Additional file 1 of Blood tissue Plasminogen Activator (tPA) of liver origin contributes to neurovascular coupling involving brain endothelial N-Methyl-D-Aspartate (NMDA) receptors
Creators: name: Furon, Jonathane
name: Yetim, Mervé
name: Pouettre, Elsa
name: Martinez de Lizarrondo, Sara
name: Maubert, Eric
name: Hommet, Yannick
name: Lebouvier, Laurent
name: Zheng, Ze
name: Ali, Carine
name: Vivien, Denis
Publisher: name: figshare
Year: 2024
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: Chemical Sciences not elsewhere classified
subject: Science Policy

description: Additional file 1: Figure S1. Recording of physiological parameters during whisker stimulations. Mice were mechanically ventilated (120 BPM, 10 ml/kg). Blood samples were collected by a femoral catheter and mean arterial pressure (MAP) was measured. Pharmacological treatments were injected via a tail vein catheter. Data are mean with SD, n=8. Figure S2. Parabiosis model and chimerism validation methods. A: Schematic representation of experimental study design for parabiosis surgery and chimerism validation. B-D: Parabiosis chimerism validation by IV injection of glucose (100µl, 1.2g/kg) at T0 in tail vein of donor mice, glycemia (mg/dl) of both donor and recipient mice was measured during 1 hour, at different time points following parabiosis surgery (D5, n=5, figure B; D15, n=7, figure C; D22, n=7, figure D). E: Representative MRI acquisition of parabionts after IV DOTA-gd injection (200µl, 0.01mMol/ml) in donor mice to validate parabiosis model. T1-weighted image (red) represents DOTA- Gd signal and was superposed on anatomical T2-weighted image. F: Quantification of DOTA-Gd distribution between parabionts at different time points. Data are mean with SD for each group. *p &lt; 0.05, **p &lt; 0.005 and ***p &lt; 0.001 from T0, 2-way ANOVA test. Figure S3. Mean arterial blood pressure and blood gases before and after injection of B2Rag. A: Mean arterial pressure (MAP, mmHg) before (-B2Rag) and after IV injection of 30 µg/kg of B2Rag (+B2Rag). Data are Mean with values for each mouse. B-C: Measurement of arterial blood gas before (-B2Rag, C) and after IV injection of 30 µg/kg of B2Rag (+B2Rag, D). pCO2, pO2, O2sat and pH were measured with 100µl of blood on RAPIDLab® 348EX Blood Gas System. Data are mean with values for each mouse and reference range for these measurements. *p &lt; 0.05 from - B2Rag, Mann-Whitney test, n=4 per group. Figure S4. Partial hepatectomy does not influence arterial blood pressure. Mean arterial pressure (MAP, mmHg) before and 24h after hepatectomy in tPAWT mice. Data are Mean with values for each mouse. Mann-Whitney test, n=5 per group. Figure S5. Mean arterial blood pressure and blood gases before and after injection of Ang-II in mice. A: Mean arterial pressure (MAP, mmHg) before (-Ang-II) and 25 minutes after IV infusion of 1 µg/kg/min of Ang-II (+Ang-II). Data are Mean with values for each mouse. B-C: Measurement of arterial blood gas before (-Ang-II, C) and 25 minutes after IV infusion of 1 µg/kg/min of Ang-II (+Ang-II, D). pCO2, pO2, O2sat and pH were measured with 100µl of blood on RAPIDLab® 348EX Blood Gas System. Data are mean with values for each mouse and reference range for these measurements. *p &lt; 0.05 from -Ang-II, Mann-Whitney test, n=4 per group. Figure S6. Intravenous administration of angiotensin-II (Ang-II) does not influence NVC in a tPA dependent manner. Figure S7. Hydrodynamic transfection of pLIVE-Cre-GFP in VECad-CreΔtPA mice. A: Construction scheme of plasmid pLIVE-Cre-GFP. The cDNA of Cre-GFP was PCR amplified from a pCAG-Cre-GFP plasmid and fused into multiple cloning sites of the pLIVE plasmid between BamHI and NotI. B: Experimental schematic representation of hydrodynamic transfection of empty-pLIVE or pLIVE-Cre-GFP. 48h after transfection of the plasmid, livers from transfected VECad-CreΔtPA mice were collected. C: Epifluorescence images of liver slices from transfected VECad-CreΔtPA mice with empty-pLIVE or pLIVE- Cre-GFP (scale bar = 500µm). Immunostaining reveals GFP (green), Cre-recombinase (red) and actin filaments with phalloidin (grey), confirming the transfection of the plasmid in hepatocytes. Images were digitally captured using a camera (CoolSNAP; Photometrics) and/or with an inverted confocal microscope (SP5, Leica). Images werevisualized respectively with Metavue 5.0 software (Molecular Devices, USA) and LAS AF lite software (LEICA). Figure S8. Deletion of tPA in hepatocytes does not influence NVC in VECad-CreΔtPA mice. Figure S9. tPA expression in wild type (tPAWT) and tPA deficient mice (tPANull). A: Representative photomicrographs (n=5) of hippocampal tissue sections subjected to immunostainings raised against tPA (red), CD31 (green) and DAPI staining (blue); scale bar 100µm and 50µm in the magnified inserts. B: Quantification of tPA fluorescent intensity in hippocampus of tPAWT and tPANull mice. C: Representative photomicrographs of hippocampal tissue sections with plat (gene encoding for tPA; green) revealed by in situ hybridization (ISH), immunostaining raised against CD31 (red) and DAPI (blue) staining; scale bar 100µm. D: Quantifications of plat positive cells in the hippocampus of tPAWT (N=1; n=30) and tPANull mice (N=1; n=30). Figure S10. tPA expression in wild type (tPAWT) and tPA deficient mice (tPANull). A: Representative photomicrographs of hippocampal tissue sections with plat (in situ hybridization for the mRNA encoding tPA; green) and neurons labelled using neurotrace (red) stainings; scale bar 100µm. B: Quantifications of plat positive cells in hippocampus in tPAWT (N=1; n=85) and tPANull (N=1; n=40) mice. C: Representative photomicrographs of cortical tissue sections with plat (in situ hybridization for the mRNA encoding tPA; green) and neurons labelled using neurotrace (red) stainings; scale bar 100µm. D: Quantifications of plat positive cells in the cortex of tPAWT (N=1; n=100) and tPANull (N=1; n=40) mice. Figure S11. Conditional deletion of GluN1 in VeCad-Cre conditional knockout mice. A: Representative photomicrographs (n=5) of hippocampal tissue sections for grin1 (in situ hybridization of the gene encoding for the GluN1 subunit of NMDA receptor; green), immunostainings against CD31 (red) and DAPI (blue) staining; scale bar 100µm. B: Quantifications of grin1 positive cells in VEcad-CreWT (N=1; n=30) and VEcad-Cre ΔGluN1 (N=1; n=30) mice. Figure S12. Conditional deletion of tPA in VeCad-Cre conditional knockout mice. A: Representative photomicrographs (n=5) of hippocampal tissue sections for plat (in situ hybridization of the gene encoding for tPA; green), immunostainings against CD31 (red) and DAPI (blue) staining; scale bar 100µm. B: Quantifications of plat positive cells in VEcad-CreWT (N=1; n=25) and VEcad-CreΔtPA (N=1; n=30) mice.