Inhibition of mTOR signaling Confers Protection against Cerebral Ischemic Injury in Acute Hyperglycemic Rats

Abstract

Hyperglycemia is known to exacerbate neuronal death resulted from cerebral ischemia. The mechanisms are not fully understood. The mammalian target of rapamycin (mTOR) pathway regulates cell growth, division and apoptosis. Recent studies suggest that activation of mTOR may mediate ischemic brain damage. The objective of the present experiment is to explore whether mTOR mediates ischemic brain damage in acute hyperglycemic animals. Rats were subjected to 10 min of forebrain ischemia under euglycemic, hyperglycemic and rapamycin-treated hyperglycemic conditions. The rat brain samples were collected from the cortex and hippocampi after 3h and 16h of reperfusion. The results showed that hyperglycemia significantly increased neuronal death in the cortex and hippocampus and the exacerbation effect of hyperglycemia was associated with further activation of mTOR under control and/or ischemic conditions. Inhibition of mTOR with rapamycin ameliorated the damage and suppressed hyperglycemia-elevated p-MTOR, p-P70S6K and p-S6. In addition, hyperglycemia per se increased the levels of cytosolic cytochrome c and autophagy marker LC3-II, while rapamycin alleviated these alterations. It is concluded that activation of mTOR signaling may play a detrimental role in mediating the aggravating effect of hyperglycemia on cerebral ischemia.

Keywords: Cerebral ischemia; Hyperglycemia; Pathology; Rapamycin; Rat.; mTOR.

Figure 1

Histopathological outcomes in the neocortex (A,B) and hippocampal CA1 region (C,D) after cerebral ischemia and reperfusion (I/R) in euglycemic (EG), hyperglycemic (HG) and rapamycin treated hyperglycemic (HG+RAPA) animals. A and C, representative microphotographs showing histological outcomes in the cortex (A) and hippocampal CA1 (C) area; B and D, summarized percentage of death in each group in the cortex (B) and CA1 (D). Euglycemic ischemia moderately increased neuronal death, which was significantly enhanced by hyperglycemia. Celestine blue and acid fuchsin staining. Arrows indicate dead neurons. Bar = 50 μm. Data are presented as means ± s.d. ΔΔ p< 0.01 versus sham group within each group. * p<0.05 and **p<0.01 versus EG at the same reperfusion endpoint. # p<0.05 and ## p<0.01 HG versus HG+RAPA. Statistical annotations are the same for Figures 2-4.

Figure 2

Western blot analyses of p-mTOR, p-P70S6K and p-S6 in the cytosolic fractions of the cortical and hippocampal samples. A and C, representative Western blots in the cortex (A) and hippocampus (C). B and D, summarized target band densities of the cortical (B) and hippocampal (D) samples. Euglycemic ischemia activated the mTOR and its downstream proteins in the cortex and/or hippocampus. Hyperglycemia per se increased the levels of detected mTOR pathway proteins and ischemia maintained the high levels of these proteins in hyperglycemic animals. Rapamycin suppressed the hyperglycemia-elevated mTOR pathway proteins in control and after ischemia and reperfusion injury. I/R, ischemia and reperfusion; EG, euglycemia; HG, hyperglycemia; HG+RAPA, rapamycin treated hyperglycemic animals.

Figure 3

Western blot analyses of cytochrome c in the cytosolic fractions of the cortical and hippocampal samples. A and C, representative Western blots in the cortex (A) and hippocampus (C). B and D, summarized cytochrome c band optical densities of the cortical (B) and hippocampal (D) samples. Euglycemic (EG) ischemia increased the cytosolic cytochrome c. Hyperglycemia (HG) increased the baseline cytochrome c levels. After ischemia and reperfusion, the cytochrome c further increased in hyperglycemic animals compared with the control but not significantly higher than euglycemic ischemic samples. Rapamycin treatment in hyperglycemic animals (HG+RAPA) significantly reduced the levels of cytochrome c in the both control and ischemic samples.

Figure 4

Western blot analyses of LC3-II in the cytosolic fractions of the cortical and hippocampal samples. A and C, representative Western blots in the cortex (A) and hippocampus (C). B and D, summarized LC3-II band optical densities of the cortical (B) and hippocampal (D) samples. Euglycemic ischemia increased the cytosolic LC3-II. Hyperglycemia increased the baseline levels of LC3-II and maintained the high levels after ischemia and reperfusion. Rapamycin treatment significantly reduced the levels of LC3-II in the both hyperglycemic control and hyperglycemic ischemic samples.