OroFacial Pain Assessment Device (OPAD)

The Orofacial Pain Assessment Device (OPAD) was developed by Neubert and colleagues (2005) as an operant system of pain assessment that relies on voluntary behavior. Popular pain batteries offer a unidimensional assessment of pain. Pain responses involve executive functioning and other experiences, thus, relying simply on reflex and innate responses do not provide the complete picture. Further, pain management drugs can have sedative effects and may also affect psychomotor abilities in addition to providing pain relief. Therefore, a conflict-based paradigm proves to be a more sensitive method that enables in-depth analysis of pain.


The OPAD system is composed of Peltier-based thermode, and metal wires that allow assessment of thermal and mechanical pain sensitivity. The starved animal is placed in the operant chamber where it can access the food reward only when it contacts the thermal and mechanical stimuli. Essentially, the subject is tasked with choosing to tolerate the pain to gain the reward. Unlike traditional pain assays that are typically based on reflex behaviors, the OPAD allows the subject to choose its own pain threshold in order to attain the reward. This reward-conflict paradigm offers better face, content, and predictive validity. Additionally, the OPAD also encompasses psychological and affective dimensions of pain as observed in humans.

Other pain assessment devices include the Electric von Frey Filament, the Tail Flick Test, and the Hargreaves Plantar Test. (For more click here).


  • The OPAD consists of a clear acrylic cage with a metal base
  • Cage dimension: Mouse 10 x 10 x 20 cm (L x W x H), Rat 20 x 20 x 20 cm (L x W x H). Custom dimension is available
  • Dual Peltier-controlled temperature thermodes or mechanical protrusions
  • Feces and urine tray – removable for feces and urine removal
  • White Noise
    • Manual start / stop white noise as ambient noise
    • Mechanical Stimulus
      • Two rows of nickel-titanium wires (Diameters of 0.010” and 0.007”) were added to the opening to the
      • reward
        Conduct-OPAD Software
        • The software provides the front end for user to configure protocols and run experiments
        • Up to 16 cages simultaneously with a single computer and an instance of the software. Historical data are saved
        • The software allows users to configure protocols. For example setup up the multi-step temperature (target temperature, ramp duration, target temperature duration)
        • Data to collect:
        • Lick: number of licks, duration of each lick, latency to the first lick, longest lick, shortest licks, frequency of licks
        • Contact: number of contacts, duration of each contact, latency to the first contact, longest contact, shortest contact.
        • Ratio: The lick/face ratio (L/F: reward licking events divided by the number of stimulus contacts. Each time there is a licking event contact is being made) is a measure of nociception on the OPAD
        • Reward
          • One Maze Engineers’ reward bottle having a metal spout and lick detection
          • Access to the reward is possible only with simultaneous contact with the thermodes on the animal’s buccal region
          • (Optional) Measure the reward intake in gram
          • Thermal Stimulus
            • Distance between the thermodes can be varied to accommodate the size of the animal.
            • Temperature range from lab room temperature (say 25°C) to 75°C
            • Take advantage of Neuralynx, Ethovision Integration, SMS and Email integration with the Conductor Science Software. No I/O Boxes Required

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              Price & Dimensions


              $ 5900

              Per Month



              2.1 Origin

              Most pain assays used in preclinical research rely on simple reflex or innate behaviors to measure pain. These methods, though simple and objective, do not take into consideration other factors associated with pain behavior. Neubert et al. (2005) aimed to develop a system that would allow a better assessment of pain and associated behaviors. The Orofacial Pain Assessment Device was designed to provide the subject with a conflict to either experience the pain to gain reward or to avoid the aversive stimuli and gain no reward. This system enabled the observation of behaviors that involved different levels of the nervous system. Thus, the OPAD assay improves the translatability of the observation for human applications.

              Neubert, Rossi, Malphurs, Vierck, and Caudle’s (2006) investigation revealed that at temperatures 42° C and 45° C significant allodynic and hyperalgesic effects induced by capsaicin could be observed. Another study by Rossi, Vierck, Caudle, and Neubert (2006) employed the OPAD to investigate the cold sensitivity of male and female rats. When subjects were evaluated at above freezing temperatures of 24° C, 10° C, and 2°C, the difference in behavioral performances of males and females could be observed. However, statistical significance was reached only at 10° C temperature. Males were observed to lick less often and consume less milk in comparison to the females.

