Acquisition of cerebral ischemic tolerance following preconditioning results from synergistic, multifactorial processes that promote cellular survival. However, to date, the endogenous neuroprotective window following preconditioning is transient and lasts only several days. We have recently developed new mouse models of induced tolerance, whereby neuroprotection against transient focal stroke, as well as reductions in cerebrovascular inflammation following TNFalpha, are extended to 4 weeks following the last of a series of repetitive hypoxic preconditioning (RHP) stimuli1, 2. The first objective of this study was to determine if such long-term tolerance (LTT) could also be achieved against permanent focal stroke. Male ND4/Swiss Webster mice were subjected to RHP (9 episodes of systemic hypoxia of varied duration [2 or 4 hours] and severity [11 or 8% O2] over a 15-day period), or to a single period of hypoxic preconditioning (SHP) (4 h @ 8%), previously shown to be protective against permanent middle cerebral artery occlusion (MCAO) injury at 24 h. Two weeks later, all animals were subjected to permanent MCAO by a craniotomy approach. RHP resulted in a statistically significant reduction (38%; p<0.05) in ischemic injury, whereas SHP showed no long-term neuroprotective effects. Thus, as with transient MCAO, LTT can protect against brain injury caused by permanent MCAO. Given that the window of neuroprotection with LTT coincides temporally with a reduced inflammatory response to TNFalpha, the second objective of this study was to identify specific proteins whose expression would be increased for a protracted period of time following RHP, and that had the capacity to modulate diverse neuronal survival and inflammation pathways. SIRT1 is a member of the Sir2 family of class III histone deacetylases, whose activity is associated with enhanced cell survival in models of aging3, 4. Recent studies suggest that SIRT1 activation prevents axonal degeneration following injury5, which may be mediated through the regulation of DNA damage responses (inhibition of p53 activity) and/or transcriptional silencing (for example inhibition of the pro-inflammatory transcription factor NF-κB)6, 7. We subjected male ND4/Swiss Webster mice to RHP and immunoblotted for SIRT1 in whole cell lysates generated from cortical samples obtained at various times after RHP. We demonstrated a 2-fold increase in SIRT1 protein expression immediately after the last RHP stimulus, which remained elevated for at least 2 weeks. These studies indicate that RHP results in a sustained upregulation of SIRT1 protein coincident with the period of LTT. The modulation of postischemic inflammatory and apoptotic pathways by SIRT1 may contribute to the unprecedented protection associated with LTT. These novel signaling pathways triggered by RHP may serve as molecular targets for therapeutics designed to reduce brain injury in stroke.
