We are grateful to Drs. Steven G. Shimada and Robert H. LaMotte (Yale University School of Medicine, Connecticut) from whose methods (reference 4) this technique was adapted. We also thank Mr. Kentaro Miyahara for creating the mouse illustrations. This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan (Nos. 15H02501 and 15H05928 to M.T., and No. 16K21691 to H.K.) and Uehara Memorial Foundation (to H.K.).&#160;We appreciate the introduction of the cheek injection method by Prof. LaMotte in Yale University.

All procedures involving the care and use of animals were approved by the institutional Animal Care and Use Committee of the National Institute of Natural Sciences (16A074), and carried out in accordance with the guidelines of the National Institute for Physiological Sciences.
NOTE: The original protocol was reported by Steven G. Shimada and Robert H. LaMotte4, and the present report describes these methods with several modifications, including the addition of a screen to prevent the animals from seeing each other, the absence of a mirror, the number of mice per recording, mouse strain, cage size, and recording time.
1. Cheek Injection Model
<ol>
	<li>Experiment preparation
	<ol>
		<li>Cage
		<ol>
			<li>Set four cages under a camera as shown in Figure 1. Set a screen to prevent the mice from seeing each other. Put in each mouse to each cage and set an acrylic lid to prevent escape of mice. Note that recordings should be performed in an environment in which temperature, humidity and sound are controlled. 
			NOTE: Behaviors can also be recorded without a lid if the cage is sufficiently high.</li>
		</ol>
		</li>
		<li>Needle and syringe
		<ol>
			<li>Use a 29 G or 30 G needle attached to a 0.5 mL syringe for the injection of test substances. If the test substance solution is highly viscous, larger needles are required. However, please note that large needles pose additional physical side effects. 
			NOTE: A needle connected to a syringe with a polyethylene tube can be used.</li>
		</ol>
		</li>
		<li>Video camera
		<ol>
			<li>Use a video camera having sufficiently large frame rates (30-60 frames/s) with relatively high resolution for the measurement of scratch bouts and wiping behaviors.</li>
		</ol>
		</li>
		<li>Software for video analysis
		<ol>
			<li>Use a video editing software for the video playback and analysis. Note that a fast-forward mode and a frame-by-frame playback mode are needed for precise analysis.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58943/58943fig1.jpg" /> 
Figure 1: Video camera and cage set-up. Four acrylic cages with acrylic lids, for preventing escape of the mice, and a screen for preventing contagious itch are used. <a href="https://www.jove.com/files/ftp_upload/58943/58943fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Injection and recording
	<ol>
		<li>One day prior to the experiment, shave the fur on the mouse&#39;s cheek with hair clippers (Movie 1). Before shaving, do not restrain the mice. 
		NOTE: CD-1 or C57BL/6 mice were mostly used in this model4,5. In this article, male 6-12 week-old C57BL/6N mice were used. Note that sensitivity to the chemicals could be different between the various mouse strains3,6,7.</li>
		<li>One day after shaving, move the mouse to the recording cage for 1 h to acclimatize it to the recording conditions. If a topical application is used, apply it during this acclimation period. 
		NOTE: In this report, 10 &#956;L of 2% (w/v) diphenhydramine hydrochloride salt in saline was topically applied 30 min prior to recording.</li>
		<li>Set a video camera above the cage, and start the video recording before the test substance injection. Inject a 10 &#956;L solution of the test substance intradermally into the shaved cheek under awake conditions (Movie 2). After the injection, return the mouse to the recording cage. 
		NOTE: In this report, 50 &#956;g/10 &#956;L histamine, 40 &#956;g/10 &#956;L capsaicin or vehicle (7% tween 80 in saline) were used as test substances.</li>
		<li>Close the lid of the cage and continue video recording for 30 min. 
		NOTE: The total recording time will depend on the experimental conditions.</li>
	</ol>
	</li>
	<li>Behavioral analysis
	<ol>
		<li>Count scratch bouts and wiping behaviors, respectively. Mice raise a hind leg towards the cheek and scratch several times over one or a few seconds, then put their leg down. This series of actions is counted as one scratch bout. Scratching of other areas should not be counted. Mice use a single foreleg for wiping and both forelegs for grooming. These actions should be distinguished. Only wiping behaviors are counted as pain-related behaviors. 
		NOTE: Each action is shown in Figure 2, Movie 3, Movie 4, and Movie 5.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58943/58943fig2.jpg" /> 
Figure 2: Typical mouse activity during recording. Panels A-C are illustrations representing wiping behavior with the right foreleg (A), scratching behavior with the right hind leg (B) and grooming behavior with both forelegs (C). Mice raise a hind leg towards the cheek and scratch several times over one or a few seconds, then put that leg down. This series of actions is counted as one scratch bout. Scratching of other areas should not be counted. Mice use a single foreleg for wiping and both forelegs for grooming. These actions should be distinguished. Only wiping behaviors are counted as pain-related behaviors. <a href="https://www.jove.com/files/ftp_upload/58943/58943fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Neck Injection Model
<ol>
	<li>Injection and recording
	<ol>
		<li>One day prior to the experiment, shave the fur on the nape of the mouse&#8217;s neck with hair clippers after approximately 5% isoflurane inhalation for 2-3 min.</li>
		<li>One day after shaving, move the mouse to the recording cage for 1 h to acclimatize it to the recording conditions.</li>
		<li>Set a video camera above the cage (Figure 1), and start the video recording before the test substance injection. Inject a 10 &#956;L solution of the test substance intradermally into the shaved neck under awake conditions. After the injection, return the mouse to the recording cage. 
		NOTE: In this article, 50 &#956;g/10 &#956;L histamine, 10 &#956;g/10 &#956;L or 40 &#956;g/10 &#956;L capsaicin, or vehicle (7% tween 80 in saline) were used as test substances.</li>
		<li>Close the lid of the cage and continue video recording for 30 min. 
		NOTE: Total recording time will depend on the experimental conditions.</li>
	</ol>
	</li>
	<li>Behavioral analysis
	<ol>
		<li>Count scratch bout behaviors. Mice raise a hind leg towards the neck and scratch several times over one or a few seconds, then put that leg down. This series of actions is counted as one scratch bout. Scratching of other areas should not be counted. 
		&#8203;NOTE: The action of scratching is similar to that in the cheek injection model, except that the location of the scratching is shifted to the neck.</li>
	</ol>
	</li>
</ol>

