apr 020 pe  (Alomone Labs)

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Changes of C3d/S100A10 levels, and pan-reactive and A1/A2 specific gene expression in reactive astrocytes after treatment with microglia-conditioned media (MCM) and P2Y 1 R-ANT. (a), (b). Representative images (a) and quantitative data (b) of western blots showing protein levels of GFAP, C3d, S100A10, and CSPG in non-OGD astrocytes, OGD astrocytes, OGD astrocytes treated with MCM, OGD astrocytes treated with P2Y 1 R-ANT, and OGD astrocytes treated with P2Y 1 R-ANT and MCM. β-actin was used as an internal control. * P < 0.05 vs. non-OGD astrocyte, # P < 0.05 vs. OGD astrocytes. (c). Venn diagram of mRNAs with upregulated (fold change ≥ 1.5) and downregulated (fold change ≤ 0.67) expressions in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. (d). Heatmap of the entire mRNA expression in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. (e). Heatmaps comparing the mean expression of pan-reactive, A1-specific, and A2 specific genes in OGD astrocytes; OGD astrocytes treated with P2Y 1 R-ANT (1 mM); and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. N = 4/group. Values are the mean ± SD. GFAP = glial fibrillary acidic protein, CSPG = chondroitin sulfate proteoglycans, OGD = oxygen–glucose deprivation, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglial conditioned medium

Article Snippet: The primary antibodies used in this study were rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba-1) (1:800; Wako), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:200; MBL), goat polyclonal anti-GFAP (1:200; Abcam), goat polyclonal anti-C3d (1:25, R&D Systems), rabbit polyclonal anti-S100A10 (1:50, Proteintech), rabbit polyclonal anti-P2Y 1 receptor antibody (1:100, Alomone Labs), rabbit polyclonal anti-nuclear factor-κβ (NF-κB) (1:200; Abcam), mouse monoclonal anti-TNF-α (1:200; GeneTex), mouse monoclonal pNFH (SMI31)(1:200; BioLegend), and mouse MAP2 (1:500; Merck).

Techniques: Expressing, Western Blot, Control

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Change in inflammatory gene expression and pathway analysis. (a). Top 20 significant canonical pathways of the core analysis in IPA of most highly expressed genes in OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM relative to OGD astrocytes. Blue bars: negative z-score; orange bars: positive z-score; gray bars: no activity pattern available; white bars: activity of zero. (b), (c). Heatmap of mRNA-related Neuroinflammation Signaling expression, and quantitative analysis of representative mRNA-related Neuroinflammation Signaling in OGD astrocytes, OGD astrocytes treated with P2Y 1 R-ANT (1 mM), and OGD astrocytes treated with P2Y 1 R-ANT (1 mM) and MCM. * P < 0.05 vs. OGD astrocyte, # P < 0.05 vs. OGD astrocytes treated with P2Y 1 R-ANT. (d). Signaling pathway predicted by analyzing changes using IPA software in mRNA expression in the OGD astrocytes treated with P2Y 1 R-ANT and MCM relative to OGD astrocytes, or OGD astrocytes treated with P2Y 1 R-ANT. The functional networks were generated via IPA (QIAGEN Inc., https://www.qiagenbio-informatics.com/products/ingenuity-pathway-analysis ). N = 4/group. Values are the mean ± SD. IPA = Ingenuity Pathway Analysis, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglial conditioned medium, MAPK = mitogen-activated protein kinase, NF-κB = nuclear factor-κβ, TNF-α = tumor necrosis factor, IL-1β = interleukin-1β, NOX = nitrogen oxides, CASP8 = Caspase 8

Article Snippet: The primary antibodies used in this study were rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba-1) (1:800; Wako), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:200; MBL), goat polyclonal anti-GFAP (1:200; Abcam), goat polyclonal anti-C3d (1:25, R&D Systems), rabbit polyclonal anti-S100A10 (1:50, Proteintech), rabbit polyclonal anti-P2Y 1 receptor antibody (1:100, Alomone Labs), rabbit polyclonal anti-nuclear factor-κβ (NF-κB) (1:200; Abcam), mouse monoclonal anti-TNF-α (1:200; GeneTex), mouse monoclonal pNFH (SMI31)(1:200; BioLegend), and mouse MAP2 (1:500; Merck).

