Journal: bioRxiv

Article Title: Microglia control cerebral blood flow and neurovascular coupling via P2Y12R-mediated actions

doi: 10.1101/2021.02.04.429741

Figure Lengend Snippet: a) In vivo two-photon imaging reveals increased microglial process motility (arrowheads) to repeated (3x) CCAo in CX3CR1 tdTomato mice (1 st denotes first-order capillary). b) Automated morphological analysis demonstrates reduced number of branching-, and ending nodes of microglial processes ipsilaterally in CX3CR1 GFP/+ mice 24h after 3x CCAo compared to the contralateral side (contra) and sham animals in the cerebral cortex. c) Schematics of the experimental protocol of 3x CCAo. Microglia were selectively eliminated from the brain by PLX5622. Repeated CCA occlusions were performed in control and microglia depleted mice by pulling away the CCA with a silk thread. Representative perfusion (1 st and 3 rd rows)-, and difference LSCI images (2 nd and 4 th rows) showing cortical perfusion changes in response to 3x CCAo in control and microglia depleted mice. Dashed lines indicate the MCA2 territory in both the ipsilateral (white arrowheads) and contralateral (empty arrowheads) hemisphere corresponding to the quantification shown in panel d. Venous sinuses were excluded from the analysis. d) CBF responses of the MCA2 area (as shown by arrowheads) to 3x CCA occlusion. CBF responses are shown as percentage of baseline. A significant CBF reduction is seen in the absence of microglia in both hemispheres. Data are expressed as mean ± SEM. n=9-12 mice, *p control vs depleted, Mann-Whitney test (a); branching-ending nodes of n=386-388 sham, n=197 contralateral n=134 ipsilateral cells from n=3 sham and n=3 CCAo mice. Kruskal-Wallis test followed by Dunn’s multiple comparisons test (b); two-way ANOVA followed by Sidak’s multiple comparison test (d),.

Article Snippet: For microglia process motility measurements in response to 3x CCAo, the galvo-scanning light path with 16x water immersion objective (Nikon CFI75 LWD 16x W, NA 0.8) was used to acquire 4 images Z-stacks with 8.5 μm step size, 150-200 μm below the dura, at 500 × 500 pixel resolution.

Techniques: In Vivo, Imaging, Control, MANN-WHITNEY, Comparison

Journal: bioRxiv

Article Title: Microglia control cerebral blood flow and neurovascular coupling via P2Y12R-mediated actions

doi: 10.1101/2021.02.04.429741

Figure Lengend Snippet: a) Outline of the experimental protocol. CCAo was performed with a silk thread. b) Representative difference images show altered haemodynamic responses in CX3CR1 GFP/+ and CX3CR1 GFP/GFP mice in both hemispheres. Dashed lines show the MCA2 area both in the ipsilateral (white arrowheads)-, and in the contralateral hemisphere (empty arrowheads) corresponding to quantitative analysis on panel c. c) CX3CR1 GFP/+ mice show significantly impaired CBF recovery in both hemispheres from the 2 nd CCA occlusion compared to control and to CX3CR1 GFP/GFP mice. However, no significant difference is seen between CX3CR1 GFP/GFP and control mice. n=6-5-5 mice. d) Cross section CLSM image (left) and 3D reconstruction (right) of two blood vessels, showing CX3CR1 GFP/+ expressing microglia (yellow) that form contact with tomato lectin-positive endothelial cells (magenta). Microglia and perivascular macrophages (PVMs, between astrocytic endfeet and endothelial layer, orange in 3D model, marked by arrows) are both expressing CX3CR1 GFP/+ (yellow). e) CLSM images show that PVMs (CD206-labeling, red) can be positive for CX3CR1 (colocalisation marked by arrowheads), but negative for P2Y12R (cyan). Inserts show microglial processes contacting a CD206-positive PVM. f) CX3CR1 GFP/+ expressing microglia contact GFAP-labeled astrocytic endfeet. CD206-positive perivascular macrophages reside between astrocytic endfeet and lectin-labeled endothelial cells (magenta). g) PVMs were eliminated from the brain by ICV liposomal clodronate injection before LSCI measurements. h) No difference in CBF is seen between clodronate-treated and control mice after 3x CCAo. Data are presented as mean ± SEM. n=5-5 mice * p control vs CX3CR1 GFP/+ , # p control vs Cx3CR1 GFP/GFP , × p CX3CR1 GFP/GFP vs CX3CR1 GFP/+ ; two-way ANOVA followed by Tukey’s multiple comparison test (c, h). Scale bars: d , 5 μm; e , 3 μm f , 20 μm.

