This work was supported by a grant from the Fondation Chiropratique du Qu&#233;bec and the Natural Sciences and Engineering Research Council of Canada (MP: grant #06659). The contribution of HK was supported by the Universit&#233; du Qu&#233;bec &#224; Trois-Rivi&#232;res (PAIR program). The contribution of BP was supported by the Fonds de recherche du Qu&#233;bec en Sant&#233; (FRQS) and the Fondation Chiropratique du Qu&#233;bec. The contribution of TP was supported by the Natural Sciences and Engineering Research Council of Canada. The contribution of NE and EK was supported by the Fondation Chiropratique du Qu&#233;bec. The contribution of MP was supported by the FRQS.

The experimental protocol was approved by the animal care committee of Universit&#233; du Qu&#233;bec &#224; Trois-Rivi&#232;res and conformed to the Guidelines of the Canadian Council on Animal Care and the Guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain (IASP). The present study used six male Wistar rats (body weight: 320-450 g; age: 18-22 weeks). The animals were obtained from a commercial source (see Table of Materials). Data from these rats are from the larger sample of a previous study30.
1. Experimental preparation
<ol>
	<li>House the animals in a temperature-controlled room in standard animal facilities with access to food and water ad libitum and a light-dark cycle of 14 h-10 h. Ensure that all animals are in good health on the day of the experiments.</li>
	<li>Generate the chronic back pain animal model following the steps below.
	<ol>
		<li>To induce chronic back pain, perform an intramuscular injection of Complete Freund Adjuvant (CFA) into the back muscles following the previous reports14,30,31.</li>
		<li>Anesthetize the animal using isoflurane (4% for induction and 2%-2.5% for maintenance).</li>
		<li>Using a 27 G needle, inject 150 &#181;L of a ready-to-use water-in-oil emulsion of CFA (see Table of Materials) into the paraspinal muscles unilaterally or bilaterally, depending on the protocol needs.</li>
		<li>Keep the injection needle in place for at least 3 min after completing the injection. For animals in the control group, use the same procedures30, but inject a solution of sterile physiological saline solution (150 &#181;L, 0.9%) instead of CFA.</li>
	</ol>
	</li>
	<li>Fabricate the test cage.
	<ol>
		<li>Make a test cage for two animals that comprises one chamber for each animal. 
		NOTE: For the present study, each chamber has the following dimensions: length x width x height: 50 x 20 x 7 cm (see Table of Materials).</li>
		<li>Mount the two contiguous chambers on four 33 cm long Plexiglass legs. Use transparent Plexiglass for the walls of the chambers, but use black Plexiglass to separate chambers to prevent animals from seeing each other.</li>
		<li>Use stainless-steel mesh made of 1 mm wire with an 8 mm inter-wire distance to make the floor and ceiling of the test cage (Figure 1).</li>
	</ol>
	</li>
</ol>
2. Back Mechanical Sensitivity (BMS) test
<ol>
	<li>Familiarize the animal with the test cage 30 min/day for 5-7 consecutive days prior to the first test. Repeat the test as needed.</li>
	<li>Anesthetize the animals using 2% isoflurane31 (see Table of Materials).</li>
	<li>In a prone position under isoflurane anesthesia, shave the back hair of the area of interest (from T6 to L6 vertebral levels) using an animal hair trimmer (see Table of Materials). For repeated measures, shave the back hair every 3 days on a day without behavioral assessment to ensure that stimuli are always applied directly to the skin. Draw a black mark on the skin with a permanent marker to ensure that filaments are always applied to the same area when repeating the test on different days.</li>
	<li>On the testing day, put the animals in the test cage for 15-30 min prior to the test until the animal is calm.</li>
	<li>During the test, apply Von Frey filaments (0.07, 0.16, 0.4, 0.6, 1, 2, 4, 6, 10, 15, and 26 g) perpendicularly to the back, always beginning with the 2 g filament and using the up-down method15 (see Table of Materials). Approach the back of the animal slowly with the filament from behind the animal.
	<ol>
		<li>Apply the filament only when the animal is awake, standing on its four paws, and not moving. Apply the filament for 2 s bilaterally to the area of interest, 10 mm from the spinous process (Figure 2), every 15-30 s. 
		NOTE: A response is considered positive if the animal exhibits one or more of the following behaviors during or immediately after the filament is applied: (1) muscle twitching, (2) arching (back extension), (3) rotation of the neck to look at the back, (4) scratching or licking the back, and (5) escaping.</li>
	</ol>
	</li>
	<li>As described previously15, if no response is observed with the application of a filament, apply the next filament with a higher force in the series. If a response is observed, use the next filament with a lower force in the series. Continue this procedure until four readings are obtained after the first behavioral change (response after a series of &#34;no response&#34; or no response after a series of &#34;response&#34;).</li>
	<li>Once data collection is completed, calculate the value representing 50% of the mechanical threshold, as described by Chaplan et al.15, using this formula: 
	50%&#160;threshold (g) = 10(Xf+k&#948;)/10 000 
	&#8203;NOTE: In this formula, &#34;Xf&#34;&#160;is the Handle Mark of the last von Frey filament that was used. &#34;k&#34; is the tabular value based on the animal&#39;s response pattern15, and &#34;&#948;&#34; is the mean of Handle Mark&#39;s increments between Von Frey filaments. Depending on the experimental design and experimental needs, only one side of the spine may be assessed to report one threshold, or two sides may be evaluated, and thresholds are reported separately or as a mean. Refer to Supplementary Table 1 for the calculation template32.</li>
</ol>
3. Animal recovery
<ol>
	<li>After the intramuscular injection is completed, discontinue anesthesia and place the animal alone in a standard housing cage for recovery.</li>
	<li>During the recovery period, examine the animal&#39;s behavior and do not leave it unattended.</li>
	<li>Confirm that the animal recovers from anesthesia and moves normally within 5 min. Then, return the animal to its usual housing cage with the other animals. 
	NOTE: At the end of the experiment, the animal is perfused through the heart with a 10% formalin solution, under deep isoflurane anesthesia (5%). The back muscles in the injected area are then extracted for histology and confirmation of inflammatory changes.</li>
</ol>

