The authors wish to thank all members of the Orofacial Physiology and Perceptual Analysis Lab (OPPAL; Etter, Director) and the Sensory Evaluation Center (Hayes, Director), both located at Penn State.

All procedures were approved by the Institutional Review Board for conducting human research at The Pennsylvania State University and were consistent with the Declaration of Helsinki.
1. Set-up
<ol>
	<li>Display all monofilaments with target points between 0.008g (lowest factory setting) and 15 g (a priori ceiling determined during pilot testing).
	<ol>
		<li>Set out the monofilaments with target forces of 0.008, 0.02, 0.04, 0.07, 0.16, 0.4, 0.6, 1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10.0, and 15.0 g.</li>
	</ol>
	</li>
	<li>Set the monofilaments out on the table so each target level can be easily read by the experimenter.</li>
	<li>Seat the participant in a comfortable chair with a glass of water within arm&#39;s reach.</li>
	<li>Instruct the participant that when it is time, they will be asked to close their eyes and stick out their tongue.
	<ol>
		<li>Identify the approximate testing location at midline tongue. The testing location should be approximately 10 mm posterior to the tongue tip.</li>
		<li>Encourage the participant to stop regularly and take a sip of water approximately every 5-10 trials or after the first incorrect response for either testing protocol, detection or discrimination. 
		CAUTION: The tongue should gently protrude from mouth such that the tongue tip meets the inferior edge of the lower lip. The participant should not be straining to stick their tongue out as far as possible or it will fatigue quickly and could alter testing results.</li>
	</ol>
	</li>
</ol>
2. Detection threshold estimates
<ol>
	<li>Instruct participant, &#34;For this test, you will hear me say &#39;Trial 1&#39; and &#39;Trial 2&#39;. You will feel a point of pressure in one of these trials. If you think you feel a point of pressure in trial 1, put up 1 finger. If you think you feel a point of pressure in trial 2, put up 2 fingers. If you&#39;re not sure, make your best guess. Pressure will always be applied in one of the two trials.&#34;</li>
	<li>Identify the starting point for testing: Determine the starting point for all participants for the detection threshold estimates. Published protocol begins at 1.0 g. Researchers strongly suggest the experimenter begins at a suprathreshold level to be sure that the majority of participants can easily identify the first target force.</li>
	<li>Tell participant to close their eyes and start the trials
	<ol>
		<li>Say Trial 1 and pause. Say Trial 2 and pause. Be sure to deliver the pressure in one of these trials such that the monofilament is pressed to the tongue surface until the monofilament buckles. Hold for a second and release.
		<ol>
			<li>Use a random number generator to create a series of 1 or 2. Follow this series to randomly present the stimulus in Trial 1 or Trial 2 (See Supplementary Files 3 &#38; 4). Do not follow a pattern.</li>
			<li>Be deliberate and intentional when applying the pressure such that the stimulus is always clearly delivered in Trial 1 or Trial 2 with a 1-s pause before and after when the Trial is said and when the stimulus is delivered.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Record the participant&#39;s response (See Supplementary Files 1-4 for examples).</li>
	<li>Continue testing using a 3-down/1-up decision rule to select the next stimulus.
	<ol>
		<li>Move up to the next monofilament in the series after any incorrect or &#34;missed&#34; response.</li>
		<li>Ensure that the participant gets three correct responses in a row for the experimenter to move down to the next monofilament in the series.</li>
	</ol>
	</li>
	<li>Stopping point
	<ol>
		<li>Stop testing when a participant has reached his/her stopping point. This is defined as the point when a participant has crossed over or received the test stimulus from the same target monofilament a total of five times.</li>
		<li>If the participant reaches the lowest available testing target (0.008g), continue delivering this target for 5 consecutive sets before stopping.</li>
		<li>If the participant reaches the highest testing target (15 g) and cannot correctly identify the target trial, discontinue testing.</li>
