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Assessment of Nerve Injury-induced Mechanical Hypersensitivity in Rats using an Orofacial Operant Pain Assay

Published: July 26, 2022 doi: 10.3791/64221


Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results by addressing the cognitive and motivational aspects of pain in rodents. This paper presents a protocol for assessing mechanical hypersensitivity following chronic constriction injury of the infraorbital nerves (CCI-ION) in rats using an orofacial operant pain system. Before CCI-ION surgery, rats were trained in an orofacial pain assessment device (OPAD) to drink sweetened condensed milk while making facial contact with the metal spiked bars and lick-tube.

In this assay, rats can choose between receiving milk as a positive reinforcer or escaping an aversive mechanical stimulus that is produced by a vertical row of small pyramid-shaped spikes on each side of the reward access hole. Following 2 weeks of training in the OPAD and before the CCI-ION surgery, baseline mechanical sensitivity data were recorded for 5 days for each rat during a 10 min testing session. During a session, the operant system automatically records the number of reward bottle activations (licks) and facial contacts, contact duration, and latency to the first lick, among other measures.

Following baseline measurements, rats underwent either CCI-ION or sham surgery. In this protocol, mechanical hypersensitivity was quantified by measuring the number of licks, latency to the first lick, the number of contacts, and the ratio of licks to facial contacts (L/F). The data showed that CCI-ION resulted in a significant decrease in the number of licks and the L/F ratio and an increase in the latency to the first lick, indicating mechanical hypersensitivity. These data support the use of operant-based pain assays to assess mechanical pain sensitivity in preclinical pain research.


Chronic pain affects millions of Americans annually1. Unfortunately, chronic pain is challenging to treat, as existing therapies are relatively ineffective at mitigating chronic pain and often have undesired side effects with long-term use2,3,4. Traditional preclinical pain assays, such as the von Frey assay, rely on reflexive outcomes or pain-stimulated responses5. While the von Frey assay has been used for decades to measure mechanical allodynia, it is susceptible to several confounding factors, notably experimenter bias6. The use of von Frey testing for evaluating orofacial pain is also problematic due to the degree of restraint needed to secure the animal's head to successfully test the facial area, which may produce undesired stress effects, such as enhancing pain or, conversely, stress-induced analgesia.

Pain-stimulated behaviors are also susceptible to false-positive outcomes7 and do not account for the affective component of pain, which is integral to the human pain experience8. Therefore, there is a growing interest in using operant pain models that assess pain-depressed behaviors that encompass both the sensory and affective components of pain to improve the content and predictive validity in preclinical testing. The operant orofacial pain assessment assay described here is based on a reward-conflict paradigm9,10,11. In this assay, the rodent can choose between receiving a positive reinforcer and subjecting itself to a nociceptive stimulus or foregoing the reward and avoiding the nociceptive stimulus, thereby controlling the amount of pain it experiences. Unlike traditional pain assays, the operant-based assay is experimenter-independent and is not susceptible to false-positive outcomes due to untoward sedative effects.

Noxious sensations from the head and face are carried by the ophthalmic, maxillary, and mandibular branches of the trigeminal nerve. Injury or inflammation of the trigeminal nerve increases the sensitivity of sensory neurons to thermal or mechanical stimuli12,13,14,15. Operant-based orofacial pain assays provide an automated measurement of thermal or mechanical orofacial pain transmitted by the trigeminal nerve in rodents11,12,16,17,18. Stimulation with non-noxious and noxious stimuli is an important distinction between testing thermal and mechanical allodynia and hyperalgesia in the orofacial region with the OPAD, as they may represent manifestations of different underlying mechanisms.

In the orofacial thermal assay, animals press their face against smooth thermodes to access the reward. The thermodes can be set to various cool, warm, and hot temperatures, thereby allowing the assessment of behavior under neutral or nociceptive conditions. In the orofacial mechanical assay, animals press their face against spiked bars during operant testing; as these spikes cause some level of discomfort, rodents might drink less when their faces touch the spikes versus the smooth surfaces of the thermodes. Thus, the operant orofacial mechanical assay can assess the effect of varying degrees of mechanical nociceptive stimulation. We have previously demonstrated that the OPAD is a useful and reliable method to assess acute thermal9, as well as acute mechanical19, nociception and hyperalgesia.

