TT reduces the permeability of BSCB after SCI
We used an impactor to generate a model of SCI (Fig. 1a). Meanwhile, EB staining and statistical analysis of spinal cord water content were used to detect the function of BSCB. The results showed that the water content increased significantly after SCI, and TT could reduce the oedema caused by SCI (Fig. 1b-c) (M vs. S: P7 < 0.001, P14 < 0.001; TM vs. M: P7 =0.008, P14 < 0.001). The amount of EB exudation increased significantly after SCI compared with that of group S, suggesting BSCB leakage (M vs. S: P7 < 0.001, P14 < 0.001). After TT training, infiltration of the BSCB was significantly improved. (Fig. 1d-e) (TM vs. M: P7 < 0.001, P14 < 0.001). The fluorescence intensity of EB after SCI was much higher than that in the S group. However, the fluorescence level of the TM group was significantly lower than that of the M group (Fig. 1f-g) (TM vs. M: P7 < 0.001, P14 < 0.001). All these data indicate that TT can inhibit BSCB disruption.
Fig. 1 TT reduces the permeability of BSCB after SCI. (a) SCI modelwas performed by NYU Impactor (10g × 20cm). (b, c) Representative quantification data of spinal cord water content in S M TM groups, columns represent mean ± SD, n=5. (d, e) Represent EB dye permeabilized into spinal cord after SCI and quantification of the amount of Evan’s Blue (ug/g), n =5. (f-g) Representative fluorescent images of Evans Blue Dye extravasation and quantification of the fluorescence intensity, n=5. #p < 0.05 as M group versus S group, *p < 0.05 as TM group versus M group. (#p, *p < 0.05; ##p, **p < 0.01; ###p, ***p < 0.001)
TT decreased tissue structure damage and improved functional recovery after SCI
At 7 d and 14 d after injury, histomorphological differences in T9-T11 levels were observed by HE staining (Fig. 2a). The arrangement of tissues in group S was normal, the grey matter and white matter were destroyed to varying degrees in group M, which was accompanied by the death of multiple neurons (Fig. 2e); the results in group TM was significantly better than that in group M. The quantitative analysis of the cavity area showed the same results (Fig. 2b) (TM vs. M: Coronal section, P7 =0.002, P14 =0.023; Sagittal section, P7 =0.008, P14 =0.002). The functional recovery of the S, M and TM groups was evaluated by BBB scores at 6 h, 1 d, 3 d, 7 d, and 14 d after SCI (Fig. 2c). The BBB scores of the TM group were significantly higher than those of the M group at 7 d and 14 d after SCI (TM vs. M: P7 =0.036, P14=0.012). We further tested the relationship between apoptosis after SCI with TT (Fig. 2d). The number of apoptotic cells in the epicenter increased significantly after SCI. Compared with the M group, the number of apoptotic cells in the TM were significantly decreased (M vs. s: P<0.001; TM vs. M: P=0.007). These results show that TT can significantly improve functional recovery and tissue preservation.
Fig. 2 TT decreased tissue structure damage and improved functional recovery after SCI. (a) HE staining at 7 d and 14 d after SCI. Scale bars are 500 µm. (b) Quantification of the size of cavity area, columns represent mean ± SD, n=5. (c) BBB scores in S, M, TM groups. (d) TUNEL staining in the epicenter. (e) Quantitative estimation positive cells. Columns represent mean ± SD, n= 5. #p < 0.05 as M group versus S group, *p < 0.05 as TM group versus M group. (#p, *p < 0.05; ##p, **p < 0.01; ###p, ***p < 0.001)
TT prevents the loss of tight junction(TJ) and adhesion junction (AJ) proteins
To further determine the effect of TT on BSCB protein, AJ protein (β-Catenin, p120-Catenin), TJ protein (Claudin-5, and Occludin) and ZO-1, were examined by Western blot. According to the results (Fig. 3a), the expression of TJ and AJ protein decreased to some extent after SCI compared to the levels of the control (M vs. S: β-Catenin, p120-Catenin, Claudin-5, Occludin ZO-1, P < 0.001). However, the rats treated with TT showed high TJ and AJ protein expression at 7 d (Fig. 3b) (TM vs. M: β-Catenin, P =0.037; p120-Catenin, P = 0.003; Claudin-5, P < 0.001; Occludin, P =0.004; ZO-1, P < 0.001). Using Occludin/CD31/Hoechst (Fig. 3c); Claudin-5/CD31/Hoechst (Fig. 3d); p120-Catenin /CD31/Hoechst (Fig. 4a) and β-Catenin /CD31/Hoechst (Fig. 4b) staining to observe the distribution of BSCB proteins after SCI, we found that TT could reduce the degradation of Claudin-5/Occludin/p120-Catenin/β-Catenin around the epicenter. These results suggest that TT can prevent the loss of TJ and AJ proteins after SCI.
