Low-intensity pulsed ultrasound therapy promotes recovery from stroke by enhancing angio-neurogenesis in mice in vivo

Figure 1. Whole-brain LIPUS therapy improves functional recovery after MCAO. (a) The timeline of the experiments. (b) Schematic drawing showing the application of whole-brain LIPUS in mice. (c) Tightrope and rotarod tests were performed on days 7, 14, 21, and 28 following MCAO or sham surgery; n = 10. *P < 0.05 (C; two-way ANOVA, D; Student’s t-test), †P < 0.05 (two-way ANOVA). (d) Cresyl violet staining was performed on day 28 following MCAO or sham surgery; n = 3 (sham), n = 8 (MCAO). The scale bar represents 100 μm. *P < 0.05 (Student’s t-test).

Figure 2. The LIPUS therapy promotes angiogenesis and upregulates angiogenesis- related molecules. (a) Schematic drawing showing the ischemic hemisphere section and 4 regions related with the lesion at (i) 40–250 μm, (ii) 250–500 μm, (iii) 500–750 μm, and (iv) 750–1000 μm from the SVZ. Representative images of ischemic hemisphere sections at 500–750 μm from the SVZ immunostained for CD31 (endothelial cell marker) on days 7, 14, and 28. The scale bar represents 50 μm. (b) Schematic drawing showing 4 lesions of interest at different distances from the SVZ. The numbers of CD31-positive vessels in the indicated lesions at days 7, 14, and 28 (n = 8 per group). (c) RT-qPCR analysis of eNOS and VEGF at day 3 (n = 14 per group). (d) Representative Western blot and quantification of phosphorylated eNOS at Ser1177 and VEGF at day 5 (n = 10 per group). *P < 0.05 (Student’s t-test). Uncropped blots are shown in Fig. S10.

Figure 3. The LIPUS therapy promotes neurogenesis and upregulates the molecules related to neuronal migration. (a) Schematic drawing showing the coronal section of the mouse brain and lesion areas of interest. Representative images of ischemic hemisphere sections immunostained for DCX on days 7, 14, and 28. The scale bar represents 100 μm. (b) Schematic drawing showing the ischemic hemisphere section and 4 regions related with the lesion at (i) 40–250 μm, (ii) 250–500 μm, (iii) 500–750 μm, and (iv) 750–1000 μm from the SVZ. The numbers of DCX-positive cells in the indicated regions at day 7, 14, and 28 (n = 8 per group). (c) RT-qPCR analysis of SDF-1, CXCR4, and BDNF at day 3 (n = 14 per group). (d) Representative Western blot and quantification of SDF-1, CXCR4 and BDNF at day 5 (n = 10 per group). *P < 0.05 (Student’s t-test). Uncropped blots are shown in Fig. S10.

Figure 4. Crucial role of eNOS for the beneficial effects of LIPUS in the MCAO model. (a) Behavioral tests were performed on days 7, 14, 21, and 28 in the Non-LIPUS and LIPUS-treated eNOS−/− mice subjected to MCAO (n = 6 per group). (b) Cresyl violet staining was performed on day 28 following MCAO (n = 6 per group). (c) Representative images in the peri-infarct lesion immunostained for CD31 on day 28. The scale bar represents 50 μm. (d) The number of CD31-positive vessels at (i) 40–250 μm, (ii) 250–500 μm, (iii) 500–750 μm, and (iv) 750–1000 μm from the SVZ on day 28 (n = 6 per group). (e) Representative images of the ischemic striatum immunostained for DCX on day 28. The scale bar represents 100 μm. (f) The numbers of DCX-positive cells at (i) 40–250 μm, (ii) 250–500 μm, (iii) 500–750 μm, and (iv) 750–1000 μm from the SVZ at day 28 (n = 6 per group). (g) Western blot analysis of VEGF and CXCR4 on day 5 (n = 6 per group). Uncropped blots are shown in Fig. S10.

Figure 5. Schematic diagram showing the proposed mechanisms underlying the beneficial effects of whole-brain LIPUS in the MCAO model in mice. The present study demonstrates that the LIPUS therapy promotes functional recovery in MCAO mouse model, and that the beneficial effects of the LIPUS therapy depend on endothelial NO synthase (eNOS). Both angiogenesis and neurogenesis were promoted by the LIPUS therapy sequentially in this order, indicating that VEGF and eNOS-induced angiogenesis subsequently upregulates neurotrophins, including SDF-1, and CXCR4, and these molecules mediate neuronal proliferation and migration. Additionally, increased vessel density supports neuronal migration structurally. Finally, neuronal functional recovery can be improved by differentiated neural stem cells.