Fig 2: The expression of Igfbp2 and Igf2 in the amygdala was analyzed using IHC in the three groups; CTL-MI, STR-HI, and STR-LI mice. IHC showed specific decreased expression of Igfbp2 and Igf2 in the CeA nucleus of STR-HI mice (A). The fluorescence signal density was quantified using the NIH Image J program, and the strength of IHC was evaluated with one-way ANOVA and Tukey’s multiple comparison tests among groups. Igfbp2 (F = 13.52) and Igf2 (F = 53.25) showed statistically significant differences among groups with one-way ANOVA. Tukey’s post tests revealed that only the STR-HI mice had a significant decrease in two proteins in the CeA (B). Data are the means ± SEM (n = 3). a, P < 0.05 between CTL-MI and STR-HI mice; c, P < 0.05 between STR-HI and STR-LI mice. Scale bars are 200 μm

Fig 3: IGFBP-2 protein in adult mouse brain. IGFBP-2 is detectable in the olfactory bulb, cortex and cerebellum of adult mice. The olfactory bulb has the highest levels of IGFBP-2 compared to the rest of the tissue. (A) Western blot of IGFBP-2 in brain lysates. (Rcb - recombinant protein, OB - olfactory blulb, CTX - cortex, CB - cerebellum). (B) IGFBP-2 ELISA using brain lysates. Results are mean ± S.D. n = 5; *p < 0.001 by ANOVA, olfactory bulb versus cortex or cerebellum.

Fig 4: IGFBP-2 Expression Following Hypoxic-Ischemic Injury. Brain sections of mice from either the Sham group or the MCAO group were used to visualize IGFBP-2 in neurons, astrocytes and microglia. All images were taken of cells in the cortex that formed the penumbra. (A, B) Neurons are co-labeled with anti-NeuN and anti-IGFBP-2. (C) Astrocytes are co-labeled with anti-GFAP and anti-IGFBP-2. (D) Microglia are co-labeled with anti-Iba-1 and anti-IGFBP-2. Bars represent 20 μm.

Fig 5: IGFBP-2 protein levels following hypoxic-lschemic injury. IGFBP-2 levels of sham animals are not significantly different between the left and the right hemispheres for the brain regions analyzed (A, C). For the brain regions analyzed, IGFBP-2 levels are not significantly different between the contralateral and stroke hemispheres 24 h post-stroke (B). IGFBP-2 levels on the stroke hemisphere increase significantly (*) in the penumbra (5-fold) and core (3-fold) when compared to the contralateral hemisphere 72 h post-stroke (D). For statistical analysis, for each brain region, IGFBP-2 levels of the two hemispheres were compared. (*) on the graphs denotes the brain regions where the statistical analysis revealed a significant difference between the stroke and the contralateral hemisphere. Results are mean ± S.D. n = 5; *p < 0.05 by ANOVA contralateral hemisphere vs. stroke hemisphere.