	</ol>
	</li>
	<li>Estimate approximately 5-10 min to complete testing to determine a detection threshold estimate for midline tongue. 
	NOTE: Stopping points apply regardless of whether the participant gets that last trial correct or incorrect.</li>
</ol>
3. Discrimination Threshold Estimates
<ol>
	<li>To complete discrimination threshold testing, follow a similar protocol to detection threshold testing.</li>
	<li>Instruct participant, &#34;For this test, you will still hear me say &#39;Trial 1&#39; and &#39;Trial 2&#39; but this time you will feel a point of pressure in both trials. I want you to identify which trial contained the stronger or harder point of pressure. If you think the harder/stronger pressure was in trial 1, put up 1 finger. If you think you the harder/stronger pressure was in trial 2, put up 2 fingers. If you&#39;re not sure, make your best guess.&#34;</li>
	<li>Identify a starting point
	<ol>
		<li>Begin test at three monofilament levels above the stopping point for their estimated detection threshold. Use the target stimulus and the monofilament immediately below it in force as the two discrimination stimuli.</li>
		<li>Follow this example: If the participant reached a stopping point of 0.008 g, start his/her discrimination testing at 0.07g as the target stimulus and the monofilament directly below it (0.04 g) as the second stimulus delivered.</li>
	</ol>
	</li>
	<li>Tell the participant to close their eyes and start the trials.
	<ol>
		<li>Deliver the two stimuli after clearly stating Trial 1 or Trial 2. Again, this should be randomized, to avoid a pattern.</li>
	</ol>
	</li>
	<li>Record the participant&#39;s response (See Supplementary Files 1-4 for examples).</li>
	<li>Continue testing using the same 3-down/1-up and stopping point as used in the protocol for Detection Thresholds.
	<ol>
		<li>Continue testing with 0.008g with no stimulus delivered in the second trial if the participant reaches 0.008 g during discrimination testing (lowest manufactured level).</li>
		<li>Offer the participant a drink of water if they miss two target stimuli in a row.</li>
	</ol>
	</li>
	<li>Estimate approximately 5-10 min to complete a discrimination threshold estimate for midline tongue.</li>
</ol>
4. Scoring
<ol>
	<li>Record all target stimuli and participant response data from threshold estimate testing.
	<ol>
		<li>Record the stopping point for each individual for both detection and discrimination</li>
		<li>Record the number of trials it took for the participant to successfully complete testing</li>
		<li>Record the participant&#39;s threshold average by finding the first time the participant was given the eventual stopping point stimulus. Add all the target stimuli delivered between the first instance of the stopping point target force and the last and divide by the total number of trials. This gives a threshold estimate average that takes in to account the variability within a participant.</li>
	</ol>
	</li>
	<li>Set up data recording sheets using one of the two suggested options. Find examples of completed sheets using the same values can be found in the Supplementary Materials. The first option (Supplementary Files 1 &#38; 2) provides a data sheet and completed example in which the participant&#39;s responses and threshold estimate can be easily visualized and recorded on one sheet. The second option (Supplementary Files 3 &#38; 4) provides a data sheet with randomized order for presenting the test stimuli. A separate sheet is needed for each test - detection or discrimination.</li>
</ol>
5. Cleaning equipment
<ol>
	<li>Do not close the monofilaments during testing or immediately following testing until they are cleaned.</li>
	<li>Place the nylon end of the monofilament between an alcohol prep pad (70% alcohol) and gently squeeze the alcohol pad together while pulling the monofilament through.</li>
	<li>Leave the monofilament in the open position, repeat the cleaning process on each monofilament used.</li>
	<li>Allow the monofilaments to air dry in the open position.</li>
	<li>Close the monofilaments and store them in a clean and dry environment, away from direct sunlight, as described in the manufacturer&#39;s directions.</li>
</ol>

