neutralization of BAK using ultraviolet C (UVC) radiation as an effort to reduce BAK toxicity potential. METHODS: BAK solutions were irradiated with a germicidal UVC lamp at various doses. Human corneal epithelial cells (HCEC) were then exposed to the UVC-irradiated BAK solutions for 5 minutes. After exposure, the cultures were assessed for metabolic activity using PrestoBlue; for cell viability using confocal microscopy with viability dyes; and for tight junction proteins using immunofluorescence staining for zonula occludens (ZO)-1. RESULTS: UVC radiation reduced BAK toxicity on cell metabolic activity in a dose-dependent manner. When the solution depth of BAK was 1.7 mm, the UVC doses needed to completely neutralize the toxicity of BAK 0.005% and 0.01% were 2.093 J/cm2 and 8.374 J/cm2, respectively. The cultures treated with UVC-neutralized BAK showed similar cell metabolic activity and cell viability to those treated with phosphate buffered saline (PBS) (p = 0.806 ∼ 1.000). The expression of ZO-1 was greatly disturbed by untreated BAK; in contrast, ZO-1 proteins were well maintained after exposure to UVC-neutralized BAK. CONCLUSIONS: Our study demonstrates that the cell toxicity of BAK can be neutralized by UVC radiation, which provides a unique way of detoxifying BAK residues. This finding may be of great value in utilizing the antimicrobial efficacy of BAK (e.g. fighting against SARS-CoV-2) while minimizing its potential hazards to human health and the environment.

Entry of Epidemic Keratoconjunctivitis-Associated Human Adenovirus Type 37 in Human Corneal Epithelial Cells.

Purpose: Ocular infection by human adenovirus species D type 37 (HAdV-D37) causes epidemic keratoconjunctivitis, a severe, hyperacute condition. The corneal component of epidemic keratoconjunctivitis begins upon infection of corneal epithelium, and the mechanism of viral entry dictates subsequent proinflammatory gene expression. Therefore, it is important to understand the specific pathways of adenoviral entry in these cells. Methods: Transmission electron microscopy of primary and tert-immortalized human corneal epithelial cells infected with HAdV-D37 was performed to identify the means of viral entry. Confocal microscopy was used to determine intracellular trafficking. The results of targeted small interfering RNA and specific chemical inhibitors were analyzed by quantitative PCR, and Western blot. Results: By transmission electron microscopy, HAdV-D37 was seen to enter by both clathrin-coated pits and macropinocytosis; however, entry was both pH and dynamin 2 independent. Small interfering RNA against clathrin, AP2A1, and lysosome-associated membrane protein 1, but not early endosome antigen 1, decreased early viral gene expression. Ethyl-isopropyl amiloride, which blocks micropinocytosis, did not affect HAdV-D37 entry, but IPA, an inhibitor of p21-activated kinase, and important to actin polymerization, decreased viral entry in a dose-dependent manner. Conclusions: HAdV-D37 enters human corneal epithelial cells by a noncanonical clathrin-mediated pathway involving lysosome-associated membrane protein 1 and PAK1, independent of pH, dynamin, and early endosome antigen 1. We showed earlier that HAdV-D37 enters human keratocytes through caveolae. Therefore, epidemic keratoconjunctivitis-associated viruses enter different corneal cell types via disparate pathways, which could account for a relative paucity of proinflammatory gene expression upon infection of corneal epithelial cells compared with keratocytes, as seen in prior studies.

Excess Transforming Growth Factor-α Changed the Cell Properties of Corneal Epithelium and Stroma.