              Since the OPAD relies on affective responses, Rossi and Neubert (2008) used it to evaluate the effects of environmental enrichment on thermal sensitivity. When 7-week-old male hairless Sprague-Dawley rats were maintained in an enriched environment that included objects like cardboard boxes, hammock, exercise wheel, and other enrichments, an effect on thermal sensitivity could be seen. In comparison to standard cage-maintained rats, enriched rats showed better performance in the OPAD at temperature 2° C, 24° C, and 45°C. However, though enriched environment can potentially be effective in improving tolerance, the effects do not occur at strongly aversive or pain threshold temperatures.

              2.2 Developments

              The initial design of the OPAD only employed thermal stimulations. Nolan, Hester, Bokrand-Donatelli, Caudle, and Neubert (2011), further adapted the OPAD to include nickel-titanium wires to act as mechanical pain stimuli. The wires also conducted electricity, thereby, allowing the experimenters to detect and record actual facial contacts. This addition of a removable mechanical stimulation allowed experimenters to evaluate the pain response to different combinations of stimuli.

              Pain relief in humans is influenced by transient receptor potential (TRP) cation channels involved in the perception of hot and cold pain. TRPV1 and TRPM8/TRPA1 were altered by capsaicin and menthol administration by Anderson, Jenkins, Caudle, and Neubert (2014) to investigate the effects of these channels on nociceptive behaviors. OPAD performance observations showed that the co-activation of TRPM8/TRPA1 and TRPV1 made cool temperatures less nociceptive.

              Ramirez’s et al. (2015) investigation looked into doses of analgesic that cause minimal trauma in laboratory animals. For their investigation, buprenorphine efficacy was evaluated in male and female Sprague-Dawley rats. Subjects were treated with either 0.01 mg/kg or 0.005mg/kg doses of buprenorphine before being subjected to either capsaicin cream or surgical incision-induced pain in the check region. The two pain models did not differ much from each other. OPAD performances showed that only at dose 0.03 mg/kg of buprenorphine resulted in antinociceptive effects in females, while the effects could be observed at both doses in males.

              2.3 Recent Developments

              Nag and Mokha (2016) tested the performances of male and ovariectomized (OVX) Sprague-Dawley rat in the OPAD. The investigation aimed to understand the effects of the activation of the trigeminal α2-adrenoceptor on nociception and hyperalgesia. Clonidine treated (0.875 or 1.75 μg/5 μl) subjects showed a dose-dependent effect on antinociception. A similar effect was also observed in carrageenan treated groups that were administered clonidine.  However, the effects of clonidine were attenuated in 17β-estradiol 3-benzoate administered OVX group. The investigation was able to highlight the sex-dependent antinociception and antihyperalgesia effects of α2-adrenoceptor. Further, the effects of estrogen on pain control were also shown suggesting the need for sex-specific drugs treatments.

              Effect of diets on pain control was evaluated by Bowden et al. (2017). The study involved assessment of cocoa flavanols on orofacial pain of male and female Sprague-Dawley rats. It was observed that the subjects that were maintained on cocoa-enriched diets had better pain thresholds when evaluated in the OPAD. The observations led to the suggestion that diet modifications can be potentially used for pain management.

              Apparatus & Equipment

              The OPAD consists of a clear acrylic cage with a metal base. The chamber base has an additional metal grate. On one side of the chamber, an opening to the reward feeder is lined with a pair of Peltier-based thermode and nickel-titanium wires to provide thermal stimulations and mechanical stimulations to the orofacial region, respectively. The distance between the thermodes can be varied to accommodate the size of the animal. Further, the temperature (4° to 75°C temperature range) and experimental parameters of the set-up can be controlled and managed using the associated software and the LCD screen. The apparatus is equipped with a reward bottle having a metal spout. The access to the reward is possible only with simultaneous contact with the thermode or mechanical protrusions. The device allows automated data recording.

