A Comprehensive Review of Experimental Models of Stress: Pragmatic Insight Into Psychoneuroimmunology
ABSTRACT
Psychoneuroimmunology is a scientific discipline exploring the interconnectedness of the nervous system, emotion state, and immune system. The current review examines the distinct mechanisms through which the mind and body interact when subjected to stress. Manifestations of psychoneuroimmunological stress encompass symptoms such as depression, aggression, fear, and social withdrawal, which can exert a profound impact on physiological well-being. Some observations suggest that humans and nonhuman animals exhibit similar stress-related symptoms, aiding in the identification of pharmacological pathways and potential clinical implications of therapeutic interventions. Animal stress models are predicated on varying approaches aimed at eliciting a motivational state to navigate and confront aversive circumstances. The current review describes the diverse stress induction models that have been investigated internationally, incorporating an ethological perspective that involves evaluating innate and unpunished behaviors through methodologies like the elevated plus maze, elevated zero maze, light-dark box, and open field test. Additionally, conditioned operant conflict tests, such as the Vogel conflict test, fall under the purview of learning and punishment models. This category encompasses classic conditioning models like fear conditioning, psychosocial models such as social defeat, and physical and chronic unpredictable stress paradigms. In this review, we critically evaluate existing cognitive and behavioral frameworks underpinning the development and perpetuation of stress-related disorders, while also elucidating the impact of immune system responses on the mental and physical health of animals. The primary objective of this review is to elucidate the array of animal models employed in previous research and the testing protocols used to assess animal performance in stress induction scenarios, with the ultimate aim of reducing mortality rates among research animals.
Abbreviations
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- CSC
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- chronic subordinate colony
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- GLP
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- germin-like proteins
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- TW
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- trauma witness
1 Introduction
The brain is a vital organ in the human body that develops via the environmental and genetic interactions [1, 2]. The cortex and hippocampal region of the brain play significant roles in harmonizing adverse emotional response that are triggered by psychological distress [3, 4]. The body encounters stress somatically and psychologically, which results in alterations of electrical, pathological, and dopamine pathways, which are involved in the development of numerous neurological and mental illnesses. These somatic and psychological responses are accompanied by electrophysiological responses and induce synaptic plasticity alterations during development. This neurocognitive adjustment causes alterations in the shape and functionality of receptors in different brain regions that affect neuronal neurotransmitter release including excitatory and inhibitory changes [5]. The presence of stressors makes it difficult for organisms to break equilibrium. During childhood and adolescence, plasticity is not at its optimum and the brain circuits that regulate energy balance and stress reactions in an individual are subject to change [6]. In an adolescent or growing baby, stressful experiences must be all-encompassing to promote previously established brain patterns. The frequency and intensity of stress may increase the risk of extended neurotransmitter systems and behavioral factors undergoing alterations [7]. The impact of traumatic events that are imperceptible or psychological depends on the individual's age and gender, the type and intensity of the stress response, how frequently it occurs, and whether the individual recognizes the stress [8]. The conserved transcriptional response to adversity that is adaptive before traumatic injury starts is induced by the connection between the central nervous system and peripheral immune system. This interconnection attracts attention and functions as a social-environmental signaling system. Additionally, this mechanism may activate the conserved transcriptional response to adversity in the absence of any concrete warnings [9, 10]. Previous research has shown that the period between youth and adulthood involves substantial ongoing activity of occupational employment; additionally, an increase in inflammatory activity usually precedes this activity, which has been tied to external expectations [11]. Sleepiness, anxiety, depression, and schizophrenia are neurodegenerative disorders that have been greatly influenced by stress. The emergence of rapid life changes in contemporary societies mean that individuals face more stress. Unexpected pregnancies, divorce, unemployment, serving in the military, and environmental damage are sources of anxiety in many individual's lives [12]. The current review focuses on stress-related physiological and psychological changes caused by stimulation of the nervous system. Stress-related neurological problems can significantly impair a person's quality of life and socioeconomic behavior. Depression can be examined as a hallmark of widespread mental illness, including symptoms like pervasive depression, a loss of passion for life, fatigue, trouble sleeping, and in severe cases, suicidal ideation [13, 14]. The World Health Organization predicts that by 2030 depression will surpass cancer as the most expensive medical condition, despite the fact that it consistently ranks low [15]. To survive in contemporary society, the human body must routinely adapt and go through internal and external changes. To prolong lifespan via homeostasis, the human body carefully manages internal information such as body temperature and oxygen levels. To maintain homeostasis, various hormones, including cortisol, adrenaline, and noradrenaline are released in the body. These hormones also stimulate the central and autonomic nervous systems, enabling individuals' awareness and maintenance of daily rhythms. Because these physiological mediators are equally activated and inactivated in the absence of external stressors, when they are released, immunological and metabolic parameters are altered in ways that are both protective and flexible. Hence, when a strategy is ineffective and the physiological response is apparent for an extended period of time, an individual's health and well-being are affected [16].