Purpose: This study is to investigate the corneal anomaly caused by excess transforming growth factor-α (TGF-α) during mouse development. Methods: Bitransgenic KeraRT/TGF-α mice, generated via cross-mating tetO-TGF-α and KeraRT mice, were induced to overexpress TGF-α by doxycycline commencing at embryonic day 0 or postnatal day 0 to different developmental stages. Bitransgenic mice with doxycycline induction were defined as TGF-αECK mice (TGF-α excess expression by corneal keratocytes). Mouse eyes were examined by hematoxylin and eosin staining, immunofluorescent staining and transmission electron microscopy. Protein and RNA from mouse cornea were subjected to western blotting and real-time quantitative polymerase chain reaction. Results: In TGF-αECK mice, TGF-α overexpression resulted in corneal opacity. Excess TGF-α initially caused corneal epithelial hyperplasia and subsequent epithelium degeneration as the mouse developed, which was accompanied by gradually diminished K12 expression from the periphery of corneal epithelium and increased K13 expression toward the corneal center. Interestingly, K14 was detected in all layers of corneal epithelium of TGF-αECK mice, whereas it was limited at basal layer of controls. Transmission electron microscopy showed desmosome loss between corneal epithelial cells of TGF-αECK mice. In TGF-αECK mice, keratocan expression was abolished; α-SMA expression was increased while expression of Col1a1, Col1a2, and Col5a1 was diminished. Cell proliferation increased in the corneal epithelium and stroma, but not in the endothelium of TGF-αECK mice. Conclusions: Excess TGF-α had detrimental effects on corneal morphogenesis during mouse development in that it changed the cell fate of corneal epithelial cells to assume conjunctival phenotypic expression of K13, and keratocytes to myofibroblast phenotype.

Carboxymethyl Cellulose-Coated Tacrolimus Nonspherical Microcrystals for Improved Therapeutic Efficacy of Dry Eye.

Dry eye (DE) is a highly prevalent ocular surface disease which affects the quality of life and results in low working efficiency. Frequent instillation is required due to low bioavailability of conventional eye drops. The aim of this study is to develop a novel formulation of tacrolimus (TAC), routinely prescribed for DE, by combination of the microcrystal technology and layer-by-layer assembly. First, nonspherical tacrolimus microcrystals (TAC MCs) are synthesized by antisolvent-induced precipitation. These TAC MCs are modified by alternate deposition of poly(allylamine hydrochloride) (PAH) and carboxymethyl cellulose (CMC) subsequently to obtain CMC-coated TAC MCs (TAC-(PAH/CMC)3 ). The resultant formulations are evaluated in vivo in a mouse DE model induced by an intelligently controlled environmental system. Compared with commercially available TAC eye drops and the TAC MCs counterpart, TAC-(PAH/CMC)3 exhibits superior therapeutic performance with reduced drug instillation frequency, which is attributed to the nonspherical geometry of MCs, the lubricant, mucoadhesive effect of CMC, and the anti-inflammatory function of TAC. Therefore, TAC-(PAH/CMC)3 represents a better option for the management of DE.

Corneal damage and its recovery after instillation of preservative-free versus preserved latanoprost eye drops.

Purpose: Latanoprost ophthalmic solution is highly effective as a therapeutic agent for glaucoma and is applied worldwide. However, harmful effects on the corneal surface have been reported regarding the commercially available latanoprost ophthalmic solution. Corneal surface toxicity may be caused by the added preservative of the ophthalmic solution. In order to ascertain whether latanoprost itself can damage the cornea or if this is solely due to the added preservatives, this study attempted to determine the corneal changes that occur at different time periods following usage of preservative-free versus preserved latanoprost eye drops.Materials and methods: Preservative-free latanoprost eye drops (Monoprost®) or preserved latanoprost eye drops (Xalatan®) containing 0.02% benzalkonium chloride (BAC) were instilled in the corneas of rabbits. For each of the two different eye drop solutions, the rabbits used in this experiment were divided into three exposure groups: 1 minute, 24 hour, and 1 week groups. Corneal transepithelial electrical resistance (TER) and scanning electron microscopy (SEM) were examined immediately (1 minute) after instillation, at 24 hours after instillation, and at 24 hours after 1 week of daily instillations of latanoprost. Hank's balanced salt solution was used in the control group.Results: The mean corneal TER of the control group was 933.8 ± 279.0 Ω cm2. In preservative-free latanoprost instilled corneas, there was no significant decrease in the TER or morphological changes at any of the time points, with the relative TER values of 117 ± 38%, 100 ± 34%, and 93 ± 21% for 1 minute, 1 day, and 1 week time points, respectively. In preserved latanoprost instilled corneas, SEM showed that only the immediate group exhibited superficial cell damage and a significant decrease in the corneal TER compared to the controls and other time points and to the immediate preservative-free latanoprost corneas. In the preserved latanoprost groups, the relative TER values were 18 ± 5%, 110 ± 28%, and 92 ± 10%, for the three respective observation time points.Conclusions: Preservative-free latanoprost can be safely instilled to the corneal epithelium. Latanoprost with 0.02% BAC has an immediate deleterious impact on the corneal epithelium; however, it disappears within 24 hours after instillation.

