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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurobiology+of+itch.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scratching+behavior+induced+by+pruritogenic+but+not+algesiogenic+agents+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Itch-scratch+responses+induced+by+opioids+through+central+mu+opioid+receptors+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+differentiation+between+itch+and+pain+in+mouse.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysophosphatidic+acid-induced+itch+is+mediated+by+signalling+of+LPA5+receptor,+phospholipase+D+and+TRPA1/TRPV1.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Itch-associated+responses+of+afferent+nerve+innervating+the+murine+skin:+different+effects+of+histamine+and+serotonin+in+ICR+and+ddY+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scratching+behavior+in+various+strains+of+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+surfactant-induced+pruritus:+involvement+of+histamine+released+from+epidermal+keratinocytes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+injection+of+phospholipases+A2+inhibitors+modulates+allodynia+after+facial+carrageenan+injection+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antinociceptive+effect+of+CNS+peroxynitrite+scavenger+in+a+mouse+model+of+orofacial+pain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Itch-scratch+responses+induced+by+lysophosphatidic+acid+in+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysophosphatidic+acid+is+a+potential+mediator+of+cholestatic+pruritus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TGR5+receptor+mediates+bile+acid-induced+itch+and+analgesia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysophosphatidic+acid+directly+activates+TRPV1+through+a+C-terminal+binding+site.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+itch-+and+pain-related+behavioral+responses+and+&#181;-opoid+modulation+in+mice.">Differential itch- and pain-related behavioral responses and &#181;-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 border="0" cellpadding="0" cellspacing="0" style="width:572px;" width="573">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">capsaicin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">M2028</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">diphenhydramine hydrochloride</td>
			<td style="width:71px;">Wako</td>
			<td style="width:119px;">044-19772</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">histamine</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">H7125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">iMovie</td>
			<td style="width:71px;">Apple</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">lysophosphatidic acid</td>
			<td>Avanti Polar Lipids</td>
			<td style="width:119px;">325465-93-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">myjector</td>
			<td style="width:71px;">Terumo</td>
			<td style="width:119px;">ss-05M2913</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tween-80</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P4780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">video camera</td>
			<td style="width:71px;">Panasonic</td>
			<td style="width:119px;">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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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>

Watch this Scientific Journal Video about Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors at JoVE.com

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

12.0K Views.  Exploratory Research Center on Life and Living Systems (ExCELLS). 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.

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

Assessment of Social Interaction Behaviors

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and stability of the networks and relationships that define societies. For animals, such as mice, recognition of conspecifics may be important for maintaining social hierarchy and for mate choice 1.
A variety of neuropsychiatric disorders are characterized by disruptions in social behavior and social recognition, including depression, autism spectrum disorders, bipolar disorders, obsessive-compulsive disorders, and schizophrenia. Studies of humans as well as animal models (e.g., Drosophila melanogaster, Caenorhabditis elegans, Mus musculus, Rattus norvegicus) have identified genes involved in the regulation of social behavior 2. To assess sociability in animal models, several behavioral tests have been developed (reviewed in 3). Integrative research using animal models and appropriate tests for social behavior may lead to the development of improved treatments for social psychopathologies.
The three-chamber paradigm test known as Crawley's sociability and preference for social novelty protocol has been successfully employed to study social affiliation and social memory in several inbred and mutant mouse lines (e.g. 4-7). The main principle of this test is based on the free choice by a subject mouse to spend time in any of three box's compartments during two experimental sessions, including indirect contact with one or two mice with which it is unfamiliar. To quantitate social tendencies of the experimental mouse, the main tasks are to measure a) the time spent with a novel conspecific and b) preference for a novel vs. a familiar conspecific. Thus, the experimental design of this test allows evaluation of two critical but distinguishable aspects of social behavior, such as social affiliation/motivation, as well as social memory and novelty. "Sociability" in this case is defined as propensity to spend time with another mouse, as compared to time spent alone in an identical but empty chamber 7. "Preference for social novelty" is defined as propensity to spend time with a previously unencountered mouse rather than with a familiar mouse 7. This test provides robust results, which then must be carefully analyzed, interpreted and supported/confirmed by alternative sociability tests. In addition to specific applications, Crawley's sociability test can be included as an important component of general behavioral screen of mutant mice.

Here we describe a detailed protocol for examination of sociability in mice by using Crawley's sociability and preference for social novelty test. We describe the advantages and possible applications for this procedure, including critical details important for correct interpretation of the results.

Social interactions are a fundamental and adaptive component of the biology of numerous species. Social recognition is critical for the structure and ...

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static1,2 or record extracellular neuronal activity during a movement.3,4 While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

A technique is described to quantify the in vivo physiological response of mammalian neurons during movement and correlate the physiology of the neuron with neuronal morphology, neurochemical phenotype and synaptic microcircuitry.

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor ...

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

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.  ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

A Mouse Model to Study Brain Physiology During Cardiac Arrest

Mouse Cardiac Arrest Model for Brain Imaging and Brain Physiology Monitoring During Ischemia and Resuscitation

Most cardiac arrest (CA) survivors experience varying degrees of neurologic deficits. To understand the mechanisms that underpin CA-induced brain injury and, subsequently, develop effective treatments, experimental CA research is essential. To this end, a few mouse CA models have been established. In most of these models, the mice are placed in the supine position in order to perform chest compression for cardiopulmonary resuscitation (CPR). However, this resuscitation procedure makes the real-time imaging/monitoring of brain physiology during CA and resuscitation challenging. To obtain such critical knowledge, the present protocol presents a mouse asphyxia CA model that does not require the chest compression CPR step. This model allows for the study of dynamic changes in blood flow, vascular structure, electrical potentials, and brain tissue oxygen from the pre-CA baseline to early post-CA reperfusion. Importantly, this model applies to aged mice. Thus, this mouse CA model is expected to be a critical tool for deciphering the impact of CA on brain physiology.

Author Spotlight: A Unique Mouse Model of Asphyxia-Induced Cardiac Arrest 

This protocol demonstrates a unique mouse model of asphyxia cardiac arrest that does not require chest compression for resuscitation. This model is useful for monitoring and imaging the dynamics of brain physiology during cardiac arrest and resuscitation.

Most cardiac arrest (CA) survivors experience varying degrees of neurologic deficits. To understand the mechanisms that underpin CA-induced brain injury ...

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

Summary

Explore More Videos

Chapters in this video

Assessment of Social Interaction Behaviors

Physiological, Morphological and Neurochemical Characterization of Neurons Modulated by Movement

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

A Novel In Vitro Model of Blast Traumatic Brain Injury

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

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Mouse Cardiac Arrest Model for Brain Imaging and Brain Physiology Monitoring During Ischemia and Resuscitation