Superficial Corneal Epithelium Research Articles

Resveratrol increases tear production and ocular pain after corneal abrasion in male, but not female, rats using a photorefractive keratectomy model.

Photorefractive keratectomy (PRK) is an alternative to LASIK and can cause intense acute pain that is often not relieved by standard treatments. To assess potential therapeutics for this type of acute pain, appropriate preclinical models are needed. We describe a preclinical corneal abrasion rat model that simulates the initial stages of PRK surgery and demonstrates similar pain and tear dysfunction as seen clinically. We used both behavioral and homeostatic assays to determine the therapeutic potential of resveratrol on pain and tear production. Studies were conducted in male and female Sprague-Dawley rats. Heptanol was applied to one eye and the superficial corneal epithelium was removed, mimicking the abrasion used in PRK. Spontaneous pain was assessed with orbital tightening (OT) scores for 7 days. Topical resveratrol increased OT scores sex-specifically in abraded males, but not females, at 72h and 1 week after abrasion. Resveratrol increased tear production in abraded males, with no effect in abraded females. There was no correlation between OT score at 1 week and tear production measurements, demonstrating no relationship between spontaneous ocular pain and tear dysfunction in this model. These findings demonstrate the usefulness of our corneal abrasion preclinical PRK model for the assessment of ocular pain therapeutics and indicate that topical resveratrol may not be useful for managing PRK-induced pain.

Hyaluronan-Cholesterol Nanogels for the Enhancement of the Ocular Delivery of Therapeutics.

The anatomy and physiology of the eye strongly limit the bioavailability of locally administered drugs. The entrapment of therapeutics into nanocarriers represents an effective strategy for the topical treatment of several ocular disorders, as they may protect the embedded molecules, enabling drug residence on the ocular surface and/or its penetration into different ocular compartments. The present work shows the activity of hyaluronan-cholesterol nanogels (NHs) as ocular permeation enhancers. Thanks to their bioadhesive properties, NHs firmly interact with the superficial corneal epithelium, without penetrating the stroma, thus modifying the transcorneal penetration of loaded therapeutics. Ex vivo transcorneal permeation experiments show that the permeation of hydrophilic drugs (i.e., tobramycin and diclofenac sodium salt), loaded in NHs, is significantly enhanced when compared to the free drug solutions. On the other side, the permeation of hydrophobic drugs (i.e., dexamethasone and piroxicam) is strongly dependent on the water solubility of the entrapped molecules. The obtained results suggest that NHs formulations can improve the ocular bioavailability of the instilled drugs by increasing their preocular retention time (hydrophobic drugs) or facilitating their permeation (hydrophilic drugs), thus opening the route for the application of HA-based NHs in the treatment of both anterior and posterior eye segment diseases.

Effect of therapeutic UVC on corneal DNA: Safety assessment for potential keratitis treatment

PurposeAntimicrobial ultraviolet C (UVC) has proven efficacy in vitro against keratitis isolates and has potential to treat corneal infection if safety can be confirmed. MethodSafety of 265 nm, 1.93 mW/cm2 intensity UVC (15–300 s exposures) was investigated in vitro via cyclobutane pyrimidine dimer (CPD) formation in DNA of human cultured corneal epithelial cells; ex vivo, by evaluating UVC transmissibility as a function of porcine corneal thickness; and in vivo, by evaluating CPD induction in the mouse cornea following UVC exposure. ResultsA single exposure of 15 s UVC did not induce significant CPD formation (0.92 ± 1.45%) in vitro relative to untreated control (p = 0.93) whereas 300 s exposure caused extensive CPD formation (86.8 ± 13.73%; p < 0.0001). Cumulative exposure to 15 s UVC daily for 3 days induced more CPD (14.6 ± 8.2%) than a single equivalent 45 s exposure (8.3 ± 4.0%) (p < 0.001) but levels returned to baseline within 72 h (p = 0.29), indicating highly efficient DNA repair. Ex vivo, UVC transmission decreased sharply with increasing corneal thickness, confirming UVC effects are limited to the superficial corneal layers. In vivo evaluations demonstrated no detectable CPD after three consecutive daily 15 s UVC exposures, whereas a single 300 s exposure induced extensive CPD formation in superficial corneal epithelium. ConclusionUp to three daily doses of 15 s UVC, in vivo, appear safe with respect to CPD formation. Ongoing research exploring UVC as a possible treatment for microbial keratitis is warranted.

