Pain and distress in animal research

Ashley Sciocchetti -

Imagine waking up in the morning, eating a bowl of cereal, and driving to work. However, along the way, you turn too sharply around a corner and drive into a tree, hitting your head in the process. Although some exams indicate you have a concussion and mild cognitive impairment, fortunately, your injuries don’t seem severe and the doctor believes the recovery prospect is hopeful. They schedule weekly check-ups to ensure the recovery process continues as planned, during which you are asked to list any symptoms and perform simple cognitive tasks to check your learning capability. Anything out of the ordinary is documented, including headaches, dizziness, and signs of being overly stressed to name a few. This monitoring of symptom progression is what allows your doctor to give you the best recovery possible. 

Detailed medical documentation, commonly utilized when recovering from an injury, helps identify a wide range of variables that may influence a phenomenon, including physical, mental, and emotional factors. However, much of the preclinical research that leads to medical advancements uses animal models as subjects. Documenting pain levels and emotional feelings in animals is considerably more difficult than it is for humans, which can skew the results. In addition to animals not being able to talk, pain itself is subjective, and noxious experiences can be caused by a variety of pain sources. A recent study investigated the role of the rodent analog of an inflammatory mediator cell called interleukin-1, or IL-1, in a mouse brain recovering from a head injury [1]. Previous studies had demonstrated evidence of IL-1 modulation of behavioral changes and cognitive impairment following injury, so this study sought to further investigate this role by knocking out the IL-1 gene, which means the gene was not expressed. To induce the injury itself, researchers anesthetized the mice and hit a target location of the skull in a controlled procedure. The mice woke up, were monitored extensively, and put through an aversive stimuli learning test, in which a shock was administered if they stepped on a particular side of a cage. The goal was to record the time it took them to learn to avoid the zone that induced a shock. The researchers found that mice with the IL-1 knockout had significantly reduced expression of inflammatory cells and also increased ability to avoid the shock zone compared to the controls. This suggests the gene can modulate recovery following head injury [1]. Although this finding seems important, how might the pain and distress experienced by the mice have influenced the data collected? Not only did they receive an unexpected head injury, but they were also forced to undergo experimental procedures and techniques that induced more pain. These experiences could have negatively impacted their ability to learn to avoid the shock, potentially decreasing the reliability of the findings.

In contrast, when humans suffer from a head injury, they typically know how it occurred and can recover with the proper support if learning was impaired. There is no shock induced if they do not learn a concept. This is not to say humans don’t experience “punishment” for failing to heal. They may have limited medical leave or lack access to the healthcare needed to fully recover. However, documentation of pain and distress that may affect behavior and wellbeing is generally more difficult for animals than it is for humans. Consideration of potential confounding variables such as pain and distress is critical to ensure the reliability of the results and the optimal quality of life of the animals.

Animal welfare and IACUC

The Institutional Animal Care and Use Committee (IACUC) refers to a group composed of researchers, veterinarians, non-science, and community members who oversee the ethical guidelines for the use of animals in research at a specific institution [2]. In the United States, IACUCs are governed by the Animal Welfare Act and related federal regulations [3]. Labs proposing the use of animal subjects must work with a veterinarian to determine proper care for animals before, during, and after experimentation. Experimental procedures must provide reasoning for the use of animals that are then reviewed by the veterinarian, with whom the researcher then develops a care plan accordingly. At the end of an experiment, animals must be euthanized in a way considered humane, such as CO2 anesthesia followed by cervical dislocation. Anesthesia refers to the removal of consciousness, which is different from analgesia, which reduces pain [4]. Animals are also monitored throughout the experiment, and veterinarians are consulted if abnormal amounts of pain and distress are observed. Each situation requires a unique protocol and procedure to mitigate the pain and distress experienced by laboratory animals [3]. However, the various methods used to quantify pain and distress introduce substantial ambiguity as to whether the guidelines truly accomplish this. 

What is pain?

Colloquially, the term “pain” encompasses a wide range of feelings of discomfort. However, researchers have developed mechanisms for measuring different sources of pain, which are important to consider when performing animal experimentation. Acute pain from a known sensory stimulus is first detected by pain receptors - called nociceptors - in the dermis, which is the inner layer of skin. These signals are then transmitted into the spinal cord via a sensory neuron tract known as the dorsal root, and from there to three functional regions of the brain involved in pain processing. These are known as the amygdala, cingulate cortex, and somatosensory cortex [5]. This pathway can be likened to a highway, with information transmitted rapidly and unidirectionally until reaching “exits” into specific regions of the brain. Although nociceptive pain may be induced in studies, such as the foot shocking procedure, much of the pain tends to involve more complex mechanisms [6]. 