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The authors Nicole M. Etter, Scott P. Breen, and Maya I.M. Alcala have no conflicts to disclose; Breen and Alcala were students at the time the work was performed and are now employed in the food industry. The authors John E. Hayes and Gregory R. Ziegler have each received speaking or consulting fees from non-profit organizations and corporate clients in the food industry. Additionally, the Sensory Evaluation Center (Hayes, Director) at Penn State routinely conducts taste tests for the food industry to facilitate experiential learning for students. None of these organizations have had any role in study conception, design or interpretation, or the decision to publish these data.

In previous studies, researchers observed that periodic rewetting of the tongue was an important step. For example, participants who did not regularly rewet the tongue demonstrated poorer sensory acuity. While no attempts were made to systematically determine the optimal rewetting interval, experience suggests experimenters should ask participants to rewet the tongue every few trials by bringing the tongue back into the mouth and/or having a small sip of water. Although work by Verrillo and colleagues found that vibrotactile detection thresholds were not impacted by skin hydration, these studies were completed on the hand, forearm, and cheek - not the tongue23. Because the tongue is consistently bathed in saliva, the change from wet to dry could alter sensory acuity. In fact, in work assessing salivary production and flavor appreciation in older adults, researchers found a relationship between decreased salivation and perception of umami flavor24. Aside from lingual moisture, changes in point pressure detection and discrimination threshold estimates can be impacted by a number of individual factors, including advancing age22 or changes in cognition25 which may impact attention to task. Additionally, some previous studies have either excluded smokers or asked participants to refrain from smoking for a period of time prior to testing26.
A blindfold was not used in this protocol as prior experience suggested participants find it distracting to be blindfolded with their mouths open and tongue out. In pilot testing of the protocol, participants were startled by the stimuli or repeatedly told researchers they couldn&#39;t concentrate. Therefore, participants were simply told to close their eyes. If a participant opened their eyes during a set of stimuli presentations, that set can be repeated. Other studies in the field have chosen to use a blindfold20; however, use of a blindfold may not significantly change the results when comparing across studies11.
During initial trials of this protocol, researchers selected a ceiling of 15.0 g. Although available monofilaments go up to 300 g, a ceiling was selected a priori to prevent the risk of damaging the skin. Additionally, when testing an area like the tongue for which there is no internal skeletal structure, application of monofilaments higher than 15 g were found to move the entire tongue muscle which could activate sensory nerve endings in surrounding locations17. In the representative data sample, many healthy young adults were able to sense the 0.008 g target monofilament - the lowest manufactured Von Frey hair monofilament. Alternatively, Cochet-Bonnet contact aesthesiometers have been used to measure sensitivity of the cornea. It is possible this device may provide an opportunity to test at levels less than 0.008 g; however, there are unresolved questions related to calibration of Cochet-Bonnet devices and ability to share findings across studies27.
This protocol is adapted from recent work by Etter and colleagues17 with a few key differences. In Etter&#39;s original protocol, threshold estimates were not averaged across trials. This may have missed some of the variability in participant responses. Additionally, Etter&#39;s original protocol was focused on using the tongue for speech-related movements, and therefore assessed point pressure of tongue at the right and left lateral edges just posterior to tongue tip versus midline tongue locations that may be more important for texture appreciation and swallowing28,29.
Future applications of this testing protocol may include studies of individuals with a variety of central and/or peripheral nervous system damage, as well as those with damage to the oral mucosa either from trauma, tumors, or post radiation therapy. For example, researchers recently evaluated individuals post-stroke who were experiencing oral dysphagia and noted increase thresholds, or decreased sensation in people with stroke as compared to healthy controls30. Additionally, in a review paper by Kaplan and colleagues, patients with xerostomia for a variety of medical reasons reported changes in mastication and swallowing, decreased salivary flow rates, and changes to oral mucosa31. Future work assessing the impact of these changes on tactile point pressure assessments would be beneficial to the field.
Finally, using this protocol does not require any mark to be made to the tongue to ensure exact testing location is repeated with each trial. This was originally done to increase clinical application of this protocol to medical and outpatient clinic locations. Reliability testing by Etter colleagues demonstrated high test-retest results without marking the tongue17. However, it is possible to mark the tongue using a dye to ensure a consistent target location as illustrated in a recent study by Santagiuliana and colleagues26. The appropriateness of using food dye to mark the tongue may vary depending on testing location and research question.