1.1 Effects of Stress on the Body
Individuals' responses to neurological behavior are typically triggered by situations such as fear, the instinct to fight, or the impulse to flee. When faced with bullying or similar stress-inducing situations, the body initiates a stress response to prepare for and prevent potential harm. This response involves the release of specific chemicals tailored to the body's immediate needs. Consequently, individuals experience various physiological changes, including increased heart rate, blood pressure, breathing rate, and blood flow to the skeletal muscles. These bodily adjustments serve to protect the individual from imminent danger and provide temporary support by enhancing endurance, speed, and aggression to ensure survival. Additionally, these mechanisms reduce pain perception and temporarily alter brain activity [17]. Stress has become a significant factor in jeopardizing human health in contemporary developing societies, primarily because of its frequent association with routine environmental factors such as work demands, ongoing conflicts, and persistent communication. Individuals experiencing chronic stress often exhibit heightened brain activity, leading to increased reactivity in stressful situations. Moreover, this condition can impede normal brain function, potentially resulting in cognitive impairment. Common symptoms of stress-related afflictions encompass physical manifestations like fatigue, headaches, memory issues, allergies, and various other health complications [18]. Persistent stress has negative effects on the brain and body because it disrupts the appropriate immune system balance, which is important for maintaining health and endurance. The immune system fights against conditions like cancer that begin in the cells of the body, and initiates healing processes, as well as protecting against environmental contamination originating from external sources [19]. A robust immune system is crucial for immediate requirements such as survival and maintaining cardiovascular function. In the event of a sudden cessation of immune function, an individual would succumb within an hour, leading to rapid infection by various pathogens and bacteria [20]. Invasive cancer cells can cause problems in the body, and protecting the immune system requires killing the mutated cells that cause abnormal functioning. Because the battle against exogenous microbes is ongoing, it is necessary to maintain the stability of the immune system, which is essential for well-being and strength. The immune system must be strong enough to resist vigorously when foreign substances negatively impact the body, externally and internally [21].
1.2 Impacts of the Brain on the Body
Medications for treating neurological illnesses operate under the premise that the body is affected by thoughts, deeds, and emotions, which impact how well an individual feels. The state of an individual's body mirrors the way they feel about themselves, which also influences how they behave [22]. There is a relationship between an individual's health and their level of affection which is determined by the most advanced neurological organ, the brain. The brain interacts with many different organ systems, glands, and tissues, and serves as the focal point for all of our thoughts and behaviors [18]. When a person is under severe stress, the body struggles to maintain a proper immunological and hormone control system which leads to physical sickness affecting the organ systems. The nervous system may have an impact on conditions like cardiac heart failure, gastrointestinal issues, migraine, asthma, and other problems like high blood pressure, headaches, cardiovascular illnesses, arthritis, autoimmune diseases, cancer, and allergies. Figure 1 illustrates how the sympathetic nervous system adrenal medullary system and the hypothalamic–pituitary–adrenal axis are affected by stress [23]. In response to stressful circumstances, hormones like norepinephrine and adrenaline are released, which also causes the release of lymphocytes. Furthermore, the effectiveness of these hormones is diminished when stress responses have been triggered, and endorphins are produced during a mental state of euphoria [24]. Excessive stress triggers the release of cortisol and glucocorticoid hormones, leading to detrimental effects on the nervous system. Interference of the immune system by external factors results in adverse cellular impacts, including a reduction in T- and B-cells, which are associated with a decrease in immune function. Notably, these alterations have been linked to increased susceptibility to cancer. Furthermore, studies suggest that energy levels can exert both beneficial and detrimental influences on the body's ability to combat diseases and other health conditions [25].
