We thank Lesley McCollum, PhD, from Liwen Bianji (Edanz) (www.liwenbianji.cn), for editing the English text of a draft of this manuscript.

The experimental protocol was approved by the Institutional Animal Care and Use Committee at Yantai University (No. YTDX20220425). The procedures followed the National Institutes of Health Guidelines for the Use of Laboratory Animals.
1. Animals
<ol>
	<li>Use ICR female mice weighing 20-25 g for the experiments.</li>
	<li>Allow the animals to acclimate to the environment of the experimental room for 3 days before starting the experiments.</li>
	<li>Maintain the experimental room with a 12-h light-dark cycle, and provide the mice with free access to water and chow.</li>
</ol>
2. Preparation of carrageenan solution
<ol>
	<li>Weigh 0.1 g of carrageenan. Add 10 mL of sterilized normal saline to a tube.</li>
	<li>Add 0.1 g of carrageenan to the tube and vortex the tube for 30 s.</li>
	<li>Collect the 1% sterilized carrageenan solution.</li>
	<li>Prepare the carrageenan solution before the experiments.</li>
</ol>
3. Intraplantar injection of carrageenan
<ol>
	<li>Use a syringe (0.05 mL, 29G x &#189; inch, 0.33 mm &#215; 12.7 mm) to inject the 1% carrageenan solution.</li>
	<li>Shake the carrageenan solution before drawing it into the syringe. Make sure that there is no bubble in the syringe.</li>
	<li>Disinfect the injection site (hind paw) with 75% alcohol.</li>
	<li>Inject 30 &#181;L6,7 of 1% carrageenan solution subcutaneously into the plantar surface of the hind paw. For the control group mice, inject 30 &#181;L of normal saline.
	<ol>
		<li>Insert the needle diagonally upwards into the hind paw foot pad. Maintain the stability of the syringe.</li>
		<li>After injection, withdraw the syringe slowly and press on the injection site for 2 s to prevent leakage.</li>
	</ol>
	</li>
</ol>
4. Hot plate test
<ol>
	<li>Conduct the hot plate test 3 h after the intraplantar injection.</li>
	<li>Clean and disinfect the surface of the hot plate.</li>
	<li>Start the hot plate apparatus. Click the Temperature Mode button. Click CONSTANT to set the temperature to 55 &#177; 0.2 &#176;C.</li>
	<li>Click Start experiment&#160;button and click REACH. Take a mouse from the accommodation container. Gently grasp its trunk. Place the mouse in the center of the hot plate. Click START.</li>
	<li>Observe and record the time to lick the left hind paw (for mice with single-hind-paw injection) or hind paw (for mice with both-hind-paw injection) in response to the heat stimulation. Press the pedal of the hot plate to see the time on the hot plate screen. Define pain threshold as the time from placing the mouse on the hot plate to licking the hind paw.</li>
	<li>Set a 60 s limit for the hot plate test4,8,9. Record a pain threshold of 60 s if the limit is reached. 
	NOTE: Clean the hot plate apparatus with 75% alcohol before placing the next mouse to avoid possible bias due to residual odor from the previous mouse.</li>
</ol>
5. Preparation of 1.5 mg/mL celecoxib suspension
<ol>
	<li>Take 1 capsule containing 200 mg celecoxib. Get the content of the capsule and grind it into a powder.</li>
	<li>Add 0.5% sodium carboxymethyl cellulose. The final volume is 13.3 mL. This is the store suspension of celecoxib.</li>
	<li>Take 1 mL of the store suspension celecoxib. Add 9.0 mL of 0.5% sodium carboxymethyl cellulose to obtain 1.5 mg/mL of celecoxib suspension.</li>
	<li>Administer 0.2 mL/10 g of the celecoxib suspension intragastrically to the mice. 
	NOTE: Select gavage needle with 4.0-4.5 cm in length for mice weighing 20-25 g.</li>
</ol>
6. Evaluating the modified inflammatory pain model
<ol>
	<li>Randomly divide ICR female mice into three groups: control, model, and celecoxib groups.</li>
	<li>Inject 30 &#181;L of 1% carrageenan solution subcutaneously into the plantar surface of the hind paw (single-hind-paw or both-hind-paw). For the control group mice, inject 30 &#181;L of normal saline.</li>
	<li>After the injection of carrageenan or normal saline, perform the treatment immediately. Intragastrically administer celecoxib at a dose of 30 mg/kg for the mice in the celecoxib group. For the mice in the control and model groups, intragastrically administer 0.5% sodium carboxymethyl cellulose.</li>
	<li>Conduct the hot plate test (section 2) at 3 h after the injection of carrageenan.</li>
</ol>
7. Statistical methods
<ol>
	<li>Present the experimental results as mean &#177; standard deviation. Conduct a normality distribution test with the Kolmogorov-Smirnov statistical goodness of fit test.</li>
	<li>Analyze the comparison between the two groups using an unpaired t-test. Perform multiple group comparisons using a one-way analysis of variance followed by Dunnett&#39;s test. Consider the value of p &#60; 0.05 statistically significant.</li>
</ol>

