Basic Biology of Aging at the University of Washington

Functional Assessments Core

Specific Aims. This Core will provide a range of functional assays that are highly relevant to studies of aging and healthspan:

  1. Cellular Function: We will make readily available flow cytometry, cell sorting, confocal image cytometry and laser capture microdissection instrumentation and techniques to allow investigators to assess cellular function and physiology in studies of the basic biology of aging and to purify subsets of cells so that their molecular characterization (including functional genomics) can be more accurately performed.

  2. Animal Physiology and Function: We will provide a comprehensive range of tests to help Center investigators evaluate animal health and healthspan by measuring the functional status of various organ and physiological systems that are important in the health/physiological performance of an animal and that are shown to change with age in mice and humans.

  3. Pathology: Will will provide expertise and ability to conduct comprehensive pathological analyses of mice used in studies of the biology of aging, including genetic, dietary or pharmacologic interventions. These data provide (a) morphological diagnoses of all tissues (b) grades for neoplastic and non-neoplastic lesions (c) summary measures of disease burden including the prevalence and severity of both neoplastic and non-neoplastic diseases, total disease burden, comorbidities; (d) the possible effect of pathological lesions on overall survival and healthspan; and (e) the probable cause or causes of death.

Research Strategy
Significance. Physiological assessment will play a major role in assisting investigators in the characterization of genetically engineered mouse models, mice with dietary manipulation (e.g., dietary restriction) or pharmacologic interventions that alter healthspan and/or lifespan.
Approach. We will apply the three specific aims described to characterize relevant and innovative mouse models of aging.

Aim 1 approach (Cellular Function). For the past 14 years the former Cytometry Core was focused on providing flow cytometry and cell sorting services in support of our community of biology of aging-related research investigators. This activity included new assay development for use in aging research (refs 1-19). While this goal remains, cytometry activities are now a subset of the new Functional Assessments Core instituted in 2009 that is more broadly targeted to a range of Functional Assessments. For the Cytometry Aim, this Core continues to provide assays of cellular function that are otherwise poorly available, require special expertise to perform and interpret, or both. In addition, this core provides access to confocal image cytometry and laser capture microdissection for assessment of aging-relevant cellular function. The range of applications for confocal analysis extends from localization and quantitation of intracellular targets and gene products to measurement of telomere length. Finally, both laser capture microdissection and flow cytometry play important roles in purification of cell subsets for subsequent molecular analyses of function, including those using microarrays and proteomics, as applied by Core C. Dr. Rabinovitch, Core Director, is an internationally recognized expert in applying cytometric technologies to cell cycle and functional cell measurements and developing new assays and software in their support. In addition to numerous publications in this area, he is editor of the Physiologic Assays Unit of Current Protocols in Cytometry (J Wiley & Sons).

The Cytometry Core employs a state-of-the-art high speed (>108 cells/hr) cell sorter, the Influx, from Cytopeia (Seattle, WA). This is a 3-laser, 12-parameter instrument, with full integration of computer control and the highest speed sorting capacity of any commercial instrument. A PALM laser catapult microdissection instrument is owned by the Department of Pathology and available for our shared use. In addition, Biorad and Leica conventional and two-photon Confocal microscopes and Applied Precision Deltavision deconvolution microscope are available for use as an inter-departmental shared resource within a shared use facility in the same wing of the building that houses our Cytometry Facility.

Publications by Center Investigators during the last funding cycle that have relied on Cytometry Core services are cited in the bibliography, with the faculty investigators noted in bold (refs 20-49). As can be seen, we continued to develop and/or apply a range of Cytometry assays important to aging-related research. To summarize, these include:

Multiparameter cytometric assays to monitor physiological changes during aging, cell injury, apoptosis and necrosis. Multiparameter assays of physiological changes in cells have been a focal point of the Cytometry Core. The measurement of mitochondrial membrane potential has become important in assessing mitochondrial health and function and the contribution of mitochondrial opening of transition pores to early initiation of apoptosis (8, 11). The mitochondrial membrane sensitive dye CMXRosamine has been used in conjunction with assays of glutathione, NADPH, cytosolic oxidant levels, the level of mitochondrial membrane lipid cardiolipin (by nonyl acridine orange fluorescence) and cell cycle parameters to quantitate and delineate events occurring during apoptosis (7, 8, 11). We have also used a number of highly informative reporters of cellular and mitochondrial ROS (H2O2 and superoxide), including DCFDA, hydroethidine and Mitosox (mitochondrial ROS) (Kennedy, Rabinovitch, Kaeberlein, Rostomily, Swisshelm).

