Background
Ischemic tolerance can be induced in the experimental animal models of focal cerebral ischemia by a transient exposure to various sublethal metabolic stresses prior to the onset of stroke. One such preconditioning technique, hypoxia, significantly improves the outcomes following occlusion of middle cerebral artery (MCAO) in rats, but the understanding of the processes involved in endogenous neuroprotection is lacking. We have previously shown that cell cycle processes are altered following focal ischemia as indicated by changes in expression, an increase in BrdU incorporation and up regulation of the cell cycle inhibitors, p16 and p21. These findings are supported by studies showing that cerebral ischemia simulates expression of certain cell cycle related processes and that their inhibition results in reduction of infarct volumes. Since cyclin dependent kinase 5 (CDK5) has been implicated in the pathogenesis of other neurodegenerative diseases, we examined the expression of the CDK5 in post-ischemic brain tissues of the animals with and without hypoxic preconditioning.
Design/Methods
Adult male Wistar rats were exposed to either a continuous 2 hours or twelve 20 min/hour episodes of 10% normobaric hypoxia in computer software regulated hypoxic chamber. Control animals were also placed in the chamber without change in oxygen tension. Middle cerebral artery was occluded with monofilament technique for 2 hours and animals were sacrificed at 8, 24 or 48 hours of reperfusion. Volumes of infarction were determined either by TTC or cresyl violent at 24 hours of reperfusion following 2 hours of MCAO in animals with and without intermittent hypoxia. Coronal brain sections through the area supplied by middle cerebral artery were stained with antibodies to CDK5 and co-localized with neuronal marker NeuN.
Results
Intermittent hypoxia reduced volumes of infarction by 35% when compared to the non-conditioned controls, which was not significantly different from the decrease of damage in the single exposure group. While CDK5 staining was increased following hypoxia only, its expression was also increased in postischemic neurons within all the regions in the territory of the occluded artery, including the penumbra and the ischemic core at 8, 24 and 48 hours of reflow. Both intermittent and continuous hypoxic preconditioning reduced the number of neurons that express CDK5 in the penumbra, but not the ischemic core.
Conclusions
Our study demonstrates that intermittent hypoxia which might be better tolerated than a continuous hypoxic exposure induces a similar endogenous neuroprotective effect and increases CDK5 expression. CDK5 staining is increased at early and late times of ischemic reperfusion in the areas where neurons are destined to die, while hypoxic preconditioning reduced the appearance of CDK5 in the penumbra following ischemia. While CDK5 is essential for neuronal growth and development it obviously has pathological functions. One possible explanation is that the expression in CDK5 in both hypoxia and/or ischemia may be attributed to different activators (e.g. p35 vs p25) following the two events. Therefore, we postulate that CDK5 related processes might mediate both hypoxia induced ischemic tolerance and ischemic neuronal death.