Dual Hind Paw Injection for Enhanced Analgesic Testing

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S., Vetter, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+used+to+evaluate+pain+behaviors+in+rodents.">Methods used to evaluate pain behaviors in rodents.</a> Front Mol Neurosci. 10, 284 (2017).</li><li>Men&#233;ndez, L., Lastra, A., Hidalgo, A., Baamonde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unilateral+hot+plate+test:+a+simple+and+sensitive+method+for+detecting+central+and+peripheral+hyperalgesia+in+mice.">Unilateral hot plate test: a simple and sensitive method for detecting central and peripheral hyperalgesia in mice.</a> J Neurosci Methods. 113 (1), 91-97 (2002).</li><li>Li, T., 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=Rotigotine-loaded+microspheres+exerts+the+antinociceptive+effect+via+central+dopaminergic+system.">Rotigotine-loaded microspheres exerts the antinociceptive effect via central dopaminergic system.</a> Eur J Pharmacol. 910, 174443 (2021).</li><li>Kambur, O., 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=Inhibitors+of+catechol-+O+-methyltransferase+sensitize+mice+to+pain.">Inhibitors of catechol- O -methyltransferase sensitize mice to pain.</a> Br J Pharmacol. 161 (7), 1553-1565 (2010).</li><li>Micheli, L., 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=Efficacy+of+Posidonia+oceanica+extract+against+inflammatory+pain:+In+vivo+studies+in+mice.">Efficacy of Posidonia oceanica extract against inflammatory pain: In vivo studies in mice.</a> Mar Drugs. 19 (2), 48 (2021).</li><li>McCarson, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+inflammation:+Carrageenan-+or+complete+Freund's+adjuvant+(CFA)-induced+edema+and+hypersensitivity+in+the+rat.">Models of inflammation: Carrageenan- or complete Freund's adjuvant (CFA)-induced edema and hypersensitivity in the rat.</a> Curr Protoc Pharmacol. 70, 5.4.1-5.4.9 (2015).</li><li>Fehrenbacher, J. C., Vasko, M. R., Duarte, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+Inflammation:+Carrageenan-+or+Complete+Freund's+Adjuvant+(CFA)-Induced+Edema+and+Hypersensitivity+in+the+Rat.">Models of Inflammation: Carrageenan- or Complete Freund's Adjuvant (CFA)-Induced Edema and Hypersensitivity in the Rat.</a> Curr Protoc Pharmacol. Chapter 5 (Unit 5.4), (2012).</li><li>Asl, M. K., Nazariborun, A., Hosseini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analgesic+effect+of+the+aqueous+and+ethanolic+extracts+of+clove.">Analgesic effect of the aqueous and ethanolic extracts of clove.</a> Avicenna J Phytomed. 3 (2), 186-912 (2013).</li><li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analgesic+effects+of+glycoproteins+from+Panax+ginseng+root+in+mice.">Analgesic effects of glycoproteins from Panax ginseng root in mice.</a> J Ethnopharmacol. 148 (3), 946-950 (2013).</li><li>Zia, F. Z., 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=Are+psychedelic+medicines+the+reset+for+chronic+pain?+Preliminary+findings+and+research+needs.">Are psychedelic medicines the reset for chronic pain? Preliminary findings and research needs.</a> Neuropharmacology. 233, 109528 (2023).</li><li>Sa&#322;at, K., Furga&#322;a, A., Sa&#322;at, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+cebranopadol,+a+dually+acting+nociceptin/orphanin+FQ+and+opioid+receptor+agonist+in+mouse+models+of+acute,+tonic,+and+chemotherapy-induced+neuropathic+pain.">Evaluation of cebranopadol, a dually acting nociceptin/orphanin FQ and opioid receptor agonist in mouse models of acute, tonic, and chemotherapy-induced neuropathic pain.</a> Inflammopharmacology. 26 (2), 361-374 (2018).</li><li>Wang, J., 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=Anti-inflammatory+and+analgesic+actions+of+bufotenine+through+inhibiting+lipid+metabolism+pathway.">Anti-inflammatory and analgesic actions of bufotenine through inhibiting lipid metabolism pathway.</a> Biomed Pharmacother. 140, 111749 (2021).</li><li>Gong, L., 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=Antinociceptive+and+anti-inflammatory+potentials+of+Akebia+saponin+D.">Antinociceptive and anti-inflammatory potentials of Akebia saponin D.</a> Eur J Pharmacol. 845, 85-90 (2019).</li><li>Deng, J., 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=Comparison+of+analgesic+activities+of+aconitine+in+different+mice+pain+models.">Comparison of analgesic activities of aconitine in different mice pain models.</a> PloS One. 16 (4), e0249276 (2021).</li><li>Penn, N. 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The authors do not have any potential conflicting interests to disclose.

