Ccl5 mRNA expression is upregulated and miR-324-5p is downregulated in the peri-infarct cortex of MCAO mice.

(A) Illustration of the animal experimental protocol. (B) Schematic diagram of the brain indicating IC, IP, and CP cortical regions analyzed in this study. (C and D) qRT-PCR analysis of Ccl5 mRNA (C) and miR-324-5p (D) expression in the IP and CP regions on D1, D3, and D7 after MCAO, and in sham mice (n=6). (E) Representative images of combined FISH for Ccl5 mRNA or miR-324-5p and immunofluorescence for GFAP or IBA1 in the IP region on D3 after MCAO. Scale bar: 100 μm. (F and G) Colocalization rate of Ccl5 mRNA probe (F) or miR-324-5p probe (G) with GFAP-positive astrocytes or IBA1-positive microglia (n=10). Statistical comparisons in C and D were performed by two-way ANOVA with Tukey’s post-hoc test. In F and G, comparisons between astrocytes and microglia were made by unpaired Student’s t-test.

CCL5 inhibition by intracerebral antibody delivery and CCR5 blockade attenuate ischemic stroke injury.

(A) ELISA quantification of CCL5 concentrations in the IP and CP regions of BSA-, CCL5 antibody-, rCCL5-, and MVC-treated mice on D3 and D7 after MCAO (n=6). (B and C) Representative TTC-stained brain sections (B) and infarct volume (C) of BSA-, CCL5 antibody-, rCCL5-, and MVC-treated mice on D3 after MCAO (n=6). Scale bar: 1cm. (D) Longa scores in BSA-, CCL5 antibody-, and rCCL5-treated mice, and sham controls, from D0 to D7 after MCAO (n=12). (E) Rotarod performance of BSA-, CCL5 antibody-, and rCCL5-treated mice, and sham controls, on D0, D3, and D7 after MCAO (n=7). (F) Representative immunofluorescence images of GFAP and IBA1 co-labeling in the IP cortex of BSA-, CCL5 antibody-, and rCCL5-treated mice, and in the CP cortex of BSA-treated mice, on D3 and D7 after MCAO. Scale bar: 50 μm. (G and H) Surface area of GFAP-positive astrocytes in the IP region of BSA-, CCL5 antibody-, and rCCL5-treated mice on D3 (G) and D7 (H) after MCAO (n=10). (I and J) Surface area of IBA1-positive microglia in the IP region of BSA-, CCL5 antibody-, and rCCL5-treated mice on D3 (I) and D7 (J) after MCAO (n=10). (K) Representative tracings of Golgi-stained cortical neurons illustrating dendritic morphology in the IP cortex of BSA-, CCL5 antibody-, and rCCL5-treated mice, and in the CP cortex of BSA-treated mice, on D3 after MCAO. Scale bar: 50 μm. (L) Sholl analysis of the dendritic branch complexity in the IP region of BSA-, CCL5 antibody-, and rCCL5-treated mice on D3 after MCAO (n=10). (M) Representative images of basal dendritic spines in the IP cortex of BSA-, CCL5 antibody-, and rCCL5-treated mice, and in the CP cortex of BSA-treated mice, on D7 after MCAO. Arrows, mushroom spines; black arrowheads, stubby spines; white arrowheads, thin spines. Scale bar: 10 μm. (N and O) Total spine density (N) and mushroom spine density (O) in the IP and CP regions of BSA-, CCL5 antibody-, and rCCL5-treated mice on D7 after MCAO (n=10). Stacked columns represent total spine density, with colored segments indicating the proportions of mushroom, stubby, and thin spines. Statistical comparisons in A, D, E, and L were performed by two-way ANOVA with Tukey’s post-hoc test; all other panels by one-way ANOVA with Tukey’s post-hoc test.

Intracerebral miR-324-5p agomir delivery reduces ischemic stroke injury.