Toxicity profiles of fixed-combination eye drops for glaucoma therapy using cultivated human corneal epithelial sheets.

PURPOSE: We aimed to investigate the toxicity of 6 fixed-combination drugs for glaucoma therapy using human corneal epithelial sheets (HCES). STUDY DESIGN: Experimental. MATERIALS AND METHODS: We used 6 kinds of commercially available fixed-combination drugs: latanoprost/carteolol (LAT/CAR), latanoprost/timolol (LAT/TIM), tafluprost/timolol (TAF/TIM), travoprost/timolol (TRA/TIM), brinzolamide/timolol (BRZ/TIM), and dorzolamide/timolol (DRZ/TIM) including different preservatives. The cell viability and barrier function of the HCES after exposure to the eye drops for 10 or 30 minutes were assessed using the WST-1 assay and transepithelial electrical resistance (TEER) measurements, respectively. The HCES were also evaluated using hematoxylin and eosin (HE) staining and transmission electron microscopy. RESULTS: The cell viability significantly decreased in the HCES treated with LAT/TIM or DRZ/TIM after 10 and 30 minutes and in those treated with BRZ/TIM after 30 minutes. The barrier function increased significantly in the HCES treated with LAT/CAR. Histologically, the HCES were damaged after treatment with LAT/TIM, BRZ/TIM, or DRZ/TIM for 30 minutes. Transmission electron microscopy indicated narrow intercellular spaces and multiple intercellular junctions in the HCES treated with LAT/CAR, TAF/TIM, or TRA/TIM. The HCES treated with DRZ/TIM, BRZ/TIM, or LAT/TIM contained cytoplasmic vacuoles and collapsed cellular structures. CONCLUSION: Glaucoma fixed-combination eye drops demonstrated a different toxic effect on the cell viability, barrier function, and morphologic changes of HCES.

Development and Characterization of an Acellular Porcine Small Intestine Submucosa Scaffold for Use in Corneal Epithelium Tissue Engineering.

Purpose: To produce an acellular small intestine submucosa (SIS) that would be a suitable scaffold for corneal epithelium tissue engineering.Methods: The SIS was decellularized by immersion in 0.1% (wt/vol) sodium dodecyl sulfate (SDS). The efficacy of acellularization was confirmed by histological observation and DNA quantification. The mechanical properties were evaluated by uniaxial tensile testing. ELISA was performed to assess the growth factor contents. The cytotoxicity of SIS scaffolds and extracts to rabbit corneal epithelial cells was determined by CCK-8 assay. We also investigated the inflammatory reaction of SIS implanted subcutaneously in a rat. The biocompatibility was studied by rabbit interlamellar corneal transplantation and reseeding assay with cornea-derived cells. Immunofluorescent staining was used to detect the expression of CK3, ZO-1 and K13.Results: Histological analyses showed that complete cell removal was achieved, and the DNA quantity, which reflects the presence of cellular materials, was significantly diminished in acellular SIS. Collagen fibers were properly preserved and appeared in an orderly fashion. The tissue structure, the mechanical properties and the growth factor contents within the acellular SIS were well retained. The CCK8 assay demonstrated that the acellular SIS scaffolds and extracts had no cytotoxicity to rabbit corneal epithelial cells. There was no sign that an immune reaction occurred with acellular SIS implanted subcutaneously in a rat. In fact, in vivo implantation to rabbit interlamellar stromal pockets showed good biocompatibility. We also observed that clusters of rabbit corneal epithelial cells were growing well on the surface of the SIS in vitro and the distinctive CK3, ZO-1 for corneal epithelial cells was detected.Conclusions: The decellularized SIS retained the major structural components. The matrix is biocompatible with cornea-derived cells and might be a suitable scaffold for corneal epithelium tissue engineering.