Pain is a protective response triggered by tissue damage or injury. However, it also has a negative impact on quality of life10. Therefore, the development of effective analgesics and exploration of pain mechanisms remain active areas of research. The methods for evaluating pain play a key role in the development of analgesic drugs. Single-hind-paw injection of carrageenan is a classic method for the hot plate test. The modified model induced by both-hind-paw injection of carrageenan we present he...

Pain is a public health problem worldwide. It is triggered by noxious thermal, mechanical, or chemical stimulation. It is difficult to perform pain studies in humans because of ethical concerns. Therefore, using animal pain models is a key approach to understanding pain mechanisms and research treatments1. The hot plate test has been widely used for the assessment of thermal nociception and hyperalgesia2. Currently, the model of inflammatory pain used for the hot plate test is induced with an intraplantar injection of carrageenan in one hind paw. For example, to investigate the antinociceptive effect of rotigotine-loaded microspheres, an animal model of inflammatory pain was prepared by a single-hind-paw injection3. To assess the effects of catechol-o-methyl-transferase inhibitors on thermal nociception in mice, carrageenan was administered into the plantar region of the right hind paw4. To investigate the analgesic role of the extract of Posidonia oceanica (L.) Delile, an inflammatory pain model was prepared in mice by intraplantar injection of carrageenan in the right hind paw5. However, the findings from our laboratory showed that a mouse with the single-hind-paw injection of carrageenan would lift its paw to avoid thermal nociception, which inaccurately extends the animal's response to a thermal stimulus in the hot plate test. Thus, we hypothesized that the inflammatory pain model with the single-hind-paw injection of carrageenan could not accurately reflect the thermal pain threshold. Therefore, we optimized the method by injecting carrageenan into both hind paws to establish a modified inflammatory pain model. 
The mice subjected to both-hind-paw injection of carrageenan had no way to avoid the thermal stimulus for both hind paws at the same time. The pain threshold was defined as the time from placing the mouse on the hot plate to the mouse licking either hind paw. The results confirmed that the modified inflammatory pain model is more suitable for evaluating the nociceptive temperature threshold. This may improve the efficacy of the hot plate test for the research and development of analgesic drugs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AL204 electronic balance</td>
			<td>Yuheng Battery Co., Ltd&#160;</td>
			<td>YH-400A</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxymethylcellulose</td>
			<td>Tianjin Chemical Co., Ltd&#160;</td>
			<td>20210326</td>
			<td></td>
		</tr>
		<tr>
			<td>Carrageenan</td>
			<td>Sigma, MO, USA</td>
			<td>29H0715</td>
			<td></td>
		</tr>
		<tr>
			<td>Celecoxib</td>
			<td>Pfizer Inc.</td>
			<td>8142838</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate&#160;</td>
			<td>UGO Ltd. Co., Italy</td>
			<td>PB-200&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>ICR female mice</td>
			<td>Jinan Pengyue&#160; Co., Ltd.</td>
			<td>SCXK20220007</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>Kelun Pharmaceutical Co., Ltd&#160;</td>
			<td>H51021157</td>
			<td></td>
		</tr>
	</tbody>
</table>

a modified inflammatory pain model to study the analgesic effect in mice

The hot plate test is widely used to evaluate analgesic effects on inflammatory pain in mice. A commonly used model of inflammatory pain was induced with an intraplantar injection of carrageenan in one hind paw. However, the findings from our laboratory showed that mice with a single-hind-paw injection of carrageenan lifted their paws to avoid thermal nociception during the hot plate test. Because of this response,&#160;previous injection method cannot accurately reflect the thermal pain threshold. Thus, we investigated a new method to avoid this issue. In the present study, we modified the previous method by injecting carrageenan into both hind paws to establish the model of inflammatory pain. The results demonstrated that both-hind-paw injection with carrageenan was sensitive and a better method to induce inflammatory pain when using the hot plate test than single-hind-paw injection. On the basis of these findings, we designed further experiments in which mice with either both-hind-paw or single-hind-paw injection of carrageenan were treated intragastrically with celecoxib (30 mg/kg). The results of the hot plate test showed that celecoxib augmented the thermal pain threshold in mice with both-hind-paw injection of carrageenan but not in mice with single-hind-paw injection of carrageenan. In summary, we developed a superior method to induce a model of inflammatory pain to evaluate analgesic effect.