Co-expression of SARS-CoV-2 entry genes in the superficial adult human conjunctival, limbal and corneal epithelium suggests an additional route of entry via the ocular surface

PurposeThe high infection rate of SARS-CoV-2 necessitates the need for multiple studies identifying the molecular mechanisms that facilitate the viral entry and propagation. Currently the potential extra-respiratory transmission routes of SARS-CoV-2 remain unclear. MethodsUsing single-cell RNA Seq and ATAC-Seq datasets and immunohistochemical analysis, we investigated SARS-CoV-2 tropism in the embryonic, fetal and adult human ocular surface. ResultsThe co-expression of ACE2 receptor and entry protease TMPRSS2 was detected in the human adult conjunctival, limbal and corneal epithelium, but not in the embryonic and fetal ocular surface up to 21 post conception weeks. These expression patterns were corroborated by the single cell ATAC-Seq data, which revealed a permissive chromatin in ACE2 and TMPRSS2 loci in the adult conjunctival, limbal and corneal epithelium. Co-expression of ACE2 and TMPRSS2 was strongly detected in the superficial limbal, corneal and conjunctival epithelium, implicating these as target entry cells for SARS-CoV-2 in the ocular surface. Strikingly, we also identified the key pro-inflammatory signals TNF, NFKβ and IFNG as upstream regulators of the transcriptional profile of ACE2+TMPRSS2+ cells in the superficial conjunctival epithelium, suggesting that SARS-CoV-2 may utilise inflammatory driven upregulation of ACE2 and TMPRSS2 expression to enhance infection in ocular surface. ConclusionsTogether our data indicate that the human ocular surface epithelium provides an additional entry portal for SARS-CoV-2, which may exploit inflammatory driven upregulation of ACE2 and TMPRSS2 entry factors to enhance infection.

Evaluation of voriconazole anti-Acanthamoeba polyphaga in vitro activity, rat cornea penetration and efficacy against experimental rat Acanthamoeba keratitis.

Acanthamoeba keratitis (AK) is a sight-threatening infectious disease. Its effective and safe medical therapy remains highly debated. Recently, voriconazole, a monotriazole with noted in vitro activity against a large variety of fungi, has been successfully used both topically and systemically to treat human AK cases. To measure anti-Acanthamoeba polyphaga in vitro activity, anti-rat AK efficiency and rat cornea penetration of eye-drop and oral voriconazole. A. polyphaga was maintained in axenic cultures. In vitro, amoebicidal and cysticidal activities of voriconazole were measured using an XTT assay. AK lesions of Sprague Dawley rats were scored from grade 0 to grade 3. For 21 days, from day 7 post-infection, voriconazole (1% solution) eye drops were instilled or voriconazole was administered by gavage (60 mg/kg/day). After killing, superficial corneal epithelium scrapings were cultured and analysed by PCR, and eye-globe histology was performed. Cornea and plasma concentrations were determined using 2D HPLC separation and tandem MS. In vitro, voriconazole inhibited trophozoite proliferation with an IC50 value of 0.02 mg/L and an IC90 value of 2.86 mg/L; no cysticidal effect was found. In AK rats, eye drops reduced clinical worsening from day 7 to day 14 post-infection and oral voriconazole was not effective. Voriconazole cornea concentrations were directly dependent on the frequency of eye-drop instillations, which resulted in lower plasma concentrations, whilst oral voriconazole resulted in lower cornea concentrations. Present data underline the need for high-frequency eye-drop instillation regimens for efficient AK therapy.

