Murine Cornea Research Articles

TGF-β1-activated type 2 dendritic cells promote wound healing and induce fibroblasts to express tenascin c following corneal full-thickness hydrogel transplantation.

We showed previously that 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide hydrochloride (EDC) cross-linked recombinant human collagen III hydrogels promoted stable regeneration of the human cornea (continued nerve and stromal cell repopulation) for over 4years. However, as EDC cross linking kinetics were difficult to control, we additionally tested a sterically bulky carbodiimide. Here, we compared the effects of two carbodiimide cross linkers-bulky, aromatic N-cyclohexyl-N0-(2-morpholinoethyl)-carbodiimide (CMC), and nonbulky EDC-in a mouse corneal graft model. Murine corneas undergoing full-thickness implantation with these gels became opaque due to dense retro-corneal membranes (RCM). Corneal epithelial cytokeratin 12 and alpha smooth muscle actin indicative of functional tissue regeneration and wound contraction were observed in RCM surrounding both hydrogel types. However, quantitatively different levels of infiltrating CD11c+ dendritic cells (DC) were found, suggesting a hydrogel-specific innate immune response. More DC infiltrated the stroma surrounding EDC-N-hydroxysuccinimide (NHS) hydrogels concurrently with higher fibrosis-associated tenascin c expression. The opposite was true for CMC-NHS gels that had previously been shown to be more tolerising to DC. In vitro studies showed that DC cultured with transforming growth factor β1 (TGF-β1) induced fibroblasts to secrete more tenascin c than those cultured with lipopolysaccharide and this effect was blocked by TGF-β1 neutralisation. Furthermore, tenascin c staining was found in 40- to 50μm long membrane nanotubes formed in fibroblast/DC cocultures. We suggest that TGF-β1 alternatively activated (tolerising) DC regulate fibroblast-mediated tenascin c secretion, possibly via local production of TGF-β1 in early wound contraction, and that this is indirectly modulated by different hydrogel chemistries.

Corneal nerve healing after in situ laser nerve transection.

PurposeWe have previously reported that lamellar dissection of the cornea transects stromal nerves, and that regenerating neurites form a dense net along the surgical plane. In these experiments, we have disrupted the stromal nerve trunks in situ, without incising the cornea, to determine the regeneration events in the absence of a surgical plane.MethodsThy1-YFP mice were anesthetized and in vivo images of the corneal nerves were obtained with a wide-field stereofluorescent microscope. A far infrared XYRCOS Laser attached to 20X objective of an upright microscope was used to perform in situ transection of the stromal nerves. 3 types of laser transections were performed (n = 5/group): (i) point transection (a single cut); (ii) segmental transection (two cuts enclosing a segment of nerve trunk); and (iii) annular transection (cuts on all nerve trunks crossing the perimeter of a 0.8 mm diameter circular area centered on the corneal apex). Mice were imaged sequentially for 4 weeks thereafter to assess nerve degeneration (disappearance or weakening of original fluorescence intensity) or regeneration (appearance of new fluorescent fronds). Beta-3-tubulin immunostaining was performed on corneal whole-mounts to demonstrate nerve disruption.ResultsThe pattern of stromal nerves in corneas of the same mouse and in corneas of littermates was dissimilar. Two distinct patterns were observed, often within the same cornea: (i) interconnected trunks that spanned limbus to limbus; or (ii) dichotomously branching trunks that terminate at the corneal apex. Point transections did not cause degeneration of proximal or distal segment in interconnected trunks, but resulted in degeneration of distal segment of branching trunks. In segmental transections, the nerve segment enclosed within the two laser cuts degenerated. Lack of beta-3 tubulin staining at transection site confirmed nerve transection. In interconnected trunks, at 4 weeks, a hyperfluorescent plaque filled the gap created by the transection. In annular transections, some nerve trunks degenerated, while others regained or retained fluorescence.ConclusionsInterconnected stromal nerves in murine corneas do not degenerate after in situ point transection and show evidence of healing at the site of disruption. Presence or absence of a surgical plane influences corneal nerve regeneration after transection.