              Training Protocol

              Clean the Orofacial Pain Assessment Device thoroughly before and after every use and between subjects. Appropriately illuminate the arena. Ensure that the presence of unnecessary external stimuli is minimized. In addition to the OPAD system’s data acquisition, tracking and video system such as the Noldus EthoVision XT can also be used.


              Fast the subjects for an appropriate period before training in the OPAD. Place the subject in the chamber and set the thermodes to a non-aversive temperature. Allow the subjects enough time to explore the apparatus. Perform at least 3 trials per week for 2 weeks or until consistent behavior is observed.

              Operant procedures are best applied to hairless rodents. In case the rodents have facial hair, it is highly recommended that appropriate methods are used to remove it. Ensure that only the buccal hair and not vibrissae pad/whiskers are removed to avoid affecting the subject’s behavior. Perform the hair removal procedure 1 to 2 days prior to the testing for accurate results. Apply the pain treatments before the testing.

              Orofacial Pain Assessment

              Fill the reward liquid in the reward bottle. Place the reward bottle on the stand such that the subject must make contact with the thermodes on its buccal region to be able to lick the spout. Place the subject in the chamber. Adjust the position of the reward bottle further if the subject’s vibrissa touches it, or the buccal regions do not touch the thermodes. Set the thermodes to the desired temperature and begin the trial set to a predetermined trial length. Repeat the trials as required.


              The initial Operant Pain Assessment Device (Neubert et al., 2005) was designed only to assess thermal sensitivity. The device involved a pair of thermodes which were constructed using grounded aluminum tubing lining the opening to the reward feeder. Thermal stimulations were induced by pumping water heated or cooled to the required stimulus temperature via flexible polyethylene tubing.


              Nolan et al. (2011) modified the OPAD design to include a mechanical component. The mechanical component allowed the opportunity to assess mechanical allodynia, hyperalgesia, and pain in the orofacial region using a single device. Two rows of nickel-titanium wires were added to the opening to the reward. The investigators tested the effects of varying the diameters (0.010” and 0.007”) of the wires on the performances. Baseline performances at 37° C and 48° C showed that the thicker wire significantly decreased licking/facial contact ratio in comparison to the 0.007” diameter wire. The effects of the thickness of the wire were further assessed after capsaicin and morphine treatments. The thinner wire did not prove to be aversive with performances after the treatments being comparable to baseline performances at 37° C stimulus. Though capsaicin treatment did not significantly decrease the licking/facial contact ratio, morphine treatment led to significantly increased licking/facial contact ratio compared to baseline when subjects were tested with the 0.010” wire.


              The mechanical component of the OPAD was further modified by Rohrs et al. (2015).  Instead of using rows of nickel-titanium wires, they made use of a 360-degree array of 0.010” diameter looped stainless steel wires. Additionally, the subject was forced to fully engage with the mechanical stimuli by moving the reward bottle further away as it continued to move its head through the stimuli. Thus, the animal is subjected to increasing intensities of mechanical stimulations. This adaption was intended only effectively to assess mechanical sensitivity by minimizing animal avoidance behavior.

              Literature Review

              Investigation of the analgesic effects of hangeshashinto on oral ulcer-induced pain

              Hitomi et al. (2016) investigated the analgesic effects of hangeshashinto, a traditional Japanese medicine, using male Sprague-Dawley rats. Oral ulcers were created by treating anesthetized subjects with 50% acetic acid-soaked filter paper placed in the labial fornix region of the inferior incisors for 30 seconds. Subjects received topical application of hangeshashinto or a control solution for 5 minutes before being subjected to the OPAD. The temperature cycles in the OPAD started from 33° C for 3 minutes to 45° C for 4 minutes followed by temperature ramping for 30 seconds and again returning to 33° C for 2 minutes followed by temperature ramping for 30 seconds. A significant increase in the licking/contact level in comparison to pre-acetic acid treatment could be observed in the hangeshashinto group when compared to controls.