A mouse dry eye model induced by topical administration of the air pollutant particulate matter 10

AimTo introduce a novel dry eye mouse model induced by topical administration of the air pollutant particulate matter 10 (PM10). MethodA total of 60 male BALB/c mice were used in this study and divided into two groups: group A (PBS eye drops, n=30) and group B (PM10 eye drop group, n=30). Each treatment was dosed four times a day, every time 50ul with the concentration of 5mg/ml PM10, for 14 consecutive days in the right eye. The clinical manifestations of dry eye were measured before therapy and 4, 7 and 14days post-treatment respectively, which included the tear volume, tear break-up (BUT) time, corneal fluorescein staining, rose bengal staining, Lissamine Green staining and inflammatory index. Eye samples were collected on D14 and examined by histologic light microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM), corneal cytokeration 10 (K10) immunnostaining, and tumor necrosis factor-α (TNF-α), NF-κB-p65 and NF-κB Western Blot analysis. ResultsAt 0d, 7d and 14d, there were no statistical changes in tear volume, BUT after treatment (P>0.05) with PBS in group A. In group B, all items showed statistical differences at each time point (P<0.05). At 14d after therapy, the fluorescein staining score of group B was higher than group A (P<0.05). The score of rose bengal staining and Lissamine Green staining in group B was also higher than that in group A (P<0.05). The number of mean layers of corneal epithelial cells in the group A was significantly lower than that in the group B (P<0.05). TEM and SEM revealed that the number of corneal epithelial microvilli were drastically reduced in group B. The number of corneal chondriosome/desmosomes was also reduced in group B by TEM. PM10 induced apoptosis in the superficial and basal corneal epithelium, and leaded to abnormal differentiation and proliferation of the ocular surface with higher expression levels of K10 and reduced number of goblet cells in the conjunctival fornix in group B. PM10 significantly increased the levels of TNF-α, NF-κB-p65 and NF-κB in the cornea. ConclusionPM10 can damage the tear film function and cause the destruction of the structural organization of ocular surface in mice. Topical administration of PM10 in mice induces ocular surface changes that are similar to those of dry eye in humans, representing a novel model of DES.

Amniotic membrane transplantation - analysis of structural characteristics in amniotic membrane transplant and corneal ulcers

AIM: To analyze the structural characteristics in the cornea and the amniotic membrane (AM) with the help of laser scanning in-vivo confocal microscopy (IVCM) and to evaluate the morphometric changes in the cornea and the integration pattern of amniotic membrane into the host tissue using anterior-segment optical coherence tomography (AS-OCT) in patients with persistent corneal defect treated with amniotic membrane transplantation (AMT). MATERIALS AND METHODS: Nine eyes of six consecutive patients (mean age 44.9 ± 8.7 years) with corneal defects and stromal thinning unresponsive to topical treatment were enrolled in this study. Transplantation of cryopreserved amniotic membrane was performed. The time of healing of the corneal defect, morphometric analysis of the cornea and transplanted amniotic membrane using AS-OCT and the structural characteristics in the amniotic membrane grafts and corneal ulcers assessed by in vivo confocal microscopy were evaluated. RESULTS: Successful results after amniotic membrane transplantation (AMT) were observed in 8 of the 9 eyes (91.2%). On the 2nd day after transplantation, IVCM showed that under the amniotic membrane a new epithelium with large, flat, immature cells of the superficial corneal epithelium with an average density of 598.4 ± 66.38cells/mm2 was observed. The basal cells also showed immaturity and their average density was 1804 ± 93.32cells/mm2. In the corneal stroma edema and activated corneal cells were visualized. The average corneal epithelium thickness increased to 24.60 ± 2.07μm, the average density of epithelial cells increased to 657.6 ± 78.41cells/mm2, while the mean basal epithelium cells density was 2541 ± 540.13 cells/mm2. AS-OCT showed that the preoperative corneal thickness at the ulcer was 418.91 ± 96.56 μm. On the 2nd day after the surgery, the amniotic membrane (AM) thickness was measured to be 268 ±105 μm. On the 8th day it was two times smaller and was measured to be 123 ±39 μm. On the 25th day post-surgery the corneal thickness was measured at 494.03 ± 67.35 μm. In two of the cases integration of AM was found. CONCLUSIONS: AMT leads to recovery of corneal defects unresponsive to conservative treatment. The amniotic membrane graft is effective in promoting re-epithelization, nonetheless, it can also integrate into the host corneal tissue which results in an increase in corneal thickness, stabilization of the epithelium and reduction of the subjective signs. The integration of amniotic membrane into the damaged cornea proves that AMT is an effective and irreplaceable procedure for difficult-to-treat anterior ocular surface diseases.

