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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This article covers the procedures to induce stress responses using physical restraint stress in mice and rats. Additional considerations that should be observed when selecting and using restraint stress in rodent models are discussed.

Abstract

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the restraint stress procedure. Restraint stress has been used for decades to investigate changes in physiology, genetics, neurobiology, immunology, and other systems impacted by stress. Due to the ease of performing the procedure, low cost, and numerous modifications to scale for the intensity and duration of stress exposure, a vast literature of studies has used restraint stress in mice and rats. As one example, this study presents previously published data showing the impact of restraint stress in transgenic mice on plasma corticosterone levels and optogenetically-induced norepinephrine release. Acute restraint stress increased plasma corticosterone levels, yet this effect was blunted in mice following repeat restraint stress. However, stimulated norepinephrine release in the bed nucleus of the stria terminalis was increased only in the repeat restraint stress group. These data highlight important considerations of restraint parameters on dependent measures. Additional descriptions of restraint stress in rats are also included for comparison. Finally, the influence of the parameters of the restraint (e.g., acute vs. chronic) and characteristics of the animal subjects (strain, sex, age) are discussed.

Introduction

Due to the universal experience of stress, investigations into the mechanisms of altered responses following stress exposures have been a consistent area of research for several decades1. These investigations have begun to parse out aspects of stress that may be beneficial to increase adaptability and responsiveness to acute stressors from stress experiences that induce maladaptive alterations of physiological and behavioral functions, often resulting from prolonged exposure to repeated and/or unpredictable stressors. Many of these responses to stressors impact the brain function, the hypothalamic-pituitary-adrenal (HPA) axis, the sympathetic and parasympathetic branches of the autonomic nervous system, and the immune system. Exposure to stressors initiates the increased release of corticotropin-releasing factor (CRF) to increase adrenocorticotropic hormone (ACTH) and glucocorticoid hormones, such as cortisol in primates and many other mammals and corticosterone in rodents2. Additionally, epinephrine and norepinephrine are released throughout the periphery and brain as part of the sympathetic nervous system response3. These chemical signals then interact with a number of physiological systems, such as immune function, metabolism, reproduction, and neuronal reactivity4.

Stressful experiences are associated with a myriad of conditions, including increases in anxiety and depression, alterations in learning and memory, impairments in decision-making and executive function, and susceptibility to substance use disorder, among others. Each of these effects is dependent on both parameters of the stressor and the characteristics of the individual experiencing stress4. The nature of the stressor, as acute or chronic and as controllable or unpredictable, substantially alters the outcomes observed. These aspects of stress appear consistent between human experiences and animal models. Thus, to model stress in animal studies, careful consideration must be used to select parameters of stress exposure that are appropriate for the species, sex, and condition.

Although a number of stressors are available for studies with rodents (including psychosocial stressors like social isolation stress or social defeat, pharmacological stressors like cortisol or yohimbine administration, physical stressors like footshock and restraint, and multimodal stressors that combine or alternate between conditions), the focus of this manuscript will be on the use of restraint stress in mice and rats. Restraint stress might be chosen over other stressors because it is easy to perform, inexpensive, adjustable to various sizes of rodents, scalable to both acute and long-term periods, and rarely results in adverse harm to the subject5. Although this manuscript focuses on the lasting changes induced by restraint stress exposure, some studies use restraint in rodents to examine mechanisms and behaviors that occur during restraint, including active coping behaviors involved in escaping the stressor6,7,8,9. The goal of this manuscript is to provide methodological considerations to assess the impact of restraint stress on mice or rat subjects.

Protocol

The experiments and protocols have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of North Carolina at Chapel Hill (mice) and Fairfield University (rats) and follow the guidelines of NIH10.

1. Restraint stress in mice

NOTE: Mice offer a number of advantages when studying the effects of stress on physiology and neuronal systems. With the prevalence of transgenic mouse lines, mechanistic investigations into the interactions between genes and stress exposures are readily feasible. Whether using transgenic or wild-type mice, consideration of the background strain is recommended. Additionally, due to their size and social housing, mice offer an advantage in space utilization over other species.