This paper reports the use of a newly developed version of the OPAD to assess mechanical nociception and hypersensitivity. Additionally, by way of validation, we demonstrate the ability of CCI-ION to induce chronic neuropathy that results in a predictable response in the OPAD. Also detailed is how to use the OPAD and its associated software to rapidly obtain and analyze rodent behavioral data.


All experimental procedures were approved by the University of Florida Institutional Animal Care and Use Committee and complied with the standards stated in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Here, the assessment of mechanical hypersensitivity using the OPAD is described using a rat model of neuropathic orofacial pain. A schematic of the timeline used in the study is shown in Figure 1. All behavioral assessments were performed by female experimenters.

1. Animals

  1. House female Sprague-Dawley rats (n = 8/group, 150-200 g) in pairs in a temperature-controlled room (22 °C ± 1 °C) with a 12 h:12 h light-dark cycle. Provide food and water ad libitum. Keep the rats in the facility for 5 days for acclimation before the experiments.
  2. Perform the operant pain assays on the same day of the week and time (9 a.m.-11 a.m.).
  3. At the end of the experiments, euthanize the rats by decapitation following isoflurane anesthesia.

2. Setting up the OPAD

  1. Place milk-drip trays, Plexiglas cages, and metal flooring grates on the OPAD. Attach wiring to the cages. Slip the bottle holder onto the metal pole at the back of the device.
  2. Prepare a 2:1 ratio of water to sweetened condensed milk as the reward solution by opening a can of sweetened condensed milk and pouring it into a 1 L beaker. Add ~600 mL of tap water to 300 mL of milk. Initially stir the solution using a spoon, and then use a stir bar and the hot-plate stirrer. Then, fill the reward bottles with the milk solution and keep the stock milk solution at 4 °C.
    NOTE: Cover the stock milk solution with plastic food wrap. Warm the milk solution before each use. Stock milk solution in the fridge can coagulate after a week. When it coagulates, it might occlude the lick tube. Therefore, discard it and prepare a new stock solution.
  3. Place the reward milk bottles on the bottle holder and adjust them so the spout can be reached by the animal. Tighten the left-side knob of the holder to secure the bottle in place.
  4. Turn on the cages using the switch on the front panel.

3. Setting up a protocol and creating an experiment file

NOTE: First, set up the protocol to run the experiment. The protocol describes how ANY-maze software performs the experiment.

  1. Open the software. Type the password. Click Log me on or hit Enter.
  2. Click New empty experiment | Protocol menu.
    1. Select the mode this protocol will use and name the protocol. Under Apparatus, click Unnamed Protocol, click the select the mode this protocol will use section, and, under Equipment-Specific Modes, select OPAD mechanical cage mode, and click OK. Then, name the protocol (e.g., OPAD mechanical).
    2. Add OPAD cages.
      1. Under Apparatus, click Apparatus | Add item found at the top of the Protocol pane | New OPAD cage | Add all connected OPAD cages.
        NOTE: Before adding the cages, ensure all the cages are turned on.
    3. Add experiment test stages.
      1. Under Testing, click Stages | First stage and name Stage (e.g., Baseline Day 1). Type 10 min for the test duration. To add more stages, click Add item found at the top of the Protocol pane | New Stage.
        NOTE: Each stage refers to the session when an assay is performed. For example, for 10 days of training, 10 stages are needed. The test duration can be increased or decreased based on the experimental design.
    4. Assign treatment groups.
      1. Under Additional Information, click Treatment groups. Check Use treatment groups | The user will manually assign the animals to their groups.
        NOTE: The referenced software (see the Table of Materials) also allows animals to be assigned randomly or in a specific order. Experiments can be run blind. To see the assigned treatment groups, uncheck Run experiments blind.
    5. Assign animal identifications (ID).
      1. Click the Protocol menu; under Additional Information, click Animal ID and check Use my IDs to refer to animals.
  3. Click the Experiment menu.
    1. Type an experiment title.
    2. Name the treatments by clicking View treatments, and type the treatment names (e.g., Treatment 1: CCI-ION, Treatment 2: sham).
    3. Add animals and assign treatments and animal IDs by clicking View animals | Add animals, enter the number of animals that will be tested, and click OK. Wait for the list of animals to appear and add animal IDs and treatments for each rat.
      NOTE: A status list appearing next to animal ID will be set to Normal at the beginning of the study. Animals can later be removed from the testing schedule by changing their status to either Retired or Deleted.
  4. Save the protocol by clicking Protocol menu | Save protocol found at the top of the Protocol pane. Type File name and the software (ANY-maze) password and click Save.
    NOTE: Saved protocols can be reused for new experiments.
  5. Save the experiment file by clicking File | Save, type the software password, and click Save.

