Inducible Models of Bone Loss

Casey R. Doucette1, Clifford J. Rosen1

1 Maine Medical Center Research Institute, Scarborough, Maine
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo140071
Online Posting Date:  December, 2014
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Abstract

Bone is an essential organ that not only confers structural stability to the organism, but also serves as a reservoir for hematopoietic elements and is thought to affect systemic homeostasis through the release of endocrine factors as well as calcium. The loss of bone mass due to an uncoupling of bone formation and bone resorption leads to increased fragility that can result in devastating fractures. Further understanding of the effects of environmental stimuli on the development of bone disease in humans is needed, and they can be studied using animal models. Here, we present established and novel methods for the induction of bone loss in mice, including manipulation of diet and environment, administration of drugs, irradiation, and surgically induced hormone deficiency. All of these models are directly related to human cases, and thus, can be used to investigate the causes of bone loss resulting from these interventions. © 2014 by John Wiley & Sons, Inc.

Keywords: bone loss; mouse models; calorie restriction; high‐fat diet; rosiglitazone; cold exposure; irradiation; ovariectomy

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Chronic Sympathetic Nervous System Activation by Long‐Term Cold Exposure
  • Basic Protocol 2: Acute Sympathetic Nervous System Activation by Short‐Term Cold Exposure
  • Alternate Protocol 1: Acute Sympathetic Nervous System Activation by BRL‐37344 Administration
  • Basic Protocol 3: 30% Calorie Restriction as a Partial Model of Anorexia Nervosa
  • Basic Protocol 4: High‐Fat Diet Administration as a Model of Diet‐Induced Obesity
  • Basic Protocol 5: Rosiglitazone Administration in the Diet as a Model of Thiazolidinedione‐Induced Bone Loss
  • Alternate Protocol 2: Rosiglitazone Administration by Gavage as a Model of Thiazolidinedione‐Induced Bone Loss
  • Basic Protocol 6: Irradiation in Mice as a Model of Radiation‐Induced Bone Loss
  • Basic Protocol 7: Ovariectomy in Mice as a Model of Postmenopausal Bone Loss
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Chronic Sympathetic Nervous System Activation by Long‐Term Cold Exposure

  Materials
  • Mice (gender‐ and age‐matched with littermate controls), at least 10 per group for statistical analysis
  • Lubricant (e.g., Priority Care Sterile Lubricating Jelly, Heartland Veterinary Supply)
  • Housing facility for mice, including:
    • Cages (1 per mouse), containing appropriate bedding, food, and water
    • Room held at 18°C on a 14/10‐ or 12/12‐hr light/dark cycle
    • Room held at 4°C on a 14/10‐ or 12/12‐hr light/dark cycle
    • Room held at room temperature (22°C) on a 14/10‐ or 12/12‐hr light/dark cycle
    • Room held at 30°C on a 14/10‐ or 12/12‐hr light/dark cycle (optional)
  • Rectal thermometer: RET‐3 rectal probe (Physitemp Instruments) and MicroTherma 2T hand held thermometer (ThermoWorks)
  • Additional reagents and equipment for euthanizing mice (Donovan and Brown, ), collecting tissues (Antal et al., ), and performing longitudinal body composition analysis and endpoint analyses (Esapa et al., )

Basic Protocol 2: Acute Sympathetic Nervous System Activation by Short‐Term Cold Exposure

  Materials
  • Mice (gender‐ and age‐matched with littermate controls), at least 10 per group for statistical analysis
  • Lubricant (e.g., Priority Care Sterile Lubricating Jelly, Heartland Veterinary Supply)
  • Housing facility for mice, including:
    • Cages (1 per mouse), containing appropriate bedding, food, and water
    • Room held at 4°C on a 14/10‐ or 12/12‐hr light/dark cycle
    • Room held at room temperature (22°C) on a 14/10‐ or 12/12‐hr light/dark cycle
  • Rectal thermometer: RET‐3 rectal probe (Physitemp Instruments) and MicroTherma 2T hand‐held thermometer (ThermoWorks)
  • Additional reagents and equipment for euthanizing mice (Donovan and Brown, ), collecting tissues (Antal et al., ), and performing longitudinal body composition analysis and endpoint analyses (Esapa et al., )

Alternate Protocol 1: Acute Sympathetic Nervous System Activation by BRL‐37344 Administration

  Additional Materials
  • 0.1 mg/ml BRL‐37344 sodium salt hydrate (Sigma, cat. no. B169) in phosphate‐buffered saline (PBS; Alanza)
  • Scale, sensitive to 0.01 g
  • 1‐ml syringe with 25‐gauge, 5/8‐in. needle, sterile

