Preliminary Data

In this are you will find:

 

Survival Curve

survival curve

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      Median lifespan data 

      To identify genetic mechanisms that regulate aging, median lifespans data were used to perform QTL analysis.  Haplotype Association Mapping analysis (HAM) has been shown to significantly reduce the amount of time and resources required to identify QTL genes from the traditional QTL analysis method. Median lifespan data were scanned across a dataset containing 70,000 genome-wide panel of SNPs. Two strains were considered to have the same inferred haplotype if and only if their genetic patterns were identical across all three adjacent SNPs. To measure the strength of associationbetween genotype and median lifespan, we computed regression-based test statistics.

      Download genome-wide panel dataset (.zip 1.73 MB) 

      Immune aging markers study

      The 6-month old population of mice consisted of 200 females and 200 males (total 400 mice). Row data from miceof each strain(n=8-16) were recorded using FACScan software and subsequently analyzed using FlowJo software. Four and five color flow cytometry analyses were used to evaluate each individual lymphocyte cell subset including: T cell, B cell, NK, NKT, as well as monocytes and granulocytes. Table 5 includes description of lymphoid cell subsets measured in 6-mo old mice.

      The frequency of each peripheral blood leukocyte subset was calculated for each mouse, averaged per group and presented as percentage of total leukocytes or percentage of total lymphocytes). Due to the sex-related differences in immune system between males and females particularly for some autoimmune-prone mouse strains, the row data were analyzed as mean value ± standard deviation for each gender.

      Preliminary results Peripheral blood cells from 27 inbred mouse strains were stained and immunophenotyped using 4-colour FACSscan. Data obtained from one time point (6-mont old mice) were assembled, rewired and analyzed. Results are summarized in Table 4 (A and B), and 5 (A and B) showing the percentages of PB leukocyte and lymphocyte cell subsets.

      In the resent work we have provided complete information regarding relative proportions of PB leukocyte and lymphocyte cell subsets in large cohort of genetically diverse mouse strains. The analysis of PB leukocyte proportions in 6-mo old mice presented in Table 4A and 4B show that the relative percentage of lymphocytes, granulocytes and monocytes were different among the 27 inbred strains. The total leukocyte percentage was low C3H/HeJ, KK/HlJ, MRL/MpJ (females and males); C3H/HeJ, KK/HlJ, MRL/MpJ, NON/HlLtJ (males), and high in RIIIS/J, C57BR/cdJ, C57L/J (both females and males). The proportion of monocytes was the relatively low in RIIIS/J (2.12% females and 2.82% males) and as expected high in several autoimmune-prone mouse strains including: MRL/MpJ (14.3% females and 12.3% males), NON/LtJ (12.28% females and 24.4% males), and NZW/LacJ (12.05% females). Interestingly, the proportions of granulocytes (defined as Gr-1+ cells) were lowest in RIIIS/J mice (2.8% females and 8.65% males) and highest in NON/LtJ mice (29.22% females and 34.36% males).

      We also analyzed PB leukocyte proportions in mature 6-mo old mice (younger mice, 2-3 months, were not included in the study). We found strain differences in major PB lymphocyte proportions at this age. Percentages of B220+ cells were high in C57BL/10J (82.67% females) and C57Bl/6L (82.01% males) and low in SJL/J mice (16.75% females). The proportions of different T cell subsets (helper CD4 T cells and cytotoxic CD8 T cells) were highly variable among the strains suggesting that PB lymphocyte proportions are genetically regulated. As expected. NON/LtJ had the lowest frequency of both CD4 (2.69% males) and CD8 T cells (1.87% males) due to the mutation on chromosome 17 present in this strain, which leads to reduced T cell number (T cell lymphopenia). In contrast, SJL/J, PWD/PhJ and RIIIS/J mice had the highest proportion of both CD4 and CD8 T cells (for details see Table 5A and B). Naïve CD4 and CD8 T cells were analyzed, defined as CD62LlowCD44high (naïve) and CD62LhoghCD44low (memory) cell subsets. Results showed substantial difference in the proportions of naïve and memory cell subsets among the strains. Additionally, NK and NK T cells cell were tested. NK and NKT cells represent one of the first arms of immune defense against pathogens and tumor immunosurveillance. The results showed that majority of the inbred strains included in the study (6 mo old) had normal percentages of NK (5%) and NK T (1%) cells with NZO/H1LtJ having the highest NK percentage (13.11% females and 13.75 males) and AKR/J having the highest NK T percentage (5.46% females and 3.03% males).

