Chronic Sleep Deprivation Impairs Visual Functions via Oxidative Damage in Mice.

Sleep deprivation (SD) is a global public health burden, and has a detrimental role in the nervous system. Retina is an important part of the central nervous system; however, whether SD affects retinal structures and functions remains largely unknown. Herein, chronic SD mouse model indicated that loss of sleep for 4 months could result in reductions in the visual functions, but without obvious morphologic changes of the retina. Ultrastructural analysis by transmission electron microscope revealed the deterioration of mitochondria, which was accompanied with the decrease of multiple mitochondrial proteins in the retina. Mechanistically, oxidative stress was provoked by chronic SD, which could be ameliorated after rest, and thus restore retinal homeostasis. Moreover, the supplementation of two antioxidants, α-lipoic acid and N-acetyl-l-cysteine, could reduce retinal reactive oxygen species, repair damaged mitochondria, and, as a result, improve the retinal functions. Overall, this work demonstrated the essential roles of sleep in maintaining the integrity and health of the retina. More importantly, it points towards supplementation of antioxidants as an effective intervention strategy for people experiencing sleep shortages.

Berberine ameliorate inflammation and apoptosis via modulating PI3K/AKT/NFκB and MAPK pathway on dry eye.

BACKGROUND: Dry eye disease (DED) is a multifactorial disease in ocular surface, and inflammation plays an etiological role. Berberine (BBR) has shown efficacy in treating inflammatory diseases. Yet, there was no adequate information related to the therapeutic effects of BBR for DED. PURPOSE: To detect the effects and explore the potential mechanisms of BBR on DED. STUDY DESIGN: In vitro, in vivo study and network pharmacology analysis were involved. METHOD: The human corneal epithelium cells viability was evaluated with different concentrations of BBR. Dry eye murine model was established by exposing to the desiccating stress, and Ciclosporin (CSA), BBR eye drops or vehicle were topical administration for 7 days. The phenol red cotton tests, Oregon-green-dextran staining and Periodic acid-Schiff staining were performed and evaluated the dry eye after treatment. Inflammation and apoptosis levels of ocular surface were quantified. The potential targets related to berberine and dry eye were collected from databases. The Protein-Protein interaction network analysis and GO & KEGG enrichment analysis were realized by STRING database, Metascape platform and Cytoscape software to find core targets and signaling pathways. The SchrÖdinger software was used to molecular docking and PyMOL software to visualization. Finally, the levels of PI3K/AKT/NFκB and MAPK pathways were detected. RESULT: The data revealed BBR could rescue impaired HCE under hyperosmotic conditions. In addition, BBR eye drops could ameliorate dry eye. And BBR eye drops suppressed the inflammatory factors and CD4+T cells infiltration in conjunctiva. Besides, BBR eye drops protected ocular surface by avoiding the severe apoptosis and decreasing the level of MMP-3 and MMP-9. 148 common targets intersection between BBR and dry eye were found via network pharmacology analysis. Core proteins and core pathways were identified through PPI and GO&KEGG enrichment analysis. Molecular docking displayed excellent binding between BBR and those core targets. Finally, in vivo study verified that BBR eye drops had a therapeutic effect in dry eye by inhibiting PI3K/AKT/NFκB and MAPK pathways. CONCLUSION: The research provided convincing evidence that BBR could be a candidate drug for dry eye.

Limbal Stem Cell Dysfunction Induced by Severe Dry Eye via Activation of the p38 MAPK Signaling Pathway.