Any time we eat or drink, we determine the acceptability of a food based on multiple sensory percepts like taste, smell, and texture. Texture is not merely a physical property of the food; rather it arises from interactions of the food with the somatosensory (pressure and touch) system in the mouth. Flavor is the integrated percept that arises from multiple neuronal inputs, including taste, smell and oral touch1. Perception of food flavor, including pressure and texture information, is a key driver of food choice. Both common sense and data from numerous studies suggest people eat what they like2. However, in practice, this relationship between texture and food selection is more nuanced, as individuals avoid what they dislike3. Food selection is a behavior based on both cognitive choices and prior sensory experiences. Individual differences in smell and taste have a demonstrated ability to influence ingestive behavior4 with large variation across individuals to the point of influencing chronic diet choices5. Thus, two people may eat the same food but react very differently to it in regard to the degree of liking of that food. However, the degree to which this food preference is driven by individual differences in oral somatosensory function, including tactile and texture appreciation, remains understudied. In fact, the influences and mechanisms of oral point pressure and texture perception are much less understood relative to other oral sensory systems. Recent data suggests there may be important individual differences in oral tactile sensation abilities6,7,8. Because oral touch information is internalized and individual in nature9, it could drive individual preferences and influence food choices.
Measurements of peripheral nerve function through cutaneous pressure assessments require activation of slowly and rapidly adapting mechanoreceptors in the skin, including Merkel cells, Meissner corpuscles, Ruffini corpuscles and Pacinian corpuscles - with a high representation of slowly adapting mechanoreceptors in the face, lips, and oral mucosa that are responsive to pressure and slow stretch10,11. One relatively time and cost effective means of assessing oral tactile sensation is through the use of point-pressure assessments using Von Frey Hair (VFH) monofilaments. VFH monofilaments are widely used to assess peripheral nerve function through point pressure detection across the body, but specifically in glabrous (non-hairy) skin, including fingertip, hands, and feet12,13,14,15,16. In fact, testing with VFH monofilaments has demonstrated high test-retest reliability in the lips, tongue, and feet in healthy young, aging, and disordered populations16,17.
Assessing oral point-pressure detection and discrimination is potentially one part of a full assessment of a person&#39;s individual oral touch profile. Better understanding of individual oral touch responses could inform food choice preferences in healthy and disordered populations. A complete assessment of individual oral touch and texture perception could allow clinicians to improve recommendations for healthy aging adults as well as people with specialized (dysphagia) diets to meet their individual nutrition needs necessary for health and wellness. Select clinical populations with dysphagia, as well as typically aging adults, may require altered food textures to achieve adequate and safe nutrition; however, these clinical populations may also reject foods based on texture and mouth feel preferences18,19. Better understanding of the mechanisms underpinning food preferences that drive food choices, eating behavior, and diet compliance may provide novel targets for intervention, both at a systems level and an individual level.
The purpose of this assessment protocol is to characterize individual differences in oral point pressure (OPP) sensitivity by establishing detection and discrimination threshold estimates at midline tongue. This protocol uses Von Frey Hair monofilaments, commercially-available devices, to complete a relatively cost and time efficient assessment. Quantitative assessments of oral point pressure somatosensation using Von Frey hair monofilaments and this protocol were recently shown to be reliable in a cohort of healthy, young adults17 for lateral edge of the lips and tongue as a prelude to future work in speech and speech disorders. However, the present protocol and recent work has focused on midline tongue due to its involvement with manipulating foods for tasting and safe and efficient swallowing20.

