The measurement of spontaneous ongoing pain in rodents is a multiplex issue and a subject of extensive and longstanding debate. Considering the need to align available rodent models with clinically ...relevant forms of pain, it is of prime importance to thoroughly characterize behavioral outcomes in rodents using a portfolio of measurements that are not only stimulus‐dependent but also encompass voluntary behavior in unrestrained animals. Moreover, the temporal course and duration of behavioral tests should be taken into consideration when we plan our studies to measure explicit chronic pain, with a particular emphasis on performing longitudinal studies in rodents. While using rodents as model organisms, it is also worth considering their circadian rhythm and the influence of the test conditions on the behavioral results, which are affected by social paradigms, stress and anxiety. In humans, general wellbeing is closely related to pain perception, which also makes it necessary in rodents to consider modulators as well as readouts of overall wellbeing. Optimizing the above parameters in study design and the new developments that are forthcoming to test the affective motivational components of pain hold promise in solving inconsistencies across studies and improving their broad applicability in translational research. In this review, we critically discuss a variety of behavioral tests that have been developed and reported in recent years, attempt to weigh their benefits and potential limitations, and discuss key requirements and challenges that lie ahead in measuring ongoing pain in rodent models.
Spontaneous ongoing pain tests in rodents can be classified into two groups of ‘non‐reflexive measures’ and ‘free‐choice tests’. The figure summarizes the current available tests and measurements to monitor behavioral changes that are discussed in this review and are under extensive and longstanding debate.
•Translational research requires the use of relevant preclinical pain models and assessment tools.•Clinically relevant behavioral tests should focus on restoring function by reducing pathological ...pain in the absence of disruptive side effects.•Grimace, Operant Behavior, Wheel Running, Burrowing, Nesting, Home Cage Monitoring, Gait Analysis, CPP and CPA are described.•Analysis of the advantages and limitations of these tests will help researchers identify appropriate tests for their particular goal.
The primary objective of preclinical pain research is to improve the treatment of pain. Decades of research using pain-evoked tests has revealed much about mechanisms but failed to deliver new treatments. Evoked pain-tests are often limited because they ignore spontaneous pain and motor or disruptive side effects confound interpretation of results. New tests have been developed to focus more closely on clinical goals such as reducing pathological pain and restoring function. The objective of this review is to describe and discuss several of these tests. We focus on: Grimace Scale, Operant Behavior, Wheel Running, Burrowing, Nesting, Home Cage Monitoring, Gait Analysis and Conditioned Place Preference/ Aversion. A brief description of each method is presented along with an analysis of the advantages and limitations. The pros and cons of each test will help researchers identify the assessment tool most appropriate to meet their particular objective to assess pain in rodents. These tests provide another tool to unravel the mechanisms underlying chronic pain and help overcome the translational gap in drug development.
Background
There is an urgent need to develop and incorporate novel behavioral tests in classically used preclinical pain models. Most rodent studies are based upon stimulus-evoked hindpaw ...measurements even though chronic pain is usually a day and night experience. Chronic pain is indeed a debilitating condition that influences the sociability and the ability for voluntary tasks, but the relevant behavioral readouts for these aspects are mostly under-represented in the literature. Moreover, we lack standardization in most behavioral paradigms to guarantee reproducibility and ensure adequate discussion between different studies. This concerns not only the combination, application, and duration of particular behavioral tasks but also the effects of different housing conditions implicating social isolation.
Results
Our aim was to thoroughly characterize the classically used spared nerve injury model for 12 weeks following surgery. We used a portfolio of classical stimulus-evoked response measurements, detailed gait analysis with two different measuring systems (Dynamic weight bearing (DWB) system and CatWalk), as well as observer-independent voluntary wheel running and home cage monitoring (Laboras system). Additionally, we analyzed the effects of social isolation in all behavioral tasks. We found that evoked hypersensitivity temporally matched changes in static gait parameters, whereas some dynamic gait parameters were changed in a time-dependent manner. Interestingly, voluntary wheel running behavior was not affected in spared nerve injury mice but by social isolation. Besides a reduced climbing activity, spared nerve injury mice did not showed tremendous alterations in the home cage activity.
