Journal: Brain : a journal of neurology

Article Title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

doi: 10.1093/brain/awr338

Figure Lengend Snippet: Figure 3 Three hours of hypoxia and 6 h of reperfusion lead to significant endothelial cell death in vitro in rat brain microvascular endothelial cells (RBMVEC; A). Non-specific gap junction blocker carbenoxolone, non-specific hemichannel blocker LaCl3, and connexin43 (Cx43) mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment, while scrambled peptide did not have any protective effects. The number of viable cells was expressed as percentage of the control without hypoxia. Hypoxia and reperfusion also lead to significant propidium iodide dye uptake into primary rat brain microvascular endothelial cells indicating open hemichannels (B). Carbenoxolone, LaCl3 and connexin43 mimetic peptide significantly prevented dye uptake compared to no treatment, indicating hemichannel closure, while scrambled peptide did not have any effect. Stars denote statistical significance when compared to the control group or compared between groups in brackets; P 5 0.05.

Article Snippet: Rat brain microvascular endothelial cells (R840K-05a, Cell Applications) were plated into 24-well plates (1 105 cells/well) in rat brain microvascular endothelial cell growth media (R819K-500, Cell Applications) and allowed to settle for 16 h. Medium was then removed and replaced with Dulbecco’s Modified Eagle’s Medium/F12 containing 0.5% foetal bovine serum and 1% glutamine.

Techniques: In Vitro, Control

Journal: The ocular surface

Article Title: Herpes Simplex Virus-1 infection in Human Primary Corneal Epithelial Cells is Blocked by a Stapled Peptide that targets Processive DNA Synthesis

doi: 10.1016/j.jtos.2020.11.001

Figure Lengend Snippet: (Left Panel) Inhibition of HSV-1 infection of human primary corneal epithelial cells by S-pep7B. Following 1 h absorption of HSV-1 onto human primary corneal epithelial cells, the cells were washed thoroughly to remove any unabsorbed virus and then treated with S-pep7B at different dilutions. At 72 h post-treatment, virus titers in the culture media were determined by plaque reduction assays using Vero cells. (Right Panel) S-pep7B cytotoxicity in the primary cells was determined after 24 h by measuring ATP content. All data represents mean±SD obtained from two independent experiments in duplicate.

Article Snippet: These human primary corneal epithelial cells were grown in serum-free corneal epithelial cell medium (ATCC® PCS-700–030TM) supplemented with corneal epithelial cell growth kit (ATCC® PCS-700–040TM) according to the manufacturer’s instruction.

Techniques: Inhibition, Infection, Virus

Journal: Molecular Vision

Article Title: Corneal epithelial basement membrane assembly is mediated by epithelial cells in coordination with corneal fibroblasts during wound healing

doi:

Figure Lengend Snippet: Confirmation of cell morphology using light microscopy and immunocytochemistry A : Phase-contrast microscopy of rabbit corneal fibroblasts, myofibroblasts, and epithelial cells. Mag.:100×. B : Immunocytochemistry (ICC) for myofibroblast markers in cornea- and bone marrow-derived myofibroblasts. Myofibroblasts cultured in transforming growth factor (TGF) beta-1 for 14 days had ICC for vimentin, alpha-smooth muscle actin (SMA), desmin, and vinculin. Both cornea- and bone marrow-derived cells expressed vimentin, alpha-SMA, desmin, and vinculin, indicating they were vimentin-alpha smooth muscle actin-desmin (VAD)-positive myofibroblasts (Chaurasia et al., 2009). ICC with the corresponding isotypic control antibodies was also performed for each marker. Blue is 4′,6-diamidino-2-phenylindole (DAPI) staining of the nuclei. Mag.: 200×.

Article Snippet: Meanwhile, the rabbit corneal epithelial cells ( ; Rb630–05; Cell Applications, San Diego, CA and N-6048; Cell Biologics, Chicago, IL) were trypsinized and resuspended at 4.1 × 10 6 cells/ml in epithelial medium I, which was a mixture of a corneal epithelial cell medium (Rb221–500; Cell Applications), 264 ng/ml of stem cell factor (Cat. #300–07; Peprotech), and 10 ng/ml of endothelin-3 (Cat. #MBS405523; MyBioSource, San Diego, CA).

