Modeling Chronic Stress

In order to survive, our bodies need to constantly adapt to environmental challenges. This process of regulation is called homeostasis^[1] and describes physiological mechanisms by which organisms continuously adjust their internal state in response to external changes.

Stress is a normal and adaptive response by the body to any potential threat, and is critical for any living organism to function properly. However, when this response persists in time, particularly in vulnerable individuals, its long-term effects contribute to the development of stress-related diseases.

Genetic predisposition^{[2, 3]}, gender^[4], and environmental factors^[5] are the three main aspects that influence the magnitude of a stress response. In general, once the body recognizes physical or psychological stressors, the central nervous system evokes multiple coping strategies to deal with specific situations.

An immediate response of fight or flight is triggered so the body can increase its chances of survival (the body is ready for action), or when active coping is not possible, the body maintains a vigilant state (active inhibition). Depending on the situation, our brain comes up with one of the two patterns. Interestingly, differences between individuals can also determine a variation in response to the same situation,^[6] meaning we don’t all respond to stress in the same way.

The General Adaptation Syndrome

Once the “threat” message reaches the central nervous system, the brain will command an organized series of events to cope with this alert. This process describes the stress response and it is represented by three distinct stages first described by Hans Selye in his theory of the General Adaptation Syndrome.^[7]

1st Stage: Alarm Reaction

Any external stimulus perceived as threatening by our sensory system will evoke a stress response. Within the brain, the Amygdala ⎯ an integrative center for processing emotions, especially threats or fear⎯  receives this information and in turn, it sends an alert message to the hypothalamus. Through the autonomic nervous system, the hypothalamus will command the involuntary effects that will take place in the rest of the body.

The autonomic system is divided into sympathetic and parasympathetic nervous systems and both will control the acute and prolonged coping mechanisms of the stress response.^[7]

At first, autonomic nerves from the sympathetic system will produce and release Epinephrine (Adrenaline) in the adrenal medulla (the center of the adrenal glands). This hormone will make energy available through the release and mobilization of glucose and fats from body reserves. In addition, it will redistribute resources to muscles and vital organs through increased heart rate, blood pressure, respiratory rate, and oxygen uptake capacity by the lungs.

Finally, the immune system will become reactive in order to protect the body from any potential damage. For this reason, immune cells like macrophages move to vulnerable tissues to combat pathogens and contribute to healing.

These effects are automatic, transitory, and undetectable, meaning they are actually out of our control and occur before we are visually self-conscious of any danger.

2nd Stage: Resistance

A second response is induced by the hypothalamic-pituitary-adrenal (HPA) axis controlled by the parasympathetic nervous system. Here, the hypothalamus produces corticotropin-releasing factor (CRH) which directly stimulates the pituitary gland. This gland in turn releases adrenocorticotropic hormone (ACTH), leading to the production of cortisol by the adrenal cortex (the outer part of the adrenal glands).^[8]

Cortisol will maintain an acute stress response, keeping the body alert to fight or flight. But once the threat disappears, levels of cortisol are reduced and the body returns to a “basal” state.

3rd Stage: Exhaustion

This stage is the result of a continuous perceived threat (real or not) or a prolonged one in time. Thus, the response to stress becomes maladaptive and sustained production of cortisol leads to alterations in the nervous, cardiovascular, endocrine, and immune systems, contributing to the development of multiple diseases.^{[9, 10]}

This continuous stimulation drives, among others, a higher risk of atherosclerosis, high blood pressure, cardiac failure, diabetes mellitus complications, anxiety, depression, and relative immunosuppression.

Three main variables must be taken into account in order to study the effects of stress in an experimental model: physiological traits (age and sex), exposure time (acute or chronic), and habituation to the stress response. The concept of habituation in stress physiology refers to a reduced response of the organism following repeated exposure to a stimulus^[11] which invalidates any experimental study.

Once these variables are determined, it is necessary to select specific stressors. Mostly known as processive or systemic,^[12] their use in research has evolved with time.

1. Processive stressors: these describe environmental factors capable of inducing a stress response. This group activates the hypothalamus mainly by limbic pathways, brain structures associated with emotion, motivation, and arousal. They include the use of restrainers to produce immobilization in animals (i.e., rodents), novel environments, noise or dark/light exposure, handling, and isolation. Since habituation of the HPA axis can easily occur with these types of stressors, they’re commonly used in combination to be considered valid.
2. Systemic stressors: their origin is physiological and can rapidly activate the hypothalamus through the brainstem. This structure is vital for autonomic functions like those occurring in a fight or flight response. These stressors represent an immediate threat for the animal and they are not susceptible to habituation.  They include the use of foot-shock, forced swimming test, limited exposure to extreme temperatures, and water or food deprivation.

A. Chronic Mild Stress Model (Unpredictable/Intermittent Stress)

This widely used model consists of randomly and continuously exposing the rodents to different and unpredictable stressors for a specific period of time (which can go from days to weeks).

