Gene and protein expression during anoxia and re-oxygenation in crucian carp tissues

Anoxia related diseases like stroke and heart infarction are among the most common causes of death and debilitation in humans. The crucian carp (Norwegian: karuss, Latin: Carassius carassius) is unique among vertebrates in that it can survive without oxygen (anoxia) for several months. This is an adaptation to life in small lakes and ponds in Northern Europe that get ice- and snow-covered in the winter, resulting in oxygen-free water. Thus, in nature crucian carp are exposed naturally to anoxia for several months every winter, and anoxia is a natural physiological condition for this species. In the laboratory, we have seen that it tolerates at least two weeks of anoxia at 8°C without increased mortality. We use this fish as a model for finding mechanisms that promote anoxia tolerance and the ability to survive re-oxygenation. In the present project, we are interested in how particularly the brain and heart in crucian carp defend themselves against anoxic and re-oxygenation damage. We will therefore measure the expression (mRNA and protein) of key components in crucian carp tissues, especially the brain and heart, making a comparison between normoxia, anoxia and re-oxygenation. A special focus will be on genes we have found to be changing in a transcriptome study we have done previously. Crucian carp are collected (nylon cages - ruse) in a local pond near Oslo (NOTE: this fish is very common, and locally considered an invasive species in Norway). The fish will be exposed to anoxia and normoxia under dark conditions (mimicking the situation in nature) for one week (at ca 8°C), after which they will be sacrificed by a stunning blow to the head, and brain, heart and other tissues (liver, red muscle, kidney) are rapidly removed and no surgical procedures are involved. A subset of anoxic fish will be exposed to re-oxygenation for one day and one week, before sampling. Another subset of the normoxic and anoxic fish will be netted, lightly anaesthetised and receive an intraperitoneal injection of mitoB or saline, for examination of reactive oxygen species (ROS) production. The normoxic fish will be sampled after three days of additional normoxia (the controls) and the anoxic fish sampled after one and three days of re-oxygenation. In another subset of fish, oxygen uptake (MO2) and critical oxygen tension (PO2crit) after the exposure to normoxia and anoxia will be measured to investigate if differences in metabolism carries over into the re-oxygenation, and these measurements can then be related to findings at the gene and protein level. These parameters will also be compared in crucian carp originating from two or more different locations, as it can be hypothesised that habitats varying in temperature and oxygen level can have led to differences in these parameters between populations having been separated for a long time.