Cytometric assay of cell cycle and proliferative survival. Cell cycle analysis is a basic tool of cytometry, using DNA dyes and augmented by Brdu or EdU. In addition, the proliferative survival assay was developed by the Core to provide a rapid and sensitive alternative to classical clonogenic survival assays. It has a great advantage in cases where cells clone poorly (e.g. Werner Syndrome fibroblasts), are technically difficult to clone (e.g., lymphoblastoid cells), or when only few cells are available for the assay. The assay uses BrdU incorporation as an indicator of proliferation, and calibrates surviving cell numbers using an internal particle control (4-6). We have also shown that this assay can be performed simultaneously with immunofluorescence staining to identify cell subsets (5). (Poot, Monnat, Loeb, Rabinovitch, SIderova, Vincent, Swisshelm)

Assays of senescence, DNA damage and DNA repair. These assays are quantitated by both flow and image cytometry and include use of antibodies to p16, Dec 1, phosphorylated Histone H2AX, WRN, mre11, BLM, and Rad51 (Maizels, Risques, Ladiges, Loeb, Monnat, Risques, Brentnall, Rabinovitch).

Quantitation of telomere length by confocal microscopy. Fluorescence in situ hybridization (FISH) with a PNA telomere probe provides an accurate and specific indication of telomere length. This has conventionally been done in cultured mitotic or interphase cells. We have shown that this method can be applied to tissue sections and that this is especially useful when the telomere lengths of cellular subsets within tissues are to be determined (18, 39). (Risques, Rabinovitch, Brentnall, Monnat, Oshima, Martin)

Quantitation of rates of telomere attrition by flow cytometry. While the intensity of Telomere PNA FISH can be readily quantitated in leukocyte subsets by flow cytometry, Center investigators who are interested in telomere maintenance and telomere attrition required an assay that could quantify rates of telomere attrition in successive cell cycles. We helped Drs. Potter and Wener develop such an assay and demonstrated that it can be adapted to measure the rate of telomere length reduction per cell division (44).

Confirmation of telomere length by qPCR. Because of the wide interest in telomere length by investigators studying the biology of aging, we have facilitated the confirmation of a higher throughput assay of telomere length by use of qPCR. This method, originally developed by R. Cawthon, has a low CV in our hands (<7%), made possible in part by use of a Corbett Research Rotogene thermocycler previously purchased by the Core (40-43). (Risques, Rabinovitch, Brentnall, Oshima, Martin)

Assays of DNA damage by alkaline unwinding. A flow cytometric assay of DNA damage based on alkaline unwinding was developed and shown to have excellent sensitivity and reproducibility (17). This assay has been applied to aging research to examine DNA damage in response to oxidative challenge in cells from control and transgenic mice and cells from Werner Syndrome (Preston, Monnat, Potter, Rabinovitch)

Sorting with preservation of RNA for microarray studies. Flow cytometry can be a rapid and efficient method of purifying cell subsets for molecular analyses, including gene expression and proteomics, particularly in cases where admixtures of cells may yield insensitive, confusing or misleading answers. The high-speed sorter can process in excess of 108 cells/hr, providing adequate numbers for molecular assays even when the desired cells are relatively rare. In particular, we have shown that epithelial cells can be labeled with anti-cytokeratin antibodies after fixation with RNAlater and that high quality RNA can be recovered after sorting, even when no special treatment (elimination of RNAases) of the cell sorter is used (19).

Quantitation of Gene Activity. Use of fluorescent protein reporters in a broad range of colors has enabled confirmation and quantitation of gene expression. In some cases, this is combined with sorting in order to enrich sub-populations for subsequent biological or molecular assays. (Yablonka-Reuveni)
Measuring changes in immune cell subsets with age. Changes in proportions of naïve and memory T cells (CD4, CD8 and their subsets) with age is a well documented “biomarker” of aging that can be applied in studies of aging and healthspan. The method uses conventional multiparameter immunolabeling. (Wener, Rabinovitch, Ladiges)

Aim 2 approach (Animal Physiology and Function). Aim 2 is designed to provide physiological assessment of mouse lines for investigators involved in aging research with a focus on cardiac function, metabolism, physical activity and cognition. In late 2009, we were able to formally incorporate this physiologic testing into services of a redesigned Core B, utilizing an array of testing apparatus that have been progressively added to our Center and dedicated to the Biology of Aging. Most importantly, this equipment is located within or adjacent to the room housing aging mouse colonies so that aging mice can be readily examined longitudinally without compromising their SPF status. This has been made possible in large part by successful application for Nathan Shock Center Administrative Supplement funds, augmented or matched by institutional funds, in 2003, 2005, 2008 and 2009. These high end instruments, provide important research tools for Shock investigators working with mice, in that they provide measures of the impact of genetic and pharmacologic interventions on health and performance as a function of animal age. The importance of such “healthspan” measurements in aging studies has become more widely recognized in recent years and a large use of this new Core is by a 2009 Administrative Supplement to our Center to study healthspan measures in mouse models of aging, in cooperation with Michigan and UTHSCSA Nathan Shock Centers.