4. Training and baseline testing sessions

NOTE: Bring rats to the room at least 15 min before the test if the behavioral testing room is at the same animal housing facility. If they are transported to a testing room outside the animal facility, give the rats 1 h to acclimatize to the room.

  1. Before baseline recordings, train the rats in the OPADs for 2 weeks (5 days/week, 10 min/day) to press their faces against the metal spiked bars to receive the milk solution.
    NOTE: A representative image of spiked bars and a rat performing the assay is shown in Figure 2.
  2. Set up the OPAD equipment.
  3. Turn on the cages using the switch on the front panel. Look for the green light on the cage, which means that the cage is ready to test.
  4. Double-click the saved experiment file to open. Type the password. Click Log me on or hit Enter.
  5. Wait for the Tests menu to show up. On the left side of the screen, make a note of the number of animals and the corresponding cage (e.g., Animal 1 will be tested in cage 1), the stage that will be run on that day, and the testing status ("ready"). On the right side of the screen, observe the chart of each animal that shows the numbers of licks and contacts.
  6. Observe the screen of the cages that displays the ID of the animal to be tested. Place each rat into the corresponding cage and press the button on the cage twice. Note that the green light will turn into an orange light once testing starts, and a warning sound will be heard when the testing session is over.
  7. For the first 2 days of training, place milk bottles completely into the cage to allow the rats to drink milk without contacting the stimulus.
  8. On days 3-8 of training, once the animals start drinking, move the bottles progressively backward to encourage the rats to press their faces against the spiked bars.
  9. On days 9-10 of training, once the animals fully press against the spiked bars and the licking numbers are consistent (a minimum of 500 licks during the 10 min testing sessions), note the location of the milk bottle for each animal and use this distance for baseline recordings.
  10. Following 2 weeks of training, collect data from the noted milk bottle distance for 5 days as the baseline (10 min/day).

5. Induction of orofacial neuropathic pain and evaluation of mechanical hypersensitivity

NOTE: Following baseline measurements, rats underwent CCI-ION surgery, which involved bilateral ligation of the ION, as previously described20. Control rats had sham surgery. CAUTION: Waste isoflurane must be scavenged through charcoal canisters. Scalpel blades and needles must be disposed of in biohazard waste.

  1. Anesthetize the rat in the induction chamber with a mixture of O2 (1 L/min) and 4% isoflurane and maintain the anesthetic state with a specialized nose cone for the duration of the surgery.
  2. Place the anesthetized rat on a surgical workbench and restrain it. Maintain the body temperature at 37 °C using a heating pad. Apply ophthalmic ointment to the eyes to prevent them from drying out.
  3. Perform the surgical procedure under a stereo microscope. Open the mouth using retractors and retract the lip using a small clip.
  4. Make a small incision between the dorsal gum and lip using a scalpel blade (#15). Gently cut away soft tissue using the tip of the scalpel blade to reveal a branch of the ION.
  5. Place two chromic gut (#5-0) ligatures around the ION using a blunt, bent syringe needle.
  6. Close the wound using tissue adhesive.
  7. For the sham surgery, expose the ION using the same procedure but do not ligate the nerve.
  8. After surgery, provide milk-softened rodent chow for 2 days to encourage eating and prevent dehydration.
  9. Test the rats in the OPAD the day after surgery for 3 consecutive days and then 3 days/week (e.g., every Tuesday, Thursday, and Friday) for the following weeks until the lick numbers reach their baseline values.
    ​NOTE: The duration of the CCI-ION-induced mechanical sensitivity can depend on the sex, the strain of the rodent used, and the experimenter's performance. Thus, it might not be accurate to indicate a certain duration to test animals. Hence, testing until lick numbers reach baseline values is more accurate.