Techniques: Expressing, Activity Assay, Software, Functional Assay, Generated

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Significance of AEVs derived from anti-inflammatory astrocytes to the peri-infarct area after MCAO. (a). The experimental scheme of isolating AEVs from OGD astrocytes treated with MCM and P2Y 1 R-ANT and their application for rats subjected to MCAO and cultured cortical neurons. (b), (c). Modified neurological severity score ( b ) and latency to fall off the rotarod at 56 days after MCAO ( c ) in PBS treatment, treatment with AEVs derived from OGD astrocytes (100 µg), and OGD astrocytes treated with MCM and P2Y 1 R-ANT (100 µg) in rats subjected to MCAO. N = 6–7/group. Values are the mean ± SD. (d), (e). Double immunofluorescent confocal images and quantitative data of the peri-infarct area at 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing C3d + area (green) ( d ) and S100A10 + area (green) ( e ), and co-localized with GFAP + area (yellow). Merge ratio of C3d /GFAP ( d ), and S100A10/GFAP ( e ). N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. * P < 0.05 vs. PBS-treated rats, # P < 0.05 vs. rats treated with AEVs derived from OGD astrocytes. Scale bar = 100 μm AEVs = astrocytic extracellular vesicles, MCAO = middle cerebral artery occlusion, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglia-conditioned medium, GFAP = glial fibrillary acidic protein, OGD = oxygen–glucose deprivation

Article Snippet: The primary antibodies used in this study were rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba-1) (1:800; Wako), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:200; MBL), goat polyclonal anti-GFAP (1:200; Abcam), goat polyclonal anti-C3d (1:25, R&D Systems), rabbit polyclonal anti-S100A10 (1:50, Proteintech), rabbit polyclonal anti-P2Y 1 receptor antibody (1:100, Alomone Labs), rabbit polyclonal anti-nuclear factor-κβ (NF-κB) (1:200; Abcam), mouse monoclonal anti-TNF-α (1:200; GeneTex), mouse monoclonal pNFH (SMI31)(1:200; BioLegend), and mouse MAP2 (1:500; Merck).

Techniques: Derivative Assay, Cell Culture, Modification

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Expression of microRNAs (miRNAs) in AEVs and inflammatory regulation in peri-infarct glial scars. (a). Heatmap of miRNA profiles on AEVs derived from OGD astrocytes and OGD astrocytes treated with P2Y 1 R-ANT and MCM. (b). Quantitative analysis of representative miRNAs related to ‘Inflammatory Response’ in AEVs derived from OGD astrocytes treated with P2Y 1 R-ANT and MCM, relative to AEVs derived from OGD astrocytes. N = 4/group. Values are the mean ± SD. * P < 0.05 vs. AEVs derived from OGD astrocytes. (c), (d). Double immunofluorescent confocal images and quantitative data of the peri-infarct area 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing TNFα + area (green) ( c ) and NF-κB + area (green) ( d ), and co-localized with GFAP + area (yellow). N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. * P < 0.05 vs. PBS-treated rats. Scale bar = 100 μm AEVs = astrocytic extracellular vesicles, P2Y 1 R-ANT = P2Y 1 receptor antagonist

Article Snippet: The primary antibodies used in this study were rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba-1) (1:800; Wako), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:200; MBL), goat polyclonal anti-GFAP (1:200; Abcam), goat polyclonal anti-C3d (1:25, R&D Systems), rabbit polyclonal anti-S100A10 (1:50, Proteintech), rabbit polyclonal anti-P2Y 1 receptor antibody (1:100, Alomone Labs), rabbit polyclonal anti-nuclear factor-κβ (NF-κB) (1:200; Abcam), mouse monoclonal anti-TNF-α (1:200; GeneTex), mouse monoclonal pNFH (SMI31)(1:200; BioLegend), and mouse MAP2 (1:500; Merck).

