Aging Aging

222 Miquellipoperoxides and malonaldehyde. Further, mtDNA may be unable to counteractthe chronic oxygen stress because, in contrast to the nuclear genome, itlacks histone protection and excision repair.If oxidative injury is not limited to organellar membranes (as proposed byearly free radical theory advocates) but also occurs in mtDNA, the organellesthat have suffered oxidation-related genome mutation, inactivation, or loss willbe unable to rejuvenate themselves by normal replication. Further, there willbe an impaired renewal of the macromolecules coded by that genome, namelythe 13 hydrophobic polypeptides of the electron transport chain and of ATPsynthase as well as the mitochondrial rRNAs and tRNAs. This may lead to aprogressive decline in mitochondrial function with decrease in ATP productionand ATP-dependent protein synthesis.It is very important from a clinical viewpoint that, because most cellularenergy is produced in mitochondria through the process of oxidative phosphorylation,the age-related dysfunction and loss of these organelles must result inbioenergetic decline. This may trigger apoptotic death (8) and play a key rolein the senescent decrease of physiological performance and in the pathogenesisof some age-related degenerative diseases of the somatic tissues mainly composedof fixed-postmitotic cells such as the cardiac and skeletal muscle and thecentral nervous system (CNS).In our opinion, the preceding summary on the causes and effects of mitochondrialaging justifies the present compilation of very detailed and practicalinstructions for the electron microscopic study of mitochondria regarding tissuefixation, sectioning, staining, and quantitative morphological observationof the organelles. We hope that this information will be especially useful toresearchers interested in furthering the understanding of the role of mitochondriaas promoters or targets in the senescence of diverse cellular types andanimal models. The techniques described may also interest the workers exploringthe modulation of the rate of animal aging by genetic manipulation, underfeeding,and pharmacological and antioxidant treatments.2. Materials1. Anesthetic solution: 2,2,2-Tribromoethanol (200 mg/kg body weight) in 10%ethanol solution.2. Perfusion solution: Prepare 0.1 M sodium cacodylate buffer at pH 7.4. If necessary,adjust the solution at the desired pH value by adding some drops of 0.1 NHCl (see Note 1). Dissolve 2% paraformaldehyde in cacodylate (Note 2). Theweighed amount of paraformaldehyde is warmed in about half the final volumeof cacodylate buffer at a temperature of 55–60°C. This solution is then cooledunder running tap water before addition of glutaraldehyde. Add 5% glutaraldehyde(Note 3) and bring the fixation solution to the final volume. As shown in