              Investigation of placebo-induced analgesia

              Nolan, Price, Caudle, Murphy, and Neubert (2012) utilized a rat model of conditioned analgesia to evaluate the different aspects of placebo-induced analgesia using the OPAD. The OPAD sessions lasted 20 minutes and were separated by 48 hours. Hairless male Sprague-Dawley rats were treated with 1 mg/kg morphine (MOR) or phosphate buffered saline (PBS) before being subjected to two sessions with the thermode set to 48 °C. Following the two sessions, the subjects were once again tested in the OPAD with the groups receiving either PBS or naloxone hydrochloride dihydrate (NXL, 5 mg/kg) injections. Drug administration was always performed 30 minutes prior to exposure to OPAD. Analysis of the performances revealed that morphine-treated animals showed a placebo effect when evaluated with PBS administration. Further, the morphine-treated group on evaluation with naloxone showed slightly less response in comparison to PBS only treated group. Thus, suggesting a reversal effect on placebo resulting response enhancement.

              Characterization of bilateral trigeminal constriction injury

              Rossi et al. (2013) assessed the neuropathic pain resulting from bilateral trigeminal constriction injury in the OPAD. Hairless Sprague-Dawley rats either received bilateral chronic constriction injury (CCI) or sham operation of the infraorbital portion of the trigeminal nerve. Following injury and habituation, subjects were evaluated at 10° C, 37° C, and 48° C thermode temperatures in 20-minute sessions based on one of the following weekly schedules: three times at 10° C and once at 37° C; twice at 10° C, once at 37° C and 48° C; or once each at 10° C, 37° C, and 48° C. The CCI-treated group showed nocifensive behaviors such as upward head-tilt which was assumed to be a response to mechanical allodynia. Performance analysis of responses at 10° C and 37° C temperatures, revealed an increase in face contacts in the CCI-treated group to match the lick frequency prior to the injury. As such no significant effect of injury could be observed, though aversive behaviors were observed at 48° C in the CCI group. 

              Investigation of sex difference in thermal pain sensitivity

              Vierck, Acosta-Rua, Rossi, and Neubert (2008) evaluated male and female thermal pain sensitivity in a variety of apparatuses including the OPAD. Subjects were selected from hooded Long-Evans and hairless Sprague-Dawley strains. Initial training of the subjects in the OPAD was performed at 37° C and testing was performed at thermode temperatures set to 45° C, 48° C, or 52° C.  Performance analysis revealed that in comparison to the females, the males were hypersensitive to nociceptive heat stimulation of the face. The males were observed to feed on the reward in short bouts to minimize the duration of contact with the thermodes.

              Investigation of the effects of botulinum neurotoxin type A on IoNC-induced thermal hyperalgesia

              The effects of administration of highly purified 150-kDa Botulinum neurotoxin type A (BoNT/A) on infraorbital nerve constriction (IoNC)-induced thermal hyperalgesia was investigated by Kumada et al. (2011). Male Sprague-Dawley rats were divided into sham surgery and IoNC surgery groups. The IoNC was performed by ligation of infraorbital nerve without occluding circulation through the superficial vasculature. The sham group only had the infraorbital nerve exposed but not constricted. Subjects were bilaterally administered purified BoNT ⁄ A as a single intradermal injection in variable doses in the snout at the center of the whisker pad or back of the neck. Performance in the OPAD was evaluated at thermode temperatures of 24° C and 45° C. Compared to baseline, performance after IoNC, a significant decrease in the ratio of contact duration ⁄contact number was observed. At 45° C IoNC rats showed an increase in the frequency of thermode contact while the duration of contact was decreased. Further, a dose-dependent effect of BoNT/A on the reversal of thermal hyperalgesia could be observed when administered in the snout. At a 200-pg dose of BoNT/A complete reversal of thermal hyperalgesia symptoms could be observed.

