In this model the rats or mice were given food and water ad libitum. The Texas A&#38;M University Baylor College of Dentistry Institutional Animal Care and Use Committee approved all the experimental protocols. Below specific settings are shown in italics and are utilized specifically for the rat TMJ arthritis model. Mice can also be utilized in this model and alternative tooth pain and myogenic orofacial pain animal models can be used as well42,43.
1. Software Settings
<ol>
	<li>Load the Animal Monitor software for the feeder units onto the computer.</li>
	<li>The Animals Monitor software is now opened by clicking on the icon and under the file menu selection choose the &#34;configuration&#34; pull down option.</li>
	<li>In the &#34;Animal Monitor Configuration&#34; window uncheck the box entitled &#34;Pellet Delivered Input&#34; (Figure 1A). 
	Note: This box is usually checked at the factory by default. Deselect this option. Once the investigator deselects this option the results are being recorded by removal of a pellet from the trough rather than when a pellet is being dispensed. 
	Note: When the &#34;Automatic File Naming&#34; box is checked the software will automatically name the files (Figure&#160;1A). This box is usually checked at the factory by default.</li>
	<li>Set the timer lights to turn on at 06:00 (6:00 am) and to turn off at 20:00 (8:00 pm). 
	Note: the hardware for the feeders units was modified so that the lights within the boxes are not controlled by the software but are wired instead to an isolated 24 hr&#160;timer. Thus, the &#34;House Light&#34; indicated on the configuration software was not functional in these examples.</li>
	<li>Select the edit pull down menu and choose Experiment. A window with the title &#34;Box 01 -&#34; appears (Figure&#160;1B).</li>
	<li>Input the file name under which the data will be saved within this window. 
	Note: if no file name is entered the data files will be automatically named by the software. Details of the experiment can be added in this window and saved with the data files. The information on the window can also be saved and used for subsequent experiments.</li>
	<li>Enter a number greater than the total time of the experiment in the entry box entitled &#34;Experiment Length (Days):&#34;. 
	Note: This will ensure that the software saves the data until the experiment is complete, a mistake can be in setting this value too short, which will stop the software from recording the data even though animals are still in the feeding modules.</li>
	<li>Enter 24 in the entry box entitled &#34;Number of Hours in a Day&#34;. 
	Note: the length can be modified to the experimenter&#39;s specifications.</li>
	<li>Enter 10 in the box entitled &#34;Meal Period End Criteria (min):&#34; 
	Note: for rats a meal was defined using a 10 min end of meal criterion based on previous studies48 (i.e.&#160;a meal was bracketed before and after by a 10 min period of no pellets being taken) and the minimum meal size was set at three pellets per meal in this software package.</li>
	<li>Enter 45 in the box entitled &#34;Pellet Size (mg)&#34;.&#160;If an individual wanted to use mice input 20 within this window.</li>
	<li>For rats add 45 mg rodent chow pellets to the feeder dispenser hopper. 
	Note: for mice add 20 mg rodent chow pellets to the chow hopper.</li>
	<li>In the &#34;Phase&#34; section of the Experiment window enter the term Day in the &#34;Name&#34; field and in the &#34;# of Hours&#34; field type 24 (Figure 1B). 
	Note: below the &#34;Phase&#34; section there are two large open fields. The first large open field will be populated by the text entered in the previous &#34;Phase&#34; and &#34;Name&#34; fields.</li>
	<li>The next large open field will have the &#34;Day Phase&#34; header Enter in the field entitled &#34;Name&#34; the word Light and enter in the field entitled &#34;Percent&#34; the number 60. 
	Note: this entered text will populate the large field below.</li>
	<li>Next enter in the field entitled &#34;Name&#34; the word Dark and enter in the field entitled &#34;Percent&#34; the number 40.&#160;Note: with these entries 60% of the day will be attributed to the light phase and 40% will be attributed to the dark phase. When the software calculates the meal patterns this information will be used. These settings typical for cycling female animals that are kept on a 14:10 light/dark cycle.</li>
	<li>Select the &#34;Set All Boxes Like This&#34; button. Save this information and then hit OK.</li>
	<li>The &#34;Start Boxes&#34; screen appears, select the feeders to activate and hit OK (Figure&#160;1C).</li>
