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Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction.
- Source :
-
Journal of visualized experiments : JoVE [J Vis Exp] 2016 Jan 12 (107), pp. e53207. Date of Electronic Publication: 2016 Jan 12. - Publication Year :
- 2016
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Abstract
- Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still unclear. Our laboratory has developed a rat model of post-MI syndrome that displays impaired cardiac functions, neuronal loss in the limbic system, cognitive deficits and behavioral signs of depression. At the neuronal level, caspase-3 activation mediates post-MI apoptosis in different limbic regions, such as the amygdala - peaking at 3 days post-MI. Cognitive and behavioral impairments appear 2-3 weeks post-MI and these correlate statistically with measures of caspase-3 activity. The protocol described here is used to induce MI, collect amygdala tissue and measure caspase-3 activity using spectrofluorometry. To induce MI, the descending coronary artery is occluded for 40 min. The protocol for evaluation of caspase-3 activation starts 3 days after MI: the rats are sacrificed and the amygdala isolated rapidly from the brain. Samples are quickly frozen in liquid nitrogen and kept at -80 °C until actual analysis. The technique performed to assess caspase-3 activation is based on cleavage of a substrate (DEVD-AMC) by caspase-3, which releases a fluorogenic compound that can be measured by spectrofluorometry. The methodology is quantitative and reproducible but the equipment required is expensive and the procedure for quantifying the samples is time-consuming. This technique can be applied to other tissues, such as the heart and kidneys. DEVD-AMC can be replaced by other substrates to measure the activity of other caspases.
Details
- Language :
- English
- ISSN :
- 1940-087X
- Issue :
- 107
- Database :
- MEDLINE
- Journal :
- Journal of visualized experiments : JoVE
- Publication Type :
- Academic Journal
- Accession number :
- 26862955
- Full Text :
- https://doi.org/10.3791/53207