Concept: Corneal epithelium
Wounds naturally produce electric signals which serve as powerful cues that stimulate and guide cell migration during wound healing. In diabetic patients, impaired wound healing is one of the most challenging complications in diabetes management. A fundamental gap in knowledge is whether diabetic wounds have abnormal electric signaling. Here we used a vibrating probe to demonstrate that diabetic corneas produced significantly weaker wound electric signals than the normal cornea. This was confirmed in three independent animal models of diabetes: db/db, streptozotocin-induced and mice fed a high-fat diet. Spatial measurements illustrated that diabetic cornea wound currents at the wound edge but not wound center were significantly weaker than normal. Time lapse measurements revealed that the electric currents at diabetic corneas lost the normal rising and plateau phases. The abnormal electric signals correlated significantly with impaired wound healing. Immunostaining suggested lower expression of chloride channel 2 and cystic fibrosis transmembrane regulator in diabetic corneal epithelium. Acute high glucose exposure significantly (albeit moderately) reduced electrotaxis of human corneal epithelial cells in vitro, but did not affect the electric currents at cornea wounds. These data suggest that weaker wound electric signals and impaired electrotaxis may contribute to the impaired wound healing in diabetes.
Abstract Purpose: To investigate the effects of low level Electromagnetic Field (low level-EMF) exposure, as frequently encountered in daily life, on the yesmal rat cornea using histological and stereological method. Methods: Twenty-two adult male Wistar rats were randomly divided into two groups: study group (n=11) and control group (n=11). Rats in the study group were exposed to 2.45 GHz Microwave (MW) radiation (11.96±0.89V/m), 0.25 W/kg specific absorption rate (SAR) for 2 hours each day for 21 days. The corneal thickness and the anterior epithelium corneal thickness were measured using two different methods. Results: Using the histological method, the mean corneal thicknesses in the control and study group were 278.9±54.5 µm, and 272.4±85.6 µm, respectively. There was no statistically significant difference between the groups (p>0.05). The anterior corneal epithelium thickness was 28.1±4.9 µm in the control group and 31.7±5.5 µm in the study group. There were statistically differences between the groups with regard to the thickness of anterior epithelium (p<0.05). In the measurement made by the stereological method, the percentage of the cornea occupied by anterior corneal epithelium was 15.94% in the control group and 17.9% in the study group. Despite the fact that there was a relation between increased anterior epithelial area (AEA) and radiation exposure, no statistically significant relationship in area fraction of each compartment was found between the control and study groups. Conclusions: Results of this preliminary study show that exposure to MW radiation might cause alterations in the rat cornea.
Introduction: Topical ocular anesthetics are generally well tolerated in clinical settings but have great potential for abuse if used by patients at home. This abuse can lead to significant ocular complications. Topical ocular anesthetic abuse can lead to superficial punctate keratitis, persistent epithelial defects, stromal/ring infiltrates, corneal edema, endothelial damage and ocular inflammation, even when used in a dilute concentration. Patient characteristics may include a healthcare association and/or psychiatric illness. In these instances, patients often do not admit to anesthetic use and are often initially treated for acanthamoeba keratitis. Local anesthetics are thought to cause direct toxicity to the corneal epithelium, stroma and endothelium. This in turn may lead to release of antigens and from there an inflammatory response in the form of infiltrate and edema. It is thought that preservatives in anesthetics may play a further role in toxicity. Areas covered: The authors provide a brief history on topical ocular anesthetics and review the most recent literature on reported ocular toxicities of topical anesthetics. Expert opinion: Practitioners must maintain high suspicion for topical ocular anesthetic abuse to identify it early. Topical ocular anesthetic abuse is often misdiagnosed as acanthamoeba keratitis. Early identification is one of the most important factors for a successful outcome. It is also imperative to give proper alternative pain control to avoid topical anesthetic abuse.
Using rabbit corneal epithelial cells (RCECs), the transport of a nonsteroidal anti-inflammatory drug (NSAID) [(3) H]ketoprofen across the cornea was investigated with the aim of revealing the mechanism of uptake.