We are proposing: 1) Imaging including ultrasound scanning, ultrasound echocardiography and

quantitative magnetic resonance (QMR) imaging; 2) Motor function assessment including rotarod,

grip strength, physical activity and running wheel; 3) Cognitive assessment including Barnes water tread maze, passive avoidance, and fear conditioning; 4) Indirect calorimetry. Publications by Center Investigators that have already relied on the Animal Function Assessment services during the past funding cycle (both while they were managed in Core A, and now that they are moved to Core B) are cited in the bibliography, with the faculty investigators noted in bold (refs 50-70).

Ultrasound scanning. Ultrasound scanning is a non-invasive procedure that allows assessment of internal organ structure and size such as heart, liver, kidney or uterus (pregnancy). The mice are initially anesthetized by placing them in a flow-through system containing 3-4% isoflurane in a 100% oxygen mix. Following loss of consciousness, the mice are placed on a modified mask assembly that allows a continuous flow of 1-2% isoflurane in an oxygen mix. The mice can breathe spontaneously, and the depth of anesthesia is monitored by continuous recording of heart rate. The mice will be taped to an isothermal pad that is maintained at 37C because anesthetized mice have difficulty maintaining body temperature. Scanning is then performed as determined by the experimental protocol using an Accuson-CV70 unit (Siemens) apparatus. This instrument was purchased with an Administrative supplement to the Center in 2003.

Ultrasound echocardiography. Echocardiography is a non-invasive procedure that allows assessment of both systolic and diastolic function, and will be performed serially on the mice to determine temporal changes. The mice are initially anesthetized as above, and the depth of anesthesia is monitored by continuous recording so that the mouse heart rate (from a surface electrocardiogram) does not fall below 400, as the accuracy of the images obtained depends on a near physiological heart rate.. From a transthoracic approach, two-dimensional targeted M-mode echocardiographic recordings are obtained with the Accuson-CV70 unit.
Using echocardiography we have been successful in documenting the changes in parameters of left ventricular myocardial index (LVMI), left atrial dimension, fractional shortening, diastolic function (Ea/Aa) and myocardial performance index (MPI) with age of C57Bl mice (52,53). Using these parameters, cardiac aging has been shown to be retarded in mice expressing catalase targeted to mitochondria (53) and in mice with disruption of protein kinase A (56). Further studies are underway in the laboratories of Kennedy, Ladiges, and Rabinovitch.

QMR. Body composition will be measured by quantitative magnetic resonance (QMR), which quantifies tissue mass by measuring nuclear magnetic resonance signals (voltage) proportional to total number of hydrogen nuclei, their relaxation times and diffusion coefficients. The QMR instrument (Echo Medical Systems, Houston, TX) was purchased by the Center in 2008 and can be used to noninvasively measure body composition in a conscious mouse. A single measurement takes only 2 minutes. For a typical experiment, each animal undergoes 3- to 5-replicate measurements. The readouts include body mass, percent lean body mass, percent fat mass and bone mass. For example, we have data collected from mice showing a significant difference in fat mass and lean body mass between PKA RII beta null mice and WT littermates in males but not females. Interestingly, mutant males, but not females, have an extended lifespan (56)

Rotarod. Rotarod performance has been measured since 2005 using a dedicated computerized rotarod system to test the ability of mice to maintain balance on a rotating rod. Up to four mice are placed on the rod within their individual lanes in the rotarod enclosure. Seven photo beams are embedded in each of the four lanes of the rotarod enclosure. When the mouse falls from the rotating rod, the photo beams are broken and the rotarod software records the mouse’s latency to fall. Three successive runs are performed per day for three days. Therefore, not only is there an evaluation of motor function, there is also an assessment of performance learning. We have found that a sample size of N=10 per cohort provides 88% power to detect a difference of 70 seconds in latency for the rotarod test between groups, when measured on just one day, and greater that 99% when measured over three days, alpha = 0.05 [two tailed t-test]

Grip strength. We have used grip strength apparatus to evaluate muscle function since 2005. We measure susceptibility to muscle fatigue in vivo using a modified grip strength protocol based on published protocols. This protocol relies on the instinctive tendency of mice to grasp with the forelimbs. The mouse is gently pulled back by the tail, while grasping a pull bar connected to a force transducer. We record the peak pull force for repeated replicates until a drop in peak force is observed, indicating muscle fatigue. Pilot experiments establish the number of pulls necessary to measure significant fatigue. Fatigue will be expressed as the sum of the last two pulls divided by the sum of the first two pulls, such that no decrement in force yields a ratio of 1 and total fatigue (complete inability to generate force) yields a ratio of 0. The extent of fatigue in treated and transgenic mice will be compared with that for WT controls after the same number of pulls. This protocol has been used extensively in the literature to measure in vivo muscle fatigue in mouse muscles. This test is currently in use in studies by Marcinek, Ladiges and Rabinovitch.