<table><tbody><tr><td>plastic cup for drinking water</td><td>various</td><td></td><td></td></tr><tr><td>Aesthesio Tactile Sensory Evaluator Kit</td><td>DanMic Global LLC, San Jose CA</td><td>514000-20C</td><td></td></tr></tbody></table>

assessment of midline lingual point-pressure somatosensation using von frey hair monofilaments

Detection and discrimination threshold estimates for oral point pressure are assessed using Von Frey Hair monofilaments. Consistent with previously published protocols, threshold estimates are determined using a two-interval forced choice (2-IFC) paradigm with a three down/one up approach. Detection threshold estimates determine the mean force in which a participant can identify the presence of pressure. During the detection threshold procedure, the participant is instructed to choose which of two sequentially presented observation intervals contained the tactile test stimulus. If the participant performs three correct detections in a row (i.e., 3 &#39;hits&#39;), the researcher decreases the stimulus to the next lower target force level. With one incorrect detection (a &#39;miss&#39;), the researcher increases the force delivered to the next higher level. This threshold estimation approach is known as a 3-down/1-up adaptive staircase. Reponses are recorded on a paper ballot, and a participant&#39;s estimated threshold is defined as the geometric mean of five reversals. During the discrimination threshold procedure, the participant is asked to make a choice between two serially presented stimuli as to which is the &#34;harder&#34; or &#34;stronger&#34; pressure. The same scoring of &#39;hits&#39;, &#39;misses&#39;, and stopping points are used. Detection and discrimination testing for oral point pressure at tongue midline takes approximately 20 min to complete. Using these commercially-available clinical tools, individual touch sensation profiles for the midline tongue can be achieved in a relatively time and cost effective means.

When interpreting the results, it is important to remember that lower threshold estimates indicate greater sensory acuity. The use of a forced choice procedure helps separates criterion response bias (i.e., a willingness to say yes) from underlying sensitivity of the system. A lower threshold indicates an individual can perceive a lighter stimulus than those with a higher threshold estimate. Higher threshold estimates indicate decreased sensory acuity. A higher threshold estimate indicates an individual requires a larger input to cognitively perceive the stimulus. Higher threshold estimates could be due to difficulty in cognitively completing the task16,21 or due to peripheral damage associated with advancing age or disorder to the cutaneous surface of the tongue or peripheral nerves22.
Data is still being collected to further develop normative data from a sample of nominally healthy adults. However, a sample of data on individual differences in oral somatosensory function was recently published7. Combining previously published data with additional data, a total of 111 healthy participants (34M:77F; mean 32.1 years, range 18 to 68 years) completed at least one portion of the detection and/or discrimination threshold estimate protocol described here.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Detection</td>
			<td colspan="2">Discrimination</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">(n= 51)</td>
			<td colspan="2">(n= 107)</td>
		</tr>
		<tr>
			<td>Location</td>
			<td>M (SD)</td>
			<td>Range</td>
			<td>M (SD)</td>
			<td>Range</td>
		</tr>
		<tr>
			<td>Midline Tongue</td>
			<td>0.0157 (0.022)</td>
			<td>0.008-0.16</td>
			<td>0.600 (1.812)</td>
			<td>0.02-15</td>
		</tr>
	</tbody>
</table>
Table 1: Detection and discrimination threshold estimates for pressure point sensitivity on midline tongue. The geometric mean, standard deviation and range are provided from our representative sample. Fifty one participants completed detection threshold estimate testing (10M:41F, mean 37.1 years). A total of 107 participants completed discrimination threshold estimate testing (31M: 76F, mean 31.9 years).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60656/60656fig1.jpg" /> 
Figure 1: This figure presents data for midline lingual point pressure detection threshold estimate (g) by participant age (years). Note the y-axis ranges from 0.0 - 0.25 g. Participants across this healthy age span demonstrated low thresholds for point-pressure detection. <a href="https://www.jove.com/files/ftp_upload/60656/60656fig1large.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/60656/60656fig2.jpg" /> 
Figure 2: This figure provides data for midline lingual point pressure discrimination threshold estimate (g) by participant age (years). Note the y-axis ranges from 0.0 - 16.0 g. Participants across this healthy age span demonstrated increased variability in discrimination threshold estimates as compared to detection threshold estimates. <a href="https://www.jove.com/files/ftp_upload/60656/60656fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Materials 
Two potential options for setting up data recording sheets are available in the Supplementary Files. Data can easily be recorded and visualized using a one page data collection sheet (Supplementary files 1 &#38; 2). On this sheet, both detection and discrimination data can be recorded in their respective sections. The list of Von Frey Hair monofilament targets in grams (g) and the color of the pen are included on the left hand side. An asterisk (*) has been placed next to the 1.000 g target in the &#39;Detection&#39; section to remind testers of the starting point for testing using this protocol. The trial numbers are listed across the bottom to easily identify the total number of trials needed. The authors suggest following the key listed on the bottom right to mark correct and incorrect participant responses. A second data recording option is provided in Supplementary Files 3 &#38; 4. The series number is noted in the column marked #. The target or test stimuli is recorded in grams in the column labeled &#39;Target (g)&#39;. The next two columns are labeled &#39;Trial 1&#39; and &#39;Trial 2&#39; to indicate in which the trial the tester should deliver the test stimuli. This pattern was created using a random number generator. Experimenters will present the test stimuli in the trial with the &#34;X&#34; in it. For example, in the first series, the target stimuli would be delivered in the second trial because the &#34;X&#34; is under the Trial 2 heading. Finally, in the last column, the experimenter can record the participant&#39;s response as correct (Y) or incorrect (N). This column can also be used to note the number of presentations of that target to aide in identifying the stopping point.
Supplementary File 1. <a href="https://www.jove.com/files/ftp_upload/60656/supplementaryfile1.pdf" target="_blank">Please click here to view this file.</a>
Supplementary File 2. <a href="https://www.jove.com/files/ftp_upload/60656/supplementaryfile2.pdf" target="_blank">Please click here to view this file.</a>
Supplementary File 3. <a href="https://www.jove.com/files/ftp_upload/60656/supplementaryfile3.pdf" target="_blank">Please click here to view this file.</a>
Supplementary File 4. <a href="https://www.jove.com/files/ftp_upload/60656/supplementaryfile4.pdf" target="_blank">Please click here to view this file.</a>

