This study is funded by the Facial Pain Research Foundation.

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
<ol>
	<li>House female Sprague-Dawley rats (n = 8/group, 150-200 g) in pairs in a temperature-controlled room (22 &#176;C &#177; 1 &#176;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.</li>
	<li>Perform the operant pain assays on the same day of the week and time (9 a.m.-11 a.m.).</li>
	<li>At the end of the experiments, euthanize the rats by decapitation following isoflurane anesthesia.</li>
</ol>
2. Setting up the OPAD
<ol>
	<li>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.</li>
	<li>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 &#176;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.</li>
	<li>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.</li>
	<li>Turn on the cages using the switch on the front panel.</li>
</ol>
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.
<ol>
	<li>Open the software. Type the password. Click Log me on or hit Enter.</li>
	<li>Click New empty experiment | Protocol menu.
	<ol>
		<li>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).</li>
		<li>Add OPAD cages.
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>Add experiment test stages.
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>Assign treatment groups.
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>Assign animal identifications (ID).
		<ol>
			<li>Click the Protocol menu; under Additional Information, click Animal ID and check Use my IDs to refer to animals.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Click the Experiment menu.
	<ol>
		<li>Type an experiment title.</li>
		<li>Name the treatments by clicking View treatments, and type the treatment names (e.g., Treatment 1: CCI-ION, Treatment 2: sham).</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Save the experiment file by clicking File | Save, type the software password, and click Save.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Set up the OPAD equipment.</li>
	<li>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.</li>
	<li>Double-click the saved experiment file to open. Type the password. Click Log me on or hit Enter.</li>
	<li>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 (&#34;ready&#34;). On the right side of the screen, observe the chart of each animal that shows the numbers of licks and contacts.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Following 2 weeks of training, collect data from the noted milk bottle distance for 5 days as the baseline (10 min/day).</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>Place the anesthetized rat on a surgical workbench and restrain it. Maintain the body temperature at 37 &#176;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.</li>
	<li>Perform the surgical procedure under a stereo microscope. Open the mouth using retractors and retract the lip using a small clip.</li>
	<li>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.</li>
	<li>Place two chromic gut (#5-0) ligatures around the ION using a blunt, bent syringe needle.</li>
	<li>Close the wound using tissue adhesive.</li>
	<li>For the sham surgery, expose the ION using the same procedure but do not ligate the nerve.</li>
	<li>After surgery, provide milk-softened rodent chow for 2 days to encourage eating and prevent dehydration.</li>
	<li>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. 
	&#8203;NOTE: The duration of the CCI-ION-induced mechanical sensitivity can depend on the sex, the strain of the rodent used, and the experimenter&#39;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.</li>
</ol>
6. Cleaning up the device
<ol>
	<li>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.</li>
	<li>Turn off the cages using the switch on the front panel.</li>
	<li>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.</li>
	<li>Wipe the metal spiked bars, testing device, and lab benches using 70% isopropyl alcohol. 
	&#8203;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.</li>
</ol>
7. Data analysis
<ol>
	<li>Double-click on the experiment file to open it.</li>
	<li>Click on the Results menu. Select which measures (i.e., lick, contact) or test days to see.</li>
	<li>Click Text or Graph or Statistical found at the top of the Results panel to see a text, graph, or statistical analysis report.</li>
	<li>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.</li>
	<li>To change or add more variables to see, click Select data, select the measures, and click View spreadsheet.</li>
	<li>Statistical analysis
	<ol>
		<li>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.</li>
		<li>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 (&#60;500 licks) prior to surgery.</li>
		<li>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&#39;s multiple comparisons or &#352;&#237;d&#225;k&#39;s multiple comparisons tests where appropriate. 
		NOTE: P &#60; 0.05 was considered statistically significant. Data were presented as mean &#177; standard error of the mean (SEM).</li>
	</ol>
	</li>
</ol>

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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&#160;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.

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.

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&#39;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.

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

assessment of nerve injury-induced mechanical hypersensitivity in rats using an orofacial operant pain assay

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.