              Investigation of the effects of cocoa flavanols on pain control

              Hairless male and female Sprague-Dawley rats were divided into cocoa diet and control diet groups. The cocoa diet group received 10% g/g cocoa research diet while an isocaloric, cocoa-free diet was given to the controls. Capsaicin cream was applied bilaterally over the masseter muscles area of the cheeks to induce acute neurogenic inflammation. Cocoa-enriched diet group showed higher operant index. Males on cocoa diet showed higher pain index at 37°C while females had higher pain index at thermode temperatures of 18°C and 44°C, in comparison to control diet group. In comparison to control-diet male rats and cocoa-diet female after capsaicin-induced pain, males fed cocoa-diet showed higher pain controls at 37°C and 44°C. A significant effect of cocoa diet at 44°C thermode temperature was observed in capsaicin-treated female rats. (Bowden et al., 2017)

              Data Analysis

              The Orofacial Pain Assessment Device uses a reward-conflict paradigm to quantify pain behavior. The simple device allows assessment of pain parameters based on responses to temperatures as well as mechanical stimuli. Thus, allowing a comparison of pain responses within a single system. The apparatus allows the comparison of performances between treated versus untreated groups, and the performances of different disease and lesion groups.

              The following measures can be obtained in the OPAD:

              • Number of licks (successful attempts)
              • Number of contact with thermode/wire
              • Reward intake (g)
              • Face contact duration/Event ratio
              • Reward/Stimulus contact ratio
              • Temperature of the thermode during a session
              • Latency to approach the reward
              • Time spent in contact with thermode/wire

              Strengths & Limitations


              The Operant Orofacial Assessment Devices allows assessment of mechanical and thermal pain sensitivity within a single system. The set-up allows direct comparisons of the performances in both modalities under the same experimental parameters. Unlike traditional pain assays, the OPAD relies on the subject’s voluntary behavior. Further, since the device doesn’t require any restraining, it is relatively less stressful for the subject. The investigator-independent nature of the OPAD offers an effective analysis of pain parameters that is not influenced by investigator bias. The device also encompasses psychological and affective dimensions of pain, thus, providing sensitive measures of pain that are reproducible. The simplicity of the system makes it easily adaptable for different investigations.


              Since the OPAD system relies on voluntary behaviors, training and testing can be time-consuming. Further, subjects that are not motivated enough may not perform the task. The reliance on voluntary behavior also may result in task duration differences within a group, which can impact the accuracy of the data. The task requires the subjects to be fasted prior to training. This introduces an appetitive factor into the performance. The thermal simulations are effective on hairless skin. Thus, subjects with fur or hair must have it removed prior to testing which adds additional labor. The rows of wires may not equally apply the mechanical stimuli. Subjects may distribute the effects of the mechanical stimuli by tilting their head. Pain sensitivities of subjects can vary based on their strain, age, and gender. Further, external factors such as olfactory, auditory or visual stimuli can affect task performances.

              Summary & Key Points

              • The Operant Pain Assessment Device is used to assess mechanical and thermal pain sensitivity in rodents.
              • The OPAD is based on voluntary behaviors rather than reflex or innate responses to stimuli.
              • The reward-conflict paradigm applied in the OPAD provides a sensitive measure of pain thresholds.
              • The device incorporates psychological and affective dimensions of pain.
              • The OPAD does not require restraining nor applies forced aversive stimuli. Thus, the test is less stressful in comparison to other assays.
              • The OPAD is investigator independent and allows for reproducible results.
              • Performances in the OPAD may be affected by factors such as strain, age, and other external stimuli.


              Anderson, E. M., Jenkins, A. C., Caudle, R. M., & Neubert, J. K. (2014). The effects of a co-application of menthol and capsaicin on nociceptive behaviors of the rat on the operant orofacial pain assessment device. PLoS One, 9(2): e89137. doi: 10.1371/journal.pone.0089137.

              Anderson, E. M., Mills, R., Nolan, T. A., Jenkins, A. C., Mustafa, G., …, Neubert, J. K. (2013). Use of the Operant Orofacial Pain Assessment Device (OPAD) to measure changes in nociceptive behavior. Journal of Visual Experiments, (76):e50336. doi: 10.3791/50336.

              Bowden, L. N., Rohrs, E. L., Omoto, K., Durham, P. L., Holliday, L. S., …, Neubert JK. (2017). Effects of cocoa-enriched diet on orofacial pain in a murine model. Orthodontics & Craniofacial Research, 20 Suppl 1:157-161. doi: 10.1111/ocr.12149.

