Disulfiram Ophthalmic Solution Inhibited Macrophage Infiltration by Suppressing Macrophage Pseudopodia Formation in a Rat Corneal Alkali Burn Model.

FROUNT is an intracellular protein that promotes pseudopodia formation by binding to the chemokine receptors CCR2 and CCR5 on macrophages. Recently, disulfiram (DSF), a drug treatment for alcoholism, was found to have FROUNT inhibitory activity. In this study, we investigated the effect of DSF eye drops in a rat corneal alkali burn model. After alkali burn, 0.5% DSF eye drops (DSF group) and vehicle eye drops (Vehicle group) were administered twice daily. Immunohistochemical observations and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed at 6 h and 1, 4, and 7 days after alkali burn. Results showed a significant decrease in macrophage accumulation in the cornea in the DSF group, but no difference in neutrophils. RT-PCR showed decreased expression of macrophage-associated cytokines in the DSF group. Corneal scarring and neovascularization were also suppressed in the DSF group. Low-vacuum scanning electron microscopy imaging showed that macrophage length was significantly shorter in the DSF group, reflecting the reduced extension of pseudopodia. These results suggest that DSF inhibited macrophage infiltration by suppressing macrophage pseudopodia formation.

A Novel Multi-Observation System to Study the Effects of Anterior Ocular Inflammation in Zinn's Zonule Using One Specimen.

Zinn's zonule is a fragile and thin tissue, and little is known about its pathogenesis. The aim of this study was to develop an experimental setup for a comprehensive analysis of Zinn's zonule. Rats were divided into two groups: a control group (n = 4) and an alkali injury group (n = 4). Seven days after injury, the eyes were enucleated, the anterior eye was dissected and embedded in gelatin, and macroscopic observations were made. The gelatin specimens were then embedded in paraffin and observed in detail by low-vacuum scanning electron microscopy, immunofluorescence, and quantitative reverse transcription polymerase chain reaction (RT-qPCR). The results show qualitative changes in Zinn's zonules in both macroscopic and microscopic observations. In addition, macrophage infiltration and increased matrix metalloproteinase 2 (MMP2) expression were observed in the injured group, consistent with the RT-qPCR results. The experimental system in this study allowed us to capture the morphological and molecular biological changes of Zinn's zonule and to gain insight into its pathogenesis. In conclusion, this study presents a new experimental setup for the comprehensive analysis of the rat Zinn's zonule. The results suggest that this system can be used in the future to study and analyze a variety of paraffin-embedded tissues and specimens.

Micromorphological observation of HLE cells under knockdown of Fascin using LV-SEM.

Liver cancer is one of the most prevalent cancers in Japan with hepatocellular carcinoma (HCC) as the major histological subtype. Successful novel treatments for HCC have been reported; however, recurrences or metastasis may occur, which results in poor prognoses and high mortality of HCC patients. Fascin, an actin-bundling protein, regulates cell adhesion, migration, and invasion. Its overexpression positively correlates with poor prognosis of malignant tumors, and Fascin is considered as one of the tumor biomarkers and therapeutic target proteins. In this study, we attempted to reveal the relationship between Fascin and HCC using HLE, one of the human HCC cell lines. We performed the study with classical immunocytochemistry and recently developed techniques, such as wound-healing assay, spheroid cultivation, and low-vacuum scanning electron microscopy (LV-SEM). Non-Fascin-knockdown (FKD) cell spheroid had a regular spherical appearance with tight cell-cell connections, while FKD cell spheroid had an irregular shape with loose cell-cell connections. Cells of non-FKD spheroid presented fibrous protrusions on the cell surface, contrarily, cells of FKD spheroids showed bulbous-shaped protrusions. Morphological observation of FKD and non-FKD HLE spheroids were performed using LV-SEM. Our study may help to reveal the roles of Fascin in the process of HCC formation and its malignancy.

Evaluation of ultrastructural alterations of glomerular basement membrane and podocytes in glomeruli by low-vacuum scanning electron microscopy.