Purpose: We evaluated the effect of poly(ADP-ribose) (PARP) inhibition with 1,5-isoquinolinediol (ISO) on the corneal epithelial innervation in diabetic rats. Methods: ISO (3 mg/kg, intraperitoneal) or vehicle was administered to streptozotocin-induced diabetic rats for 4 weeks. Epithelial innervation, epithelial wound healing, and corneal sensation were evaluated in diabetic rats (DM rats), diabetic rats treated with ISO (DM-ISO rats), and non-diabetic (non-DM) rats. The density of epithelial innervation was calculated separately as nerve terminals and sub-basal nerve plexus by analyzing the images of whole mount corneas. The healed area of epithelial defect were measured at 0, 18, and 36 h after creating a 4 mm wound on the cornea. Corneal sensitivity test was conducted using a Cochet-Bonnet handheld esthesiometer. Additionally, PARP1 and poly(ADP-ribosyl)ated polymers (pADPr) as its products, were identified in trigeminal ganglions (TGs) by Western blot analysis and immunofluorescence staining. Results: In DM rats, the density of nerve terminals (5.57 ± 0.94%) and sub-basal nerve plexus (22.08 ± 1.78 mm/mm2) was significantly reduced in comparison with that in DM-ISO rats (8.64 ± 1.42%, 30.82 ± 2.01 mm/mm2) or and non-DM rats (9.02 ± 1.14%, 34.77 ± 4.45 mm/mm2). The healed area (%) of the epithelial defects at 18 and 36 h was significantly smaller in DM rats (23.8 ± 5.2%, 53.2 ± 4.6%) than in DM-ISO rats (43.2 ± 1.4%, 75.8 ± 2.2%) and non-DM rats (48.1 ± 8.6%, 86.1 ± 3.3%). Corneal sensitivity decreased in DM rats (51.1 ± 0.3 mm), but not in DM-ISO rats (57.8 ± 0.2 mm). There were no differences in any parameters between DM-ISO and non-DM rats. Conclusions: Diabetic corneas showed loss of epithelial innervation along with delayed epithelial healing and decreased corneal sensitivity. PARP inhibition alleviated these diabetes-induced alterations in the corneal epithelium.
Herpes simplex virus-1 (HSV-1) causes lifelong recurrent pathologies without a cure. How infection by HSV-1 triggers disease processes, especially in the immune-privileged avascular human cornea, remains a major unresolved puzzle. It has been speculated that a cornea-resident molecule must tip the balance in favor of pro-inflammatory and pro-angiogenic conditions observed with herpetic, as well as non-herpetic, ailments of the cornea. Here, we demonstrate that heparanase (HPSE), a host enzyme, is the molecular trigger for multiple pathologies associated with HSV-1 infection. In human corneal epithelial cells, HSV-1 infection upregulates HPSE in a manner dependent on HSV-1 infected cell protein 34.5. HPSE then relocates to the nucleus to regulate cytokine production, inhibits wound closure, enhances viral spread, and thus generates a toxic local environment. Overall, our findings implicate activated HPSE as a driver of viral pathogenesis and call for further attention to this host protein in infection and other inflammatory disorders.
MicroRNAs are powerful gene expression regulators, but their corneal repertoire and potential changes in corneal diseases remain unknown. Our purpose was to identify miRNAs altered in the human diabetic cornea by microarray analysis, and to examine their effects on wound healing in cultured telomerase-immortalized human corneal epithelial cells (HCEC) in vitro. Total RNA was extracted from age-matched human autopsy normal (n=6) and diabetic (n=6) central corneas, Flash Tag end-labeled, and hybridized to Affymetrix® GeneChip® miRNA Arrays. Select miRNAs associated with diabetic cornea were validated by quantitative RT-PCR (Q-PCR) and by in situ hybridization (ISH) in independent samples. HCEC were transfected with human pre-miR™miRNA precursors (h-miR) or their inhibitors (antagomirs) using Lipofectamine 2000. Confluent transfected cultures were scratch-wounded with P200 pipette tip. Wound closure was monitored by digital photography. Expression of signaling proteins was detected by immunostaining and Western blot. Using microarrays, 29 miRNAs were identified as differentially expressed in diabetic samples. Two miRNA candidates showing the highest fold increased in expression in the diabetic cornea were confirmed by Q-PCR and further characterized. HCEC transfection with h-miR-146a or h-miR-424 significantly retarded wound closure, but their respective antagomirs significantly enhanced wound healing vs. controls. Cells treated with h-miR-146a or h-miR-424 had decreased p-p38 and p-EGFR staining, but these increased over control levels close to the wound edge upon antagomir treatment. In conclusion, several miRNAs with increased expression in human diabetic central corneas were found. Two such miRNAs inhibited cultured corneal epithelial cell wound healing. Dysregulation of miRNA expression in human diabetic cornea may be an important mediator of abnormal wound healing.
