The Hunt for Why You Hurt – Improvements Needed in Animal Research of Chronic Pain

/ lionstalkscience

By Corinne Augusto

Chronic pain can develop after tissue or nerve injury, yet the mechanisms underlying the chronification of acute pain continue to elude researchers and clinicians alike. Patients with chronic pain can have their lives upended by their pain, and are susceptible to comorbidities like depression1–3 and opioid use disorder.4 Their pain may be disabling,5 hindering their ability to work or do basic tasks of living for themselves and their families. Despite 50 million U.S. adults having chronic pain,5 we still can’t predict who will develop chronic pain. Why does one person who fractures a rib recover completely, while someone else with the same injury feels the pain linger on?

To tackle this biological mystery, scientists are on the hunt for biomarkers – physiological “fingerprints” – that could predict a person’s susceptibility to chronic pain (“prognostic biomarkers”), or that could provide objective evidence of chronic pain (“diagnostic biomarkers”).6 Critical improvements in the methods of how we conduct animal research will improve this hunt.

 To improve human health, we must understand human disease. To understand human disease, we must accurately model disease.

While controversial, rodent research remains an invaluable tool in biomedical research, allowing us to ask questions and explore answers that aren’t feasible or ethical in human participants. Over the years, numerous rodent models of chronic pain have been developed. Models of nerve damage, such as streptozotocin (STZ) injection7 for diabetic neuropathy or the Chronic Constriction Injury (CCI)8 for traumatic neuropathy, are commonly used due to their unremitting, stable, and long-lasting pain induction. These nerve damage models reproduce the symptoms characteristic of human nerve damage, such as allodynia (the perception of innocuous stimuli as painful) and hyperalgesia (an increased sensitivity to painful stimuli), as well as depression- and anxiety-like symptoms.9–11 However, there is an important limitation to any animal model of chronic pain: the detection of the ongoing pain – critical to proving that the animal model is successful – is overly reliant on a small handful of behavioral measures.

Namely, the success of animal chronic pain models is usually demonstrated through evoked pain behavior testing. Evoked pain behavior testing relies on reflexive responses to stimuli, usually by stimulating the hind paws or the tail with devices such as Von Frey filaments12 or a Hot Plate.13 While these tests are crucial to confirming the presence of existing pain, they do not capture the full experience of chronic pain. While preclinical models of chronic pain cannot recapitulate economic hardship or social stigma, creative, new methods can capture secondary symptoms of chronic pain and examine physiological changes over time more cheaply than clinical studies.

Improvement #1: Use clinical research techniques in animal models of chronic pain.

Exploring beyond the patient-reported experience of chronic pain, clinical researchers have used non-behavioral, minimally invasive techniques like neuroimaging and blood collection to find objective physiological biomarkers of chronic pain. Human functional magnetic resonance imaging (fMRI) studies have repeatedly shown that both brain activity14,15 and functional connectivity at cognitive rest16–20 are altered in patients with chronic pain compared to healthy individuals. Additionally, several human studies have shown that as pain chronifies, the brain activity in response to rating the ongoing spontaneous pain shifts from sensory circuits to emotional circuity.21,22 In other words, the chronification of pain neurobiologically is pain shifting from a new sensory experience to a constant burden of suffering.

In contrast, there are very few preclinical chronic pain neuroimaging studies, particularly in rodents. While preclinical fMRI is an expensive technique compared to traditional evoked behavioral testing for pain detection, the translational utility of pre- and post-injury scans is currently undervalued. Animal models can create specific, reproducible injuries that often result in chronic pain. The same animal can be scanned prior to any injury, and for multiple post-injury timepoints, for a fraction of the cost of scanning a human patient longitudinally. A preclinical neuroimaging biomarker causally linked to the development of chronic pain could provide a necessary, objective physiological underpinning to the “chronification” of acute pain. It is this part of the chronic pain experience – the time where acute pain gets stuck for some patients but resolves in others – that is difficult to understand mechanistically. The possibility of a non-invasive imaging “fingerprint” of chronic pain in neural connectivity is an exciting avenue of clinical research, and preclinical scientists should join the hunt with their animal models. The combined results of preclinical and clinical neuroimaging studies, each compensating for the unique deficits of the other, provide the best possible chance of finding clinically relevant neuroimaging biomarkers of chronic pain.  

Improvement #2: Utilize new equipment to explore complex behaviors in animals with chronic pain.

One exciting new method is spontaneous activity monitoring, using devices like the PhenoTyper (Noldus Information Technology).23 The PhenoTyper is a recording environment where rats or mice can live for days at a time. An embedded infrared camera in the ceiling captures all activity within the cage, sending this information to EthoVision software (Noldus Information Technology) for live behavioral tracking and analysis. EthoVision can characterize numerous behaviors over the course of the recording, including eating and drinking, general activity, and resting.24

Experimenters can set up the PhenoTyper environment to conduct specific behavioral tasks, or to simply record spontaneous, voluntary behavior. Importantly, the PhenoTyper can independently conduct this behavioral analysis, recording behaviors without an experimenter present in the room. This eliminates a large source of bias in behavioral pain testing. Unchangeable characteristics of an experimenter, such as the scent of their biological sex,25 can have unforeseen and unintended consequences on the behavioral output of the evoked pain behavior tests, like the Von Frey. By removing the experimenter, the PhenoTypers can classify behaviors without the interference of an experimenter’s scent or noise, allowing rodents to behave more naturally.

Using techniques like the PhenoTypers, chronic pain researchers can examine their animals’ “experience” of chronic pain beyond reflexive evoked pain responses. Researchers can calculate resting duration, walking speed, jumping frequency, and other activity variables as proxies of “disability” in rodents with chronic pain compared to control rodents. Paired with other metrics of movement quality (e.g., the CatWalkTM XT26,27), these variables could be relevant and comparable to the symptoms surveyed in human patients by such surveys as the McGill Pain Questionnaire,28 the Chronic Pain Grade Scale,29 the Brief Pain Inventory,30,31 or the Pain Disability Questionnaire.32

Also, by having the space for two fluid bottles, the PhenoTyper environment can be used to perform Sucrose Preference testing, a measure of rodent anhedonia (a key symptom of depression, wherein patients exhibit a loss of pleasure and interest in things that used to excite them).33 By pairing the PhenoTyper data with other tests of psychological distress (e.g., anxiety-like behavior with an Elevated Zero Maze34,35), researchers can characterize the broader emotional experience of chronic pain in their rodent models, capturing variables relevant to the human distress captured by surveys like the Pain Catastrophizing Scale.36 This new characterization could lead to improvements in our pain induction models, leading to more faithful models of chronic pain. Additionally, tests of new pain medications could employ spontaneous behavior tracking to monitor drug success beyond just reducing pain on evoked pain tests. Undesirable side effects could be easier to capture with multi-day, 24-hour monitoring.

Conclusion

While new bench technologies like organoids or “organs on a chip” may someday replace animal models of disease altogether, animals still provide the best in vivo, behavior-capable models of human disease. However, our techniques for investigating our animal models must continuously improve if we wish to uncover more mechanisms of disease and discover treatments for human patients. Extending our animal pain detection techniques beyond reflexive pain behaviors is vital to thoroughly investigating the disabling and distressing mystery of chronic pain.

TL; DR:

  • We are currently unable to predict who will develop chronic pain following an injury
  • Rodent animal models offer unique insights into how chronic pain progresses
  •  New equipment can monitor the behaviors of rodents for days at a time independent of a researcher, which can reduce bias

Reference

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