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The authors declare no competing interests or relationships that may lead to any conflicts of interest.

Critical steps 
The BMS test is a simple method to assess mechanical sensitivity in the back of rats, either at one time point or repeatedly over days or weeks, when changes are expected to occur (pain models) or after pharmacological or non-pharmacological intervention. Critical issues of the method include the test cage, whose dimensions must ensure that the rat is comfortable but does not move too much. The animal&#39;s back must stay accessible through the mesh ceiling for reproducible mechanical stimulation. To limit variability in the threshold assessment, the back area under investigation must be shaved so that mechanical stimuli are applied directly to the skin. In addition, the skin needs to be marked for mechanical stimuli to be applied to the same area. Lastly, the experimenter must approach the filament to the skin from behind the animal to avoid being seen by the animal.
Compared with the Von Frey test used for assessing the mechanical sensitivity at the hind paw15,16, the mechanical force needed to produce a positive response in the BMS test is lower. The filaments used for the test should be selected carefully. Using the following filaments should cover most experimental needs (0.07, 0.16, 0.4, 0.6, 1, 2, 4, 6, 10, 15, and 26 g) and prevent facing a ceiling or a floor effect. In this case, the 2 g filament is used for the first application. This can be adapted to experimental needs as long as the calculation is adjusted accordingly.
Modifications and troubleshooting 
During a pilot experiment, the ideal area for the test was determined. Because of the shape of the rat&#39;s body, the thoracolumbar region is the most accessible area in the test cage. If there is no reason for the test to be performed in other regions of the spine, this is the area of choice for applying mechanical stimuli. The lumbar area is also easily accessible. When deciding which area to test, it must be kept in mind that the filament must be applied perpendicularly to the surface and bend properly to deliver the pre-determined calibrated force.
Limitations 
The experimenter must be trained to observe the behaviors associated with the test. The five positive responses include muscle twitching, arching, neck rotation to look at the back, licking or scratching the back, and escaping30. While most of these responses are easily observable, muscle twitching is sometimes subtle for stimuli of a lower force. Also, the rat may move spontaneously in the cage, so this must not be confounded with escaping, which occurs specifically when the filament is applied. To avoid confounding both behaviors, the experimenter must wait for the animal to be calm for at least a few seconds.
Significance and potential applications 
Several rodent models of back pain are used in current investigations8. Surprisingly, however, no standardized behavioral test was validated to assess mechanical sensitivity in back pain models. This is critical to confirm that animals with presumed back pain present local hypersensitivity to nociceptive stimuli, and to monitor sensitivity during interventions designed to relieve back pain. The BMS test presented here provides a simple and accessible solution for these purposes. Although it was developed for rats30, it may be adapted to other laboratory animals in the future.