An example of a single rat&#39;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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64221/64221fig01.jpg" /> 
Figure 1: Schematic representation of the study design. Abbreviations: OPAD = orofacial pain assessment device; CCI-ION = chronic constriction injury of the infraorbital nerves. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64221/64221fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64221/64221fig02.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64221/64221fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64221/64221fig03.jpg" /> 
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 =&#160;chronic constriction injury of the infraorbital nerves; AS = after surgery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64221/64221fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64221/64221fig04.jpg" /> 
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)&#160;an&#160;increase in latency to first lick on surgery week (week 0) and 1 week after surgery (**p &#60; 0.01, *p &#60; 0.05: after surgery weeks vs. baseline. #p &#60; 0.05: CCI-ION vs. sham). There was no significant decrease in the sham group (n = 7, p &#62; 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 &#60; 0.01, *p &#60; 0.05: after surgery weeks vs. baseline. # p &#60; 0.05: CCI-ION vs. sham). In the graphs, the red line represents the CCI-ION group, and the blue line represents the sham group. The data are presented as mean &#177; SEM. Significant differences were analyzed by two-way repeated-measures ANOVA followed by &#352;&#237;d&#225;k&#39;s or Dunnett&#39;s multiple comparison tests, as appropriate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64221/64221fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay at JoVE.com

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

This protocol describes the assessment of mechanical hypersensitivity in a rat model of neuropathic orofacial pain using an operant-based orofacial pain assessment device.

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results ...

assessment-nerve-injury-induced-mechanical-hypersensitivity-rats

Nanyang Technological University

Research

JoVE Journal

Behavior

3.5K Views.  University of Florida, Gainesville. Het orofaciale pijnbeoordelingsapparaat, of OPAD, is een waardevol hulpmiddel voor preklinisch pijnonderzoek omdat het een operant beloningsconflictparadigma gebruikt. Dit omvat nociceptie, leren en motivatie, die belangrijk zijn in de menselijke pijnervaring en verbeterde translationele validiteit. Een belangrijk voordeel van de OPAD ten opzichte van traditionele reflexgebaseerde testen is dat het volledig geautomatiseerd is, waardoor het niet meer nodig is om dieren te beperken en gerelatee...

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

Use of the Operant Orofacial Pain Assessment Device (OPAD) to Measure Changes in Nociceptive Behavior

We present an operant system for the detection of pain in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) assesses pain behaviors in a more clinically relevant way by not relying on reflex-based measures of nociception. Food fasted, hairless (or shaved) rodents are placed into a Plexiglas chamber which has two Peltier-based thermodes that can be programmed to any temperature between 7 &deg;C and 60 &deg;C. The rodent is trained to make contact with these in order to access a reward bottle. During a session, a number of behavioral pain outcomes are automatically recorded and saved. These measures include the number of reward bottle activations (licks) and facial contact stimuli (face contacts), but custom measures like the lick/face ratio (total number of licks per session/total number of contacts) can also be created. The stimulus temperature can be set to a single temperature or multiple temperatures within a session. The OPAD is a high-throughput, easy to use operant assay which will lead to better translation of pain research in the future as it includes cortical input instead of relying on spinal reflex-based nociceptive assays.

Use of the Operant Orofacial Pain Assessment Device OPAD to Measure Changes in Nociceptive Behavior

We present a user-friendly, high-throughput operant system for the evaluation of pain behaviors in awake, conscious rodents. The Orofacial Pain Assessment Device (OPAD) can assess pain through a reward/conflict paradigm thus providing a more humane way of testing. This protocol will yield more clinically relevant and translational data from rodents.

We present an operant  system for the detection of pain in awake, conscious rodents. The Orofacial  Pain Assessment Device (OPAD) assesses pain behaviors ...

Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents

A lengthening in meal duration can be used to measure an increase in orofacial mechanical hyperalgesia having similarities to the guarding behavior of humans with orofacial pain. To measure meal duration unrestrained rats are continuously kept in sound attenuated, computerized feeding modules for days to weeks to record feeding behavior. These sound-attenuated chambers are equipped with chow pellet dispensers. The dispenser has a pellet trough with a photobeam placed at the bottom of the trough and when a rodent removes a pellet from the feeder trough this beam is no longer blocked, signaling the computer to drop another pellet. The computer records the date and time when the pellets were taken from the trough and from this data the experimenter can calculate the meal parameters. When calculating meal parameters a meal was defined based on previous work and was set at 10 min (in other words when the animal does not eat for 10 min that would be the end of the animal&#39;s meal) also the minimum meal size was set at 3 pellets. The meal duration, meal number, food intake, meal size and inter-meal interval can then be calculated by the software for any time period that the operator desires. Of the feeding parameters that can be calculated meal duration has been shown to be a continuous noninvasive biological marker of orofacial nociception in male rats and mice and female rats. Meal duration measurements are quantitative, require no training or animal manipulation, require cortical participation, and do not compete with other experimentally induced behaviors. These factors distinguish this assay from other operant or reflex methods for recording orofacial nociception.