Re-epithelialization and remodeling of decellularized corneal matrix in a rabbit corneal epithelial wound model.

This study investigated the in vivo correlation between re-epithelialization and remodeling of a decellularized corneal matrix prepared by a high-hydrostatic pressure (HHP) method in rabbits. Decellularized corneal matrices were transplanted in a 6-mm-diameter recipient corneal interlamellar pocket with a 2 mm epithelial defect. The time course of graft status in rabbits was examined daily for 6 months by biomicroscopy and scored for clarity and re-epithelialization, after which the rabbits were sacrificed for histological analysis. Fluorescein staining revealed that the corneal epithelial cells had migrated onto the decellularized corneal matrix. Histological analysis revealed that the implanted decellularized corneal matrix was completely integrated with the recipient rabbit cornea and the stratified corneal epithelia consisting of multiple layers were regenerated, similar to that in the normal cornea. The recipient keratocytes infiltrated into the decellularized corneal matrix at 6 months after the operation and the decellularized corneal matrix was gradually remodeled into the recipient tissue. Transmission electron microscopy revealed that the ultrastructure of the decellularized corneal matrix was rearranged, similar to the normal cornea. These findings suggest that the decellularized corneal matrix serves as a template for remodeling. The decellularized corneal matrix obtained through HHP is a useful graft for corneal tissue regeneration.

Effects of explant size on epithelial outgrowth, thickness, stratification, ultrastructure and phenotype of cultured limbal epithelial cells.

PURPOSE: Transplantation of limbal stem cells is a promising therapy for limbal stem cell deficiency. Limbal cells can be harvested from either a healthy part of the patient's eye or the eye of a donor. Small explants are less likely to inflict injury to the donor site. We investigated the effects of limbal explant size on multiple characteristics known to be important for transplant function. METHODS: Human limbal epithelial cells were expanded from large versus small explants (3 versus 1 mm of the corneal circumference) for 3 weeks and characterized by light microscopy, immunohistochemistry, and transmission electron microscopy. Epithelial thickness, stratification, outgrowth, ultrastructure and phenotype were assessed. RESULTS: Epithelial thickness and stratification were similar between the groups. Outgrowth size correlated positively with explant size (r = 0.37; P = 0.01), whereas fold growth correlated negatively with explant size (r = -0.55; P < 0.0001). Percentage of cells expressing the limbal epithelial cell marker K19 was higher in cells derived from large explants (99.1±1.2%) compared to cells derived from small explants (93.2±13.6%, P = 0.024). The percentage of cells expressing ABCG2, integrin ß1, p63, and p63α that are markers suggestive of an immature phenotype; Keratin 3, Connexin 43, and E-Cadherin that are markers of differentiation; and Ki67 and PCNA that indicate cell proliferation were equal in both groups. Desmosome and hemidesmosome densities were equal between the groups. CONCLUSION: For donor- and culture conditions used in the present study, large explants are preferable to small in terms of outgrowth area. As regards limbal epithelial cell thickness, stratification, mechanical strength, and the attainment of a predominantly immature phenotype, both large and small explants are sufficient.

Inhibition of microRNA-129-5p expression ameliorates ultraviolet ray-induced corneal epithelial cell injury via upregulation of EGFR.

Existing evidence has highlighted the effect of ultraviolet light radiation leading to corneal epithelium impairment. During this study, we aim to investigate the effect of microRNA-129-5p (miR-129-5p) on the wound healing process of corneal epithelial cells (CECs) induced by ultraviolet rays in mice by targeting epidermal growth factor receptor (EGFR). First, mouse models of ultraviolet ray-induced CEC injury were established and intrastromally injected with different mimic, inhibitor, and short interfering RNA (siRNA) to detect the effect of miR-129-5p on CEC injury. Subsequently, the corneal tissues were obtained to detect the antioxidant ability and EGFR-positive expression rate. The dual-luciferase reporter gene assay was used to test whether EGFR could directly target miR-129-5p. To further investigate the specific mechanism of miR-129-5p and EGFR in CEC injury, CECs were cultured and transfected with miR-129-5p mimic, miR-129-5p inhibitor, siRNA-EGFR, and miR-129-5p inhibitor + siRNA-EGFR. miR-129-5p has been proven to directly target EGFR. Inhibition of miR-129-5p is able to increase the antioxidant capacity, EGFR-positive rate and the expressions of EGFR, B-cell lymphoma-2, zonula occluden-1, occludin, and keratinocyte growth factor-2, but decrease the expression of vascular endothelial growth factor, BCL2-associated X protein, interleukin (IL)-1ß, and IL-4. Inhibition of miR-129-5p arrests cells at the S and G2 phases and decreases apoptosis. Our study provides evidence stating that inhibiting miR-129-5p and upregulating EGFR could aid in the repair of mice CEC injury induced by ultraviolet radiation. Therefore, inhibition of miR-129-5p might provide a basic theory in the repair of CEC injury caused by ultraviolet rays.