Fig. 3 TT prevent the loss of TJ and AJ protein. (a, b) Represent western blots and quantification data of TJ and AJ protein in each group, columns represent mean ± SD, n=5. (c-d) Double staining of Occludin/CD31/Hoechst and Claudin-5/CD31/Hoechst. Red: Occludin /Claudin-5; Green: CD31; Blue: Hoechst. Scale bar, 50 µm. #p < 0.05 as M group versus S group, *p < 0.05 as TM group versus M group. (#p, *p < 0.05; ##p, **p < 0.01; ###p, ***p < 0.001)
Fig. 4 (a, b) Double staining of p120-Catenin /CD31/Hoechst and β-Catenin/CD31/Hoechst. Red: p120-Catenin/β-Catenin; Green: CD31; Blue: Hoechst. Scale bar, 50 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article)
TT promotes angiogenesis after SCI
We further detected the expression of VEGF protein 7 d in the ischemic penumbra (The shaded part in Fig. 5a) after SCI. Compared with its expression in group S, the expression of VEGF decreased significantly after SCI (M vs. S: P =0.029). At the same time, TT significantly upregulated the expression of VEGF (Fig. 5b-c) (TM vs. M: P =0.039). Blood vessels that co-labeled with 5-bromo-2-deoxyuridine (BrdU) and laminin (Laminin) in spinal cord tissue were quantitatively analysed. As shown in Fig. 5d-e, angiogenesis in the M group was significantly higher than it was in the S group at 7 d after injury (M vs. S: P <0.001). Additionally, the neovascularization density of the TM group was increased compared with that in the M group, suggesting that TT can effectively promote angiogenesis (TM vs. M: P <0.001).
Fig. 5 TT promotes angiogenesis after SCI. (a) Schematic diagram of sampling and positioning (b, c) Representative western blots and quantification data of VEGF/Tubulin, columns represent mean ± SD, n=5. (e) Double staining of Laminin(green)/Brdu (red) of sections from the spinal cord in each group rats. Scale bars are 20 μm. (d) Quantification data of number of Brdu and Laminin co-stained cells, columns represent mean ± SD, n=5. #p < 0.05 as M group versus S group, *p < 0.05 as TM group versus M group. (#p, *p < 0.05; ##p, **p < 0.01; ###p, ***p < 0.001)
TT inhibits the expression of MMP-2/9 after SCI
The expression level of MMP-2/9 protein was detected by Western blot. The results showed that TT could significantly inhibit the upregulation of MMP-2/9 after SCI (Fig. 6a-c) (TM vs. M: MMP-2, P <0.001; MMP-9, P =0.002). In addition, to determine the defects of vascular ECs, we analysed them by TEM. In group S, the vascular ECs of spinal cord showed a tight connection of rivet-like structure, and a series of electronic dense bands appeared between the plasma membranes, forming a relatively closed vascular lumen, and the intercellular TJ was partially opened after SCI. Compared with group S in the same period, the structure of BSCB in group M was disordered, the electron dense zone of TJ was lighter, the ECs were atrophied and scar tissue was formed around the blood vessels. On the contrary, the rats in the TM group had more electronic dense bands, and the ultrastructural changes were between S and M (Fig. 6d).
Fig. 6 TT inhibits the expression of MMP-2 and MMP-9 after SCI. (a-c) Represent western blots and quantification data of MMP-2/9 in each group, columns represent mean ± SD, n=5. #p < 0.05 as M group versus S group, *p < 0.05 as TM group versus M group. (d) TEM showed the EC-EC junctions in S, M, TM. Arrows indicate TJ electron dense band and the open TJ, scale bars are 1.0 µm. (#p, *p < 0.05; ##p, **p < 0.01; ###p, ***p < 0.001)