Fig 2: Immunofluorescence staining indicates changes in Ngb and activated p38 levels in the ischemic stroke brain. (a,d) Representative images of Nissl staining of the areas that were selected for quantifying Ngb and p-p38 in the ipsilateral brain area 1 day (a–c) and 3 days (d–f) post-stroke, respectively. Black solid line and yellow-labeled boxes (1 × 1 mm2 square field) outline the core and penumbra (C and P) infarct regions. (b,c,e,f) Representative images and quantification of Ngb (green) and p-p38 (red) staining in the ipsilateral area post-tMCAO in mice. Treatment with PCE resulted in significant increases in Ngb and decreases in activated p38 in comparison with the vehicle-treated tMCAO group. Data are presented as mean ± SEM. White scale bar = 20 μm. ***, ### p < 0.001, ** p < 0.01, *, # p < 0.05. Statistical significance was determined using one-way ANOVA, followed by Tukey’s multiple comparisons test (1 day post-stroke, n = 5; 3 days post-stroke, n = 4).

Fig 3: PCE treatment increases Ngb levels in neurons in the ischemic area. Representative immunofluorescence images and quantification of colocalization with Ngb (red) and NeuN (green) staining in the ipsilateral area. Immunofluorescence images of activated p38 (white) are also shown as a reference. The numbers of Ngb-expressing neurons decreased after ischemic stroke. PCE treatment exhibited increased numbers of Ngb-expressing neurons. Colocalization of Ngb and neurons is indicated by white arrows. Data are presented as mean ± SEM. White scale bar = 20 μm. * p < 0.05. Statistical significance was determined using one-way ANOVA, followed by Tukey’s multiple comparison test (n = 4).

Fig 4: PCE and PCCs induce Ngb transcriptional activity and increase Ngb protein levels in treated neuronal cells. (a) Increased Ngb promoter activity levels from PCE and PCCs by luciferase reporter assay in SKNSH cells. SKNSH cells were treated with various concentrations of PCE (10−8−10−6 g/mL, n = 10) and PCCs (10−8−10−6 M, n = 10) for 1 day. Results are expressed as the relative luciferase value (Veh, n = 20). (b) Levels of Ngb mRNA were increased by PCE (10−7−10−5 g/mL) and PCCs (10−6 M) in SKNSH cells. The ratio of target gene expression to that of GAPDH housekeeping gene was determined by qPCR as a quantitative measure of gene expression. Ngb mRNA levels were significantly increased by 10−5 g/mL PCE and leucoside (Veh, n = 18; PCE, n = 13; PCCs, n = 8). (c) Protein levels of Ngb were induced by PCE (10−7−10−5 g/mL) and selected PCCs (10−6 M) in primary neuronal cells (n = 4). The mean values in triplicate experiments are presented as SEM. *** p < 0.001, ** p < 0.01, * p < 0.05 compared with the vehicle control, determined by the Holm–Sidak method following a one-way analysis of variance.

Fig 5: Representative OCT and histology images of the fundus of the optic nerve head (ONH) from primate number 6 showing the optic nerve head (ONH) structural changes. (A,B) No difference in baseline cup disc ratio (CDR, black dotted lines). (C–H) At 10 months, CDR in the eye treated with neuroglobin (Ngb) has a smaller CDR (C) compared to untreated eyes with EG, which is corroborated by the OCT findings (E,F) that show increased LCD (yellow arrows) and thinner MRW (red arrows) in the untreated eye (F). This is also reflected in the histology of the ONH (G,H), where there are similar findings of increased LCD and thinner MRW (H) in untreated EG eyes. Ngb, neuroglobin; BMO, Bruch’s membrane opening; ILM, internal limiting membrane; MRW, minimum rim width; ALC, anterior lamina cribrosa; and LCD, lamina cribrosa depth.