Aging Research Using Mouse Models

Cheryl L. Ackert‐Bicknell1, Laura C. Anderson2, Susan Sheehan2, Warren G. Hill3, Bo Chang2, Gary A. Churchill2, Elissa J. Chesler2, Ron Korstanje2, Luanne L. Peters2

1 Present address: University of Rochester, Department of Orthopaedics and Rehabilitation, Rochester, New York, 2 The Jackson Laboratory, Bar Harbor, Maine, 3 Laboratory of Voiding Dysfunction, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo140195
Online Posting Date:  June, 2015
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Abstract

Despite the dramatic increase in human lifespan over the past century, there remains pronounced variability in “health‐span,” or the period of time in which one is generally healthy and free of disease. Much of the variability in health‐span and lifespan is thought to be genetic in origin. Understanding the genetic mechanisms of aging and identifying ways to boost longevity is a primary goal in aging research. Here, we describe a pipeline of phenotypic assays for assessing mouse models of aging. This pipeline includes behavior/cognition testing, body composition analysis, and tests of kidney function, hematopoiesis, and immune function, as well as physical parameters. We also describe study design methods for assessing lifespan and health‐span, and other important considerations when conducting aging research in the laboratory mouse. The tools and assays provided can assist researchers with understanding the correlative relationships between age‐associated phenotypes and, ultimately, the role of specific genes in the aging process. © 2015 by John Wiley & Sons, Inc.

Keywords: mouse; phenotyping; lifespan; health‐spanm; age‐related disease

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Assessment of Bladder Function—Urinary Incontinence Test
  • Basic Protocol 2: Behavioral Indicators of Health‐Span: Open Field, Novel Object Exploration and Recognition, Continuous Spontaneous Alternation, and Tail Suspension
  • Alternate Protocol 1: Grip Strength as a Measure of Neuromuscular Function
  • Basic Protocol 3: Assessment of Kidney Function
  • Basic Protocol 4: Blood Collection for Assessment of Hematological, Immunological and Metabolic Parameters
  • Basic Protocol 5: Assessment of Skeletal and Body Composition
  • Basic Protocol 6: Morphological Eye Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Assessment of Bladder Function—Urinary Incontinence Test

  Materials
  • Mouse urine or dye solution (for calibration)
  • Whatman filter paper sheets (#1 or #540, 30 cm diameter)
  • Cardboard template of same dimensions as mouse cage
  • Paper cutter or scissors
  • Pencil
  • Digital camera, preferably one capable of taking RAW or TIFF images
  • Computer‐based imaging software (e.g., ImageJ from NIH, http://imagej.nih.gov)
  • Clean duplex cages (one per mouse for the 4‐hr duration of the test)
  • Masking tape
  • Cage cards
  • Resealable plastic bags
  • UV transilluminator
  • Spreadsheet program (e.g., MS Excel)

Basic Protocol 2: Behavioral Indicators of Health‐Span: Open Field, Novel Object Exploration and Recognition, Continuous Spontaneous Alternation, and Tail Suspension

  Materials
  • Mice to be tested
  • For open field testing:
    • Opaque Plexiglas box (39 × 39 × 39 cm) with a dark gray floor, illuminated at 43 ± 4 lux in a 10 × 15–foot room
    • Video camera hooked to a computer monitor
    • Real‐time video‐tracking software (e.g., Noldus Ethovision XT)
    • Timer
    • Holding cage
  • For novel object exploration and recognition:
    • Opaque Plexiglas box (39 × 39 × 39 cm) with a dark gray floor, illuminated at 43 ± 4 lux in a 10 × 15–foot room
    • Video camera hooked to a computer monitor
    • Real‐time video‐tracking software (e.g., Noldus Ethovision XT)—The Multiple Body Points module is used to differentiate nose‐point and tail‐base in the novel object exploration and recognition tasks
    • Two novel object types with at least two objects of each type per test arena (see step 2b)
    • Timer
    • Holding cage
  • For continuous spontaneous alternation:
    • The Med Associates T‐maze for mice is configured with 14.5‐in. runways, a square hub (ENV‐334U) for the T configuration or a triangular hub (ENV‐333U) for the Y configuration, and manually operated guillotine doors (ENV‐339U)
    • Blue floor inserts (Med Associates, MED‐RAMM‐BI) to better facilitate video tracking of multiple mouse coat colors
    • Appropriate visual cues (see text above step 1c, below)
    • Video camera hooked to computer monitor
    • Real‐time video tracking software (e.g., Noldus Ethovision XT)
    • Timer
    • Holding cage
  • For tail‐suspension test:
    • Horizontal ring‐stand bar
    • 15‐ml conical polypropylene centrifuge tube
    • Hacksaw
    • 640 grit sandpaper or gentle heat source
    • Timer
    • Holding cage
    • Masking tape
    • Video camera hooked to computer monitor
    • Real‐time video tracking software (e.g., Noldus Ethovision XT)