In vivo 3D measurement of moxifloxacin and gatifloxacin distributions in the mouse cornea using multiphoton microscopy.

Moxifloxacin and gatifloxacin are fourth-generation fluoroquinolone antibiotics used in the clinic to prevent or treat ocular infections. Their pharmacokinetics in the cornea is usually measured from extracted ocular fluids or tissues, and in vivo direct measurement is difficult. In this study multiphoton microscopy (MPM), which is a 3D optical microscopic technique based on multiphoton fluorescence, was applied to the measurement of moxifloxacin and gatifloxacin distribution in the cornea. Intrinsic multiphoton fluorescence properties of moxifloxacin and gatifloxacin were characterized, and their distributions in mouse cornea in vivo were measured by 3D MPM imaging. Both moxifloxacin and gatifloxacin had similar multiphoton spectra, while moxifloxacin had stronger fluorescence than gatifloxacin. MPM imaging of mouse cornea in vivo showed (1) moxifloxacin had good penetration through the superficial corneal epithelium, while gatifloxacin had relatively poor penetration, (2) both ophthalmic solutions had high intracellular distribution. In vivo MPM results were consistent with previous studies. This study demonstrates the feasibility of MPM as a method for in vivo direct measurement of moxifloxacin and gatifloxacin in the cornea.

Stem Cell-Like Characteristics of Corneal Epithelial Cells

Purpose: The adult corneal epithelium is maintained by a population of limbal stem cells (LSCs), transmembrane protein prominin, regarded as stem cell marker was investigated on mouse corneal tissue, to study weather contains CD133-expressing cells and their distribution. Methods: Enucleated mouse eyes were embedded in OCT and cryosections were performed for mmunohistochemical studies using the avidin-biotin-peroxidase complex (ABC) procedure. Meanwhile, dissected mouse corneas were analyzed by westernblot. Results: In the adult mouse, 13A4 immunoreactivity was detected at the apical side of superficial corneal epithelium, including the limbus region, but not by stroma and endothelium. 115 KDa protein was approved in corneal tissue by Westernblot. Conclusions: The stem cell activity does not occur along the limbus but presumably presented by small portion of corneal epithelial cells which may hold a similar properties of stem cells.

Confocal Microscopy of Corneal Crystals in a Patient With Hereditary Tyrosinemia Type I, Treated With NTBC

To describe the confocal microscopic findings in a patient with hereditary tyrosinemia type I (HT-I) treated with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) who developed corneal crystals. In this case study, we describe the confocal microscopic findings in a boy, who was diagnosed with HT-I at the age of 4 months. At 16 years of age, he developed painful corneal lesions in both eyes. On slit-lamp examination, whorl-like branching epithelial corneal lesions were found, staining faintly with fluorescein. His NTBC treatment was stopped and reintroduced at a lower dose after 1 month. The lesions clearly regressed, leaving only mild residual epithelial scarring, without fluorescein staining and without pain. Confocal microscopy was performed in the acute painful stage and in the asymptomatic convalescent stage 5 months later. Confocal microscopy confirmed the presence of slender birefringent spiky crystals in the very superficial corneal epithelium. In the asymptomatic convalescent phase, the crystals clearly persisted on confocal microscopy, although they were barely visible on slit-lamp examination. This is the first in vivo demonstration by confocal microscopy of corneal crystals present in a patient with proven type I tyrosinemia, under NTBC treatment.