Transcriptional Profiling of Daily Patterns of mRNA Expression in the C57BL/6J Mouse Cornea

ABSTRACTPurpose: The purpose of this study was to determine how the transcriptome of the murine cornea adapts to diurnal changes in physiology.Methods: C57BL/6J mice were maintained under a 12-h light/12-h dark (LD) cycle for two weeks. Corneas were collected from euthanized mice at Zeitgeber time (ZT) 0, 3, 6, 9, 12, 15, 18, and 21. Total RNA was extracted and subjected to RNA sequencing (RNA-Seq). A JTK_CYCLE algoithm and other software tools were used to analyze the transcriptional data to determine the periodicity, rhythmicity, and amplitude of the transcripts. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the enrichment of cycling transcripts.Results: Approximately 24% of the total transcripts from the murine corneal genome were rhythmically expressed over an LD cycle. GO analysis showed that these cycling genes are primarily involved in cellular and metabolic processes. A KEGG pathway analysis identified 6 branches and 44 pathways that encode the gene outputs necessary for basic cellular functions and processes. More importantly, most of the rhythmic genes between the day and night are enriched in their own unique pathways in addition to some common pathways. Furthermore, most of the rhythmic gene expression was concentrated in the 12-h and 24-h periods. A comparative analysis of GO and KEGG showed large differences in metabolic processes, but not cellular processes. Finally, the murine cornea also rhythmically expressed 11 canonical components of circadian clock genes over an LD cycle at the transcriptional level.Conclusions: One fourth of the corneal transcriptome follows a rhythmic expression pattern involved in basic molecular and cellular mechanisms. This implies that the time of day contributes significantly to the overall temporal organization of the corneal transcriptome.

Analysis of leukocyte populations and nerves in developing murine corneas

Abstract The avascular cornea is the most densely innervated tissue in the body and is endowed with resident bone marrow-derived cells. We have identified a novel subset of resident corneal plasmacytoid dendritic cells (pDCs). We have shown pDCs support corneal nerves through release of nerve growth factor (NGF). This study was to examine the presence of pDCs in the developing cornea. We examined corneas from E17, P2, and 8 week adult C57BL/6 mice. Corneas were stained for the pan-leukocyte marker CD45, the pDC marker plasmacytoid dendritic cell antigen-1 (PDCA-1), the pan-neuronal marker bIII-tubulin. Corneal CD45+ and PDCA-1+ cells and nerves were quantified. Significance was assigned by ANOVA and post-hoc Tukey test. All data is reported as mean ± standard deviation. Corneal CD45+ leukocyte density increased from E17 (65.4±14 cells/mm2) to P2 pups (301.9±19 cells/mm2, p<0.05). Both E17 and P2 corneas had lower CD45+ density when compared to adults (345±34 cells/mm2, p<0.05). We observed pDCs in the anterior stroma proximal to corneal nerves in E17, P2, and adult corneas. In E17, few pDCs were present in the peripheral cornea (1.5±2.6 cells/mm2), with none in the central cornea. In P2, pDCs were found in the periphery (68.19±8.99 cells/mm2), but not central corneas. The density of pDCs in adult peripheral (82.2±5.1 cells/mm2) and central (51.6±4.9 cells/mm2) corneas was greater than E17 (p<0.001) and P2 (p<0.05) corneas. Nerve density increased from E17 (45.43±6.75 mm/mm2) when compared to P2 (83.15±.21.6 mm/mm2 p<0.05) and to adult (151.6±3.25 mm/mm2 p<0.05) corneas. Our data show pDC density increased from embryonic, to neonatal, and adult corneas. Concurrently corneal nerve density increased during development from embryonic to adult mice.

Neutrophil extracellular traps (NETs) contribute to pathological changes of ocular graft-vs.-host disease (oGVHD) dry eye: Implications for novel biomarkers and therapeutic strategies

PurposeTo investigate the role of neutrophil extracellular traps (NETs) and NET-associated proteins in the pathogenesis of oGVHD and whether dismantling of NETs with heparin reduces those changes. MethodsOcular surface washings from oGVHD patients and healthy subjects were analyzed. Isolated peripheral blood human neutrophils were stimulated to generate NETs and heparinized NETs. We performed in vitro experiments using cell lines (corneal epithelial, conjunctival fibroblast, meibomian gland (MG) epithelial and T cells), and in vivo experiments using murine models, and compared the effects of NETs, heparinized NETs, NET-associated proteins and neutralizing antibodies to NET-associated proteins. ResultsNeutrophils, exfoliated epithelial cells, NETs and NET-associated proteins (extracellular DNA, Neutrophil Elastase, Myeloperoxidase, Oncostatin M (OSM), Neutrophil gelatinase-associated lipocalin (NGAL) and LIGHT/TNFSF14) are present in ocular surface washings (OSW) and mucocellular aggregates (MCA). Eyes with high number of neutrophils in OSW have more severe signs and symptoms of oGVHD. NETs (and OSM) cause epitheliopathy in murine corneas. NETs (and LIGHT/TNFSF14) increase proliferation of T cells. NETs (and NGAL) inhibit proliferation and differentiation of MG epithelial cells. NETs enhance proliferation and myofibroblast transformation of conjunctival fibroblasts. Sub-anticoagulant dose Heparin (100 IU/mL) dismantles NETs and reduces epithelial, fibroblast, T cell and MG cell changes induced by NETs. ConclusionNETs and NET-associated proteins contribute to the pathological changes of oGVHD (corneal epitheliopathy, conjunctival cicatrization, ocular surface inflammation and meibomian gland disease). Our data points to the potential of NET-associated proteins (OSM or LIGHT/TNFSF14) to serve as biomarkers and NET-dismantling biologics (heparin eye drops) as treatment for oGVHD.

