Clocks and hypoxia: Linking oxygen to time
1.Purpose
Diving mammals display a high tolerance towards lack of oxygen (hypoxia), a condition that they experience during breath-hold diving. They therefore serve as a unique model to study and understand evolutionary adaptive mechanisms that protect mammalian tissues and organs from hypoxia injuries, which are known to kill or disable millions of humans worldwide on an annual basis. In rodents, tolerance to hypoxia is dependent on the time of day and the molecular basis for this is partially known. Daily activity data of free-living hooded seals show that their diving depth varies with time of day and we hypothesize that, in a similar way as in rodents, their hypoxia tolerance is synchronized with behaviour, by way of their circadian clock. To investigate this further, we must first establish whether circadian rhythmicity persists in seals, under constant conditions.
We will use captive hooded seals that are instrumented with data loggers that monitor activity and body temperature (both representing commonly measured outputs of the circadian clock). Experiments will be performed in light-controlled approved seal facilities at UiT. By alternating between periods with simulated natural light conditions and periods of constant light/darkness, it will be possible to identify whether rhythms of activity and body temperature persist under constant photic conditions, and are therefore driven by an endogenous/circadian clock.
Once the live-animal studies are complete we will harvest tissue postmortem, for cell-culturing that will enable us to use cell respirometry and genomics/transcriptomics/proteomics to further study the adaptive hypoxia tolerance mechanisms and their link to the circadian clock.
2.Distress
A body temperature logger (Anipill) will be fed to the seal via its normal diet (herring) causing no distress. Activity will be monitored via an Actiwatch glued to the fur, this is only mildly distressing due to the necessary restraint, via syringe delivered sedation, of the animal for the glueing procedure. Light regime manipulations will cause minimal distress.
3.Expected benefit
Diving mammals display a high tolerance towards lack of oxygen (hypoxia), a condition that they experience during breath-hold diving. They, therefore, serve as a unique model to study and understand evolutionary adaptive mechanisms that protect mammalian tissues and organs from hypoxia injuries, which are known to kill or disable millions of humans worldwide on an annual basis. Understanding the mechanisms underlying the greatly enhanced hypoxia tolerance of diving mammals, including its possible links to circadian rhythms, may, thus, benefit medical science.
4.Animals
Hooded seals
5.3Rs
Reduction: We will use only 6 hooded seals, which is close to the minimum number required for statistical analyses.
Refinement: We will accustom pups to human contact and handling and offer enrichments throughout their time in captivity. Combining this with biologging methods (Anipill, Actiwatch) that allow data collection from undisturbed and freely moving animals in controlled semi-natural settings, will reduce confounding effects of stress and improve data quality.
Replacement: The study of basic circadian organization must be performed using live animals of appropriate species. By utilizing animals postmortem for development of cell-culturing techniques we may replace future live-animal usage.
Diving mammals display a high tolerance towards lack of oxygen (hypoxia), a condition that they experience during breath-hold diving. They therefore serve as a unique model to study and understand evolutionary adaptive mechanisms that protect mammalian tissues and organs from hypoxia injuries, which are known to kill or disable millions of humans worldwide on an annual basis. In rodents, tolerance to hypoxia is dependent on the time of day and the molecular basis for this is partially known. Daily activity data of free-living hooded seals show that their diving depth varies with time of day and we hypothesize that, in a similar way as in rodents, their hypoxia tolerance is synchronized with behaviour, by way of their circadian clock. To investigate this further, we must first establish whether circadian rhythmicity persists in seals, under constant conditions.
We will use captive hooded seals that are instrumented with data loggers that monitor activity and body temperature (both representing commonly measured outputs of the circadian clock). Experiments will be performed in light-controlled approved seal facilities at UiT. By alternating between periods with simulated natural light conditions and periods of constant light/darkness, it will be possible to identify whether rhythms of activity and body temperature persist under constant photic conditions, and are therefore driven by an endogenous/circadian clock.
Once the live-animal studies are complete we will harvest tissue postmortem, for cell-culturing that will enable us to use cell respirometry and genomics/transcriptomics/proteomics to further study the adaptive hypoxia tolerance mechanisms and their link to the circadian clock.
2.Distress
A body temperature logger (Anipill) will be fed to the seal via its normal diet (herring) causing no distress. Activity will be monitored via an Actiwatch glued to the fur, this is only mildly distressing due to the necessary restraint, via syringe delivered sedation, of the animal for the glueing procedure. Light regime manipulations will cause minimal distress.
3.Expected benefit
Diving mammals display a high tolerance towards lack of oxygen (hypoxia), a condition that they experience during breath-hold diving. They, therefore, serve as a unique model to study and understand evolutionary adaptive mechanisms that protect mammalian tissues and organs from hypoxia injuries, which are known to kill or disable millions of humans worldwide on an annual basis. Understanding the mechanisms underlying the greatly enhanced hypoxia tolerance of diving mammals, including its possible links to circadian rhythms, may, thus, benefit medical science.
4.Animals
Hooded seals
5.3Rs
Reduction: We will use only 6 hooded seals, which is close to the minimum number required for statistical analyses.
Refinement: We will accustom pups to human contact and handling and offer enrichments throughout their time in captivity. Combining this with biologging methods (Anipill, Actiwatch) that allow data collection from undisturbed and freely moving animals in controlled semi-natural settings, will reduce confounding effects of stress and improve data quality.
Replacement: The study of basic circadian organization must be performed using live animals of appropriate species. By utilizing animals postmortem for development of cell-culturing techniques we may replace future live-animal usage.