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.

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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

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JoVE Journal

Behavior

3.5K Views.  University of Florida, Gainesville. This protocol describes the assessment of mechanical hypersensitivity in a rat model of neuropathic orofacial pain using an operant-based orofacial pain assessment device.

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 ...

A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste reactivity with the behavioral aspects of operant self-administration of drugs. Under very brief general anaesthesia, rats are implanted with an intraoral (IO) cannula that allows delivery of test solutions directly in the oral cavity. Animals are then tested in operant self-administration chambers whereby they can press a lever to receive IO infusions of test solutions. IO self-administration has several advantages over experimental procedures that involve drinking a solution from a spout&#160;or operant responding for solid pellets or solutions delivered in a receptacle. Here, we show that IO self-administration can be employed to study self-administration of high fructose corn syrup (HFCS). Rats were first tested for self-administration on a progressive ratio (PR) schedule, which assesses the maximum amount of operant behavior that will be emitted for different concentrations of HFCS (i.e.&#160;8%, 25%, and 50%). Following this test, rats self-administered these concentrations on a continuous schedule of reinforcement (i.e.&#160;one infusion for each lever press) for 10 consecutive days (1 session/day; each lasting 3 hr), and then they were retested on the PR schedule. On the continuous reinforcement schedule, rats took fewer infusions of higher concentrations, although the lowest concentration of HFCS (8%) maintained more variable self-administration. Furthermore, the PR tests revealed that 8% had lower reinforcing value than 25% and 50%. These results indicate that IO self-administration can be employed to study acquisition and maintenance of responding for sweet solutions. The sensitivity of the operant response to differences in concentration and schedule of reinforcement makes IO self-administration an ideal procedure to investigate the neurobiology of voluntary intake of sweets.

The current study evaluates a novel procedure for assessing the reinforcing effects of palatable solutions in laboratory rats: intraoral self-administration. To this end, operant responding (i.e.&#160;lever pressing) for intraoral infusions of sweet solutions at different concentrations was measured on continuous and progressive ratios schedules of reinforcement.

This paper describes a novel method for studying the bio-behavioral basis of addiction to food. This method combines the surgical component of taste ...

Operant Procedures for Assessing Behavioral Flexibility in Rats

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these processes is the ability to adjust behavior in response to changes in one&#8217;s environment (i.e., behavioral flexibility). These processes are essential to normal human behavior, and may be disrupted in diverse neuropsychiatric conditions, including schizophrenia, alcoholism, depression, stroke, and Alzheimer&#8217;s disease. Understanding of the neurobiology of executive functions has been greatly advanced by the availability of animal tasks for assessing discrete components of behavioral flexibility, particularly strategy shifting and reversal learning. While several types of tasks have been developed, most are non-automated, labor intensive, and allow testing of only one animal at a time. The recent development of automated, operant-based tasks for assessing behavioral flexibility streamlines testing, standardizes stimulus presentation and data recording, and dramatically improves throughput. Here, we describe automated strategy shifting and reversal tasks, using operant chambers controlled by custom written software programs. Using these tasks, we have shown that the medial prefrontal cortex governs strategy shifting but not reversal learning in the rat, similar to the dissociation observed in humans. Moreover, animals with a neonatal hippocampal lesion, a neurodevelopmental model of schizophrenia, are selectively impaired on the strategy shifting task but not the reversal task. The strategy shifting task also allows the identification of separate types of performance errors, each of which is attributable to distinct neural substrates. The availability of these automated tasks, and the evidence supporting the dissociable contributions of separate prefrontal areas, makes them particularly well-suited assays for the investigation of basic neurobiological processes as well as drug discovery and screening in disease models.

The ability to assess executive functions such as behavioral flexibility in rats is useful for investigating the neurobiology of cognition in both intact animals and disease models. Here we describe automated tasks for assessing strategy shifting and reversal learning, which are particularly sensitive to disruptions in prefrontal cortical networks.

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these ...

