Uncontrolled pain remains a public health epidemic, with countless people suffering from a multitude of disorders that cost society billions of dollars annually. The American Pain Society recently released a statement indicating that translational approaches to pain relief (i.e., bench to bedside) would have "immediate and profound benefits" . Despite significant advances made to our understanding of molecular pain mechanisms, few novel analgesic therapies have managed to reach clinical practice . This failure to translate from bench to beside is in part due to the use of inefficient behavioral assays in animal models of pain. Many assays typically require that the investigator both apply the pain stimulus and evaluate a reflexive response by the animal. This can be both time consuming and highly subjective. The use of these assays to model and quantify pain in animals is a major bottle-neck in the development of new analgesics, and providing obstacles in the validation and optimization of clinical treatment strategies.
The use of mice as the preferred model for pain testing provides the opportunity to utilize genetically-altered (i.e., knock-out) strains to study specific targets related to pain processing. The drawback of behaviorally testing mice relates to difficulties in producing fast and reliable results using an animal known for being skittish and jumpy. In this study, we have overcome these traits using our operant test paradigm by presenting a system whereby the investigator is removed from the field and the animal tests itself. We build on our prior work completed in rat models of orofacial pain [17, 19, 27].
As part of our battery of behavioral assessment assays, we evaluate the effects of a variety of factors (e.g., drugs) on general activity to determine if these factors could have an impact on the ability of an animal to perform the operant task. For example, we previously demonstrated that doses of morphine (≥ 2.5 mg/kg) produced significant reduction of rearing behavior in rats . Another factor that may affect performance relates to inherent differences between strains of animals, as some strains may be relatively more active or inquisitive. Therefore, prior to testing the different mice strains (SKH1-Hrhr, C57BL/6J, TRPV1 k.o.) on the operant device, we evaluated them using the rearing assay. We found that the general activity for these three strains was not so much a factor, as they all had similar responses. In fact the rearing duration/event outcome was virtually identical for each strain by the end of two week acclimation period. We concluded that the general activity or exploratory behavior differences between these strains of mice would not likely influence their ability to complete the operant task. The rearing data indicates a potential habituation to the environment for all strains over the two-week test schedule, with each strain displaying decreased events and duration. However, in contrast, we found that the operant results improved with each session; indicating that motivation and cognitive factors are encouraging the animal to complete the task.
When we decided to modify the existing rat operant unit to test mice, we searched for a mouse that was equivalent to the hairless Sprague-Dawley (S.D.) rat that we routinely use and found that the hairless SKH1-Hrhr mouse strain. While the SKH1-Hrhr mouse is typically used in dermatological studies, it is not the typical strain used in pain studies. Here we demonstrated that this strain indeed follows a normal thermal stimulus response, as compared to the wild-type C57BL/6J mice. Additionally, they respond in the appropriate fashion following capsaicin-induced pain with decreased operant licking behavior, and then with morphine-rescue, with return of licking events to baseline levels in the presence of capsaicin. As demonstrated in Figure 3, they are quick to learn the task, therefore minimizing training time. We find this hairless strain to be extremely docile and easy to handle, more convenient to use, as we do not need to shave them in order to test them. The video clip provides a real-time demonstration of the ease of using these animals. Note how quickly the animals are independently and successfully completing the task once placed in the box. Given these traits, the SKH1-Hrhr mice are certainly appropriate and ideal for use in future pharmacological studies.
Trigeminal nociceptors project to the nucleus caudalis and synapse with second-order neurons in the superficial layers, which are organized in the same way as the dorsal horn of the spinal cord, with the nucleus caudalis being laminated like the dorsal horn in spinal cord [33–36]. In fact, the nucleus caudalis extends and merges with the spinal dorsal horn in the cervical spinal cord  and has been termed the medullary dorsal horn . Neurons within this superficial region have been shown to respond to cooling, cold, warming, and hot stimuli [39–42] and chemical irritants [42, 43]. As it is considered a correlate to the spinal cord dorsal horn, the trigeminal spinal nucleus and trigeminal sensory system provides a relevant region with respect to the understanding of pain mechanisms in general [44, 45]. Given this, we targeted this region using the ultrapotent TRPV1 agonist, RTX, in order to assess changes in orofacial pain.
We found that w.t. C57BL/6J mice treated with RTX had a significant decrease in the ability to sense hot (48°C) and very hot (55°C) stimulus temperatures. This contrasts the response of the TRPV1 k.o. mice, as they were only significantly affected at the hottest (55°C) temperature. These results appear to be inconsistent with the work completed by Caterina, et al., as they demonstrated a significant difference at high temperature stimuli, such as 55°C on the hot plate assay, when comparing the TRPV1 k.o. versus the w.t. mice . This is interesting because the thermal assay difference between the reflex and operant tasks may explain this discrepancy. For example, for the reflex-based hot-plate assay, the difference between the k.o. and w.t. groups may be a function of the wild-type mice being more sensitive to a rapid temperature increase, so the response may be a function of the temperature difference detection plus the thermal pain producing the response. In addition, when within-group effects were evaluated for temperature, there was a decreased latency for both the TRPV1 k.o. and w.t. mice. While these authors did not report on this comparison, there appears to be a significant decrease in hot plate latency in the TRPV1 k.o. group as the stimulus moves to higher temperatures. This is consistent with what we found using the operant test, with a significant decrease in licking outcome at the 55°C stimulus. Collectively, these results indicate that while RTX is specific for the TRPV1 receptor, other receptors co-expressed (e.g., TRPV2) on the same neurons with TRPV1 may be susceptible to the RTX-lesioning effects within the nucleus caudalis. Another possibility is that a population of A-δ fibers expressing TRPV1 is also lesioned following RTX-treatment in the w.t. mice.
When we evaluated the pain sensitivity of these mice using an unlearned-reflexive measure such as the capsaicin eye-wipe assay, the RTX-treated C57BL/6J mice appeared to respond identically as the TRPV1 k.o. mice. This brings up an interesting observation that this particular type of assay is sufficient for evaluating a gross-impairment in the nociceptive signaling pathway. However, it cannot tease out subtle differences uncovered using the operant assay, such as the different responses of these two groups at 55°C. This additional information may be relevant for the development of novel analgesics.
In conclusion, we have successfully used the operant orofacial assay to evaluate and characterize thermal pain sensitivity in mice, thus providing a revolutionary step in the ability to model and study pain. Pain is ultimately experienced as a culmination of complex information from the periphery (including the location, intensity, quality, and time course). While in vitro studies can provide insight into the individual components, adequate behavioral assessment of animal models is requisite for understanding the integration of these components into the perception of pain. These findings demonstrate that operant methods, which reflect physiological and cerebral processing of pain, can provide insights not possible with reflex-based testing alone. Such insight may enhance our understanding of pain and ultimately lead to the ability to treat uncontrollable. This replicates what occurs with humans, whereby a person may need to choose between tolerating pain in order to receive some reward (e.g., going to a nice dinner with a migraine headache versus not going to a bad restaurant with the same type of headache). As pain spans any number of diseases, ranging from diabetes to cancer, providing a means of quickly identifying new analgesic agents would provide a tremendous societal benefit, and this mouse operant system provides such a way.