Figure 1.
Evaluation of infarct volume and neurological scores in CIRI model rats.
(A) TTC-stained brain sections showing the ischemic region (white) and the infarct area (red). (B) Infarct volume in the five experimental groups. (C) Neurological outcomes assessed by the mNSS. The three treatment groups (A, EA, and ED) demonstrated significant improvements in neurological function compared with MCAO model group M. Quantitative data (n = 8 animals per group) are given as the mean ± the SD. △P<0.05 vs. sham group S; ☆P<0.05 vs. MCAO model group M; *P<0.05 vs. acupuncture group A; ΦP<0.05 vs. electroacupuncture group EA; ΟP<0.05 vs. edaravone group ED.
Figure 2.
Evaluation of histopathological changes and neuronal damage in the ischemic penumbra and the core zone by H&E and NeuN staining.
(A, B, C) H&E staining showing gross histopathological changes in normal, ischemic penumbra and the ischemic core at 24 h after reperfusion. The black arrows in (B, C) indicate significant nuclear shrinkage, and the red arrows indicate neuronal vacuolization. Scale bar = 50 µm.
Figure 3.
Evaluation of neuronal loss in the ischemic penumbra and the core zone by NeuN staining.
NeuN immunohistochemical staining showing neuronal nuclei changes in the ischemic penumbra and the core zone at 24(black arrows) and neuronal vacuolization (red arrows) in group M. Scale bar = 50 µm.
Figure 4.
Effects of acupuncture and electroacupuncture on mRNA expression levels of MMP2, AQP4, and AQP9.
Analysis of MMP2, APQ4, and APQ9 mRNA expression levels by qPCR. GAPDH was used as an internal control. Lower mRNA expression levels of MMP2, AQP4, and AQP9 were exhibited in group S vs. groups M, A, EA, and ED. The mRNA expression levels of all three target proteins were significantly lower in groups A and EA than in group M. Quantitative data (n = 3) are given as the mean ± the SD. △P<0.05 vs. group S; ☆P<0.05 vs. group M; *P<0.05 vs. group A; ΦP<0.05 vs. group EA; ΟP<0.05 vs. group ED.
Figure 5.
Colocalization of MMP2 and AQP4 with GFAP and CD34 in the ischemic penumbra.
Immunofluorescence labeling for the MMP2, AQP4, GFAP, and CD34 expression levels in the ischemic hemisphere of MCAO model group M rats. MMP2 (red, A) and GFAP (green, B) staining and the MMP2/GFAP merge (C) show MMP2 expression on astrocyte endfeet; MMP2 (red, D) and CD34 (green, E) staining and the MMP2/CD34 merge (F) show MMP2 expression on endothelial cells; AQP4 (red, G) and GFAP (green, H) staining and the AQP4/GFAP merge (I) show AQP4 expression on astrocyte endfeet; and AQP4 (red, J) and CD34 (green, K) staining and the AQP4/CD34 merge (L) show AQP4 expression on endothelial cells. The yellow staining (arrows) in panels (C), (F), (I) and (L) indicates the colocalization of both antigens in the merged images. Scale bar = 50 µm.
Figure 6.
Protein expression of MMP2, AQP4, and AQP9 in the ischemic penumbra and the core zone.
MMP2, AQP4, and AQP9 immunoreactivity (A, B, D, E, G, and H) and integrated optical density of MMP2/AQP4/AQP9 labeling (C, F, and I). Quantitative data (n = 6) are given as the mean ± the SD. △P<0.05 vs. group S; ☆P<0.05 vs. group M; *P<0.05 vs. group A; ΦP<0.05 vs. group EA; ΟP<0.05 vs. group ED. Arrows indicate the immunoreactive area for each target protein. Scale bar in (A) = 50 µm.
Figure 7.
Western blot analysis of MMP2, AQP4, and AQP9 protein expression in the lesion boundary zone of the ipsilateral hemisphere.
(A) Western blot analysis of MMP2, AQP4, and APQ9. (B–D) Relative intensity of MMP2 (B), APQ4 (C), and AQP9 (D) in each of the five rat groups. Relative intensity is defined as the intensity of the target protein normalized to that of β-actin. Quantitative data (n = 4) are given as the mean ± the SD. △P<0.05 vs. group S; ☆P<0.05 vs. group M; *P<0.05 vs. group A; ΦP<0.05 vs. group EA; ΟP<0.05 vs. group ED.
Figure 8.
Quantification of MPO+ and CD68+ cells in the ischemic penumbra and the core zone.
Representative photomicrographs showing immunohistochemical staining for MPO and CD68 in the ischemic penumbra (A, D) and the core zone (B, E) at 24 h after reperfusion. Brown spots indicate MPO+ cells, the majority of which were present within the ischemic core vs. the ischemic penumbra for all five rat groups. (B, E) The majority of CD68+ cells were also present in the core zone. (C, F) Bar graphs showing the quantification of MPO+ and CD68+ cells. The numbers of MPO+ and CD68+ cells were significantly higher in both the ischemic penumbra and the core zone for group M vs. the other four groups, whereas no significant differences were found between groups A and EA. Quantitative data (n = 6) are given as the mean ± the SD. △P<0.05 vs. group S; ☆P<0.05 vs. group M; *P<0.05 vs. group A; ΦP<0.05 vs. group EA; ΟP<0.05 vs. group ED. Arrows in (A), (B), (D), and (E) show the MPO+ and CD68+ immunoreactive cells. Scale bar = 50 µm.