	<li>Next the Animal Monitor Run-Time windows will appear with the meal pattern data (Figure&#160;1D). 
	Note: monitor and record the &#34;# of Pellets Dispensed&#34; from this window to determine the current health of the rat. A healthy male rat weighing around 300 g will typically eat between 300-800&#160;45 mg pellets a day.</li>
	<li>Files are generated daily and automatically saved that have a .CSV extension. Open these files to retrieve meal pattern data such as food intake, meal number, meal duration, meal size, or intermeal interval. The intervals for these meal patterns can be calculated for the entire day or for a phase of the day such as the Dark and Light phase. As noted above, the settings are for a 14:10 light/dark cycle. The raw data of when each pellet was removed from the trough is also recorded as a raw .CSV&#160;file. 
	Note: In older versions of the software a minimum meal size of 3 pellets is not used in the calculations for generating the .CSV file.&#160; Moreover, to get the average meal duration using the older software you must subtract 10 min from the values in the Average Meal Duration column of the .CSV file. 
	When the software is operating the operator can manually select the file pull down menu option and daily select &#34;save raw data&#34;. This will save the raw data for a 24 hr period rather than for the entire experiment. This raw data can be processed by alternative software at the user&#39;s discretion. 
	Note: in the results shown we used alternative software to include a minimum meal size of 3 pellets.</li>
</ol>
2. Meal Duration Assay
<ol>
	<li>Place individual rats in the sound-attenuated chambers equipped with photobeam computer-activated pellet feeders. 
	Note: in these feeding units there are graduated water bottles and waste pans in which a sheet of thick absorbent paper is placed. In the feeder dispenser hopper 45 mg rodent chow pellets can be added for rats or 20 mg rodent chow pellets can be added for mice. Chow pellets are dispensed into a V shaped feeding trough and at the bottom of this trough is a photobeam. A pellet dispensed into the trough will be detected by breaking this photobeam. Once a rat removes this pellet from the feeder trough the photobeam is restored and this signals the computer to drop another pellet. Restoration of the photobeam also triggers the computer to record the date and time and keeps a running tally of the pellets dispensed. This tally of pellets is then analyzed to determine food intake, meal number, meal duration, meal size, or intermeal interval during any part of the day using Med Assoc. Inc. software. Again the raw .CSV&#160;data file can be analyzed by outside software39,40,49-51.</li>
	<li>Record the total number of pellets eaten, the amount of water consumed and the weight of the animals to discern the general health of the rats during the experimentation.</li>
	<li>Rinse water bottles and fill with fresh water daily and add chow to the feeder hopper when needed.</li>
	<li>Dump waste pan and the thick absorbent paper beneath the cage daily and blow the dust from moving part of the feeder daily using high pressure air. 
	Note: personal protection equipment (e.g.&#160;gowns, gloves, masks, and masks) is required.</li>
	<li>Remove floors, waste pans and water bottles after the experiment is completed and wash these components. Also, remove the feeding electronics from the caging wash by hand or in a dishwasher.</li>
</ol>
3. Induction of TMJ Arthritis
<ol>
	<li>Place animals in the feeders at least 4 days before experimentation. 
	Note: this data will be reported as predays to obtain a baseline feeding behavior. Then the animals are removed from the feeder for treatment. One type of treatment was to induce an arthritic TMJ. For this model rats are injected with complete Freund&#39;s adjuvant (CFA) at 08:00 (i.e.&#160;beginning of the light phase) after the rats are anesthetized with isoflurane (5% flow).</li>
	<li>Inject 250 mg of CFA in 50 L bilaterally into the periarticular space of each TMJ. 
	Note: in the example (Figure&#160;2) 250 mg of CFA in 50 L was injected into each TMJ, but doses as low as 10 mg in volumes as small as 15 &#956;l are effective over shorter periods of time52.</li>
	<li>Inject control rats TMJ with 50 L of 0.9% saline. Note: all animals were mobile within 5 min or less after induction of anesthesia. In the event that a smaller dose of CFA is given in a lesser volume the control rats would receive this same volume of saline.</li>
</ol>

