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A unique class of intrinsically photosensitive retinal ganglion cells in mammalian retinae has been recently discovered and characterized. These neurons can generate visual signals in the absence of inputs from rods and cones, the conventional photoreceptors in the visual system. These light sensitive ganglion cells (mRGCs) express the non-rod, non-cone photopigment melanopsin and play well documented roles in modulating pupil responses to light, photoentrainment of circadian rhythms, mood, sleep and other adaptive light functions. While most research efforts in mammals have focused on mRGCs in retina, recent studies reveal that melanopsin is expressed in non-retinal tissues. For example, light-evoked melanopsin activation in extra retinal tissue regulates pupil constriction in the iris and vasodilation in the vasculature of the heart and tail. As another example of nonretinal melanopsin expression we report here the previously unrecognized localization of this photopigment in nerve fibers within the cornea. Surprisingly, we were unable to detect light responses in the melanopsin-expressing corneal fibers in spite of our histological evidence based on genetically driven markers and antibody staining. We tested further for melanopsin localization in cell bodies of the trigeminal ganglia (TG), the principal nuclei of the peripheral nervous system that project sensory fibers to the cornea, and found expression of melanopsin mRNA in a subset of TG neurons. However, neither electrophysiological recordings nor calcium imaging revealed any light responsiveness in the melanopsin positive TG neurons. Given that we found no light-evoked activation of melanopsin-expressing fibers in cornea or in cell bodies in the TG, we propose that melanopsin protein might serve other sensory functions in the cornea. One justification for this idea is that melanopsin expressed in Drosophila photoreceptors can serve as a temperature sensor.
In vitro screening and testing of drugs and devices is necessary, but in vitro conditions differ greatly from those found in vivo. These differences can lead to false promises of efficacy, or can hide problems of tissue compatibility. Models with ex vivo tissues can be highly valuable bridges which provide relevant matrices for testing [1], [2], [3], [4], [5], [6], [7], [8], [9]. Ex vivo tissue models which are closer both biochemically and biophysically can provide useful feedback in a more time- and cost-efficient manner. Herein we describe an ex vivo corneal model for use in drug delivery testing and corneal infection modeling [10]. The protocol covers the tissue harvesting, sterilization, inoculation, and bacterial load quantification. We envision that the model can be used to study bacterial physiology on metabolizable matrices and to study the direct effects of microbial colonization on the cornea's integrity and clarity.•Devitalized cornea.•Non-submersed conditions.•Contact lens compatible.
The ocular surface is always attacked by oxidative stress, and cornea epithelial cells are supposed to have their own recovery system against oxidative stress. Therefore we hypothesized that tears supply key molecules for preventing oxidative stress in cornea. The potential target key molecule we focused is selenoprotein P (SeP). SeP is a carrier of selenium, which is an essential trace element for many animals, for oxidative stress metabolism in the organism, and was extremely expressed in lacrimal gland. An experiment was performed with SeP eye drops in a rat dry eye model, prepared by removing the lacrimal glands. The anticipated improvement in corneal dry eye index and the suppression of oxidative stress markers were observed in SeP eye drop group. Furthermore, the concentration of SeP was significantly higher in dry eye patients compared with normal volunteers. Collectively, we concluded that tear SeP is a key molecule to protect the ocular surface cells against environmental oxidative stress.
Tumors of the conjunctiva and cornea comprise a large and varied spectrum of conditions. These tumors are grouped into two major categories of congenital and acquired lesions. The acquired lesions are further subdivided based on origin of the mass into surface epithelial, melanocytic, vascular, fibrous, neural, histiocytic, myxoid, myogenic, lipomatous, lymphoid, leukemic, metastatic and secondary tumors. Melanocytic lesions include nevus, racial melanosis, primary acquired melanosis, melanoma, and other ocular surface conditions like ocular melanocytosis and secondary pigmentary deposition. The most frequent nonmelanocytic neoplastic lesions include squamous cell carcinoma and lymphoma, both of which have typical features appreciated on clinical examination. The caruncle displays a slightly different array of tumors compared to those elsewhere on the conjunctiva, as nevus and papilloma are most common, but oncocytoma and sebaceous gland hyperplasia, adenoma, and carcinoma can be found. In this report, we provide clinical description and illustration of the many conjunctival and corneal tumors and we discuss tumor management.
