Figure 10. Effects of TIIA on oxidative stress markers after SCI. SCI induced the decrease of SOD activity at 24 h after SCI, but treatment with TIIA increased SOD 65 activity (A). The MDA level was significantly increased in the tissues from the SCI group at 24 h after SCI. TIIA treatment inhibited SCI-induced increase in MDA level (B). Bars represent means 6 SEM. **p,0.001 vs. sham group, #p,0.05 vs. SCI group. doi:10.1371/journal.pone.0038381.g010 the cells in all groups at this time point. The results showed that a few TUNEL-positive stained cells were detected in the two sham groups (Fig. 8A and B). Moreover, SCI induced more TUNEL- positive cells in the grey and white matter in the SCI group (Fig. 8C), but TIIA resulted in a marked reduction in the number of these cells compared with the SCI group (Fig. 8D). The results of the quantitative analysis are shown in Fig. 8E. These data suggest that TIIA could reduce SCI-induced cell apoptosis in the spinal cord. Effects of TIIA on apoptosis-related factors after SCI To further confirm the effect of TIIA on apoptosis, we measured two apoptosis-related factors. First, Bcl-2, an inhibitive factor of apoptosis was subjected to western blot analysis. The results showed that SCI resulted in a significant reduction in the expression of Bcl-2 at Day 1 and Day 3 in the SCI group (Fig. 9A and B). However, in the TIIA/SCI group, the level of Bcl-2 was significantly increased compared with the SCI group (Fig. 9A and B). No difference in Bcl-2 expression was observed at the site of insult in the tissues on Day 7 after SCI (Fig. 9B). Second, we tested the change in caspase-3 expression, which is a component of the cysteine protease family that plays a key role in apoptosis. Compared with the rats in the sham and sham/TIIA groups, SCI induced a marked increase in caspase-3 at each time point (Fig. 9C and D). In contrast, cleaved caspase-3 was attenuated at Day 3 and Day 7 in the SCI/TIIA group compared with the SCI group (Fig. 9C and D). Effects of Tanshinone IIA in Adult Rats after SCI Effects of TIIA on oxidative stress markers after SCI SOD and MDA are anti-oxidant and a pro-oxidant markers of oxidative stress, respectively, and were measured at 24 h after SCI. In the SCI group, SCI caused a significant decrease in the SOD activity and an increase in the MDA level. Compared with the SCI group, TIIA treatment after SCI dramatically rescued the SOD activity and significantly decreased the MDA level, indicating that TIIA played an important anti-oxidative role after SCI (Fig. 10A and B). Discussion In this study, we demonstrated that TIIA exhibited a protective effect against SCI in rats. The administration of TIIA promoted motor function recovery of the hind limbs, ameliorated histopath- ological changes, inhibited the expression of proinflammatory factors and NF-kB and MAPK pathways, regulated SCI-induced apoptosis and apoptosis-related factors, and reduced the extent of oxidative stress in rats subjected to mechanical compressive insult by an NYU impactor. To simulate the major events of SCI in rats, various experimental models have been developed [20]. One of the most widely used models is contusive injury. In this model, a rapid insult through dropping a weight onto the exposed surface of the dorsal spinal cord results in an immediate and transient contusion, which generates the primary damage. This device stimulates various levels of injury through correlative intensity weight, from moderate to severe, and even to complete paraplegia. The model’s success is measured by the degree of movement in the bilateral hind limbs; a successful model occurs when the bilateral hind limbs have no movement [23]. In our experiment, we used this model to assess the therapeutic effect of TIIA on SCI. Tanshinone (Danshen), is an herbal drug derived from the dried root of Salvia miltiorrhiza Bunge, and has been used either alone or in combination with other herbal materials to clinically prevent or treat many diseases in China and other countries [12,21]. TIIA is one of the key components of Danshen. Previous studies suggest that TIIA might exhibit anti-inflammatory, anti-apoptotic and anti-oxidative capacities. We also assessed the therapeutic effects of TIIA in the SCI model. Based on the BBB score, TIIA promoted the recovery of locomotor function. Several studies demonstrate evidence of edema, hyperemia and incompact structures in the injured tissue [24]. We also observed the same results in the rat model of SCI, and found a significant effect of TIIA against SCI. Astrocytes are activated upon SCI and are involved in the formation of a glial scar. Astrocytes also inhibit regeneration in the CNS. Moreover, activated astrocytes increase the production of intermediate filaments, such as GFAP (glial fibrillary acidic protein) and can secrete other molecules to restrict axonal growth. Together, this evidence showed that activated astrocytes exhibit a detrimental effect upon SCI [25]. Our data showed that GFAP- positive cells were increased after SCI, and TIIA administration significantly reduced the number of these positive cells. As a result, we conclude that TIIA effectively attenuated the number of activated astrocytes and glial scar formation. Pro-inflammatory cytokines, such as TNF-a, IL-1b and IL-6, are key mediators and participate in the development of SCI [26,27]. Nitric oxide (NO), which is mainly regulated through iNOS, also contributes to secondary damage after SCI [28]. Moreover, there are several pathways to modulate these inflam- matory factors, such as NF-kB and MAPKs pathways [29–31]. Consistent with other studies [32–36], we detected increased mRNA and protein levels of pro-inflammatory cytokines and the activation of inflammation related pathways in SCI-induced PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e38381