<ol><li>Ikoma, A., Steinhoff, M., Stander, S., Yosipovitch, G., Schmelz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neurobiology+of+itch%5BTitle%5D)+AND+%22Nature+Reviews+Neuroscience%22%5BJournal%5D)">The neurobiology of itch.</a> Nature Reviews Neuroscience. 7 (7), 535-547 (2006).</li>
<li>Kuraishi, Y., Nagasawa, T., Hayashi, K., Satoh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scratching+behavior+induced+by+pruritogenic+but+not+algesiogenic+agents+in+mice%5BTitle%5D)+AND+%22European+Journal+of+Pharmacology%22%5BJournal%5D)">Scratching behavior induced by pruritogenic but not algesiogenic agents in mice.</a> European Journal of Pharmacology. 275 (3), 229-233 (1995).</li>
<li>Kuraishi, Y., Yamaguchi, T., Miyamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Itch-scratch+responses+induced+by+opioids+through+central+mu+opioid+receptors+in+mice%5BTitle%5D)+AND+%22Journal+of+Biomedical+Science%22%5BJournal%5D)">Itch-scratch responses induced by opioids through central mu opioid receptors in mice.</a> Journal of Biomedical Science. 7 (3), 248-252 (2000).</li>
<li>Shimada, S. G., LaMotte, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+differentiation+between+itch+and+pain+in+mouse%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Behavioral differentiation between itch and pain in mouse.</a> Pain. 139 (3), 681-687 (2008).</li>
<li>Kittaka, H., Uchida, K., Fukuta, N., Tominaga, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lysophosphatidic+acid-induced+itch+is+mediated+by+signalling+of+LPA5+receptor%2C+phospholipase+D+and+TRPA1%2FTRPV1%5BTitle%5D)+AND+%22The+Journal+of+Physiology%22%5BJournal%5D)">Lysophosphatidic acid-induced itch is mediated by signalling of LPA5 receptor, phospholipase D and TRPA1/TRPV1.</a> The Journal of Physiology. 595 (8), 2681-2698 (2017).</li>
<li>Maekawa, T., Nojima, H., Kuraishi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Itch-associated+responses+of+afferent+nerve+innervating+the+murine+skin%3A+different+effects+of+histamine+and+serotonin+in+ICR+and+ddY+mice%5BTitle%5D)+AND+%22The+Japanese+Journal+of+Pharmacology%22%5BJournal%5D)">Itch-associated responses of afferent nerve innervating the murine skin: different effects of histamine and serotonin in ICR and ddY mice.</a> The Japanese Journal of Pharmacology. 84 (4), 462-466 (2000).</li>
<li>Inagaki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scratching+behavior+in+various+strains+of+mice%5BTitle%5D)+AND+%22Skin+Pharmacology+and+Applied+Skin+Physiology%22%5BJournal%5D)">Scratching behavior in various strains of mice.</a> Skin Pharmacology and Applied Skin Physiology. 14 (2), 87-96 (2001).</li>
<li>Inami, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Topical+surfactant-induced+pruritus%3A+involvement+of+histamine+released+from+epidermal+keratinocytes%5BTitle%5D)+AND+%22Journal+of+Pharmacology+and+Experimental+Therapeutics%22%5BJournal%5D)">Topical surfactant-induced pruritus: involvement of histamine released from epidermal keratinocytes.</a> Journal of Pharmacology and Experimental Therapeutics. 344 (2), 459-466 (2012).</li>