The cornea is the transparent outermost surface of the eye, consisting of a stratified epithelium, a collagenous stroma and an innermost single-cell layered endothelium and providing 2/3 of the refractive power of the eye. Multiple diseases of the cornea arise from genetic defects where the ultimate phenotype can be influenced by cross talk between the cell types and the extracellular matrix. Cell culture modeling of diseases can benefit from cornea organoids that include multiple corneal cell types and extracellular matrices. Here we present human iPS cell-derived organoids through sequential rounds of differentiation programs. These organoids share features of the developing cornea, harboring three distinct cell types with expression of key epithelial, stromal and endothelial cell markers. Cornea organoid cultures provide a powerful 3D model system for investigating corneal developmental processes and their disruptions in diseased conditions.
The corneal stroma consists of orthogonally stacked collagen-fibril lamellae that determine the shape of the cornea and provide most of the refractive power of the eye. We have applied electromechanical reshaping (EMR), an electrochemical platform for remodeling cartilage and other semirigid tissues, to change the curvature of the cornea as a potential procedure for nonsurgical vision correction. EMR relies on short electrochemical pulses to electrolyze water, with subsequent diffusion of protons into the extracellular matrix of collagenous tissues; protonation of immobilized anions within this matrix disrupts the ionic-bonding network, leaving the tissue transiently responsive to mechanical remodeling. Re-equilibration to physiological pH restores the ionic matrix, resulting in persistent shape change of the tissue. Using ex vivo rabbit eyes, we demonstrate here the controlled change of corneal curvature over a wide range of refractive powers with no loss of optical transparency. Optical coherence tomography (OCT), combined with second-harmonic generation (SHG) and confocal microscopy, establish that EMR enables extremely fine control of corneal contouring while maintaining the underlying macromolecular collagen structure and stromal cellular viability, positioning electrochemical vision therapy as a potentially simple and ultralow-cost modality for correcting routine refractive errors.
Corneal wound healing studies have a long history and rich literature that describes the data obtained over the past 70 years using many different species of animals and methods of injury. These studies have lead to reduced suffering and provided clues to treatments that are now helping patients live more productive lives. In spite of the progress made, further research is required since blindness and reduced quality of life due to corneal scarring still happens. The purpose of this review is to summarize what is known about different types of wound and animal models used to study corneal wound healing. The subject of corneal wound healing is broad and includes chemical and mechanical wound models. This review focuses on mechanical injury models involving debridement and keratectomy wounds to reflect the authors' expertise.
The eyes are highly susceptible to the oxidative stress induced by ultraviolet B (UVB, wavelength between 280 ∼ 320 nm), which could cause severe damage to the cornea. Fullerenols are effective antioxidants to alleviate UVB-induced injury, while their application for the eyes is still rare. In present study, we investigated the protective performance and mechanism of fullerenols on cornea under UVB radiation in vivo and in vitro. The synthesized fullerenols exhibited broad-spectrum free radical scavenging properties (applicable to both reactive oxygen species (ROS) and reactive nitrogen species (RNS)) and photo-stability. When compared with another widely used antioxidant glutathione (GSH), the administration of fullerenols markedly decreased the injured area, corneal edema, cell death, and increased the cell proliferation in UVB-induced rat cornea. The effects of fullerenols were confirmed in UVB-exposed human corneal epithelial cells (hCECs), where elevated cell viability and proliferation, decreased oxidative free radical production, repaired mitochondrial dysfunction and DNA lesions were observed. RNA sequencing (RNA-Seq) analysis demonstrated that fullerenol alleviated UVB-induced corneal injury through down-regulation of oxidative stress-related genes and up-regulation of proliferation-associated genes. Our results demonstrate the suitability of fullerenols as a potential exogenous treatment in ameliorating UVB-induced cornea damage.
The cornea has an important role in vision, is highly innervated and many neurotransmitter receptors are present, e.g., muscarine, melatonin, and dopamine receptors. γ-aminobutyric acid (GABA) is the most important inhibitory neurotransmitter in the retina and central nervous system, but it is unknown whether GABA receptors are present in cornea. The aim of this study was to determine if GABA receptors are located in chick cornea.