Conclusion
This is the first longitudinal study providing detailed insights into various voluntary behavioral parameters related to pain and highlights the importance of social environment on spontaneous non-evoked behaviors in a mouse model of chronic neuropathy. Our results provide fundamental considerations for future experimental planning and discussion of pain-related behavioral changes.
Background
Chronic pain is often accompanied by comorbidities like anxiety and depression. The temporal correlations, as well as the underlying mechanisms of these reciprocal correlations, are ...unclear. Moreover, preclinical studies examining emotional behaviour are very controversial, and a chronological analysis of anxiety‐like behaviour in mouse pain models considering both genders has not been performed so far.
Methods
We used several behavioural tests to assess and validate anxiety‐like behaviour in complete Freund's adjuvant (CFA) and spared nerve injury (SNI) pain models in C57BL/6 mice. Among these were the elevated plus maze test, open field test, hole‐board test and light‐dark test. Additionally, we included a late stage analysis of depression‐like behaviour using the forced swim test. All tests were applied once for each cohort of mice. Importantly, we used C57BL/6N mice of both genders; we investigated the effect of social isolation, the impact of pain induction to either the right or left hind limb and also investigated C57BL/6J mice.
Results
The validity of test conditions was confirmed using the anxiogenic drugs Yohimbine and Pentylenetetrazol. Anxiety‐like behaviour was analysed throughout the time period when mice exhibited hypersensitivity to mechanical stimuli. We did not observe any consistent alteration in anxiety‐like behaviour at any of the investigated time points between 1 and 14 days following CFA‐induced inflammation or 3 and 84 days following SNI surgery using different behavioural tests.
Conclusions
Inflammatory and neuropathic pain conditions do not primarily evoke anxiety‐ and depression‐like behavioural alterations within the herein investigated time period.
Significance
Anxiety‐like behaviour is not primarily altered following CFA and SNI in C57BL6 mice, irrespective of the gender, mouse sub‐strain, housing conditions or affected body side within the herein investigated time period.
Painful neuropathy is a frequent complication in diabetes. Proopiomelanocortin (POMC) is an endogenous opioid precursor peptide, which plays a protective role against pain. Here, we report ...dysfunctional POMC-mediated antinociception in sensory neurons in diabetes. In streptozotocin-induced diabetic mice the Pomc promoter is repressed due to increased binding of NF-kB p50 subunit, leading to a loss in basal POMC level in peripheral nerves. Decreased POMC levels are also observed in peripheral nervous system tissue from diabetic patients. The antinociceptive pathway mediated by POMC is further impaired due to lysosomal degradation of μ-opioid receptor (MOR). Importantly, the neuropathic phenotype of the diabetic mice is rescued upon viral overexpression of POMC and MOR in the sensory ganglia. This study identifies an antinociceptive mechanism in the sensory ganglia that paves a way for a potential therapy for diabetic neuropathic pain.
Rodent behavior is affected by different environmental conditions. These do not only comprise experimental and housing conditions but also familiarization with the experimenter. However, specific ...effects on pain-related behavior and chronic pain conditions have not been examined. Therefore, we aimed to investigate the impact of different housing conditions, using individually ventilated and standard open top cages, inverted day-night cycles, and experimenter familiarization on male mice following peripheral neuropathy using the spared nerve injury (SNI) model. Using a multimodal approach, we evaluated evoked pain-related- using von Frey hair filaments, measured gait pattern with the CatWalk system, assessed anxiety- and depression-like behavior with the Elevated plus maze and tail suspension test, measured corticosterone metabolite levels in feces and utilized an integrative approach for relative-severity-assessment. Mechanical sensitivity differed between the cage systems and experimenter familiarization and was affected in both sham and SNI mice. Experimenter familiarization and an inverted day-night cycle reduced mechanical hypersensitivity in SNI and sham mice. SNI mice of the inverted day-night group displayed the slightest pronounced alterations in gait pattern in the Catwalk test. Anxiety-related behavior was only found in SNI mice of experimenter-familiarized mice compared to the sham controls. In addition, familiarization reduced the stress level measured by fecal corticosteroid metabolites caused by the pain and the behavioral tests. Although no environmental condition significantly modulated the severity in SNI mice, it influenced pain-affected phenotypes and is, therefore, crucial for designing and interpreting preclinical pain studies. Moreover, environmental conditions should be considered more in the reporting guidelines, described in more detail, and discussed as a potential influence on pain phenotypes.