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Assessment of Midline Lingual Point-Pressure Somatosensation Using Von Frey Hair Monofilaments

This work describes a standard method to assess tactile sensation at the midline of the tongue tip. Using Von Frey Hair (VFH) monofilaments, this protocol provides estimates of detection and discrimination threshold estimates for oral point pressure (OPP).

Detection and discrimination threshold estimates for oral point pressure are assessed using Von Frey Hair monofilaments. Consistent with previously ...

assessment-midline-lingual-point-pressure-somatosensation-using-von

Research

JoVE Journal

Behavior

8.2K Views.  The Pennsylvania State University. Dit protocol is belangrijk omdat het onderzoekers in staat stelt om gemakkelijk te beoordelen punt druk sensatie op de middellijn tong geeft ons inzicht in elk individu zintuiglijke profiel. Deze techniek kan overal worden uitgevoerd met behulp van eenvoudige, draagbare apparatuur. Deze apparaten zijn gemakkelijk te gebruiken en gemakkelijk te reinigen en zijn ook relatief tijd- en kostenefficiënt.Het doel van het gebruik van deze techniek is om te begrijpen hoe punt druk sensatie ver...

Video: Assessment of Midline Lingual Point-Pressure Somatosensation Using Von Frey Hair Monofilaments

Measurement of Vibration Detection Threshold and Tactile Spatial Acuity in Human Subjects

Tests that allow the precise determination of psychophysical thresholds for vibration and grating orientation provide valuable information about mechanosensory function that are relevant for clinical diagnosis as well as for basic research. Here, we describe two psychophysical tests designed to determine the vibration detection threshold (automated system) and tactile spatial acuity (handheld device). Both procedures implement a two-interval forced-choice and a transformed-rule up and down experimental paradigm. These tests have been used to obtain mechanosensory profiles for individuals from distinct human cohorts such as twins or people with sensorineural deafness.