BACKGROUND: Low-vacuum scanning electron microscopy (LV-SEM) is applied to diagnostic renal pathology. METHODS: To demonstrate the usefulness of LV-SEM and to clarify the optimal conditions of pathology samples, we investigated the alterations of glomerular basement membrane (GBM) and podocytes in control and experimental active Heymann nephritis (AHN) rats by LV-SEM. RESULTS: On week 15 following induction of AHN, spike formation on GBM with diffuse deposition of IgG and C3 developed. Using LV-SEM, diffuse crater-like protrusions were clearly noted three-dimensionally (3D) on surface of GBM in the same specimens of light microscopy (LM) and immunofluorescence (IF) studies only after removal coverslips or further adding periodic acid-silver methenamine (PAM) staining. These 3D ultrastructural findings of GBM surface could be detected in PAM-stained specimens by LV-SEM, although true GBM surface findings could not be obtained in acellular glomeruli, because some subepithelial deposits remained on surface of GBM. Adequate thickness was 1.5-5 µm for 10% formalin-fixed paraffin-embedded (FFPE) and 5-10 µm for the unfixed frozen sections. The foot processes and their effacement of podocytes could be observed by LV-SEM using 10%FFPE specimens with platinum blue (Pt-blue) staining or double staining of PAM and Pt-blue. These findings were obtained more large areas in 2.5% glutaraldehyde-fixed paraffin-embedded (2.5%GFPE) specimens. CONCLUSION: Our findings suggest that LV-SEM is a useful assessment tool for evaluating the alterations of GBM and podocytes in renal pathology using routine LM and IF specimens, as well as 2.5%GFPE specimens.

Low-voltage SEM of air-sensitive powders: From sample preparation to micro/nano analysis with secondary electron hyperspectral imaging.

Powder materials are used in all corners of materials science, from additive manufacturing to energy storage. Scanning electron microscopy (SEM) has developed to meet morphological, microstructural and bulk chemical powder characterization requirements. These include nanoscale elemental analysis and high-throughput morphological assays. However, spatially localized powder surface chemical information with similar resolution to secondary electron (SE) imaging is not currently available in the SEM. Recently, energy filtered (EF-) SEM has been used for surface chemical characterization by secondary electron hyperspectral imaging (SEHI). This review provides a background to existing powder characterization capabilities in the low voltage SEM provided by SE imaging, EDX analysis and BSE imaging and sets out how these capabilities could be extended for surface chemical analysis by applying SEHI to powders, with particular emphasis on air and beam sensitive powder surfaces. Information accessible by SEHI, its advantages and limitations, is set into the context of other chemical characterization methods that are commonly used for assessing powder surface chemistry such as by Auger electron spectroscopy (AES) and X-ray photoelectron spectroscopy (XPS). The applicability of existing powder preparation methods for SEM to SEHI is also reviewed. An alternative preparation method is presented alongside first examples of SEHI characterization of powder surfaces. The commercial powder materials used as examples were carbon-fiber/polyamide composite powder feedstock (CarbonMide®) used in additive manufacturing and powders consisting of lithium nickel cobalt oxide (NMC). SEHI is shown to differentiate bonding present at carbonaceous material surfaces and extract information about the work function of metal oxide surfaces. The surface sensitivity of SEHI is indicated by comparison of pristine powders to those with surface material added in preparation. A minimum spatial localization of chemical information of 55 nm was achieved in differentiating regions of NMC surface chemistry by distinct SE spectra.

Exploiting Plasma Exposed, Natural Surface Nanostructures in Ramie Fibers for Polymer Composite Applications.

Nanoscale surface morphology of plant fibers has important implications for the interfacial bonding in fiber-polymer composites. In this study, we investigated and quantified the effect of plasma-surface modification on ramie plant fibers as a potential tool for simple and efficient surface modification. The extensive investigation of the effects of plasma treatment of the fiber surface nano-morphology and its effect on the fiber-polymer interface was performed by Low-Voltages Scanning Electron Microscopy (LV-SEM), infrared spectroscopy (FT-IR) analysis, fiber-resin angle measurements and mechanical (tensile) testing. The LV-SEM imaging of uncoated plasma treated fibers reveals nanostructures such as microfibrils and elementary fibrils and their importance for fiber mechanical properties, fiber wettability, and fiber-polymer matrix interlocking which all peak at short plasma treatment times. Thus, such treatment can be an effective in modifying the fiber surface characteristics and fiber-polymer matrix interlocking favorably for composite applications.