6. Cleaning up the device

  1. When testing is finished, quit the software by clicking the x icon at the top-right corner and wait for the data to be saved automatically.
  2. Turn off the cages using the switch on the front panel.
  3. Unplug the wires from the metal flooring grates. Remove and wash the milk-drip trays, Plexiglas cages, metal flooring grates, and bottle holders with dish soap. Put everything on the drying rack.
  4. Wipe the metal spiked bars, testing device, and lab benches using 70% isopropyl alcohol.
    ​NOTE: The instruments need to be handled with care. Use soft brushes while cleaning the milk bottles and licking tubes. Dirty equipment can lead to bacteria buildup.

7. Data analysis

  1. Double-click on the experiment file to open it.
  2. Click on the Results menu. Select which measures (i.e., lick, contact) or test days to see.
  3. Click Text or Graph or Statistical found at the top of the Results panel to see a text, graph, or statistical analysis report.
  4. To see the raw data, click on the Data menu. Click Save at the top of the Data panel to save the data as a spreadsheet or click Send to receive it through e-mail.
  5. To change or add more variables to see, click Select data, select the measures, and click View spreadsheet.
  6. Statistical analysis
    1. Automatically derive the number of licks and contacts and the latency to the first lick from the software and export the data from the software to a spreadsheet.
    2. Calculate the L/F ratio as an index of hypersensitivity by dividing the number of licks by the number of contacts21,22,23.
      NOTE: In this study, one of the rats in the sham group was excluded from the study due to low licking numbers (<500 licks) prior to surgery.
    3. Analyze the statistical significance of the differences between L/F, the number of licks and the contacts, and the latency to the first lick via two-way repeated measures ANOVA followed by Dunnett's multiple comparisons or Šídák's multiple comparisons tests where appropriate.
      NOTE: P < 0.05 was considered statistically significant. Data were presented as mean ± standard error of the mean (SEM).

Representative Results

An example of a single rat's licks on the reward bottle and contacts with the metal spiked bars at baseline and 2 weeks, 4 weeks, and 6 weeks after surgery is presented in Figure 3. During the non-noxious periods, rats generally have long sessions of drinking (e.g., at baseline and recovery after CCI-ION: week 6 in the image), and, following CCI-ION, the lick numbers decrease as they cannot maintain facial contact with the spiked bars for a long duration (Figure 3A), with no significant changes in the periods of drinking in the sham group (Figure 3B).

Rats with CCI-ION had a significant decrease in the number of licks until 4 weeks after surgery and an increase in the latency to first lick on surgery week (week 0) and 1 week after surgery compared to baseline. There was no significant change in the sham group (Figure 4A,B). CCI-ION produced a decrease in the number of contacts, but this difference was not significant (Figure 4C). CCI-ION also caused a significant decrease in the L/F, and the decrease for the CCI-ION group was greater than the decrease for the sham group (Figure 4D).

These results indicate that, following CCI-ION, rats show less reward milk drinking behavior, and it takes them a while to make the first lick, indicating a nocifensive behavior. However, CCI-ION does not impact their desire to reach the milk. In addition, the decrease in L/F of rats with CCI-ION indicates mechanical hypersensitivity, as L/F is higher during non-painful conditions.

