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: Saline, 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: Saline, Injection, Staining, Microscopy, MANN-WHITNEY

Journal: Molecular medicine reports

Article Title: Hypoxia-induced increases in A549/CDDP cell drug resistance are reversed by RNA interference of HIF-1α expression.

doi: 10.3892/mmr.2011.604

Figure Lengend Snippet: Figure 2. The effects of hypoxia and HIF-1α siRNA on the expression of MDR1 and MRP mRNAs in A549/CDDP cells. (A and C) The expression of MDR1 and MRP mRNA was measured by RT-PCR. M, DNA bp ladder; lane 1, control group; lane 2, hypoxia-treated group; lane 3, hypoxia- and control siRNA‑treated group; lane 4, hypoxia- and HIF-1α siRNA-treated-group. (B and D) Quantification of results shown in (A and C). MDR1, multidrug resis tance-1; MRP, multidrug resistance-associated protein; HIF-1α, hypoxia-inducible factor 1α; siRNA, small interfering RNA; RT-PCR, reverse transcription polymerase chain reaction.

Article Snippet: Antibodies to HIF-1α, MDR1 and MRP were purchased from Beijing Zhongshan Golden Bridge Biotechnology Company (China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Small Interfering RNA, Reverse Transcription, Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: Hypoxia-induced increases in A549/CDDP cell drug resistance are reversed by RNA interference of HIF-1α expression.

doi: 10.3892/mmr.2011.604

Figure Lengend Snippet: Figure 5. The effects of hypoxia and HIF-1α siRNA on expression of MRP protein in A549/CDDP cells. (A-D) The expression of MRP protein was ana lyzed by immunocytochemistry. (A) Control group; (B) hypoxia-treated group; (C) hypoxia- and control siRNA-treated group; (D) hypoxia- and HIF-1α siRNA‑treated group. (E) Grading of positive protein staining in all 4 groups. HIF-1α, hypoxia-inducible factor 1α; siRNA, small interfering RNA; MRP, multidrug resistance‑associated protein.

Article Snippet: Antibodies to HIF-1α, MDR1 and MRP were purchased from Beijing Zhongshan Golden Bridge Biotechnology Company (China).

Techniques: Expressing, Immunocytochemistry, Control, Staining, Small Interfering RNA