Here, we present protocols to determine vibration detection thresholds and tactile acuity using psychophysical methods in man.

Tests that allow the precise determination of psychophysical thresholds for vibration and grating orientation provide valuable information about ...

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

Medicine

A Protocol of Manual Tests to Measure Sensation and Pain in Humans

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study demonstrates a quantitative sensory testing protocol using techniques to measure tactile sensation and pain threshold for pressure and heat using portable and easily accessed equipment. These techniques and equipment are ideal for new laboratories and clinics where cost is a concern or a limiting factor. We demonstrate measurement techniques for the following: cutaneous mechanical sensitivity on the arms and legs (von-Frey filaments), radiant and contact heat sensitivity (with both threshold and qualitative assessments using the Visual Analog Scale (VAS)), and mechanical pressure sensitivity (algometer, with both threshold and the VAS). The techniques and equipment described and demonstrated here can be easily purchased, stored, and transported by most clinics and research laboratories around the world. A limitation of this approach is a lack of automation or computer control. Thus, these processes can be more labor intensive in terms of personnel training and data recording than the more sophisticated equipment. We provide a set of reliability data for the demonstrated techniques. From our description, a new laboratory should be able to set up and run these tests and to develop their own internal reliability data.

The goal of this procedure is to demonstrate a battery of quantitative techniques for sensory and pain measurement in humans. The equipment and techniques described are commonly found in pain clinics or are easy to obtain.

Numerous qualitative and quantitative techniques can be used to test sensory nerves and pain in both research and clinical settings. The current study ...

Determining heat and mechanical pain threshold in inflamed skin of human subjects

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific nociceptive and inflammatory biochemicals in humans. In this article we will show pain-testing paradigms that are often used in tandem with microdialysis procedures. Combining pain tests with microdialysis provides the critical link between behavioral and biochemical data that allows identifying key biochemicals responsible for generating and propagating pain.
Two models of evoking pain in inflamed skin of human study participants are shown. The first model evokes pain with aid of heat stimuli. Heat evoked pain as described here is predominantly mediated by small, non-myelinated peripheral nociceptive nerve fibers (C-fibers). The second model evokes pain via punctuated pressure stimuli. Punctuated pressure evoked pain is predominantly mediated by small, myelinated peripheral nociceptive nerve fibers (A-delta fibers). The two models are mechanistically distinct and independently examine nociceptive processing by the two major peripheral nerve fiber populations involved in pain signaling.
Heat pain is evoked with aid of the TSA II, a commercially available thermo-sensory analyzer (Medoc Advanced Medical Systems, Durham, NC). Stimulus configuration and delivery is handled with aid of specific software. Thermodes vary in size and shape but in principle consist of a metal plate that can be heated or cooled at various rates and for different periods of time. Algorithms assessing heat-evoked pain are manifold. In the experiments shown here, study participants are asked to indicate at what point they start experiencing pain while the thermode in contact with skin is heated at a predetermined rate starting at a temperature that does not evoke pain. The thermode temperature at which a subject starts experiencing pain constitutes the heat pain threshold.
Mechanical pain is evoked with punctuated probes. Such probes are commercially available from several manufacturers (von Frey hairs). However, the accuracy of von Frey hairs has been criticized and many investigators use custom made punctuated pressure probes. In the experiments shown here eight custom-made punctuated probes of different weights are applied in consecutive order, a procedure called up-down algorithm, to identify perceptional deflection points, i.e., a change from feeling no pain to feeling pain or vice versa. The average weight causing a perceptional deflection constitutes the mechanical pain threshold.

Algorithms assessing heat and mechanical pain thresholds in experimentally inflamed skin of human study subjects are shown. The two pain testing paradigms independently examine nociceptive processing by the two major peripheral nerve fiber populations transmitting pain, i.e., non-myelinated C fibers and small myelinated A-delta fibers.

In a previous article in the Journal of Visualized Experiments we have demonstrated skin microdialysis techniques for the collection of tissue-specific ...