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

An Operant Intra-/Extra-dimensional Set-shift Task for Mice

Alterations in executive control and cognitive flexibility, such as attentional set-shifting abilities, are core features of several neuropsychiatric diseases. The most widely used neuropsychological tests for the evaluation of attentional set-shifting in human subjects are the Wisconsin Card Sorting Test (WCST) and the CANTAB Intra-/Extra-dimensional set shift task (ID/ED). These tasks have proven clinical relevance and have been modified and successfully adapted for research in animal models. However, currently available tasks for rodents present several limitations, mainly due to their manual-based testing procedures, which are hampering translational advances in psychiatric medicine. To overcome these limitations and to better mimic the original version in primates, we present the development of a novel operant-based two-chamber ID/ED "Operon" task for rodents. We demonstrated the effectiveness of this novel task to measure different facets of cognitive flexibility in mice including attentional set formation and shifting, and reversal learning. Moreover, we show the high flexibility of this task in which three different perceptual dimensions can be manipulated with a high number of stimuli cues for each dimension. This novel ID/ED Operon task can be an effective preclinical tool for drug testing and/or large genetic screening relevant to the study of executive dysfunction and cognitive symptoms found in psychiatric disorders.

Attentional set-shifting cognitive abnormalities are a core feature of many psychiatric diseases. Here, we present the development of a novel automatic system for the effective study of attentional set-shifting abilities, executive functions and other cognitive abilities in mice with high translational validity to human studies.

Alterations in executive control and cognitive flexibility, such as attentional set-shifting abilities, are core features of several neuropsychiatric ...

Determining Pain Detection and Tolerance Thresholds Using an Integrated, Multi-Modal Pain Task Battery

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify potentially suitable therapeutic populations. The need to use a comprehensive battery of pain models is highlighted by studies whereby only a single pain model, thought to relate to the clinical situation, demonstrates lack of efficacy. No single experimental model can mimic the complex nature of clinical pain. The integrated, multi-modal pain task&#160;battery presented here encompasses the electrical stimulation task, pressure stimulation task, cold pressor task, the UVB inflammatory model which includes a thermal task and a paradigm for inhibitory conditioned pain modulation. These human pain models have been tested for predicative validity and reliability both in their own right and in combination, and can be used repeatedly, quickly, in short succession, with minimum burden for the subject and with a modest quantity of equipment. This allows a drug to be fully characterized and profiled for analgesic effect which is especially useful for drugs with a novel or untested mechanism of action.

Human pain models are valuable tools used to assess the analgesic potential of novel compounds and predict their clinical efficacy, especially when used in an integrated manner. Although implementation of these models is complex, with proper execution, the pain models described in this protocol can provide predictive and reliable results.

Human pain models are useful in the assessing the analgesic effect of drugs, providing information about a drug&#39;s pharmacology and identify ...

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and human studies to investigate the cortical processing of nociceptive information. These laser-evoked brain potentials (LEPs) consist of several transient responses that are time-locked to the onset of laser stimuli. However, the functional properties of the LEP responses are still largely unknown, due to the lack of a sampling technique that can simultaneously record neural activities at the surface of the cortex (i.e., electrocorticogram [ECoG] and scalp electroencephalogram [scalp EEG]) and inside the brain (i.e., local field potential [LFP]). To address this issue, we present here an animal protocol using freely moving rats. This protocol is composed of three main procedures: (1) animal preparation and surgical procedures, (2) a simultaneous recording of ECoG and LFP in response to nociceptive laser stimuli, and (3) data analysis and feature extraction. Specifically, with the help of a 3D-printed protective shell, both ECoG and LFP electrodes implanted on the rat&#39;s skull were securely held together. During data collection, laser pulses were delivered on the rat&#39;s forepaws through gaps in the bottom of the chamber when the animal was in spontaneous stillness. Ongoing white noise was played to avoid the activation of the auditory system by the laser-generated ultrasounds. As a consequence, only nociceptive responses were selectively recorded. Using the standard analytical procedures (e.g., band-pass filtering, epoch extraction, and baseline correction) to extract stimulus-related brain responses, we obtained results showing that LEPs with a high signal-to-noise ratio were simultaneously recorded from ECoG and LFP electrodes. This methodology makes the simultaneous recording of ECoG and LFP activities possible, which provides a bridge of electrocortical signals at the mesoscopic and macroscopic levels, thereby facilitating the investigation of nociceptive information processing in the brain.