1.3 Processes for Reducing Stress
Stress is a fundamental component of life that can have both beneficial and detrimental effects [26]. Optimal changes can be catalyzed by positive stress symptoms, while negative stress symptoms appear when there is an imbalance between a person's desires and perception of their abilities. This situation can place an individual under pressure to divulge information because stress can be hazardous to a person's health if they are unable to manage their emotions or find a way to cope [27]. Stress can result from persistent negative reactions. Tension can become a significant problem if there is pressure for the individual to respond quickly, and neurological drugs can be used to treat this condition, indicating that suppressing stress in the body can improve well-being. Using psychoimmunomodulatory techniques, a person can change their perspective regarding stress-related issues and gain control over the outcome. Because individuals have the ability to maintain their stress reactions and behaviors, they can endure and manage stress-related obstacles [28]. An individual may be better able to control their emotions and respond to stressful events if they can recognize the behaviors that are associated with stress. Stress has no detrimental consequences for a person's physical or psychological health; rather, the way in which a person responds to stressful conditions depends on how they interpret stress. Thus, individuals must practice restraint when faced with challenges [29].
If strategies are used to lessen stress, individuals may benefit psychologically and experience a better standard of living. Using this approach, medical personnel can adapt some of their cognitive strategies from daily existence to assist patients [18]. Through strengthening the immune system and its connection to the brain, patients' recovery can be promoted using aromatherapy and certain essential oils. The effects of aromatherapy may provide insight into the relationship between immune function and the brain. Research indicating the effectiveness of psychoneuroimmunological methods has led the western medical establishment to accept this therapy as a cutting-edge method [30].
2 Stress Models
It is widely recognized that depression and other symptoms of chronic stress can develop in humans and nonhuman animals [31]. Therefore, it is crucial to validate various animal stress models as representations of the behavioral and physiological responses observed in humans, to gain a comprehensive understanding of stress-induced psychiatric disorders [14]. Rodent animal models have been created to replicate physiological mechanisms observed in humans, facilitating research into the molecular underpinnings of diseases and potential therapeutic interventions [32, 33]. Furthermore, the utilization of animal models has proven to be a common and valuable practice while studying stress-related behaviors in humans is ethically or practically infeasible. With approximately 90% genetic similarity between rats and humans, animal models are considered to provide valuable tools in neuroscience and related biomedical fields, and a growing number of research groups are focused on this approach [34]. Figure 2 shows a variety of animal stress models that have been developed to investigate the antistress effects of drugs derived from natural and synthetic sources. Research on stress has focused on creating innovative treatment options and exploring the mechanisms of stress responses by utilizing effective animal reference models. An ideal animal model for stress should accurately predict all aspects of the stress response and naturally replicate disease development. Psychoneuroimmunological research has demonstrated the significant impact of future medical decisions on health and well-being by elucidating the intricate relationships between the mind, brain, and body. It is overly simplistic to attribute the core components of any disease solely to emotional responses, because competence in this field has not yet been fully established, indicating that emotions are not the sole cause of disease genesis. Over the past 50 years, the availability of animal models for various diseases has significantly increased, making the selection of an appropriate animal model for specific research tasks a challenging endeavor. The choice of animal model is influenced by the nature of the experiments, the research methods employed, researcher expertise, and awareness of the ethical considerations associated with using animals as models. Emphasis should be placed on developing approaches that can effectively address false-positive and false-negative results, as well as biases introduced by local laboratory conditions. Despite their limitations, animal models play a crucial role in enhancing our understanding of stress-related diseases, as highlighted in this review.