Biology

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain perception in rodents. Withdrawal thresholds can be measured first at baseline and then following drug, venom, injury, allergen,&#160;or otherwise evoked inflammation by applying an accurate force on very specific areas of the skin. An electronic von Frey apparatus allows precise assessment of mouse hind paw withdrawal thresholds that are not limited by the available filament sizes in contrast to classical von Frey measurements. The ease and rapidity of measurements allow for incorporation of assessment of tactile sensitivity outcomes in diverse models of rapid-onset inflammatory and neuropathic pain as multiple measurements can be taken within a short time period. Experimental measurements for individual rodent subjects can be internally controlled against individual baseline responses and exclusion criteria easily established to standardize baseline responses within and across experimental groups. Thus, measurements using an electronic von Frey apparatus represent a useful modification of the well-established classical von Frey filament-based assays for rodent mechanical allodynia that may also be applied to other nonhuman mammalian models.

Electronic von Frey measurements are an effective and improved alternative to classical von Frey filaments, as they allow rapid and precise measurement of changes in rodent mechanical withdrawal thresholds to continuously applied pressure stimuli before and after an inflammatory event.

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

Neuroscience

Assessment of Spatial Lingual Tactile Sensitivity using a Gratings Orientation Test

Individual thresholds by R-index estimates are calculated using a gratings orientation test (6 different tools of increasing grating size from 0.20-1.25 mm) to assess spatial lingual tactile sensitivity. During the experiment, the subjects are blindfolded and asked to specify the orientation of the grating (either horizontal or vertical) placed on the tongue. R-index is based on Signal Detection Theory (SDT), and it is an estimated probability of correctly identifying a target stimulus (the signal, e.g., the correct orientation) compared to an alternative stimulus (the noise, e.g., the incorrect orientation). Once the R-index values for each subject and each tool dimension are calculated, it is possible to derive the individual threshold by interpolating the two R-indices immediately below and above the established cut-off (typically 75%) based on one-sided R-index critical values. This procedure can be helpful in the medical field to study the association between oral tactile sensitivity, speech clarity, and swallowing disorders, as well as in sensory and consumer studies to explore individual variation in texture perception, food preferences, and eating behavior.

This work illustrates a standard procedure and threshold determination by the R-index to assess spatial lingual tactile sensitivity using a gratings orientation test.

Individual thresholds by R-index estimates are calculated using a gratings orientation test (6 different tools of increasing grating size from 0.20-1.25 ...

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 ...

Somatosensory Event-related Potentials from Orofacial Skin Stretch Stimulation

Cortical processing associated with orofacial somatosensory function in speech has received limited experimental attention due to the difficulty of providing precise and controlled stimulation. This article introduces a technique for recording somatosensory event-related potentials (ERP) that uses a novel mechanical stimulation method involving skin deformation using a robotic device. Controlled deformation of the facial skin is used to modulate kinesthetic inputs through excitation of cutaneous mechanoreceptors. By combining somatosensory stimulation with electroencephalographic recording, somatosensory evoked responses can be successfully measured at the level of the cortex. Somatosensory stimulation can be combined with the stimulation of other sensory modalities to assess multisensory interactions. For speech, orofacial stimulation is combined with speech sound stimulation to assess the contribution of multi-sensory processing including the effects of timing differences. The ability to precisely control orofacial somatosensory stimulation during speech perception and speech production with ERP recording is an important tool that provides new insight into the neural organization and neural representations for speech.

This paper introduces a method for obtaining somatosensory event-related potentials following orofacial skin stretch stimulation. The current method can be used to evaluate the contribution of somatosensory afferents to both speech production and speech perception.

Cortical processing associated with orofacial somatosensory function in speech has received limited experimental attention due to the difficulty of ...

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 &#181;g/kg, respectively) and concluded that a critical dose of RTX (50 &#181;g/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to ...

Assessment of Midline Lingual Point-Pressure Somatosensation Using Von Frey Hair Monofilaments

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