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.

Accurate detection and grading of pterygium through smartphone by a fusion training model.

BACKGROUND/AIMS: To improve the accuracy of pterygium screening and detection through smartphones, we established a fusion training model by blending a large number of slit-lamp image data with a small proportion of smartphone data. METHOD: Two datasets were used, a slit-lamp image dataset containing 20 987 images and a smartphone-based image dataset containing 1094 images. The RFRC (Faster RCNN based on ResNet101) model for the detection model. The SRU-Net (U-Net based on SE-ResNeXt50) for the segmentation models. The open-cv algorithm measured the width, length and area of pterygium in the cornea. RESULTS: The detection model (trained by slit-lamp images) obtained the mean accuracy of 95.24%. The fusion segmentation model (trained by smartphone and slit-lamp images) achieved a microaverage F1 score of 0.8981, sensitivity of 0.8709, specificity of 0.9668 and area under the curve (AUC) of 0.9295. Compared with the same group of patients' smartphone and slit-lamp images, the fusion model performance in smartphone-based images (F1 score of 0.9313, sensitivity of 0.9360, specificity of 0.9613, AUC of 0.9426, accuracy of 92.38%) is close to the model (trained by slit-lamp images) in slit-lamp images (F1 score of 0.9448, sensitivity of 0.9165, specificity of 0.9689, AUC of 0.9569 and accuracy of 94.29%). CONCLUSION: Our fusion model method got high pterygium detection and grading accuracy in insufficient smartphone data, and its performance is comparable to experienced ophthalmologists and works well in different smartphone brands.

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.

The cGAS-STING pathway-dependent sensing of mitochondrial DNA mediates ocular surface inflammation.

The innate immune response is the main pathophysiological process of ocular surface diseases exposed to multiple environmental stresses. The epithelium is central to the innate immune response, but whether and how innate immunity is initiated by ocular epithelial cells in response to various environmental stresses in ocular surface diseases, such as dry eye, is still unclear. By utilizing two classic experimental dry eye models-a mouse ocular surface treated with benzalkonium chloride (BAC) and a mouse model with surgically removed extraorbital lachrymal glands, as well as dry eye patient samples-along with human corneal epithelial cells (HCE) exposed to hyperosmolarity, we have discovered a novel innate immune pathway in ocular surface epithelial cells. Under stress, mitochondrial DNA (mtDNA) was released into the cytoplasm through the mitochondrial permeability transition pore (mPTP) and further activated the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, aggravating downstream inflammatory responses and ocular surface damage. Genetic deletion or pharmacological suppression of STING and inhibition of mtDNA release reduced inflammatory responses, whereas mtDNA transfection supported cytoplasmic mtDNA-induced inflammatory responses by activating the cGAS-STING pathway. Our study clarified the cGAS-STING pathway-dependent sensing of mitochondrial DNA-mediated ocular surface inflammation, which elucidated a new mechanism of ocular surface diseases in response to multiple environmental stresses.

MAP4Ks inhibition promotes retinal neuron regeneration from Müller glia in adult mice.

Mammalian Müller glia (MG) possess limited regenerative capacities. However, the intrinsic capacity of mammalian MG to transdifferentiate to generate mature neurons without transgenic manipulations remains speculative. Here we show that MAP4K4, MAP4K6 and MAP4K7, which are conserved Misshapen subfamily of ste20 kinases homologs, repress YAP activity in mammalian MG and therefore restrict their ability to be reprogrammed. However, by treating with a small molecule inhibitor of MAP4K4/6/7, mouse MG regain their ability to proliferate and enter into a retinal progenitor cell (RPC)-like state after NMDA-induced retinal damage; such plasticity was lost in YAP knockout MG. Moreover, spontaneous trans-differentiation of MG into retinal neurons expressing both amacrine and retinal ganglion cell (RGC) markers occurs after inhibitor withdrawal. Taken together, these findings suggest that MAP4Ks block the reprogramming capacity of MG in a YAP-dependent manner in adult mammals, which provides a novel avenue for the pharmaceutical induction of retinal regeneration in vivo.

Can the cGAS-STING Pathway Play a Role in the Dry Eye?