Staphylococcus aureus alpha-hemolysin impairs corneal epithelial wound healing and promotes intracellular bacterial invasion

Colonization by Staphylococcus aureus (S. aureus) has been implicated in many infectious and wound healing disorders. This study was performed to characterize the pathogenic role of S. aureus alpha-hemolysin (alpha-toxin) in corneal epithelial wound healing and infectious keratitis in the setting of a corneal wound. The effect of wild-type and isogenic Hla mutant (α-hemolysin gene deleted) S. aureus bacteria and conditioned media on corneal epithelial wound healing was tested in vitro using a scratch assay and in vivo using a murine epithelial debridement model. The invasiveness of wild-type and Hla mutant S. aureus was evaluated in vitro in human corneal epithelial cells and in vivo in a murine model of infectious keratitis following total epithelial debridement. S. aureus and its conditioned media significantly delayed epithelial wound closure both in vitro (P < 0.05) and in vivo (P < 0.05). The effect of S. aureus on wound healing was significantly diminished with the Hla mutant strain (P < 0.05). Likewise, compared to the wild-type strain, the Hla mutant strain demonstrated significantly reduced ability to invade corneal epithelial cells in vitro (P < 0.05) and infect murine corneas following total epithelial debridement in vivo (P < 0.05). In conclusion, S. aureus alpha-hemolysin plays a major role in the pathologic modulation of corneal epithelial wound healing and the intracellular invasion of the bacteria. Limiting colonization by S. aureus and/or blocking alpha-hemolysin may provide a therapeutic approach for corneal wound healing and infectious disorders.

SOX2 Regulates P63 and Stem/Progenitor Cell State in the Corneal Epithelium.

Mutations in key transcription factors SOX2 and P63 were linked with developmental defects and postnatal abnormalities such as corneal opacification, neovascularization, and blindness. The latter phenotypes suggest that SOX2 and P63 may be involved in corneal epithelial regeneration. Although P63 has been shown to be a key regulator of limbal stem cells, the expression pattern and function of SOX2 in the adult cornea remained unclear. Here, we show that SOX2 regulates P63 to control corneal epithelial stem/progenitor cell function. SOX2 and P63 were co‐expressed in the stem/progenitor cell compartments of the murine cornea in vivo and in undifferentiated human limbal epithelial stem/progenitor cells in vitro. In line, a new consensus site that allows SOX2‐mediated regulation of P63 enhancer was identified while repression of SOX2 reduced P63 expression, suggesting that SOX2 is upstream to P63. Importantly, knockdown of SOX2 significantly attenuated cell proliferation, long‐term colony‐forming potential of stem/progenitor cells, and induced robust cell differentiation. However, this effect was reverted by forced expression of P63, suggesting that SOX2 acts, at least in part, through P63. Finally, miR‐450b was identified as a direct repressor of SOX2 that was required for SOX2/P63 downregulation and cell differentiation. Altogether, we propose that SOX2/P63 pathway is an essential regulator of corneal stem/progenitor cells while mutations in SOX2 or P63 may disrupt epithelial regeneration, leading to loss of corneal transparency and blindness. Stem Cells 2019;37:417–429

Epithelial Membrane Protein-2 (EMP2) Antibody Blockade Reduces Corneal Neovascularization in an In Vivo Model.