Air pollutant particulate matter 2.5 induces dry eye syndrome in mice.

In this study, we explored the effects of particulate matter 2.5 (PM2.5) eye drops on the ocular surface structure and tear function in mice and established a novel animal model for dry eye research. We found that, following treatment with PM2.5, the tear volume and, the tear film break-up time showed statistical differences at each time point (P < 0.05). The FL score of the PM2.5-treated group was higher than that of others (P < 0.05). The average number of corneal epithelial layer cells in groups A and B was significantly lower than that in group C (P < 0.05). Scanning electron microscopy and transmission electron microscopy revealed that the number of corneal epithelial microvilli and corneal desmosomes was drastically reduced in group C. PM2.5 induced apoptosis in the corneal superficial and basal epithelium and led to abnormal differentiation and proliferation of the ocular surface with higher expression levels of Ki67 and a reduced number of goblet cells in the conjunctival fornix in group C. PM2.5 significantly increased the levels of TNF-α, NF-κB p65 (phospho S536), and NF-κB in the cornea. Thus, the topical administration of PM2.5 in mice induces ocular surface changes that are similar to those of dry eye in humans, representing a novel model of dry eye.

Dendritic cell maturation in the corneal epithelium with onset of type 2 diabetes is associated with tumor necrosis factor receptor superfamily member 9.

Type 2 diabetes mellitus is characterized by a low-grade inflammation; however, mechanisms leading to this inflammation in specific tissues are not well understood. The eye can be affected by diabetes; thus, we hypothesized that inflammatory changes in the eye may parallel the inflammation that develops with diabetes. Here, we developed a non-invasive means to monitor the status of inflammatory dendritic cell (DC) subsets in the corneal epithelium as a potential biomarker for the onset of inflammation in type 2 diabetes. In an age-matched cohort of 81 individuals with normal and impaired glucose tolerance and type 2 diabetes, DCs were quantified from wide-area maps of the corneal epithelial sub-basal plexus, obtained using clinical in vivo confocal microscopy (IVCM). With the onset of diabetes, the proportion of mature, antigen-presenting DCs increased and became organized in clusters. Out of 92 plasma proteins analysed in the cohort, tumor necrosis factor receptor super family member 9 (TNFRSF9) was associated with the observed maturation of DCs from an immature to mature antigen-presenting phenotype. A low-grade ocular surface inflammation observed in this study, where resident immature dendritic cells are transformed into mature antigen-presenting cells in the corneal epithelium, is a process putatively associated with TNFRSF9 signalling and may occur early in the development of type 2 diabetes. IVCM enables this process to be monitored non-invasively in the eye.

Role of α1-adrenoceptor subtypes on corneal epithelial thickness and cell proliferation in mice.

Adrenergic stimuli are important for corneal epithelial structure and healing. The purpose of the present study was to examine the hypothesis that the lack of a single α1-adrenoceptor (α1-AR) subtype affects corneal epithelial thickness and cell proliferation. Expression levels of α1-AR mRNA were determined in mouse cornea using real-time PCR. In mice devoid of one of the three α1-AR subtypes (α1A-AR-/-, α1B-AR-/-, α1D-AR-/-) and in wild-type controls, thickness of individual corneal layers, the number of epithelial cell layers, and average epithelial cell size were determined in cryosections. Endothelial cell density and morphology were calculated in corneal explants, and epithelial cell proliferation rate was determined with immunofluorescence microscopy. Moreover, the ultrastructure of the corneal epithelium was examined by transmission electron microscopy. Messenger RNA for all three α1-AR subtypes was expressed in whole cornea and in corneal epithelium from wild-type mice with a rank order of abundance of α1A ≥ α1B > α1D. In contrast, no α1-AR mRNA was detected in the stroma, and only α1B-AR mRNA was found in the Descemet endothelial complex. Remarkably, corneal epithelial thickness and mean epithelial cell size were reduced in α1A-AR-/- mice. Our findings suggest that the α1A-AR exerts growth effects in mouse corneal epithelial cells.