Techniques: Light Microscopy, Immunocytochemistry, Microscopy, Derivative Assay, Cell Culture, Control, Marker, Staining

Journal: Molecular Vision

Article Title: Corneal epithelial basement membrane assembly is mediated by epithelial cells in coordination with corneal fibroblasts during wound healing

doi:

Figure Lengend Snippet: Schematic workflow for preparing 3D corneal organotypic cultures. Rabbit corneas were procured and processed on the day of arrival approximately 24 h after enucleation. The corneas were punched centrally using a 9-mm trephine in a sterile Petri dish containing phosphate buffered saline (PBS) with 1× penicillin-streptomycin antibiotic solution. The epithelial and endothelial layers, along with the basement membranes (BMs), were removed under a dissection microscope using a scalpel blade. The corneal stroma was cut into ~2-mm 2 pieces and incubated overnight in an enzyme digestion medium containing collagenase and hyaluronidase. The stromal cell pellet collected after a brief centrifugation contained the corneal keratocytes, which were incubated in a fibroblast medium containing 40 ng/ml of fibroblast growth factor (FGF-2). Once the desired confluency was reached, 50% of the cells were treated with a myofibroblast medium containing 20 ng/ml of TGF beta-1, and the remaining 50% were maintained in the fibroblast medium. The cells were mixed with collagen type-1 to prepare the cellular layer, which was loaded above the acellular layer and coated onto the insert of six-well Transwell plates (Corning, NY). The cellular layer was incubated undisturbed for 4 days at 37°C in 5% carbon dioxide (CO 2 ). On Day 5, epithelial cells procured from commercial sources were prepared as a cell suspension and added to each insert, which was then incubated for 2 days in epithelial medium I at 37°C. Subsequently, the media was removed from both the inside and outside of the insert and replaced with fresh epithelial growth medium II only to the outside of the insert. The epithelial cells were then exposed to the air–liquid interface for 18 days, and the medium was replaced every 2 days. At the end of 18 days of incubation, a 3D corneal construct with different combinations of stromal cells along with multi-layered epithelial cells was generated.

Article Snippet: Meanwhile, the rabbit corneal epithelial cells ( ; Rb630–05; Cell Applications, San Diego, CA and N-6048; Cell Biologics, Chicago, IL) were trypsinized and resuspended at 4.1 × 10 6 cells/ml in epithelial medium I, which was a mixture of a corneal epithelial cell medium (Rb221–500; Cell Applications), 264 ng/ml of stem cell factor (Cat. #300–07; Peprotech), and 10 ng/ml of endothelin-3 (Cat. #MBS405523; MyBioSource, San Diego, CA).

Techniques: Sterility, Saline, Dissection, Microscopy, Incubation, Centrifugation, Suspension, Construct, Generated

Journal: Molecular Vision

Article Title: Corneal epithelial basement membrane assembly is mediated by epithelial cells in coordination with corneal fibroblasts during wound healing

doi:

Figure Lengend Snippet: Comparison of laminin alpha 5, perlecan and nidogen-1 expression in different 3D cultures. A : Triplex immunohistochemistry (IHC) for laminin alpha-5, perlecan, and nidogen-1 in 3D corneal organotypic cultures. The corneal fibroblast and epithelial (CFib + Epi) organotypic cultures showed expression of laminin alpha-5 (green), perlecan (red), and nidogen-1 (yellow) at the interface between the epithelial cells and the fibroblasts, suggesting a well-assembled epithelial BM. In corneal myofibroblast and epithelium (CMyo + Epi) cultures, there was a high expression of perlecan, a lower expression of laminin alpha-5, and no expression of nidogen-1 detected. It is important to note that the expression was observed only in the stroma, and the overlying epithelial BM was not assembled when myofibroblasts were co-cultured with epithelial cells. When the epithelial cells were cultured alone above the collagen matrix (Epi only control), laminin alpha-5 was predominantly expressed throughout the epithelium, perlecan expression was low, and no expression of nidogen-1 was detected. Blue is DAPI staining of cell nuclei. Mag.: 200×. B : A graph showing the percentage assembly of individual epithelial BM components, comparing between the groups of 3D cultures. Error bars represent mean ± SEM. Each BM component was compared between the groups using one-way ANOVA, and p < 0.05 was considered statistically significantly different.