A team of researchers investigated the role of the dorsomedial prefrontal cortex (dmPFC) in the regulation of pain chronicity. The dmPFC integrates sensory information with cognitive processes and is at least partially responsible for the experience of emotions. Twice a day for six days, the researchers used an experimental technique called optogenetics to selectively activate nociceptive neurons in the dmPFC [6]. This activation stimulated the sensation of pain without inducing any real injury. Unlike humans, mice cannot describe their level of anxiety, so the researchers instead observed them for “anxiety-like behaviors,” such as paw licking or scratching, which are indicative of psychological stress. Additionally, researchers paired one region of the cage with the administration of morphine to evaluate whether dmPFC activation led to an increased preference for the region associated with morphine. Surprisingly, the mice did not exhibit anxious behaviors until days 10 and 11 post-activation. However, heightened activation of the dmPFC reduced the propensity of the mice to explore as early as day 3. Furthermore, experimental mice spent more time in the region of the cage paired with morphine on day 3, suggesting pain could have affected their behavior even before they began exhibiting signs of distress. To rule out the possibility that activation of the dmPFC triggered anxiety rather than pain, the researchers also inhibited the nociceptive neurons, observing reduced anxiety-like behaviors [6]. The delayed anxiety response observed in this study could be important if not taken into account, especially for behavioral studies using procedures that cause long-term pain. 

Measurement of pain

When people go to the doctor to address an ailment, the doctor often asks the patient to rank their pain on a scale of 1 to 10. However, lab animals such as rodents cannot easily be asked to answer such a question, so pain is often quantified using various methods. When called in to check on the animals, veterinarians often use the grimace test, which relies on alteration of facial characteristics to quantify the pain level [7]. Quantification is typically done by a human observer using a standardized chart with different facial expressions [8]. However, variation between observers at different times decreases the translatability of the results. In addition, while grimacing in response to pain is consistent across many animal species, it does not always occur in the presence of pain.

There are also several methods to determine how much of a given stimulus evokes a quantifiable response. One method of measuring pain in rodents is through the von Frey test, which measures the stimulus needed to evoke a “painful” response 50% of the time [9]. The stimulus varies but includes contact with a hot plate, pressure, and even sound. Another method of pain measurement is the operant test, which evaluates an animal’s activity level compared to their baseline activity level [7]. Open field is one operant test used to evaluate an animal’s response to anxiety-inducing stimuli [10]. A camera or tracker is used to record the locomotive activity of a rodent within a given space. Anxiety tends to decrease the time spent in the central area of the box, while increasing movement in the peripheral space [10]. A similar method is the use of an intracranial self-stimulation test, in which the animal is injected with an electrode that will provide non-painful brain stimulation when the animal presses a certain trigger. The test assumes animals in pain will press the trigger more often to receive pain relief. The consideration of pain-induced depressive effects is potentially useful in the establishment of anesthetic procedures for behavioral tests, but this method is infrequently used due to the equipment required. In addition, the animal’s response to the use of anesthetics is not necessarily guaranteed to fully be a result of pain relief. It could also be an indication of reward behavior, since the animal could press the trigger more frequently to receive anesthetic even without pain. Future research is needed to investigate whether the use of anesthetics interferes with operant behavior and whether this procedure for measuring pain and anxiety can be made accessible to labs using animals [7].

Pain management

Research studies that induce nociceptive pain as a side effect of a procedure, such as surgery, often attempt to alleviate pain using anesthetics [2]. However, veterinary anesthetic guidelines are constantly changing, and the development of dosage guidelines based on weight is based on exhibited responses to pain, which is not always clear. When developing experimental procedures, researchers often choose anesthetic dosages based on similar experiments, which may not account for variables affecting the efficacy of the dose [4]. For example, one study observed various pain sensitivities in different common mouse strains in response to a given dose of the opiate buprenorphine [11]. Other animal-specific factors to consider in dosage include sex, age, and weight [4]. Failure to account for discrepancies based on strains could lead to the experience of greater pain, which is both morally and scientifically problematic. 

The delivery mechanism of the anesthetic is also important. Inhaled anesthetics have quick uptake and can easily be regulated throughout a procedure [4]. However, inhaled anesthetics do not offer pain relief past the duration of administration, so their use could cause the animal to experience unnecessary pain if not paired with an analgesic. Injectable anesthetics have risks associated with a greater likelihood of fatal overdosages. Therefore, some procedures use a combination of various drugs to optimize pain relief and mitigate the risk of adverse side effects [4]. It is important for each factor that could potentially influence the efficacy of the anesthetic to be considered during experimental design to prevent unnecessary harm or undesirable results.

