Name: meal duration as a measure of orofacial nociceptive responses in rodents
Uploaded: 2014-01-10T13:00:00
Description: Watch this Scientific Journal Video about Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents at JoVE.com

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
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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 A recent unpublished study suggested that von Frey filament testing had greater sensitivity than meal duration measurements, showing a significant change for a longer period but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.

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			<td height="42" style="height:42px;width:335px;">Animal Monitor software</td>
			<td style="width:92px;">Med Assoc. Inc</td>
			<td style="width:137px;">SOF-710</td>
			<td style="width:335px;">East Fairfield, VT</td>
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			<td height="24" style="height:24px;">Dustless Precision Pellets, Rodent, Grain-Based&#160;</td>
			<td style="width:92px;">Bio-Serv</td>
			<td>F0165</td>
			<td style="width:335px;">45 mg pellets, 50,000/box</td>
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		<tr height="24">
			<td height="24" style="height:24px;">Dustless Precision Pellets, Rodent, Grain-Based&#160;</td>
			<td style="width:92px;">Bio-Serv</td>
			<td style="width:137px;">FO163</td>
			<td style="width:335px;">20 mg pellets</td>
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			<td height="63" style="height:63px;">Complete Freund&#39;s Adjuvant</td>
			<td style="width:92px;">Chondrex, Inc.</td>
			<td style="width:137px;">7001</td>
			<td style="width:335px;">No loger provides the 5 mg/ml concentration.&#160; Can use CFA from other sources as long as the investigator consistently uses this source</td>
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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 ...

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