Article Snippet: Meanwhile, the rabbit corneal epithelial cells ( ; Rb630–05; Cell Applications, San Diego, CA and N-6048; Cell Biologics, Chicago, IL) were trypsinized and resuspended at 4.1 × 10 6 cells/ml in epithelial medium I, which was a mixture of a corneal epithelial cell medium (Rb221–500; Cell Applications), 264 ng/ml of stem cell factor (Cat. #300–07; Peprotech), and 10 ng/ml of endothelin-3 (Cat. #MBS405523; MyBioSource, San Diego, CA).

Techniques: Comparison, Expressing, Immunohistochemistry, Cell Culture, Control, Staining

Journal: Molecular Vision

Article Title: Corneal epithelial basement membrane assembly is mediated by epithelial cells in coordination with corneal fibroblasts during wound healing

doi:

Figure Lengend Snippet: Comparison of laminin beta 3 and collagen IV expression in different 3D cultures. A : Duplex IHC for laminin beta-3 and collagen type IV in corneal organotypic cultures. The CFib + Epi cultures showed high expression of laminin beta-3 (green) in basal and more superficial epithelial layers. Collagen type IV (red) at the interface of the epithelial cells and fibroblasts indicates a normally regenerated epithelial BM. However, CMyo + Epi cells in organotypic cultures showed no evidence for epithelial BM generation, although myofibroblasts were found to produce collagen type IV. In Epi only controls, laminin beta-3 is expressed throughout the epithelium, with low expression of collagen type IV. Blue is DAPI staining of cell nuclei. Mag.: 200×. B : Graph showing percentage formation of laminin beta-3 and collagen IV compared between the groups of 3D cultures. Error bars represent mean ± SEM. One-way ANOVA was used to compare between the groups, and p < 0.05 was considered statistically significantly different.

Article Snippet: Meanwhile, the rabbit corneal epithelial cells ( ; Rb630–05; Cell Applications, San Diego, CA and N-6048; Cell Biologics, Chicago, IL) were trypsinized and resuspended at 4.1 × 10 6 cells/ml in epithelial medium I, which was a mixture of a corneal epithelial cell medium (Rb221–500; Cell Applications), 264 ng/ml of stem cell factor (Cat. #300–07; Peprotech), and 10 ng/ml of endothelin-3 (Cat. #MBS405523; MyBioSource, San Diego, CA).

Techniques: Comparison, Expressing, Staining

Journal: Molecular Vision

Article Title: Corneal epithelial basement membrane assembly is mediated by epithelial cells in coordination with corneal fibroblasts during wound healing

doi:

Figure Lengend Snippet: IHC for laminin alpha-5, perlecan, and nidogen-1 in CMyo + Epi organotypic cultures. Triplex IHC did not detect epithelial BM posterior to the epithelium in this example culture or any other corneal myofibroblast, bone marrow-derived myofibroblast, or mixed corneal-bone marrow myofibroblast organotypic culture with corneal epithelium. Also, no stratified epithelium was detected in these cultures. This indicates that the myofibroblasts derived from both cornea and bone marrow—either alone as CMyo (100%)/BMyo (100%) or combined (CMyo 50% + BMyo 50%)—when incubated with corneal epithelial cells could not assemble BM. Rather, spheroid-like epithelial masses were present in many cultures. Myofibroblasts were observed throughout the matrix. Blue is DAPI staining of the nuclei. Mag.: 200×.