Expression of Phosphorylated Heat Shock Protein 27 During Corneal Epithelial Wound Healing

The 27-kDa heat shock protein (HSP27) has been implicated in wound healing in multiple tissues. We investigated the expression and localization of phosphorylated HSP27 during epithelial wound healing in the murine cornea. Corneas of 8- to 10-week-old C57BL6 mice were wounded by epithelial debridement (n = 40). Unwounded corneas served as controls (n = 3). After 3, 7, and 14 days, phosphorylated HSP27 localization in wounded corneas was observed by confocal immunohistochemistry and double immunogold labeling transmission immunoelectron microscopy. Western blot analysis was performed to determine expression levels of phosphorylated HSP27 in scraped epithelia. Phosphorylated HSP27 localization was also separately performed with confocal immunohistochemistry 8 hours after epithelial debridement to investigate the early epithelial wound-healing process. In unwounded corneas, phosphorylated HSP27 was localized only to the superficial epithelium. In contrast, phosphorylated HSP27 was localized in the basal and superficial epithelia 3 days after corneal epithelial wounding. After 7 and 14 days, HSP27 localization was similar to that in unwounded controls. Expression levels of phosphorylated HSP27 were greater in wounded corneal epithelia on day 3 than in unwounded controls and on day 14. After 8 hours, phosphorylated HSP27 expression was prominent in the leading edge of migrating corneal epithelium. Constitutive expression of phosphorylated HSP27 is limited to the superficial corneal epithelium in unwounded murine corneas. Changes in HSP27 epithelial distribution and expression levels after corneal epithelial wounding suggest that phosphorylated HSP27 plays a role in early phase of corneal epithelial wound healing.

Corneal Epithelial Opacity in Dysfunctional Tear Syndrome

To compare the appearance of the superficial corneal epithelium in patients with dysfunctional tear syndrome (DTS) and that of an asymptomatic control group using laser scanning confocal microscopy and to determine the correlations between confocal microscopic findings and clinical severity parameters. Prospective case-control study. Thirty-one patients with newly diagnosed DTS and 21 asymptomatic control subjects were evaluated for this study. Subjects with DTS were classified into 4 levels of clinical severity (DTS 1 through 4) based on the Delphi dry eye panel report criteria. The Heidelberg Retina Tomograph 2 Rostock Cornea Module (Heidelberg Engineering GmbH, Heidelberg, Germany) laser scanning confocal microscope was used to image the superficial corneal epithelium. Areas of single or multiple opaque superficial epithelial cells were measured as a percentage of the 400 x 400-microm(2) field area in 4 randomly selected confocal images from each eye. Spearman correlations between the confocal findings and severity of symptoms, visual acuity, and ocular surface signs were calculated. The mean area of opaque superficial corneal epithelial cells was significantly greater in DTS patients than in normal subjects (P < .0001). Significant differences were observed between the DTS severity groups and the control group (P < .001), except for the DTS 1 group. The area of opaque cells significantly increased with level of clinical severity. The confocal findings showed significant correlation with clinical severity parameters, including blurred vision symptoms (r = 0.86; P = .0001), best-corrected visual acuity (Spearman r = 0.4; P = .03), conjunctival lissamine green staining scores (Spearman r = 0.4; P = .026), corneal fluorescein staining scores (Spearman r = 0.5; P = .002), and videokeratoscopic surface regularity index (Spearman r = 0.5; P = .02). Morphologic changes in the superficial corneal epithelium of DTS patients detected by laser scanning confocal microscopy correlates with blurred vision symptoms and objective severity parameters. Objective confocal image analysis of the superficial corneal epithelium may prove useful for classifying DTS severity and for monitoring the efficacy of therapies.

Meesmann Corneal Dystrophy Associated With Epithelial Basement Membrane and Posterior Polymorphous Corneal Dystrophies

To report a rare case of bilateral and symmetric Meesmann corneal dystrophy concurrent with bilateral epithelial basement membrane dystrophy and bilateral but asymmetric posterior polymorphous corneal dystrophy in a patient of Armenian origin. Complete ophthalmologic examination was performed on a 6-year-old boy from Armenia who was diagnosed with bilateral symmetric Meesmann corneal dystrophy combined with bilateral epithelial basement membrane dystrophy and bilateral but asymmetric posterior polymorphous corneal dystrophy. This case was observed and treated for 24 years. On slit-lamp biomicroscopy, the patient showed bilateral multiple intraepithelial cystic lesions, bilateral irregularly shaped grayish-white opacities in the superficial corneal epithelium, and bilateral but asymmetric transparent vesicles surrounded by gray halos at the level of the Descemet membrane and the endothelium. This case is reported because of the unusual occurrence of Meesmann corneal dystrophy with other corneal dystrophies.