Magnetic resonance (MRS) and optical spectroscopic (OS) in vivo measurements of mitochondrial function. Application of this state of the art approach is now funded by an RC2 grant from the NIA to Drs. Conley and Marcinek to provide these tools for aging studies. These assays can measure in vivo mitochondrial coupling (ATP/O2 or P/O) and capacity (O2max), cell glycolysis and ATP level, and cellular and vascular oxygenation (PO2). Drs. Conley and Marcinek will make these assays readily available to Shock Center users during the next 2 years (see letter of support in Section 1, earlier in this application), after which we will jointly evaluate their incorporation into Core B services.

Physical activity. We use a photo beam activity system instrumented to a standard mouse cage (San Diego Instruments), purchased in 2006. We can assess both nocturnal and daylight activity as the number of ambulations per hour. However, we have found that a simple 5 minute activity paradigm can distinguish genotypes of mice at old ages. We assess activity for a five-minute period on three consecutive days and use a sample size number N=11 per genotype per sex as determined by power analysis calculations.

Running wheel. We have recently (2009) obtained a bank of 60 in-cage low-profile running wheels that are connected to computers running monitoring software to assess the speed, distance and time the mice engage in running behavior (Med Associates, Inc., St. Albans, VT). The software tracks each mouse individually and allows complex multivariate analysis to be performed upon the physical performance of the mice in the study. The animals are examined across various time periods from hours to days. The inclination and capacity of mice to engage in voluntary spontaneous activity is a viable measure of their general physical aptitude.

Barnes water tread maze. Mice tend to poke their noses into holes, especially to explore dark holes, which may lead to escape routes away from a lighted, open field with wading levels of water. One of the holes leads to a dark “escape” box located just below the circular platform. Cues are placed around the inside rim wall. Day 1. Training phase. Put mouse in escape box for 2 min, repeat. Put mouse in escape hole, repeat. Put mouse outside escape hole, repeat. Put mouse in center of circular platform, repeat. If it does not find the escape hole in 3 minutes, repeat the above steps. Days 2,3,4. Testing phase. Put mouse in center, always pointing in the same direction each time. Allow 3 minutes to find escape hole and enter escape box. It if does not find escape box in 3 minutes, put mouse in escape box, then place mouse in center again to start over. Repeat test 4 more times, for a total of 5 evaluations. Measurements: Number of errors (number of times head poked in wrong hole). Time to enter the correct escape hole. Day 5. Probe trial test phase. Change the location of the escape tube and box, and repeat testing procedure as described for the testing phase. The purpose of this phase is to determine if the mice are using spatial memory by returning to the original escape hole.

Passive avoidance. Cognition is measured by a passive avoidance task using a Gemini Avoidance System, a paradigm consisting of a step-through, two-chambered apparatus (San Diego Instruments, San Diego, CA) where the mouse is placed in the front chamber, and after a 1 minute ‘orientation period’ a guillotine door is raised, allowing the mouse to enter the second, darker chamber. The door is automatically lowered, prohibiting the mouse from escaping to the lighted side, and a 0.25 mA foot shock is applied to floor grids for 1 second. Following this single training trial, the mouse is returned to its home cage to await retention testing 24 hours, 7 days, or 28 days later. Cognition testing is accomplished by placing the mouse into the front chamber in exactly the same manner as on the training trial. The latency to enter the darker chamber is then measured. All animals will be tested during the same time period each day, i.e., between 1:00 and 4:00 PM.. Latencies shorten as days pass and the mice "forget" the prior adverse stimulus. Using day 7 latency data obtained from young and old mice (Strong et al., 2002), we estimate that 20 mice per group are sufficient to detect a 40% to 50% difference in latency between cohorts of mice.

Fear conditioning. This paradigm consists of a novel test chamber with stainless steel bars on the base (San Diego Instruments, San Diego, CA). The novel chamber, along with a mild foot shock (approximately 0.25 mA), are used as conditioning cues to the mouse. The freezing behavior is tracked by motion sensors and recorded within the software running the device.