2.1 In Silico Studies of Stress in Plant Models
The promoter regions of genes encoding rice germin-like proteins (GLPs) have been examined using In Silico methods to identify transcription factor binding sites associated with biotic and abiotic stress. GLPs belong to the cuprin superfamily and are regulated by glycoproteins. Initially identified as a marker for the regeneration process in wheat grain germination, the gene germin was discovered to be related to this process. GLPs have been identified in various angiosperm, gymnosperm, and moss species. These proteins are referred to as participants because they share structural similarities with gemin proteins and belong to the same protein family. Each plant species has distinct biochemical and physiological processes. Numerous members of the GLP family are involved in responding to both biotic and abiotic stressors [35].
A significant proportion of the global population consumes rice (Oryza sativa) as a primary staple food. Various sequencing technologies have been utilized to conduct research aiming to differentiate each GLP family within rice and other crops, with the resulting data being made publicly available in databases [36]. Rice has been reported to contain a larger number of Oryza sativa GLP members. The expression of various Oryza sativa GLP members in response to different abiotic stresses has also been studied across various reproductive stages. Additionally, computational analyses were conducted on two OsGLP families, namely OsRGLP1 and OsRGLP2. Notably, distinct characteristics of certain OsGLP genes were identified [37, 38]. Various transcription factors, such as NAC, WRKY, bHLH, bZIP, MYB, and AP2/ERF, are influenced by the biotic and abiotic adaptations observed in different plant species. The transcription factors NAC, WRKY, bHLH, bZIP, MYB, and AP2/ERF were examined in silico within 1.5 kb promoter regions of various OsGLP genes. OsGLP8-11 was found to have the highest number of tested transcription factors in the promoter region, whereas OsGLP5-1 had the lowest number of tested transcription factors in the promoter area, among the several OsGLP gene promoters. Pathogenic analysis of multiple OsGLP genes led to the identification of four additional subfamilies. These findings highlight the influence of several OsGLP gene promoters on socio-emotional development. Additionally, the results support the notion that gene duplication and other polymorphisms in transcriptional regulators play a critical role in the diversification of OsGLP genes and their promoters for enhancing biotic and abiotic stress tolerance in rice [39].
2.2 In Vitro Study of Stress
Li et al. reported a brand-new member of the Cistanches herba family. In the chronic unpredictable stress (CUS) model rat paradigm, Cistanche tubulosa exhibited antidepressant effects that functioned through re-establishing the equilibrium of gut flora. Normal CUS-induced depression rat models were used to analyze the metabolic profile of C. tubulosa in in vitro and in vivo which controls the biological processes such as immunological modulation, anti-inflammation, and control of neurodegeneration [40, 41]. In normal and CUS rats, in vitro modeling utilizing ultra-performance liquid chromatography quadruple time of flight mass spectrometry (UPLC-Q-TOF-MS) has been used to determine the gastrointestinal metabolism of C. tubulosa extract [42]. To evaluate the metabolism of C. tubulosa extract in rats, the same approach and timing were used through urination. Similarly, in vivo and in vitro research techniques have been used to detect additional metabolites in normal and CUS rats, respectively [43]. Furthermore, in in vitro models, this extract has been assimilated to aglycones, abasement materials of phenylethanoid glycosides (PhGs), and iridoid glycosides in normal rats and in the intestinal microbiota of chronically unpredictable stressed animals [44]. In rats, urine and feces are the most prevalent aglycone metabolites and abasement by-products of phenylethanoid and iridoid glycosides of phase 2 [45]. Contrary to stressed rats, rat gut microbiota needed a relatively small amount of food to produce secondary glycoside and aglycone. Because of hydrolysis, this irregular glycoside was found to be produced in the intestinal flora of stressed and anxious rats. All these findings are useful for understanding the genesis of the metabolic process, therapeutic mechanism, and inhibitory properties of C. tubulosa's herbal treatments [46].
