Additional file 1 of Synergistic effect of sildenafil combined with controlled hypothermia to alleviate microglial activation after neonatal hypoxia–ischemia in rats
Creators: name: Julien, Pansiot
name: Zinni, Manuela
name: Bonnel, Natacha
name: El Kamouh, Marina
name: Odorcyk, Felipe
name: Peters, Lea
name: Gautier, Emilie-Fleur
name: Leduc, Marjorie
name: Broussard, Cédric
name: Baud, Olivier
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer

description: Additional file 1: Figure S1. Segmentation of the brain slices according to Paxinos and Watson Atlas (1998)* used for immunohistochemistry. Quantifications were performed on a section nearby the maximal lesion size around Bregman -3.36mm. * Paxinos G and Watson C. 1998. The rat brain in stereotaxic coordinates, 4th ed. San Diego, CA, USA; Academic press. Figure S2. Quantification of cell-specific staining within segmented brain area, from a standardized threshold. Imaging processing for assessing immunostaining using the Fiji software in total hemisphere and four delimited brain regions: hippocampus, S1 cortex, perirhinal cortex and thalamus. Quantifications were performed after images were converted into black and white 8-bit signal and according to a predefined standardized threshold. Figure S3. Assessment of cell purity after magnetic antibody-based cell sorting. A: CD11b/c positive cells showed a large majority of Cx3cr1positive cells and less than 1% astrocytic contamination. B: ACSA-1 positive cells showed a large proportion of GFAP positive astrocytes and less than 5% contamination from oligodendroglial lineage. RDC means recurrent DNA double-strand break clusters observed in neural stem/progenitor cells. Figure S4. Body weight gain between P10 and P13 rats subjected to HI at P11 and kept either on normothermia (HI, n = 78) compared to sham (n = 26), or treated by hypothermia alone (HT, n = 41), Sildenafil ip alone (Sild, n = 48), and the combined treatment (Sild + HT, n = 46). Body weight changes for each day compared to P10 are expressed mean ± SD. Weight gain in Sham group (controls) was compared in each experimental groups using one-way ANOVA with Dunnet’s multiple comparisons test (***: p &lt; 0.001; ****: p &lt; 0.0001). Figure S5. Gene expression polarization in Iba1-positive MG/Mf cells sorted from the ipsilateral hemisphere of P13 rats subjected to HI at P11 and kept either on normothermia (HI, n = 41) compared to sham (n = 12), or treated by hypothermia alone (HT, n = 19), Sildenafil ip alone (Sild, n = 20), and the combined treatment (Sild + HT, n = 18). Detailed gene expression of several pro-inflammatory (A) and immunoregulatory/anti-inflammatory markers (B). Quantified results are mean ± SD. Sham vs untreated (NT) HI animals were first compared using a non-parametric Mann Whitney t-test (*: p &lt; 0.05; **: p &lt; 0.01; ****: p &lt; 0.0001). Then, each treatment was compared to untreated animals (HI) using a Kruskal–Wallis test followed by a Dunn’s multiple comparison test when appropriate (#: p &lt; 0.05; ###: p &lt; 0.001; ####: p &lt; 0.0001). Figure S6. Gene expression polarization in Iba1-positive MG/Mf cells sorted from the contralateral hemisphere of P13 rats subjected to HI at P11 and kept either on normothermia (HI, n = 41) compared to sham (n = 12), or treated by hypothermia alone (HT, n = 19), Sildenafil ip alone (Sild, n = 20), and the combined treatment (Sild + HT, n = 18). Detailed gene expression of several pro-inflammatory (A) and immunoregulatory/anti-inflammatory markers (B). Quantified results are mean ± SD. Sham vs untreated (NT) HI animals were first compared using a non-parametric Mann Whitney t-test (*: p &lt; 0.05). Then, each treatment was compared to untreated animals (HI) using a Kruskal–Wallis test followed by a Dunn’s multiple comparison test when appropriate (#: p &lt; 0.05; ###: p &lt; 0.001; ####: p &lt; 0.0001). Figure S7. Gene expression polarization in GFAP-positive astrocytes sorted from the ipsilateral hemisphere of P13 rats subjected to HI at P11 and kept either on normothermia (HI, n = 41) compared to sham (n = 12), or treated by hypothermia alone (HT, n = 19), Sildenafil ip alone (Sild, n = 20), and the combined treatment (Sild + HT, n = 18). Detailed gene expression of A1 (A) and A2 astrocytic markers (B). Quantified results are mean ± SD. Sham vs untreated (NT) HI animals were first compared using a non-parametric Mann Whitney t-test (*: p &lt; 0.05; **: p &lt; 0.01; ***: p &lt; 0.001). Then, each treatment was compared to untreated animals (HI) using a Kruskal–Wallis test followed by a Dunn’s multiple comparison test when appropriate (###: p &lt; 0.001). Figure S8. Gene expression polarization in GFAP-positive astrocytes sorted from the contralateral hemisphere of P13 rats subjected to HI at P11 and kept either on normothermia (HI, n = 41) compared to sham (n = 12), or treated by hypothermia alone (HT, n = 19), Sildenafil ip alone (Sild, n = 20), and the combined treatment (Sild + HT, n = 18). Detailed gene expression of A1 (A) and A2 astrocytic markers (B). Quantified results are mean ± SD. Sham vs untreated (NT) HI animals were first compared using a non-parametric Mann Whitney t-test (**: p &lt; 0.01). Then, each treatment was compared to untreated animals (HI) using a Kruskal–Wallis test followed by a Dunn’s multiple comparison test (all comparisons are not significant). Figure S9. Over-representation analysis (ORA) using GeneOntology/biological process non redundant of the proteome of MG/Mf cells sorted from the ipsilateral hemisphere of P13 rats subjected to HI at P11 without and with neuroprotective treatment Sild + HT. This ORA analysis was performed to identify the enriched genesets inversely regulated by HI and in HI-injured animals treated by Sild + HT. GO pathways defined by logFC &gt; 1,3, p-value &lt; 0.05 and FDR &lt; 0.25 were considered significantly up or downregulated. Four genesets were identified as inversely deregulated (highlighted in yellow). Figure S10. Neutrophilic infiltration in the lesion site. Quantification of MPO + cells within S1 cortical brain area, and typical pictures of labelled cells in Sham and HI animals (A). Data are expressed as mean ± SD. Sham vs untreated (NT) HI animals were compared using a non-parametric Mann Whitney t-test (*: p &lt; 0.05). Linear regression between lesion size and MPO + cells density in each animals (B). Table S1: Primers sequences used for RT-qPCR. Table S2. Analyses of main endpoints according to sex and interaction between sex and each variable analyzed. Quantified results are mean ± SD or SEM. Sham vs untreated (NT) HI animals were first compared using a non-parametric Mann Whitney t-test (*: p &lt; 0.05; **: p &lt; 0.01; ***: p &lt; 0.001; ****: p &lt; 0.0001). Then, each treatment was compared to untreated animals (HI) using a Kruskal–Wallis test followed by a Dunn’s multiple comparison test when appropriate (#: p &lt; 0.05; ##:p &lt; 0.01; ###: p &lt; 0.001). Two-way ANOVA was used to assess interaction between sex and treatment. Table S3. ORA analyses looking for deregulated biological processes in the HI group compared to the Sham group, depending on various the statistical thresholds. Table S4. ORA analyses looking for deregulated biological processes in the HI + hypothermia group (HT) compared to the HI untreated group, depending on various the statistical thresholds. Table S5. ORA analyses looking for deregulated biological processes in the HI + Sildenafil group (Sild) compared to the HI untreated group, depending on various the statistical thresholds. Table S6. ORA analyses looking for deregulated biological processes in the HI + Hypothermia + Sildenafil group (SildHT) compared to the HI untreated group, depending on various the statistical thresholds. Table S7. GSEA analysis against reactome pathways looking for deregulated biological processes in the HI untreated group compared to the Sham group. Table S8. GSEA analysis against reactome pathways looking for deregulated biological processes in the HI + Hypothermia group compared to the HI untreated group. Table S9. GSEA analysis against reactome pathways looking for deregulated biological processes in the HI + Sildenafil group compared to the HI untreated group. Table S10. GSEA analysis against reactome pathways looking for deregulated biological processes in the HI + Hypothermia + Sildenafil group compared to the HI untreated group.