<li>Vos, B. P., Hans, G., Adriaensen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Behavioral+assessment+of+facial+pain+in+rats%3A+face+grooming+patterns+after+painful+and+non-painful+sensory+disturbances+in+the+territory+of+the+rat%27s+infraorbital+nerve%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Behavioral assessment of facial pain in rats: face grooming patterns after painful and non-painful sensory disturbances in the territory of the rat's infraorbital nerve.</a> Pain. 76 (1-2), 173-178 (1998).</li>
<li>Yeo, J. F., Ong, W. Y., Ling, S. F., Farooqui, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intracerebroventricular+injection+of+phospholipases+A2+inhibitors+modulates+allodynia+after+facial+carrageenan+injection+in+mice%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Intracerebroventricular injection of phospholipases A2 inhibitors modulates allodynia after facial carrageenan injection in mice.</a> Pain. 112 (1-2), 148-155 (2004).</li>
<li>Yeo, J. F., Ling, S. F., Tang, N., Ong, W. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antinociceptive+effect+of+CNS+peroxynitrite+scavenger+in+a+mouse+model+of+orofacial+pain%5BTitle%5D)+AND+%22Experimental+Brain+Research%22%5BJournal%5D)">Antinociceptive effect of CNS peroxynitrite scavenger in a mouse model of orofacial pain.</a> Experimental Brain Research. 184 (3), 435-438 (2008).</li>
<li>Hashimoto, T., Ohata, H., Momose, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Itch-scratch+responses+induced+by+lysophosphatidic+acid+in+mice%5BTitle%5D)+AND+%22Pharmacology%22%5BJournal%5D)">Itch-scratch responses induced by lysophosphatidic acid in mice.</a> Pharmacology. 72 (1), 51-56 (2004).</li>
<li>Kremer, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lysophosphatidic+acid+is+a+potential+mediator+of+cholestatic+pruritus%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Lysophosphatidic acid is a potential mediator of cholestatic pruritus.</a> Gastroenterology. 139 (3), 1008-1018 (2010).</li>
<li>Alemi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+TGR5+receptor+mediates+bile+acid-induced+itch+and+analgesia%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">The TGR5 receptor mediates bile acid-induced itch and analgesia.</a> Journal of Clinical Investigation. 123 (4), 1513-1530 (2013).</li>
<li>Nieto-Posadas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lysophosphatidic+acid+directly+activates+TRPV1+through+a+C-terminal+binding+site%5BTitle%5D)+AND+%22Nature+Chemical+Biology%22%5BJournal%5D)">Lysophosphatidic acid directly activates TRPV1 through a C-terminal binding site.</a> Nature Chemical Biology. 8 (1), 78-85 (2011).</li>
<li>Akiyama, T., Carstens, M. I., Carstens, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+itch-+and+pain-related+behavioral+responses+and+%C2%B5-opoid+modulation+in+mice%5BTitle%5D)+AND+%22Acta+Dermato-Venereologica%22%5BJournal%5D)">Differential itch- and pain-related behavioral responses and µ-opoid modulation in mice.</a> Acta Dermato-Venereologica. 90 (6), 575-581 (2010).</li>
</ol>

