Molecular mechanisms of anoxia and re-oxygenation tolerance in crucian carp and goldfish
1) Anoxia related diseases like stroke and heart infarction are among the most common causes of death and debilitation in humans. Anoxia is also a problem in the context of organ transplantation. The crucian carp (Carassius carassius) and the closely related goldfish (Carassius auratus) are unique among vertebrates in that they can survive without oxygen (anoxia) for several months. In nature, crucian carp are exposed naturally to anoxia for several months every winter, and anoxia is a natural physiological condition for this species. We use the crucian carp as a model for finding mechanisms that promote anoxia tolerance and the ability to survive re-oxygenation, and are interested in the molecular regulation and mechanisms involved in the anoxic response. We will therefore measure the expression (mRNA and protein) of key components in crucian carp tissues, such as the brain and heart, making a comparison between normoxia, anoxia and re-oxygenation. We will also investigate cell-type specific gene expression, epigenetic regulation, compare the transcriptomic response between crucian carp and goldfish, and investigate the hypothesis that the crucian carp excrete succinate, a metabolite that in mammals becomes toxic during re-oxygenation due to its role in production of reactive oxygen species.
2) This study does not include any surgical procedures. The anoxia exposure is mimicking what these fish experience naturally every winter in their ponds, and can be considered mild- to moderately stressful. When the fish are netted it is a very short-term stress (seconds) that is identical to the handling of any aquarium fish.
3) Strokes and heart infarctions are among the most common causes of death and debilitation in humans, and lack of oxygen is also a problem in the context of organ transplantation. One approach to better understand the mechanisms that defend tissues against lack of oxygen (anoxia) and reperfusion damage is to study animals that have evolved natural protection against this.
4) For each standard exposure and sampling experiment, 80 fish will be used. We expect to complete one in 2021, two in 2022 and 2023, one in 2024 and 2025, giving a total use 560 crucian carp. We will conduct one exposure experiment on goldfish to obtain samples for RNAseq (40 fish), to compare with crucian carp. Lastly, we will conduct an experiment where we will measure the levels of succinate in the water during exposure to detect whether succinate is excreted by crucian carp (60 fish).
5) There are no cell-lines available from anoxia tolerant vertebrates, and only looking at cells in isolation will not provide answers to the scientific question asked. We use the minimum number of animals possible to assure that effects of the treatments can be statistically examined and differences are likely to be detected, and several tissues are harvested from each individual. For the sampling, we want to avoid putting the fish in water containing anaesthetics since that is possibly more stressful than the immediate effect of the blow to the head.
2) This study does not include any surgical procedures. The anoxia exposure is mimicking what these fish experience naturally every winter in their ponds, and can be considered mild- to moderately stressful. When the fish are netted it is a very short-term stress (seconds) that is identical to the handling of any aquarium fish.
3) Strokes and heart infarctions are among the most common causes of death and debilitation in humans, and lack of oxygen is also a problem in the context of organ transplantation. One approach to better understand the mechanisms that defend tissues against lack of oxygen (anoxia) and reperfusion damage is to study animals that have evolved natural protection against this.
4) For each standard exposure and sampling experiment, 80 fish will be used. We expect to complete one in 2021, two in 2022 and 2023, one in 2024 and 2025, giving a total use 560 crucian carp. We will conduct one exposure experiment on goldfish to obtain samples for RNAseq (40 fish), to compare with crucian carp. Lastly, we will conduct an experiment where we will measure the levels of succinate in the water during exposure to detect whether succinate is excreted by crucian carp (60 fish).
5) There are no cell-lines available from anoxia tolerant vertebrates, and only looking at cells in isolation will not provide answers to the scientific question asked. We use the minimum number of animals possible to assure that effects of the treatments can be statistically examined and differences are likely to be detected, and several tissues are harvested from each individual. For the sampling, we want to avoid putting the fish in water containing anaesthetics since that is possibly more stressful than the immediate effect of the blow to the head.