2.3 In Vivo Study of Stress
2.3.1 Intruder Chronic Psychosocial Stress
The intruder chronic psychosocial stress approach entails segregating four to six rats in each cage into two distinct groups and placing them in neighboring cages. Rats are housed together for a period of 1 week across all groups to facilitate familiarization and the establishment of social hierarchies [47]. Over a period of 6 weeks, two rats were placed alternately in each of the two cages, leading to a random rotation schedule that induced stress. The uncertainty stemming from this altered protocol resulted in the emergence of social hierarchies among the rats, necessitating their adaptation to the new stressful setting. Subsequently, the behavior of the rats was analyzed in comparison to that of humans, because both species commonly experience changes in living arrangements and workplaces. This shared characteristic was found to elevate corticosterone levels and blood pressure in both humans and rats [48].
2.3.2 Restraint Stress
In studies using restraint stress, rats are placed in a small Plexiglas enclosure with access to air through their noses, causing them stress. The size of the Plexiglas cylinder was adjusted according to the weight of each animal. To induce chronic stress over a period of 7–14 days, the animals' activity was reduced by placing them individually in a double-layered Ziploc bag and sealing the edges with tape. Acute stress was induced by employing the same method for a duration of 2 h [49].
2.3.3 Chronic Subordinate Colony Housing (CSC) Paradigm
In the CSC paradigm, long-term stress is deliberately induced in mice that are housed continuously for a period of 19 days [50]. When a resident male mouse is kept in a large cage, it behaves aggressively in comparison to the CSC mouse. Observations of the activity levels of the CSC mice are recorded, including instances of pursuit, mounting, and attacks. Using this methodology, it has been observed that CSC mice exhibit submissive behaviors and protective responses, such as engaging in flight reflexes when faced with unfair treatment [51].
2.3.4 Trauma Witness (TW)
Experiencing a frightening incident can induce psychiatric symptoms, such as posttraumatic stress disorder. In the TW paradigm, the TW rat is kept outside a cage containing resident rats and observe a social defeat situation. In this paradigm, TW rats exhibit signs of social apprehension. When the invader rats are introduced, the TW rats were kept outside the resident cage to examine their social defeat reaction; however, when TW rats were exposed to any stress associated with social defeat response they froze, as other studies have confirmed and documented. Social defeat is typically observed three times, with a 5-min separation period to increase stress in TW rats during the observation period. Similarly, for social defeat control activities, a fresh invader rat in a cage was placed behind a wire barrier for the control group. TW control rats' group was allowed out of its cage once a day for 30 min. As a result, rats experienced heightened anxiety and depressive-like tendencies as well as learning and memory deficits. Compared with intruder rats, TW rats behave better because they had been in a social context [52].
2.3.5 Early Life Maternal TW
In the early life maternal TW paradigm, young rats are exposed to similar conditions of observing social rejection experienced by their mother. Mother rats are placed in a confined space to experience social defeat, in an apparatus containing six distinct small witness box enclosures. The rat pups are exposed to their mother's social defeat when confronted with an unfamiliar aggressive male rat for a total duration of 45 min spread over 7 days (three times intervals, followed by 5 min of rest, and 10 min of social defeat) [53]. In one study, the young rats were confined in their enclosure for 1 month before the commencement of behavioral evaluation. To minimize exposure, the female rat was isolated without a male counterpart, while the pups were kept nearby. Subsequently, the pups were placed in a transparent cage for half an hour daily over the course of 7 days. By the 60th day after birth, both male and female rats were found to demonstrate behaviors that were indicative of depression. The mother rat, having endured these repeated stressors, began to exhibit symptoms of anxiety and depressive despair [54].
