br Material and methods br Results

Material and methods
Results
Discussion Nimodipine administration inhibited the level of AMP-activated protein kinase (AMPK) phosphorylation, a sensor of cellular energy status that directs metabolic Hyper Assembly Cloning to support cellular growth and survival. CCH influences the oxygen and glucose supply in the brain which often results in metabolic alterations and oxidative stress. A variety of signaling molecules have been found to regulate metabolism in response to distinct metabolic stress (Paiva et al., 2011). And AMPK has been extensively demonstrated as an important metabolic regulator activated during hypoxia, oxidative, stress, glucose deprivation, and ischemia. AMPK responds to changes in intracellular adenine nucleotide levels, being activated by an increase in AMP/ADP relative to ATP. Activation of AMPK increases the rate of catabolic (ATP generating) pathways and decreases the rate of anabolic (ATP utilising) pathways (Carling, 2017). And phosphorylates TSC1/2 to activate autophagy (Simon et al., 2017). Since phosphorylation stimulates the catalytic activity of AMPK (Gwinn et al., 2008), the increased relative levels of p-AMPK suggested that AMPK was activated in the hippocampus of 2VO rats. In mammals, calcium/calmodulin dependent protein kinase kinase (CaMKK) β and liver kinase B1 (LKB1) have been identified as the two upstream kinases in the cascade (Woods et al., 2005; Woods et al., 2003). Intracellular calcium was increased in cerebral hypufusion rats (Min et al., 2013). That increase intracellular Ca2+ activate AMPK via phosphorylation of Thr172 by the calmodulin-dependent protein kinase CaMKKb (Woods et al., 2005). Nimodipine, an L-type calcium channel antagonist, inhibited intracellular Ca2+ generation. Hence, Nimodipine administration inhibited excessive autophagy by decreasing the level of AMPK phosphorylation. Cerebral hypofusion may bring a potent cellular stress, in which AMPK phosphorylation triggers autophagy. Autophagy is induced during different stress responses, including starvation, oxidative stress and hypoxia (Rami et al., 2008; Yang et al., 2014) and is associated with the pathological mechanisms involved in many diseases. The presence of autophagosomes in dying cells has implicated autophagy in the cell death process. Indeed, excessive autophagic activity may destroy portions of the cytosol and organelles, leading to a collapse of all cellular functions. Studies have confirmed that autophagy activated in the hippocampus of VaD induced by cerebral hypofusion (Liu et al., 2014). Autophagy dysfunction occurred early after CCH and played an important role in neuronal deterioration and cognitive decline (Zou et al., 2017). Activation of autophagy initiation remained elevated beginning at 2 weeks after 2VO in the cortex and at 4 weeks in the hippocampus, and autophagy played an important role in the processes of neuronal injury and cognitive decline (Zou et al., 2017). In the present study, autophagy is markedly activated along with neuronal deterioration, cognitive decline after 5 weeks of 2VO. LC3B, a microtubule-associated protein that is lipidated upon activation of autophagy (Jiwa et al., 2010). Moreover, one study reported that in cerebral hypofusion rat, the induction of autophagy was linked to the localization of LC3B in dystrophic axons. Nimodipine significantly decreased autophagy after 4 weeks of administration consistent with the reduced p-AMPK aggregation. Meanwhile, neuronal degeneration in the nimodipine group was significantly improved. These findings indicate that nimodipine may modulate energy sensing AMPK to reduce excessive autophagy and protect hippocampal neurons form degeneration induced by 2VO. Nimodipine administration also inhibited the eIF2α/ATF4 singnaling which is a crucial evolutionarily conserved adaptive pathway during cellular stresses including hypoxia and ischemia (Evans et al., 2016; Manwani and McCullough, 2013). While, the eIF2a/ATF4 pathway, activated by different forms of stress and part of a biological process known as the integrated stress response, directs an autophagy gene transcriptional program in response to amino acid starvation or endoplasmic reticulum stress (B'Chir et al., 2013). The AMPK and eIF2a/ATF4 pathway role in different phase of autophagy. The eIF2α/ATF4 singnaling is essential for energy deficit stress-induced autophagy gene expression. eIF2a is part of the multimeric eIF2 complex that initiates mRNA translation (Baird and Wek, 2012). eIF2 binds GTP and Met-tRNAi and transfers Met-tRNA to the 40S subunit to form the 43S preinitiation complex (Sokabe and Fraser, 2014; Kimball, 1999). eIF2 promotes a new round of translation initiation by exchanging GDP for GTP, a reaction catalyzed by eIF2B (Kimball, 1999). Phosphorylation of eIF2α inhibits translation and global protein synthesis but increases the cap independent translation of certain mRNAs, such as activating transcription factor 4 (ATF4) (Vattem and Wek, 2004). ATF4 induces genes involved in autophagy (B'Chir et al., 2013). The over-activation of eIF2α/ATF4 singnaling and p-AMPK occurs in pyramidal cells under cerebral hypofusion condition and is interconnected. After nimodipine administration, expression of eIF2α, ATF4 and p-AMPK decreased in the same cells. Meanwhile, the nimodipine group exhibited more improvement in neuronal degeneration and autophagy. Thus, the inhibition of eIF2α/ATF4 signals is mandatory for neuroprotection.