Severe dry eye (SDE) can cause grievous damage to the ocular surface and result in vision impairment and even blindness. To investigate the fate of limbal stem cells in SDE and the underlying mechanism, the current study established an SDE rat model by removing the extraorbital and infraorbital lacrimal glands and maintaining them in a low-humidity environment. One month after the surgery, aqueous tear secretion was reduced dramatically, blood vessels invaded into the central cornea, and inflammatory cells infiltrated into the limbal stroma. The expressions of keratin 12 and paired box gene 6 were down-regulated dramatically, while those of keratin 10, small proline-rich protein 1b, and mucin 5AC were up-regulated in the corneal epithelium of the SDE rats. Cell proliferation in the limbal epithelium was up-regulated, while the stem/progenitor marker adenosine 5'-triphosphate-binding cassette member 2 and the limbal epithelial colony-forming efficiency were decreased in the SDE condition. Furthermore, the p38 mitogen-activated protein kinase signaling pathway was activated in the limbal corneal epithelium of SDE rats. The abnormal differentiation and stemness loss in the corneal epithelium could be reversed upon treatment with a p38 inhibitor in a SDE in vivo model and in vitro hyperosmolar corneal epithelial culture conditions. These data suggest that SDE can lead to limbal stem cell dysfunction, and p38 mitogen-activated protein kinase signaling pathway activation plays an essential role in this process.

T-Cell Repertoire Analysis in the Conjunctiva of Murine Dry Eye Model.

Purpose: Dry eye is closely related to the activation and proliferation of immune cells, especially T cells. However, the determination of the preferential T-cell clonotypes is technically challenging. This study aimed to investigate the characterization of T-cell receptor (TCR) repertoire in the conjunctiva during dry eye. Methods: A desiccating stress animal model was established using C57/BL6 mice (8-10 weeks, female). After 7 days of stress stimulation, the slit-lamp image and Oregon-green-dextran staining were used to evaluate the ocular surface injury. Periodic acid-Schiff staining was used to measure the number of goblet cells. Flow cytometry was used to detect the activation and proliferation of T cells in the conjunctiva and cervical lymph nodes. Next-generation sequencing was used to detect the αß TCR repertoire of the conjunctiva. Results: The αß TCR diversity increased significantly in the dry eye group, including the higher CDR3 amino acid length, marked gene usage on TCR V and J gene segments, extensive V(D)J recombination, and distinct CDR3 aa motifs. More important, several T-cell clonotypes were uniquely identified in dry eye. Furthermore, these perturbed rearrangements were reversed after glucocorticoid administration. Conclusions: A comprehensive analysis of the αß TCR repertoire in the conjunctiva of the dry eye mouse model was performed. Data in this study contributed significantly to the research on dry eye pathogenesis by demonstrating the TCR gene distribution and disease-specific TCR signatures. This study further provided some potential predictive T-cell biomarkers for future studies.

Primary Culture of Porcine Retinal Pigment Epithelial Cells.

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Downregulation of p38 MAPK Signaling Pathway Ameliorates Tissue-Engineered Corneal Epithelium.

Tissue-engineered corneal epithelium transplantation is effective treatment for severe limbal stem cell deficiency (LSCD), while epithelial terminal differentiation, tans-differentiation, and insufficient stem cell during construction affect the quality of tissue-engineered corneal epithelium. In this study, we applied SB203580 in the culture medium to downregulate the p38 mitogen-activated protein kinase (MAPK) signaling pathway during construction of tissue-engineered corneal epithelium. With application of SB203580, tissue-engineered corneal epithelium showed enhanced strength and condensed structure. The expression of progenitor cell markers ATP-binding cassette sub-family G member 2, tumor protein p63, keratin 14, and Wnt family member 7A was increased, differentiation markers keratin 12, paired box 6, keratin 10, and keratin 13 and trans-differentiation markers actin alpha 2, smooth muscle and snail family transcriptional repressor 1 was decreased, while cell junction markers claudin 1 and cadherin 1 was increased in the tissue-engineered corneal epithelium. The Wnt/catenin beta 1 signaling pathway was upregulated in the epithelium after p38 MAPK inhibition. Transplantation of tissue-engineered corneal epithelium treated with SB203580 to rabbit LSCD model showed faster wound healing and improved epithelial quality. We conclude that downregulation of p38 MAPK signaling pathway helps maintain the stemness and prevent terminal differentiation and abnormal differentiation of corneal epithelial cells during epithelium construction process, and thus can improve the quality of tissue-engineered corneal epithelium. Impact statement Downregulation of p38 MAPK signaling pathway helps maintain the self-renewal of limbal stem cells and prevents terminal differentiation and abnormal differentiation of corneal epithelial cells. Small molecules modulating p38 MAPK signaling pathway ameliorate tissue-engineered corneal epithelium.