Low back pain (LBP) is the leading cause of disability worldwide, which has dramatic personal, economic, and social consequences1,2,3,4. Every year, approximately 37% of the population is affected by LBP5. LBP usually resolves within a few weeks but recurs in 24%-33% of individuals, becoming chronic in 5%-10% of cases2. To understand the mechanisms and impacts of LBP as well as the effects of different therapeutic interventions, several animal models of LBP have been used, mimicking clinical conditions or some components of LBP6. These mouse and rat models can be classified in one or more of the following categories: (1) discogenic LBP7,8,, (2) radicular LBP8,9,10,11, (3) facet joint osteoarthritis12, and (4) muscle-induced LBP13,14. Since the pain cannot be measured directly in non-human species, numerous tests have been developed to quantify pain-like behaviors in these models8. These tests assess behaviors evoked by a noxious stimulus (mechanical force15,16,17, thermal stimulation18,19,20,21,22,23,24,25) or produced spontaneously26,27,28,29.
The methods using mechanical stimuli include the Von Frey test15,16 and the Randall-Selitto Test17. Methods using heat stimuli include the tail flick test18, hot plate test19, Hargreaves test20, and thermal probe test21. Methods using cold stimuli include the cold plate test22, acetone evaporation test23, and cold plantar assay24. Methods for spontaneous behaviors include the grimace scales26, burrowing27, weight-bearing and gait analysis28, as well as an automated behavioral analysis29. Despite these numerous available tests, none of them is designed specifically for back pain models.
The objective of this study is to lay down a simple and accessible test to assess mechanical sensitivity in the back of rats. The technique is largely based on the Von Frey test applied to the plantar surface of the hind paw15,16. The basic principle of the Von Frey test is to use a series of monofilaments to the region of interest, delivering constant pre-determined forces. A response is considered positive if the rat shows a nocifensive behavior. The mechanical threshold can then be calculated based on the filaments that evoked responses. In the present study, a simple and accessible method adapted from the Von Frey test is provided to determine mechanical sensitivity in the back of rats.

<table><tbody><tr><td>Aerrane (isoflurane, USP) - Veterinary Use Only</td><td>Baxter</td><td>NDC 10019-773-60</td><td>Inhalation Anaesthetic ; DIN 02225875, for inducing anasthesia</td></tr><tr><td>Complete Freund Adjuvant (CFA)</td><td>Fisher Scientific</td><td>#77140</td><td>Water-in-oil emulsion of Complete Freund Adjuvant (CFA) with killed cells of &lt;em&gt;Mycobacterium butyricum&lt;/em&gt;.</td></tr><tr><td>Male Wistar Rats</td><td>Charles River Laboratories</td><td></td><td>body weight: 320&amp;ndash;450 g; age: 18-22 weeks.</td></tr><tr><td>Penlon Sigma Delta Vaporizer</td><td>Penlon</td><td>990-VI5K-SVEEK</td><td>Penlon Sigma Delta Vaporizer used for anasthesia</td></tr><tr><td>Sharpie Permanent Marker</td><td>Sharpie</td><td>BC23636</td><td>Permanent Marker, Fine Point, Black</td></tr><tr><td>Test cage</td><td>Custom-made</td><td></td><td>Width: 20 cm;&amp;nbsp; Length: 50 cm; Height from the bottom to the top: 40 cm; Height from the bottom mesh to the top of the cage: 7 cm; Wall thickness: 5 mm; Mesh: 1 mm wire with an 8 mm inter-wire distance&amp;nbsp;&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Von Frey Filaments</td><td>Aesthesio, Precise Tactile Sensory Evaluator</td><td>514000-20C</td><td>Filaments from 0.07 g to 26 g</td></tr><tr><td>Wahl Professional Animal, ARCO Cordless Pet Clipper, Trimmer Grooming&amp;nbsp;</td><td>Wahl</td><td>Kit #8786-1201</td><td>Animal hair trimmer, for shaving purposes, zero blade&amp;nbsp;</td></tr></tbody></table>

back mechanical sensitivity assessment in the rat for mechanistic investigation of chronic back pain