The authors have nothing to disclose.

In this article, a cheek injection model is introduced and shown to be useful for the analysis of pain and itch-related behaviors.
A previous report showed that algesic substances induce scratching behaviors in a mouse neck injection model4. In contrast, capsaicin reduced scratch behaviors in other neck injection experiments (Figure 3). Therefore, the effect of pain on scratching behavior remains unclear. It is thus necessary to develop a method to concurrently investigate pain and itch through behavioral tests. By using a cheek injection model, the effect of a substance on both pain and itch can be evaluated.
It has previously been reported that intracellular LPA directly activates TRPV1 and TRPA1 as a mechanism of LPA-induced itch5. Accordingly, LPA-induced itch could be histamine independent. A previous study also showed that a cheek injection model can be used for the evaluation of histamine-independent itch16. Therefore, this model can be used for both histamine-dependent and -independent itch.
One of the critical steps in this experiment is the injection. Because this is an acute behavioral test, anesthesia cannot be used in the injection step as it might affect these behaviors. Therefore, the mouse needs to be held securely in order to perform a precise injection. When the hold on the animal is not secure, especially when injecting strong algogens, the mice may struggle, leading to an inaccurate injection volume and depth, followed by difficulty in interpreting the results. When handling a mouse, pull the skin on the nape between the thumb and forefinger to inhibit movement of the mouse.
Another critical step is the behavioral analysis. Because scratching is very rapid, the video needs to be watched carefully. Moreover, wiping can be difficult to distinguish from grooming. Therefore, the use of positive control chemicals such as capsaicin is recommended during the first experiment. Recording and replay conditions can also affect counting of both behaviors. If it is difficult to clearly observe the behaviors, mirrors can be used around the cages to enhance the viewing angles4.
It should be noted that the cheek injection model may show reduced quantitative accuracy for the tested chemicals than in the neck injection model. As shown in Figure 3 and Figure 4, the same amount of histamine induced more scratching behaviors in the neck model than in the cheek model, and showed smaller variation in the neck injection model. Once a tested substance has been shown to be a pruritogen with low algesic effects with the cheek injection model, the neck injection model is recommended for additional quantitative analysis. Even with these limitations, the cheek injection model is useful for the precise evaluation of test substances, especially in the field of itch research.

Itch was originally characterized as a sensation which induces scratching behaviors to remove harmful materials from the skin surface. However, itch has been the focus of therapies for unpleasant sensations caused by many diseases such as atopic dermatitis, neurogenic lesions, and cholestasis1. In these cases, itch is a serious unpleasant sensation similar to pain. Therefore, itch represents an important research target. Scratching behavior is the primary indicator of itch in animal experiments, and scratching behaviors can be induced in mice by injecting pruritogens into the skin on their neck2,3. However, a previous study showed that capsaicin, an algogen, also induces scratching behaviors in a neck injection model4, making it difficult to discriminate itch from pain in this model. &#160;In contrast, it has been found that capsaicin reduced scratching behaviors in a neck injection model, indicating that the effect of pain on scratching behaviors is complicated and depends on the experimental conditions.&#160; Therefore, simultaneous measurements of pain and itch-related behaviors would enable the precise analysis of pruritogens and algogens. Steven G. Shimada and Robert H. LaMotte succeeded in simultaneous measurements of pain and itch-related behaviors by changing the injection site from the neck to the cheek4. In the cheek injection model, capsaicin induced wiping, but not scratching behaviors, while histamine, a pruritogen, only induced scratching behaviors. Therefore, this model enables the independent assessment of itch and pain, making cheek injection a useful model for itch research. When trying to determine if a test substance is a pruritogen and/or an algogen, this model provides more information than the neck injection model. This article describes a practical method for performing cheek injection and counting the bouts of scratching or wiping, and shows the utility of this method for evaluating scratching behaviors induced by lysophosphatidic acid (LPA).

