In vivo analysis of cell death with age and estrogen deficiency in the female rat heart

Open Access
Eldred, Stephanie
Area of Honors:
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Donna Hope Korzick, Thesis Supervisor
  • Stephen Jacob Piazza, Honors Advisor
  • myocardial infarction
  • postmenopausal women
  • coronary heart disease
  • ischemia/reperfusion injury
  • apoptosis
  • autophagy
  • necrosis
  • cell death
Women after menopause are more prone to myocardial infarctions (MI) compared to males, and postmenopausal women have a twofold increased risk for cardiovascular disease compared to premenopausal women. This suggests a potential cardioprotective role for estrogen (E2) in women. However, research regarding hormone replacement therapy in older women has been inconclusive concerning its efficacy. Three types of cell death are seen following MI: necrosis, apoptosis and autophagy. However, the role each plays in ischemia/reperfusion (I/R) injury following MI is unknown. This knowledge is critical to the development of new treatments and therapies to counteract the effects of E2-deficiency in aged females. Purpose: This study focuses on quantifying and characterizing the different forms of cell death following MI with age-associated E2-deficiency in Fischer 344 female rats. A secondary goal was the optimization of the triphenyltetrazolium chloride (TTC) staining protocol to accurately assess necrotic cell death using an in vivo model of ischemia. Methods: In vivo coronary artery ligation (CAL; 55 min I, 2 to 6 hrs R) was used to generate MI in three groups of female rats: aged ovary-intact (23-24 mo, n=8), aged ovariectomized (OVX; 23-24 mo, n=8) and adult ovary-intact (6-7 mo, n=8). Subsequent tissue analysis was performed for each type of cell death. Area at risk was assessed using Evan’s Blue dye (1%), necrotic cell death using 1% TTC staining, apoptotic cell death using DNA laddering and terminal deoxynucleotidyl transferase dUTP nick end labeling, and autophagy was assessed through western blot analysis using the protein markers LC3I:LC3II, cathepsin D, Beclin-1 and Atg5. Results: An optimized TTC protocol was generated and includes the perfusion of 1% Evan’s Blue dye (200-300 ìl) followed by ten minutes of TTC staining at 37oC. Following I/R, necrosis increased with age, apoptosis decreased with age and OVX, and autophagy increased with age. Conclusion: These results suggest that necrosis and autophagy play a larger role in cell death following I/R with age-associated E2-deficiency.