Dry eye is one of the most common ocular surface diseases in the world and seriously affects the quality of life of patients. As an immune-related disease, the mechanism of dry eye has still not been fully elucidated. The cGAS-STING pathway is a recently discovered pathway that plays an important role in autoimmune and inflammatory diseases by recognizing dsDNA. As an important signal to initiate inflammation, the release of dsDNA is associated with dry eye. Herein, we focused on the pathophysiology of the immune-inflammatory response in the pathogenesis of dry eye, attempted to gain insight into the involvement of dsDNA in the dry eye immune response, and investigated the mechanism of the cGAS-STING pathway involved in the immune-inflammatory response. We further proposed that the cGAS-STING pathway may participate in dry eye as a new mechanism linking dry eye and the immune-inflammatory response, thus providing a new direction for the mechanistic exploration of dry eye.

Sleep deprivation induces corneal epithelial progenitor cell over-expansion through disruption of redox homeostasis in the tear film.

Sleep deficiency, a common public health problem, causes ocular discomfort and affects ocular surface health. However, the underlying mechanism remains unclear. Herein, we identified that short-term sleep deprivation (SD) resulted in hyperproliferation of corneal epithelial progenitor cells (CEPCs) in mice. The expression levels of p63 and Keratin 14, the biomarkers of CEPCs, were upregulated in the corneal epithelium after short-term SD. In addition, SD led to elevated levels of reactive oxygen species (ROS), and subsequent decrease in antioxidant capacity, in the tear film. Exogenous hydrogen peroxide (H2O2) could directly stimulate the proliferation of CEPCs in vivo and in vitro. Topical treatment of antioxidant L-glutathione preserved the over-proliferation of CEPCs and attenuated corneal epithelial defects in SD mice. Moreover, the activation of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway is essential to ROS-stimulated cell proliferation in CEPCs. However, long-term SD ultimately led to early manifestation of limbal stem cell deficiency.

Obstructive Sleep Apnea Affects Lacrimal Gland Function.

PURPOSE: To determine the effect of obstructive sleep apnea syndrome (OSA) on lacrimal gland function and its mechanism. METHODS: Male mice aged seven to eight weeks were housed in cages with cyclic intermittent hypoxia to mimic OSA, and the control group was kept in a normal environment. Slit-lamp observation, fluorescein staining, and corneal sensitivity detection are used to assess cornea changes. Tear secretion was detected by phenol red cotton thread, and the pathological changes of lacrimal gland were observed by hematoxylin and eosin staining, oil red O staining, cholesterol and triglyceride kits, immunofluorescence staining, immunohistochemical staining, real-time polymerase chain reaction, transmission electron microscopy, and Western blot. RESULTS: Studies revealed a decreased tear secretion, corneal epithelial defects and corneal hypersensitivity. Myoepithelial cell damage, abnormal lipid accumulation, reduced cell proliferation, increased apoptosis and inflammatory cell infiltration in the lacrimal gland were also seen. Hifα and NF-κB signaling pathways, moreover, were activated, while Pparα was downregulated, in the lacrimal glands of OSA mice. Fenofibrate treatment significantly alleviated pathological changes of the lacrimal gland induced by OSA. CONCLUSION: OSA disturbs the Hifα/Pparα/NF-κB signaling axis, which affects lacrimal gland structure and function and induces dry eye.

Single-Cell Transcriptomics Identifies a Unique Entity and Signature Markers of Transit-Amplifying Cells in Human Corneal Limbus.