We developed a technique that simultaneously records both electrocorticography and local field potentials in response to nociceptive laser stimuli from freely moving rats. This technique helps establish a direct relationship of electrocortical signals at the mesoscopic and macroscopic levels, which facilitates the investigation of nociceptive information processing in the brain.

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and ...

Multi-Modal Signals for Analyzing Pain Responses to Thermal and Electrical Stimuli

The assessment of pain relies mostly on methods that require a person to communicate. However, for people with cognitive and verbal impairments, existing methods are not sufficient as they lack reliability and validity. To approach this problem, recent research focuses on an objective pain assessment facilitated by parameters of responses derived from physiology, and video and audio signals. To develop reliable automated pain recognition systems, efforts have been made in creating multimodal databases in order to analyze pain and detect valid pain patterns. While the results are promising, they only focus on discriminating pain or pain intensities versus no pain. In order to advance this, research should also consider the quality and duration of pain as they provide additional valuable information for more advanced pain management. To complement existing databases and the analysis of pain regarding quality and length, this paper proposes a psychophysiological experiment to elicit, measure, and collect valid pain reactions. Participants are subjected to painful stimuli that differ in intensity (low, medium, and high), duration (5 s / 1 min), and modality (heat / electric pain) while audio, video (e.g., facial expressions, body gestures, facial skin temperature), and physiological signals (e.g., electrocardiogram [ECG], skin conductance level [SCL], facial electromyography [EMG], and EMG of M. trapezius) are being recorded. The study consists of a calibration phase to determine a subject&#8217;s individual pain range (from low to intolerable pain) and a stimulation phase in which pain stimuli, depending on the calibrated range, are applied. The obtained data may allow refining, improving, and evaluating automated recognition systems in terms of an objective pain assessment. For further development of such systems and to investigate pain reactions in more detail, additional pain modalities such as pressure, chemical, or cold pain should be included in future studies. Recorded data of this study will be released as the &#8220;X-ITE Pain Database&#8221;.

This article focuses on the experimental elicitation of pain through heat (thermal) and electrical stimulation while recording physiological, visual, and paralinguistic responses. It aims at collecting valid multimodal data for analyzing pain based on its intensity, quality, and duration.&#160;

The assessment of pain relies mostly on methods that require a person to communicate. However, for people with cognitive and verbal impairments, existing ...

Fear Incubation Using an Extended Fear-Conditioning Protocol for Rats

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the relationships between aversive events and otherwise neutral stimuli. The most-widely utilized procedures for studying emotional memories entail fear conditioning in rats. In these tasks, the unconditioned stimulus (US) is a footshock presented once or several times across single or several sessions, and the conditioned response (CR) is freezing. In a version of these procedures, called cued fear conditioning, a tone (conditioned stimulus, CS) is paired with footshocks (US) during the training phase. During the first test, animals are exposed to the same context in which training took place, and freezing responses are tested in the absence of footshocks and tones (i.e., a context test). During the second test, freezing is measured when the context is changed (e.g., by manipulating the smell and walls of the experimental chamber) and the tone is presented in the absence of footshocks (i.e., a cue test). Most cued fear conditioning procedures entail few tone-shock pairings (e.g., 1-3 trials in a single session). There is a growing interest in less common versions involving an extensive number of pairings (i.e., overtraining) related to the long-lasting effect called fear incubation (i.e., fear responses increase over time without further exposure to aversive events or conditioned stimuli). Extended fear-conditioning tasks have been key to the understanding of fear incubation&#8217;s behavioral and neurobiological aspects, including its relationship with other psychological phenomena (e.g., post-traumatic stress disorder). Here, we describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

We describe an extended fear-conditioning protocol that produces overtraining and fear incubation in rats. This protocol entails a single training session with 25 tone-shock pairings (i.e., overtraining) and a comparison of conditioned freezing responses during context and cue tests 48 h (short-term) and 6 weeks (long-term) after training.

Emotional memory has been primarily studied with fear-conditioning paradigms. Fear conditioning is a form of learning through which individuals learn the ...

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.