Figure 1
Figure 1: Schematic representation of the study design. Abbreviations: OPAD = orofacial pain assessment device; CCI-ION = chronic constriction injury of the infraorbital nerves. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Representative image of spiked bars and a rat performing the assay. Spiked bars are made of stainless steel metal. The length of the entire bar is 7 cm. The height of the spikes is 0.3 cm. The distance between the spikes is 0.5 cm. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Representative contact attempts and licking data of a single CCI-ION- and sham-operated rat during the standard 10 min testing session at baseline and 2 weeks, 4 weeks, and 6 weeks after surgery. Abbreviations: CCI-ION = chronic constriction injury of the infraorbital nerves; AS = after surgery. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Development of mechanical hypersensitivity following CCI-ION in Sprague-Dawley rats. (A) Rats with CCI-ION (n = 8) had a significant decrease in licking numbers until 4 weeks after surgery and (B) an increase in latency to first lick on surgery week (week 0) and 1 week after surgery (**p < 0.01, *p < 0.05: after surgery weeks vs. baseline. #p < 0.05: CCI-ION vs. sham). There was no significant decrease in the sham group (n = 7, p > 0.05). (C) CCI-ION or sham surgery did not produce any significant change in the number of contacts. (D) Rats with CCI-ION showed a significant decrease in L/F on surgery week and 3 weeks after and exhibited a decreasing trend 2 weeks after surgery. Compared to the sham group rats, this decrease was significantly higher in CCI-ION rats and started 1 week after surgery and continued until 3 weeks after surgery. There was no significant difference in the sham group (**p < 0.01, *p < 0.05: after surgery weeks vs. baseline. # p < 0.05: CCI-ION vs. sham). The data are presented as mean ± SEM. Significant differences were analyzed by two-way repeated-measures ANOVA followed by Šídák's or Dunnett's multiple comparison tests, as appropriate. Please click here to view a larger version of this figure.


Pain triggered by innocuous mechanical stimulation of the face and intraoral mucosa is a prominent feature of orofacial pain conditions, including trigeminal neuralgia and temporomandibular joint disorders24,25. Although trigeminal neuropathic pain is clinically well-described, the assessment of neuropathic nociceptive behaviors in rodents is challenging. Pain assays measuring reflexive behaviors are the most frequently used methods in preclinical pain research. However, testing apparatus-related stress, the inability to assess the affective state, and experimenter bias raise concerns regarding the usefulness and validity of reflex assays26.

This study introduces the assessment of mechanical sensitivity in the orofacial region of rats, demonstrating its sensitivity to CCI-ION using an operant-based pain assay. The same operant system can also be used to test the mechanical sensitivity of mice. It should be noted that mouse and rat strains can exhibit variation in their response to CCI-ION, and, thus, the levels of mechanical hypersensitivity can differ. Based on our experience, Sprague-Dawley rats typically develop a stable mechanical hypersensitivity 2 weeks after CCI-ION, they start recovering 4 weeks after CCI-ION, and, after 6 weeks of CCI-ION, we see recovery from the surgery.

In this protocol, mechanical hypersensitivity was quantified by measuring the number of licks and the contacts, L/F, and latency to the first lick. The data demonstrated that CCI-ION resulted in decreases in L/F and the number of licking responses and increases in the latency to the first lick response, indicating that animals were unwilling to press their faces against spiked bars due to increased orofacial pain sensitivity.

OPAD is a reward-conflict assay in which animals must endure nociceptive stimuli to access a palatable reward. Licking behavior in the assay could be affected by appetitive behavior. In addition, in this study, we used rats that had facial hair. Based on prior experience with operant pain assays, among rodents, hairless strains are better for detecting facial contacts16; however, at the time of publication, hairless rat strains were no longer commercially available. This can be considered a limitation of the study. As we also only used female Sprague-Dawley rats, sex- and strain-related differences in pain responses should be anticipated.

There are also some critical steps for ensuring optimal outcomes with the assay. Accurate lick and contact data must appear as solid red and white blocks in the referenced software, respectively (see Figure 3). The distance between the spikes and the milk bottle is crucial for the success of the experiment. If the tip of the milk bottle is too far forward, the animal will not make contact with the spikes, and the software will not correctly register contacts or lick numbers. Conversely, if the milk bottle is too far back, contacts will register, but the animal will not be able to reach the milk. During training sessions, lick data might appear as solid white blocks, as the tip of the milk bottle is too far forward. It changes into red solid blocks once the milk bottle is pushed backward. For some reason, if lick data start appearing as white blocks from the distance that was noted, pushing the bottle a little and moving the milk holder slightly downward/upward might help.