PurposePathologic corneal neovascularization is a major cause of blindness worldwide, and treatment options are currently limited. VEGF is one of the critical mediators of corneal neovascularization but current anti-VEGF therapies have produced limited results in the cornea. Thus, additional therapeutic agents are needed to enhance the antiangiogenic arsenal. Our group previously demonstrated epithelial membrane protein-2 (EMP2) involvement in pathologic angiogenesis in multiple cancer models including breast cancer and glioblastoma. In this paper, we investigate the efficacy of anti-EMP2 immunotherapy in the prevention of corneal neovascularization.MethodsAn in vivo murine cornea alkali burn model was used to study pathologic neovascularization. A unilateral corneal burn was induced using NaOH, and subconjunctival injection of either anti-EMP2 antibody, control antibody, or sterile saline was performed after corneal burn. Neovascularization was clinically scored at 7 days postalkali burn, and eyes were enucleated for histologic analysis and immunostaining including VEGF, CD31, and CD34 expression.ResultsAnti-EMP2 antibody, compared to control antibody or vehicle, significantly reduced neovascularization as measured by clinical score and central cornea thickness, as well as by histologic reduction of neovascularization, decreased CD34 staining, and decreased CD31 staining. Incubation of corneal limbal cells in vitro with anti-EMP2 blocking antibody significantly decreased EMP2 expression, VEGF expression and secretion, and cell migration.ConclusionsThis work demonstrates the effectiveness of EMP2 as a novel target in pathologic corneal neovascularization in an animal model and supports additional investigation into EMP2 antibody blockade as a potential new therapeutic option.

The facilitation of corneal permeability of cyclosporine A loaded on chitosan-graft-cyclodextrin copolymers vector

Objective To investigate the corneal permeability of cyclosprin A (CsA) loaded on polymeric vector after topical application. Methods The grafted copolymer chitosan-graft-cyclodextrin (CS-g-CD) was synthesized, and the physicochemical structures of the polymer were investigated using nuclear magnetic resonance spectroscopy (NMR) and fourier transform infrared spectroscopy (FT-IR). A novel CsA eye drop was prepared using the grafted copolymer as carrier material.The physicochemical properties of eye drop, including drug-loading content, osmotic pressure and viscosity were investigated by high performance liquid chromatography-mass spectrometry (HPLC-MS), osmotic pressure gauge and viscometer, respectively.New Zealand albino rabbits were randomly divided into intact cornea CsA group, epithelium debrided CsA group and epithelium debrided control group.The corneal epithelia of the left eyes was debrided in the cornea epithelium debrided group.Cornea irritation test was performed on New Zealand albino rabbits.The aqueous humor was taken and the corneas were collected at 0.5 hour and 1 hour after instilled.The concentration of CsA was measured by HPLC-MS.Cy5 labeled vector loaded with Coumarin 6 served as model copolymers system, the penetration capabilities of the double fluorescent labeling copolymers system were monitored in vivo using two-photon scanning fluorescence microscopy on murine corneas after topical application.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results The polymer of CS-g-CD was successfully synthesized and confirmed using NMR and FT-IR.The drug loading of CsA in eye drop solution was 0.06 %; the osmotic pressure was 305 mOsmol/kg and the viscosity was 36.5 cP.The CsA drug delivery system had a reversible temperature-sensitive drug release behavior and had no obvious irritation on the eyes of New Zealand rabbits.One hour after treatment, the concentration of CsA in the cornea and aqueous humor of epithelium debrided CsA group was (5.88±1.46)μg/g and (149.19±3.93)ng/ml, respectively, which was significantly higher than (3.98±0.95)μg/g and (30.25±11.43)ng/ml in epithelium debrided control group (both at P<0.05); the concentration of CsA in the aqueous humor of intact cornea CsA group was (7.23±1.31)ng/ml, which was significantly lower than that in epithelium debrided CsA group (P<0.05). Polymer vectors were mainly retained in the corneal epithelium, and coumarin 6 gradually diffused into the deep corneal stroma with time. Conclusions The grafted copolymer can load CsA, and the eye drop can effectively overcome the corneal barrier and increase the corneal permeability of CsA. Key words: Cyclosporine A; Immunosuppressive agents; Corneal barrier; Fluorescent double labeling; Corneal tissue distribution

The Chemokine Receptor CXCR4 Mediates Recruitment of CD11c+ Conventional Dendritic Cells Into the Inflamed Murine Cornea.

