Journal: Disease Models & Mechanisms

Article Title: Interleukin-1 regulates myeloid cell trafficking and cerebral blood flow following intracerebral haemorrhage

doi: 10.1242/dmm.052306

Figure Lengend Snippet: Myeloid cells dominate the acute response to ICH and contribute to early damage. (A) Single cells were isolated from the right hemisphere of naïve, sham-operated and collagenase-induced ICH mice 24 h post-surgery and immunophenotyped using flow cytometry. (B) Left: gating strategy used during flow cytometry analysis. Right: cell counts are shown from the following populations: CD45 int CD11b + microglia, CD45 hi CD11b + Ly6G + neutrophils, CD45 hi CD11b + Ly6G − Ly6C hi monocytes (Mo), CD45 hi CD11b + Ly6G − Ly6C lo monocytes/macrophages (Mϕ), CD45 hi CD11b − CD45R + B cells, and CD45 hi CD11b − CD3 + T cells. Data presented as mean+s.e.m., n =5, two independent experiments. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001, **** P <0.0001, determined by one-way ANOVA with Tukey's post-hoc test. (C) Formalin-fixed paraffin-embedded post-mortem human brain tissue from age- and region-matched control (left) and ICH (right) cases were immunostained for the myeloid cell marker CD11b (green) and DAPI (blue), with red blood cell (RBC) autofluorescence shown in red. One representative image from three cases per group shown. Scale bars: 100 µm (50 μm in insets). (D) White blood cells were isolated from whole blood of wild-type (WT) ( n =7) and chemokine receptor 2 ( Ccr2 ) −/− ( n =9) male littermate mice. Top left: CD115 + monocytes and Ly6G + neutrophils as a percentage of all CD45 + immune cells. Bottom left: representative flow plots of the circulating monocyte compartment of each genotype. Right: the amount of Ly6C hi monocytes as a percentage of total circulating monocytes from each genotype. ns, not significant; **** P <0.0001, determined by unpaired two-tailed t -test. (E) Following a 3 day training period on the rotarod assay, WT and Ccr2 −/− littermates were subjected to collagenase-induced ICH and tested on the rotarod again 24 h later. Data presented as net difference in latency to fall from baseline performance, n =7-9, four independent experiments. * P <0.05, determined by unpaired two-tailed t -test.

Article Snippet: Following single-cell isolation, myeloid cells were purified using CD11b + magnabeads (130-093-636, Miltenyi) according to the manufacturer's protocol.

Techniques: Isolation, Flow Cytometry, Formalin-fixed Paraffin-Embedded, Control, Marker, Two Tailed Test

Journal: Disease Models & Mechanisms

Article Title: Interleukin-1 regulates myeloid cell trafficking and cerebral blood flow following intracerebral haemorrhage

doi: 10.1242/dmm.052306

Figure Lengend Snippet: IL-1 produced by mononuclear phagocytes controls acute recruitment of myeloid cells during ICH. (A) Mice were subjected to collagenase-induced ICH and culled at 4 h ( n =5) and 24 h ( n =4) post-ICH. 20 µm coronal brain sections were immunostained for either IL-1α (green, top row) or IL-1β (green, bottom row) together with Iba1 (red) and DAPI (blue). Dashed lines represent haematoma border, arrows define haematomal IL-1β+ cells, and insets are higher-magnification images of respective white boxes. Scale bars: 50 µm. (B) Quantification of IL-1 + cells. (C) 8 µm sections of formalin-fixed paraffin-embedded post-mortem human brain tissue from age- and region-matched control (top) and acute ICH (bottom) cases were immunostained for Iba1 (red), IL-1β (green) and DAPI (blue). Red blood cell (RBC) autofluorescence can be seen in white. One representative image from three patients per group is shown. Scale bars: 100 µm. Insets are higher-magnification images of the areas within the white boxes. (D) Representative flow plot of cells isolated from brains of mice injected with central (10 µg intrastriatal) and peripheral (100 mg kg −1 subcutaneous) IL-1 receptor antagonist (IL-1Ra) ( n =4) or placebo ( n =5) and subjected to ICH, two independent experiments. (E) Cell counts are shown from the following populations: CD45 hi CD11b − lymphocytes, CD45 int CD11b + microglia, CD45 hi CD11b + myeloid cells, CD45 hi CD11b + Ly6G + neutrophils, CD45 hi CD11b + Ly6G − Ly6C hi monocytes (Mo), CD45 hi CD11b + Ly6G − Ly6C lo monocytes/macrophages (Mϕ). Data presented as mean+s.e.m. ns, not significant; * P <0.05, ** P <0.01, *** P <0.001, determined by unpaired two-tailed t -test.

Article Snippet: Following single-cell isolation, myeloid cells were purified using CD11b + magnabeads (130-093-636, Miltenyi) according to the manufacturer's protocol.

Techniques: Produced, Formalin-fixed Paraffin-Embedded, Control, Isolation, Injection, Two Tailed Test

Journal: Journal of neuroinflammation

Article Title: Microglia either promote or restrain TRAIL-mediated excitotoxicity caused by Aβ 1-42 oligomers.

doi: 10.1186/s12974-024-03208-2

Figure Lengend Snippet: Fig. 6 Microglial repopulation strongly reduces Aβ-induced neurotoxicity. (A) Experimental design for microglial repopulation in brain slice culture. (B) Expression of microglial genes at baseline, after depletion and after repopulation. Each data point represents the average of two technical replicates from different culture wells. 2-way ANOVA. F3,34=7.4, p = 0.0006. Tukey’s multiple comparisons test. ****p < 0.0001 Control vs. Depletion, ####p < 0.0001, Deple tion vs. Repopulation. N = 3–6/group. (C) Depiction of microglial isolation by CD11b + MACS. (D) Expression of microglial genes after MACS isolation in the microglial fraction and the remaining cells. 2-way ANOVA, F2,24=136.9, p < 0.00001****p < 0.0001 Sidak’s post-test. (E) Microglia were isolated from HEBSCs containing either native or RM by MACS. RM showed increased expression of BDNF compared to native microglia. (F) Represented image of PI staining in HEBSCs with either naïve or RM treated with Aβ1−42 (1µM) for 4 days. (G) Quantification of robust increases in neurotoxicity after Aβ1−42 treatment in HEBSCs with naïve, but not repopulated microglia. 1-way ANOVA, F2,37=21.72, p < 0.0001. N = 10–20 slices/group

Article Snippet: Samples were incubated with FcR blocking reagent (10 μL; Miltenyi Biotec, Cat. #130-092-575) for 10 min at 4 °C followed by incubation with 10 μL CD11b microbeads (Miltenyi Biotec, Cat. #130-105-634) at 4 °C for 15 min.

Techniques: Slice Preparation, Expressing, Control, Isolation, Staining