Journal: bioRxiv

Article Title: Disrupted Cerebral Peri-Microvascular Glycogen Promotes Capillary Constrictions and Aggravates Ischemia in Mice

doi: 10.1101/2022.08.24.505172

Figure Lengend Snippet: CD13 positive capillary pericyte mediated constrictions were evaluated in Swiss albino, wild-type (WT) and GYS1 Nestin-KO mice. (A) Experiments performed in adult Swiss albino male and female mice intracerebroventricularly (i.c.v) administered with saline (vehicle) or DAB (as indicated in figure) sacrificed after 30 mins, 1h, 3h, 6h, 9h and 24h (shown left to right respectively). Lycopersicon Esculentum Lectin (upper panel-green) and CD13 (middle panel-red) double labelling (merged as lower panel) reveals increased microvascular constrictions 30 minutes after DAB injections shown by arrows. Images represent 3D reconstruction of 40-μm z-stack. Scale bars, 10 μm. (B) Microvascular constrictions in ipsilateral and contralateral hemispheres were quantified semi-stereologically. DAB (i.c.v) injections result in robust increase of constrictions after 30 minutes, persists for 6 hours. Although number of constrictions started to decline after 9 hours, impact of DAB on these constrictions are not fully reversible even after 24 hours. Quantification of CD13+ pericyte mediated microvascular constrictions in ten fields fields for each hemisphere spanning MCA territory (240 μm × 160 μm) per animal (n = 3 animals per group). Two-way analysis of Kruskal-Wallis and Mann-Whitney U, n = 3; *P < 0.05. Black: ipsilateral hemisphere, Orange: Contralateral hemisphere. (C) Adult naïve wild-type (WT) and GYS1 Nestin-KO mice are sacrificed, and brain sections are labeled with Lycopersicon Esculentum Lectin (upper panel-green) and CD13 (middle panel-red and merged as lower panel). GYS1 Nestin-KO mice demonstrate higher number of microvascular constrictions as shown by arrows. Images represent 3D reconstruction of 40-μm z-stack. Scale bars, 10 μm. (D) CD13+ pericyte mediated microvascular constrictions in adult naïve wild-type (WT) and GYS1 Nestin-KO mice are quantified. Transgenic mice have higher number of constrictions. Quantification of CD13+ pericyte mediated microvascular constrictions in ten fields for each hemisphere spanning MCA territory (240 μm × 160 μm) per animal (n = 3 animals per group). Mann-Whitney U, n = 3; *P < 0.05.

Article Snippet: The sections were blocked in 1% bovine serum albumin containing 0.3% Triton-X, 10% normal goat serum (NGS) (0.3% TBS-T / 1% BSA, 10% NGS) solution for 1 hour at room temperature and then incubated with primary antibodies (anti-glycogen antibodies ESG1A9 and IV58B6 (courtesy of Dr. Hitoshi Ashida and Dr. Otto Baba), CD13 (Acris Antibodies GmBH, AM26636AF-N) for pericytes) in 0.3% TBS-T overnight at + 4°C.

Techniques: MANN-WHITNEY, Labeling, Transgenic Assay

Journal: bioRxiv

Article Title: Disrupted Cerebral Peri-Microvascular Glycogen Promotes Capillary Constrictions and Aggravates Ischemia in Mice

doi: 10.1101/2022.08.24.505172

Figure Lengend Snippet: Permanent MCAo experiments performed in Saline, DAB injected WT, naïve WT and GYS1 Nestin-KO mice (n = 3 animals per group). (A) Representative images taken from Cresyl-violet stained sections of ischemic mice via phase contrast microscopy (as indicated in figure) that underwent 2-hour MCAo. Red dots are placed over the border between core and peri-infarct areas. Scale bars, 500 μm. (B) Infarct volumes after 2-hour MCAo are measured then quantified with volume correction (Swanson, JCBFM 1990). Ischemic infarct volumes after MCAo were significantly higher in DAB treated mice than saline treated groups (Two-way analysis of Mann-Whitney U, n = 3; *P < 0.05). Infarct volumes of GYS1 Nestin-KO group were also higher when compared to wild type (Two-way analysis of Mann-Whitney U, n = 3; *P < 0.05). (C) Quantification of microvascular constrictions after MCAo in Saline, DAB injected WT, naïve WT and GYS1 Nestin-KO mice (n = 3 animals per group). Quantification of CD13+ pericyte mediated microvascular constrictions are held in ten fields fields for each hemisphere spanning MCA territory (240 μm × 160 μm) per animal. Ischemia further resulted in higher number of microvascular constrictions in peri-microvascular glycogen disrupted mice (DAB injected and GYS1 Nestin-KO ) compared to ischemic controls (Two-way analysis of Mann-Whitney U, n = 3; *P < 0.05). Black: ipsilateral hemisphere, Orange: Contralateral hemisphere. (D) Lycopersicon Esculentum Lectin (upper panel-green) and CD13 (middle panel-red) double labelling (merged as lower panel) reveals increased microvascular constrictions (arrows) 2 hours after MCAo in Saline, DAB injected WT, naïve WT and GYS1 Nestin-KO mice (left to right, respectively). Images represent 3D reconstruction of 40-μm z-stack. Scale bars, 10 μm. (E) Lycopersicon Esculentum Lectin (left panel-green) and CD13 (middle panel-red) double labelling (merged as right panel) in i.c.v DAB+ L/D-lactate injected mice. Double labelling shows that L-lactate (upper panel) reverses the DAB’s impact on CD13+ pericyte mediated constrictions contrary to its enantiomer D-Lactate (lower panel). Images represent 3D reconstruction of 40-μm z-stack. Scale bars, 10 μm. (F) Quantification of CD13+ pericyte mediated microvascular constrictions in i.c.v DAB+ L/D-lactate injected mice compared to controls (n = 3 animals per group). L-lactate can reverse the DAB-induced constrictions; however, D-lactate demonstrates similar number of microvascular constrictions with DAB (Two-way analysis of Mann-Whitney U, n = 3; *P < 0.05). Black: ipsilateral hemisphere, Brown: Contralateral hemisphere.