  1. Restraint stress exposure
    1. Randomly assign mice to homecage control, acute restraint stress, or chronic restraint stress groups.
    2. Determine the length of stress exposure. For acute stressors, use a single session lasting 5 min up to 12 h confined in the restraint apparatus. For chronic stressors, ensure that the session matches the length of the acute stressor but is repeated daily for as short as 3 days and as long as 21 days.
      NOTE: Because mice will be without access to food and water while in the restraint conditions, consider whether homecage control animals should also lack access to food and water.
    3. Modify 50 mL conical tubes to add ventilation holes.
      1. Use a power drill with a 1/8" bit to evenly space holes around each conical tube to ensure airflow throughout all areas of the tube.
      2. Place these holes to allow the mouse access to air regardless of which direction the mouse is facing in the tube. Ensure no sharp edges remain after modification.
        NOTE: These modified conical tubes should be effective for most adult mice (~20-40 g) but may not restrain small/young mice or larger strains.
    4. In a testing room, separate from animal housing and behavioral testing, place the restraint-assigned animal into the modified conical tube with the cap attached to confine the mouse in the tube for the designated length of restraint.
      NOTE: The specifics of the testing room design can vary based on the space available in the facility; the room could include fume hoods and other laboratory equipment and/or laboratory tables/benches or be an empty procedure room. However, regardless of the room setup, the restraint room environment should remain consistent over repeated restraint exposures to avoid context-induced alterations in stress responses11.
    5. Place the restraint tube horizontally on a flat surface. If needed, secure the tube with laboratory tape or a pipette basin/reagent reservoir so it does not roll with the mouse inside.
      NOTE: Under stress conditions, mice produce ultrasonic vocalizations. Consider whether the study design requires sound isolation from other restrained subjects12. Corticosterone and other markers of stress responses follow circadian rhythms. As such, considerations regarding the lighting conditions and time of stressor should be made. Across stress groups, restraint should occur at a consistent time of day, preferably early in the light cycle13,14,15. For the study presented here, mice were restrained for approximately 3 h following lights on.
    6. Monitor each animal during restraint stress at least every 20 min. Visually determine whether the animal is displaying unusual responses (slow breathing, lack of movement). If observed, remove the mouse from the restraint tube, contact a veterinarian, and exclude the mouse from the study.
    7. Return the mouse to the home cage and provide access to food and water.

2. Restraint stress in rats

NOTE: As larger rodents, rats have advantages to consider when investigating the effects of stress. Like mice, rats have various strains with differential responses to stress exposure16,17. Additionally, the increased size of rats makes rats easier to use with certain behavioral measures, e.g., drug self-administration.

  1. Restraint stress exposure
    1. Randomly assign the rat to homecage control, acute restraint stress, or chronic restraint stress groups.
    2. Determine the length of stress exposure. For acute stressors, use a single session lasting 5 min up to 12 h confined in the restraint apparatus. For chronic stressors, ensure that the session matches the length of the acute stressor but is repeated daily for as short as 3 days and as long as 21 days.
      NOTE: Because rats will be without access to food and water while in the restraint condition, consider whether homecage control animals should also lack access to food and water.
    3. For a commercially available restraint tube, follow the steps described below.
      NOTE: Commercially available restraint tubes for rats are available. These devices have a variety of designs, including metal wire, containers, and plastic/plexiglass tubes with rounded and flat-bottom options. Each option is simple to use.
      1. Insert the rat into the device, then add a plug adjusted to the subject's size, and lock it in place. When placed in the restraint tube, ensure that the rat is snugly confined to prevent head-to-tail turns but can breathe easily.
        NOTE: These devices are designed for restraining rats and do not need modification for respiration. This study uses the tailveiner restrainer (Table of Materials), which includes suction cups on a platform under the tube to secure the device. An additional benefit of this device is that the tail is accessible outside of the device for control of the subject during insertion and removal from the tube or tail vein blood draw during restraint.
    4. In a testing room separate from animal housing and behavioral testing, place restraint-assigned animas into the restraint tube for the designated length of restraint. Place the restraint tube horizontally on a flat surface.
      NOTE: Under stress conditions, rats also produce ultrasonic vocalizations. Consider whether the study design requires sound isolation from other restrained subjects.
    5. Monitor each animal during restraint stress at least every 20 min. Visually determine whether the animal is displaying unusual responses (slow breathing, lack of movement). If observed, remove the rat from the restraint tube, contact a veterinarian, and exclude the rat from the study.
    6. Return the rat to the home cage and provide access to food and water.