Although acellular corneas have been reported to be a potential substitute for allogeneic cornea transplantation to treat corneal injury, severe corneal injury is hard to repair due to inflammation and neovascularization. The use of the amniotic membrane as a graft in ocular surface reconstruction has become widespread because of the anti-inflammatory and anti-angiogenic properties of amniotic epithelial cells (AECs). Our objective was to construct a tissue-engineered cornea (TEC) composed of an acellular porcine cornea (APC) and AECs to repair severe corneal injury. Corneal cells were completely removed from the prepared APC, and the microstructure, mechanical properties, and stability of a natural porcine cornea (NPC) was maintained. In vitro, MTT and flow cytometry analyses showed that the APC did not negatively affect cell viability and apoptosis. In vivo, corneal pocket and subcutaneous transplantation demonstrated that the APC was incapable of trigging accepted immune response. AECs isolated from the human amniotic membrane have proliferation potential and present healthy morphology before 6 passages. After 7 days of culture on the surface of the APC, the AECs were stratified into 5-6 layers. We found that the AECs reconstituted the basement membrane that had been disrupted by the decellularization process. ELISA results showed that after culturing the TEC, the culture medium contained anti-inflammatory and anti-angiogenic growth factors, such as MIF, IL6, Fas-L, and PDEF. Finally, the results of lamellar keratoplasty to treat an alkali burn showed that the transplanted TEC was transparent and completely inoculated into the host cornea. However, the transplanted APC was degraded due to host rejection. Therefore, we conclude that a TEC composed of AECs and an APC holds great potential for the repair of severe corneal injury.
To assess the influence of donor, environment, and logistical factors on the results of virological testing of blood samples from cornea donors.Data from 670 consecutive cornea donors were analyzed retrospectively. Logistic regression analysis was used to assess the influence of different factors on the results of virological testing of blood samples from cornea donors.The mean annual rate of donors with serology-reactive or not evaluable result was 14.8% (99 of 670) (range 11.9%-16.9%). The cause of donor death by cancer increased the risk of serology-reactive or not evaluable result (P = .0300). Prolonged time between death and post mortem blood removal was associated with a higher rate of serology-reactive or not evaluable result (P < .0001). Mean monthly temperature including warmer months, differentiating between septic and aseptic donors, sex, and donor age had no significant impact on the results of virological testing of blood samples from cornea donors.The cause of donor death by cancer and a prolonged time between death and post mortem blood removal seem to be mainly responsible for serology-reactive or not evaluable result of blood samples from cornea donors. The percentage of discarded corneas caused by serology-reactive or not evaluable result may be reduced by shortening the period of time between death and post mortem blood removal.
The domestic swine eye resembles the human eye both anatomically and physiologically. Xenotransplantation of the swine cornea to humans in need of full keratoplasty shows promise as a potential therapeutic strategy to restore vision in individuals with advanced corneal disease, especially those residing in developing nations. That said, we characterized the morphology of corneas from miniature swine, which are smaller in size, easier to handle, and more cost-effective compared to domestic swine. Eyes (N = 15) were harvested from miniature swine from different age groups: 1 month (N = 3), 2 month (N = 3), 4 month (N = 3), 8 month (N = 3), as well as 24 month old adult domestic swine (N = 3). They were immediately submerged in fixative and processed for histological examination at the light and transmission electron microscopic level. Gross anatomic measurements of the cornea were significantly less (P value ≤ 0.05) in miniature swine versus domestic swine. Corneal strata exhibited morphological characteristics similar to the domestic swine cornea. Adult miniature swine corneas show similar overall corneal thickness at 8 months of age versus domestic swine. Miniature swine exhibit similar corneal morphology with the domestic pig and humans, with the exception of Bowman's layer, which is absent in pigs. Therefore, miniature pigs may be a useful resource of corneal tissue for humans in need of full keratoplasty, as well as serve as a large eye model for ophthalmology residents to develop surgical skills and for development and testing of ocular therapeutic strategies that translate to humans. Anat Rec, 301:1955-1967, 2018. © 2018 Wiley Periodicals, Inc.
The cornea is a transparent, avascular, and highly specialized connective tissue that provides the majority of light refraction in the optical system of the eye. The human cornea is composed of several layers interacting in a complex manner and possessing specific functions, like eye protection and optical clearness. Only few proteomic studies of mammalian cornea have been performed leading to the identification of less than 200 proteins in human corneas. The present study explores the proteome of the intact normal human cornea using a shot-gun nanoLC-MS/MS strategy and an LTQ Orbitrap mass spectrometer. A total of 2070 distinct corneal proteins were identified from five human cornea samples, which represents a 14-fold improvement in the number of proteins identified so far for human cornea. This enlarged dataset of human corneal proteins represents a valuable reference library for further studies on cornea homeostasis and pathophysiology. Network and gene ontology analyses were used to determine biological pathways specific of the human cornea. They allowed the identification of subnetworks of putative importance for corneal diseases, like a redox regulation and oxidative stress network constituted of aldehyde and alcohol dehydrogenases, most of them being described for the first time in human cornea.
