In the world of stroke and heart disease research, it is understood that a great deal of the damage from ischemia (reduction in blood flow) is due not only to the lack of oxygen when blood flow is cut off but also results from the restoration of blood flow and oxygen to the tissue. When oxygen floods back into the system, the mitochondrial electron transport chain functions inefficiently, resulting in a burst of reactive oxygen species (ROS). These highly reactive compounds may continue damaging proteins, lipids and DNA for hours to days after the initial ischemic insult. But animals that hibernate,or go for extended periods without oxygen, experience and then recover from greatly reduced respiration, heart rates and blood flow, and thus provide a natural model of ischemia and recovery without apparent tissue damage. Hatchling painted turtles (Chrysemys picta elegans) that hibernate in shallow natal nest chambers during their first winter after hatching not only hibernate but may actually survive by supercooling or actual freezing. This led Patrick Baker and his colleagues at Miami University to wonder how well frozen or supercooled turtles handled what could be the extremely stressful period of thawing and restoration of blood flow, with its potential for ROS production.
Turtle eggs hatched in the laboratory were gradually cold acclimated through the autumn down to 4°C. Then the team exposed the hatchlings to 48-h bouts of supercooling (down to –6.0°C in an environment free of nucleating agents), freezing (down to –2.5°C, with ice to induce nucleation) or hypoxia, before allowing the youngsters to recover for 24 h and taking tissue samples to see how the turtles responded to the icy conditions.
Knowing that animals that experience hypoxia switch to anaerobic respiration and generate lactate, the team measured the hatchlings plasma lactate levels and found that all of the hatchlings experienced hypoxia or anoxia (little or no oxygen).
The team also measured total antioxidant capacity in the hatchlings' plasma and brain and liver tissues; rather than measure levels of individual antioxidants, Baker and his colleagues opted to measure the overall ability of the tissues to prevent free radical accumulation when hydrogen peroxide was added to the reaction medium. They also looked for markers of oxidatively damaged proteins and lipids. The scientists found no significant increase in damaged lipids or proteins in any group compared with control animals kept at 4°C, nor were there differences in total antioxidant capacity of the tissues. This implies that the youngsters' ability to fight oxygen free radicals is not upregulated during hypoxia or cooling, but that intrinsic antioxidant levels are sufficient to fight ROS damage.
Baker and co-workers found that the antioxidant capacity of the hatchling painted turtles is comparable to that of several other animals, including a freeze-tolerant and an anoxia-tolerant frog, and the laboratory mouse (which as a warm-blooded mammal could be expected to have a high antioxidant capacity). Interestingly, the hatchlings' antioxidant capacity was higher than in the adult anoxia-tolerant turtle Trachemys scripta, which also can withstand anoxia and reoxygenation without damage, although this may be due in part to the suppression of ROS formation in addition to antioxidant levels. Additional experiments are planned to determine if the differences between hatchlings and adults are ontogenetic or taxonomic.
But at least we know this much: when it comes to resisting oxidative stress, these turtle babies are supercool!