Article Snippet: The sections were blocked in 1% bovine serum albumin containing 0.3% Triton-X, 10% normal goat serum (NGS) (0.3% TBS-T / 1% BSA, 10% NGS) solution for 1 hour at room temperature and then incubated with primary antibodies (anti-glycogen antibodies ESG1A9 and IV58B6 (courtesy of Dr. Hitoshi Ashida and Dr. Otto Baba), CD13 (Acris Antibodies GmBH, AM26636AF-N) for pericytes) in 0.3% TBS-T overnight at + 4°C.

Techniques: Injection, Staining, Microscopy, MANN-WHITNEY

Journal: JCI Insight

Article Title: Brd4 modulates diet-induced obesity via PPAR γ -dependent Gdf3 expression in adipose tissue macrophages

doi: 10.1172/jci.insight.143379

Figure Lengend Snippet: ( A ) PCA of RNA-Seq data derived from CD11b + ATMs of WT and Brd4 -CKO mice fed a HFD for 20 weeks. ( B ) Volcano plot of mRNA-Seq analysis in ATMs as indicated in A . Brown dots represent genes increased in ATMs of Brd4 -CKO mice vs. WT mice (fold change ≥ 2, adjusted P value ≤ 0.001, calculated by raw count value). Blue dots represent genes decreased in ATMs of Brd4 -CKO mice vs. WT mice (fold change ≥ 2, adjusted P value ≤ 0.001, calculated by raw count value). Gray dots represent genes without significantly altered expression. Clusters of significantly altered genes (fold change ≥ 2, adjusted P value ≤ 0.001, calculated by raw count value) were identified using gene ontology terms ( C ) and KEGG pathways ( D ). ( E ) Heat map of the relative expression levels (scaled Z -score) of cytokine-cytokine receptor interaction-related genes clustered in ( D ). ( F ) mRNA levels of Gdf3 in CD11b + ATMs isolated from WT or Brd4 -CKO mice fed a HFD for 20 weeks. ( G ) Left panel: Gdf3 or F4/80 IHC staining of eWAT of WT or Brd4-CKO mice fed a HFD for 20 weeks. Right panel: statistical analysis of Gdf3-positive or F4/80-positive area percentage, the ratio of Gdf3-positive cells in F4/80-positive cells in eWAT of WT and Brd4 -CKO mice fed a HFD. Data are mean and SD and are determined by an unpaired 2-tailed Student’s t test. n = 3 mice. ** P < 0.01. Brd4 -CKO, myeloid lineage-specific Brd4 knockout; HFD, high-fat diet–induced; eWAT, epididymal WAT; ATMs, adipose tissue macrophages; PCA, principal component analysis.

Article Snippet: Four-μm continuous sections were prepared for immunostaining with Gdf3 antibody (AF958, R&D Systems) or F4/80 antibody (BM4008, Origene).

Techniques: RNA Sequencing Assay, Derivative Assay, Expressing, Isolation, Immunohistochemistry, Knock-Out

Journal: Journal of Neuroinflammation

Article Title: Lack of macrophage migration inhibitory factor in mice does not affect hallmarks of the inflammatory/immune response during the first week after stroke

doi: 10.1186/1742-2094-8-75

Figure Lengend Snippet: Primary antibodies

Article Snippet: Rat anti-Mac2, biotinylated , Acris Antibodies , CL049B , 1:500.

Techniques: Transduction

Journal:

Article Title: Herpes Simplex Virus UL12.5 Targets Mitochondria through a Mitochondrial Localization Sequence Proximal to the N Terminus ▿ §

doi: 10.1128/JVI.02087-08

Figure Lengend Snippet: Residues downstream of M185 are crucial for mitochondrial localization of UL12.5. (A) UL12.5-eGFP colocalizes with mitochondria in transfected cells. HeLa cells transfected with a UL12.5-eGFP expression plasmid were visualized by fluorescence microscopy after staining with MitoTracker Red. (B) HeLa cells transfected with plasmids encoding eGFP or the indicated UL12.5-eGFP fusion proteins were examined by live cell imaging as described for panel A. UL12.5-R → A-eGFP: UL12.5-eGFP bearing R → A substitutions at residues 188, 192, 196, 199, and 200. Bars = 10 μm.

Article Snippet: Plasmids containing UL12.5 sequences M185 to L214, M215 to R245, and M185 to R245 fused to the N terminus of eGFP in the pEGFP-C1 vector (Clontech) were constructed as follows.

Techniques: Transfection, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Staining, Live Cell Imaging