Anterior-Segment Optical Coherence Tomography and Scanning Electron Microscopy to Evaluate Corneal Epithelial Changes in Patients Undergoing Glaucoma Therapy.

PURPOSE: To measure corneal epithelial thickness (CET) in patients with glaucoma using anterior-segment optical coherence tomography and to evaluate CET changes in relation to corneal epithelial microvilli analyzed by scanning electron microscopy (SEM). METHODS: Twenty-two eyes (16 patients) being treated with preservative-containing topical medications and 12 normal eyes underwent anterior-segment optical coherence tomography imaging using RTVue-100. The CET maps generated corresponded to a 6-mm diameter area of cornea that was divided into 17 sectors. We compared the CETs of each sector obtained in the glaucomatous group with those obtained in the control group. RESULTS: Glaucomatous eyes were divided into 2 groups based on the number of microvilli on SEM: group 1 (6 eyes) = grades 1 and 2 at SEM (range: 500-3000) and group 2 (10 eyes) = grades 3 and 4 at SEM (range: 0-500). Four CET sectors were significantly thinner in group 1 than in normal eyes: central (P = 0.012), superior (P = 0.005), temporal paracentral (P = 0.003), and temporal midperipheral (P = 0.023). No significant differences were observed between group 2 and normal eyes. CET sectors were significantly thinner in group 1 than in group 2 only in the superior (P = 0.024) and superior-temporal paracentral (P = 0.020) sectors. CET progressively increased in patients with glaucoma as the number of corneal epithelial microvilli decreased. CONCLUSIONS: CET and corneal epithelial microvilli are new parameters with which to evaluate early stages of corneal epithelial changes during glaucoma therapy. In advanced stages of corneal epithelial damage, SEM evaluation reveals ultrastructural epithelial changes that may not be observed on CET measurements.

Amyloid Beta Deposition Could Cause Corneal Epithelial Cell Degeneration Associated with Increasing Apoptosis in APPswePS1 Transgenic Mice.

OBJECTIVE: To investigate the expression of amyloid precursor protein (APP) and amyloid beta (Aß) in cornea and further explore the pathological and ultrastructural changes in corneal epithelium in APPswePS1 transgenic mice. METHODS: Twelve wild type mice were grouped into control group and twelve TgAPPswePS1 mice at least 8 months old were grouped into the young experiment group (Tg-8M group), and another twelve transgenic mice at least 15 months old were selected into the aged experiment group (Tg-15M group). The pathological degeneration, ultrastructural changes, and the expression of APP, Aß deposition, and the TUNEL reaction in corneal epithelial cells were observed. Western blot analysis was performed to determine expression levels of APP and Aß with scraped epithelial debridement. All the results were quantified and analyzed. RESULTS: In transgenic mice, the H&E-stained cornea sections demonstrated histopathological changes in corneal epithelial cells with irregular arrangement and the number of cell layers decreased, while normal structure observed in controls. In Tg-15M group, the corneal epithelial cell displaced a significant number of intracellular vacuoles with 1-2 cell layers left. Transmission electron microscopy (TEM) further confirmed the dramatic degeneration in corneal epithelium, the microvilli suffered degenerative changes and found with typical fingerpoint-like morphology in controls; however, microspike-like in Tg-15M group, and the number of microvilli decreased considerabely. An APP-positive immunoreaction was detected with a diffuse pattern in the corneal epithelial cells layer, about 3.122 ± 0.596 and 7.372 ± 0.936 fold changes in Tg-8M and Tg-15M groups, respectively, as compared with controls. On corneal flatmount, Aß deposition found a diffuse pattern in the cytoplasm by fluorescence staining in TgAPPswePS1 with significantly increasing as compared with the controls, but no plaque was found. The apoptosis of TUNEL cells were observed in TgAPPswePS1 mice and increased 16.329 ± 3.542 fold changes in Tg-15M group as compared with controls. CONCLUSION: The APP expression and Aß deposition might cause cornea epithelial cells degeneration in TgAPPswePS1 mice, associated with apoptosis in basal lamina cells.