Article Snippet: Meanwhile, the rabbit corneal epithelial cells ( ; Rb630–05; Cell Applications, San Diego, CA and N-6048; Cell Biologics, Chicago, IL) were trypsinized and resuspended at 4.1 × 10 6 cells/ml in epithelial medium I, which was a mixture of a corneal epithelial cell medium (Rb221–500; Cell Applications), 264 ng/ml of stem cell factor (Cat. #300–07; Peprotech), and 10 ng/ml of endothelin-3 (Cat. #MBS405523; MyBioSource, San Diego, CA).

Techniques: Derivative Assay, Incubation, Staining

Journal: Molecular Vision

Article Title: Corneal epithelial basement membrane assembly is mediated by epithelial cells in coordination with corneal fibroblasts during wound healing

doi:

Figure Lengend Snippet: Transmission electron microscopy (TEM) showing epithelial BM assembly. A : The presence of a well-orchestrated, multi-layered stratified epithelium when cultured in the presence of fibroblasts in the stroma (Mag.: 890×). B : The presence of fibroblasts embedded and dispersed in the collagen matrix (Mag.: 890×). C , D : The presence of a well-assembled epithelial BM when co-cultured with corneal fibroblasts, as indicated by arrows directly beneath the epithelial cells (Mag.: 30,000×).

Article Snippet: Meanwhile, the rabbit corneal epithelial cells ( ; Rb630–05; Cell Applications, San Diego, CA and N-6048; Cell Biologics, Chicago, IL) were trypsinized and resuspended at 4.1 × 10 6 cells/ml in epithelial medium I, which was a mixture of a corneal epithelial cell medium (Rb221–500; Cell Applications), 264 ng/ml of stem cell factor (Cat. #300–07; Peprotech), and 10 ng/ml of endothelin-3 (Cat. #MBS405523; MyBioSource, San Diego, CA).

Techniques: Transmission Assay, Electron Microscopy, Cell Culture

Journal: Molecular Vision

Article Title: Corneal epithelial basement membrane assembly is mediated by epithelial cells in coordination with corneal fibroblasts during wound healing

doi:

Figure Lengend Snippet: Comparison of rabbit corneas at different time points after Photorefractive Keratectomy surgeries. A , B : Triplex IHC for vimentin, SMA, and nidogen-1 in corneas at different time points after PRK surgeries. Triplex IHC for mesenchymal marker vimentin (green), myofibroblast marker alpha-SMA (red), and epithelial BM marker nidogen-1 (magenta) was observed in rabbit corneas at different time points after −3D PRK. At 1 and 2 weeks after PRK, there was a patchy expression of nidogen-1 with irregular cellular arrangement of epithelial cells. There was evidence for few vimentin-positive fibroblasts at both of these time points, and vimentin-positive fibrocytes could also be present in the anterior stroma. At 4 weeks after PRK, there was an increase in the number of vimentin-positive cells in the anterior stroma. In parallel, there was an increase in the percentage of epithelial BM regenerated to around 80–90%. This suggests a strong association of fibroblasts with epithelial BM regeneration at 4 weeks after PRK injury. At 6 weeks after PRK, when the percentage of epithelial BM regeneration was further increased, the corneal fibroblast numbers had decreased. At 8 weeks after PRK, most vimentin-positive cells disappeared from the anterior stroma, when the epithelial BM was fully regenerated. No alpha-SMA-positive cells were observed at any of the time points tested, indicating there was no fibrosis in these corneas. Blue is DAPI staining of the nuclei. Mag.: 200×.

Article Snippet: Meanwhile, the rabbit corneal epithelial cells ( ; Rb630–05; Cell Applications, San Diego, CA and N-6048; Cell Biologics, Chicago, IL) were trypsinized and resuspended at 4.1 × 10 6 cells/ml in epithelial medium I, which was a mixture of a corneal epithelial cell medium (Rb221–500; Cell Applications), 264 ng/ml of stem cell factor (Cat. #300–07; Peprotech), and 10 ng/ml of endothelin-3 (Cat. #MBS405523; MyBioSource, San Diego, CA).

Techniques: Comparison, Marker, Expressing, Staining