There is nothing to disclose.

TMJ patients with orofacial pain report increased pain with increased chewing time, such that, the chewing cycle lengthens the longer the individual has been chewing45,53-56. Our behavioral assay allows for similar testing in rats and mice when measuring meal duration39. A recent unpublished study suggested that von Frey filament testing had greater sensitivity than meal duration measurements, showing a significant change for a longer period but von Frey filament testing can have a reflex response c...

Animal models have been used to study pain and nociception associated with orofacial damage and or inflammation1,2, but a lack of appropriate animal models results in an incomplete understanding of the mechanisms. Although current models help us to understand various mechanisms involved in acute and chronic orofacial pain, there are strengths and weaknesses to these animal models.
Many models measure behavioral nociceptive responses for short durations. Face grooming is a known behavioral response following constriction of facial nerves3. Others studies measured facial rubbing with the ipsilateral fore or hindpaw, as well as, flinching of the head after administering formalin injections into the temporomandibular joint (TMJ) or lip4-7. Head withdrawal latencies is another model for measuring nociceptive behavior where a modified tail flick analgesia meter is used to quantitate the nociceptive response (i.e.&#160;head withdrawal) after applying heat to the shaved vibrissae pad of a rat8. Digastric and masseter muscle activity has also been recorded as a correlate of pain after glutamate injections into the TMJ9. Another study has measured changes in sleep parameters to assess nociceptive responses in male and female rats with an inflamed TMJ, these parameters included sleep latency, rapid-eye-movement (REM), percentage of non-REM sleep, and percentage of REM sleep10. Most animal models that measure behavioral nociceptive responses utilize a short time frame, i.e.&#160;minutes to hours per day11-14. In addition, most animal models testing occurs during the light phase and in a nocturnal animal, like a rat, this can cause stress which can confound the nociceptive results15-18. The above assays measure nociceptive response in varying orofacial conditions but for short duration and hence can only be used to study acute disorders. An alternative assay has used facial expression as a measure of nociception of moderate duration, but this methodology can be subjective19.
To evaluate persistent or chronic orofacial nociception some have used the application of a von Frey filament on the surface of the skin to assess mechanical sensitivity of animals subjected to nerve constriction or TMJ inflammation3,20. Liverman et al. 2009 measured withdrawal responses using graded monofilaments following CFA injections into the masseter muscle of rats 21,22. Yamazaki et al.&#160;2008 injected the TMJ with CFA and then over 14 days quantified nociceptive behaviors to mechanical or heat or cold stimulation applied over the TMJ region. Unfortunately, these nociceptive behavioral assays involve animal restraint, which produce stress hormones, learning or alternative behaviors that may interfere with the measured outcomes.
Models to measure nociception in teeth utilize the jaw opening reflex but this method can be unreliable23 or imprecise24. Electromyographic activity has been used to measure tooth nociception25, but this method typically requires that the animal be unconscious, although in one study tooth nociception was investigated in freely moving rats26. In 2008, Khan studied the relationship between dental nociception and masticatory function using a sensitive strain gauge27 but this bite duration model requires restraining the animal from normal activity 28. Bite force is a reliable measure of tooth pain in humans but because rats require training and/or restraint to measure bite force a source of stress is introduced which can produce findings with questionable physiological significance29-31
Some limitations of restraint and stress can be overcome by using an operant design to assess nociceptive behaviors. One operant model uses avoidance of an uncomfortable temperature to evaluate and characterize orofacial nociception32-35. This reward-conflict model is based on a reward of sweetened milk to induce the rodent to position its face voluntarily against a heated or cooled thermal probe34,36. However, the test requires animal training, but a strength of the assay is the data is collected in an automated fashion.&#160;
Still another animal model used nociception-induced gnawing dysfunction as an index of orofacial nociception37. However, the rodent is confined to a tube and its only escape is to gnaw through a dowel to exit. An advantage of this model is that it measures jaw function after acute or chronic jaw injury in mice. However, the rodent is confined, which adds a confounding alternate competing behavior, i.e.&#160;escape, which would be stressful and thus could influence the nociception assay results.
Meal duration has been used to measure nociception in animals with TMJ arthritis38-41, tooth pulp exposure42, and muscle damage43. A rodent who experienced orofacial nociception ate more slowly after the animal initiated a meal. Patients experiencing TMJ pain also take longer to chew their food and the cycle length shortens when TMJ pain is diminished44-46. The lengthening of meal duration when TMJ pain is present is expected to be a &#34;guarding behavior&#34;, operationally defined as nociceptive behavior47.
Meal duration measures TMJ nociception using a noninvasive method for up to 19 days in male and female rats and 6 days (longest period tested) in male mice and could be described as a biological marker of nociception38-41. In support that meal duration measures nociceptive responses, nociception can be reduced by pharmacological intervention causing the animal&#39;s meal duration to return to normal38,40,41. This was also confirmed when nociceptive neurons were destroyed using capsaicin; after nerve destruction the animals meal duration was not increased following injection of CFA into the TMJ 40.
Below is the protocol on how to obtain and statistically analyze meal duration data.