First studies were performed by Katz in the 80’ to validate the use of the Open Field test as a tool to study levels of stress and depression-like symptoms in rats.

The open field test is based on the paradigm that open spaces can expose rodents to different threats (i.e. predators). During the protocol, a subject is placed in the center of the cage with a camera placed above and the experimenter will evaluate for how long (time moving) and how much (total distance traveled) the animal moves. A useful variation of this protocol is the novel recognition test where a new object is introduced in the cage. The idea is to quantify the amount of time the animal spends exploring the new object during the assay, compared to objects already placed in the cage during the familiarization period.

Katz’s team applied the use of processive (isolation and noise exposure) and systemic stressors (footshock, tail pinch, and forced swimming) to then evaluate the locomotion activity of rats during the open field test. Their research showed that unpredictable exposure of rodents to severe stressors resulted in a decreased locomotion behavior, suggesting higher levels of stress.^[13]

Due to ethical implications, Willner and colleagues modified Katz’s work by removing painful stimuli to focus mainly on “micro-stressors” like food and water deprivation. The aim was to evaluate how animals responded to rewards (sugar) after stress exposure.^[14] As a result, they not only minimized the levels of stress (as much as possible) but they also showed “stressed subjects” had a reduced sucrose consumption, suggesting an anhedonic state in rodents (a reduced response to pleasure or lack of motivation) for the first time.

The chronic mild stress model is by far the most useful tool in research since it can closely simulate certain clinical symptoms (i.e. Anhedonia) associated with chronic stress. Such symptoms can be ameliorated with the use of specific treatments (like antidepressants or anxiolytics) and they mostly show the same physiological consequences seen in humans.

Other stressors commonly used in this model are:

1.    Elevated plus, T, and zero mazes

These different mazes are based on the same paradigm as the open field test. Because rodents avoid open spaces, they rather stay longer in the closed part of these mazes.

• Elevated plus maze
• Elevated T maze
• Elevated zero maze

2.    Light/dark assay

The light/dark box is a cage with two different chambers: one with black walls and the other one bright and illuminated. As in the mazes, rodents would spend more time on the dark side to avoid any risks. Thus, the experimenter would quantify the number of times the animal goes from one chamber to the other one and how long it stays in each.

This tool has been mostly used to evaluate the effects of drugs like anxiolytics. However, certain studies have described the use of it to evaluate the response of animals after exposition to certain stressors.^[15].

3.    Restraint/immobilization

These stressors are easy, inexpensive, and painless tools to study stress response in rodents.^[16] Restraint can be done either manually by a manipulator or mechanically by using specific devices which can vary in material and size.

Normally they consist of a cylindrical tube with a ventilation hole in one of the sides where the animal will be kept for a period of time. On the other hand, immobilization protocols require the restriction of upper and/or lower limbs and head movement.

Animals can be restrained for long periods but once the peak response is reached, they can adapt to the tube or device, meaning its use is limited and it should be alternated with other stressors.

4.    Electric footshock stress

This stressor consists of a cage with a metal grid floor connected to a shock generator, by which the manipulator gives foot shocks to the rodent with variable intensity and duration. This tool includes physical and emotional components that induce behavioral and pathological changes in the animals. Because of the mechanisms involved in the response, animals don’t habituate. Moreover, it doesn’t cause any structural damage. Together with other stressors, they are frequently used to study anxiety-like disorders.

Over time, the original footshock has been modified so a stress response can also be studied under operant conflict models, a method of learning based on associating a specific behavior with a consequence (either positive or negative). The best example is the Vogel test,^[17] a conflict test where animals deprived of water will receive shocks with any attempts of water consumption. This leads the rodents to reduced drinking behavior.

5.    Forced swimming

In this procedure, the rodent is placed in a transparent tank filled with water from where there’s no escape, with the goal of measuring how active the animal is (mobility behavior) to avoid a stressful environment.^[18,19] The subject (after a constant effort and multiple failures) adopts a posture of immobilization, keeping only the necessary movement to maintain its nose above the water.

Some variants of this tool include exposure of the animals to extreme temperatures, usually cold water in the tank. Because the hypothalamus detects the changes in body temperature, a stress response caused by cold exposure can rapidly activate the HPA axis. This can be done alone (water immersion) or in combination with the forced swimming test.

6.    Noise exposure

When objects in the environment vibrate, they create waves of energy that are transmitted through the air. This phenomenon is caught by the ear and the perception is what we know as sound. The vibration in the air generates positive and negative pressure that alternate. The frequency at which this occurs is measured in Hertz (waves per second) and the change in the level of pressure (how much we hear) is measured in Decibels (dB).

Noise is an unpleasant sound capable of triggering a stress response. Especially in rodents, auditory stimuli drive the perception of an animal’s environment, giving them a sense of the existence (or absence) of a threat by context evaluation. In animal experimentation, white noise − a noise presenting the same frequency throughout a period of time is used in this protocol at different intensities and duration.