Several points might also be considered as limitations of the orofacial operant pain system described here. The training of the rodents is necessary and takes weeks. Before each testing session, food restriction is necessary in mice but not in rats. Unfasted mice have been shown to have low and inconsistent licking numbers compared to fasted mice27. Since the OPAD system is a reward-conflict model, it might be affected by the appetitive behavior of the animals or by a drug that affects appetite. Having multiple apparatus is also advantageous for reducing the overall time to test the animals, which might increase costs. However, orofacial operant pain assays are still advantageous over conventional reflex-based assays, as they allow testing of multiple animals at the same time and limit animal-experimenter interaction.

Operant conditioning during pain states modifies human and animal behavior according to their consequences28. Using a reward-conflict model is, therefore, advantageous for evaluating pain conditions because it allows the animals to perform operant responses. This is more clinically relevant because the characteristics of operant behaviors involve intention, motivation, and, typically, cortical processing29. As animals voluntarily approach the reward bottle and can freely withdraw from the spiked bars at any time, this integrates higher centers of the brain and allows for evaluation of the affective-motivational states related to pain10. Thus, operant pain assays provide superior data when assessing pain and analgesics in vivo. They also help understand the nociceptive processes in the trigeminal system, thereby contributing to the advancement of the orofacial pain field.


John K. Neubert and Robert M. Caudle are the inventors of the OPAD. Stoelting Co. is the manufacturer of the OPAD, and Richard Mills is an employee of Stoelting. The publication fee has been paid by Stoelting Co. Richard Mills, John K. Neubert, and Robert M. Caudle are previous owners of Velocity Laboratories, a company that provides fee-for-service behavioral testing using operant pain assays.


This study is funded by the Facial Pain Research Foundation.


Name Company Catalog Number Comments
ANY-maze Video Tracking Software Stoelting 60000
Bottle cleaning brushes ANY ANY Different size brushes for bottles and tubes
Chromic gut suture size 5-0 Ethicon 687-G
Dish soap ANY ANY Liquid
Dish sponge ANY ANY
GraphPad Prism version 9.3.1  GraphPad Software, San Diego, CA
Hotplate magnetic stirrer Benchmark Scientific H4000-HS
Isoflurane Patterson Veterinary 07-893-8440 Pivetal
Isopropyl alcohol Fisher Scientific 60-001-56
Ophthalmic ointment Dechra Puralube Vet Ointment, petrolatum ophthalmic ointment
Operant Pain Assessment Device (OPAD) System Stoelting 67500
Oxygen tank Medical
Paper towel ANY ANY
Plastic food wrap ANY ANY
Polygon stir bars Fisher Scientific 14-512-124
Reusable glass Berzelius beakers (1 L) Fisher Scientific FB1021000
Scalpel blade #15 FST 10015-00
Small animal anesthesia system VetFlo VetFlo-1205S
Sprague-Dawley rats, female Charles River Laboratories,  USA
Stereo boom microscope Omano OM2300S-GX4
Sweetened condensed milk Borden  Eagle Brand
Tissue adhesive 3M Vetbond 1469SB
Water circulating heating pad and pump Gaymar Model TP-500



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Donertas-Ayaz, B., Brice-Tutt, A. C., Malphurs, W. L., Montgomery, D., Mills, R. H., Neubert, J. K., Caudle, R. M. Assessment of Nerve Injury-induced Mechanical Hypersensitivity in Rats using an Orofacial Operant Pain Assay. J. Vis. Exp. (185), e64221, doi:10.3791/64221 (2022).More

Donertas-Ayaz, B., Brice-Tutt, A. C., Malphurs, W. L., Montgomery, D., Mills, R. H., Neubert, J. K., Caudle, R. M. Assessment of Nerve Injury-induced Mechanical Hypersensitivity in Rats using an Orofacial Operant Pain Assay. J. Vis. Exp. (185), e64221, doi:10.3791/64221 (2022).

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