Lschemic preconditioning in the protection of global cerebral ischemia in rats by reducing endoplasmic reticulum stress

Abstract

Objective To investigate the effect of inhibition of endoplasmic reticulum stress （ERS） in ischemic preconditioning-induced cerebral ischemia tolerance. Methods A total of 120 adult male Sprague- Dawley rats were randomly allocated into three groups： sham operation, global cerebral ischemic and ischemic preconditioning groups （ischemlc preconditioning for 3 minutes, and global cerebral ischemia for 15 minutes after 2 days）. Three time points （day 1, day 3 and day 7） were set. Sugawara method was used to observe the changes of neurological behavior in rats. TUNEL staining was used to observe the conditions of cortical neuronal apoptosis. Immunofluorescence staining and Western blot analysis were used to detect the expression levels of ERS-related protein CHOP, GRP78, and caspase-12. Results The neurological behavior score showed that the sham operation group did not have neurological deficits. Both the global cerebral ischemic group and the ischemic preconditioning group had obvious neurological deficits, and they improved gradually with the passage of time, but after modeling, the neurological scores at each time point in the global cerebral ischemic were significantly lower than those in the ischemic preconditioning group： at day 1 ： 11.00 ± 0. 63 vs. 14. 33 ± 0. 33 （t = 21.74, P = 0. 001）; at day 3：12.17±0.31 vs. 15.17±0.48 （t=27.93, P=0.000）; at day 7： 14.67±0.49vs. 16. 33 ±0. 33 （t =7. 81, P=0. 020）. TUNEL staining showed that at day 7 after ischemia, the positive cell count per mm2 in the sham operation, global cerebral ischemic and ischemic preconditioning groups were 4. 83 ± 1.85vs. 395.67± 43.43 and 146.17± 27.38 respectively （F= 23.62, P= 0.001）. The ischemic preconditioning group was significantly lower than that in the global cerebral ischemic group （P = 0. 001 ）. Immunofluorescence staining showed that at day 7 after ischemia, the numbers of positive cells of CHOP （26. 50±3.89vs. 82. 33±4.25, P=0.000）, GRP78 （15.00±2.02vs. 35.67±2.99; t=0.000）, and caspase-12 （22. 33 ± 2. 76 vs. 66. 50 ± 7. 25; P = 0. 000） in the ischemic preconditioning group were significantly less than those in the global cerebral ischemic group. Western blotting showed that at day 7 after ischemia, the expression levels of CHOP （1.22 ± 0. 38 vs. 3.22 ± 0. 51; t = 24. 50, P= 0. 001）, GRP78 （1.78±0.45vs. 3.16±0.76; t=14.29, P=0.005）, and caspase-12 （2. 89±0.53vs. 5.96±0.67; t= 77. 73; P=0. 000） in the ischemic preconditioning group were significantly lower than those in the global cerebral ischemic group. Conclusions Ischemic preconditioning demonstrated a neuroprotective effect for the second lethal ischemia, its mechanism may be associated with the relief of ERS and downregulation of ERS- related protein. Key words: Brain Ischemia; Ischemic Preconditioning; Endoplasmic Reticulum; Transcription Factor CHOP; Molecular Chaperone GRP78; Caspase 12; Apoptosis; Disease Models, Animal; Rats