Below-level central neuropathic pain (CNP) affects a large proportion of spinal cord injured individuals. To better define the dynamic changes of the spinal cord neural network contributing to the ...development of CNP after spinal cord injury (SCI), we characterized the morphological and behavioral correlates of CNP in female C57BL/6 mice after a moderate T11 contusion SCI (50 kdyn) and the influence of moderate physical activity. Compared with sham-operated animals, injured mice developed mechanical allodynia 2 weeks post injury when tested with small-diameter von Frey hair filaments (0.16 g and 0.4 g filament), but presented hyporesponsiveness to noxious mechanical stimuli (1.4 g filament). The mechano-sensory alterations lasted up to 35 days post injury, the longest time point examined. The response latency to heat stimuli already decreased significantly 10 days post injury reaching a plateau 2 weeks later. In contrast, injured mice developed remarkable hyposensitivity to cold stimuli. Animals that underwent moderate treadmill training (2 × 15 minutes; 5 d/wk) showed a significant reduction in the response rate to light mechanical stimuli as early as 6 days after training. Calcitonin gene-related peptide (CGRP) labeling in lamina III-IV of the dorsal horn revealed significant increases in CGRP-labeling density in injured animals compared with sham control animals. Importantly, treadmill training reduced CGRP-labeling density by about 50% (P < 0.01), partially reducing the injury-induced increases. Analysis of IB4-labeled nonpeptidergic sensory fibers revealed no differences between experimental groups. Abnormalities in temperature sensation were not influenced by physical activity. Thus, treadmill training partially resolves signs of below-level CNP after SCI and modulates the density of CGRP-labeled fibers.
Deep brain temperature detection by hypothalamic warm-sensitive neurons (WSNs) has been proposed to provide feedback information relevant for thermoregulation. WSNs increase their action potential ...firing rates upon warming, a property that has been presumed to rely on the composition of thermosensitive ion channels within WSNs. Here, we describe a synaptic mechanism that regulates temperature sensitivity of preoptic WSNs and body temperature. Experimentally induced warming of the mouse hypothalamic preoptic area in vivo triggers body cooling. TRPM2 ion channels facilitate this homeostatic response and, at the cellular level, enhance temperature responses of WSNs, thereby linking WSN function with thermoregulation for the first time. Rather than acting within WSNs, we—unexpectedly—find TRPM2 to temperature-dependently increase synaptic drive onto WSNs by disinhibition. Our data emphasize a network-based interoceptive paradigm that likely plays a key role in encoding body temperature and that may facilitate integration of diverse inputs into thermoregulatory pathways.
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•Temperature in the hypothalamic preoptic area (POA) tracks body temperature in mice•TRPM2 participates in warmth detection in the POA in vivo•Presynaptic TRPM2 detects temperature increases to activate warm-sensitive neurons•Warming enhances inhibitory transmitter release via TRPM2 to mediate disinhibition
Kamm, Boffi, et al. found that the ion channel TRPM2 mediates detection of warm temperatures in the mouse hypothalamus. Specifically, TRPM2 enhances the thermosensitivity of hypothalamic warm-sensitive neurons (WSNs) via a presynaptic mechanism. This study highlights the role of WSNs and TRPM2 in body temperature homeostasis under hot environments.
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