While a great deal of research has focused on the induction of hibernation in mammals and how they are able to survive long periods with a drastically reduced metabolic rate and blood flow, recent research is beginning to focus on the other end of the equation, asking, `How do hibernating animals survive reoxygenation and increased blood flow as they increase metabolic rates back to normal?' Oxygen consumption and respiratory rates in hibernators may increase to as much as 300% of resting cenothermic (normal body temperature)levels during the shivering stage of arousal thermogenesis. In most animals, a sudden increase in blood flow or increase in oxygen supply, after a period of low perfusion, results in the production of highly reactive oxygen (ROS) and reactive nitrogen species. When ROS levels exceed cellular antioxidant capacity, tissue injury results as the high-energy compounds interact with DNA, proteins and lipids.
Hibernating ground squirrels have been shown to increase the activities of antioxidant enzymes and plasma ascorbate during hibernation, presumably in preparation for the intense metabolic activity of arousal and large fluxes in blood flow and temperature. The recent study by Peter Osborne and Masaaki Hashimoto in Behavioral Brain Research extends these findings by profiling concentrations of the antioxidants ascorbate, glutathione and urate in the hamster brain during hibernation, arousal and between hibernation bouts. Using very slow flow microdialysis, in which a probe with a semi-permeable membrane tip is inserted into the striatum (an area of the brain rich with the neurotransmitters glutamate and dopamine, and therefore highly vulnerable to oxidative damage) and perfused with artificial cerebral spinal fluid, they were able to measure changes in the brain extracellular fluid independent of temperature. Total brain tissue and plasma antioxidant levels were also recorded.
They found that brain tissue levels, striatal extracellular (ECF)concentrations, and plasma levels of the antioxidants are differentially regulated during hibernation and during the metabolically demanding transition from hibernation to cenothermia. Surprisingly, mid- and forebrain tissue levels of ascorbate did not change from hibernation to cenothermia, even though plasma and ECF levels varied significantly. Plasma and ECF ascorbate increased significantly during hibernation (to more than 400% over cenothermia) and then decreased upon arousal, presumably because of the scavenging of free radicals generated during the reperfusion period. The authors hypothesize that ascorbate increased in the ECF, but not the whole tissue, because uptake into the cells decreased while uptake from the plasma into the ECF continued. ECF glutathione concentrations were low during hibernation and increased during arousal and cenothermia, while levels remained fairly constant under each condition in tissues and plasma.
By contrast, ECF urate levels were regulated at a constant level during hibernation and cenothermia (with a transient doubling of ECF levels during shivering thermogenesis), despite a 50% reduction in intracellular concentrations during hibernation. Because urate, along with superoxide and hydrogen peroxide, is produced as a metabolite of xanthine activity, this increase can be viewed as a marker of ROS production as well as a means of ROS defence.
Looking at work done on several different species, the authors point out that the only common pattern of arousal metabolism is significant oxidation of ascorbate from the extracellular and plasma spaces, while brain tissue content of ascorbate and glutathione remains constant; whether this is because the brain is insulated from extracellular changes or is able to tightly regulate antioxidant levels is yet unknown. Very slow flow microdialysis, then, allows a real-time look at the metabolically demanding events of hibernation and arousal, which may provide investigators with a way to address these questions. And by looking at animals adapted for metabolic depression and arousal, it may be possible to separate protective regulatory mechanisms from the background pathological events observed in other mammalian brains.