Author Spotlight: Enhanced Method for Evaluating Analgesic Effects &amp;#8212; Dual Hind Paw Carrageenan Injection in Mice

A Modified Inflammatory Pain Model to Study the Analgesic Effect in Mice

Pain has a negative impact on quality of life. The method for evaluating pain plays a key role in developing analgesic drugs. The present study tried to provide a reliable and superior method to evaluate analgesic effects.The hot plate test is widely used to evaluate analgesic effects on inflammatory pain in mice. A commonly used method of inflammatory pain was induced with an intraplantar injection of carrageenin in one hind paw. We found that mice with a single hind paw carrageenin injection lifted their paws to avoid the during the hot plate test.Because of this response, the injection method cannot accurately the mouse's pain threshold. Thus, we modified the previous method by injecting carrageenin into both hind paws to establish the model of inflammatory pain. The results demonstrated that both hind paw injection with carrageenin are sensitive, and the better method to induce inflammatory pain when used in the hot plate test than single hind paw injection.In summary, we developed a superior method to induce a model inflammatory pain to evaluate analgesic effects. The present experiment used only ICR mice. To test the purposes in the future study, we will investigate whether this modified method accurately and consequently detects the pain associated in the other mouse's stress.

The pain threshold in inflammatory pain models 
The pain threshold of the inflammatory pain model with single-hind-paw injection of carrageenan was 32.23 &#177; 11.11&#160;s, whereas the pain threshold of the control group was 27.73 &#177; 7.08&#160;s. Compared with the control group, the pain threshold of the single-hind-paw injection model did not show a significant reduction (p &#62; 0.05, Figure 1A). The pain threshold of the inflammatory pain model with both-hind-p...

Here, we present a modified inflammatory pain model with the both-hind-paw carrageenan injection to evaluate the analgesic effect.

The hot plate test is widely used to evaluate analgesic effects on inflammatory pain in mice. A commonly used model of inflammatory pain was induced with ...

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Preparation of Mice and Carrageenan Solution for Creating a Model of Inflammatory Pain

Begin by using ICR female mice weighing 20 to 25 grams for the experiments. Allow the animals to acclimate to the experimental room environment for three days before starting the experiments. To prepare carrageenan solution, weigh 0.1 grams of carrageenan, add 10 milliliters of sterilized normal saline to a tube, add 0.1 grams of carrageenan to the tube, and vortex the tube for 30 seconds.Then collect the 1%sterilized carrageenan solution.

Establishing a Modified Inflammatory Pain Model with Both-Hind-Paw Carrageenan Injection

To begin, take a syringe to inject the prepared 1%Carrageenan solution. Shake the Carrageenan solution and draw it into the syringe while ensuring that there is no bubble in the syringe. Then, disinfect the injection site with 75%alcohol.Insert the needle diagonally upwards into the hind paw foot pad while maintaining the stability of the syringe to inject 30 microliters of 1%Carrageenan solution. After injection, withdraw the syringe slowly and press on the injection site for two seconds to prevent leakage. Clean and disinfect the surface of the hot plate.Start the hot plate apparatus and click the Temperature Mode button. Then, click Constant to set the temperature to approximately 55 degrees Celsius. Click Start Experiment button and click Reach.Now, take a mouse from the accommodation container. Gently grasp its trunk and place it in the center of the hot plate. Then, click Start.Observe and record the time to lift the left hind paw or hind paw in response to the heat stimulation. Press the pedal of the hot plate to see the time on the hot plate screen. Then set a 60 second limit for the hot plate test.Record a pain threshold of 60 seconds if the limit is reached. Take one capsule, containing 200 milligrams of Celecoxib. Get the content of the capsule and grind it into a powder.To prepare the store suspension of Celecoxib, add 0.5%sodium carboxy methylcellulose to make a final volume of 13.3 milliliters. Then take one milliliter of the store suspension of Celecoxib and add 9.0 milliliters of 0.5%sodium carboxy methylcellulose to obtain Celecoxib suspension. Administer 0.2 milliliters per 10 grams of the Celecoxib suspension, intragastrically to the mice.Randomly divide ICR female mice into three groups, control, model and Celecoxib groups. Inject 30 microliters of 1%Carrageenan solution subcutaneously into the planter surface of the hind paw. After the injection, intragastrically administer Celecoxib at 30 milligrams per kilogram for the mice in the Celecoxib group.Conduct the hot plate test at three hours after Carrageenan injection. The pain threshold of the single hind paw injection model did not show a significant reduction, compared with the control group. The pain threshold of the both hind paw injection model decreased significantly compared with the control group.In the single hind paw injection model, the pain threshold of the Celecoxib group did not significantly increase, compared to the model group. However, in the both hind paw injection model, the pain threshold of the Celecoxib group showed a significant increase.

468 Views.  Yantai University.  Here, we present a modified inflammatory pain model with the both-hind-paw carrageenan injection to evaluate the analgesic effect.

Dual Hind Paw Injection for Enhanced Analgesic Testing

Summary