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

The method was used in a previous study, in which full data and statistics were presented to compare back mechanical sensitivity between CFA and control rats30. Representative individual data (mean of left and right thresholds) from six rats included in the previous study are presented in Figure 3 and Table 1. At baseline, mechanical sensitivity was similar between groups. Intramuscular injection of CFA in the lumbar muscles caused a marked increase in mechanical sensitivity (decreased threshold) from 7 days to 28 days after CFA injection. In contrast, the control (CTL) rats did not show this change. As shown in Figure 3, variability was observed within and between animals, as expected with this type of behavioral assessment. However, hypersensitive CFA rats showed decreased variability. Based on the previous study30, 16 animals (8 CFA and 8 CTL) is sufficient to detect a significant effect between groups over time (&#951;2p = 0.38) for 5 time points.
In this study, the presence of chronic inflammatory changes in the muscles injected with CFA was confirmed by histological examination (Figure 4)30. Also, mechanical hypersensitivity was observed at the hind paw with a standard Von Frey test, in addition to the back (Figure 5)30. In previous studies with the same back pain model, we showed increased spontaneous pain behavior and neuroinflammatory and neurophysiological changes14,31. Indeed, licking behaviors were increased in CFA compared with control rats during the formalin test, and single-unit responses to noxious stimulation of the sciatic nerve were altered in the right amygdala31. In addition, NF-kB protein expression was increased in the spinal cord of CFA compared with control rats14. Together, the results of these studies validate this chronic back pain model, and the present study visually demonstrates how to confirm the presence of mechanical hypersensitivity in the back of this rat model.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63667/63667fig01.jpg" /> 
Figure 1: Back Mechanical Sensitivity (BMS) test cage. (A) Schematic drawing of the test cage. (B) Custom-made test cage comprising two chambers, one for each animal. (C) Lateral view of the test cage with a rat in one of the chambers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63667/63667fig01large.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/63667/63667fig02.jpg" /> 
Figure 2: Back Mechanical Sensitivity assessment. The experimenter approaches the animal from behind and applies the Von Frey filament to the area of interest, 10 mm laterally from the spinous process. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63667/63667fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63667/63667fig03.jpg" /> 
Figure 3: Individual examples of back mechanical sensitivity. Back mechanical sensitivity in CFA and control (CTL) rats, at Baseline and 7, 14, 21, and 28 days after the intramuscular injection of CFA or saline, respectively. Individual data are shown by gray (CTL) and black (CFA) filled circles. Horizontal bars indicate the means. Error bars denote the standard error of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63667/63667fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63667/63667fig04.jpg" /> 
Figure 4: Histological confirmation of chronic muscle inflammation. Individual examples of back muscles from CFA rats and controls30. (A) Healthy back muscle from a control rat 14 days after intramuscular injection of saline. (B-C) Back muscles from two CFA-treated rats showed chronic inflammation 14 days after intramuscular CFA injection, with a clear leukocyte infiltration. Hematoxylin-eosin coloration was used for staining the muscle slices. Scale bar = 250 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63667/63667fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63667/63667fig05.jpg" /> 
Figure 5: Mechanical hypersensitivity in CFA rats30. Time course of mechanical sensitivity over 4 weeks, following either CFA (n = 8) or saline (n = 8) injection into the back muscles (L5-L6 level). (A-B) Mechanical sensitivity under the left and right hind paws. Mechanical thresholds significantly reduced in CFA compared with control rats (P &#60; 0.01) over time. This effect was not significantly different between left and right hind paws (P = 0.7). For both hind paws combined, the Tukey HSD test revealed lower mechanical thresholds in CFA compared with control rats, from 1 week to 4 weeks after injection (all P&#39;s &#60; 0.03). Time courses for separate hind paws are shown for illustration purposes only (interaction not significant, see results for details). (C-D) Mechanical sensitivity in the back. Mechanical thresholds significantly reduced in CFA compared with control rats (P &#60; 0.001) over time. This effect was not significantly different between left and right assessment sites (P = 0.3). For left and right assessment sites combined, the Tukey HSD test revealed a lower mechanical threshold in CFA compared with control rats, from 1 week to 4 weeks after injection (all P&#39;s &#60; 0.05). Time courses for separate hind paws are shown for illustration purposes only (interaction not significant, see results for details). In panel (D), the individual data of one CFA rat is not shown at the baseline (9.6 g) for illustration purposes. Shaded areas represent baseline assessment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63667/63667fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Rat</td>
			<td>Group</td>
			<td>Baseline</td>
			<td>Day 7</td>
			<td>Day 14</td>
			<td>Day 21</td>
			<td>Day 28</td>
		</tr>
		<tr>
			<td>1</td>
			<td></td>
			<td>2.34</td>
			<td>0.29</td>
			<td>0.12</td>
			<td>0.29</td>
			<td>0.29</td>
		</tr>
		<tr>
			<td>2</td>
			<td>CFA</td>
			<td>1</td>
			<td>0.48</td>
			<td>0.05</td>
			<td>0.48</td>
			<td>0.08</td>
		</tr>
		<tr>
			<td>3</td>
			<td></td>
			<td>1.26</td>
			<td>0.05</td>
			<td>0.05</td>
			<td>0.05</td>
			<td>0.19</td>
		</tr>
		<tr>
			<td>Mean &#177; SD</td>
			<td></td>
			<td>1.53 &#177; 0.58</td>
			<td>0.27 &#177; 0.18</td>
			<td>0.07 &#177; 0.03</td>
			<td>0.27 &#177; 0.18</td>
			<td>0.19 &#177; 0.09</td>
		</tr>
		<tr>
			<td>4</td>
			<td></td>
			<td>1.59</td>
			<td>2.61</td>
			<td>0.64</td>
			<td>3.26</td>
			<td>2.45</td>
		</tr>
		<tr>
			<td>5</td>
			<td>CTL</td>
			<td>1.15</td>
			<td>0.63</td>
			<td>3.41</td>
			<td>2.3</td>
			<td>1.29</td>
		</tr>
		<tr>
			<td>6</td>
			<td></td>
			<td>0.43</td>
			<td>1.26</td>
			<td>0.77</td>
			<td>0.32</td>
			<td>2.09</td>
		</tr>
		<tr>
			<td>Mean &#177; SD</td>
			<td></td>
			<td>1.06 &#177; 0.48</td>
			<td>1.50 &#177; 0.83</td>
			<td>1.61 &#177; 1.28</td>
			<td>1.96 &#177; 1.22</td>
			<td>1.94 &#177; 0.48</td>
		</tr>
	</tbody>
</table>
Table 1: Individual examples of back mechanical sensitivity in CFA and control rats.
Supplementary Table 1: Determination of mechanical threshold. This template table is used to calculate the mechanical threshold. The pattern of responses (X/O) is noted, and the values needed for the calculation are entered for Xf and k only, corresponding to the Handle Marking of the last filament that was used for the test and to the k-value associated with the response pattern, in this case, XX followed by OOXXO. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63667/63667_Supplementary Table 1.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Back Mechanical Sensitivity Assessment in the Rat for Mechanistic Investigation of Chronic Back Pain at JoVE.com