Stress and pain

Anesthetics and analgesics are primarily used to reduce physical pain, but psychological distress has also been shown to influence the pain response. Stress causes the release of aptly named “stress” hormones such as cortisol in humans and corticosterone in rodents [12]. These hormones work to increase emotional thinking while suppressing higher levels of cognitive function [12]. This is important to take into account during studies using animal models because altered cognitive processes may affect behavior. 

One study investigating the role of stress in pain sensitivity evaluated how a stressor affected the perception of pain among rats with and without endometriosis [13] [14]. Rats were placed on a small plot of plastic in the middle of a tub of water for a given amount of time for several days, and then made to undergo a hot plate test in which researchers recorded the amount of time it took the rat to withdraw its paw from a hot plate. Interestingly, rats with endometriosis exhibited an inhibition in the speed at which they withdrew their paws, suggesting they perceived less pain. Even further, the surgical procedure that induced endometriosis itself seemed to increase pain sensitivity [14]. Overall, the stress-induced reduction in pain sensitivity further demonstrates the potential for stressors to impact results.

Housing regulations

Another potential factor impacting animal behavior in research is their housing conditions. IACUCs often mandate the use of minimally equipped housing environments to reduce the potential for lab-to-lab variation [15]. However, new research delving into the effects of housing enrichment on animal behavior suggests that lack of enrichment may unintentionally influence the behavior as well. A recent study investigated the effects of an enriched housing environment on PTSD-like behavior in adult male rats [16]. Compared to rats raised in standard living environments, rats placed in a cage with toys such as rat wheels and play tubes for six hours a day exhibited increased locomotor activity but decreased fear-related behaviors such as freezing in response to a fear conditioned test. The rats raised with environmental enrichment also had a greater ratio of histone acetyltransferases (HATs) to histone deacetylases (HDACs) in the hippocampus, a region of the brain essential for memory processing, and a decreased ratio in the amygdala, which plays a major role in the fear response. HATs and HDACs help increase and decrease gene expression, respectively, so these results suggest the modulation of gene expression may be responsible for decreased fear behaviors [16]. A similar study indicated reduced anxiety-like behaviors in males and depressive-like behaviors in females [17]. However, both studies used methods such as fear based conditioning to measure anxiety and forced swimming tests to measure depression [17]. Whether or not fear-based tests are the most effective for investigating anxiety and depression is controversial. Forced swim tests, for example, do not directly measure depression, but rather infer that the amount of time a rodent swims serves as a proxy for their motivation to live. 

Some studies have investigated the benefits of enriched housing on cognitive performance. A meta analysis of rodent models of dementia indicated decreased cognitive impairment and anxiety for rodents reared in enriched environments [18]. Additional research should delve further into the relationship between different types of housing enrichment and rodent behavior.

Non-rodent models

Although rodents constitute as much as 90% of animals used for medical research, other animal models include fish, insects, roundworms, frogs, cats, pigs, dogs, and even primates [19]. Each species has specific IACUC regulations that do not always reflect how they may experience pain or distress.

Cephalopods, a group of marine invertebrates with advanced cognitive function, have been particularly popular in the field of neuroscience because of their highly developed nervous systems and their ability to adapt to environmental stressors [20]. Their unusually high cognitive function has led to the implementation of research regulations similar to those for vertebrates. However, much of the research on mechanisms of pain and distress has been performed using vertebrate models, and those done on invertebrate models have largely focused on octopi and squid [21]. One study investigated the effects of exposing cuttlefish, a species of cephalopods, to acetic acid. This noxious stimulus led to an increase in grooming behaviors believed to be indicative of pain, which was mitigated by injection of the analgesic Lidocaine. This study demonstrates the importance of improving our understanding of the pain and stress response in all species used in animal research because their behaviors often differ [21].

