Animals and grouping

In total, 108 specific-pathogen-free (SPF) male Sprague-Dawley rats (each weighing 280±20 g) were purchased from Guangdong Medical Experimental Animal Centre (no. 44007200017130). The rats were housed in the Experimental Animal Centre of Jinan University. The temperature of the feeding environment was 20–24°C with a relative humidity of 50%. The rats were fed a normal diet. The experiments were performed under the supervision and assessment of the Laboratory Animal Ethics Committee of Jinan University (permit no. 20160629001). All procedures were in line with the Statute on the Administration of Laboratory Animals approved by China’s Council 1988 and the ethical guidelines of the International Association for the Study of Pain. The rats were randomly divided into three groups in accordance with a random number table: normal group (Normal, n=18), TBI model group (TBI, n=36) and manual acupuncture-treated TBI group (TBI+MA, n=36). Eighteen rats were allocated to the Normal group in view of the smaller degree of variance anticipated (based on our previous studies) in keeping with the 3Rs principle of reduction. Then, each group was subdivided into 3-day (3d), 7-day (7d) and 14-day (14d) groups.