The setup includes a noise generator (0-26 kHz) connected to speakers placed above the cage. Some parameters able to generate a significant stress response in rodents go from 90 to 114 dB for a max period of 30 min.^[20]

7.    Stress-induced by circadian rhythm changes

Influenced by a sleep-wake cycle, the levels of cortisol fluctuate throughout the day. This hormone reaches its peak early in the morning and gradually decreases, with the lowest level at midnight. Alterations in this rhythm directly affect the homeostasis of any individual and in rodents manipulation of the day-night cycle triggers a stress response.^[22]

The protocol consists of exposing the mice to light during their dark phase of the cycle (“night time”) and then to dark during the light phase (“day time”). Some variations describe multiple cycles during the day (going from 1 to 3 hours) and it is suggested to be used together with other stressors since continuous exposure can result in habituation.

8.    Isolation

Animal models of stress including maternal deprivation and social isolation are environmental experiences that can be used as early life stressors in rodents. Their effects include major reactivity to stress, social behavior, and neurochemical alterations^[23] with long-lasting repercussions in adult life.

Protocols can vary from early isolation (starting on the second day of postnatal life) to later in life (from 3 weeks to 3 months). In this experimental procedure, pups are isolated individually in single cages for at least 1 hour. Once the period is over, they are placed back with the dams in their cages. Depending on the hypothesis of the study, this stressor can be used alone (mostly in the early postnatal period)^[21] or together with other stressors later in life.

B. Models of Social Stress

Mammals (and other animal species) spend most of their lives close to their conspecifics. They adapt to a social structure, follow complex rules, and accept hierarchy levels to guarantee their survival. Yet, these structures are highly dynamic and they constantly change throughout time, meaning the individuals from a specific group will be subjected to constant adaptation. Because of this, social stimuli could be perceived as a source of stress.^[24]

Animal models of social stress are mostly performed in adult male rodents and they include either a single (and intermittent) or chronic exposure to another subject. This is because male dominance is more evident in male mammals than females, although aggressive behavior can also occur in females during maternal care.^[24]

1.    Social defeat stress

This test evaluates the stress response of a naive animal to a dominant conspecific. In this protocol, a male mouse (the intruder) is placed in the cage of a resident mouse (the aggressor), usually larger, older, and with previous experience in confrontation.  Before the trials, an active female will be placed in the resident’s cage to co-house.

Chronic social defeat stress follows the resident/intruder paradigm where a naive mouse is introduced in the resident’s cage whose response is to attack (by biting, grooming, or chasing). This action evokes a defensive (or evasive) response from the intruder. The experimenter will record the time in which the intruder holds a social defeat posture expressed by:

• reactive immobility (paws on the ground)
• avoidance (position of the body opposite to the aggressor) or escape
• defensive stance (the mouse stands erect)

After the confrontation, the males will be separated in the same cage for a limited period of time. Since the cage is divided by a perforated barrier; visual, auditory, and olfactory contact can be maintained and the response to stress is prolonged.

2.    Intermittent defeat stress

In this variation of the social defeat stress, the male rodents are housed together allowing visual, olfactory, and auditory stimuli to be perceived but not the tactile ones. In multiple intervals, the barrier that divides the cage is removed to provoke a confrontation that leads to the defeat of one of the members.

However, once the barrier is placed back, every sensory stimulus except for physical contact, will be kept so exposure to the stressors is somehow chronic.

3.    Visible Burrow System

To better replicate “natural habitats”, males and females can be grouped to form colonies. These environments can vary in design but they usually consist of a large animal cage with tunnels that simulate burrow systems^[25] −common in rodents, and the inclusion of food and water.

The paradigm is based on how certain rodents can gain control of resources (survival) and increase their levels of reproduction (conservation of their species) by confrontation with their pairs.  This design allows studying not only social defeat but social dominance and group behavior.

Instability and social disruption models are variants of the burrow system where the experimenter evaluates how colonies already settled react to either the introduction of new members in their colony or an aggressive male respectively.

4.    Crowding exposure

In social crowding experiments, a significant number of rodents are placed to co-house in a conventional cage, reducing the amount of space each animal has. This protocol attempts to evaluate the behavior animals have to proximity (number of interactions), rather than subordination. Previous studies have shown this social stressor is able to induce body weight loss and higher levels of stress hormones.^[26]

During a conventional protocol, groups of up to 20 mice are placed together in one cage for several hours (it can vary from two to twelve hours per day) with food and water available. Variations in the experiment described the inclusion of either males or females and age groups.

By simulating specific conditions, models of stress are useful tools in studying the mechanisms underlying multiple diseases in humans.

As a result, the resources and techniques available to study stress have increased and evolved significantly over time. This variety means that choosing the appropriate model during the experimental design stage is more important than ever.

Animal models of stress are very complex studies, with many factors and limitations to be considered. To guarantee their validation they must be reproducible and they should effectively represent the disease or condition being studied.

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