Analogous neuroprotection induced by resveratrol and ischemic preconditioning in CA1 region of hippocampus after ischemia

Introduction

Resveratrol, trans-3,5,4″-trihydroxystibene, is a naturally occurring phytoalexin produced by variety of plants in response to stress. Resveratrol is proven to have cardioprotective, anti-cancer, anti-aging and neuroprotective effects. Recent studies demonstrated either resveratrol treatment or caloric restriction in yeast activate Sir2 (Silent information regulator 2) ¹ . Yeast Sir2 has homology to mammalian SIRT1- located in the nucleus, requires co-factor NAD and determines cell fate by deacetylating transcription factor-p53. Employing the organotypic hippocampal slice culture present study was carried out to investigate: (1) the efficacy of resveratrol pre-treatment (RT) for neuroprotection; and (2) similarities between the mechanisms of induction of neuroprotection by ischemic preconditioning (IPC) and RT.

Methods

Hippocampal slices were obtained from 9–11 days old Sprague Dawley rats and cultured for 14–15 days. Slices were exposed to IPC (15 min of oxygen glucose deprivation (OGD)) or 1 h of RT (75,200 μM) followed by ‘test’ ischemia (40 min of OGD) 48 h later. To characterize role of SIRT1, slices were exposed to sirtinol (10, 50, and 100–μM) for 48 h of reperfusion after IPC/RT. Parameters like quantification of neuronal death in CA1 region of hippocampus and SIRT1 estimation were carried out. For SIRT1 assay slices were collected following IPC/RT/vehicle treatment-sham at 30 min / 48 h intervals. Propidium iodide (PI) fluorescence images were obtained using a SPOT CCD camera and were digitized using SPOT advanced software. Percentage of relative optical intensity was used as an index of cell death. Results are expressed, as mean ± SD. Statistical significance was determined with an ANOVA and a Bonferroni's post-hoc test.

Results

The RT (75–μM) protected the CA1 neurons against subsequent lethal OGD (p < 0.001) and mimics IPC. Higher dosage RT could not mimic IPC. Blocked of SIRT1 activation by sirtinol after IPC /RT abolished neuroprotection. PI fluorescence of RT (75 μM), sirtinol treated (10 μM), IPC and ischemic groups were 38.04 ± 8.81 (n =7), 87.13 ± 3.82 (n =7), 34.13 ± 19.25 (n = 20) and 63.5 ± 8.96 (n = 5), respectively. A proposed mechanism by which resveratrol protects cell survival is via the SIRT1. Thus, we measured SIRT1 activity in slices. The RT was able to stimulate SIRT1 activity by about 85% (n = 4, P < 0.05) at 30 min after treatment when compared with the sham (n = 7). However, RT (n = 3) induced activation in SIRT1 activity was abolished at 48 h. In contrast to RT, SIRT1 activity did not change in the IPC group at 30 min of reperfusion (n = 4) as compared to the sham. SIRT1 activity increased by 81 % (n = 4, p < 0.05), as compared to sham, when measured 48 h after IPC.

Conclusion

Resveratrol pre-treatment induce neuroprotection in CA1 hippocampal neurons to subsequent ischemia. Analogues neuroprotection induced by IPC/RT occur via activating novel SIRT1 pathway. IPC/RT and calorie restriction induced increase in life expectancy might share common pathway.