Article Figures & Data
- Figure 1
Pertussis toxin (PT) increases stroke severity in intracerebral haemorrhage (ICH) mice. ICH was induced by injection of 0.0375 U collagenase. After surgeries, mice were treated with PT immediately by intraperitoneal injection at a dose of 250 ng or 500 ng or vehicle. Mice were subjected to neurological assessment and MRI scanning until day 7 after ICH. (A, B) Cumulative data illustrate the neurological assessments of ICH mice and sham mice receiving PT or vehicle from day 1 to day 7 after surgery, including modified Neurological Severity Score (mNSS) (A) and rota-rod test (B). n=8 mice per group from three independent experiments. Data were expressed as mean±SEM; *p<0.05; **p<0.01. (C) Representative MR images show lesion area and haematoma area in ICH mice receiving PT or vehicle. Red lines delineate lesion area, yellow shadows represent haematoma area and perihaematomal oedema volume was calculated by subtracting the haematoma volume from lesion volume. (D) Cumulative data show the lesion volume and perihaematomal oedema volume of ICH mice receiving PT or vehicle. n=4 mice per group from two independent experiments. Data were expressed as mean±SEM; *p<0.05.
- Figure 2
Pertussis toxin (PT) enhances brain inflammation after intracerebral haemorrhage (ICH). ICH in mice was induced using 0.0375 U collagenase injection. PT treatment was given immediately after ICH by intraperitoneal injection, at a dose of 500 ng. (A, B) At day 1, day 3 and day 7 after ICH, representative bioluminescence images and quantification analysis show reactive oxygen species generation in sham and ICH mice receiving PT or vehicle. n=3 mice per group from two independent experiments. (C) Brain tissues were obtained from ICH mice receiving PT or vehicle. Sham-operated mice receiving PT or vehicle were used as control. Brain homogenates were analysed by a Mouse XL Cytokines Array kit. Heat map and cluster analysis show the expression of inflammatory factors in brain homogenates from sham and ICH mice with indicated treatments. A heat map was generated and the relative pixel intensity of spots signal is indicated by the representative colour code (red, upregulated; blue, downregulated). (D) Bar graphs show the top significantly dysregulated factors. n=6 mice per group. The data were calculated as mean±SEM; *p<0.05; **p<0.01.
- Figure 3
Pertussis toxin (PT) promotes brain-infiltrating leucocytes after intracerebral haemorrhage (ICH). ICH was induced by 0.0375 U collagenase injection. At day 1, day 3 or day 7 after ICH, single cell suspensions were isolated from brain tissues of ICH mice treated with PT or vehicle. (A) Gating strategy of brain-infiltrating immune cells including neutrophils (CD45highCD11b+Ly6G+), CD4+ T cells (CD45highCD3+CD4+), CD8+ T cells (CD45highCD3+CD8+), B cells (CD45highCD3−CD19+), and NK cells (CD45highCD3−NK1.1+). (B) Quantification of lymphocytes, monocytes and neutrophils in the brain of sham and ICH mice receiving PT or vehicle at indicated time points. Day 1 after ICH, n=3 mice per group from two independent experiments; day 3 and day 7 after ICH, n=5 mice per group from three independent experiments. The data were calculated as mean±SEM; *p<0.05.
- Figure 4
Pertussis toxin (PT) increases the permeability of blood–brain barrier after intracerebral haemorrhage (ICH). ICH was induced by 0.0375 U collagenase injection. (A) Representative dorsal surfaces and quantification analysis show the Evans Blue leakage of brain at day 3 after surgeries. n=6 mice per group from three independent experiments. (B, C) Representative immunostaining for CD31 (green) and claudin-5 (red) and MMP-9 (red) in ipsilateral hemisphere from ICH and sham mice treated with 500 ng PT or vehicle at day 3 after onset. White lines delineate lesion area. Scale bar: 50 µm, insert: 25 µm. (D, E) Quantification of claudin-5 and MMP-9 coverage in sham mice and peri-lesion area of ICH mice receiving PT or vehicle at the indicated time. n=9 sections from 3 mice each group. Data were expressed as mean±SEM; *p<0.05; **p<0.01.
- Figure 5
Pertussis toxin (PT) exacerbates neurological deficits and brain infarction after cerebral ischaemia and reperfusion. Mice were treated with 500 ng PT or vehicle immediately by intraperitoneal injection after 60 min of middle cerebral artery occlusion (MCAO) and reperfusion. Neurological deficits, lesion volume and the cerebral blood flow (CBF) were evaluated in MCAO mice. (A) Summarised results show the modified Neurological Severity Scores and the rota-rod test latency of MCAO mice and sham mice receiving PT or vehicle at the indicated times. n=6 mice per group from three independent experiments. (B) Representative MR images show infarct area (outlined in red) in MCAO mice and sham mice receiving PT or vehicle. Bar graphs show the infarct lesion at day 3 after ischaemia/reperfusion. n=4 mice per group. (C) Images of CBF in MCAO mice that received PT or vehicle treatment. Quantification of reduced blood flow in the ipsilateral hemisphere. The data were calculated as mean±SEM; *p<0.05, **p<0.01.
- Figure 6
Pertussis toxin (PT) augments brain inflammation after brain ischaemia and reperfusion. (A, B) Visualisation of reactive oxygen species generation, in vivo bioluminescence imaging and quantification of signal strength in sham and middle cerebral artery occlusion (MCAO) mice given PT or vehicle at day 3 after ischaemia and reperfusion. n=3 mice per group from two independent experiments. (C) Counts of central nervous system–infiltrating immune cell subsets were measured using flow cytometry on day 3 after reperfusion. Representative flow cytometry plots show the gating strategy of leucocyte subpopulations isolated from the brain. (D) Summarised results show the cell counts of the indicated subsets in the brain of sham and MCAO mice receiving PT or vehicle. n=4 mice per group. The data were calculated as mean±SEM; *p<0.05, **p<0.01.
- Figure 7
Pertussis toxin (PT) enhances the permeability of blood–brain barrier after ischaemia. (A) Photographs of the dorsal surfaces of brain display Evans Blue extravasation at day 3 after ischaemia and reperfusion, and quantification of Evans Blue dye leakage at day 3 after surgery in the indicated groups. (B, C) Representative images show staining of CD31 (green) and claudin-5 (red) and MMP-9 (red) in brain sections from middle cerebral artery occlusion (MCAO) and sham mice at day 3 after ischaemia and reperfusion. Infarct areas are outlined in white dotted line. Scale bar: 50 µm, insert: 25 µm. (D, E) Summarised results show the claudin-5 and MMP-9 positive areas covered to the total endothelial surface area (CD31) in the sham mice and peri-infarct of MCAO mice at day 3 after MCAO. n=3 mice in each group from three independent experiments. The data were calculated as mean±SEM; **p<0.01.
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