EMT-Related Gene Signature Predicts the Prognosis in Uveal Melanoma Patients.

Background: Uveal melanoma (UVM) is the most common primary intraocular malignancy in adults. Epithelial-mesenchymal transition (EMT) is an essential regulator of the UVM's immune microenvironment. However, the precise role of EMT in UVM remains to be explored and the development of a related treatment strategy is urgently needed. Methods: Multiomics data and clinical information for TCGA-UVM were used to identify the EMT subtypes and analyze their regulatory role in the immune microenvironment in UVM. A machine-learning method based on the identified subtypes was utilized to construct the EMT feature-based prognostic model. External validation cohorts GSE84976 and GSE22138 were employed to validate the model's robustness. Immunotherapy cohort IMvigor210 was used to explore the model's potential to predict immunotherapy responsiveness. Results: Two EMT subtypes were identified in UVM. The role of EMT in shaping the immune microenvironment and regulating cancer-immunity circle of UVM was analyzed. A robust prognostic model was presented and validated to predict patient prognosis. The model also predicted patient's immune features and immunotherapy responsiveness. Conclusion: The EMT-mediated immune features in UVM were illustrated, providing a reliable model to facilitate precise UVM treatment. This research may assist in decision-making during clinical UVM therapy.

Novel Cell Culture Paradigm Prolongs Mouse Corneal Epithelial Cell Proliferative Activity in vitro and in vivo.

It has been a long-standing challenge to obtain from cell cultures adequate amounts of mouse corneal epithelial cells (mCEC) to perform transplantation surgery. This limitation is attributable to the passage dependent declines in their proliferative activity. We describe here development of a novel 6C medium that contains six different modulators of different signaling pathways, which control proliferative mCEC activity. Its usage shortens the time and effort required to obtain epithelial sheets for hastening healing of an epithelial wound in an experimental animal model. This serum-free 6C medium contains:Y27632, forskolin, SB431542, DAPT, IWP-2, LDN-193189 and also DermaLife K keratinocyte calcium. Their inclusion inhibits rises in four specific markers of epithelial mesenchymal transdifferentiation:ZEB1/2, Snail, ß-catenin and α-SMA. This medium is applied in a feeder-free air-lifted system to obtain sufficient populations of epithelial progenitor cells whose procurement is facilitated due to suppression of progenitor epithelial cell transdifferentiation into epithelial-mesenchymal cells. Diminution of this decline in transdifferentiation was confirmed based on the invariance of P63, K14, Pax6, and K12 gene expression levels. This cell culture technique is expected to facilitate ex vivo characterization of mechanisms underlying cell fate determination. Furthermore, its implementation will improve yields of progenitor mouse corneal epithelial cells, which increases the likelihood of using these cells as a source to generate epithelial sheets for performing transplantation surgery to treat limbal stem cell deficiency in a clinical setting. In addition, the novel insight obtainable from such studies is expected to improve the outcomes of corneal regenerative medicine.

Canonical Wnt Signaling Drives Myopia Development and Can Be Pharmacologically Modulated.

Purpose: The purpose of this study was to evaluate the role of the canonical Wnt signaling in the development of the myopia. Methods: Plasma from adult patients with myopia, myopic animal models including the adenomatous polyposis coli (APC) gene mutation mouse model, and the form deprivation (FD) induced mouse model of myopia were used. Niclosamide, a canonical Wnt pathway inhibitor, was orally administrated in animal models. Plasma levels of DKK-1 were determined by using enzyme-linked immunosorbent assay. Refraction, vitreous chamber depth (VCD), axial length (AL), and other parameters, were measured at the end of the FD treatment. Canonical Wnt signaling changes were evaluated by Western blot analysis and immunostaining analysis. Results: Plasma level of Wnt inhibitor DKK-1 was markedly decreased in patients with myopia. Meanwhile, the canonical Wnt pathway was progressively activated during myopia development in mice. Moreover, inhibition of canonical Wnt signaling by niclosamide in mouse models markedly reduced lens thickness (LT), VCD, and AL elongation, resulting in myopia inhibition. Conclusions: Dysregulation of canonical Wnt signaling is a characteristic of myopia and targeting Wnt signaling pathways has potential as a therapeutic strategy for myopia.