Techniques: Expressing, Derivative Assay

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Axonal outgrowth after AEV treatment and hindering by TNF-α (a), (b). Double immunofluorescent confocal images and quantitative data of the peri-infarct area 56 days after MCAO with intracerebral administration of PBS, 100 µg AEVs derived from OGD astrocytes, and 100 µg AEVs derived from OGD astrocytes treated with MCM and P2Y 1 R-ANT, showing pNFH + axons (green) ( a ) and MAP2 cells + (green) ( b ), with GFAP. N = 5/group (three sections per rat, and total of 15 samples in each group). Values are the mean ± SD. Scale bar = 100 μm. (c). Representative time-lapse microscopic images and quantitative data of primary cortical neurons in a microfluidic chamber showing axonal elongation (distance from yellow arrow to red arrow) in OGD neurons, OGD neurons treated with 1 ng/µl of TNF-α, and OGD neurons treated with 10 ng/µl of TNF-α. N = 3/group. Values are the mean ± SD. Scale bar = 20 μm. Quantitative data of axonal elongation per 30 min prior to 96 h after OGD. * P < 0.05, OGD neurons treated with 1 ng/µL of TNF-α vs. OGD neurons; # P < 0.05, OGD neurons treated with 10 ng/µL of TNF-α vs. OGD neurons; $ P < 0.05, OGD neurons treated with 10 ng/µL of TNF-α vs. OGD neurons treated with 1 ng/µL of TNF-α. AEVs = astrocytic extracellular vesicles, MCAO = middle cerebral artery occlusion, OGD = oxygen–glucose deprivation, P2Y 1 R-ANT = P2Y 1 receptor antagonist, MCM = microglia-conditioned medium, GFAP = glial fibrillary acidic protein, pNFH = phosphorylated neurofilament heavy chain

Article Snippet: The primary antibodies used in this study were rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba-1) (1:800; Wako), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:200; MBL), goat polyclonal anti-GFAP (1:200; Abcam), goat polyclonal anti-C3d (1:25, R&D Systems), rabbit polyclonal anti-S100A10 (1:50, Proteintech), rabbit polyclonal anti-P2Y 1 receptor antibody (1:100, Alomone Labs), rabbit polyclonal anti-nuclear factor-κβ (NF-κB) (1:200; Abcam), mouse monoclonal anti-TNF-α (1:200; GeneTex), mouse monoclonal pNFH (SMI31)(1:200; BioLegend), and mouse MAP2 (1:500; Merck).

Techniques: Derivative Assay

Journal: Molecular Neurobiology

Article Title: Astrocytic Extracellular Vesicles Regulated by Microglial Inflammatory Responses Improve Stroke Recovery

doi: 10.1007/s12035-023-03629-9

Figure Lengend Snippet: Diagram depicting the findings of the present study. Microglia and inhibition of P2Y 1 R regulate reactive astrocytes by transforming C3d/S100A10 expression and suppressing neuroinflammation. AEVs derived from reactive astrocytes with anti-inflammatory properties possessing miR-146a-5p regulate glial scars by suppressing NF-κB and TNF-α, which is permissive for axonal outgrowth and improves stroke recovery

Article Snippet: The primary antibodies used in this study were rabbit polyclonal anti-ionized calcium-binding adapter molecule 1 (Iba-1) (1:800; Wako), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (1:200; MBL), goat polyclonal anti-GFAP (1:200; Abcam), goat polyclonal anti-C3d (1:25, R&D Systems), rabbit polyclonal anti-S100A10 (1:50, Proteintech), rabbit polyclonal anti-P2Y 1 receptor antibody (1:100, Alomone Labs), rabbit polyclonal anti-nuclear factor-κβ (NF-κB) (1:200; Abcam), mouse monoclonal anti-TNF-α (1:200; GeneTex), mouse monoclonal pNFH (SMI31)(1:200; BioLegend), and mouse MAP2 (1:500; Merck).

Techniques: Inhibition, Expressing, Derivative Assay

Journal: eLife

Article Title: Purinergic regulation of vascular tone in the retrotrapezoid nucleus is specialized to support the drive to breathe