2.3.6 Single Prolonged Stress
The single prolonged stress method involves subjecting animals to significant stressors. The animals are placed in separate Ziploc bags with taped edges for a 2-h period of immobilization. The animals are then required to swim in a cylindrical water tank for 20 min. Subsequently, a 15-min recovery period is provided before the animals are released. The animals then undergo anesthesia and are left undisturbed for 7 days to allow researchers to observe the development of posttraumatic stress disorder symptoms without increasing mortality rates [55].
2.3.7 Nonhuman Primate Models
Stress in nonhuman adults can be caused by psychological events such as an animal being taken from its social habitat, placed in a new social setting, or subjected to encounters with members of the superior or superior group. In humans, adolescence is a critical stage at which drug use often begins, and is marked by significant neurobiological changes that can make individuals more vulnerable to substance use disorders. Because early life stress is a key factor in increasing this risk, it is important to examine how stress affects the brain's response to drugs. Nonhuman primate models provide valuable insights into these processes, helping researchers develop prevention strategies for at-risk populations [56].
3 Parameters for Measuring Stress Developed in Rodent Models
3.1 Ethological (Unconditioned) Behavior-Based Model/Exploration-Based Models
Ethological behavior-based models were utilized to analyze the superficial restriction which also formed owing to logical augmentation, inducement, laboratory settings, and what is going on in our surroundings, such as fear or avoidance [57]. These models have higher criterion for validity, allowing for more implementation of behavioral changes caused by diverse procedures. This procedure can be used to provide a new environment to reduce fear, curiosity, and conflict avoidance (Table 1). The unconditioned behavior model was first reported by Montgomery, who used the Y-maze apparatus consisting of one closed arm and two open arms [58]. The results revealed a high proportion of movement of the rat in the closed arm because of fear and avoidance of open areas. Hence, to minimize avoidance behavior between social environments, different animal model experiments have been carried out to develop novel procedures and minimize the animal death rate [59].
| Parameters for measuring stress developed | |
|---|---|
| Unconditioned test | Conditioned test |
|
|
3.1.1 Elevated Plus Maze
The elevated plus maze involves a ground-level apparatus comprising two closed arms that are parallel to close arm and the other side of two open arms. The premise of this experimental method is that when rodents explore new environments they naturally avoid exposed, bright, and elevated regions. Exposure to common anxiety-reducing medications, including benzodiazepines, heightens the tendency of mice to study these arms, increasing excretion and corticosterone levels, and causing physical stress as a result of confinement in the open arms [55]. In considering the performance of a mouse in this paradigm, various factors, such as living circumstances, circadian cycle fluctuation, illumination levels, handling and stress exposure, and characteristics of the maze equipment must be taken into account. Unlike rats, mice do not become more tense in isolated housing. The existence of distinct social organization patterns within a species is likely to depend on the cause of increasing tension during stressful circumstances like foot shock, social defeat, or predator exposure [57]. Re-exposure to this device has a detrimental effect on animals' exploratory behavior in the open arms of the maze and negates the overall calming effect of benzodiazepine. Hence, although the process exhibited some traditional assessment while employing various groups to conduct this parameter, it also had some risk appraisal towards open arms, such as head dipping, which gave the sensitivity to this experiment [59].
3.1.2 Elevated Zero Maze
The elevated zero maze is a modified version of the elevated plus maze apparatus, and both traditional and recently developed ethological criteria can be used for the investigation of drug effects. It also removes the veiled clue of the rodent's position that was present in the raised plus labyrinth's center [60]. It also included a circular path gadget that was elevated above the ground, replacing the open and brightly lit environment with one that was closed and softly lit. The uniform nature of the open and closed areas of the circular path are considered to reduce issues related to the central area of the elevated plus maze. The number of entries into the open area of the elevated zero maze within a 5-min period are reported to be associated with anxiety. As environmental activity has substantial consequences on rat's performance, this approach was done to verify the avoidance-based activity, in which video recording sessions were evaluated in the absence of an investigator [61].