PurposeThe cornea contains distinct populations of antigen-presenting cells (APCs), including conventional dendritic cells (cDCs). Little is known about the molecular mechanisms involved in cDCs homing and recruitment into the naïve and inflamed cornea. The purpose of this study was to investigate the presence of CXCR4 and its ligand CXCL12 in the murine cornea and its role in cDC migration during corneal inflammation.MethodsThe expression of CXCR4 and CXCL12 in naïve and suture-inflamed murine corneas was assessed by whole-mount staining, flow cytometry, and quantitative PCR. The role of CXCR4 in recruitment into inflamed corneas was investigated using adoptive transfer of cDCs blocked with neutralizing antibody against CXCR4.ResultsWe show the chemokine receptor CXCR4 to be expressed on 51.7% and 64.8% of total corneal CD11c+ cDCs, equating to 98.6 ± 12.5 cells/mm2 in the peripheral and 64.7 ± 10.6 cells/mm2 in the central naïve cornea, respectively. Along with a 4.5-fold increase in CXCL12 expression during inflammation (P < 0.05), infiltrating cDCs also expressed CXCR4 in both the peripheral (222.6 ± 33.3 cells/mm2; P < 0.001) and central cornea (161.9 ± 23.8 cells/mm2; P = 0.001), representing a decrease to 31.0% and 37.3% in the cornea, respectively. Further, ex vivo blockade (390.1 ± 40.1 vs. 612.1 ± 78.3; P = 0.008) and local blockade (263.5 ± 27.1 vs. 807.5 ± 179.5, P < 0.001) with anti-CXCR4 neutralizing antibody resulted in a decrease in cDCs homing into the cornea compared with cells pretreated with isotype controls.ConclusionsOur results demonstrate that corneal CXCL12 plays a direct role in CXCR4+ cDC recruitment into the cornea. The CXCR4/CXCL12 axis is therefore a potential target to modulate corneal inflammatory responses.

Impact of topical corticosteroid pretreatment on susceptibility of the injured murine cornea to Pseudomonas aeruginosa colonization and infection

Research with animal models of Pseudomonas aeruginosa keratitis has shown that use of a topical corticosteroid alone against an established infection can significantly increase the number of colonizing bacteria or worsen clinical disease. Moreover, retrospective analysis has suggested that corticosteroid use in humans is associated with an increased risk of keratitis in eyes with pre-existing disease. Thus, while corticosteroids are often used to reduce ocular inflammation in the absence of infection, the risk of opportunistic infection remains a concern. However, the effect of corticosteroids on the intrinsic barrier function of uninfected corneas is unknown. Here, we tested if short-term topical corticosteroid treatment of an uninfected murine cornea would increase susceptibility to P. aeruginosa colonization or infection after epithelial injury. Topical prednisolone acetate (1%) was administered to one eye of C57BL/6 mice three times a day for 3 days; control eyes were treated with sterile PBS. Prior to inoculation with a cytotoxic P. aeruginosa corneal isolate strain 6206, corneas were subject to superficial-injury by tissue paper blotting, or scratch-injured followed by 12 h of healing. Previously we have shown that blotting renders mouse corneas susceptible to P. aeruginosa adhesion, but not infection, while 12 h healing reduces susceptibility to infection after scratching. Corneas were evaluated at 48 h for bacterial colonization and microbial keratitis (MK). To monitor impact on wound healing, corneal integrity was examined by fluorescein staining immediately after scarification and after 12 h healing. For both the tissue paper blotting and scratch-injury models, there was no significant difference in P. aeruginosa colonization at 48 h between corticosteroid-pretreated eyes and controls. With the blotting model, one case of MK was observed in a control (PBS-pretreated) cornea; none in corticosteroid-pretreated corneas. With the 12 h healing model, MK occurred in 6 of 17 corticosteroid-pretreated eyes versus 2 of 17 controls, a difference not statistically significant. Corticosteroid-pretreated eyes showed greater fluorescein staining 12 h after scarification injury, but this did not coincide with increased colonization or MK. Together, these data show that short-term topical corticosteroid therapy on an uninfected murine cornea does not necessarily enhance its susceptibility to P. aeruginosa colonization or infection after injury, even when it induces fluorescein staining.

Universal Corneal Epithelial-Like Cells Derived from Human Embryonic Stem Cells for Cellularization of a Corneal Scaffold.