Phenotypic and transcriptomic changes in the corneal epithelium following exposure to cigarette smoke.

Cigarette smoke extract (CSE), a complex mixture of compounds, contributes to a range of eye diseases; however, the underlying pathophysiological responses to tobacco smoke remain ambiguous. The purpose of the present study was to evaluate the cigarette smoke-induced phenotypic and transcriptomic changes in the corneal epithelium with a view to elucidating the likely underlying mechanism. Accordingly, for the first time, we characterized the genome-wide effects of CSE on the corneal epithelium. The ocular surface of the mice in the experimental groups was exposed to CSE for 1 h per day for a period of one week, while mice in the control group were exposed to preservative-free artificial tears. Corneal fluorescein staining, in vivo confocal microscopy and scanning electron microscopy were performed to examine the corneal ultrastructure. Transcriptome sequencing and bioinformatics analysis were performed followed by RT-qPCR to validate gene expression changes. The results indicate that CSE exposure disrupted the structural integrity of the superficial epithelium, decreased the density of microvilli, and compromised the corneal epithelial barrier intactness. RNA-seq revealed 667 differentially expressed genes, and functional analysis highlighted the enhancement of several biological processes such as antioxidant activity and the response to oxidative stress. Moreover, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that glutathione metabolism and drug metabolism cytochrome P450 were the most relevant pathways contributing to the effects of CSE on the corneal epithelium. Protein-protein interaction (PPI) network analysis illustrated that GCLC, NQO1, and HMOX1 were the most relevant nodes. In conclusion, the present study indicates that CSE exposure induces changes in the phenotype and genotype of the corneal epithelium. The antioxidant response element is essential for counteracting the effects of cigarette smoke on this tissue layer. These results shed novel insights into how cigarette smoke damages this ocular surface.

Comprehensive strategy improves the genetic diagnosis of different polycystic kidney diseases.

Polycystic kidney disease (PKD) is known to occur in three main forms, namely autosomal dominant PKD (ADPKD), autosomal recessive PKD (ARPKD) and syndromic PKD (SPKD), based on the clinical manifestations and genetic causes, which are diagnosable from the embryo stage to the later stages of life. Selection of the genetic test for the individuals with diagnostic imaging reports of cystic kidneys without a family history of the disease continues to be a challenge in clinical practice. With the objective of maintaining a limit on the time and medical cost of the procedure, a practical strategy for genotyping and targeted validation to resolve cystogene variations was developed in our clinical laboratory, which combined the techniques of whole-exome sequencing (WES), Long-range PCR (LR-PCR), Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to work in a stepwise approach. In this context, twenty-six families with renal polycystic disorders were enrolled in the present study. Thirty-two variants involving four ciliary genes (PKD1, PKHD1, TMEM67 and TMEM107) were identified and verified in 23 families (88.5%, 23/26), which expanded the variant spectrum by 16 novel variants. Pathogenic variations in five foetuses of six families diagnosed with PKD were identified using prenatal ultrasound imaging. Constitutional biallelic and digenic variations constituted the pathogenic patterns in these foetuses. The preliminary clinical data highlighted that the WES + LR PCR-based workflow followed in the present study is efficient in detecting divergent variations in PKD. The biallelic and digenic mutations were revealed as the main pathogenic patterns in the foetuses with PKD.

Novel Biallelic DNAH1 Variations Cause Multiple Morphological Abnormalities of the Sperm Flagella.

