Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay

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. No pre- or post-operative analgesia was used in the procedure as it can change the time course of the neuropathy. 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. Check the anesthetic depth by pinching the toe and start the procedure when the toe withdrawal reflex is no longer observed.
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 contacts^21,22,23.
      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).