PurposeWe generated universal corneal epithelial cells (CEC) from human embryonic stem cells (hESC) by genetically removing human leukocyte antigens (HLA) class I from the cell surface.MethodsThe serum-free, growth factor-free, and defined medium E6 was used to differentiate hESC to CEC. Decellularized murine corneas were recellularized with hESC-derived CEC. Using CRISPR/Cas9, β-2-microglobulin (B2M) was deleted in hESC to block the assembly of HLA class-I antigens on the cell surface to generate B2M−/− CEC.ResultsE6 alone was sufficient to allow hESC differentiation to CEC. A time-course analysis of the global gene expression of the differentiating cells indicates that the differentiation closely resembles the corneal development in vivo. The hESC-CEC were highly proliferative, and could form multilayer epithelium in decellularized murine cornea, retain its transparency, and form intact tight junctions on its surface. As reported before, B2M knockout led to the absence of HLA class-I on the cell surface of hESC and subsequently derived CEC following stimulation with inflammatory factors. Moreover, B2M−/− CEC, following transplantation into mouse eyes, caused less T-cell infiltration in the limbal region of the eye than the wild-type control.ConclusionsCEC can be derived from hESC via a novel and simple protocol free of any proteins, hESC-CEC seeded on decellularized animal cornea form tight junctions and allow light transmittance, and B2M−/− CEC are hypoimmunogenic both in vitro and in vivo.Translational RelevanceB2M−/− hESC-CEC can be an unlimited and universal therapy for corneal repair in patients of any HLA type.

Interaction of mannose binding lectin and other pattern recognition receptors in human corneal epithelial cells during Aspergillus fumigatus infection

Mannose binding lectin (MBL), a member of C-type lectin superfamily, plays a significant role in the innate immune system as one of the Pattern-Recognition Receptors (PRRs). This study investigated the relationship between MBL and other PRRs including Dectin-1and TLR2, and detected the reaction of MBL towards the expression of cytokines in human corneal epithelial cells (HCECs) and murine corneas upon Aspergillus fumigatus (A. fumigatus) hyphae infection. We found that MBL was significantly up-regulated in C57BL/6 corneas infected with A. fumigatus and HCECs incubated with A. fumigatus hyphae. Moreover, both of mRNA and protein levels of Dectin-1, TLR2, IL-1β and TNF-α were increased dramatically to peak point with the pretreatment of exogenous MBL, while decreased significantly with the pretreatment of MBL monoclonal antibody in HCECs. Pretreatment with laminarin and TLR2 neutralizing antibody decreased both of mRNA and protein levels of MBL and proinflammatory mediators (IL-1β and TNF-α) in A. fumigatus hyphae infected HCECs. These data demonstrate that A. fumigatus introduce the expression of MBL in human corneas, C57BL/6 corneas and HCECs. MBL, Dectin-1 and TLR2 interact with each other upon the treatment of A. fumigatus, and MBL contributes to the innate immune responses by regulating proinflammatory mediators, such as IL-1β and TNF-α.

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

IL-1R and MyD88 Contribute to the Absence of a Bacterial Microbiome on the Healthy Murine Cornea.

Microbial communities are important for the health of mucosal tissues. Traditional culture and gene sequencing have demonstrated bacterial populations on the conjunctiva. However, it remains unclear if the cornea, a transparent tissue critical for vision, also hosts a microbiome. Corneas of wild-type, IL-1R (-/-) and MyD88 (-/-) C57BL/6 mice were imaged after labeling with alkyne-functionalized D-alanine (alkDala), a probe that only incorporates into the peptidoglycan of metabolically active bacteria. Fluorescence in situ hybridization (FISH) was also used to detect viable bacteria. AlkDala labeling was rarely observed on healthy corneas. In contrast, adjacent conjunctivae harbored filamentous alkDala-positive forms, that also labeled with DMN-Tre, a Corynebacterineae-specific probe. FISH confirmed the absence of viable bacteria on healthy corneas, which also cleared deliberately inoculated bacteria within 24 h. Differing from wild-type, both IL-1R (-/-) and MyD88 (-/-) corneas harbored numerous alkDala-labeled bacteria, a result abrogated by topical antibiotics. IL-1R (-/-) corneas were impermeable to fluorescein suggesting that bacterial colonization did not reflect decreased epithelial integrity. Thus, in contrast to the conjunctiva and other mucosal surfaces, healthy murine corneas host very few viable bacteria, and this constitutive state requires the IL-1R and MyD88. While this study cannot exclude the presence of fungi, viruses, or non-viable or dormant bacteria, the data suggest that healthy murine corneas do not host a resident viable bacterial community, or microbiome, the absence of which could have important implications for understanding the homeostasis of this tissue.

Tetramethylpyrazine in a Murine Alkali-Burn Model Blocks NFκB/NRF-1/CXCR4-Signaling-Induced Corneal Neovascularization.

