Postinjury Inhibition of miR-181a Promotes Restoration of Hippocampal CA1 Neurons after Transient Forebrain Ischemia in Rats

Hippocampal increased GFAP expression in the CA1 pyramidal layer following forebrain ischemia. A, Fluorescent labeling of GFAP after forebrain ischemia in the hippocampus (50× magnification). B, Enlarged (200×) example of CA1 pyramidal layer at 14 d after forebrain ischemia. C, Postinjury quantification of GFAP⁺ cells in CA1. N = 3-6/group, *p < 0.05 compared with sham, #p < 0.05 compared with every group. Error bars are mean ± SEM.

Effect of postinjury miR-181a antagomir treatment on CA1 NeuN and DCX precursor expression and long-term CA1 neuronal recovery following forebrain ischemia. A, Fluorescently labeled (green, 6-FAM) miR-181a antagomir is detected in ipsilateral hippocampus (solid arrowhead), but not in contralaterally injected hippocampus (empty arrowhead). Magnified views of contralateral and ipsilateral CA1 following injection of 6-FAM-labeled miR-181a antagomir. B, C, Examples (B) and quantification (C) of ipsilateral hippocampal NeuN fluorescent labeling 14 d after forebrain ischemia in postinjury MM-con-treated and antagomir-treated animals. D, E, Examples (D) and quantification (E) of ipsilateral hippocampal NeuN fluorescent labeling at 28 d postinjury in MM-con-treated and antagomir-treated animals. F, G, Examples (F) and quantification (G) of ipsilateral CA1 DCX⁺ cells 14 d after forebrain ischemia with MM-con or antagomir treatment 2 h or 7 d after forebrain ischemia. N = 5-6 for each condition. *p < 0.05 compared with sham, #p < 0.05 compared with every group. Error bars are mean ± SEM.

Postinjury CDON expression in CA1 following forebrain ischemia. A, Immunofluorescent labeling of CDON expression (green) and all nuclei (DAPI) in CA1 14 d after forebrain ischemia with MM control treatment or 2 h or 7 d postinjury antagomir treatment. B, Quantification of CDON in CA1 14 d after forebrain ischemia with the three treatment groups. N = 4 for each condition, *p < 0.05 difference versus sham. Error bars are mean ± SEM. n.s. = not significant.

Fate-mapping postinjury DCX expression following forebrain ischemia. A, Diagram and map of Cre-Lox GFAP/DCX fate-mapping plasmid. A floxed GFAP promoter EGFP (green) reporter and a DCX promoter controlling Cre and mCherry (red) reporter are included on the same plasmid. In the absence of DCX activity, astrocytes fluoresce green. With DCX activation, mCherry is expressed and EGFP expression is simultaneously terminated. B, C, Sham animals display very little new EGFP expression in CA1 after transfection, and no mCherry. D, E, Following forebrain ischemia (7 d), colocalization of EGFP/c-Tomato is evident in CA1, suggesting de novo expression of DCX in GFAP-expressing cells. The graphs indicate relative expression of DCX⁺ only, GFAP⁺ only, and DCX/GFAP⁺ double-positive cells. Error bars are mean ± SEM.