Auricularia cornea is a widely cultivated edible fungus with substantial nutritive value. This study aimed to enrich the multifunctional bionutrient element selenium in A. cornea to improve its quality and explore the accumulation of selenium in the fungus using high-throughput RNA-Seq technology. In general, the treatment group with a 100 µg/g supply of selenium outperformed the other treatment groups in terms of high yield, rich crude polysaccharides and a high total selenium concentration. Additional evidences demonstrated the budding and mature phases were two typical growth stages of A. cornea and were important for the accumulation of selenium. Therefore, the budding and mature phase tissues of A. cornea in the treatment group with a 100 µg/g supply of selenium were used for transcriptome analysis and compared to those of a control group that lacked additional selenium. A total of 2.56 × 105 unigenes from A. cornea transcriptome were assembled and annotated to five frequently used databases including NR, GO, KEGG, eggNOG and SwissProt. GO and KEGG pathway analysis revealed that genes involved in metabolic process and translation were up-expressed at the budding stage in response to selenium supplementation, including amino acid metabolism, lipid metabolism, ribosome. In addition, the differential gene expression patterns of A. cornea suggested that the up-expressed genes were more likely to be detected at the budding stage than at the mature stage. These results provide insights into the transcriptional response of A. cornea to selenium accumulation.
Hyaluronan (HA) is a major constituent of the extracellular matrix (ECM) that has high viscosity and is essential for maintaining tissue hydration. In the cornea, HA is enriched in the limbal region and is a key component of the limbal epithelial stem cell niche. HA is upregulated after injury participating in the formation of the provisional matrix, and has a key role in regulating the wound healing process. This study investigated whether changes in the distribution of HA before and after injury affects the biomechanical properties of the cornea in vivo.
The composition and location of professional antigen presenting cells (APC) varies in different mucosal surfaces. The cornea, long considered an immune-privileged tissue devoid of APCs, is now known to host a heterogeneous network of bone marrow-derived cells. Here, we utilized transgenic mice that express enhanced green fluorescent protein (EGFP) from the CD11c promoter (pCD11c) in conjunction with immunohistochemical staining to demonstrate an interesting stratification of APCs within non-inflamed murine corneas. pCD11c(+) dendritic cells (DCs) reside in the basal epithelium, seemingly embedded in the basement membrane. Most DCs express MHC class II on at least some dendrites, which extend up to 50 µm in length and traverse up 20 µm tangentially towards the apical surface of the epithelium. The DC density diminishes from peripheral to central cornea. Beneath the DCs and adjacent to the stromal side of the basement membrane reside pCD11c(-) CD11b(+) putative macrophages that express low levels of MHC class II. Finally, MHC class II(-)pCD11c(-) CD11b(+) cells form a network throughout the remainder of the stroma. This highly reproducible stratification of bone marrow-derived cells is suggestive of a progression from an APC function at the exposed corneal surface to an innate immune barrier function deeper in the stroma.
The objective of this study was to evaluate which hyperelastic model could best describe the non-linear mechanical behavior of the cornea, in order to characterize the capability of the non-linear model parameters to discriminate structural changes in a damaged cornea. Porcine corneas were used, establishing two different groups: control (non-treated) and NaOH-treated (damaged) corneas (n = 8). NaOH causes a chemical burn to the corneal tissue, simulating a disease associated to structural damage of the stromal layer. Quasi-static uniaxial tensile tests were performed in nasal-temporal direction immediately after preparing corneal strips from the two groups. Three non-linear hyperelastic models (i.e. Hamilton-Zabolotskaya model, Ogden model and Mooney-Rivlin model) were fitted to the stress-strain curves obtained in the tensile tests and statistically compared. The corneas from the two groups showed a non-linear mechanical behavior that was best described by the Hamilton-Zabolotskaya model, obtaining the highest coefficient of determination (R2 > 0.95). Moreover, Hamilton-Zabolotskaya model showed the highest discriminative capability of the non-linear model parameter (Parameter A) for the tissue structural changes between the two sample groups (p = 0.0005). The present work determines the best hyperelastic model with the highest discriminative capability in description of the non-linear mechanical behavior of the cornea.
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