3.1.3 Elevated T Maze
The elevated T maze apparatus contains three arms. One of the arms has a sideway that is closed and perpendicular to the other two arms. The apparatus is elevated above the ground. The day before testing begins, animals are subjected to the open arm for a 30-min period. In subsequent training sessions, forced exposure has been found to slow the animal's response time to the arm. This demonstrates how behavior is dependent on fresh activity and in one manner, drives from interruption [60]. After being pre-exposed for 24 h in an open arm, the animals are tested to document the acquisition of inhibitory avoidance. Each animal is placed at the end of a closed arm, and the amount of time it takes for all four paws to emerge is then noted. After 30 s, a follow-up measurement is taken. Each animal is then held at the end of an open arm and a 30-s interval is timed from the instant it emerges from the arm with all four paws extended. Normally, the avoidance test and the escape phase last 300 s [60]. Hence, this model can be used to measure the two behavioral responses in the same animal (i.e., conditioned behavior represented by inhibitory avoidance in the open arm and unconditioned behavior represented by escape behavior when the animal is placed at the edge of the arm).
3.1.4 The Light-Dark Box
In the light-dark box, animals are allowed to wander freely around an unfamiliar environment divided into two parts: protected [in the dark] and not protected [in light]. This method is designed to reflect the inherent tension between the exploration impulse and avoidance behavior regarding the light compartment. When benzodiazepines or other anxiety-relieving drugs are given, animals have been found to spend a long period of time in a small space. Finally, this test analyzes the curious records of animals as well as stimulants amphetamines, which gives inaccurate positive findings, and the foundation of rats' inherent resentment in the presence of light [62].
3.1.5 The Hole-Board Test
The hole-board test apparatus is constructed from a square platform with several floor holes. Rodents peer through these openings, exhibiting a behavior called head dipping [63]. Observation of this behavior is used to measure the level of curiosity and anxiety. In one study, when an anxiety medication was tested on rats in a familiar setting, the biphasic effects on head dipping were exacerbated and there was no increase in activity when rats were exposed to an unfamiliar environment [64].
3.1.6 Social Interaction Test
The social interaction test does not require animals to be trained. To start the procedure, the duration of social engagement is monitored and pairs of rats are allowed to move around and communicate in a space. Each pair's behavior throughout time has a direct impact on the length of the social engagement. As a result, each pair is counted as one data unit (i.e., if one rat is used for the treatment and the other is designated as the control, the other rat in the pair is then used as the dependent measure). Increased social interaction time in rats, but not altered motor activity is a behavioral sign of reduced anxiety [65]. Hence, this test differs from other methods and provides a necessary component that can prohibit the involvement of disinclination and appetite conditions.
3.2 Ethological (Conditioned) Behavioral-Based Model/Conditioned Operant Conflict Test/The Geller-Seifter and Vogel Conflict Test
In the Geller-Seifter test, animals are taught to pull a lever independently to obtain sugary drinks at varied intervals while being denied nourishment for 24 h. Light is provided to animals, which acts as a signaling stimulus and influences improved lever-pressing behavior. Furthermore, the animal faces a conflict between drinking water and receiving an electric shock that is simultaneously delivered. In the control group, animals are reported to become less inclined to pull the lever. Administration of anxiolytic drugs has been found to increase the likelihood that the animal complies with punishment. According to Vogel conflict test, animals were dehydrated for 24 h before training them to detect water bottles in the test box's water [66]. Animals were exposed once more the next day for a total of 24 h in the same box with a stainless-steel grid floor. The sensor-controlled electrical circuit was fastened to the steel grid floor and the animals were connected to the water nozzle. As a result, animal received a small foot shock (0.5 mA). Pharmacological medication exhibits conflict-free characteristics which elevates the proportion of licking after punishment [64]. Despite the potential usefulness of these paradigms for examining the effects of anxiolytic drugs, the Geller-Seifter and Vogel conflict test are vulnerable to interference from various factors, such as hunger, pain, thirst, learning and memory, which may affect the validity of the results.