Alternate Protocol 1: Grip Strength as a Measure of Neuromuscular Function

  Additional Materials (also see protocol 2)
  • Grip‐strength meter with digital peak force display (Columbus Instruments; see Fig.  ).

Basic Protocol 3: Assessment of Kidney Function

  Materials
  • Mice to be tested
  • Reagents for AU680 microalbumin assay:
    • Mouse albumin (Sigma‐Aldrich, cat. no. A3139)
    • Beckman protein diluent (Beckman Coulter, cat. no. 442825)
    • Beckman Coulter microalbumin calibrator (cat. no. ODR3024)
    • Tris buffer, 95 mmol/liter (pH 7.6)
    • Normal saline: 0.9% NaCl
  • Reagents for AU680 creatinine assay:
    • Sodium hydroxide solution (final concentration 120 mmol/liter)
    • Picric acid (final concentration 2.9 mmol/liter)
    • Beckman Coulter AU System creatinine reagent (cat. no. OSR6178)
  • Beckman Coulter urine chemistry calibrator (cat. no. DR0091)
  • 1.5‐ml microcentrifuge tubes (one per animal)
  • Weigh boats (one per animal)
  • Chemistry analyzer (e.g., the Beckman Coulter AU680)
  • Graphing software (e.g., Excel, GraphPad)
  • Goat anti‐human albumin antiserum (OSR6167 Beckman Coulter)
  • Test tubes, 12 to 16 mm in diameter, or sample cups (Beckman Coulter, cat. no. AU1063)
  • Beckman AU680 clinical chemistry analyzer (Beckman Coulter)
  • Beckman Coulter AU System microalbumin reagent (cat. no. OSR6167)

Basic Protocol 4: Blood Collection for Assessment of Hematological, Immunological and Metabolic Parameters

