Endothelial cell Pannexin1 overexpression impairs ischemic stroke outcome in a sex-dependent manner

Figures

• 

  Figure 1.. Panx1 is overexpressed successfully in ECs of cerebral arteries of both female and male mice. (A) Cartoon of the inserted human isoform of Panx1 (hPanx1) with a FLAG tag in the ROSA 26 locus to induce conditional overexpression of Panx1. (B) PCA from Cre− R26-hPanx1Tg control mice (EC Panx1 Intact) and CreERTT2+ R26-hPanx1Tg (EC Panx1 OvExp) were stained for hPanx1 using the anti-Flag tag antibody (red). Internal elastic lamina is identified by its autofluorescence (green) and nuclei were identified using DAPI (blue). Western blot (C) and quantification (D) for Panx1 protein expression levels from ECs isolated from the brain of female and male Cre− R26-hPanx1Tg control mice (EC Panx1 Intact) and CreERTT2+ R26-hPanx1Tg mice (EC Panx1 OvExp). B-Tubulin (red) was used to normalize Panx1 expression (green) for quantification. Quantification of Panx1 protein expression is relative to levels in control (EC Panx1 Intact) female mice. EC Panx1 Intact: N = 9 females and 6 males. EC Panx1 OvExp: N = 8 females and 7 males. Two-way ANOVA with Tukey’s multiple comparisons test. *p<0.05.

• 

  Figure 2.. EC Panx1 levels do not impact peripheral vascular myogenic tone, systemic blood pressure and heart rate. Pressure myography was used to measure percent myogenic tone (A-B), active diameter (C-D), and passive diameter (E-F) of isolated third-order mesenteric arteries from female and male EC Panx1 overexpressing and control mice. EC Panx1 Intact: N = 6 females and 7 males. EC Panx1 OvExp: N = 6 females and 5 males. Two-way ANOVA with repeated measures and Sidak’s multiple comparisons post hoc test. Radiotelemetry was used to collect average mean arterial pressure (MAP) and heart rate from female (G) and male (H) EC Panx1 overexpressing and control mice. EC Panx1 Intact: N = 5 females and 6 males. EC Panx1 OvExp: N = 6 females and 6 males. Student’s T test. *p<0.05.

• 

  Figure 3.. EC Panx1 overexpression increases myogenic tone in cerebral arteries only in female mice. Pressure myography was used to measure percent myogenic tone, active diameter, and passive diameter of isolated PCAs from female and male EC Panx1 overexpressing and control mice. EC Panx1 Intact: N = 9 females and 9 males. EC Panx1 OvExp: N = 11 females and 8 males. Two-way ANOVA/Sidak’s multiple comparisons post hoc test. *p<0.05; **p<0.01.

• 

  Figure 4.. Overexpression of EC Panx1 significantly increased infarct volume and impaired cerebral blood flow recovery 24h after ischemic stroke in females. 45-minute MCA occlusions were performed on EC Panx1 overexpressing and control mice, which were confirmed with reduced CBF, measured by LSCI, at the end of the MCA occlusion in both sexes. (A and C). Infarct volumes were quantified using TTC staining in females (B and E) and males (D and F). CBF was measured using LSCI 24 hr prior to MCAO (baseline), at the end of the MCA occlusion (MCAO), and 24 hr post-MCAO/reperfusion in females (G) and males (H). EC Panx1 Intact: N = 8 females and 8 males. EC Panx1 OvExp: N = 7 females and 9 males. Student’s T test (A-D) and Two-way ANOVA/Sidak’s multiple comparisons post hoc test (G and H). *p<0.05; **p<0.01; ****p<0.0001.