Sperm motility is vital to human reproduction, and malformed sperm flagella can cause male infertility. Individuals with multiple morphological abnormalities of the flagella mostly have absent, short, coiled, bent, and/or irregular-caliber flagella. In this study, a patient with male infertility underwent a physical examination along with his wife. Genetic testing was performed by whole-exome sequencing of the couple, and Sanger sequencing was performed for validation. Novel biallelic variations in the DNAH1: (NM_015512.4) gene consisting of c.1336G>C (p.E446Q) and c.2912G>A (p.R971H) were identified. In silico structural analysis revealed that the amino acid residues affected by the variation were evolutionarily conserved, and the variant p.R971H influenced the stability of the DNAH1 protein. Morphological studies of the patient's sperm showed defects in its flagella. Results of Papanicolaou staining and scanning electron microscopy demonstrated coiled and short flagella with multiple anomalies. Transmission electron microscopy of the sperm flagella showed that the inner dynein arm and radial spoke were absent, and the dense fiber and microtubule doublets were displaced. Quantitative PCR of the mRNA of the patient's sperm showed that the expression of DNALI1 was dramatically reduced. Collectively, these findings elucidated the genetic cause of the family's infertility and provided insight into the functioning of the DNAH1 gene.

IL-1/IL-1R signaling induced by all-trans-retinal contributes to complement alternative pathway activation in retinal pigment epithelium.

The underlying mechanisms of complement activation in Stargardt disease type 1 (STGD1) and age-related macular degeneration (AMD) are not fully understood. Overaccumulation of all-trans-retinal (atRAL) has been proposed as the pathogenic factor in both diseases. By incubating retinal pigment epithelium (RPE) cells with atRAL, we showed that C5b-9 membrane attack complexes (MACs) were generated mainly through complement alternative pathway. An increase in complement factor B (CFB) expression as well as downregulation of complement regulatory proteins CD46, CD55, CD59, and CFH were observed in RPE cells after atRAL treatment. Furthermore, interleukin-1ß production was provoked in both atRAL-treated RPE cells and microglia/macrophages. Coincubation of RPE cells with interleukin-1 receptor antagonist (IL1Ra) and atRAL ameliorated complement activation and downregulated CFB expression by attenuating both p38 and c-Jun N-terminal kinase (JNK) signaling pathways. Our findings demonstrate that atRAL induces an autocrine/paracrine IL-1/IL-1R signaling to promote complement alternative pathway activation in RPE cells and provide a novel perspective on the pathomechanism of macular degeneration.

Transepithelial corneal cross-linking assisted by two continuous cycles of iontophoresis for progressive keratoconus in adults: retrospective 5-year analysis.

PURPOSE: The aim of this study is to compare the long-term effects of transepithelial corneal crosslinking with two continuous cycles of iontophoresis (EI-CXL) and conventional corneal crosslinking (C-CXL) in adults with progressive keratoconus. METHODS: A retrospective analysis was conducted in adults who underwent C-CXL or EI-CXL between 2013 and 2015. Visual acuity, corneal tomography, anterior segment optical coherence tomography, in vivo corneal confocal microscopy (IVCM), and endothelial cell count (ECC) were performed preoperatively and 5 years postoperatively. RESULTS: Sixty-eight patients with a mean age of (24.3 ± 3.8) years were included, 34 for each group. After CXL, UCVA or BCVA remained stable, while the spherical diopter, cylinder diopter, spherical equivalent, and Kmax significantly decreased at 1, 2, and 3 years in both groups than baseline (P < 0.05). No significant differences were found in any refractive or tomographic parameters as well as the minimal corneal thickness between groups during follow-up. At 5 years, Kmax was slightly higher in EI-CXL group (58.16 ± 6.28) than that of C-CXL group (57.46 ± 4.98). At 3 and 5 years, the minimal corneal thickness in C-CXL group was still significantly lower than baseline (P < 0.05). IVCM demonstrated the demarcation zone at a mean depth of (302.0 ± 41.7) µm after C-CXL, and at (251.2 ± 28.1) µm after EI-CXL (P < 0.001). Keratocyte repopulation was detectable at all follow-up timepoint in both groups. Postoperative complications including progression were recorded in 6 patients (11.7%) after C-CXL and 3 patients (8.8%) after EI-CXL. ECC remained stable in both groups. CONCLUSION: EI-CXL showed approximate efficacy with C-CXL in stabilizing progressive keratoconus in adults. EI-CXL has the potential to be a preferable transepithelial protocol.

Tissue engineered corneal epithelium derived from clinical-grade human embryonic stem cells.