Purpose: Differentiated from adult stem cells (ASCs), transit-amplifying cells (TACs) play an important role in tissue homeostasis, development, and regeneration. This study aimed to characterize the gene expression profile of a candidate TAC population in limbal basal epithelial cells using single-cell RNA sequencing (scRNA-seq). Methods: Single cells isolated from the basal corneal limbus were subjected to scRNA-seq using the 10x Genomics platform. Cell types were clustered by graph-based visualization methods and unbiased computational analysis. BrdU proliferation assays, immunofluorescent staining, and real-time reverse transcription quantitative polymerase chain reaction were performed using multiple culture models of primary human limbal epithelial cells to characterize the TAC pool. Results: Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters. A unique cluster (3.21% of total cells) was identified as a TAC entity, based on its less differentiated progenitor status and enriched exclusive proliferation marker genes, with 98.1% cells in S and G2/M phases. The cell cycle-dependent genes were revealed to be largely enriched by the TAC population. The top genes were characterized morphologically and functionally at protein and mRNA levels. The specific expression patterns of RRM2, TK1, CENPF, NUSAP1, UBE2C, and CDC20 were well correlated in a time- and cycle-dependent manner with proliferation stages in the cell growth and regeneration models. Conclusions: For the first time, to the best of our knowledge, we have identified a unique TAC entity and uncovered a group of cell cycle-dependent genes that serve as TAC signature markers. The findings provide insight into ASCs and TACs and lay the foundation for understanding corneal homeostasis and diseases.

Single-cell transcriptomics identifies limbal stem cell population and cell types mapping its differentiation trajectory in limbal basal epithelium of human cornea.

PURPOSE: This study aimed to uncover novel cell types in heterogenous basal limbus of human cornea for identifying LSC at single cell resolution. METHODS: Single cells of human limbal basal epithelium were isolated from young donor corneas. Single-cell RNA-Sequencing was performed using 10x Genomics platform, followed by clustering cell types through the graph-based visualization method UMAP and unbiased computational informatic analysis. Tissue RNA in situ hybridization with RNAscope, immunofluorescent staining and multiple functional assays were performed using human corneas and limbal epithelial culture models. RESULTS: Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters belonging to three lineages. A smallest cluster (0.4% of total cells) was identified as LSCs based on their quiescent and undifferentiated states with enriched marker genes for putative epithelial stem cells. TSPAN7 and SOX17 are discovered and validated as new LSC markers based on their exclusive expression pattern and spatial localization in limbal basal epithelium by RNAscope and immunostaining, and functional role in cell growth and tissue regeneration models with RNA interference in cultures. Interestingly, five cell types/states mapping a developmental trajectory of LSC from quiescence to proliferation and differentiation are uncovered by Monocle3 and CytoTRACE pseudotime analysis. The transcription factor networks linking novel signaling pathways are revealed to maintain LSC stemness. CONCLUSIONS: This human corneal scRNA-Seq identifies the LSC population and uncovers novel cell types mapping the differentiation trajectory in heterogenous limbal basal epithelium. The findings provide insight into LSC concept and lay the foundation for understanding the corneal homeostasis and diseases.

Role of CD4+ T Helper Cells in the Development of BAC-Induced Dry Eye Syndrome in Mice.

Purpose: To evaluate the role of CD4+ T helper cells in benzalkonium chloride (BAC)-induced ocular surface disorder in C57BL/6 mice. Methods: Topical 0.075% BAC was applied twice daily in C57BL/6 mice for 7 consecutive days; PBS-treated and untreated mice served as controls. Adoptive transfer of CD4+ T cells isolated from the BAC-treated mice or PBS-treated mice into nude mice was conducted to identify the roles of CD4+ T cells, with untreated nude mice as controls. Oregon green dextran staining, PAS staining, and the phenol red cotton test were carried out in these two models. The gene and protein levels of T-bet, IFN-γ, RORγt, and IL-17 were detected by quantitative RT-PCR and ELISA, respectively. The activation and subsets of CD4+ T cells were identified by double immunofluorescent staining and flow cytometry. Results: An increase in CD4+CD69+, CD4+IFN-γ+, and CD4+IL-17+ cells was induced by BAC in C57BL/6 mice. IFN-γ, IL-17, Th1, Th17, and the transcription factors T-bet and RORγt were increased in BAC-treated mice compared with control mice. In addition, ocular surface damage, including corneal barrier dysfunction, goblet cell loss, and decreased tear production, was induced by BAC. Interestingly, adoptive transfer of CD4+ T cells isolated from BAC-treated mice into nude mice resulted in ocular surface manifestations similar to those of direct topical BAC treatment of C57BL/6 mice, including increased CD4+ T cells, IFN-γ, IL-17, and ocular surface disorders. Conclusions: Topical application of BAC induced a dry-eye-like ocular surface disorder partly through the CD4+ T cell-mediated inflammatory response.

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.