      Summary and future plans Several studies demonstrate that aging is associated with decline in many physiological systems including immune system. Different investigators have observed decreased lymphocyte proliferation, decline on CD4+ T cells proportions, change in NK and macrophage proportions and activity. Our goal is to conduct immune survey of 32 inbred mouse and look for age-related changes in immune cell subsets. We found significant strain difference in major immune cells subsets at 6-mo of age. Further study will allow us to compare these results with results obtained from older mice (12-mo, 18-mo, and 24-mo). Immunosenescence is also accompanied by change in B cell lineage and increased autoantibody production. We will therefore asses the age-related changes in serum imminoglobulins including; IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA. All together, results will allow us to characterize immune system of 32 genetically diverse inbred mouse strains in the process of aging. This new information will add to the complexity of the aging immune system and define immune parameters as useful biomarkers for assessment of biological age and predictors for longevity

      Comprehensive Cage Monitoring System

      The Comprehensive Cage Animal Monitoring System (Columbus Instruments, Columbus, OH) is used to simultaneously determine calorimetric parameters, food and drink consumption, as well as ambulatory and rearing activity of individual animals. The software and hardware platform measures the volumes of O 2 consumed and CO 2 produced in a sample from each animal, thereby allowing calculation of the respiratory quotient and metabolic rate on the basis of the mass of an individual mouse. Food and water consumption is measured directly at hourly intervals. Patterns of food intake can be determined from continuously recorded bout data. Locomotor activity is measured every 10 seconds for 72-80 hrs. It is presented as the number of beam breaks within the X, Y and Z planes. Total horizontal activity counts every beam break in the X-Y plane, whereas exploratory activity is characterized by a minimum of three consecutive beam breaks in the horizontal planes. Any beam break in the Zl plane contributes to counts for rearing activity.

      Testing Routine. Thirty-two animals are placed individually in cages of similar size to the home cage. At 3, 6, 12, 18, 24 months of age animals are placed in the metabolic cages from three to four days to acclimate to a novel environment; the 12/12 light:dark cycle and temperature, and food are identical to the home environment. Animals are individually housed for the 72-80 hour test period and data only from the last 24-48 hrs will analyzed for statistical comparison. Earlier data points may be used assess acclimatization. Ground chow diet containing 6% fat content is provided (identical to home colony diet, 6% fat content, NIH formula, #5427 PMI Feeds). Water is available ad libitum. Each metabolic chamber (12.3 cm high x 10.5 cm wide x 21.0 cm long, similar to home environment) consists of a plastic grid floor and absorbent sheet for urine and fecal waste. The feeder is accessible through a tube 2.5cm in diameter, allowing mice up to 40 g to enter freely. For animals surpassing this body weight, extra food is weighed, and placed inside the cage to permit total daily consumption (after subtracting spillage) during the testing period

      Preliminary Data & Interpretation

      Ten parameters are recorded for individual animals over a three-to-four-day period. Specifically, these ten parameters include oxygen consumption, carbon dioxide production, respiratory quotient, metabolic rate (using Weir’s Equation), food and water consumption, and counts of horizontal, movements, rearing behavior, ambulation. Behavioral measurements, such as food and water consumption, can facilitate identification of neural system, or peripheral system, contribution with underlying changes with aging. Voluntary locomotor activity, including total horizontal counts, rearing, and ambulation, can be used to assess the lack of physical activity that leads to reduced energy expenditure and hence age-related weight gain. When combined together, these behavioral variations can signify the presence of general malaise, either concomitant to, or independent of locomotor dysfunction. Consumption measurements of food and water may indicate the presence of some metabolic disorders common to aging, such as diabetes mellitus and metabolic syndrome.