PURPOSE: To construct tissue engineered corneal epithelium from a clinical-grade human embryonic stem cells (hESCs) and investigate the dynamic gene profile and phenotypic transition in the process of differentiation. METHODS: A stepwise protocol was applied to induce differentiation of clinical-grade hESCs Q-CTS-hESC-1 and construct tissue engineered corneal epithelium. Single cell RNA sequencing (scRNA-seq) analysis was performed to monitor gene expression and phenotypic changes at different differentiation stages. Immunostaining, real-time quantitative PCR and Western blot analysis were conducted to detect gene and protein expressions. After subcutaneous transplantation into nude mice to test the biosafety, the epithelial construct was transplanted in a rabbit corneal limbal stem cell deficiency (LSCD) model and followed up for eight weeks. RESULTS: The hESCs were successfully induced into epithelial cells. scRNA-seq analysis revealed upregulation of ocular surface epithelial cell lineage related genes such as TP63, Pax6, KRT14, and activation of Wnt, Notch, Hippo, and Hedgehog signaling pathways during the differentiation process. Tissue engineered epithelial cell sheet derived from hESCs showed stratified structure and normal corneal epithelial phenotype with presence of clonogenic progenitor cells. Eight weeks after grafting the cell sheet onto the ocular surface of LSCD rabbit model, a full-thickness continuous corneal epithelium developed to fully cover the damaged areas with normal limbal and corneal epithelial phenotype. CONCLUSION: The tissue engineered corneal epithelium generated from a clinical-grade hESCs may be feasible in the treatment of limbal stem cell deficiency.

Onion Epithelial Membrane Scaffolds Transfer Corneal Epithelial Layers in Reconstruction Surgery.

Plants and their extracts have been used especially in China for more than ten centuries for preventing and treating disease. However, there are only few reports describing their use in animal cell culture and tissue transplantation. In this study, onion epithelial membranes (OEM) is used as scaffolds to support cultures of a variety of cells such as fibroblasts and epithelial cells notably; they maintain the phenotypic characteristics of corneal epithelial cells. This improvement includes preservation of the proliferative potential and stemness of rabbit corneal epithelial cells (RCECs). Such an outcome suggests that this cost-effective technology warrants further evaluation to determine if OEM is a viable candidate for use as scaffolds in corneal epithelial transplantation surgery. To test this possibility, rabbit corneal epithelial cells expanded on OEM are transplanted to treat corneal epithelial defects in limbal stem cell deficient rabbits. This procedure is successful because it shortens the time required for wound healing to restore losses in corneal epithelial integrity, and forms a more compact and stratified epithelium framework than the untreated group. Ultimately, should they be proven to be effective in other relevant animal model systems, their usefulness for treating wounds in a clinical setting warrants consideration.

High-fat diet induces dry eye-like ocular surface damages in murine.

PURPOSE: A high-fat diet leads to dysfunction in multiple systems of the body. Herein we investigate the effects of a high-fat diet on the ocular surface using a murine model. METHODS: Four-week-old male C57BL/6 mice were fed with a standard-fat diet (10 kcal% fat, SFD) or a high-fat diet (60 kcal% fat, HFD) for 1 or 3 months. Phenol red thread test was used to detect tear production, oregon green dextran (OGD) staining was performed to assess corneal epithelial permeability, and PAS staining was conducted to ascertain the presence of conjunctival goblet cells. Squamous metaplasia in the ocular surface and corneal epithelial barrier function were detected by immunofluorescent staining, zymography and Western blot analysis. Oxidative stress related protein expression was evaluated by immunostaining and Western blot analysis. Corneal and conjunctival cell apoptosis was determined by TUNEL assay and caspase-3 expression. RESULTS: A HFD induced obvious ocular surface damages, including decreased tear production, notable OGD staining and distinct goblet cell loss. It also resulted in corneal epithelial barrier dysfunction and significant squamous metaplasia of the corneal and conjunctival epithelia. The HFD also upregulated key factors that regulate oxidative stress in the ocular surface, and upregulated cell apoptosis in ocular surface epithelial cells. CONCLUSIONS: A HFD induces dry eye-like ocular surface damages in mice via the activation of oxidative stress and an induction of apoptosis in the cells of the ocular surface.