<table><tbody><tr><td>Capsaicin</td><td>Sigma</td><td>M2028</td><td></td></tr><tr><td>Diphenhydramine hydrochloride</td><td>Wako</td><td>044-19772</td><td></td></tr><tr><td>Histamine</td><td>Sigma</td><td>H7125</td><td></td></tr><tr><td>iMovie</td><td>Apple</td><td></td><td></td></tr><tr><td>Lysophosphatidic acid</td><td>Avanti Polar Lipids</td><td>325465-93-8</td><td></td></tr><tr><td>Myjector</td><td>Terumo</td><td>ss-05M2913</td><td></td></tr><tr><td>Tween-80</td><td>Sigma</td><td>P4780</td><td></td></tr><tr><td>Video camera</td><td>Panasonic</td><td>VX985M</td><td></td></tr></tbody></table>

cheek injection model for simultaneous measurement of pain and itch-related behaviors

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts are typically counted to evaluate itch induced by pruritogens. However, previous reports have shown that algesic substances also induce scratching behaviors in a mouse neck injection model, which is the most common test used for scratching behaviors. This finding makes it difficult to study itch in mice. &#160;In contrast, capsaicin, a common algogen, reduced scratching behaviors in some neck injection experiments. Therefore, the effect of pain on scratching behaviors remains unclear. It is thus necessary to develop a method to concurrently investigate itch and pain sensation using behavioral tests. Here, a cheek injection model is introduced which can be used to simultaneously measure pain- and itch-related behaviors. In this model, pruritogens induce scratching behaviors while algesic substances induce wiping behaviors. Using this model, lysophosphatidic acid (LPA), an itch mediator found in cholestatic patients with itch, is shown to exclusively induce itch but not pain. However, in mouse models, LPA has been reported to be both a pruritogen and an algogen. Investigation into the effects of LPA in a mouse cheek injection model showed that LPA only induced scratching, but not wiping behaviors. This indicates that LPA acts as a pruritogen similarly in mice and humans, and demonstrates the utility of a cheek injection model for itch research.

