The Effects And The Relationship Of PI3K/Akt Signal Pathway And The Caspase-8,Bid On Neuron In Rats After Traumatic Brain Injury

Objective: To study the role of PI3K/Akt signal pathway in the effects of neural protection in rats after traumatic brain injury (TBI). Detecting the changes of p-Akt,Caspase-8,Bid index by analyzing TBI and treatment with LY294002 group, TBI and treatment with DMSO group, sham operated group. For the clinical treatment of early intervention.Methods: To produce TBI, Marmarou's rat acceleration impact model was used. The LY294002 was injected intracerebroventricularly 30 min before injury. DMSO control group was injected with the same dose DMSO and PBS at the same time.Animals and experiment groups: Adult male Wistar rats were divided into three groups:(1) sham group (n=42); (2)DMSO control group (n =56); and (3) LY294002-treated group (n =56). Animals were sacrificed at 1, 3, 6 hours and 1, 2, 3, 7 days after injury.Detection method and index:①The neurological functional evaluation;②Adopt H﹠ E stain observe pathological changes of brain tissue in rats③Adopt immunochemistry to semiquantitative determination the protein expression level of p-Akt, Caspase-8, Bid in injured cortex.④Adopt western blot to quantitative determination the protein expression of p-Akt, Caspase-8, Bid in injured cortex.Morris Water Maze (MWM) testing: 30 rats were divided into three groups randomly: the sham group; DMSO control group and LY294002-treated group. N = 10, Morris Water Maze (MWM) task was performed between 7 and 9 days after injury to test spatial memory.Quantitative assay: The immunohistochemical picture were semi-quantitative analyzed with IOD values by using Image-Pro Plus 6.0 analysis system. NC membrane were quantitative analyzed by the automatic image analysis system. The average optical density was determined by the ImageJ 14.0f analysis system.Method: The data are presented as x±s, analyzed with SPSS16.0 for windows. Student's t-test and one-way ANOVA were used to determine the significance of the differences. Differences were considered significant at P<0.05.Results:1 Pathological changes: The sham-operated rat brain tissue was normal, neuron cells arranged in neat rows by H&E staining. Traumatic brain tissue in rats can be seen varying degrees of brain injury, capillary congestion, hemorrhagic focus, neuron swelling, endochylema hyoochromatic, chromatin assemble, intercellular space broadening. Some of the cells in the injured cortex showed cell death features, such as condensed nuclei or fragmentation of nuclei. Similar features were also in the LY294002 group, compared with DMSO group increased to varying degrees (Fig.7) .2 Recommending score of neural function: sham group, DMSO group and LY294002 group each had a score of 24, 15-19, 11-15 respectively. There was significant difference among DMSO group, LY294002 group and sham groups(P<0.05); there was also significant difference between DMSO group and LY294002 group (P<0.05) (Fig.1; Tab.1).3 p-Akt results in immunochemistry and western blot: The positive cells of p-Akt were stained yellow and brown has little expressed in the cell cytosols in the cortex of a sham-operated animal. In the DMSO, the expression of p-Akt was increased as early as 1 h after TBI, with the enhanced immunoreactivity of p-Akt in the cortex area. The immunoreactivity of the cortex area was strongly increased 6 h after TBI, mainly expressed 7d after TBI, The expression of p-Akt in the LY294002 group less than in the DMSO group. There were significant difference at 1h,3h between LY294002 group and DMSO group. (P<0.05).Western blot results showed the same results of immunohistochemical results (Fig.2; Fig.8;Tab.2). 4 Caspase-8 results in immunochemistry and western blot: The positive cells of Caspase-8 were stained yellow and brown has little expressed in the cell cytosols in the cortex of a sham-operated animal. In the DMSO group, the expression of Caspase-8 was increased as early as 6 h after TBI, with the enhanced immunoreactivity of Caspase-8 in the cortex area. The immunoreactivity of the cortex area was strongly increased 1d after TBI, mainly expressed 7d after TBI, The expression of Caspase-8 in the LY294002 group less than in the DMSO group. There were significant difference at 1d, 2d between LY294002 group and DMSO group. (P<0.05).Western blot results showed the same results of immunohistochemical results (Fig. 3; Fig.9;Tab.3).5 Bid results in immunochemistry and western blot: The positive cells of Bid were stained yellow and brown has little expressed in the cell cytosols in the cortex of a sham-operated animal. In the DMSO group, the expression of Bid was increased as early as 6 h after TBI, with the enhanced immunoreactivity of Bid in the cortex area. The immunoreactivity of the cortex area was strongly increased 1d after TBI, mainly expressed 7d after TBI, The expression of Bid in the LY294002 group less than in the DMSO group. There were significant difference at 1d, 2d between LY294002 group and DMSO group. (P<0.05).Western blot results showed the same results of immuno-histochemical results(Fig. 4; Fig.10;Tab.4).6 Morris water maze results: Morris water maze tests showed that obvious space learning and memorizing obstruction existed in rats after injury, delitescence prolonged obviously 7 to 10 days later. Search moving tracks shows that search strategy altered more ideally with time in the sham group than in DMSO group and LY294002 group (P<0.05); there was also significant difference between DMSO group and LY294002 group (P<0.05) (Fig.5-6; Tab.5).Conclusions:1 The activation of PI3K/Akt signal pathway can protect brain from injury after TBI.2 The p-Akt plays a neuroprotective role by inhibit its downstream Caspase-8 and Bid expression.3 The activation of PI3K/Akt signal pathway is one of the mechanisms of improvement for neurological function and long-term learning and memory abilities after injury.