<table><tbody><tr><td>Animal Monitor software</td><td>Med Assoc. Inc</td><td>SOF-710</td><td>East Fairfield, VT</td></tr><tr><td>Dustless Precision Pellets, Rodent, Grain-Based&amp;nbsp;</td><td>Bio-Serv</td><td>F0165</td><td>45 mg pellets, 50,000/box</td></tr><tr><td>Dustless Precision Pellets, Rodent, Grain-Based&amp;nbsp;</td><td>Bio-Serv</td><td>FO163</td><td>20 mg pellets</td></tr><tr><td>Complete Freund&#039;s Adjuvant</td><td>Chondrex, Inc.</td><td>7001</td><td>No loger provides the 5 mg/ml concentration.&amp;nbsp; Can use CFA from other sources as long as the investigator consistently uses this source</td></tr></tbody></table>

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.

Meal duration is a behavioral correlate of orofacial pain and meal duration measurements have been applied to animals with TMJ arthritis (Figure 2) and tooth decay (Figure&#160;3). In one experiment, rats had TMJ arthritis after administering a high 250 mg dose of CFA and this treatment induced a significant increase in meal duration for 19 days (Figure&#160;2). A lower dose of CFA (10 mg) injected into each TMJ joint produced a smaller increase in meal duration for only...

Watch this Scientific Journal Video about Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents at JoVE.com

Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents

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 ...

meal-duration-as-measure-orofacial-nociceptive-responses

Research

JoVE Journal

Behavior

7.5K Views.  Texas A&M University Baylor College of Dentistry. 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.

Video: Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents

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 ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Neuroscience

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 ...

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. 
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.

We describe a protocol to examine the development of opioid-induced hyperalgesia and tolerance in mice. Based on the measurement of thermal and mechanical nociceptive responses of na&#239;ve and morphine-treated animals, it allows to quantify the increase in pain sensitivity (hyperalgesia) and decrease in analgesia (tolerance) associated with chronic opiate administration.

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular ...

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.

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 ...

Inflammatory TMJ Pain Mouse Model: Bite Force and Von Frey Filament Assessments

Temporomandibular Joint Pain Measurement by Bite Force and Von Frey Filament Assays in Mice

Temporomandibular joint (TMJ) pain and osteoarthritis (OA) are common and debilitating disorders that impair patients' quality of life. The mechanisms driving diseases-related pain are poorly understood, and current treatments fail to provide effective and long-term therapeutic effects. Additionally, pain assessment in research, particularly orofacial pain, poses several challenges that complicate studies in both clinical and basic science settings. Therefore, we have established an inflammatory TMJ pain mouse model via intra-articular injection of CFA (Complete Freund's Adjuvant) and evaluated pain behaviors by bite force measurement and the von Frey filament test. Mice with CFA injection exhibited orofacial pain behaviors compared to PBS injection, including reduced bite force and head withdrawal threshold in the von Frey filament test. These methods are relatively easy to execute to have reproducible results and can be potentially extended to pain studies for other disease models related to TMJ disorders. Together, bite force, and the von Frey filament tests are reliable in measuring orofacial pain, as demonstrated in CFA injection-induced painful TMJOA mouse models.

This protocol describes how measuring bite force and head withdrawal threshold can capture pain behavior in Complete Freund's Adjuvant (CFA)-induced inflammatory temporomandibular joint (TMJ) pain.