Basic Protocol 3: 30% Calorie Restriction as a Partial Model of Anorexia Nervosa

  Materials
  • Mice (gender‐ and age‐matched with littermate controls), at least 10 per group for statistical analysis
  • Control diet (10% kcal from fat; Research Diets, cat. no. D12450B)
  • 30% CR diet (30% kcal caloric and fiber restriction; Research Diets, cat. no. D10012703)
  • Housing facility for mice maintained at a constant temperature and humidity on a 14/10‐ or 12/12‐hr light/dark cycle, with cages (1 per mouse) containing appropriate bedding, food, and water
  • Scale, sensitive to 0.01 g
  • Additional reagents and equipment for euthanizing mice (Donovan and Brown, ), collecting tissues (Antal et al., ), and performing longitudinal body composition analysis and endpoint analyses (Esapa et al., )

Basic Protocol 4: High‐Fat Diet Administration as a Model of Diet‐Induced Obesity

  Materials
  • Mice (gender‐ and age‐matched with littermate controls), at least 10 per group for statistical analysis
  • Control diet (10% kcal from fat; Research Diets, cat. no. D12450B)
  • Very high‐fat diet (VHFD, 60% kcal from fat; Research Diets, cat. no. D12492); store at −20°C per manufacturer's recommendation
  • Housing facility for mice maintained at a constant temperature and humidity on a 14/10‐ or 12/12‐hr light/dark cycle, with cages (1 per mouse) containing appropriate bedding, food, and water
  • Scale, sensitive to 0.01 g
  • Additional reagents and equipment for euthanizing mice (Donovan and Brown, ), collecting tissues (Antal et al., ), and performing longitudinal body composition analysis and endpoint analyses (Esapa et al., )

Basic Protocol 5: Rosiglitazone Administration in the Diet as a Model of Thiazolidinedione‐Induced Bone Loss

  Materials
  • Mice (gender‐ and age‐matched with littermate controls), at least 10 per group for statistical analysis
  • Control diet (Research Diets, cat. no. D12450B)
  • Rosiglitazone diet: high‐fat diet containing 20 mg/kg rosiglitazone maleate (Cayman Chemicals), custom made by Research Diets; store at −20°C, per manufacturer's recommendation
  • Housing facility for mice maintained at a constant temperature and humidity on a 14/10‐ or 12/12‐hr light/dark cycle, with cages (1 per mouse) containing appropriate bedding, food, and water
  • Additional reagents and equipment for euthanizing mice (Donovan and Brown, ), collecting tissues (Antal et al., ), and performing longitudinal body composition analysis and endpoint analyses (Esapa et al., )

Alternate Protocol 2: Rosiglitazone Administration by Gavage as a Model of Thiazolidinedione‐Induced Bone Loss

  Additional Materials
  • 2 mM rosiglitazone maleate/5% v/v bovine serum albumin/5% v/v dimethyl sulfoxide; dilute to final concentration of 2 μM (see Im et al., )
  • 22‐gauge feeding tubes with rounded tips (Cadence Science)
  • 1‐ml syringe

Basic Protocol 6: Irradiation in Mice as a Model of Radiation‐Induced Bone Loss

  Materials
  • Medicated water (see recipe)
  • Mice (gender‐ and age‐matched with littermate controls), at least 10 per group for statistical analysis
  • Housing facility for mice maintained at a constant temperature and humidity on a 14/10‐ or 12/12‐hr light/dark cycle with cages (1 per mouse) containing appropriate bedding, food, and water
  • Irradiator (institutionally monitored)
  • Additional reagents and equipment for euthanizing mice (Donovan and Brown, ), collecting tissues (Antal et al., ), and performing longitudinal body composition analysis and endpoint analyses (Esapa et al., )

Basic Protocol 7: Ovariectomy in Mice as a Model of Postmenopausal Bone Loss

  Materials
  • Female mice, age‐matched with littermate controls, at least 10 per group for statistical analysis
  • Isofluorane (IsoFlo; Abbott Animal Health)
  • 10% (w/v) Betadine
  • Buprenophine (Patterson Veterinary)
  • Housing facility for mice maintained at a constant temperature and humidity, on a 14/10‐ or 12/12‐hr light/dark cycle, with cages containing appropriate bedding, food, and water
  • Nose cone for administration of isofluorane
  • Electric razor (fur clipper)
  • Gauze
  • Dissecting microscope (optional)
  • Sterile surgical implements, including:
    • Moria iris forceps (two; Fine Science Tools, cat. no. 11373‐12)
    • Fine scissors (Fine Science Tools, cat. no. 04060‐11)
    • Absorbable sutures
    • Wound clips
  • 0.3‐ml syringe, with a 31‐gauge, 5/16‐in. needle
  • Heating pad
  • Additional reagents and equipment for euthanizing mice (Donovan and Brown, ), collecting tissues (Antal et al., ), and performing longitudinal body composition analysis and endpoint analyses (Esapa et al., )
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Literature Cited

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