A lengthening in meal duration represents orofacial nociceptive behavior in rodents similar to the guarding behavior of humans with orofacial pain. Eating is a behavior that requires no training or animal manipulation, requires cortical participation, and is not competing with other experimentally induced behaviors, distinguishing this assay from alternative reflex or operant measurements.

A lengthening in meal duration can be used to measure an increase in orofacial mechanical hyperalgesia having similarities to the guarding behavior of ...

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. 
The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn&#x2019;t require expert microsurgical skills.
The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. 
As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.

The Spared Nerve Injury SNI Model of Induced Mechanical Allodynia in Mice

The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared ...

Medicine

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Chronic Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical condition. However, the underlying mechanisms of CRPS-I have yet to be revealed. It is known that ischemia/reperfusion is one of the leading factors that causes CRPS-I. By means of prolonged ischemia and reperfusion of the hind limb, the rat chronic post-ischemia pain (CPIP) model has been established to mimic CRPS-I. The CPIP model has become a well-recognized animal model for studying the mechanisms of CRPS-I. This protocol describes the detailed procedures involved in the establishment of the rat model of CPIP, including anesthesia, followed by ischemia/reperfusion of the hind limb. Characteristics of the rat CPIP model are further evaluated by measuring the mechanical and thermal hypersensitivities of the hind limb as well as the nocifensive responses to acute capsaicin injection. The rat CPIP model exhibits several CRPS-I-like manifestations, including hind limb edema and hyperemia in the early stage after establishment, persistent thermal and mechanical hypersensitivities, and increased nocifensive responses to acute capsaicin injection. These characteristics render it a suitable animal model for further investigation of the mechanisms involved in CRPS-I.

Provided here is a protocol that details steps to establish an animal model of chronic post-ischemia pain (CPIP). This is a well-recognized model mimicking human complex regional pain syndrome type-I. Mechanical and thermal hypersensitivities are further evaluated, as well as capsaicin-induced nocifensive behaviors observed in the CPIP rat model.

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical ...

Back Mechanical Sensitivity Assessment in the Rat for Mechanistic Investigation of Chronic Back Pain

Low back pain is the leading cause of disability worldwide, with dramatic personal, economic, and social consequences. To develop novel therapeutics, animal models are needed to examine the mechanisms and effectiveness of novel therapies from a translational perspective. Several rodent models of back pain are used in current investigations. Surprisingly, however, no standardized behavioral test was validated to assess mechanical sensitivity in back pain models. This is critical to confirm that animals with presumed back pain present local hypersensitivity to nociceptive stimuli, and to monitor sensitivity during interventions designed to relieve back pain. The objective of this study is to lay down a simple and accessible test to assess mechanical sensitivity in the back of rats. A test cage was fabricated specifically for this method; length x width x height: 50 x 20 x 7 cm, having a stainless-steel mesh on the top. This test cage allows the application of mechanical stimuli to the back. To perform the test, the back of the animal is shaved in the region of interest, and the test area is marked to repeat the test on different days, as needed. The mechanical threshold is determined with Von Frey filaments applied to the paraspinal muscles, utilizing the up-down method described previously. The positive responses include (1) muscle twitching, (2) arching (back extension), (3) rotation of the neck (4) scratching or licking the back, and (5) escaping. This behavioral test (Back Mechanical Sensitivity (BMS) test) is useful for mechanistic research with rodent models of back pain for the development of therapeutic interventions for the prevention and management of back pain.

To develop novel therapeutic interventions for the prevention and management of back pain, animal models are required to examine the mechanisms and effectiveness of these therapies from a translational perspective. The present protocol describes the BMS test, a standardized method to assess back mechanical sensitivity in the rat.

Low back pain is the leading cause of disability worldwide, with dramatic personal, economic, and social consequences. To develop novel therapeutics, ...

Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral procedures to study trigeminal neuropathic pain in rats. To meet the specificity of trigeminal neuropathic pain syndromes, the infraorbital nerve (IoN) is subjected to a chronic constriction injury (CCI) by loosely ligating the nerve. An intra-orbital approach is presented here to expose and ligate the IoN in the orbital cavity. After IoN ligation, rats exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are indicative of persistent pain and mechanical allodynia. Two phases can be defined in the development of the behavioral changes. During the first week following IoN-CCI (phase 1), rats show an increased and asymmetric face grooming activity, i.e., with face wash strokes primarily directed to the nerve-injured IoN territory. A distinction is made between face grooming behavior that is part of a more general body grooming behavior, which remains largely unaffected by IoN-CCI, and face grooming that is neither preceded nor followed by body grooming, which is significantly increased after IoN-CCI. During this period, responsiveness to mechanical stimulation of the IoN territory is reduced. This hyporesponsiveness is abruptly replaced by an extreme hyperresponsiveness whereby even very weak stimulus intensities provoke nocifensive behavior (phase 2). The phenomenological similarities between these behavioral alterations and reported signs of facial pain (i.e., responses to noxious stimulation of the face) suggest the presence of dysesthesia/paresthesia and mechanical allodynia in the ligated IoN territory.

Chronic Constriction Injury of the Rat's Infraorbital Nerve IoN-CCI to Study Trigeminal Neuropathic Pain

This manuscript describes a rodent model to study trigeminal neuropathic pain. The methods described include surgical procedures to perform a chronic constriction injury of the rat&#8217;s infraorbital nerve and the post-surgical behavioral tests to measure the changes in spontaneous and evoked behavior that are indicative of persistent pain and allodynia.

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

Neuroscience

Establishing the Mouse Model for Trigeminal Neuropathic Pain

Chronic Constriction Injury of the Distal Infraorbital Nerve (DIoN-CCI) in Mice to Study Trigeminal Neuropathic Pain

Animal models remain necessary tools to study neuropathic pain. This manuscript describes the distal infraorbital nerve chronic constriction injury (DIoN-CCI) model to study trigeminal neuropathic pain in mice. This includes the surgical procedures to perform the chronic constriction injury and the postoperative behavioral tests to evaluate the changes in spontaneous and evoked behavior that are signs of ongoing pain and mechanical allodynia. The methods and behavioral readouts are similar to the infraorbital nerve chronic constriction injury (IoN-CCI) model in rats. However, important changes are necessary for the adaptation of the IoN-CCI model to mice. First, the intra-orbital approach is replaced by a more rostral approach with an incision between the eye and the whisker pad. The IoN is thus ligated distally outside the orbital cavity. Secondly, due to the higher locomotor activity in mice, allowing rats to move freely in small cages is replaced by placing mice in custom-designed and constructed restraining devices. After DIoN ligation, mice exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are similar to those in IoN-CCI rats, i.e., increased directed face grooming and hyperresponsiveness to von Frey hair stimulation of the IoN territory.

Author Spotlight: Utilizing Infraorbital Nerve Ligation in Mice for Investigating Trigeminal Neuropathic Pain and Treatment Strategies

Chronic constriction injury of the distal infraorbital nerve in mice induces changes in spontaneous behavior (increased face grooming activity) and nocifensive behavior in response to tactile stimulation (hyperresponsiveness to von Frey hair stimulation) that are signs of ongoing pain and allodynia and serves as a model for trigeminal neuropathic pain.

Animal models remain necessary tools to study neuropathic pain. This manuscript describes the distal infraorbital nerve chronic constriction injury ...

Induction of Neuropathic Pain in a Rat Model by Constriction of the Infraorbital Nerve

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/53167/chronic-constriction-injury-rat-s-infraorbital-nerve-ion-cci-to-study'>Source: Deseure, K., et al.&#160;Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain. J. Vis. Exp. (2015).</a>This video demonstrates a method to induce neuropathic pain in a rat model by constricting the infraorbital nerve. After exposing the infraorbital nerve and tying ligatures to constrict it, the immune response at the injury site increases the excitability of neighboring neurons, which transmit pain signals to the brain.

Source: Deseure, K., et al.&#160;Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain. J. Vis. Exp. ...

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

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