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

<ol>
	<li>Deuis, J. R., Dvorakova, L. 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. 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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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiation+of+morphine+analgesic+action+in+mice+by+beta-carotene.">Potentiation of morphine analgesic action in mice by beta-carotene.</a> Eur J Pharmacol. 284 (1-2), 191-193 (1995).</li><li>Akkol, E. K., G&#252;ven&#231;, A., Yesilada, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+study+on+the+antinociceptive+and+anti-inflammatory+activities+of+five+Juniperus+taxa.">A comparative study on the antinociceptive and anti-inflammatory activities of five Juniperus taxa.</a> J Ethnopharmacol. 125 (2), 330-336 (2009).</li></ol>

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.

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...

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

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

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 ...

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1.2K Views.  Yantai University. Here, we present a modified inflammatory pain model with the both-hind-paw carrageenan injection to evaluate the analgesic effect.

Video: Author Spotlight: Enhanced Method for Evaluating Analgesic Effects — Dual Hind Paw Carrageenan Injection in Mice

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model. While ICH
accounts for 10-15% of all strokes, there remains no specific
effective therapy. The autologous blood injection model in mice
involves the stereotaxic injection of arterial blood into the basal
ganglia mimicking a spontaneous hypertensive hemorrhage in man. The
response to hemorrhage can then be studied in vivo and the
neurobehavioral deficits quantified, allowing for description of the
ensuing pathology and the testing of potential therapeutic agents. The
procedure described in this protocol uses the double injection
technique to minimize risk of blood reflux up the needle track, no
anticoagulants in the pumping system, and eliminates all dead space
and expandable tubing in the system.

The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question the safety of anesthesia in children, implicating anesthetics as potentially toxic to the brain in development. To date, no studies have successfully elucidated the mechanism(s) by which anesthesia may be neurotoxic. Animal studies allow investigation of such mechanisms, and neonatal piglets represent an excellent model to study these effects due to their striking developmental similarities to the human brain.
This protocol adapts the use of enzyme-based microelectrode array (MEA) technology as a novel way to study the mechanism(s) of anesthesia-induced neurotoxicity (AIN). MEAs enable real-time monitoring of in vivo neurotransmitter activity and offer exceptional temporal and spatial resolution. It is hypothesized that anesthetic neurotoxicity is caused in part by glutamate dysregulation and MEAs offer a method to measure glutamate. The novel implementation of MEA technology in a piglet model presents a unique opportunity for the study of AIN.

This study explores the novel use of enzyme-based microelectrode array (MEA) technology to monitor in vivo neurotransmitter activity in piglets. The hypothesis was that glutamate dysregulation contributes to the mechanism of anesthetic neurotoxicity. Here, we present a protocol to adapt MEA technology to study the mechanism of anesthesia-induced neurotoxicity.

Every year, millions of children undergo anesthesia for a multitude of procedures. However, studies in both animals and humans have called into question ...

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In addition to its peripheral function, its involvement in brain functions has also been suggested. Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been known to induce long-term desensitization of TRPV1, and this desensitization has been an alternative approach for investigating the physiological relevance of TRPV1-expressing cells. Here we describe a protocol for intracerebroventricular (i.c.v.) treatment with RTX in mice. Procedures are described for testing nociception to peripheral TRPV1 stimulation (RTX test) and mechanical stimulation (tail pressure test) then follow. Although the nociceptive responses of mice that had been administered RTX i.c.v. were comparable to those of the control groups, RTX-i.c.v.-administered mice were insensitive to the analgesic effect of acetaminophen, suggesting that i.c.v. RTX treatment can induce supraspinal-selective TRPV1 desensitization. This mouse model can be used as a convenient experimental system for studying the role of TRPV1 in brain/supraspinal function. These techniques can also be applied to studies of the central actions of other drugs.

The transient receptor potential vanilloid type 1 (TRPV1) in the supraspinal region has been suggested to play some roles in the brain function. Described here is a protocol for intracerebroventricular injection of resiniferatoxin for supraspinal TRPV1 desensitization in mice. Procedures for some pain tests are also presented.

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...

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.

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

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Adaptation of Microelectrode Array Technology for the Study of Anesthesia-induced Neurotoxicity in the Intact Piglet Brain

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

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