Tetramethylpyrazine (TMP) is the active ingredient extracted from the Chinese herb Chuanxiong. The purpose of our study was to identify the mechanism of therapeutic TMP suppression of pathologic chemokine receptor 4 (CXCR4) transcription. C57BL/6J mice with alkali-burned corneas were treated with either TMP eye drops (1.5 mg/mL) or PBS. Corneal neovascularization (CNV) was measured and a clinical assessment was made by slit lamp microscopy. Expression of CXCR4 and the transcription factors nuclear respiratory factor-1 (NRF-1), nuclear factor kappa B (NFκB), forkhead box C1, and yin yang 1 were tracked by real-time RT-PCR and immunofluorescence staining of murine corneas. Western blot, real-time PCR, and immunofluorescence evaluated expression of related genes in human umbilical vein endothelial cells (HUVECs) after 200-μmol/L TMP treatment. In addition, plasmid transfection and chromatin immunoprecipitation assays elucidated the relationship among NRF-1, NFκB, and CXCR4. Corneas treated with TMP had smaller areas of neovascularization and scored better in clinical assessments. Injured corneas showed significantly elevated expressions of NRF-1, NFκB, and CXCR4 that were normalized in vivo by TMP treatment. Similarly, in HUVECs in vitro, TMP decreased expression of NRF-1, NFκB, and CXCR4. Overexpression of NFκB or NRF-1 raised the expression of CXCR4 in HUVECs, but not synergistically. Chromatin immunoprecipitation assays detected only NRF-1 bound to the CXCR4 promoter region, suggesting NFκB controls CXCR4 expression by upregulating NRF-1. Together, our data suggest TMP downregulates CXCR4 by repressing NRF-1 expression in CNV, likely indirectly by downregulating NFκB. Our results implicate a novel mechanism wherein TMP inhibits neovascularization via an NFκB/NRF-1/CXCR4 circuit.

KCa3.1 ion channel: A novel therapeutic target for corneal fibrosis.

Vision impairment from corneal fibrosis is a common consequence of irregular corneal wound healing after injury. Intermediate-conductance calmodulin/calcium-activated K+ channels 3.1 (KCa3.1) play an important role in cell cycle progression and cellular proliferation. Proliferation and differentiation of corneal fibroblasts to myofibroblasts can lead to corneal fibrosis after injury. KCa3.1 has been shown in many non-ocular tissues to promote fibrosis, but its role in corneal fibrosis is still unknown. In this study, we characterized the expression KCa3.1 in the human cornea and its role in corneal wound healing in vivo using a KCa3.1 knockout (KCa3.1-/-) mouse model. Additionally, we tested the hypothesis that blockade of KCa3.1 by a selective KCa3.1 inhibitor, TRAM-34, could augment a novel interventional approach for controlling corneal fibrosis in our established in vitro model of corneal fibrosis. The expression of KCa3.1 gene and protein was analyzed in human and murine corneas. Primary human corneal fibroblast (HCF) cultures were used to examine the potential of TRAM-34 in treating corneal fibrosis by measuring levels of pro-fibrotic genes, proteins, and cellular migration using real-time quantitative qPCR, Western blotting, and scratch assay, respectively. Cytotoxicity of TRAM-34 was tested with trypan blue assay, and pro-fibrotic marker expression was tested in KCa3.1-/-. Expression of KCa3.1 mRNA and protein was detected in all three layers of the human cornea. The KCa3.1-/- mice demonstrated significantly reduced corneal fibrosis and expression of pro-fibrotic marker genes such as collagen I and α-smooth muscle actin (α-SMA), suggesting that KCa3.1 plays an important role corneal wound healing in vivo. Pharmacological treatment with TRAM-34 significantly attenuated corneal fibrosis in vitro, as demonstrated in HCFs by the inhibition TGFβ-mediated transcription of pro-fibrotic collagen I mRNA and α-SMA mRNA and protein expression (p<0.001). No evidence of cytotoxicity was observed. Our study suggests that KCa3.1 regulates corneal wound healing and that blockade of KCa3.1 by TRAM-34 offers a potential therapeutic strategy for developing therapies to cure corneal fibrosis in vivo.

Cornea-Derived Mesenchymal Stromal Cells Therapeutically Modulate Macrophage Immunophenotype and Angiogenic Function.