Article Snippet: For microglia process motility measurements in response to 3x CCAo, the galvo-scanning light path with 16x water immersion objective (Nikon CFI75 LWD 16x W, NA 0.8) was used to acquire 4 images Z-stacks with 8.5 μm step size, 150-200 μm below the dura, at 500 × 500 pixel resolution.

Techniques: Control, Expressing, Labeling, Injection, Comparison

Journal: bioRxiv

Article Title: Microglia control cerebral blood flow and neurovascular coupling via P2Y12R-mediated actions

doi: 10.1101/2021.02.04.429741

Figure Lengend Snippet: a) Depleted mice show reduced extracellular brain pH and b) reduced CBF in response to hypercapnia. c-d: CBF by laser Doppler flowmetry, tissue pH by pH-selective electrode and DC potential were simultaneously assessed during 3x CCAo performed by a mechanic occluder. The induction of spreading depolarization (SD) after the 1 st occlusion was observed in 6 out of 6 microglia depleted mice and none in controls, which was associated with larger negative pH shift and progressive oligaemia by the end of the 3 rd reperfusion. e) CBF response characteristic to 3x CCAo and f) extracellular pH. g-h) Effect of hypercapnia (g) and hypoxia (h) on levels of purinergic metabolites in primary endothelial-astrocyte- and microglia cultures as measured by HPLC., Ado, adenosine. Data are expressed as mean ± SEM. n=10 and 16 hypercapnic challenges from 5 control and 8 depleted mice, respectively (a); n=6 control and n=9 depleted mice per group (b); n=6 mice per group (e,f); Mann-Whitney test (b), two-way ANOVA followed by Sidak’s multiple comparison (a,g,e,f,h).

Article Snippet: For microglia process motility measurements in response to 3x CCAo, the galvo-scanning light path with 16x water immersion objective (Nikon CFI75 LWD 16x W, NA 0.8) was used to acquire 4 images Z-stacks with 8.5 μm step size, 150-200 μm below the dura, at 500 × 500 pixel resolution.

Techniques: Control, MANN-WHITNEY, Comparison

Journal: bioRxiv

Article Title: Microglia control cerebral blood flow and neurovascular coupling via P2Y12R-mediated actions

doi: 10.1101/2021.02.04.429741

Figure Lengend Snippet: a) Outline of the experimental 3x CCAo protocol. CCAo was performed by using a silk thread. b) Representative difference images show altered perfusion in both hemispheres in response to 3x CCAo in P2Y12R KO and PSB-0739-injected mice compared to controls. Dashed lines show the MCA2 area both in the ipsilateral (white arrowheads)-, and in the contralateral hemisphere (unfilled arrowheads) corresponding to the quantitative analysis shown in panel c. c) A significant impairment in adaptation to hypoperfusion is seen both in the ipsilateral and contralateral hemispheres of P2Y12R KO mice and PSB-0739 injected mice compared to controls. n=12-12-7 mice. *p control vs P2Y12R KO mice, # p control vs PSB-0739 injected mice, two-way ANOVA followed by Tukey’s multiple comp. test (c).

Article Snippet: For microglia process motility measurements in response to 3x CCAo, the galvo-scanning light path with 16x water immersion objective (Nikon CFI75 LWD 16x W, NA 0.8) was used to acquire 4 images Z-stacks with 8.5 μm step size, 150-200 μm below the dura, at 500 × 500 pixel resolution.

Techniques: Injection, Control