Calculation of infarct volume and swelling

Experimental protocol for calculation of infarct volume and swelling, TUNEL/NeuN staining, immunofluorescence, and Western blotting is shown in Figure 2(a). Mice were sacrificed 72 h after reperfusion, and their brains were quickly removed and processed for TTC staining and calculation of cerebral infarct volume. Ischemic lesions were traced in each slice in a blinded manner, and the total volume of infarction was calculated with correction for edema using ImageJ software. The infarct volume was calculated by subtracting the area of the noninfarcted area in the ischemic hemisphere from the area of the respective contralateral hemisphere. Volume calculation with edema correction was performed blindly using the following formula: swelling (% contralateral hemispheric volume) = (contralateral hemisphere volume − noninfarct ipsilateral hemisphere volume)/contralateral hemisphere volume × 100. In this experiment, there were 12 animals in each group, and the three-day survival rate was as follows: no animals died in the sham group (100%), two mice died in the MCAO group (83.3%), two mice died in the MCAO + microRNA-494 agomir group (83.3%), two mice died in the MCAO + microRNA-494 antagomir group (83.3%), and one mouse died in the MCAO + microRNA-494 antagomir + RGFP966 group (91.7%).

MiR-494 protected the brain against mouse ischemic injury in the acute stage and recovery stage. (a) Experimental protocol. (b) FAM-labeled miR-494 agomir with green fluorescence was used to assess neuronal uptake (red) after ICV delivery. Green fluorescence was mainly distributed in neurons (arrows) in the brain tissue. (c) QuantitativePCR was performed to verify the efficacy of miR-494 agomir and antagomir transfection. n = 6/group. (d) Cerebral infarct volume and brain edema evaluated by TTC staining of coronal brain sections. n = 10–12/group. (e–f) Neuronal apoptosis in the ipsilateral cortex as detected by TUNEL/NeuN immunofluorescence double staining. n = 6 per group. (g) Immunoblot of MAP-2 in the cortex in the recovery stage. n = 6/group. MCAO + control denotes mice subjected to 45 min ischemia followed by reperfusion for three days, with control administered by ICV injection immediately after ischemia; MCAO + miR-494 denotes mice subjected to 45 min ischemia followed by reperfusion for three days, with miR-494 agomir administered by ICV injection immediately after ischemia. *P < 0.05, **P < 0.01, ***P < 0.001 vs. sham group. ^#P < 0.05 vs. MCAO + control group. One-way ANOVA with Tukey–Kramer post hoc test.