Study of the mechanism of sonodynamic therapy in a rat glioma model

Figures & data

Figure 1 Schematic diagram of the insonation device.

Figure 2 Effect of sonodynamic therapy (SDT) treatment on tumor volume and animal survival time. The tumor volume was measured with conventional light microscopy before treatment and after treatment on Days 3, 7, and 14 (A). Animal survival time was calculated as the number of days until the animal died of natural causes or were anesthetized (B). If a glioma had developed for more than 60 days, survival time was counted as 60 days.

Notes: (A) Data are presented as the mean ± standard deviation (SD) (n=8); ^#P<0.01 versus control. (B) Data are presented as the mean ± SD (n=8); *P<0.05 versus control; **P<0.01.
Abbreviations: HMME, hematoporphyrin monomethyl ether; US, ultrasound.

Figure 3 Evaluation of treatments by magnetic resonance imaging (MRI) (A–D), hematoxylin and eosin (HE) staining (E–H), and transmission electron microscopy (TEM) (I–L). Treated by sonodynamic therapy (SDT) (A, E, I), ultrasound (B, F, J), hematoporphyrin monomethyl ether (HMME) (C, G, K), and untreated control (D, H, L).

Notes: HE staining of the SDT-treated brain sections revealed that the area of irregular lamellar necrosis was clearly enlarged (E). In the center of the tumor necrosis, no cell structures were found [indicated by thick arrow]; instead, a large amount of debris and shrunken glioma cells were discovered at the boundary of necrosis and normal tumor tissue [indicated by thin arrow]. In the ultrasound-treated brain section (F) sporadic punctiform necrosis could still be observed in the transitional area between the necrosis and the normal tumor tissue, indicated by an arrow. HMME-treated (G) and untreated (H) glioma at 24 hours after treatment (×40). TEM images of glioma at 24 hours after treatment showing nuclear apoptosis (I) and necrosis (J), and treatment with ultrasound showing nuclear apoptosis (K) and necrosis (L) (×6,000).

Figure 4 Effect of sonodynamic therapy (SDT) on apoptosis detected by TUNEL assay. The images were taken at 24 hours after treatment with SDT (A), ultrasound (US) (B), and hematoporphyrin monomethyl ether (HMME) (C), or after no treatment (D). Summary of in situ apoptosis assay at different times (24 hours, 3 days, and 7 days) after the indicated treatments (E).

Notes: Data are presented as the mean ± standard deviation (n=8); *P<0.01 versus control. Apoptotic cells are indicated by the arrows.
Abbreviation: TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 5 Effect of sonodynamic therapy (SDT) treatment on the expression of protein cytochrome-c (Cyto-C) and protein caspase-3. Immunohistochemical staining with Cyto-C antibody at 24 hours after treatment with SDT (A), ultrasound (US) (B), and hematoporphyrin monomethyl ether (HMME) (C), or after no treatment (D). Summary of the effect of SDT on protein Cyto-C (E). Immunohistochemical staining with caspase-3 antibody at 24 hours after treatment with SDT (G), US (H), and HMME (I), or no treatment (J). Summary of the effect of SDT on protein caspase-3 (F).

Notes: Data are presented as the mean ± standard deviation (n=8); *P<0.01 versus control. Positively stained cells are indicated by arrow.

Figure 6 Effect of sonodynamic therapy (SDT) treatment on glioma angiogenesis. Immunohistochemical staining with CD34 antibody at 24 hours (A), 3 days (B), and 7 days (C) after treatment with SDT. Summary of the effect of SDT, US and HMME on microvessel density (MVD) evaluated by counting CD34-positive microvessels (D). Immunohistochemical staining with vascular endothelial growth factor (VEGF) antibody at 24 hours (E), 3 days (F), and 7 days (G) after treatment with SDT. Summary of the effect of SDT, US and HMME on VEGF levels (H).

Notes: Data are presented as the mean ± standard deviation (n=8); *P<0.01 versus control.
Abbreviations: US, ultrasound; HMME, hematoporphyrin monomethyl ether.