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: Expression of key protein associated apoptosis, inflammation and cell-marker among different group. Note: (1) Meanings of the combination of letters and numbers in the icon: a, b, c, d, e, f are respectively represented as proteins of Bax, Bcl-2, NF-?b, MAP-2, Olig-2 and GFAP; 1, 2, 3 are respectively represented as Sham operated Group, Model Group and MDP Group. (2)*: Compared to Model Group, P < 0.05; #: Compared to Sham operated Group, P < 0.05. Part A, C described the expression of key proteins associated apoptosis and inflammation among three groups. Expression of Bax was up-regulated in Model Group (a2), but it was inhibited by MDP (a3). For the expression of Bcl-2, the trend is completely an opposite, it was down-regulated in Model Group (b2) but boosted by MDP (b3). The trend of NF-?B is similar to that of Bax (c2, c3). Part B, D delineate the expression of cell-specific marker proteins among three groups. MAP-2 was inhibited in Model Group (d2) but encouraged by MDP (d3); For the expression of Olig-2, the trend was roughly same to MAP-2 (e2, e3); For the expression of GFAP, the trend is completely an opposite (f2, f3).

Fig 4: Expression of the neuronal dendrite marker microtubule-associated protein (MAP)2 in the dentate gyrus (DG) of cold-stressed mice. (A) MAP2 expression in the DG was detected by immunohistochemistry. Scale bar = 50 µm. (B) Representative Nissl staining of the DG region. Scale bar = 50 µm. (C) Quantification of the MAP2 expression level by densitometry. Differences between room temperature male (RTM) and cold exposure male (CEM), room temperature female (RTF) and cold exposure female (CEF), and CEM and CEF groups were evaluated by two-way analysis of variance. Results are expressed as the mean ± SD (n = 6) of four independent experiments. N.S.: not significant; ** p < 0.01. (D) Quantification of Nissl-positive cells in the DG. Differences between RTM and CEM, RTF and CEF, and CEM and CEF groups were evaluated by two-way analysis of variance. Results are expressed as the mean ± SD (n = 6) of four independent experiments. N.S.: not significant; *** p < 0.001.

Fig 5: MiR-92a-2-5p modulates the differentiation of ASD-NPCs. (A,B) Representative images of TUJ1 (green)-expressing neurons derived from ASD NC and ASD inhibitor NPCs at day 10. DAPI (blue) was used to stain nuclei. The percentage of TUJ1-labeled cells was calculated. (C,D) Representative images of MAP2 (green)-expressing neurons derived from ASD NC and ASD inhibitor NPCs at day 20. The percentage of MAP2-labeled cells was calculated. (E–G) Representative images of MAP2- (green) and VGAT (red)-expressing neurons derived from ASD NC and ASD-inhibitor NPCs at day 30. The percentage of MAP2- and VGAT-labeled cells was calculated. (H) Relative mRNA expression of SYP, PSD95, MAP2, and VGAT in ASD NC and ASD inhibitor NPCs at day 30, as determined by RT-qPCR. All data represent mean ± SEM; scale bar: 50 µm; *** p < 0.001, ** p < 0.01, * p < 0.05.