第944回千葉医学会例会・第1外科教室談話会

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Cell Death Pathways in Astrocytes with a Modified Model of Oxygen-Glucose Deprivation

<div><p>Traditional oxygen-glucose deprivation (OGD) models do not produce sufficiently stable and continuous deprivation to induce cell death in the ischemic core. Therefore, we modified the OGD model to mimic the observed damage in the ischemic core following stroke and utilized this new model to study cell death pathways in astrocytes. The PO₂ and pH levels in the astrocyte culture medium were compared between a physical OGD group, a chemical OGD group and a mixed OGD group. The mixed OGD group was able to maintain anaerobic conditions in astrocyte culture medium for 6 h, while the physical and the chemical groups failed to maintain such conditions. Astrocyte viability decreased and LDH release into in the medium increased as a function of exposure to OGD. Compared to the control group, the expression of active caspase-3 in the mixed OGD group increased within 2 h after OGD, but decreased after 2 h of OGD. Additionally, porimin mRNA levels did not significantly increase during the first 2 h of OGD, while bcl-2 mRNA levels decreased at 1 h. However, both porimin and bcl-2 mRNA levels increased after 2 h of OGD; interestingly, they both suddenly decreased at 4 h of OGD. Taken together, these results indicate that apoptosis and oncosis are the two cell death pathways responsible for astrocyte death in the ischemic core. However, the main death pathway varies depending on the OGD period.</p></div

Silica-Coated Core–Shell Structured Polystyrene Nanospheres and Their Size-Dependent Mechanical Properties

The core–shell structured PS/SiO₂ composite nanospheres were synthesized on the basis of a modified Stöber method. The mechanical properties of monodisperse nanospheres were characterized with nanoindentation on the basis of the atomic force microscopy (AFM). The surface morphologies of PS/SiO₂ composite nanospheres was scanned with the tapping mode of AFM, and the force–distance curves were measured with the contact mode of AFM. Different contact models were compared for the analyses of experimental data. The elastic moduli of PS/SiO₂ composite nanosphere (4–40 GPa) and PS nanosphere (∼3.4 GPa) were obtained with the Hertz and Johnson–Kendall–Roberts (JKR) models, respectively, and the JKR model was proven to be more appropriate for calculating the elastic modulus of PS/SiO₂ nanospheres. The elastic modulus of SiO₂ shell gradually approached a constant value (∼46 GPa) with the increase of SiO₂ shell thickness. A core–shell model was proposed for describing the relationship between PS/SiO₂ composite nanosphere’s elastic modulus and shell thickness. The mechanical properties of the composite nanospheres were reasonably explained on the basis of the growth mechanism of PS/SiO₂ composite nanospheres, in particular the SiO₂ shell’s formation process. Available research data of PS/SiO₂ composite nanospheres in this work can provide valuable guidance for their effective application in surface engineering, micro/nanomanufacturing, lubrication, and so on

mRNA levels of bcl-2 and porimin in astrocytes exposed to mixed OGD.

<p>(A) the bcl-2 mRNA relative expression and (B) porimin mRNA relative expression for 1 h, 2 h, 3 h, 4 h and 6 h were altered relative to the rates of expression in the control samples (0 h). (*) indicates a significant difference (P<0.05) from the control group.</p

The PO₂ and pH of the media among the three OGD groups.

<p>PO<b>₂</b> (A) and pH (B) of the media among the three groups measured at different OGD time points. The mixed group kept the PO<b>₂</b> at zero for 6 h in the incubation solution and mimics the ischemic core.</p

LDH leakage induced by mixed OGD.

<p>Mixed OGD induced a significant increase in LDH leakage after 1 h, 2 h, 3 h, 4 h and 6 h (F = 220.7, P = 0.001). (*) indicates a significant difference (P<0.05) from the control group (Ctrl).</p

Representative electron microscopy images of the ultrastructural changes in astrocytes induced by mixed OGD.

<p>(A) Image of a normal astrocyte (6200×). (B,I) Images of typical apoptotic cells (the red arrowhead shows chromatin condensation rendering a curved profile to the nucleus and apoptotic body formation). Apoptotic cells were found in almost all samples, particularly after 1 h (B) and 2 h (C) of OGD. (G–H) Images of typical oncotic-like cells. The co-existence of cellular swelling and vacuolization were found in the 3 h (D) and 4 h (E) OGD samples. After 6 h of OGD, the astrocytes were almost fragmental (F). Scale bars: I: 1 µm; H: 2 µm; D: 10 µm; all others: 5 µm.</p

The viable astrocyte exposed to mixed OGD.

<p>(<b>A</b>) Astrocytes exposed to mixed OGD in the control condition (Ctrl) and for 1 h, 2 h, 3 h, 4 h and 6 h were stained by HE (200×). (B) Counting 5 views for statistical analysis, we found that the amount viable astrocytes decreased as a function of time spent under OGD. (*) indicates a significant difference (P<0.05) from the control group (Ctrl).</p

Additional file 1 of Empagliflozin targets Mfn1 and Opa1 to attenuate microglia-mediated neuroinflammation in retinal ischemia and reperfusion injury

Additional file 1: Fig. S1. EMPA relieves inflammation and neuron death in the IR retina. A. Dot plot showing markers genes (rows) that uniquely mark different cell types in mouse retina (columns). The size of the dot indicates the percentage of cells expressing the gene, and the color represents the average expression level of the gene in the indicated cell types. B. Bar plots showing the GO terms enriched for the downregulated IR-DEGs (left) and upregulated EMPA-DEGs (right) of overall retinal cells. The color from light to dark indicates the statistical significance value from low to high. C. GSEA of EMPA-DEGs mapping to GO pathway neuron death. D. GSEA of IR-DEGs mapping to GO pathway neuron death. Fig. S2. EMPA suppresses microglial activation in the IR retina. A. t-SNE plot showing the distribution of microglia from three groups. B. Heatmap showing the expression of M1 and M2 markers from three groups in microglia. The color key from blue to red indicates the expression level from low to high. Fig. S3. EMPA modulates LPS-induced polarization in BV2 cells treated with LPS. A. BV2 cells were treated with indicated concentrations of EMPA. Cell viability was measured by CCK-8 assay. B. Representative immunofluorescence images of INOS in control and LPS-stimulated BV2 cells treated with or without EMPA. Scale bar: 50 µm. C. The quantity of the intensity of INOS immunofluorescence in BV2 cells (n = 3). D-E. qRT-PCR analysis of mRNA expression levels of M1 markers (CD68, CD32, COX2, INOS) and M2 markers (CCL-22, Arg1) (n = 4–6). Fig. S4. EMPA activates mitophagy in BV2 cells treated with LPS. A. The levels of mitophagy-related proteins (LC3B and P62) were analyzed through Western blots. β-actin served as the internal protein control. B. Relative densitometry quantitation of relative protein expression levels in the 3 groups of BV2 cells (n = 3). Fig. S5. Knockdown efficiency of Opa1 (A) or Mfn1 (B) si RNA. Table S1. Primary antibodies and dilutions. Table S2. qRT-PCR primer sequences

Additional file 3: Table S3. of Whole genome shotgun sequence of Bacillus amyloliquefaciens TF28, a biocontrol entophytic bacterium

General feature of genome from 22 strains of B. amyloliquefaciens (DOCX 17 kb