              Hitomi, S., Ono, K., Yamaguchi, K., Terawaki, K., Imai, R., …, Inenaga, K. (2016). The traditional Japanese medicine hangeshashinto alleviates oral ulcer-induced pain in a rat model. Archives of Oral Biology, 66:30-7. doi: 10.1016/j.archoralbio.2016.02.002.

              Kumada, A., Matsuka, Y., Spigelman, I., Maruhama, K., Yamamoto, Y., …, Oguma, K. (2011). Intradermal injection of Botulinum toxin type A alleviates infraorbital nerve constriction-induced thermal hyperalgesia in an operant assay. Journal of Oral Rehabilitation, 39(1), 63–72. doi:10.1111/j.1365-2842.2011.02236.x

              Murphy, N. P., Mills, R. H., Caudle, R. M., & Neubert, J. K. (2014). Operant assays for assessing pain in preclinical rodent models: highlights from an orofacial assay. Current Topics in Behavioral Neuroscience, 20:121-45. doi: 10.1007/7854_2014_332.

              Nag, S., & Mokha, S. S. (2016). Activation of the trigeminal α2-adrenoceptor produces sex-specific, estrogen dependent thermal antinociception and antihyperalgesia using an operant pain assay in the rat. Behavioral Brain Research, 314:152-8. doi: 10.1016/j.bbr.2016.08.012.

              Neubert, J. K., Rossi, H. L., Malphurs, W., Vierck, C. J. Jr, & Caudle, R. M. (2006). Differentiation between capsaicin-induced allodynia and hyperalgesia using a thermal operant assay. Behavioral Brain Research, 170(2):308-15.

              Neubert, J. K., Widmer, C. G., Malphurs, W., Rossi, H. L., Vierck, C.  J. Jr, & Caudle, R. M. (2005). Use of a novel thermal operant behavioral assay for characterization of orofacial pain sensitivity. Pain, 116(3):386-95.

              Nolan, T. A., Hester, J., Bokrand-Donatelli, Y., Caudle, R. M., & Neubert, J. K. (2011). Adaptation of a novel operant orofacial testing system to characterize both mechanical and thermal pain. Behavioral Brain Research, 217:477–480.

              Nolan, T. A., Price, D. D., Caudle, R. M., Murphy, N. P., & Neubert, J. K. (2012). Placebo-induced analgesia in an operant pain model in rats. Pain, 153(10):2009-16. doi: 10.1016/j.pain.2012.04.026.

              Ramirez, H. E., Queeney, T. J., Dunbar, M. L., Eichner, M. C., Del Castillo, D. I., …, Neubert, J. K. (2015). Assessment of an Orofacial Operant Pain Assay as a Preclinical Tool for Evaluating Analgesic Efficacy in Rodents. Journal of American Association for Laboratory Animal Science, 54(4):426-32.

              Rohrs, E. L., Kloefkorn, H. E., Lakes, E. H., Jacobs, B. Y., Neubert, J. K., …, Allen, K. D. (2015). A novel operant-based behavioral assay of mechanical allodynia in the orofacial region of rats. Journal of Neuroscience Methods, 248:1-6. doi: 10.1016/j.jneumeth.2015.03.022.

              Rossi, H. L., & Neubert, J. K. (2008). Effects of environmental enrichment on thermal sensitivity in an operant orofacial pain assay. Behavioral Brain Research, 187(2):478-82.

              Rossi, H. L., Jenkins, A. C., Kaufman, J., Bhattacharyya, I., Caudle, R. M., & Neubert, J. K. (2012). Characterization of bilateral trigeminal constriction injury using an operant facial pain assay. Neuroscience, 224, 294–306. doi: 10.1016/j.neuroscience.2012.08.015

              Rossi, H. L., Vierck, C. J. Jr, Caudle, R. M., & Neubert, J. K. (2006). Characterization of cold sensitivity and thermal preference using an operant orofacial assay. Molecular Pain, 2:37.

              Vierck, C. J., Acosta-Rua, A. J., Rossi, H. L., & Neubert, J. K. (2008). Sex Differences in Thermal Pain Sensitivity and Sympathetic Reactivity for Two Strains of Rat. The Journal of Pain, 9(8), 739–749. doi:10.1016/j.jpain.2008.03.008