3.3 Classic Conditioning Tests
In this method, spontaneous reactions and reflex reactions necessitate addressable memory in which neutral conditioned stimulus are repeatedly paired with an unconditional stimulus. After repeating this procedure, the conditioned stimulus alone elicits an effective response, which is referred to as a conditional emotional response [67].
3.3.1 Fear Conditioning
In this method, when a conditioned stimulus is presented again in a situation, a reaction signifying danger is triggered. This response exhibits protective characteristics, such as freezing, reflexive expression, endocrine response, and autonomic reaction. Memory of trauma, phobias, panic disorders, agoraphobia, and posttraumatic stress disorder have been examined in conditioning-based models. Administering anxiolytic medicines before the balancing of conditioned and unconditioned stimuli is believed to affect the emergence of conditioned learning behavior [68].
3.3.2 Predator-Based Models
In predator-based models, defensive mechanisms and behaviors in predators are recorded. Exposure to a live cat or its odor has been found to induce fight, freeze, risk-taking, and autonomic activation responses in dogs. Following this reaction, locomotor activity and nondefense reactions like grooming and reproduction have been found to slow down. Both stimuli resulted in protective reactions. Exposure to a live cat induced a defensive reaction, which was accompanied by more freezing behavior and ultrasonic vocalization compared with smell alone. It was found that exposure to a cat usually had a positive conditioning effect and was well-tolerated. Furthermore, cat exposure was also found to stimulate the angiogenic response observed when exposed to anxiety models [66].
4 Conclusions
This comprehensive review explored the connections between stress, psychoneuroimmunology, and animal models. We highlighted the physiological and psychological responses to stress on the body and brain. Various animal models, including ethological and conditioned tests, demonstrate the depth of research in this field. The use of in silico and in vitro studies reflects the development of methodologies to understand stress on a molecular level. Despite their limitations, animal models play a vital role in understanding stress-related disorders. Collaborative efforts have enabled research to capture the complex relationship between stress, behavior, and physiology. The potential for advancement in psychoneuroimmunology and stress-related research is vast. Understanding stress through various models can lead to the development of effective therapeutic interventions and improved understanding of stress-induced disorders. This review provides a strong foundation for future research and emphasizes the importance of exploring stress on multiple levels.
Author Contributions
Ashmun Nisha: writing–original draft (equal). Arshiya Shamim: supervision (lead), validation (lead). Aleza Rizvi: resources (equal), supervision (equal). Tarique Mahmood: visualization (equal). Bhagyashree Goswami: writing–review and editing (equal). Farogh Ahsan: software (equal). Mohammad Shariq: methodology (equal). Saba Parveen: formal analysis (equal).
Acknowledgments
The authors thank Founder and Chancellor, Prof. Syed Waseem Akhtar, Integral University and Vice-Chancellor, Prof. Javed Musarrat, Integral University, for providing an excellent research environment and facilities. The authors thank Prof. Syed Misbahul Hassan, Dean, Faculty of Pharmacy, Integral University for his motivation and support. The authors thank Chairman Mr. Ishrat Hussain Jafri and Prof. (Dr.) Udayvir Singh Sara, Director, Hygia Institute of Pharmaceutical Education and Research. The MCN provided by the institution for communicating this manuscript i: IU/R&D/2022-MCN0001730.
Ethics Statement
The authors have nothing to report.
Consent
The authors have nothing to report.
Conflicts of Interest
The authors declare no conflicts of interest.
Open Research
Data Availability Statement
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.