Efficacy and Safety of Houttuynia Eye Drops Atomization Treatment for Meibomian Gland Dysfunction-Related Dry Eye Disease: A Randomized, Double-Blinded, Placebo-Controlled Clinical Trial.

PURPOSE: To evaluate the efficacy and safety of Houttuynia eye drops (a Chinese traditional medicine) atomization treatment in meibomian gland dysfunction (MGD)-related dry eye disease (DED) patients. METHODS: A total of 240 eligible patients diagnosed with MGD-related DED were assigned either Houttuynia eye drops or placebo for atomization once daily for four weeks in a multi-center, randomized, double-blind, placebo-controlled clinical study. Primary outcome evaluations used included eye symptom score (using the Chinese Dry Eye Questionnaire), meibum quality, and tear break-up time (TBUT), while safety evaluations included adverse events (AEs), visual acuity, and intraocular pressure monitoring. Indicators were measured at baseline as well as one week, two weeks, and four weeks after treatment. RESULTS: Primary outcome measures of the Houttuynia group were improved compared with their placebo counterparts following four-week treatment. Eye symptom scores were significantly reduced relative to the baseline in the Houttuynia group (mean ± standard error of the mean, 9.00 ± 0.61) compared with the placebo group (6.29 ± 0.55; p = 0.0018). Reduction in meibum quality score in the Houttuynia group (0.91 ± 0.10) was also significantly higher compared with the placebo group (0.57 ± 0.10; p = 0.0091), while TBUT in the treatment group (6.30 ± 0.22) was also longer than in the latter (5.60 ± 0.24; p = 0.0192). No medication-related adverse events were observed. CONCLUSIONS: Atomization treatment with Houttuynia eye drops is both clinically and statistically effective for the treatment of mild to moderate MGD-related DED patients. This approach is generally safe and was tolerated well by patients.

Comparison of Treatment Effect and Tolerance of the Topical Application of Mizoribine and Cyclosporine A in a Mouse Dry Eye Model.

Purpose: To compare the treatment effects and tolerability of a topical application of mizoribine (MZR) and cyclosporine A (CsA) eye drops (Restasis; Allergan, Inc., Irvine, CA, USA) in a mouse dry eye model. Methods: C57BL/6 mice subjected to desiccating stress (DS) were treated with 0.05% MZR in phosphate-buffered saline (PBS) or Restasis eye drops four times a day for 5 days. Untreated mice served as control. Tear secretion, Oregon green dextran staining, and the conjunctival goblet cell quantity were evaluated. The apoptosis and matrix metalloproteinase 9 (MMP-9) in the ocular surface, conjunctival CD4, and T helper-related cytokines were verified. The ocular tolerance of these two drugs was evaluated by observing the mice's behavioral changes. Results: Topical administrations of MZR or Restasis both increased tear production, maintained goblet cell density, and improved corneal barrier function. Both MZR and Restasis suppressed the expression of MMP-9 and apoptosis in the ocular surface. Meanwhile, both MZR and Restasis decreased the infiltration of CD4+ T cells, reversed the production of interferon-γ, interleukin (IL)-17A, and IL-13 in conjunctiva under DS. The abovementioned efficacies between these two eye drops were not statistically significant. However, the number of scratching and wiping behaviors in the MZR-treated group was significantly less than in the Restasis-treated group. Conclusions: MZR (0.05% in PBS) could be a good competitive product for Restasis because of the comparable treatment effect in dry eye diseases and better ocular tolerability in ocular itch and pain. Translational Relevance: This study provided an immunosuppressive agent comparable to Restasis for the treatment of dry eye disease.

IL-33/ST2/IL-9/IL-9R signaling disrupts ocular surface barrier in allergic inflammation.