(A) ELISA quantification of CCL5 concentrations in the IP and CP regions of NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated mice on D3 and D7 after MCAO (n=6). (B and C) Representative TTC-stained brain sections (B) and infarct volume (C) in sham, NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated mice on D3 after MCAO (n=6). Scale bar: 1cm. (D) Longa scores in NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated mice, and sham controls, from D0 to D7 after MCAO (n=15). (E) Rotarod performance of NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated mice, and sham controls, on D0, D3, and D7 after MCAO (n=7). (F) Representative immunofluorescence images of GFAP and IBA1 co-labeling in the IP cortex of NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated mice, and in the CP cortex of NC agomir-treated mice, on D3 and D7 after MCAO. Scale bar: 50 μm. (G and H) Density of GFAP-positive astrocytes (cells per 105 μm2) in the IP and CP regions of NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated mice on D3 (G) and D7 (H) after MCAO (n=6). (I and J) Surface area of GFAP-positive astrocytes in the IP region of NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated mice on D3 (I) and D7 (J) after MCAO (n=10). (K and L) Surface area of IBA1-positive microglia in the IP and CP regions of NC agomir-,miR-324-5p agomir-, and miR-324-5p antagomir-treated mice on D3 (K) and D7 (L) after MCAO (n=10). (M) Representative tracings of Golgi-stained cortical neurons illustrating dendritic morphology in the IP cortex of NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated mice, and in the CP cortex of NC agomir-treated mice, on D3 after MCAO. Scale bar: 50 μm. (N) Sholl analysis of dendritic branch complexity in the IP region of NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated mice on D3 after MCAO (n=8). (O) Representative images of basal dendritic spines in the IP cortex of NC agomir-,miR-324-5p agomir-, and miR-324-5p antagomir-treated mice, and in the CP cortex of NC agomir-treated mice, on D7 after MCAO. Arrows, mushroom spines; black arrowheads, stubby spines; white arrowheads, thin spines. Scale bar: 10 μm. (P and Q) Total spine density (P) and mushroom spine density (Q) in the IP and CP regions of NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated mice on D7 after MCAO (n=10). Stacked columns represent total spine density, with colored segments indicating the proportions of mushroom, stubby, and thin spines. Statistical comparisons in A, D, E, and N were performed by two-way ANOVA with Tukey’s post-hoc test; all other panels by one-way ANOVA with Tukey’s post-hoc test.

CCL5 inhibition and CCR5 blockade protect cortical neurons from OGD injury in astrocyte-neuron co-cultures.

(A) Schematic of the astrocyte-neuron co-culture and OGD experimental design. (B) ELISA quantification of CCL5 concentrations in the medium of BSA-, CCL5 antibody-, rCCL5-, and MVC+rCCL5-treated co-cultures, and in un-OGD-treated controls (n=5). (C) Representative immunofluorescence images of NeuN and TUNEL co-labeling. Scale bar: 20 μm. (D and E) Proportions of apoptotic cells (D) and NeuN-positive neurons (E) in BSA-, CCL5 antibody-, rCCL5-, and MVC+rCCL5-treated co-cultures (n=10). (F and G) Representative tracings of MAP2-stained neurons (F) and Sholl analysis (G) of neuronal dendritic morphology in BSA-, CCL5 antibody-, rCCL5-, and MVC+rCCL5-treated co-cultures (n=10). Scale bar: 50 μm. (H and I) Representative immunofluorescence images of MAP2 and SYN1 co-labeling (H) and synapse puncta density (I) in BSA-, CCL5 antibody-, rCCL5-, and MVC+rCCL5-treated co-cultured neurons (n=10). Scale bar: 10 μm. Statistical comparisons in G were performed by two-way ANOVA with Tukey’s post-hoc test; asterisks positioned above indicate anti-CCL5 vs. BSA comparisons, and those positioned below indicate MVC+rCCL5 vs. BSA comparisons. All other panels were analyzed by one-way ANOVA with Tukey’s post-hoc test.

Overexpression of miR-324-5p protects cortical neurons from OGD injury in astrocyte-neuron co-cultures.

(A) ELISA quantification of CCL5 concentrations in the medium of NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated co-cultures, and in un-OGD-treated controls (n=5). (B) Representative immunofluorescence images of NeuN and TUNEL co-labeling. Scale bar: 20 μm. (C and D) Proportions of apoptotic cells (C) and NeuN-positive neurons (D) in NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated co-cultures (n=10). (E and F) Representative tracings of MAP2-stained neurons (E) and Sholl analysis (F) of neuronal dendritic morphology in NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated co-cultures (n=10). Scale bar: 50 μm. (G and H) Representative immunofluorescence images of MAP2 and SYN1 co-labeling (G) and synapse puncta density (H) in NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated co-cultured neurons (n=10). Scale bar: 10 μm. Statistical comparisons in F were performed by two-way ANOVA with Tukey’s post-hoc test; all other panels by one-way ANOVA with Tukey’s post-hoc test.

CCL5 antibody delivery and CCR5 blockade upregulate ERK/CREB signaling in the ipsilateral cortex of MCAO mice.

(A) Representative Western blot images of p-ERK, t-ERK, p-CREB, and t-CREB in cortical lysates from the IP and CP regions of BSA-, CCL5 antibody-, rCCL5-, and MVC-treated mice on D3 after MCAO. (B and C) Ratios of p-ERK to t-ERK (B), and p-CREB to t-CREB (C) in the IP and CP regions of BSA-, CCL5 antibody-, rCCL5-, and MVC-treated mice on D3 after MCAO (n=6). Protein levels were normalized to the CP region of BSA-treated group. Statistical comparisons were performed by two-way ANOVA with Tukey’s post-hoc test.

CCL5 inhibition and CCR5 blockade upregulate the neuronal ERK/CREB pathway after OGD injury.