In Vivo Confocal Microscopy of Hereditary Sensory and Autonomic Neuropathy

Purpose: To observe the morphology of the corneal cells and corneal nerve fibers in patients with type IV or V hereditary sensory and autonomic neuropathy (HSAN) by in vivo confocal microscopy and elucidate the mechanism leading to the loss of corneal sensation in this disease. Methods: In vivo confocal microscopy was performed on the central cornea of the right eye in 3 patients with HSAN (ages 17, 20, and 32 years), and their corneal morphology was compared with that of 3 healthy subjects (ages 28, 30, and 36 years). Corneal sensation was tested with a Cochet-Bonnet esthesiometer. Results: The superficial epithelial cell density was lower in the HSAN patients compared with the healthy subjects (1525, 1225, and 1250/mm2 vs. 2225, 1750, and 2500/mm2), but the basal epithelial cell density of the patients was similar to that of the healthy subjects. Nerve bundles were clearly observed in the sub-basal nerve plexus layer of the cornea in the healthy subjects, but were undetectable at the central cornea in the patients with HSAN. The corneal sensation of the patients with HSAN was much weaker than that of the healthy subjects (2.79, 40.30, and 132.50 g/mm2 vs. 1.47, 1.47, and 1.47 g/mm2). Conclusions: Superficial keratopathy accompanied with neurotrophic keratopathy and tear film instability observed clinically agrees with the large keratinized cells in the superficial corneal epithelium by in vivo confocal microscopy in these patients. Our findings suggest that the loss of corneal nerves contributes to impairment of corneal sensation in patients with type IV or V HSAN.

Comparative in vivo high‐resolution confocal microscopy of corneal epithelium, sub‐basal nerves and stromal cells in mice with and without dry eye after photorefractive keratectomy

The present study compares, using a new-generation high-resolution in vivo confocal microscope, the epithelial morphology, sub-basal nerves and stroma in two groups of mice: one exposed to normal conditions (NC) and the other to a desiccating environment (DE), following photorefractive keratectomy (PRK) with mechanical epithelial scraping. Twenty-four 4- to 8-week-old female Balb/C mice were used in this study. Twenty mice underwent bilateral corneal epithelial scraping using an electric brush prior to PRK. Then, the mice were divided in two groups: 10 mice were placed in NC. The other 10 mice were exposed to a DE for 2 weeks. Four mice served as controls. Corneas were analysed in vivo using the Rostock Cornea Module of the Heidelberg retina tomograph II. For all eyes, 20 confocal microscopic images of each layer, that is, the superficial and basal corneal epithelium, Bowman's layer, anterior and posterior stroma and the endothelium, were recorded. Epithelial and stromal cell densities and sub-basal and stromal nerves were measured and compared. There was a higher density of superficial epithelial cells in the DE group (693 +/- 148 cells/mm(2) in NC group and 443 +/- 128 cells/mm(2) in DE group; Mann-Whitney U-test; P = 0.05). Higher number of basal cells were observed in the DE group. Its density was 986 +/- 198 cells/mm(2) in NC and 1598 +/- 280 cells/mm(2) in DE group (Mann-Whitney U-test; P < 0.05). Significantly higher number of reflective structures were noted within the stroma without clearly visible nuclei in the DE group compared with the NC eyes. Additionally, higher number of beads, nerve sprouts and higher tortuosity of sub-basal nerves were observed in the DE group. No difference was observed in the endothelial cell density between the groups. Exposure of corneas to a DE after PRK with previous mechanical epithelial scraping increases epithelial turnover and is associated with a higher number of reflective structures in the stroma. Additionally increased nerve beading, nerve sprouts and tortuosity of sub-basal nerves were observed in the DE group, possibly directed to repair the alterations observed at the epithelial level.

