Three levels of study of the effects of genetic knockouts on the aging process in mice:

Determination of effects on lifespan.

This is the gold standard for assays of mechanisms of aging. This parameter can only be determined by allowing mice to live as long as they can. We are especially interested in maximum lifespan, commonly defined as the 90th percentile of life. While some gene mutations may increase both average and maximum lifespan, others may affect only average lifespan without increasing maximum life span. This type of scenario could be interpreted as the result of reducing the rate of certain midlife diseases, and not the fundamental processes of aging. Thus, it becomes imperative to be able to detect differences in maximum lifespan, in addition to mean and median lifespan.

Attenuation of age-related declines in biological functions.

We will use physiological variables validated by Millerís group (Harper et al., 2003; Harper et al., 2004), which are detectable early in life in juveniles or young adults. They are capable of influencing risks of multiple forms of functional decline and risk of multiple diseases with increasing age. These variables will include body weight, serum leptin, and serum T4 collected at four months of age. We will also include serum Igf1 since many of the genes we are working with interact directly or indirectly with the insulin/Igf1 pathway. All mice will be weighed weekly. The lower the body weight at four months and the lower the rate of growth increase from one to four months, the longer the lifespan (Miller et al., 2002). Low leptin levels are associated with increased life span. Millerís group (Harper et al., 2003) observed a negative effect of high levels of serum leptin on life expectancy in 4-month-old 4-way cross mice, which confirmed an earlier study by Barzilai and Gupta (1999). Leptin is a hormone which controls appetite based on satiation. The thyroid hormones T3 and T4 are key regulators of basal metabolic rate in mammals (Lebon et al., 2001). An increase in the levels of both hormones results in an increase in basal metabolic rate. The Miller group (Harper et al., 2003) found that high T4 levels in 4-way cross mice at four months of age were associated with increased life span. Generally, high Igf1 levels are correlated with decreased life span (Harper et al., 2003).

Reduced incidence and/or delayed onset of age-related pathologies.

For this third measure of aging, we will monitor and evaluate age associated disease using ultrasound imaging and end of life pathology. We will use ultrasound to noninvasively monitor for internal lesions with increasing age, employing an Acuson-70 mobile ultrasound unit (Siemens) that can provide a resolution of 1 to 2 millimeters. This methods can readily detect internal tumors and other space occupying lesions before they can be palpated. We can also detect certain cardiovascular lesions such as mineralization, cardiac hypertrophy, and perform echocardiography. The rationale for using ultrasound scanning is that it will allow us to compare the incidence of age-related pathology or disease at an earlier non life-threatening time point in the lifespan experiment. We will then be able to compare these early image-derived lesions with lesions observed at end of life pathological evaluation, and ultimately with lifespan and health span. Necropsies will be performed on all mice that are sacrificed in a moribund condition or found dead. Tissues will be examined microscopically by a board certified veterinary pathologist. A list of pathological lesions will be constructed for each mouse that will include both neoplastic and non-neoplastic conditions. We will then be able to calculate the tumor burden, disease burden, and severity of each lesion in each mouse. The severity of a number of lesions will be determined using grading systems because their high prevalence allows a finer dissection of the effect of genetic variation on the extent of pathology. The probable cause of death in each mouse will be determined by the severity of pathological disease. For neoplastic diseases, cases which have Grades 3 and 4 lesions are categorized as death by neoplastic lesions. For non-neoplastic diseases, cases which have a severe lesion, e.g., Grade 4, associated with other histopathological changes (pleural effusion, ascites, congestion and edema in lung) are categorized by death by non-neoplastic lesion. In some cases cause of death will be categorized as unknown.

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