      As an illustration of the data generated by CCMS, I will briefly review some preliminary data for the overall daily averages for some of the parameters, across ten different strains (please refer to the following page of figures when indicated). As seen in figure 1, body mass of the males across the ten strains did not vary greatly at 6 months of age, except for NZO/HiLtJ – a strain predisposed to maturity-onset diabesity. The comprehensive measurements of calorimetery, oxygen consumption and carbon dioxide production indicates metabolism on an organismal level. From these calorimetric measures, shown in figure 2, one can again see that the NZO/HiLtJ strain has a notably curtailed energy expenditure. Two additional variables are derived, which are crucial for interpreting and pinpointing underlying changes in physiological systems: respiratory quotient and metabolic rate. Respiratory quotient indicates which metabolic fuel sources are utilized, such as fat (level 0.7) and carbohydrate (level 1) etc., or combination thereof. As shown in figure 3, most strains utilize a mixture of metabolic fuels (range of 0.8), which is expected at this level of analysis because mice typically use fat as a source during the day and carbohydrate from the diet during the night (when eating occurs). Interestingly, strain BTBRT +tf/J is biased towards using carbohydrates, instead of fats, which suggests these mice may constantly eat to support their overall high daily metabolic output (figure 6). A more detailed analysis of circadian patterns and feeding behavior will answer these questions.

      Metabolic rate calculation is a comprehensive estimate of the amount of energy expenditure during fundamental processes to sustain life, such as basal and cellular metabolism, heat production, and movement. Together, these metabolic measurements reflect the activity and can be used to interpret whether peripheral or central mechanisms changes occur with aging. One controversy in the field of energy regulation is how to analyze the data in respect to size – as a larger animal in general has a higher total energy expenditure than a smaller one. Furthermore, it is claimed that body weight should be expressed per kilogram of lean body mass because fat tissue has relatively low metabolism and adds little to the total energy expenditure. Analyzing the data in total output, size by body mass, or estimated amount of lean body mass are all necessary comparisons for a true comprehensive understanding of energy expenditure. In my experience, which is exemplified by the preliminary data, one needs to examine the data using each perspective, as different analysis reveals difference clues about the underlying organismal physiology and morphometrics. As mentioned earlier, in figure 6, BTBRT +tf/J, clearly expends the most energy overall, even though it is not the largest mouse (figure 1). On the contrary, the heaviest strain, NZO/HiLtJ, has the next highest output, as shown in figure 6. However, when taken into consideration on a per total body mass or lean mass basis (figures 7, 8 & 9) NZO/HiLtJ strain is clearly quite metabolically efficient. Another quite interesting strain is the PWD/PhJ because it appears to be a energy wasting “Cadillac” (figures 7,8,9), which reflects the high metabolism of its lean tissue most likely in defense of heat loss due to its relatively small size. In the long-term, it would be of keen interest to examine whether strains of higher metabolic rate are predictive for longer lifespan or if this constant high metabolic output is detrimental to renal and liver physiology. As an example of lifestyle condition in humans – overeating – is LP/J (figure 4) despite being one of one of the lightest strains (figure 1). This strain appears to compensate for its increased energy intake by increasing its metabolic rate per mass (figures 7, 8, 9), its overall energy expenditure per hour (figure 6) nowhere matches a larger mouse, BTBRT +tf/J. Perhaps, LP/J holds an insight for curbing age-related weight gain.

      These examples serve to illustrate the power of organismal physiology and behavioral experiments to unravel changes in homeostatic systems, and its regulation, that are vital for a high quality of life.