3. Assessments of stress

  1. Measurement of HPA activation
    1. If appropriate, following restraint stress exposure or removal from homecage, rapidly decapitate subjects to collect trunk blood into heparinized tubes.
    2. Isolate plasma via centrifugation and store plasma samples at -80 °C. Analyze blood corticosterone using an enzyme-linked immunosorbent assay (ELISA) kit.
      NOTE: Although corticosterone is often used as a readout of the severity of stress exposure, especially acute stress, corticosterone levels do not always correlate with stress-induced behavioral changes6. Furthermore, the HPA axis response regulating corticosterone release habituates with repeated exposure to the same stressor18. Alternatively, adrenocorticotrophic hormone (ACTH) may be a more reliable measure of the intensity of an acute stressor4. It remains elevated for approximately an hour following the cessation of stressor exposure, but severe and prolonged (approximately > 2 h) stress may fatigue its production.
  2. Behavioral effects
    1. If appropriate, following restraint stress exposure or removal from homecage, assess the behavioral performance of the rodent with the appropriate behavioral task.
      ​NOTE: Further information regarding the effects of restraint on various behavioral models can be found in the discussion section.
  3. Neurobiology
    1. Alternatively or additionally, assess neurobiological function following restraint stress exposure or control. A wide range of assays can investigate restraint stress effects on cellular, synaptic, and circuitry. Depending on the assay, implant an in vivo measurement device or harvest brain tissue to perform the selected analysis.
      NOTE: The effects of stress on cell-type specific changes in protein synthesis and epigenetic modifications have been thoroughly reviewed by McEwen and colleagues1. These genetic and protein modifications induce plasticity to alter synaptic connections and circuitry. In order to conduct these studies, harvest brain tissue following stress exposure and perform genomic, proteomic, ex vivo physiology, and other analyses. Alternatively, assess neurobiological changes through in vivo measurements such as microdialysis, fast-scan cyclic voltammetry (FSCV), fiber photometry, and others. These approaches record neurochemical release and neuronal activation, both following restraint stress exposure2,19 and now during restraint8,9,20. The toolbox to assess the neurobiological mechanisms underlying the effects of restraint stress is rapidly expanding allowing new insights into the complexities of stress.

Results

In a previously published study21, the restraint stress procedures described in this manuscript were used to assess the impact of restraint stress on the regulation of cortisol and norepinephrine, falling under the broad possibilities of changes in neurobiology induced by stress mentioned in protocol 3.3. In this study, we utilized the protocol described above for the restraint of mice. Additional details that are beyond the scope of the restraint focus of this article, e.g., the rationale for bra...

Discussion

To investigate the effects of stress on physiological, neurobiological, and behavioral functions in rodent models, conditions that produce stress for rodents must be used. Among these considerations are the conditions that produce stress responses in the subject. Restraint is a validated procedure to produce physiological and behavioral responses in rodents matching stress effects in humans. Using a methodology similar to this protocol, restraint has been used to assess a number of molecu...

Disclosures

No conflicts of interest to report.

Acknowledgements

I appreciate the editorial feedback provided by Holly Rahurahu and Sam Shaffer.

Materials

NameCompanyCatalog NumberComments
50 mL General Purpose Screwcap Conical TubeGlobe Scientific6288
Disposable Pipette BasinFisher Scientific13-681-500
ELISA Kit for CorticosteroneArbor AssaysK014-H
Tailveiner Restrainer TubeBraintree ScientificRTV

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