Fig 1: C57/BL6 MOUSE
Our main area of research into ageing has focussed on performing tests of the free-radical theory of ageing. We have experimentally manipulated and measured increases in metabolic rate in two rodent models; the Short-tailed field vole Microtus agrestis and the inbred mouse strain C57/BL6.
We have then examined the effects of these measured changes in metabolic rate on oxidative stress and ultimately on lifespan. Our hypothesis is that if the free-radical theory of ageing is correct then any increase in metabolic rate (oxygen consumption) will lead to an increase in oxygen free-radical production, elevated damage to tissues (lipids, proteins and DNA) and ultimately a shortened lifespan.
|
Fig 1: C57/BL6 MOUSE |
Fig 2: Short-tailed field vole Microtus agrestis |
Experimental protocol:
Metabolic rate in voles and mice have been manipulated two ways;
1. For the first manipulation of metabolism we have compared individuals continuously exposed to the cold (7oC) with individuals maintained at 22oC. We are using sublingpairs in these manipulations to control for genetic variability to some extent. Comparisons are then made using pairwise comparison statistics.
2. We are also examining whether free-radical damage, which is expected to be elevated in the high metabolism cold exposure group, can be attenuated by feeding the rodents with food supplemented with antioxidant vitamins C (16x expected daily requirements) and vitamin E (25x expected daily requirements).
3. As a second manipulation of metabolism we have compared individuals (voles only) with access to a running wheel with non-running siblings. We have found that voles provided with wheels will run throughout the hours of darkness and run on average over 7km per night (Fig 3). By manipulating the duration of darkness we can modulate the extent to which these animals exercise so that we have the same elevation in metabolism as that produced by the cold exposure manipulation.
|
Fig 3. Running activity of voles. Click here to see larger image. |
Running wheel equipment for monitoring activity of voles. For more details click here. |
Metabolic measurements
Metabolic rate has been measured in two ways.
1. Oxygen consumption has been
measured by open-flow respirometry to determine resting metabolic
rate (RMR) and, in conjunction, carbon dioxide has been measured
to estimate the respiratory quotient (RQ).
2. We have also used the doubly-labelled water (DLW) technique to determine daily energy expenditure (DEE). DLW is a technique that uses the elimination rates of both deuterium and 18oxygen isotopes to calculate carbon dioxide production and hence DEE.
3. We have shown that our experimental manipulations (either cold exposure or wheel running) increase DEE by over 40% compared to control individuals.
Biochemical measurements
We are using several different techniques to measure oxidative stress in our experimental animals. We have used enzymatic assays to determine the activities of various antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) in various tissues. These enzymes are involved in the dismutation and decomposition of the superoxide radical and hydrogen peroxide. We have shown that long-term cold exposure leads to an increase in these enzymes in various tissues of voles.
DNA Damage
To detect and measure DNA strand breaks, single cell gel electrophoresis, also known as the comet assay is used. Lymphocytes and hepatocytes are isolated by centrifugation on a density gradient and are embedded in agarose on a microscope slide and lysed in a solution containing triton X-100 and 2.5M NaCl. The resulting nucleoids are incubated with repair endonucleases specific for certain kinds of damage. Endonuclease III (Endo III) was used to detect oxidised pyrimidines and formamidopyrimidine glycosylase (FPG) to detect damaged purines including 8-oxo-guanine. The slides are then electrophoresed under alkaline conditions. The presence of breaks in the DNA allows it to extend towards the anode, forming a comet-like image when viewed by fluorescence microscopy. The comets are then scored on a scale of zero to four depending on the percentage of DNA in the tail.
This latter work is being carried out in collaboration with Prof. Andrew Collins, Division of Cellular Integrity Rowett Research Institute, Aberdeen.
Lipid peroxidation
The thiobarbituric acid reactive substance assay (T-Bars) is used to measure lipid peroxidation. The principle of this method is that malonaldehyde, a product of lipid peroxidation, is coupled to thiobarbituric acid. This assay is widely used, technically simple and works well in animal models. Specificity is improved by using HPLC (high performance liquid chromatography).
Vitamin E concentration
Vitamin E content is determined using a reverse phase HPLC method.
This allows the measurement of vitamin e content in plasma and
tissue (liver) using fluorescence and visible detection.
Vitamin C concentration
For vitamin C content the simultaneous determination of the plasma
and tissue concentration of ascorbic acid is analysed using reversed
phase HPLC using an ion-pairing reagent with UV detection. This
work is being performed in collaboration with Dr Garry Duthie,
Division of Cellular Integrity, Rowett
Research Institute.
We have recently shown that acute exercise of 1 day or 1 week
duration appears to have little effect on either antioxidant enzyme
activities or on the levels of DNA damage in lymphocytes or hepatocytes,
and that 2 month cold exposure did increase activity of CAT and
SOD in some tissues, but did not alter T-Bars or levels of oxidative
DNA damage.
Antioxidant enzyme gene expression
More recently we have started to measure antioxidant enzyme gene expression levels using RNA oligonucleotides specific for murine SOD 1, SOD 2, CAT and GPx.
Gene expression arraying
We are using the CLONTECH stress gene array to examine patterns of gene expression of the major stress genes between cold and warm exposed animals and also young and old animals under identical temperature treatments.
Current collaborations:
We are currently involved with several collaborations outwith
Aberdeen, including one with Tilman Grune (Humboldt University,
Berlin ) looking
at the effect of acute cold exposure on proteosome activity/protein
carbonyl levels and one with Darren Talbot and Dr Martin Brand
(MRC-Dunn, Cambridge
) examining the effects of cold exposure and intraspecific metabolic
rate on the rates of proton leak across mitochondrial membranes.