Macrophages are crucial drivers of inflammatory corneal neovascularization and thus are potential targets for immunomodulatory therapies. We hypothesized that therapeutic use of cornea-derived mesenchymal stromal cells (cMSCs) may alter the function of macrophages. We found that cMSCs can modulate the phenotype and angiogenic function of macrophages. In vitro, cMSCs induce apoptosis of macrophages while preferentially promoting a distinct CD14hi CD16hi CD163hi CD206hi immunophenotype that has significantly reduced angiogenic effects based on in vitro angiogenesis assays. In vivo, application of cMSCs to murine corneas after injury leads to reduced macrophage infiltration and higher expression of CD206 in macrophages. Macrophages cocultured ("educated") by cMSCs express significantly higher levels of anti-angiogenic and anti-inflammatory factors compared with control macrophages. In vivo, injured corneas treated with cMSC-educated macrophages demonstrate significantly less neovascularization compared with corneas treated with control macrophages. Knocking down the expression of pigment epithelial derived factor (PEDF) in cMSCs significantly abrogates its modulating effects on macrophages, as shown by the reduced rate of apoptosis, decreased expression of sFLT-1/PEDF, and increased expression of vascular endothelial growth factor-A in the cocultured macrophages. Similarly, cMSCs isolated from PEDF knockout mice are less effective compared with wild-type cMSCs at inhibiting macrophage infiltration when applied to wild-type corneas after injury. Overall, these results demonstrate that cMSCs therapeutically suppress the angiogenic capacity of macrophages and highlight the role of cMSC secreted PEDF in the modulation of macrophage phenotype and function. Stem Cells 2018;36:775-784.

Bmi1+ Progenitor Cell Dynamics in Murine Cornea During Homeostasis and Wound Healing

The outermost layer of the eye, the cornea, is renewed continuously throughout life. Stem cells of the corneal epithelium reside in the limbus at the corneal periphery and ensure homeostasis of the central epithelium. However, in young mice, homeostasis relies on cells located in the basal layer of the central corneal epithelium. Here, we first studied corneal growth during the transition from newborn to adult and assessed Keratin 19 (Krt19) expression as a hallmark of corneal maturation. Next, we set out to identify a novel marker of murine corneal epithelial progenitor cells before, during and after maturation, and we found that Bmi1 is expressed in the basal epithelium of the central cornea and limbus. Furthermore, we demonstrated that Bmi1+ cells participated in tissue replenishment in the central cornea. These Bmi1+ cells did not maintain homeostasis of the cornea for more than 3 months, reflecting their status as progenitor rather than stem cells. Finally, after injury, Bmi1+ cells fueled homeostatic maintenance, whereas wound closure occurred via epithelial reorganization. Stem Cells 2018;36:562-573.

The suppression of local cytokine production in experimental models of injured cornea after stem cell treatment

PurposeLimbal stem cell deficiency (LSCD) is associated with the break of immune privilege of the cornea, a harmful local inflammatory reaction, production of cytokines and a failure of corneal regeneration. The corneal transplantation is not sufficient treatment. In the case of bilateral LSCD the only therapeutic option is an allogenic transplantation of limbal tissue or cultured limbal stem cells (LSCs) with a systemic administration of immunosuppressive medication. As an alternative, mesenchymal stem cells (MSCs) turned out to be a suitable source of autologous stem cells (SCs).MethodsIn this study MSCs were obtained from murine bone marrow. Flow cytometry was used to characterize the phenotypic markers of such MSCs. LSCs were isolated from limbal tissue by trypsin digestion. For in vitro experiments SCs were cultured in 48‐well plates for 24 hours. Excised murine corneas were added into cultures and stimulated by proinflammatory cytokines, interleukin‐1β (IL‐1β), interferon gamma and by lipopolysaccharide or were pretreated with 0.25M NaOH for 20 seconds. After 48 hours of cocultivation of corneas with SCs, the expression of genes for inflammation cytokines and growth factors in the cornea was detected using real‐time PCR. In in vivo experiments, SCs were seeded onto nanofiber scaffolds (allowed to adhere for 24 hours) and transplanted on the damaged (0.25M NaOH, 20 seconds) cornea.ResultsWe showed that LSCs and MSCs have the ability to suppress the local expression of genes for IL‐1β, tumour necrosis factor‐α, vascular endothelial growth factor and inducible NO synthase in the damaged cornea in both in vitro and in vivo models and that transplantation of SCs supports corneal regeneration.ConclusionsWe suggest that MSCs can be a suitable substitute for ocular surface regeneration in cases when autologous LSCs are absent or difficult to obtain.