Healthy eyes contain a population of limbal stem cells (LSCs) that continuously renew the corneal epithelium. However, each year, 1 million Americans are afflicted with severely reduced visual acuity caused by corneal damage or disease, including LSC deficiency (LSCD). Recent advances in corneal transplant technology promise to repair the cornea by implanting healthy LSCs to encourage regeneration; however, success is limited to transplanted tissues that contain a sufficiently high percentage of LSCs. Attempts to screen limbal tissues for suitable implants using molecular stemness markers are confounded by the poorly understood signature of the LSC phenotype. For cells derived from the corneal limbus, we show that the performance of cell stiffness as a stemness indicator is on par with the performance of ΔNP63α, a common molecular marker. In combination with recent methods for sorting cells on a biophysical basis, the biomechanical stemness markers presented here may enable the rapid purification of LSCs from a heterogeneous population of corneal cells, thus potentially enabling clinicians and researchers to generate corneal transplants with sufficiently high fractions of LSCs, regardless of the LSC percentage in the donor tissue.
Chronic inflammation is associated with a variety of pathological conditions in epithelial tissues, including cancer, metaplasia and aberrant wound healing. In relation to this, a significant body of evidence suggests that aberration of epithelial stem and progenitor cell function is a contributing factor in inflammation-related disease, although the underlying cellular and molecular mechanisms remain to be fully elucidated. In this study, we have delineated the effect of chronic inflammation on epithelial stem/progenitor cells using the corneal epithelium as a model tissue. Using a combination of mouse genetics, pharmacological approaches and in vitro assays, we demonstrate that chronic inflammation elicits aberrant mechanotransduction in the regenerating corneal epithelium. As a consequence, a YAP-TAZ/β-catenin cascade is triggered, resulting in the induction of epidermal differentiation on the ocular surface. Collectively, the results of this study demonstrate that chronic inflammation and mechanotransduction are linked and act to elicit pathological responses in regenerating epithelia.
Corneal integrity and transparency are indispensable for good vision. Cornea homeostasis is entirely dependent upon corneal stem cells, which are required for complex wound-healing processes that restore corneal integrity following epithelial damage. Here, we found that leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) is highly expressed in the human holoclone-type corneal epithelial stem cell population and sporadically expressed in the basal cells of ocular-surface epithelium. In murine models, LRIG1 regulated corneal epithelial cell fate during wound repair. Deletion of Lrig1 resulted in impaired stem cell recruitment following injury and promoted a cell-fate switch from transparent epithelium to keratinized skin-like epidermis, which led to corneal blindness. In addition, we determined that LRIG1 is a negative regulator of the STAT3-dependent inflammatory pathway. Inhibition of STAT3 in corneas of Lrig1-/- mice rescued pathological phenotypes and prevented corneal opacity. Additionally, transgenic mice that expressed a constitutively active form of STAT3 in the corneal epithelium had abnormal features, including corneal plaques and neovascularization similar to that found in Lrig1-/- mice. Bone marrow chimera experiments indicated that LRIG1 also coordinates the function of bone marrow-derived inflammatory cells. Together, our data indicate that LRIG1 orchestrates corneal-tissue transparency and cell fate during repair, and identify LRIG1 as a key regulator of tissue homeostasis.