Indirect calorimetry. Total energy expenditure is measured using the Oxymax Lab Animal Monitoring System, purchased in 2008. The main feature of the system is indirect open-circuit calorimetry, which measures a number of biological variables including oxygen consumption, CO2 production, respiratory quotient, and heat production. Each mouse will be temporarily housed for up to 48 hours in a specially designed cage that contains inlet and outlet ports through which a known flow of ambient air is being forcibly ventilated. Animals will eat pulverized food from the feeder and drink water from a sipper tube. We measure total energy expenditure by monitoring the rate of oxygen consumption and carbon dioxide production of the animals. Food and water consumption are measured by the mass of food removed from a container that resides on a precision balance and the volume of water consumed from a sipper tube connected with the chamber. We have enough cage units to test eight mice at a time. We have data using this unit with PKA C beta null mice suggesting these mice have increased metabolic rate compared to WT littermates (59). We have shown these mice to be robustly resistant to a high fat diet, with maintenance of insulin sensitivity and significantly less fat mass.

Aim 3 approach (Pathology). To determine the impact of an experimental intervention on aging, it is essential that an investigator have knowledge of how the intervention alters the pathological lesions that occur with age. Age-related pathology increases exponentially with advancing age and is largely responsible for age-related morbidity as well as mortality. Pathological information also provides investigators with insight into the potential biological/molecular mechanism(s) of the intervention. Furthermore, the pathological assessment of old animals that are included in basic studies of biological aging processes is necessary to help investigators determine whether the changes in physiological/biochemical parameters measured are associated with or are independent of underlying pathological conditions. Thus it is essential to obtain accurate and thorough pathological assessments of aging animals. The Pathology component of the Core will provide investigators in the Center with access to detailed pathological analyses of the lesions that occur with age, altered genotype and/or intervention. Dr. Piper Treuting, although new to the Core in late 2009, is a highly experienced and expert mouse pathologist who has previously been successful in assisting Center investigators to incorporate pathologic assessments into their studies (45, 50, 56, 58, 63, 65, 66). She is also an active member of our Center team working on the Administrative Supplement for Healthspan testing.

Pathological analysis. Necropsy consists of examination of external body surface, all orifices, cranial vault, external surface of the brain, the thoracic, abdominal and pelvic cavities and viscera. The following organs will be weighed prior to partitioning and fixation: brain, heart, lungs, liver, kidneys, spleen, adrenals, uterus for female, and testes for male. Any other organs or tissues with gross lesions also will be examined, measured and photographed. Tissues will be fixed immediately in 10% neutral buffered formaldehyde solution. The fixed tissues are embedded in paraffin, sectioned at 5 um and stained with hematoxylin-eosin. In addition, we will use Masson’s trichrome stain to evaluate the fibrosis of heart and periodic acid Schiff to highlight the mesangial matrix within kidney sections.

A profile of pathological lesions will be constructed for each mouse that includes the prevalence and severity of both neoplastic and non-neoplastic disease, along with the probable cause of death with assigned grades of degree of certainty (65). The total population burden for each lesion will be measured by combining the prevalence and severity of the lesion in the individual mice to establish effect of pathology on longevity. The degree of comorbidity is a metric term we will use for the number of significant disease processes present in an individual animal at death and is defined as the number of separate severe lesions in each disease category.

Future Core B plans. The general strategy will be that testing of animal and cellular function of mouse models will proceed as the mice created by Core A reach suitable age(s) to be tested. Mouse lines that are being developed as part of the Evolutionary Conserved Pathways Theme are shown in Table B of the Core A report. Other mouse models that will be requested by investigators for Functional Assessments testing will come from those shown in Table A of the Core A report. In addition, we will continue to work on the Center’s recent 2-year Supplement to measure healthspan in mice from the NIA aging colony that have differing longevity.

Core Coordination and Resource Access. Dr. Rabinovitch’s role in directing this Core includes the Coordination of Core B activities with overall Center goals and plans, as well as coordinating Drs. Ladiges’ and Wiley’s efforts. These three meet on a regularly schedule weekly basis to discuss issues related to the Core administration and coordination. Questions of Resource access are also discussed at this time, and are jointly resolved by considerations of 1) closeness/relevance to the basic biology of aging; 2) maximizing distribution of benefit across as many of the Investigators listed in Table III as possible. Unresolved access issues, and/or any disputes by investigators are immediately brought to the Executive Committee for resolution; however, this has never been necessary over the history of the Center, as mutually agreeably equitable solutions have always been found. The success of distributed resource allocation is illustrated by the range of investigators shown in this report.