Effect of Nitric Oxide on Acanthamoeba castellanii.

Purpose: Acanthamoeba keratitis is a well-known intractable corneal infectious disease. We investigated the anti-Acanthamoeba effect of exogenous nitric oxide (NO). Methods: Acanthamoeba castellanii was axenically cultured and exposed to various concentrations of NO donors, such as sodium nitrite, sodium nitroprusside (SNP), and NO-releasing silica nanoparticles (coated in branched polyethylene imine, size:100 nm), for 1 to 7 days (sodium nitrite and SNP: 0, 0.1, 1, 10, 100, and 1000 µM; silica nanoparticles: 0, 6.25, 12.5, 25, 50, and 100 µg/mL). Human corneal epithelial cells (HCECs) were cultured and exposed to sodium nitrite, SNP (0, 0.1, 1, 10, 100, and 1000 µM), and silica nanoparticles for 1, 2, and 3 days. Results: Sodium nitrite and SNP showed a dose-dependent inhibitory effect on A. castellanii viability. A more prominent inhibitory effect was observed with SNP (less than 10% of organisms survived at 7-day culture with 1000 µM) compared with sodium nitrite. However, more cytotoxicity on HCEC was observed with SNP. NO-releasing silica nanoparticles were successfully internalized into the amoebic cytoplasm and accumulated in large vacuoles. Although blank silica nanoparticles had no inhibitory effect on A. castellanii viability, NO-releasing silica nanoparticles showed a dose-dependent amoebicidal effect. Furthermore, no cystic transformation of A. castellanii was observed under a phase contrast microscope or transmission electron microscope after exogenous NO treatment. Conclusions: Our results demonstrated the anti-Acanthamoeba effect of exogenous NO. This finding suggests that NO-releasing drug platforms, including nano-carriers, can be a promising therapeutic strategy for Acanthamoeba keratitis.

New Human Organotypic Corneal Tissue Model for Ophthalmic Drug Delivery Studies.

Purpose: The purpose of the current work was to develop a physiologically relevant, in vitro human three-dimensional (3D) corneal epithelial tissue model for use in ophthalmic drug development. Methods: Normal human corneal epithelial cells were cultured at the air-liquid interface to produce the 3D corneal tissue model. Corneal barrier was determined by measuring transepithelial electrical resistance (TEER). Quantitative PCR arrays were utilized to investigate expression of 84 phase I/II metabolizing enzymes and 84 drug transporter genes. Permeability was evaluated using model compounds with a wide range of hydrophobicity, molecular weight, and excipients. Finally, different formulations of latanoprost and bimatoprost were administered and drug absorption and tissue viability and integrity were investigated. Results: Histologic assessment and TEER of the corneal tissue model revealed tissue structure, thickness, and barrier formation (1000 ± 146 Ω·cm2) comparable to native human corneal epithelium. The 3D corneal tissue expressed tight junctions, mucins, and key corneal epithelial detoxification enzymes. Drug-metabolizing enzyme and transporter gene expression in 3D corneal tissue and excised human corneal epithelium were highly correlated (r2 = 0.87). Coefficients of permeation for model drugs in the tissue model and excised rabbit corneas also showed a high correlation (r2 = 0.94). As expected, latanoprost and bimatoprost free acids had much lower permeability (Papp = 1.2 × 10-6 and 1.9 × 10-6) than the corresponding prodrugs (Papp = 2.5 × 10-5 and 5.6 × 10-5), respectively. The presence of 0.02% benzalkonium chloride in ophthalmic formulations significantly affected tissue barrier and viability. Conclusions: The newly developed 3D corneal tissue model appears to be very useful for evaluation of corneal drug permeability and safety during ophthalmic drug development.