Lifespan reflects an animal's overall health and the rate of aging. As shown in , caspase-2 knockout mice had almost the same median lifespan (950 days) as the wild-type littermates (954 days). However, from that time point on, the survival curve of caspase-2 knockout mice diverged from that of the wild-type mice (). There was a small difference in the mean lifespan between caspase-2 knockout mice (903 days) and the wild-type littermates (935 days). However, there was a large and significant difference in the maximum lifespan (mean of the last 10 percentile of the mice) between caspase-2 knockout mice and the wild-type littermates. The maximum lifespan of caspase-2 knockout mice was 117 days (~4 months or 10%) shorter than that of the wild-type mice (1137 versus 1254 days). None of the caspase-2 knockout mice lived beyond 1200 days, whereas 12.5% of wild-type littermates did. Log-rank test of the last 50 percentile of animals (i.e., animals living longer than the group's median lifespan) showed that caspase-2 knockout mice lived statistically shorter (p<0.01) than the wild-type littermates.
Lifespan A. Survival curves for all mice (n=64 for each group). B. Survival curves for last 50 percentile mice in each cohort (n=32 for each group).
Apoptosis serves as an endogenous defense mechanism against tumorigenesis. It is a widely-held view that defects in apoptosis may lead to increased tumorigenesis (Dlamini et al., 2005
). Therefore, our initial hypothesis was that caspase-2 deficiency might lead to increased tumorigenesis. However, after a comprehensive examination of various tumors in the mice at 24-26 months of age, we found that caspase-2 deficiency did not increase tumor incidence (). The overall tumor burden (the sum of different types of tumors in a mouse) was 0.44 for caspase-2 knockout mice, 0.66 for the wild-type mice, and 0.61 for the heterozygous mice. The percentage of tumor bearing mice (the percentage of mice that have one or more neoplastic lesions in an experimental group) was 39.3% for caspase-2 knockout mice, 55.2% for the wild-type mice and 48.5% for heterozygous mice. We also examined the incidence of a number of non-neoplastic pathologies/diseases including glomerulonephritis, lymphocyte infiltration, hydronephrosis, acidophilic macrophage pneumonia, brain psammoma body, angiectasia and arteritis in the mice (). Caspase-2 deficiency did not increase the incidence of these pathologies/diseases, either. Therefore, the shortened lifespan of caspase-2 knockout mice was not caused by increased tumor incidence or these non-neoplastic pathologies/diseases.
Comparative pathology of old mice (24-26 months of age)
Since the reduction of the survival rate in caspase-2 knockout mice began only at a later stage of life and the pathological study did not find the cause for the shortened lifespan of caspase-2 knockout mice, we asked whether the shortened lifespan of caspase-2 knockout mice may be due to increased rate of aging. Because there is no single definitive biological marker of aging, the change of aging rate is always assessed by examining the animal's lifespan together with a number of aging-dependent traits, most important of which are skeletal health, hair growth and subcutaneous fat content (Tyner et al., 2002
; Trifunovic et al., 2004
Mammalian bone is a dynamic tissue that is continually remodeled throughout life (Blair et al., 1993
). Through this remodeling, localized new bone formation by osteoblasts replaces bone that is resorbed by osteoclasts. One of the hallmarks of aging is a progressive loss of bone mass due to an increase in bone resorption activity relative to bone formation activity.
A number of researchers have reported that several premature aging mice had severe kyphosis (Tyner et al., 2002
; Trifunovic et al., 2004
). In their papers, the evidence was demonstrated by representative images, but not by quantitative measurement of the severity of the kyphosis. In fact, there is no accepted or consensus method for the measurement of the degree of kyphosis in mice. We tried a method described by Garcia-Cao et al. (2002)
and found it too subjective. Therefore, we chose not to use kyphosis as an indicator of aging-dependent skeletal change. The bone mass can be accurately measured. We examined bone mineral density (BMD) of the mice using DEXA scan. At 14 months of age, the BMD of caspase-2 knockout mice was not significantly different from that of wild-type littermates (). However, at the age of 24-26 months, caspase-2 knockout mice had a significantly lower BMD than wild-type mice (). To further investigate this phenotype, we performed quantitative histomorphometric analysis of the vertebral bodies of old mice (29 months of age) and found that caspase-2 knockout mice exhibited decreased trabecular bone (indicated by arrows in Figure and ) volume compared to age-matched wild-type mice (). We counted the number of osteoblasts and osteoclasts per mm of the bone surface. Aged caspase-2 knockout mice had similar numbers of osteoblasts (), but increased numbers of osteoclasts (indicated by arrows in Figure and ) compared to the wild-type mice (). These results indicate that caspase-2 knockout mice had a more severe aging-dependent bone loss at advanced ages. Furthermore, these results also suggest that the mechanism of increased bone loss in caspase-2 knockout mice may be due to enhanced bone resorption. In vivo
rates of bone resorption can also be determined by measuring the products of bone resorption in urine. Type I collagen, which accounts for approximately 90% of the organic matrix of bone, is cross-linked by specific molecules such as deoxypyridinoline (DPD). Resorption of the bone causes release of DPD into the circulation and excretion in urine (Delmas et al., 1991
). Urinary DPD levels normalized against urinary creatinine concentrations reflect rates of bone resorption in vivo
. As shown in , at 26 months of age, caspase-2 knockout mice had much higher rates of bone resorption than wild-type littermates based upon the level of DPD urinary excretion. These results demonstrated that caspase-2 deficiency enhances aging-dependent bone loss at least partially through enhancing bone resorption.
