Fig 2: Cranial irradiation inhibits cellular proliferation and neurogenesis in the rat hippocampus. Confocal micrographs of hippocampal sections labeled with BrdU (red), DCX (green), GFAP (green) and NeuN (green). (A and B) Proliferating cells labeled with BrdU. (C and D) Immature neurons labeled with BrdU and DCX. (E and F) Differentiated astrocytes labeled with BrdU and GFAP. (G and H) Recently-divided mature neurons labeled with BrdU and NeuN. All histograms represent the mean ± SEM. *P<0.05 and **P<0.01. BrdU, 5-bromodeoxyuridine; DCX, doublecortin; GFAP, glial fibrillary acidic protein; NeuN, neuronal nuclei. Green: (C) DCX, (E) GFAP, and (G) NeuN; Red: BrdU.

Fig 3: Effect of M1 and M2 on Müller cells gliosis and microglial reactivity. Representative Western blots (A) and densitometric analysis of GFAP (B) and Iba-1 (C) in controls, PEG-injected untreated, PEG-injected treated with M1 or M2 vehicles, PEG-injected treated with low or high dose of M1 as well as with low or high-dose of M2. β-actin was used as loading control. Data are plotted as mean ± SEM. Differences among groups were assessed using one-way ANOVA followed by Tukey’s multiple comparison post-hoc test (N = 6). *p < 0.01 and **p < 0.001 vs. control; § p < 0.01 and §§ p < 0.001 vs. respective vehicle; #p < 0.05 vs. M1 high.

Fig 4: Brain damage and viral tropism in the CNS of PHEV-infected mice.(A-B) The 3w and 6w BALB/c mice were inoculated with 103.96 TCID50 PHEV and samples were collected at 5 dpi for qRT–PCR and viral titer determination (n = 6). (A) Viral genome loads were monitored in different organs and blood. The limit of detection (LOD) is shown with a dashed line. (B) Infectious viral titers were detected in different organs (n = 6). (C-L) The 3w BALB/c mice were intranasally inoculated with 103.96 TCID50 PHEV. Mice were euthanized at 5 dpi, and brain samples were harvested for histopathological examination. (C-H) H&E staining of brain sections from control and PHEV-infected mice. (C-D) Lymphocytic perivascular cuffing (red arrows). (E) Dying neurons undergoing degeneration (black arrows). (F) Microglial nodules (green arrows). (G-H) Brains from mock-infected mice. (I-L) Immunofluorescence images of PHEV-infected neurons in 3w BALB/c mice. MAP-2, GFAP, IBA1, and MBP are markers for neurons, astrocytes, microglia, and oligodendroglia, respectively. Scale bars, 100 μm (C, G), 20 μm (D-F, H), and 50 μm (I-L).

Fig 5: Effects of pre-formulated mixture on oxidative markers, gliosis and microglial activation. (A) Western blots and densitometric analysis of Nrf2 and HO-1 in retinal homogenates of control and LD rats untreated or pretreated with the mixture at low or high dosage. (B) Representative images of retinal sections immunolabeled for GFAP (green) and Iba-1 (red) and counterstained with DAPI (blue). (C) Quantitative analysis of ONL thickness. (D,E) Quantitative analysis of GFAP and Iba1 immunofluorescence intensity. Scale bar, 50 µm (n = 6 retinas per group). Data are expressed as mean ± SD. Differences between groups were tested for statistical significance using one-way ANOVA followed by the Newman–Keuls multiple comparison post hoc test. * p < 0.05; *** p < 0.001 versus control; § p < 0.05; §§ p < 0.01; §§§ p < 0.001 versus LD.