Fig 1: Iba-1-positive area in CA1, CA3, and DG hippocampal regions. (a) Representative images of Iba1-positive immunostaining in CA1, CA3, and DG hippocampal areas. Scale bar—500 µm. (b) Histogram demonstrating the percentage of area covered by Iba1-positive staining in CA1, CA3, and DG regions. (c) Histogram demonstrating the percentage of area covered by Iba1-positive staining in CA1 subregions. (d) Histogram demonstrating the percentage of area covered by Iba1-positive staining in CA3 subregions. (e) Histogram demonstrating the percentage of area covered by Iba1-positive staining in DG subregions. Mean ± SEM, n = 25 (number of slices per group). One-way ANOVA with a post-hoc Tukey test, * p < 0.05, *** p < 0.001; + p < 0.05, ++ p < 0.01, +++ p < 0.001. *—compared to Veh, +—compared to LPS. Str. oriens—stratum oriens, str. pyr.—stratum pyramidale, str. luc.—stratum luciderm, str. rad.—stratum radiatum, str. lac.-mol.—stratum lacunosum–moleculare, str. mol.—stratum moleculare, str. gr.—stratum granulosum.
Fig 2: Immunoreactivity of GFAP, Iba1, and CD11b in a Sham rat and in a high-grade subarachnoid hemorrhage (SAH) rat. (A) Representation of coronal brain section,21 the black box indicates the selected area for immunofluorescence study in the right cortex. Representative immunofluorescence images of 4',6'-diamidino-2-phénylindole (DAPI; blue channel, B-D), Glial Fibrillary Acidic Protein (GFAP; red channel, B), Ionized calcium binding adapter molecule 1 (Iba1; red channel, C), and CD11b (green channel, D) of a Sham rat (upper) and a high-grade SAH rat (lower). Scale bars represent 150 µm.
Fig 3: Uptake of GR@LNPs by parenchymal microglia. Representative immunofluorescences of 6-FAM-labeled GR@LNPs (green in panels B,F, and J), iba1+ parenchymal microglia (red in panels A,E, and I), and nuclear DAPI (blue in panels C,G, and K) in rat prefrontal cortex sections from control rats treated with GR-free@LNP (panels A–D), animals treated with GR1 and GR2-antisense @LNPs (panels E–H), and negative control rats treated with GR3-nonsense@LNPs (panels I–L). White head arrows and magnifications in merge images (panels D,H, and L) show a lack of co-localizations of GR@LNPs with parenchymal microglia in the different experimental groups studied. On the contrary, yellow arrows in H and L show a 6-FAM signal in perivascular and meningeal locations, respectively. Scale bars = 15 µm.
Fig 4: (A) Microglial activation measured by Iba1-immunoreactivity optical density in PLP-a-synuclein and control mice (*p < 0.05, ***p < 0.001). (B) Correlation between the level of a-synuclein oligomers and microglial activation in the hippocampus of PLP-a-synuclein mice (R2 = 0.381, p < 0.01), the analysis includes both immunized and non-immunized mice. (C) Microglial activation measured by the number of Iba1-immunoreactive cells with activated morphology per mm2 in PLP-a-synuclein mice and controls. (D) Representative microphotographs of Iba1 immunohistochemistry in SNc of control and PLP-a-synuclein mice receiving rec47 or vehicle (counterstaining with cresyl violet). (E) Heat map representation of the percentage CD68-positive microglia out of the total Iba1-positive microglia in the four experimental groups (*p < 0.05, ***p < 0.001). (F) Representative microphotographs of CD68 immunohistochemistry in SNc of control and PLP-a-synuclein mice receiving rec47 or vehicle.
Fig 5: Microglia quantification and profiling in the MSA mice. a Sterological analysis of the number Iba-1-positive cells demonstrated no significant differences between control and MSA mice in substantia nigra after Bonferroni correction (two way ANOVA indicates a general effect of age and genotype: effect of genotype F1,23 = 6.12, p = 0.0211, effect of age F2,23 = 3.61, p = 0.0432, interaction F2,23 = 0.15, p = 0.8599). No significant differences between controls and MSA mice were detected after Bonferroni correction in the striatum (two way ANOVA indicates no general effect of age and genotype: effect of genotype F1,23 = 0.08, p = 0.7812, effect of age F2,23 = 3.04, p = 0.0671, interaction F2,23 = 0.08, p = 0.7812). In the pontine nuclei, age-related increase in the number of Iba-1-positive cells was seen in the control, but not in the MSA group after Bonferroni correction (two way ANOVA indicates a general effect of age: effect of genotype F1,21 = 1.86, p = 0.1866, effect of age F2,21 = 8.83, p = 0.0017, interaction F2,21 = 3.37, p = 0.0537). Similar effects were found in the inferior olives; however, at 15 months of age a significant difference between MSA and control mice was found after Bonferroni correction (two way ANOVA indicates a general effect of age and genotype: effect of genotype F1,22 = 9.27, p = 0.0059, effect of age F2,21 = 30.17, p < 0.0001, interaction F2,21 = 27.05, p < 0.0001) (b) Four microglia morphological profiles were distinguished (Iba1 immunohistochemistry and cresyl-violet counterstaining): Type A cells (corresponding to surveillant/homeostatic microglia, and the most abundant phenotype), characterized by a compact and regular shaped nucleus, very thin visible cytoplasm, and long and thin processes without many secondary branches. Type B cells (hyperramified) presented with ramified processes with multiple short branches, and increased amount of cytoplasm around the nucleus as compared to type A. Type C microglia (hyperthrophic) showed even larger cell body with more irregular outline, enlarged nucleus and shorter and thicker processes. Finally, type D cells (amoeboid), which resembled peripheral macrophages, were identified by their amoeboid shape, with a large nucleus and the soma merging with the processes. c The stereological counts of each microglia subtype are represented in percentages of the total Iba1+ cells counted. In the substantia nigra, the non-homeostatic activated microglia profiles (type B, C and D) were significantly more abundant in the MSA mice than in the control ones at 5 and 15 months of age. Homeostatic microglia (type A) showed a significant reduction from 2 to 5 months and from 2 to 15 months of age in the transgenic animals, while activated microglia were significantly increased from 2 to 15 months of age. In the control mice, a reduction in A cells was seen at 15 months of age (two way ANOVA indicates a general effect of age and genotype: effect of genotype F1,23 = 51.42, p < 0.0001, effect of age F2,23 = 29.42, p < 0.0001, interaction F2,23 = 19.53, p < 0.0001); (d) In the striatum, an age-related decrease in A cells, accompanied by an increase in B cells, was observed in both mouse lines. No significant differences were detected between the two at any time point, except for an increase in the percentage of D cells in the MSA mice at 15 months of age (two way ANOVA indicates a general effect of age and genotype without interaction: effect of genotype F1,23 = 8.29, p = 0.0085, effect of age F2,23 = 72.14, p < 0.0001, interaction F2,23 = 1.1, p = 0.35); (e) In the pontine nuclei, as well as in the inferior olives (f), an increase of activated microglia was visible at 15 months of age, if compared with earlier stages, in both transgenic and control mice (two way ANOVA for the pontine nuclei indicates a general effect of age: effect of genotype F1,21 = 2.65, p = 0.1184, effect of age F2,21 = 3.61, p < 0.0001, interaction F2,21 = 1.27, p = 0.3008; two way ANOVA for the inferior olives indicates a general effect of age: effect of genotype F1,21 = 0.03, p = 0.8731, effect of age F2,21 = 15.43, p < 0.0001, interaction F2,21 = 6.11, p = 0.0081)
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