Abstract BACKGROUND The treatment of diffuse brain injury during an acute period is focused on relieving degrees of secondary brain injury. Generation and development of pathological changes of secondary brain injury depend on signal conduction, so down-regulating over response of astrocyte through interfering a key link of signal conduction pathway may bring a new thinking for the treatment of diffuse brain injury. OBJECTIVE To observe the effect of over activity of extracellular signal regulated kinases 1/2 (ERK1/2) signal pathway on the response of astrocyte during an acute period of diffuse brain injury. DESIGN Completely randomized grouping and controlled animal study. SETTINGS Department of Neurosurgery, the Third Affiliated Hospital, Nanchang University; Department of Neurosurgery, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology. MATERIALS A total of 158 healthy male SD rats, of 11 weeks old, weighing 320 – 370 g, were provided by Experimental Animal Faulty, Tongji Medical College, Huazhong University of Science and Technology. Rabbit-anti-phosphorylated ERK1/2 (pERK1/2) polyclonal antibody was provided by RD rabbit-anti-glial fibrillary acidic protein (GFAP) polyclonal antibody, SP immunohistochemical kit and horseradish peroxidase (HRP)-labeled goat-anti-rabbit IgG by Santa Cruz Company; specific inhibitor U0126 of ERK1/2 signal pathway by Alexis Company. METHODS The experiment was carried out in the Laboratory of Neurosurgery, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from September 2004 to March 2006. (1) Detection of pERK1/2 expression; A total of 110 rats were randomly divided into sham operation group (n = 5), model group (n = 35), high-dosage U0126 group (n = 35) and low-dosage U0126 group (n = 35). Rats in the sham operation group were only treated with incision of epicranium and fixation of backup plate, but not hit. Rats in the model group were used to establish diffuse brain injury models based on Marmarou free falling body without drug intervention. Rats in the high- and low-dosage U0126 groups were injected into caudal vein with 0.1 and 0.05 mg/kg U0126, respectively, and then, rats were hit to establish injured models. Every 5 rats were collected from model, high- and low-dosage U0126 groups at 5, 30 minutes, 3, 12, 24, 72 hours and 7 days after diffuse brain injury to detect pERK1/2 expression in cortex of parietal lobe based on Western blot technique. (2) Distribution of pERK1/2 and positive GFAP cells in brain tissue; Another 48 rats were randomly divided into sham operation group (n = 3), model group (n = 5), high-dosage U0126 group (n = 15) and low-dosage U0126 group (n = 15). The intervention and administration were dealt as the same as those mentioned above. Every 3 rats were collected from model, high- and low-dosage U0126 groups at 30 minutes, 3, 12, 24 and 72 hours after model establishment to observe the distribution of pERK1/2 and positive GFAP cells in brain tissue which was cut from coronal section at Bregma – 4.8 mm layer with immunohistochemical staining. MAIN OUTCOME MEASURES pERK1/2 expression in cortex of parietal lobe and distribution of pFRK1/2 and positive GFAP cells in brain tissues. RESULTS (1) pERK1/2 expression: After diffuse brain injury, pERK1/2 expression in cortex of parietal lobe was rapidly increased in the model group, reached at peak at 5 minutes and then decreased gradually. But the expression was still in a high level until the 72nd hour and fallen to the basic level on the 7th day. pERK1/2 level was lower in high- and low-dosage U0126 groups than that in model group at various time points (P CONCLUSION Diffuse brain injury strongly induces the activity of ERK1/2 signal pathway and response of astrocyte; in addition, U0126 can inhibit response of glial cells during an acute period, and the effect manifests dosage dependence.