Additional file 1 of Delayed NLRP3 inflammasome inhibition ameliorates subacute stroke progression in mice
Creators: name: Bellut, Maximilian
name: Bieber, Michael
name: Kraft, Peter
name: Weber, Alexander N. R.
name: Stoll, Guido
name: Schuhmann, Michael K.
Publisher: name: figshare
Year: 2024
Subjects: subject: Medicine
subject: Genetics
subject: FOS: Biological sciences
subject: Neuroscience
subject: Physiology
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine

description: Additional file 1: Table S1. Primary antibody information. Table S2. Secondary antibody information. Figure S1. Survival Curve of the two vehicle groups. Before merging the two vehicle groups, the survival of the vehicle groups with prophylactic treatment (d0) and therapeutic treatment after 24 h of reperfusion (d1) was compared. A log-rank test was performed to assess whether significant differences exist between the two study groups. Results show that survival distributions of the two application times did not differ significantly, χ² = 0.02, p ~ 0.9. Figure S2. MAP2 staining to depict neuronal ischemic damage. NLRP3 inhibition reduces neuronal ischemic damage after prophylactic and delayed treatment 24h post stroke onset. (A) Infarct size comparison measured in percentage of the respective hemisphere (n ≥ 7) and (B) representative MAP2 stainings of vehicle-, MCC950 d0- and MCC950 d1-treated mice euthanized 7 days after tMCAO. Additionally, MAP2 staining was performed 1 day after tMCAO of vehicle treated animals to visualize infarct progression between days 1 and 7. Scale bar = 2 mm. The infarcts are circled by a blue line. Data was analyzed by 1-way ANOVA with post hoc Tukey adjustment for p values. *p &lt; 0.05; ***p &lt; 0.001. Figure S3. Cytokine gene expression downstream of NLRP3. Ipsilesional Caspase 1 and IL1b/18 gene expression levels are reduced by NLRP3 inhibition. Relative gene expression as detected by rtPCR of Caspase 1, interleukin-1β (IL1β) and interleukin-18 (IL18) (top) in the ischemic cortices and (bottom) basal ganglia of mice 7 d after 30 min transient middle cerebral artery occlusion (tMCAO) treated with either vehicle or MCC950 prophylactically (MCC d0)/ therapeutically (MCC950 d1) (n ≥ 5). Figure S4. Histological NLRP3-positivity of infiltrating immune cells. NLRP3 Co-staining of immune cells (A) Left: Percentage of NLRP3-positive cells within all CD4-positive cells of vehicle-, MCC950 d0- and MCC950 d1-treated mice within 5 ipsilesional priorly defined segments as in Figure 4. Right: Representative immunocytologic stainings (as depicted in red) of CD4-positive (red) as well as NLRP3-positive lymphocytes (green) and nuclei (DAPI, blue) in the ipsilateral hemisphere on day 7 after tMCAO in vehicle-, MCC950 d0- and MCC950 d1-treated mice using 20x objective. Scale bar = 75 μm (n ≥ 7). (B) Left: Percentage of NLRP3-positive cells within all Iba1-positive cells of vehicle-, MCC950 d0- and MCC950 d1-treated mice within 5 ipsilesional priorly defined segments as in Figure 4. Right: Representative immunocytologic stainings (as depicted in red) of Iba1-positive (red) as well as NLRP3-positive microglia (green) and nuclei (DAPI, blue) in the ipsilateral hemisphere on day 7 after tMCAO in vehicle-, MCC950 d0- and MCC950 d1-treated mice using 20x objective. Scale bar = 75 μm (n ≥ 7). (C) Left: Percentage of NLRP3-positive cells within all Ly.6g-positive cells of vehicle-, MCC950 d0- and MCC950 d1-treated mice within 5 priorly defined segments as in Figure 4. Right: Representative immunocytologic stainings (as depicted red) of Ly.6g-positive (red) as well as NLRP3-positive neutrophils (green) and nuclei (DAPI, blue) in the ipsilateral hemisphere on day 7 after tMCAO in vehicle-, MCC950 d0- and MCC950 d1-treated mice using 10x objective. Scale bar = 100 μm (n ≥ 7). Data was analyzed by 1-way ANOVA with post hoc Tukey adjustment for p values. *p &lt; 0.05, **p &lt; 0.01; ***p &lt; 0.001. Figure S5. Iba1 Western Blot. Microglia Western Blot. Iba1 protein content in cortex or basal ganglia of vehicle- and sham-treated mice after 1 d and 7 d of reperfusion. For densitometric quantification actin was used as a loading control. (n ≥ 5). Data was analyzed by 1-way ANOVA with post hoc Tukey adjustment for p values. *p &lt; 0.05, **p &lt; 0.01. Figure S6. Negative control stainings. Figure S4: Negative controls of antibodies used for immunohistochemistry. Positive (first and second antibody) and negative (second antibody only) staining controls for (A) CD4 stainings, (B) Iba1 stainings, (C) Ly.6g stainings derived from spleen and (D) GFAP stainings derived from brain. 20 x magnification. (A-B) Scale bar = 50 µm, (B-C) Scale bar = 100 µm.