A keratitis rat model for evaluation of anti- Acanthamoeba polyphagia agents

To develop a rat model of chronic Acanthamoeba polyphaga keratitis suitable for pharmacologic assessment of therapeutic agents.An A. polyphaga isolate (ATCC #50495) was grown in peptone-yeast extract-glucose medium. Five-weeks-old, Sprague-Dawley male rats were injected with 10(3) or 10(4) trophozoites in the left cornea stromal layer. A subconjunctival injection of 0.14, 0.28, or 0.57 mg long-acting betamethasone was performed weekly. At the end of experiments, rats were killed; the superficial corneal epithelium gently scraped and cultured; and globes histologically examined. Topical polyhexamethylene biguanide (PHMB), hexamidine diisethionate, and miltefosine (hexadecylphosphocholine) were administered topically as eye drops 3 times a day at concentrations of 0.02%, 0.1%, and 0.01% respectively. In vitro minimal inhibitory concentration (MIC) and fractional inhibitory concentration values were measured in A. polyphaga cultures.In infected eyes, lesions consisted of the sequential appearance within 2 weeks of edema, infiltrates, and/or abscesses. On day 35 postinfection, a combination of 10(4) parasites with a regimen of 0.28 mg/week betamethasone resulted in the highest ratio of rats with abscesses. Presence of A. polyphaga was confirmed histologically and inconsistently in cultures. In rats optimally prepared as said earlier, agents were administered on day 6 postinfection. A combination of PHMB and hexamidine diisethionate exerted a synergistic effect and was more effective than PHMB, hexamidine diisethionate, or miltefosine alone. In vitro, PHMB (MIC = 14.6 microM) and hexamidine diisethionate (MIC = 555 microM) exerted a synergistic effect (fractional inhibitory concentration = 0.06), and miltefosine exhibited antiamoebal activity (MIC = 27.4 microM).In this study, a rat model of chronic A. polyphaga keratitis was obtained and found suitable for assessment of pharmacologic agents. It provides an in vivo approach of drug resistance, pathogenicity, and physiopathologic mechanisms of chronic amoebic keratitis.

A Keratitis Rat Model for Evaluation of Anti-Acanthamoeba Polyphaga Agents

To develop a rat model of chronic Acanthamoeba polyphaga keratitis suitable for pharmacologic assessment of therapeutic agents. An A. polyphaga isolate (ATCC #50495) was grown in peptone-yeast extract-glucose medium. Five-weeks-old, Sprague-Dawley male rats were injected with 10(3) or 10(4) trophozoites in the left cornea stromal layer. A subconjunctival injection of 0.14, 0.28, or 0.57 mg long-acting betamethasone was performed weekly. At the end of experiments, rats were killed; the superficial corneal epithelium gently scraped and cultured; and globes histologically examined. Topical polyhexamethylene biguanide (PHMB), hexamidine diisethionate, and miltefosine (hexadecylphosphocholine) were administered topically as eye drops 3 times a day at concentrations of 0.02%, 0.1%, and 0.01% respectively. In vitro minimal inhibitory concentration (MIC) and fractional inhibitory concentration values were measured in A. polyphaga cultures. In infected eyes, lesions consisted of the sequential appearance within 2 weeks of edema, infiltrates, and/or abscesses. On day 35 postinfection, a combination of 10(4) parasites with a regimen of 0.28 mg/week betamethasone resulted in the highest ratio of rats with abscesses. Presence of A. polyphaga was confirmed histologically and inconsistently in cultures. In rats optimally prepared as said earlier, agents were administered on day 6 postinfection. A combination of PHMB and hexamidine diisethionate exerted a synergistic effect and was more effective than PHMB, hexamidine diisethionate, or miltefosine alone. In vitro, PHMB (MIC = 14.6 microM) and hexamidine diisethionate (MIC = 555 microM) exerted a synergistic effect (fractional inhibitory concentration = 0.06), and miltefosine exhibited antiamoebal activity (MIC = 27.4 microM). In this study, a rat model of chronic A. polyphaga keratitis was obtained and found suitable for assessment of pharmacologic agents. It provides an in vivo approach of drug resistance, pathogenicity, and physiopathologic mechanisms of chronic amoebic keratitis.