doi: 10.7554/eLife.25232

Figure Lengend Snippet: ( A ) trace of an RTN arteriole diameter show that increasing CO 2 in the perfusion media from 5% to 15% (balance air, in TTX) caused vasoconstriction under baseline conditions but not in PPADS (5 µM). ( B ) example vessel image under baseline conditions and corresponding fluorescent intensity profile plots also show that exposure to high CO 2 decreased vessel diameter. Profile plot scale bars: 2000 a.u., 10 µm. ( C ) summarized results of RTN arteriole responses to CO 2 /H + under baseline conditions (N = 34 vessels) and when P2-receptors were blocked (5 µM PPADS; N = 8 vessels), P1-receptors were blocked (10 µM 8-PT; N = 7 vessels), or ectonucleotidase activity was inhibited (100 µM POM1; N = 5 vessels). ( D ) example diameter traces show RTN arterioles constrict in response to bath application of ATP (100 µM) or the selective P2Y 2/4 receptor agonist UTPγS (0.5 µM) but dilate when P1 receptors are activated by adenosine (Ado; 1 µM). ( E ) summary data plotted as % diameter change in response to ATP (N = 7 vessels), UTP (N = 8 vessels), α,β-mATP (100 µM, preferential P2X agonist; N = 9 vessels) or adenosine (N = 9 vessels). ( F–G ), immunoreactivity for P2Y 2 ( F ) and P2Y 4 ( G ) receptors was detected as brightly label puncta near endothelial cells (DyLight 594 Isolectin B4 conjugate; IB4), arteriole smooth muscle (α-smooth muscle actin; αSMA), and astrocytes (glial fibrillary acidic protein; GFAP) associated with arterioles in the RTN (N = 3 animals). Arrows identify receptor labeling close to endothelial or smooth muscle cells and arrowhead identifies receptor labeling of astrocyte processes. Scale bar 10 µM. Hash marks designate a difference in µm from baseline as determined by RM-one-way ANOVA and Fishers LSD test or paired t-test and asterisks identify differences in CO 2 /H + -induced % change under baseline conditions vs in the presence of PPADS ( C ) or ATP vs specific agonist-induced % change ( E ) (one-way ANOVA and Fishers LSD test); one symbol = p<0.05, two symbols = p<0.01, three symbols = p<0.001, four symbols = p<0.0001. DOI: http://dx.doi.org/10.7554/eLife.25232.003

Article Snippet: Sections were then incubated overnight at RT with primary antibodies diluted in blocking solution as follows: 1:200 rabbit anti-P2Y 2 (RRID: AB_2040078 ) or P2Y 4 receptor (RRID: AB_2040080 ) (Alomone Labs, Alomone Labs, Jerusalem Israel), 1:200 chicken anti-glial fibrillary acidic protein (RRID: AB_177521 ) (Chemicon) and 1:500 mouse anti-α-smooth muscle actin (RRID: AB_262054 ) (Sigma-Aldrich, St. Louis, MO).

Techniques: Activity Assay, Labeling

Journal: eLife

Article Title: Purinergic regulation of vascular tone in the retrotrapezoid nucleus is specialized to support the drive to breathe

doi: 10.7554/eLife.25232

Figure Lengend Snippet: ( A ) diameter trace of a cortical arteriole with an example vessel image and fluorescence profile plots ( B ) show that exposure to CO 2 /H + caused vasodilation under baseline conditions and this response was blunted by PPADS (5 µM). Profile plot scale bars: 2000 a.u., 10 µm. ( C ) summary data show CO 2 /H + -induced vasodilation of cortical arterioles under bassline conditions (N = 11 vessels) but not in PPADS (N = 6 vessels). ( D–E ), immunoreactivity for P2Y 2 ( D ) and P2Y 4 ( E ) receptors was detected as brightly label puncta near endothelial cells (DyLight 594 Isolectin B4 conjugate; IB4), arteriole smooth muscle (α-smooth muscle actin; αSMA), and astrocytes (glial fibrillary acidic protein; GFAP) associated with cortical arterioles (N = 3 animals). Arrows identify receptor labeling close to endothelial or smooth muscle cells and arrowheads identifies receptor labeling of astrocyte processes. Scale bar 10 µM. ##, difference in µm from baseline (paired t test, p<0.01). **, difference in CO 2 /H + -induced % change under control conditions vs in PPADS (paired t-test, p<0.01). DOI: http://dx.doi.org/10.7554/eLife.25232.005

Article Snippet: Sections were then incubated overnight at RT with primary antibodies diluted in blocking solution as follows: 1:200 rabbit anti-P2Y 2 (RRID: AB_2040078 ) or P2Y 4 receptor (RRID: AB_2040080 ) (Alomone Labs, Alomone Labs, Jerusalem Israel), 1:200 chicken anti-glial fibrillary acidic protein (RRID: AB_177521 ) (Chemicon) and 1:500 mouse anti-α-smooth muscle actin (RRID: AB_262054 ) (Sigma-Aldrich, St. Louis, MO).

Techniques: Fluorescence, Labeling

Journal: Blood Transfusion

Article Title: Expression and function of purinergic receptors in platelets from apheresis-derived platelet concentrates

doi: 10.2450/2015.0073-15

Figure Lengend Snippet: Unchanged P2Y12 receptor function during storage of APC.

Article Snippet: Rabbit polyclonal anti-P2Y1, anti-P2Y12 and anti-P2X1 antibodies were from Alomone Labs (Jerusalem, Israel).

Techniques: