Fig 2: Ovariectomized female FBN-ARO-KO mice exhibit worse neuronal damage and cognitive deficits 7 d after GCI injury. Aa, IHC analysis for degenerating neurons by F-Jade C, a typical neurodegenerative marker, with NeuN double staining. Ab, The number of F-Jade C-positive neurons per 250 um was counted. B, MAP2 staining was performed to further evaluate hippocampal CA1 dendrite morphology following aggravated neuronal damage in FBN-ARO-KO mice. C, Barnes Maze Test was used to assess the spatial reference memory. Representative track plots of FLOX+Sham (Ca), FLOX+GCI (Cb), KO+Sham (Cc), and KO+GCI (Cd) on the probe trial were recorded. Primary escape latency (Ce), escape velocity (Cf), quadrant occupancy (Cg), and exploring errors (Ch) during probe trial were analyzed. D, Open field test was conducted to determine the locomotor ability, in which the travel distance and rearing times were recorded and analyzed. Values are mean ± SEM of determinations from each group. N = 5 for A, B; N = 8 or 9 for C, D. *p < 0.05 versus FLOX+Sham. #p < 0.05 versus FLOX+GCI. NS, no significant difference.

Fig 3: Ovariectomized female FBN-ARO-KO mice have diminished astrocyte A2 phenotype 7 d after GCI. Aa, Astrocyte PAN-reactive, A1-specific, and A2-specific phenotypes were determined by Western blot analysis with purified astrocyte lysates from ovariectomized female brains 7 d after GCI reperfusion. Ab, Levels of the examined markers for different astrocyte phenotypes were quantitatively analyzed. N = 3. B, IHC analysis for astrocyte A1 phenotype by double staining of the selected astrocyte A1 marker C3D with GFAP. Ca, IHC examination for astrocyte A2 phenotype using A2-specific marker S100A10 and GFAP double staining. Cb, Percentage changes of S100A10 in FBN-ARO-KO+GCI mice versus FLOX+GCI mice were quantified. N = 4. Values are mean ± SEM of determinations from each group. *p < 0.05 versus FLOX+Sham. #p < 0.05 versus FLOX+GCI. ND, Nondetectable.

Fig 4: Evidence that upregulation of neuronal FGF2 signaling mediates decreased astrocyte activation in ovariectomized female FBN-ARO-KO mice after GCI. Aa, Representative image of FGF2 and NeuN double staining in hippocampal CA1 region. Ab, Quantitative analysis of neuronal FGF2 levels in each group (N = 4 or 5). B, Neuronal FGF2 alterations in hippocampus were confirmed by Western blotting analysis (N = 3). C, Representative images of FGFR3 and GFAP double staining showing changes of FGFR3 expression in hippocampal CA1 astrocytes (N = 4 or 5). Da, Schematic illustration of FGFR3 neutralization by bilateral intracerebroventricular injection of FGFR3 blocking antibody in FBN-ARO-KO GCI mice. Db, The purity of isolated astrocytes from the ischemic brains was evaluated by flow cytometry analysis. E, To examine the effects of FGFR3 neutralization on astrocyte reactivity and functional restoration in FBN-ARO-KO mice after GCI, levels of GFAP, p-STAT3, BDNF, and GLT-1 in purified astrocytes were determined by Western blotting (Ea), and further quantitatively analyzed (Eb) (N = 3). Fa, IHC analysis for neuronal degeneration by F-Jade C and NeuN double staining in hippocampal CA1 region. Fb, Quantification of F-Jade C levels in CA1 pyramidal neurons, which was presented as relative intensity of each group versus FLOX+GCI (N = 4 or 5). Values are mean ± SEM of determinations from each group. *p < 0.05 versus FLOX+Sham. #p < 0.05 versus FLOX+GCI. $p < 0.05 versus FLOX+GCI. &p < 0.05 versus KO+GCI.

Fig 5: Male FBN-ARO-KO mice display attenuated astrocyte activation, compromised astrocyte functions, and worse neuronal damage at 3 and 14 d after GCI. A, B, GFAP and aromatase double staining to examine astrocyte activation and astrocytic aromatase induction at 3 d (Aa) and 14 d (Ba) after GCI. Relative changes of GFAP intensities at 3d (Ab) and 14d (Bb) were quantified. C, Astrocyte-derived BDNF (Ca), IGF-1 (Cb), and GLT-1 (Cc) levels at 3 d after GCI were explored by IHC analysis. D, Their relative intensities in KO + 3 d mice versus FLOX + 3 d mice. E, F, BDNF (Ea), IGF-1 (Eb), and GLT-1 (Ec) levels in hippocampal astrocytes at 14 d after GCI were determined with IHC, and expressed as relative changes of KO + 14 d mice compared with FLOX + 14 d mice (F). G, H, Neuronal damage in hippocampal CA1 region at 3 d (G) and 14 d (H) after GCI was assessed by NeuN and F-Jade C double staining. Values are mean ± SEM of determinations from each group. N = 4. #p < 0.05 versus FLOX + 3 d. &p < 0.05 versus FLOX + 14 d.