Abstract: Introduction There is now a substantial body of evidence which suggests that chronic stress can increase susceptibility to disease such as depression and posttraumatic stress disorder. Chronic stress induces neuronal atrophy and death in the cortex as well as in the hippocampus. The proto-oncogene protein kinase B (PKB), also known as Akt, is a central player in a signaling pathway of which many components have been linked to cellular survival. Akt as well as of its downstream targets Forkhead transcription factors (forkhead homologue in rhabdomyosarcoma, FKHR) have emerged in recent years as cardinal pathway underlying cellular survival and opposing apoptosis in neurons. But the activation of Akt/FKHRL signaling pathway after chronic stress in the brain remains poorly defined. We hypothesized that repeated immobilization stress exposure may change the levels of phosphorylation of Akt (pAkt) and FKHRL1 (pFKHRL1) in the rat hippocampus. The study was designed to analyze the effect of chronic stress on activation of Akt/FKHRL1 signaling pathway in the rat hippocampus.
Methods Twenty four male Sprague Downey rats (280 ± 20g) were purchased from Shanghai Laboratory Animal Center, Chinese Academy Sciences and were used for all experiments. Rats were randomly divided into three groups: psychologically stressed group, immobilization stressed group, and controls. Each group contained eight rats. Animals were housed in groups of 4 under standard laboratory conditions in temperature-controlled rooms (24℃), and maintained on a 12 h light/dark cycle (lights on at 08.00) with food pellets and water available ad libitum. Rats of the two stressed groups were subjected to immobilization stress or psychological stress one hour per day for 28 consecutive days, respectively. At the end of the experimental period, the animals were decapitated and their hippocampi were rapidly removed and kept at −80℃ until analysis. The protein level of Akt, FKHRL1 and phosphorylation of Akt, FKHRL1 were determined by Western-blotting. The intensities of the bands corresponding to the protein of interest were quantified using scanning densitometry and compared using t tests or one-way ANOVA as appropriate. The adrenal gland and thymus gland index were also calculated at the day after 28-day-stress modeling. Organ index = wet weight/weight of rat×100. The statistical significance was determined at p < 0.05.
Results Totally 24 rats were involved in the analysis. The avoirdupois and the organ index of the stressed groups were significantly changed comparing to the controls. There was no significant difference in Akt and FKHRL1 levels of hippocampus among three groups. The phosphorylation of Akt and FKHRL1 had significant difference among the three groups (F=13.75, p < 0.01; F=28.60, p < 0.001, respectively). The phosphorylation of Akt and FKHRL1 in immobilization stressed group was decreased than that in control group (p< 0.01). In contrast, the levels of the phosphorylation of Akt and FKHRL1 show no significant deference between the psychological stressed group and controls.
Conclusions These results suggest that chronic immobilization stress can induce more significant decrease of phospho-Akt and phospho-FKHRL1 level in the rat hippocampus. Activation of Akt/FKHRL1 signaling pathway may be an effective biological predictor for change of structure or function of hippocampus induced by chronic stress

Key words: stress, hippocampus, Akt, FKHRL1, phosphorylation