Transmission electron microscopy and autofluorescence findings in the cornea of diabetic rats treated with aminoguanidine

Background: The accumulation of advanced glycation end products (AGEs) has been implicated in the pathogenesis of diabetic keratopathy.The present study was aimed to understand if aminoguanidine (AG), an AGE inhibitor, was protective against the development of corneal complications in a diabetic rat model. Methods: Wistar rats were divided into three experimental groups: control, diabetic, and AG-treated diabetic. Diabetes was induced in rats via a single intraperitoneal injection (60 mg/kg) of streptozocin (STZ) and AG was administered in drinking water at a dose of 1 g/L. All animals were sacrificed at the end of 10 weeks and corneas from diabetic and nondiabetic rats were analyzed via transmission electron microscopy (TEM). Corneal autofluorescence measurements were also performed in all experimental groups. Results: Electron microscopic evaluation revealed that aminoguanidine treatment in diabetic rats prevented the formation of intracellular spaces between neighbouring cells in the superficial corneal epithelium. Hyperglycemia-induced degeneration of intracellular organelles and formation of cytoplasmic vacuoles in the corneal stroma was also prevented with the treatment of AG. Corneal autofluorescence detected in the diabetic group (5.98 ± 2.17 Fi/mg protein) was found to be significantly greater than the control (3.92 ± 0.56 Fi/mg protein) and the AG-treated diabetic group (4.18 ± 0.59 Fi/mg protein) ( p < 0.05). Interpretation: The presented data provide evidence that AG is preventive against corneal alterations in experimental diabetes.

Examination of murine tear film.

To define spatially any free aqueous layer in murine tear film. A pre-zeroed microelectrode was touched to the superficial corneal epithelium and then raised in steps of 1 micro m through the murine tear film into the air and then retraced along the same path. Other murine tear films were partially probed with a spatial resolution of 0.1 micro m. The reference microelectrode was placed in a fragment of 3% polyacrylamide gel equilibrated against 154 mM NaCl and located on the nasal quadrant of the scleral conjunctiva. Other murine corneas were quick frozen in melting isopentane and freeze substituted or pretreated with cetylpyridinium chloride and then examined by transmission electron microscopy. The recorded electrical profiles of the tear film were reproducible in each preparation and showed a relatively uniform positive electrical potential throughout their whole thickness, except within 0.5 micro m of the epithelial surface when the potential reversed to negative values. The thickness of mouse tear film averaged 7.4 +/- 0.8 micro m (mean +/- SD, n = 40). The electron microscope images showed the murine tear film to have a relatively uniform positive electron density throughout the thickness. Electrical profiles of the murine tear film presented no evidence of a separate free aqueous phase. The tear film is observed as an aqueous gel that includes anion-exchanging polyelectrolytes throughout most of its thickness, but within 0.5 micro m of the epithelial surface, it changes to cation-exchanging polyelectrolytes. Electron microscope images provide some supporting evidence.

Twenty percent alcohol toxicity on rabbit corneal epithelial cells: electron microscopic study.

To evaluate 20% ethanol toxicity on the rabbit corneal epithelium, ethanol-treated rabbit corneas were examined with electron microscopy. Rabbit corneas (24 eyes) were treated with 20% ethanol for 30 seconds, 1 minute, and 2 minutes by using LASEK (laser-assisted subepithelial keratectomy) instruments and then washed with sterile water. Zero time, 1, 3, 5 days after ethanol treatment, corneas were excised and examined with scanning and transmission electron microscopy (SEM and TEM). Widespread partial or total damage of microvilli, focal breaks of intercellular junction, and cellular edema was observed. The damage was more severe in corneas with longer ethanol treatment. In corneas with ethanol treatment more than 1 minute, slough of superficial corneal epithelium occurred and progressed with time. Two-minute ethanol treatment resulted in complete destruction of microvilli and significant separation of intercellular junction. These pathologic changes persisted 5 days after ethanol treatment. From these results, increasing exposure time to ethanol more than 1 minute results in significant damage to superficial corneal epithelium and prolongs its normal recovery time.