Temporomandibular joint (TMJ) pain and osteoarthritis (OA) are common and debilitating disorders that impair patients' quality of life. The mechanisms ...

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

Medicine

Dural Stimulation and Periorbital von Frey Testing in Mice As a Preclinical Model of Headache

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system. For example, they protect the brain from the skull and anchor/organize the vascular and neuronal supply of the cortex. However, the meninges are also implicated in nervous system disorders such as migraine, where the pain experienced during a migraine is attributed to local sterile inflammation and subsequent activation of local nociceptive afferents. Of the layers in the meninges, the dura mater is of particular interest in the pathophysiology of migraines. It is highly vascularized, harbors local nociceptive neurons, and is home to a diverse array of resident cells such as immune cells. Subtle changes in the local meningeal microenvironment may lead to activation and sensitization of dural perivascular nociceptors, thus leading to migraine pain. Studies have sought to address how dural afferents become activated/sensitized by using either in vivo electrophysiology, imaging techniques, or behavioral models, but these commonly require very invasive surgeries. This protocol presents a method for comparatively non-invasive application of compounds on the dura mater in mice and a suitable method for measuring headache-like tactile sensitivity using periorbital von Frey testing following dural stimulation. This method maintains the integrity of the dura and skull and reduces confounding effects from invasive techniques by injecting substances through a 0.65 mm modified cannula at the junction of unfused sagittal and lambdoid sutures. This preclinical model will allow researchers to investigate a wide range of dural stimuli and their role in the pathological progression of migraine, such as nociceptor activation, immune cell activation, vascular changes, and pain behaviors, all while maintaining injury-free conditions to the skull and meninges.

The most notable symptom of migraine is severe head pain, and it is hypothesized that this is mediated by sensory neurons innervating the meninges. Here, we present a method to locally apply substances to the dura in a minimally invasive manner while using facial hypersensitivity as an output.

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system.  ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

The mechanical conflict-avoidance assay is used as a non-reflexive readout of pain sensitivity in mice which can be used to better understand affective-motivational responses in a variety of mouse pain models.

Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using ...

Optimizing Photoneuromodulation Techniques to Evaluate the Role of Green Light-Emitting Diodes in Pain Management

Despite extensive research and the identification of numerous analgesic targets, the range of pharmacological treatments available for pain remains limited. However, a potential paradigm shift could introduce a new wave of non-pharmacological pain treatments with remarkable safety, efficacy, and tolerability. One promising area of investigation is photoneuromodulation using green light-emitting diodes (GLED, 525 nm), which have shown potential in alleviating pain in both acute and chronic conditions, leading to numerous preclinical and clinical studies exploring the efficacy of this therapy. These research projects have demonstrated how exposure to GLED enhances the activity of the endogenous opioid system in the brain and spinal cord after M-cone activation in the retina. The findings suggest that GLED may alleviate pain by modulating the descending pain pathway. In light of the compelling effects of GLED, the proliferation of photoneuromodulation investigations underscores the importance of establishing consistency in well-defined and standardized exposure protocols for preclinical and clinical trials. In preclinical studies, beneficial effects have been observed following a minimum of 2 days of exposure, with protocols involving 8 h of light at 100 lux during the 12 h light phase. In clinical trials, exposure protocols are tailored to the specific pathology under investigation. Exposure for 15 min has proven favorable in the modulation of acute post-surgical pain. For modulation of chronic pain, patients are instructed to use GLED at home for 1 to 2 h a day over 10 weeks. This article details preclinical and clinical protocols to improve reproducibility and consistency in the different studies evaluating photoneuromodulation benefits. By establishing these standardized protocols, this work aims to advance the clinical translation of GLED phototherapy as a viable non-pharmacological treatment for pain.

Recent developments in pain research highlight the potential of photoneuromodulation using green light-emitting diodes (GLED) as a non-pharmacological treatment. GLED modulates pain pathways, offering effective pain relief. This article aims to standardize and refine GLED exposure protocols, improving consistency across studies and advancing the clinical application of this therapy.

Despite extensive research and the identification of numerous analgesic targets, the range of pharmacological treatments available for pain remains ...