ative damage, and eventually results in the functional disability of the spinal cord below the injured area. In addition, oxidative stress, which results in the imbalance of the redox state and contributes to the development of SCI. Because of the complex pathogenesis of SCI, a therapy that has multiple targets will be effective. Tanshinone IIA (TIIA) is an important lipophilic diterpene extracted from Salvia miltiorrhiza BUNGE and is used in Chinese traditional herbal medicine (Danshen) for the treatment of many diseases, such as cardiovascular [12,13], cerebrovascular [14,15] and postmenopausal syndromes [16]. Previous studies show that TIIA might exert a series of biochemical effects through its anti- inflammatory [12,16–18], anti-oxidative [19] and anti-apoptotic properties [20], and has been used to prevent ischemic injury [21]. However, whether TIIA produces protective effects to alleviate traumatic injury in the spinal cord is not yet clear. In the present study, we aim to evaluate effects of TIIA on traumatic SCI. We used a weight drop device (NYU impactor) to create a rat SCI model to investigate a series of changes that occur in the spinal cord and to analyze the protective effects of TIIA. In our research, we found that pro-inflammatory factors were up- regulated after SCI, and subsequently the apoptosis process was activated. Moreover, our results demonstrated that TIIA markedly inhibited the expression of pro-inflammatory factors and related pathways, ameliorated the activation of apoptosis, and reversed the imbalance of the redox state. Materials and Methods Animal preparation Sprague-Dawley male adult rats, weighing 220–250 g each, were obtained from the Animal Center (Fourth Military Medical University, Xi’an, China). The rats were kept in a temperature- controlled house under a 12 h light/dark cycle and fed a standard laboratory diet and water ad libitum. The Animal Care and Use Committee of the Fourth Military Medical University approved all experiments in accordance with the Declaration of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985). Spinal cord injury Spinal cord injury was induced using a weight drop device as previously reported [22]. Before this surgical operation, all instruments were autoclaved for sterilization. The acclimatization of rats was achieved in the surgical room prior to the start of the experiments. All animals were anaesthetized through intraperito- neal injection of 4% sodium pentobarbital (40 mg/kg, i.p.). An Figure 1. Effects of TIIA on functional recovery after SCI. The function of hindlimb recovery was accessed from day 1 to day 10 after SCI by Basso, Beattie and Bresnahan scores. The hindlimb dysfunction was ameliorated with treatment of TIIA (20 mg/kg). Bars represent means 6 SEM. *p,0.05 vs. SCI group. doi:10.1371/journal.pone.0038381.g001 Effects of Tanshinone IIA in Adult Rats after SCI incision was made along the middle of the back, exposing the paravertebral muscles. A laminectomy was performed at the T9– T11 level, exposing the cord without damaging the dura. The exposed dorsal surface of the cord was subjected to weight drop impact using a 10-g metal rod at a height of 25 mm. Subsequently, the lesioned muscle and skin were sutured in layers, followed by a 1.0 ml subcutaneous injection of saline solution to replace the blood volume lost during the surgery. After the surgical operation, sterile gauze was wrapped around the wound. Animals were injected with penicillin (intramuscularly, 100,000 unit/per ani- mal/day) to prevent post-operative infection. To relieve the pain from surgery, we subcutaneously injected buprenorphine (0.03 mg/kg) post-surgery for 3 days. During recovery from anesthesia, the rats were placed in a room with moderate temperature and humidity and covered with a warm blanket. A standard diet and water were provided daily to the rats ad libitum. Manual bladder expression was performed twice a day until the rats were able to urinate by themselves. Throughout the process, symptoms of infection, pain, decubitus, dehydration and autopha- gia were monitored daily. Experimental design Rats were randomly divided into four groups, namely the sham group, sham/TIIA group, SCI group and SCI/TIIA group. We injected a 0.9% sterile saline (0.09 g NaCl dissolve 100 ml distill water) solution as a vehicle control. In the sham group, the animals were only subjected to laminectomy. In the sham/TIIA group, the animals were subjected to laminectomy with TIIA treatment. In the SCI group, the animals were subjected to SCI using an impactor and treated with an intraperitoneal injection of TIIA. TIIA was administered 1 h before operation (50 mg/kg). From day 1 to 7 post-SCI, TIIA was administrated (20 mg/kg) once a day at the same time. The current dose and timing of TIIA administration were applied on the basis of our preliminary experiments. At the indicated time point, the rats were sacrificed using cervical dislocation in all groups. Subsequently, the spinal cord was immediately exposed from T1 to T12, and the damaged tissue (T9 to T10) at the site of injury was cut. The time of sacrifice was determined according to the different parameters measured: motor function (BBB) score = 10 days after SCI; TUNEL staining = 3 days after SCI; western blot analysis for caspase-3 and Bcl-2 = 1, 3 and 7 days after SCI; real-time PCR analysis of proinflammatory cytokines = 6 h after SCI; and other measur- ements = 24 h after SCI. Behavioral examination A motor function recovery test (n = 6) was performed twice a day from Day 1 to Day 10 after the spinal cord was contused. The behavioral test was scored in accordance with the rules of Basso, Beattie and Bresnahan (BBB) [28], which comprise 21 criteria for the movement of lower limbs, from complete paralysis to complete mobility. These criteria are based on the accurate observation of the lower limbs, including movement, step, and coordinated motor action. Two additional investigators blinded to treatment and grade observed the mechanically contused animals. Hematoxylin-eosin staining The animals were sacrificed at 24 h after injury (n = 4 for each group) in all groups. Briefly, the rats were intracardially perfused with saline followed by treatment with ice-cold 4% paraformal- dehyde. Subsequently, the spinal cord, including the center site of injury tissue, was collected. After fixation, the tissue blocks were embedded in paraffin, and divided into 5-mm slices. Microscopic evaluation was performed (Department of Pathology, Xijing PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e38381