  Materials
  • Mice to be tested
  • Beckman Coulter chemistry calibrator (cat. no. DR0070)
  • Reagents for BUN analysis:
    • Beckman Coulter AU System urea (BUN) nitrogen reagent (cat. no. OSR6134/6234)
  • Reagents for measuring basic electrolytes (Na+, K+)
    • Beckman ISE buffer (cat. no. AUH1011)
    • Beckman ISE reference solution (cat. no. AUH1013)
  • Reagents for measuring magnesium levels:
    • Beckman Coulter AU System magnesium reagent (cat. no. OSR6189)
  • Reagents for measuring glucose:
    • Beckman Coulter AU System glucose reagent (cat. no. OSR2161)
  • Reagents for fluorescence labeling and flow cytometric analysis:
    • FACS buffer (see recipe)
    • Hemolytic Gey's buffer (see recipe)
    • Antibody cocktail for B cells and myeloid cells (Abs available from eBiosciences or PharMingen):
    • CD45R/B220—Pacific Blue clone RA3‐6B2
    • Gr‐1—APC‐Cy7 clone RB6‐8C5
    • CD11c—FITC clone N418
    • CD11b/Mac‐1—PE clone M1/70
    • F4/80—AlexaFluor 647
    • Antibody cocktail for T cells and NK cells:
    • CD3e—PE clone 145‐2C11
    • CD4—APC clone RM 4‐5
    • CD8—AlexaFluor 700 clone 53‐6.7
    • CD44—APC‐Cy7 clone IM7.8.1
    • CD62L—PE‐Cy7 clone MEL‐14
    • NKG2A/C/E—FITC clone 20d5
    • B220—Pacific Blue
    • 20 mg/ml propidium iodide in FACS buffer (see above), for dead cell exclusion
  • Cages
  • Animal balance
  • Yellow‐capped Microtainer tubes with serum separators, one for each mouse (Becton Dickinson, cat. no. 365967) and 1.5‐ml collecting tubes, labeled, for each mouse
  • Tray for holding 1.5‐ml tubes
  • Refrigerated centrifuge
  • Beckman AU680 clinical chemistry analyzer (Beckman Coulter)
  • Microhematocrit tubes: EDTA‐coated, 75 mm, 75‐μl volume (Fisherbrand)
  • Lavender‐capped Microtainer tube containing lyophilized K 2EDTA (Becton Dickinson Diagnostics, or Fisher Scientific, cat. no. 6‐5974)
  • Tray for holding serum tubes
  • Tray for holding 1.5 ml tubes
  • Wooden toothpicks
  • Siemens ADVIA 2120 hematology analyzer equipped with mouse‐specific software
  • Microhematocrit tubes: heparinized, 75 mm, 75 μl volume size (Fisherbrand)
  • Glucose strips and glucose reader
  • 12 × 75–mm polypropylene tubes (USA Scientific, cat. no. 1450‐0000FC)
  • Flow cytometer: Becton Dickinson (BD) FACScan with 5‐color upgrade (Cytek Development), or FACS Calibur (BD Biosciences)
  • FlowJo software (TreeStar, Inc.)
  • Additional reagents and equipment for blood collection from mice (Rathkolb et al., )

Basic Protocol 5: Assessment of Skeletal and Body Composition

  Materials
  • Mice for testing
  • 2% (w/v) tribromoethanol or other suitable anesthetic (check with the institution's rules and recommendations for selecting an anesthetic for small animals for non‐surgical procedures; also see Adams and Pacharinsak, )
  • Pupillary dilation solution: e.g., 1% atropine sulfate ophthalmic solution (sterile; Alcon Laboratories), or 1% cyclopentolate hydrochloride ophthalmic solution USP (sterile; Bausch & Lomb)
  • Clean mouse cages
  • Standard animal balance
  • LUNAR PIXImusII mouse densitometer (DXA; GE‐Lunar)
  • Imaging tray to hold the mouse on the instrument (purchased specifically for use with the PIXImus instrument)
  • Exogenous heat source for recovery from anesthesia, such as a water‐circulating warming pad or a slide warmer placed under the cage
  • Additional reagents and equipment for mouse anesthesia (Adams and Pacharinsak, )
  • NOTE: Subcutaneous microchips and metal ear tags used for mouse identification may not be compatible with DXA analysis. The microchips, while usually implanted in the scapular region, will migrate around the mouse, and months after implantation can be found almost anywhere subcutaneously on the dorsal side of the mouse. As the microchips are X‐ray‐opaque, the microchip will be erroneously interpreted as mineral by the PIXImus software. As these migrate around the mouse, it is not a simple matter of simply excluding the microchip from the area imaged, as a different part of the mouse will be excluded each time. Metal ear tags that are small can be used as long as these are completely excluded with the head region during analysis of the image.

Basic Protocol 6: Morphological Eye Analysis

  Materials
  • Mice for testing
  • Pupillary dilation solution: e.g., 1% atropine sulfate ophthalmic solution (sterile), Alcon Laboratories, or 1% cyclopentolate hydrochloride ophthalmic solution USP (sterile), Bausch & Lomb)
  • Slit‐lamp biomicroscope (e.g., Nikon NS‐1 slit lamp)
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Figures

Videos

Literature Cited

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