Hyperlipidemia Affects Tight Junctions and Pump Function in the Corneal Endothelium.

Hyperlipidemia impacts on various diseases, such as atherosclerosis, hypertension, and diabetes mellitus. However, its influence, if any, on ocular tissues is largely unknown. Herein, we developed hyperlipidemic murine models by feeding 4-week-old male wild-type mice with a high-fat diet and apolipoprotein E knockout (ApoE-/-) mice with a high-fat diet or standard diet to investigate the corneal endothelial change under hyperlipidemic conditions. Oil Red O staining showed an accumulation of lipid droplets in corneal endothelial cells (CECs) of hyperlipidemic mice. Other manifestations included a reduced cell density and distorted cell morphology, a disruption of the endothelial cell tight junctions and adhesion junctions, a reduced number of surface microvilli, down-regulation of Na+-K+-ATPase expression and function, activation of oxidative stress, changes in mitochondrial ultrastructure, and increased apoptosis. CEC recovery after injury, moreover, was diminished in hyperlipidemic mice; and high palmitate levels were found in the aqueous humor. In vitro hyperlipemia model, moreover, was found to be associated with dose-dependent CEC cytotoxicity, altered cell morphology, reduced pump function, and an induction of oxidative stress, leading to functional and pathologic changes in the corneal endothelium.

A New Perspective on Dry Eye Classification: Proposal by the Asia Dry Eye Society.

The 2017 consensus report of the Asia Dry Eye Society (ADES) on the definition and diagnosis of dry eyes described dry eye disease as "Dry eye is a multifactorial disease characterized by unstable tear film causing a variety of symptoms and/or visual impairment, potentially accompanied by ocular surface damage." The report emphasized the instability of tear film and the importance of visual dysfunction in association with dry eyes, highlighting the importance of the evaluation of tear film stability. This report also discussed the concept of tear film-oriented therapy, which stemmed from the definition, and which is centered on provision of insufficient components in each tear film layer and ocular surface epithelium. The current ADES report proposes a simple classification of dry eyes based on the concept of tear film-oriented diagnosis and suggests that there are three types of dry eye: aqueous-deficient, decreased wettability, and increased evaporation. It is suggested that these three types respectively coincide with the problems of each layer: aqueous, membrane-associated mucins, and lipid/secretory mucin. Although each component cannot be quantitatively evaluated with the current technology, a practical diagnosis based on the patterns of fluorescein breakup is recommended. The Asia Dry Eye Society classification report suggests that for a practical use of the definition, diagnostic criteria and classification system should be integrated and be simple to use. The classification system proposed by ADES is a straightforward tool and simple to use, only through use of fluorescein, which is available even to non-dry eye specialists, and which is believed to contribute to an effective diagnosis and treatment of dry eyes.

Application of benzonase in preparation of decellularized lamellar porcine corneal stroma for lamellar keratoplasty.

This study was to develop anovel and efficient method using endonuclease (benzonase) to preparedecellularized lamellar porcine corneal stroma (DLPCS). The DLPCS was preparedfrom native lamellar porcine corneal stroma (NLPCS) and was treated with 1000 U/ml benzonase for 5hours. We conducted the following measurements and animal transplantation tocompare DLPCS and NLPCS. The residual DNA was decreased significantly from 367.13 ± 19.96 ng/mg to 15.41 ± 0.65 ng/mg after treatment of benzonase by the detection of fluorescentnucleic acid stain. The residual benzonase was also less than detection limit.There was no significant difference in light transmittance of DLPCS comparedwith NLPCS. The extracts of DLPCS did not inhibit cell proliferation of human cornealepithelial cells, mouse fibroblast (L-929) and African green monkey kidney cell(Vero cell). The DLPCS was transplanted into the corneas of rabbit by lamellarkeratoplasty. There was no corneal melting and graft rejection been observedwithin 12 months. The images demonstrated that the repairment of corneal nervesand keratocytes of DLPCS were in indentical shape and reflection compared withnormal cornea, and no obvious inflammatory cells were observed postoperation, byin vivo confocal microscopy. We provided novel evidence that the application ofbenzonase may improve the quality of DLPCS.