Figure 2 Skeletal traits. A. Section of vertebral body of old wild-type mice (29 months of age) stained by haematoxylin and eosin. B. Section of vertebral body of old caspase-2 knockout mice stained by haematoxylin and eosin. Arrows in A and B indicate trabecular (more ...)
Figure 3 Skeletal uptake of99mTc-MDP. A. The representative micro-CT image of a wild-type mouse at the age of 29 months. B. The distribution of 99mTc-MDP in skeleton in the same mouse as detected by micro-SPECT . C. The overlay of the left and middle panel. D. (more ...)
Technetium −99m methylene diphosphonate (99m
Tc-MDP) is a widely used bone-scanning agent (Subramanian et al., 1975
). Because the diphosphonate group has a very high affinity for the inorganic matrix of the bone, 99m
Tc-MDP is concentrated mainly in the skeletal system. The uptake of 99m
Tc-MDP is particularly high at active bone remodeling sites because more bone mineral surfaces are exposed by osteoclasts. Clinically, it has been found that the total skeletal uptake of 99m
Tc-MDP is increased in osteoporotic patients due to increased bone turnover activity (Carnevale et al., 2000
). We used micro-single photon emission computed tomography (SPECT) to compare 99m
Tc-MDP uptake in male caspase-2 knockout and wild-type mice at the age of 29 months shows the micro-computed tomography (CT) image of a mouse. shows the distribution of 99m
Tc-MDP in the body as detected by micro-SPECT. is the overlay of Figures and that shows the concentration of 99m
Tc-MDP in the skeleton. Quantitative measurement revealed that caspase-2 knockout mice had a significantly higher skeletal uptake of 99m
Tc MDP than the wild-type mice (). Therefore, there results are consistent with those of bone densitometry and histomorphometry.
Hair growth declines as a function of age in mice (Harrison et al., 1988
). Hair regrowth ability has been widely used as a measurement of aging (Tyner et al., 2002
; Trifunovic et al., 2004
). To measure the hair growth ability, we shaved a dorsal segment (2×3 cm) of skin on the mouse. After 30 days, the number of mice whose hair failed to grow back was counted. The number is 40% (6 out of 15) for old caspase-2 knockout mice (24 months of age) and 10% (1 out of 10) for age-matched wild-type mice. After 48 days, all wild-type mice had the hair grown back (n=10), but there were still 21.4% (3 out of 14) of caspase-2 knockout mice whose hair failed to grow back. Therefore, the hair growth ability in old caspase-2 knockout mice is impaired relative to that in age-matched caspase-wild type mice.
Loss of body fat, especially subcutaneous fat, and body weight, is common in very old people. Due to uneven distribution, it is very difficult, if not impossible, to accurately quantify the subcutaneous fat content. As an alternative way, we measured the whole body fat content with DEXA scan and found that caspase-2 knockout mice at advanced ages had a lower body fat content than age-matched wild-type littermates ().
Body fat content. Body fat content of the mice at the age of 24-26 months measured by DEXA scan (n=28-34).
Caspase-2 is activated by oxidative stress and mediates oxidative stress-induced cell death (Lopez-Cruzan et al., 2005
; Prasad et al., 2006
). As a consequence of loosing caspase-2 function, the animals may not be able to efficiently clear oxidatively damaged cells. Liver is where a lot of oxidative reaction occurs. We hypothesized that if the oxidatively damaged cells are not promptly cleared through apoptosis, this would result in increased levels of oxidized macromolecules in the liver. Therefore, we examined the extent of global protein oxidation in the liver. The amount of cysteine residues that have not been irreversibly oxidized is inversely proportional to the cellular content of oxidized protein (). We used the assay described in to measure the cysteine oxidation in the liver of caspase-2 knockout and wild-type mice (Chaudhuri et al., 2001
). Cysteine residues on proteins were first exposed by treating proteins with dithiothreitol and 6M urea, and then labeled with 6-iodoacetamidofluorescein (6-IAF). After the removal of free 6-IAF, the 6-IAF-labeled proteins were separated on a polyacrylamide gel and their amount was quantified. A low amount of 6-IAF-labelled proteins corresponds to a high degree of irreversible cysteine oxidation. There was a general trend of an increase of protein oxidation with an increase in animal's age, which can be seen clearly in the boxed areas in and in . This is consistent with other studies (Bokov et al., 2004
). Interestingly, we found that caspase-2 knockout mice had a higher level of protein oxidation than the wild type littermates at 6 months of age (), which suggests that there are more oxidatively damaged cells in the caspase-2 knockout mice due to deficiency of caspase-2-mediated apoptosis.
Figure 5 Cysteine oxidation in caspase-2 deficient and wild-type mice. A, Schematic representation of different states of oxidation of cysteine residues in proteins. B. Schematic representation of cysteine oxidation assay. C. Global measurement of irreversible (more ...)