In the neck injection model, histamine induced strong scratching behaviors (Figure 3). Vehicle (7% tween 80 in saline) also induced scratching behaviors. A previous report showed that detergent induced itch by histamine production from keratinocytes8. Tween 80 could induce itch by similar mechanisms, whereas capsaicin reduced vehicle-induced scratching behaviors, contrary to a previous report4. It is possible that mouse strain differences could account for these different results. These findings indicate that the effect of pain on scratching behaviors is complicated. Therefore, the simultaneous measurement of pain and itch-related behaviors may enable the precise analysis of pruritogens and algogens. In the cheek injection model, capsaicin did not induce scratching behaviors and only induced wiping behaviors, which are thought to be related to pain9,10,11 (Figure 4). Capsaicin injection also increased grooming behaviors (data not shown). Following injection into the cheek, histamine mainly induced scratching behaviors with weak wiping behaviors. The application of an anti-histamine reagent, diphenhydramine, inhibited scratching. This result suggests that histamine-induced scratching behaviors are caused by the action of histamine on histamine receptors. Here, as an example, the effects of LPA in the cheek injection model are shown (Figure 5). LPA is an itch mediator found in cholestatic patients with itch. However, in mouse models, LPA was reported as both a pruritogen and an algogen12,13,14,15. Therefore, the effects of LPA were investigated in a cheek injection model and results showed that LPA induced scratching, but not wiping behaviors. This indicates that LPA is a pruritogen, but not an algogen, in mice similar to its effects in humans.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58943/58943fig3.jpg" /> 
Figure 3: Effects of histamine and capsaicin in the neck injection model. (A) Scratching behaviors induced by the injection of vehicle (7% tween 80 in saline, open square), histamine (50 &#956;g/10 &#956;L per site, filled circles) or capsaicin (10 &#956;g/10 &#956;L per site, open triangle; 40 &#956;g/10 &#956;L per site, filled triangle) into the neck were assessed every 5 min for a total of 30 min. (B) Quantitative analysis of the scratching behaviors during the 30 min period in panel A. &#8727;&#8727;P &#60; 0.01 by ANOVA followed by Bonferroni correction. Data are shown as the mean &#177; SEM (n = 6-8). <a href="https://www.jove.com/files/ftp_upload/58943/58943fig3large.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/58943/58943fig4.jpg" /> 
Figure 4: Effects of a pruritogen and an algogen in the cheek injection model. Wiping (A) and scratching (B) behaviors induced by injection of vehicle (7% tween 80 in saline, open square), histamine (50 &#956;g/10 &#956;L per site, filled circles) or capsaicin (40 &#956;g/10 &#956;L per site, filled triangle) into the right cheek of mice were assessed every 5 min for a total of 30 min. The group shown as open circles was treated with 10 &#956;L of 2% (w/v) diphenhydramine hydrochloride salt (DHHCL) percutaneously 30 min prior to histamine injection (50 &#956;g/10 &#956;L per site). DHHCl was dissolved in saline. Quantitative analysis of the wiping (C) and scratching (D) behaviors during the 30 min period in panels A and B; n = 8 animals per group. &#8727;P &#60; 0.05, &#8727;&#8727;P &#60; 0.01 by ANOVA followed by Bonferroni correction. Data are shown as the mean &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/58943/58943fig4large.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/58943/58943fig5.jpg" /> 
Figure 5: LPA induces scratching, but not wiping behaviors in the cheek injection model. Scratching (A) and wiping (B) behaviors induced by injection of vehicle (saline, open squares) or LPA (100 nmol/10 &#956;L per site, filled circles) into the right cheek of mice were assessed every 5 min for a total of 30 min. Quantitative analysis of the scratching (C) and wiping (D) behaviors during the 30 min period in panels A and B; n = 17 for vehicle and n = 20 for LPA. &#8727;P &#60; 0.05, &#8727;&#8727;P &#60; 0.01 by Welch&#39;s t test. Data are shown as the mean &#177; SEM. This figure has been modified from Kittaka et al.5. <a href="https://www.jove.com/files/ftp_upload/58943/58943fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/58943/58943movie1.jpg" /> 
Movie 1: Movie demonstrating how to shave a mouse cheek. <a href="https://www.jove.com/files/ftp_upload/58943/Yamanoi_re-Rev._Movie_1.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 2" src="/files/ftp_upload/58943/58943movie2.jpg" /> 
Movie 2: Movie demonstrating the injection into a mouse cheek. <a href="https://www.jove.com/files/ftp_upload/58943/Yamanoi_re-Rev._Movie_2.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 3" src="/files/ftp_upload/58943/58943movie3.jpg" /> 
Movie 3: Movie demonstrating typical wiping behavior. This movie is shown at 25% speed. <a href="https://www.jove.com/files/ftp_upload/58943/Yamanoi_re-Rev._Movie_3.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 4" src="/files/ftp_upload/58943/58943movie4.jpg" /> 
Movie 4: Movie of typical scratching behavior. <a href="https://www.jove.com/files/ftp_upload/58943/Yamanoi_re-Rev._Movie_4.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 5" src="/files/ftp_upload/58943/58943movie5.jpg" /> 
Movie 5: Movie of typical grooming behavior. This movie is shown at 25% speed. <a href="https://www.jove.com/files/ftp_upload/58943/Yamanoi_re-Rev._Movie_5.mp4" target="_blank">Please click here to view this video. (Right-click to download.)</a>

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Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Typically, the mouse neck injection model is used for evaluate pruritogen-induced scratch behaviors. However, the model provides information only on itch, not pain. Here, a cheek injection model is introduced in mice which can be used to simultaneously measure pain and itch-related behaviors.

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts ...

cheek-injection-model-for-simultaneous-measurement-pain-itch-related

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Neuroscience

12.1K Views.  Exploratory Research Center on Life and Living Systems (ExCELLS). Onze gegevens geven aan dat algogens krassen gedrag te verminderen, maar het effect van capsaïcine op krassen gedrag blijft onduidelijk. Daarom is een gelijktijdige meting van elke hoeveelheid pijn belangrijk. Traditionele methode bieden informatie voor alleen jeuk of pijn sensaties, maar de wang injectie model bieden informatie voor jeuk en pijn, afzonderlijk en gelijktijdig.De wang injectie kan moeilijk zijn op het eerste, want het duurt de praktijk om het krabben, afvegen, en groom...