This study was to explore a novel IL-33/ST2/IL-9/IL-9R signaling pathway that disrupts ocular surface barrier and amplifies allergic inflammation. Two murine models of experimental allergic conjunctivitis (EAC) and IL-9 topical challenge in wild type Balb/c and ST2-/- mice, and two culture models of primarily human corneal epithelial cells (HCECs) and mouse CD4+ T cells were performed. Clinical manifestations, Oregon-Green Dextran (OGD) staining, the apical junction complexes (AJCs), IL-33/ST2 and IL-9/IL-9R signaling molecules were evaluated in ocular surface and its draining cervical lymph nodes (CLNs) by RT-qPCR, immunostaining and ELISA. The typical allergic signs, enhanced OGD staining intensity, disrupted morphology of AJCs, including ZO-1, claudin 1, occludin, and E-cadherin, and the stimulated signaling of IL-33/ST2 and IL-9/IL-9R were observed in ocular mucosa and draining CLNs in EAC-Balb/c mice, but significantly reduced or eliminated in EAC-ST2-/- mice. Topical challenge of IL-9 resulted in the obvious OGD staining and disrupted ocular surface AJCs in Balb/c mice and in HCECs in vitro. IL-9 production was found to be stimulated by IL-33 in CD4+ cells from Balb/c mice in vitro. Our findings uncovered a novel phenomenon and mechanism by which ocular surface barrier integrity is disrupted in allergic conjunctivitis by IL-33/ST2/IL-9/IL-9R signaling pathway, which may amplify the allergic inflammation.

Short ragweed pollen promotes M2 macrophage polarization via TSLP/TSLPR/OX40L signaling in allergic inflammation.

This study was to explore the role and mechanism of macrophages in pollen-triggered allergic inflammation. A murine model of short ragweed (SRW) pollen-induced experimental allergic conjunctivitis (EAC), and bone marrow (BM)-macrophages cultures were used. Typical allergic manifestations and TSLP-stimulated Th2 hyperresponse were observed in ocular surface of EAC model in wild-type (WT) mice induced by SRW. The M2 phenotype markers, Arg1, Ym1 and FIZZ1, were highly expressed by conjunctiva and draining cervical lymph nodes (CLNs) of WT-EAC mice when compared with controls, as evaluated by RT-qPCR and Immunofluorescent double staining with macrophage marker F4/80. The stimulated expression of TSLPR and OX40L by macrophage was detected in conjunctiva and CLNs by RT-qPCR, double staining, and flow cytometry. M2 macrophages were found to produce TARC and MDC. In contrast, EAC model with TSLPR-/- mice did not show allergic signs and any increase of Th2 cytokines (IL-4, IL-5 and IL-13) and M2 markers. In vitro cultures confirmed that SRW extract stimulates expression of TSLPR, OX40L, TARC, MDC, and three M2 markers by BM-macrophages from WT mice, but not from TSLPR-/- mice. These findings demonstrate that SRW pollen primes macrophage polarization toward to M2 phenotype via TSLP/TSLPR/OX40L signaling to amplify allergic inflammation.

Hypoxic postconditioning activates the Wnt/ß-catenin pathway and protects against transient global cerebral ischemia through Dkk1 Inhibition and GSK-3ß inactivation.

The Wingless/Int (Wnt)/ß-catenin pathway plays an essential role in cell survival. Although postconditioning with 8% oxygen can alleviate transient global cerebral ischemia (tGCI)-induced neuronal damage in hippocampal CA1 subregion in adult rats as demonstrated by our previous studies, little is understood about the role of Wnt/ß-catenin pathway in hypoxic postconditioning (HPC)-induced neuroprotection. This study tried to investigate the involvement of Wnt/ß-catenin pathway in HPC-induced neuroprotection against tGCI and explore the underlying molecular mechanism thereof. We observed that HPC elevated nuclear ß-catenin level as well as increased Wnt3a and decreased Dickkopf-1 (Dkk1) expression in CA1 after tGCI. Accordingly, HPC enhanced the expression of survivin and reduced the ratio of B-cell lymphoma/lewkmia-2 (Bcl-2)-associated X protein (Bax) to Bcl-2 following reperfusion. Moreover, our study has shown that these effects of HPC were abolished by lentivirus-mediated overexpression of Dkk1, and that the overexpression of Dkk1 completely reversed HPC-induced neuroprotection. Furthermore, HPC suppressed the activity of glycogen synthase kinase-3ß (GSK-3ß) in CA1 after tGCI, and the inhibition of GSK-3ß activity with SB216763 increased the nuclear accumulation of ß-catenin, up-regulated the expression of survivin, and reduced the ratio of Bax to Bcl-2, thus preventing the delayed neuronal death after tGCI. Finally, the administration of LY294002, an inhibitor of PI3K, increased GSK-3ß activity and blocked nuclear ß-catenin accumulation, thereby decreasing survivin expression and elevating the Bax-to-Bcl-2 ratio after HPC. These results suggest that activation of the Wnt/ß-catenin pathway through Dkk1 inhibition and PI3K/protein kinase B pathway-mediated GSK-3ß inactivation contributes to the neuroprotection of HPC against tGCI.-Zhan, L., Liu, D., Wen, H., Hu, J., Pang, T., Sun, W., Xu, E. Hypoxic postconditioning activates the Wnt/ß-catenin pathway and protects against transient global cerebral ischemia through Dkk1 inhibition and GSK-3ß inactivation.

