Fig 2: Peripheral immune cell infiltration. (A~D) Graphs showing the number of CCR2+ and Ly6C+ cells in the microglia (CD11b+/CD45low/int) and macrophage (CD11b+/CD45high) populations in non-lesioned (NL) and lesioned animals at 24 hpi and 3 dpi in WT and GFAP-IL10Tg mice. (E, F) Graphs showing the quantification of CCR2+/Ly6C+ inflammatory monocytes in non-lesioned (NL) and TBI-lesioned animals at 24 hpi and 3 dpi in WT and GFAP-IL10Tg mice. Inflammatory monocytes exhibited a significant increase in transgenic animals at 3 dpi. In WT animals: “a” indicates significant vs NL and in GFAP-IL10Tg animals: “a’” indicates significant vs NL. (G~J) Representative images showing the number of myeloperoxidase (MPO)+ cells (arrowheads) in the penumbra of TBI-lesioned WT (H) and GFAP-IL10Tg animals (J) at 24 hpi. Toluidine blue staining was used as a nuclei marker. (K) Graph showing the quantification of MPO+cells in WT and GFAP-IL10Tg animals from 24 hpi to 7 dpi. Note that, at 24 hpi, GFAP-IL10Tg animals presented a significantly lower number of MPO+ cells than did WT. In WT animals: “a” indicates significant vs 24 hpi. In GFAP-IL10Tg animals: “a’” indicates significant vs 24 hpi. (L~O) Representative images showing the number of CD3+ T lymphocytes (arrowheads) in the penumbra of both WT (L, M) and GFAP-IL10Tg animals (N, O) at 3 dpi and 7 dpi. (P) Graph showing the quantification of CD3+ T lymphocytes in WT and GFAP-IL10Tg animals at 3 dpi and 7 dpi. Note that,GFAP-IL10Tg presented a higher number of CD3+ lymphocytes than did WT at 3 dpi. All values are represented as mean±SEM (£p=0.1, *p=0.05, ****p=0.0001). Scale bar=30 µm.

Fig 3: Leukocyte infiltration is monitored by analysing neutrophils (Ly6B2 staining) and T-lymphocytes (CD3 staining). Ly6B2 staining in the control animals at P7 shows a few neutrophils inside the blood vessels in the hippocampal fissure (a), in DG (b), and in the CA (c) regions. In the hippocampus the maximum density of cells is observed at 7 days after hypoxia especially in the hippocampal fissure (d); CA3 (e) and CA1 (f). In caudate-putamen an increase is observed at 7 days after hypoxia (h). In the ventricles (g) and the neocortex (i) the maximum quantity of neutrophils is observed at 3 hours after hypoxia. No significant difference was observed in the thalamus (j). CD3 staining in the control animals shows the presence of lymphocytes in the meninges (k) and in the ventricles (l). Finally, scattered cells are observed in the neocortex (m). Scale bars: 10 μm. Hf: hippocampal fissure; DG: Dentate gyrus; CA: cornu ammunis.

Fig 4: T cell infiltration after PPT. a Representative dot plot of SSC/CD3 expression of cells obtained from the lesioned WT animals. The square delimits the CD3+ cell population used in this study. Representative dot plot of CD3/CD4/CD8 cells obtained from the spleen, positive control (top), and hippocampus (bottom) of lesioned WT. b Graphs showing the number of CD4+, CD3+/CD4+/CD8+ cells, CD8+ and CD3+/CD4-/CD8− in NL and at 7dpl. c Representative histogram-plot showing the expression of Tbet, Gata3, RORgt, and Foxp3 in the population of CD3+Th-cells in comparison to the corresponding isotype control, in which the positive staining was defined. d Graphs showing the number of the different subtypes of CD4+ T-helper lymphocytes: Tbet+, Gata 3+, RORg+, and Foxp3+ in NL and at 7dpl. e Graph showing the quantification of CD3+ immunostaining cells in non-lesioned (NL) and PPT-lesioned hippocampus from 2 to 14 dpl, in WT and GFAP-IL6Tg mice. f Representative images showing CD3 immunostaining in WT and GFAP-IL6Tg mice at 7 dpl. Scale bar = 20 μm. A minimum of four WT and four GFAP-IL6Tg animals per group were used for this study. Data are represented as mean ± SD. The significances are represented as #p ≤ 0.05 vs. NL of respective group; $p ≤ 0.1 vs. NL of respective group and %p ≤ 0.05 vs. 3dpl of respective group. Significant differences between genotypes are represented as *p ≤ 0.05

Fig 5: VNS-Induced PlGF Release and Immune Response Initiation Are Mediated by α-Adrenergic Receptor Signaling(A–C) Vehicle-, phentolamine-, or propranolol-treated mice were subjected to either sham or VNS procedure. (A) SSNA (vehicle, sham versus VNS: q(17) = 4.936, ∗p < 0.05; phentolamine, sham versus VNS: q(17) = 4.753, ∗p < 0.05; propranolol, sham versus VNS: q(17) = 4.729, ∗p < 0.05) and (B and C) TH-positive staining (vehicle, sham versus VNS: q(17) = 7.480, ∗∗∗p < 0.001; phentolamine, sham versus VNS: q(17) = 7.118, ∗∗p < 0.01; propranolol, sham versus VNS: q(17) = 4.599, ∗p < 0.05) (scale bar, 100 μm) were increased in all VNS groups.(D and E) Although vehicle- and propranolol-treated mice shown a similar activation of PlGF, phentolamine treatment significantly reduced it (vehicle, sham versus VNS: q(17) = 5.856, ∗∗p < 0.01; propranolol, sham versus VNS: q(17) = 7.861, ∗∗∗p < 0.001; VNS, phentolamine versus vehicle: q(17) = 4.991, ∗p < 0.05; VNS, phentolamine versus propranolol: q(17) = 7.361, ∗∗∗p < 0.001) (scale bar, 50 μm).(F and G) A reduced area of CD3+ cells (red), delimited by B220+ cells (green), is observed in both vehicle- and propranolol-treated VNS mice. Conversely, phentolamine-treated VNS mice display a CD3+ area comparable with sham mice (vehicle, sham versus VNS: q(17) = 8.337, ∗∗∗p < 0.001; propranolol, sham versus VNS: q(17) = 4.955, ∗p < 0.05; VNS, phentolamine versus vehicle: q(17) = 6.983, ∗∗p < 0.01; VNS, phentolamine versus propranolol: q(17) = 6.688, ∗∗p < 0.01) (scale bar, 100μm).For all panels, data were obtained from n = 4 sham vehicle, n = 4 VNS vehicle, n = 4 sham phentolamine, n = 4 VNS phentolamine, n = 3 sham propranolol, and n = 4 VNS propranolol mice and are represented as mean ± SEM.