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

The orofacial pain assessment device, or OPAD, is a valuable tool for preclinical pain research as it uses an operant reward conflict paradigm. This encompasses nociception, learning, and motivation, which are important in the human pain experience and improved translational validity. A significant advantage of the OPAD over traditional reflex-based assays is that it is fully automated, which eliminates the need to restrain animals preventing related stress effects, and it also reduces experimenter bias.

Demonstrating the CCI surgery of the infraorbital nerve will be Darrice Montgomery, a laboratory technician from my lab. Begin with setting up the orofacial pain assessment device, or OPAD, by placing the milk drip trays, plexiglass cages, and metal flooring grates. Attach wiring to the cages and slip the bottle holder onto the metal pole at the back of the device.

Then, place the reward milk bottles on the bottle holder in a way that the spout can be reached by the animal. Tighten the left-side knob of the holder to secure the bottle in place. Then, switch on the front panel and turn on the cages.

To set up the protocol, open the software, enter the password, and click Log Me On or hit Enter. Click New Empty Experiment and the Protocol menu. Under Apparatus, click Unnamed Protocol and select the mode this protocol will use.

Under Equipment Specific Modes, select OPAD Mechanical Cage Mode and name the protocol. After ensuring that all the cages are turned on, add the OPAD cages by clicking on Apparatus and Add Item at the top of the protocol pane, followed by selecting New OPAD Cage and Add All Connected OPAD Cages. To add the experiment test stages, go to the Testing tab to select the stages, and First Stage tabs, then name the stage.

Add 10 minutes as the test duration. Add more stages by clicking Add Item Tab, followed by New Stage. Next, assign the groups by clicking on Additional Information and Treatment Groups.

Check Used Treatment Groups followed by selecting the option The User Will Manually Assign the Animals to Their Groups. Assign animal identifications by clicking on the Protocol menu. Under Additional Information, click on Animal ID and check the option Use My IDs to Refer to Animals.

Set the experiment by typing in experiment title before clicking View Treatments and typing the treatment names. Add animals and assign the treatments and animal IDs by clicking View Animals and Add Animals. Enter the number of animals to be tested, followed by Okay.

Once the animal list appears, add animal ID and treatment for each rat. Save the protocol by clicking the Protocol menu followed by Save Protocol. Enter the file name and software password and click Save.

Then, save the experiment file by clicking on the file, then Save, typing the software password and clicking the Save tab. After switching on the cages, look for the green light on the cage denoting the cage is ready to test. Open the saved experiment file with a double click.

Enter the password, click Log Me On, or hit Enter. On the left-side of the screen, note the number of animals and the corresponding cage, the stage running on that day and the testing status. Observe the test animal's ID on the cage's screen.

Place the rat into the cage and press the button on the corresponding cage. Note that the green light turns into an orange light once testing starts. Ensure that the animal can access the reward bottle while making sufficient contact with the mechanical spikes.

On the right-side of the screen, observe the chart of each animal showing the numbers of licks and contacts. A warning sound is heard when the testing session is over. Place the anesthetized rat on a surgical workbench and restrain it.

Maintain the body temperature at 37 degrees Celsius using a heating pad. Apply ophthalmic ointment to the eyes to prevent them from drying out. Use scalpel blade 15 to make a small incision between the dorsal gum and lip.

Remove the soft tissue using the scalpel blade and reveal the branch of the infraorbital nerves, or ION. With the help of a blunt bent syringe needle, place two 5-O chromic gut ligatures around the ION, then close the wound using tissue adhesive. The contact attempts and licking data of a single CCI ION and Sham operated rat during the standard 10 minute testing session at baseline, and two weeks, four weeks, and six weeks after surgery is presented here.

The model revealed that during the non-noxious periods rats have long sessions of drinking. After the injury, the animal cannot maintain facial contact with the spiked bars for a long duration resulting in a decreased lick number. In the Sham group, significant changes were not observed for the drinking period.

The mean number of licks between CCI ION and Sham operated rats at the baseline showed no significant differences. However, CCI ION significantly reduced licking numbers until four weeks after surgery and increased latency for the first lick during the post-surgery week and one week after nerve injury indicating mechanical hyperalgesia. There was no significant change in Sham operated rats.

While using the OPAD, optimize the distance of the milk bottle and the width of the spikes. It is important to keep this consistent to reduce errors during the data collection.