Video: Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents

A lengthening in meal duration can be used to measure an increase in orofacial mechanical hyperalgesia having similarities to the guarding behavior of humans with orofacial pain. To measure meal duration unrestrained rats are continuously kept in sound attenuated, computerized feeding modules for days to weeks to record feeding behavior. These sound-attenuated chambers are equipped with chow pellet dispensers. The dispenser has a pellet trough with a photobeam placed at the bottom of the trough and when a rodent removes a pellet from the feeder trough this beam is no longer blocked, signaling the computer to drop another pellet. The computer records the date and time when the pellets were taken from the trough and from this data the experimenter can calculate the meal parameters. When calculating meal parameters a meal was defined based on previous work and was set at 10 min (in other words when the animal does not eat for 10 min that would be the end of the animal&#39;s meal) also the minimum meal size was set at 3 pellets. The meal duration, meal number, food intake, meal size and inter-meal interval can then be calculated by the software for any time period that the operator desires. Of the feeding parameters that can be calculated meal duration has been shown to be a continuous noninvasive biological marker of orofacial nociception in male rats and mice and female rats. Meal duration measurements are quantitative, require no training or animal manipulation, require cortical participation, and do not compete with other experimentally induced behaviors. These factors distinguish this assay from other operant or reflex methods for recording orofacial nociception.

A lengthening in meal duration represents orofacial nociceptive behavior in rodents similar to the guarding behavior of humans with orofacial pain. Eating is a behavior that requires no training or animal manipulation, requires cortical participation, and is not competing with other experimentally induced behaviors, distinguishing this assay from alternative reflex or operant measurements.

A lengthening in meal duration can be used to measure an increase in orofacial mechanical hyperalgesia having similarities to the guarding behavior of ...

Behavior

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

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

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

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

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

Assessment of 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 ...

Real-Time Mouse Eye Squint Detection for Migraine Pain Tracking Using DeepLabCut

An Automated Squint Method for Time-syncing Behavior and Brain Dynamics in Mouse Pain Studies

Spontaneous pain has been challenging to track in real time and quantify in a way that prevents human bias. This is especially true for metrics of head pain, as in disorders such as migraine. Eye squint has emerged as a continuous variable metric that can be measured over time and is effective for predicting pain states in such assays. This paper provides a protocol for the use of DeepLabCut (DLC) to automate and quantify eye squint (Euclidean distance between eyelids) in restrained mice with freely rotating head motions. This protocol enables unbiased quantification of eye squint to be paired with and compared directly against mechanistic measures such as neurophysiology. We provide an assessment of AI training parameters necessary for achieving success as defined by discriminating squint and non-squint periods. We demonstrate an ability to reliably track and differentiate squint in a CGRP-induced migraine-like phenotype at a sub second resolution.

Author Spotlight: Deciphering Electrical Networks Behind Complex Brain Activities and Disorders

This protocol provides a method for tracking automated eye squint in rodents over time in a manner compatible with time-locking to neurophysiological measures. This protocol is expected to be useful to researchers studying mechanisms of pain disorders such as migraine.

Spontaneous pain has been challenging to track in real time and quantify in a way that prevents human bias. This is especially true for metrics of head ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Medicine

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Spontaneous and Evoked Measures of Pain in Murine Models of Monoarticular Knee Pain

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are necessary and useful for studying the mechanisms of pain and for evaluating efficacy of arthritis therapies. Measuring pain in animal models of arthritis is challenging. We have developed methods for measuring evoked and spontaneous pain in three models of murine arthritis. We quantitate the evoked pain responses of mice subjected to firm palpation of a painful knee. We also evaluate spontaneous pain by the proportion of weight and the amount of time placed on each of their 4 limbs after induction of arthritis pain in one knee. Joint pain in these mouse models produces a significant increase in evoked pain responses and an alteration in weight bearing. Since mice are quadrupeds, they offload the painful limb to the contralateral limb, to the forelimbs, or some combination. These methods are simple, require minimal equipment, and are reproducible and sensitive for detecting pain. They are useful for studying both disease-modifying arthritis treatments and analgesics in mice.

We have developed an evoked measure of arthritis pain and coupled it with a standardized method for measuring spontaneous pain in different murine models of chemically induced arthritis. These measures are sensitive and reproducible for different types of joint pain.

Pain is the main cause of disability from arthritis. There is currently an unmet need for adequate treatments for arthritis pain. Pre-clinical models are ...

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

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

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

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

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

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

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

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

Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

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