Acute ocular hypertension disrupts barrier integrity and pump function in rat corneal endothelial cells.

Acute ocular hypertension (AOH) frequently compromises corneal endothelial cell (CEC) function in clinical practice. This type of stress induces corneal oedema and a decrease in the corneal endothelial cell density (ECD). The anterior chamber of the right eye of Sprague-Dawley rats was irrigated with Balanced Salt Solution (BSS) for two hours, and the left eye served as a control to determine the time-dependent effects of AOH on endothelial cell morphology and function. The average intraocular pressure (IOP) increased to 82.6 ± 2.3 mmHg (normal range: 10.2 ± 0.4 mmHg) during anterior irrigation. Very soon after initiating irrigation, corneal oedema became evident and the cornea exhibited a significant increase in permeability to FITC-dextran. The peripheral ECD was significantly reduced, and the morphology of CECs became irregular and multiform. The structures of the zonula occludens-1 (ZO-1) and F-actin were severely disrupted. In addtion, Na,K-ATPase exhibited a dispersed expression pattern. Two days after irrigation, obvious CEC proliferation was observed, the ECD recovered to a normal level, and F-actin was dispersed throughout the cytoplasm. Seven days later, the CEC structure and function were nearly normalized. Based on the results obtained using this model, an acute IOP crisis exerts transient deleterious effects on CEC structure and function in rats.

Benzalkonium chloride suppresses rabbit corneal endothelium intercellular gap junction communication.

PURPOSE: Gap junction intercellular communication (GJIC) plays a critical role in the maintenance of corneal endothelium homeostasis. We determined if benzalkonium chloride (BAK) alters GJIC activity in the rabbit corneal endothelium since it is commonly used as a drug preservative in ocular eyedrop preparations even though it can have cytotoxic effects. METHODS: Thirty-six adult New Zealand albino rabbits were randomly divided into three groups. BAK at 0.01%, 0.05%, and 0.1% was applied twice daily to one eye of each of the rabbits in one of the three groups for seven days. The contralateral untreated eyes were used as controls. Corneal endothelial morphological features were observed by in vivo confocal microscopy (IVCM). Immunofluorescent staining resolved changes in gap junction integrity and localization. Western blot analysis and RT-PCR evaluated changes in levels of connexin43 (Cx43) and tight junction zonula occludens-1 (ZO-1) gene and protein expression, respectively. Cx43 and ZO-1 physical interaction was detected by immunoprecipitation (IP). Primary rabbit corneal endothelial cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing BAK for 24 hours. The scrape-loading dye transfer technique (SLDT) was used to assess GJIC activity. RESULTS: Topical administration of BAK (0.05%, 0.1%) dose dependently disrupted corneal endothelial cell morphology, altered Cx43 and ZO-1 distribution and reduced Cx43 expression. BAK also markedly induced increases in Cx43 phosphorylation status concomitant with decreases in the Cx43-ZO-1 protein-protein interaction. These changes were associated with marked declines in GJIC activity. CONCLUSIONS: The dose dependent declines in rabbit corneal endothelial GJIC activity induced by BAK are associated with less Cx43-ZO-1 interaction possibly arising from increases in Cx43 phosphorylation and declines in its protein expression. These novel changes provide additional evidence that BAK containing eyedrop preparations should be used with caution to avoid declines in corneal transparency resulting from losses in GJIC activity and endothelial function.