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

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

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

back-mechanical-sensitivity-assessment-rat-for-mechanistic

Nanyang Technological University

Research

JoVE Journal

Behavior

2.6K Views.  Université du Québec à Trois-Rivières. To develop novel therapeutic interventions for the prevention and management of back pain, animal models are required to examine the mechanisms and effectiveness of these therapies from a translational perspective. The present protocol describes the BMS test, a standardized method to assess back mechanical sensitivity in the rat.

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

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

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

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

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

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

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

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

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

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

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 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 ...

Urokinase-type Plasminogen Activator-induced Mouse Back Pain Model

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid induction of a persisting back pain model in mice are provided here using an injection of urokinase-type plasminogen activator (urokinase), a serine protease present in humans and other animals. The methodology for induction of persisting lower back pain in mice involves a simple injection of urokinase along the ligamentous insertion region of the lumbar spine. The urokinase inflammatory agent activates plasminogen to plasmin. Typically, the model can be induced within 10 min and hypersensitivity persists for at least 8 weeks.
Hypersensitivity, gait disturbance, and other standard anxiety- and depression-like measures can be tested in the persisting model.&#160;Back pain is the most prevalent type of pain. To improve awareness of back pain, the International Association for the Study of Pain (IASP) named 2021 the &#34;Global Year about Back Pain&#34; and 2022 the &#34;Global Year for Translating Pain Knowledge to Practice.&#34; One limitation of the therapeutic advancement of pain therapeutics is the lack of suitable models for testing persistent and chronic pain. The&#160;features of this model&#160;are suitable for testing potential therapeutics aimed at the reduction of back pain and its ancillary characteristics, contributing to IASP&#39;s naming 2022 as the Global Year for Translating Pain Knowledge to Practice.

Methods for simple, rapid induction of a back pain model in mice are provided here using an intraligament injection of urinary plasminogen activator.

A model of persisting lower back pain can be induced in mice with the simple methodology described herein. Step-by-step methods for simple, rapid ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

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 ...

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 ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...