(A) Schematic of the ACM-neuron co-culture experimental design for panels B-D. (B) Representative Western blot images of p-ERK, t-ERK, p-CREB, and t-CREB in lysates from BSA-, CCL5 antibody-, rCCL5-, and MVC+rCCL5-treated neurons. (C and D) Ratios of p-ERK to t-ERK (C), and p-CREB to t-CREB (D) in BSA-, CCL5 antibody-, rCCL5-, and MVC+rCCL5-treated neuronal lysates (n=6). Protein levels were normalized to the BSA group. (E) Schematic of the ACM-neuron co-culture experimental design for panels F-K. (F) Representative Western blot images of p-ERK, t-ERK, p-CREB, and t-CREB in neurons co-cultured with BSA OGD-ACM, anti-CCL5 OGD-ACM, or rCCL5 OGD-ACM. (G and H) Ratios of p-ERK to t-ERK (G), and p-CREB to t-CREB (H) in neurons co-cultured with BSA OGD-ACM, anti-CCL5 OGD-ACM, or rCCL5 OGD-ACM (n=5). Protein levels were normalized to the BSA+DMSO group. (I) Representative Western blot images of p-ERK, t-ERK, p-CREB, and t-CREB in neurons co-cultured with NC agomir OGD-ACM, miR-324-5p agomir OGD-ACM, or miR-324-5p antagomir OGD-ACM. (J and K) Ratios of p-ERK to t-ERK (J), and p-CREB to t-CREB (K) in neurons co-cultured with NC agomir OGD-ACM, miR-324-5p agomir OGD-ACM, or miR-324-5p antagomir OGD-ACM (n=5). Protein levels were normalized to the NC agomir+DMSO group. Statistical comparisons in C and D were performed by one-way ANOVA with Dunnett’s post-hoc test; all other panels by one-way ANOVA with Tukey’s post-hoc test.

Proposed model of neuroprotection by astrocytic miR-324-5p through CCL5 inhibition and regulation of neuronal CCR5/ERK/CREB signaling after ischemic stroke.

(A and B) Density of GFAP-positive astrocytes (cells per 10 µm²) in the IP and CP regions of BSA-, CCL5 antibody-, and rCCL5-treated mice on D3 (A) and D7 (B) after MCAO (n=5). (C and D) Density of IBA1-positive microglia (cells per 10 µm²) in the IP and CP regions of BSA-, CCL5 antibody-, and rCCL5-treated mice on D3 (C) and D7 (D) after MCAO (n=6). (E) Sholl analysis of dendritic branch complexity in the IP region of BSA-, CCL5 antibody-, and rCCL5-treated mice on D7 after MCAO (n=10). (F) Sholl analysis of dendritic branch complexity in the CP region of BSA-, CCL5 antibody-, and rCCL5-treated mice on D7 after MCAO (n=10). (G and H) Total spine density (G) and mushroom spine density (H) in the IP and CP regions of BSA-, CCL5 antibody-, and rCCL5-treated mice on D3 after MCAO (n=10). Stacked columns represent total spine density, with colored segments indicating the proportions of mushroom, stubby, and thin spines. Statistical comparisons in A-D, G, and H were performed by one-way ANOVA with Tukey’s post-hoc test. In E, statistical comparisons were performed by two-way ANOVA with Tukey’s post-hoc test.

(A and B) Density of IBA1-positive microglia (cells per 10 µm²) in the IP and CP regions of NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated mice on D3 (A) and D7 (B) after MCAO (n=5 in A, n=6 in B). (C) Sholl analysis of dendritic branch complexity in the IP region of NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated mice on D7 after MCAO (n=10). (D) Sholl analysis of dendritic branch complexity in the CP region of NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated mice on D7 after MCAO (n=10). (E and F) Total spine density (E) and mushroom spine density (F) in the IP and CP regions of NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated mice on D3 after MCAO (n=10). Stacked columns represent total spine density, with colored segments indicating the proportions of mushroom, stubby, and thin spines. Statistical comparisons in A, B, E, and F were performed by one-way ANOVA with Tukey’s post-hoc test. In C, statistical comparisons were performed by two-way ANOVA with Tukey’s post-hoc test.

(A) Sholl analysis of dendritic branch complexity in BSA-, CCL5 antibody-, and rCCL5-treated co-cultured neurons on D3 after OGD (n=15). (B) Representative immunofluorescence images of GFAP labeling in astrocyte-neuron co-cultures following agomir-Cy3 transfection (left panel) and agomir transfection rate at D3 and D6 in co-cultured cells (right panel). (C) Sholl analysis of dendritic branch complexity in NC agomir-, miR-324-5p agomir-, and miR-324-5p antagomir-treated co-cultured neurons on D3 after OGD (n=10). Statistical comparisons in A and C were performed by two-way ANOVA with Tukey’s post-hoc test.

Characterization of primary cortical astrocyte purity.

(A) Representative immunofluorescence images of GFAP co-labeled with Tuj1, Olig2, or Iba1 in P0 and P1 primary cortical astrocytes in vitro. Scale bar: 250 µm (low magnification) and 75 µm (high magnification). (B) Proportions of Tuj1-positive, Olig2-positive, and Iba1-positive cells among total DAPI-positive cells in P0 and P1 astrocyte cultures (n=6). Statistical comparisons between P0 and P1 were performed by unpaired t-test.