Zebrafish are another common species used for neurological research due to how similar their neural circuitry is compared to humans [22]. At the moment, zebrafish remain unprotected in Europe until they are six days old and are able to feed themselves [23]. Young zebrafish are translucent, making it easier to visualize the nervous system through their skin. However, recent research has suggested even larval zebrafish experience anxiety and fear that may modulate their response to pain [23]. A study of 5-day-old zebrafish evaluated the behavior of the fish in response to exposure to acetic acid, a noxious stimulus known to activate the nociceptors responsible for the pain sensation. The researchers compared how the response to acetic acid was affected by the stressor of brief air exposure. Zebrafish typically respond to such stimuli by swimming away. Interestingly, fish exposed to air after acetic acid had a decreased swimming velocity and length of swimming time when the noxious stimuli were introduced, suggesting the stressor interfered with their pain response to the acetic acid itself. Administration of an anesthetic drug known to reduce the stress hormone cortisol prevented this inhibited response. This finding demonstrates the need for further research investigating the role of fear and anxiety in response to pain, especially with regard to different species, and may implicate the need for significant revisions of IACUC guidelines [23]. 

Social observation of pain

Other studies have also investigated the role of social observation of pain on the behavior of animals. In one rat study, the group of rats that watched other rats receive a foot shock exhibited increased freezing behavior and increased activation of specific neurons in the anterior cingulate cortex (ACC), which integrates and helps respond to emotions. Interestingly, there was increased activation when the rats observed the direct experience of pain but not when they observed another rat experiencing fear [24]. Another study analyzed the relationship between mirror neuron activation in the ACC and insular cortex (InC) with prosocial behavior, namely the rescuing of a fellow mouse from an enclosed cage that could only be opened on the outside [25]. The InC was included in the study because of its known role in empathy in humans, and empathetic-like behavior, often termed social affective behavior due to the effect of social observation on the action of the observer, in other animals. Compared to the trials in which a toy was “stuck” inside the enclosure, mice were significantly more likely to rescue a fellow mouse and also exhibited increased activation of mirror neurons [25]. Such empathy-like behavior has also been observed in rats [26]. Results from these mirror neuron tests suggest that observation of painful experiences could increase the distress of the observer itself. 

Rats have also been shown to express social affective behavior in response to distress. A study evaluating the behavior of adult female rats when allowed to interact with either undisturbed or distressed rats demonstrated increased interaction with distressed juvenile rats and increased avoidance of distressed adults [27]. However, suppression of the insula and basolateral amygdala, which play roles in social cognition, eliminated differential preferences for stressed or undisturbed mice, suggesting these regions are critical for the observed empathetic response [27]. This study implies that even if an unpleasant procedure is performed with an animal isolated from its littermates, it could very well influence the behavior of the other animals when it returns. Some studies even rear littermates in social isolation for parts of the experiment, and evidence suggests this can disrupt defensive behaviors and decrease the ability for social recognition among littermates [28]. Humans are not the only species dependent on social interactions for healthy development. 

Future directions

Pain is difficult to quantify, especially when pain is experienced differently not only between species, but also between individuals of the same species [7]. However, consistent measurement and monitoring of pain and distress in animal models is necessary to ensure both the welfare of the animals themselves and also accurate results. Although not yet applied to lab settings, recent research has explored the potential to measure both internal and external states to more comprehensively understand how an animal feels at a given moment [29]. A team of researchers in Japan tested a system that integrated behavioral information and internal recordings such as heart rate and respiratory rate in pet hamsters [29]. Behavioral information was obtained by using video analysis to calculate center of mass, movement, body elongation, and shrinking, all of which can help indicate whether an animal is comfortable or experiencing stress. Although the hamsters were kept in a controlled and low-stress environment for the majority of the experiment, one day involved the introduction of a clapping sound to observe how the hamsters would react. The clapping sound produced short-term behavioral alteration, with the hamsters often changing direction and experiencing reductions in heart rate. Even though the video recording was capable of detecting this behavioral anomaly, there was considerable behavioral variation throughout the experiment in general [29]. Future research should investigate ways to more accurately discern between behavioral states. Regardless, this study suggests video analysis may be helpful in monitoring the well-being of animals and may be able to be applied to research using animals in the future.

Currently, there is substantial variation in the protocols and guidelines each lab or agency uses to mitigate the distress of animal subjects. As human beings, we tend to wish to provide the best care possible for the animals we work with, but without a comprehensive understanding of how pain and distress affects the wellbeing and behavior of animals, it is difficult to accomplish this. Ultimately, research into the impact of stress and pain on behavior suggests the need to revise and perhaps even standardize IACUC protocols. A revised system could further regulate procedures that induce stress and provide more comprehensive guidance on how to measure stress and anxiety. Even further, although labs tend to avoid unwanted variation by raising rodents in bare environments, it may be worth considering how lifestyle enrichment could increase not only the wellbeing and contentedness of the animal subjects, but the applicability of the results to real-life scenarios of people as well.

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