All of the experimental protocols used here were approved by the Animal Care and Use Committee of Musashino University. Male ddY mice (SLC, Shizuoka, Japan) were kept for at least 7 days under a 12-h light/dark cycle before experiments with water and food ad libitum. 5- or 6-week-old mice were used for the experiments.
1. Preparation of Drugs
<ol>
	<li>RTX 
	NOTE: Alcoholic RTX solution can cause severe skin burns and eye damage. Make sure to use rubber gloves and glasses for protection when handling. This stock solution can be used for 6 months.
	<ol>
		<li>Add 500 &#181;L of ethanol to 1 mg of RTX.</li>
		<li>Add 500 &#181;L of polyoxyethylene (20) sorbitan monooleate to the solution above and vortex well.</li>
		<li>Add 4 mL of physiological saline to the mixture and vortex well.</li>
		<li>Aliquot 40 &#181;L of the solution into 1.5-mL screw cap tubes, and store them at -40 &#176;C.</li>
	</ol>
	</li>
	<li>Acetaminophen
	<ol>
		<li>Add 20% w/v propyleneglycol solution to acetaminophen at a concentration of 30 mg/mL, and dissolve with a sonicator. Since acetaminophen may precipitate at room temperature several hours after dissolution, prepare just before use or keep the solution warm until use.</li>
	</ol>
	</li>
</ol>
2. Subcutaneous or Intracerebroventricular Injection of RTX
<ol>
	<li>Thaw the stocked solution prepared in 1.1. above and dilute it to 20 &#181;g/mL in saline or artificial cerebrospinal fluid (ACSF) consisting of (in mM): 119 NaCl, 2.5 KCl, 1 NaH2PO4, 26 NaHCO3, 11 glucose, 1.3 MgSO4, 2.5 CaCl2 equilibrated with 95% O2 and 5% CO2 (pH 7.2).</li>
	<li>Anesthetize mice with pentobarbital sodium salt (60 mg/kg, intraperitoneally), and check for loss of the righting reflex.</li>
	<li>For s.c. treatment, inject RTX (20 &#181;g/mL) into the back of the neck at a volume of 0.1 mL/10 g body weight. For the control group, inject the vehicle (10% ethanol, 10% polyoxyethylene (20) sorbitan monooleate and 80% saline) in the same way.</li>
	<li>For i.c.v. treatment, inject 5 &#181;L of RTX (20 &#181;g/mL) into the right lateral ventricle. For the control group, inject the vehicle (10% ethanol, 10% polyoxyethylene (20) sorbitan monooleate and 80% ACSF) in the same way.
	<ol>
		<li>Pass a disposable 27-G needle through a metal tube (0.8 mm I.D.) to expose the 3.0-3.5 mm tip of the needle (Figure 1A).</li>
		<li>Disinfect the head of mouse with 70% alcohol, and hold the squamosal bones of the mouse firmly with the fingers (Figure 1B). 
		NOTE: Pay attention to the positions of the squamosal protrusions, since these protrusions will serve as landmarks for injection.</li>
		<li>Move the needle laterally on the scalp, and find the sagittal suture as the needle tip is hooked on the suture.</li>
		<li>Move the tip about 1 mm to the right, then move the tip rostrally, and find the coronal suture as with 2.4.3. (Figure 1B).</li>
		<li>Insert the needle slowly and vertically, inject the RTX solution in about 10 seconds, and hold it for about 10 seconds.</li>
		<li>Withdraw the needle slowly, and return the mouse to its home cage. Bleeding is usually minimal or absent. If major bleeding occurs, use of another mouse should be considered.</li>
	</ol>
	</li>
	<li>Assign the pretreated mice as subjects for the RTX test or the tail pressure test (Step 3 and 4, respectively).</li>
</ol>
3. RTX Test
NOTE: Testing is performed between 10:00 AM and 5:00 PM. The testing room is maintained at 200 lux and 24-26 &#176;C.
<ol>
	<li>One week after pretreatments with RTX (Step 2.), transfer mice to the testing room at least 60 min prior to starting the test.</li>
	<li>Weigh and place each mouse individually in a plexiglass cage (29.5 &#215; 17.5 &#215; 13.5 cm3 height) at least 30 min prior to starting the test in order to allow it to acclimate to the environment. 
	NOTE: The order of tests should be counterbalanced across pretreatment groups.</li>
	<li>Administer acetaminophen (300 mg/kg) to the mouse intraperitoneally 20 min before the test.</li>
	<li>Hold the mouse loosely in a small cloth bag, and insert a 30-gauge needle into the heel of the right hind paw. Advance the needle subcutaneously to near the walking pads, and inject 20 &#181;L of RTX solution (0.05 &#181;g/mL).</li>
	<li>Measure the period of licking/biting behavior in theglabrous region of the affected paw in each 5-min block.</li>
</ol>
4. Tail Pressure Test
NOTE: A Randall-Selitto-type pressure meter is used to assess the threshold for acute mechanical nociception. Testing is performed between 10:00 AM and 5:00 PM. The testing room is maintained at 200 lux and 24-26 &#176;C.
<ol>
	<li>One week after pretreatments with RTX (Step 2.), transfer mice to the testing room, and weigh and place each mouse individually in a plexiglass cage.</li>
	<li>Mark the spots at 1.5 and 2.5 cm from the base of the tail.</li>
	<li>Hold the mouse loosely in a small cloth bag, and apply pressure to the spots with a blunt probe. 
	NOTE: A cutoff pressure of 250 g is imposed to avoid tissue damage.</li>
	<li>Determine the pressure required to elicit escape behavior (tail whisking, twisting, and squeaking), and calculate the nociceptive threshold by averaging the pressure determined at the two spots.</li>
	<li>Repeat steps 4.3. to 4.4. every 15 min.</li>
	<li>After obtaining the baseline, administer acetaminophen (300 mg/kg) to the mouse intraperitoneally. After administration, repeat steps 4.3. and 4.4 every 15 min.</li>
</ol>

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The authors have no conflicts of interest to declare

The most critical step in these experiments is the success of the i.c.v. injection. The i.c.v. injection technique used here is quite simple but requires some practice. Prior to experiments, practice with dyes (e.g. 0.5% trypan blue in saline) is recommended. If the injection is performed correctly, a needle mark should be evident on the coronal suture and the injected dye should be present in the contralateral ventricle and the third ventricle. Moreover, forcible insertion should be avoided during injection. If the needle tip is correctly placed on the coronal suture, the needle should penetrate the skull smoothly.
This i.c.v. technique can also be applied to awake, non-anesthetized mice, and we have reported the acute central effects of drugs examined using this technique23,24. Although the present procedure is advantageous in that no special equipment for cannulation is necessary, the i.c.v. injection can be performed only once. If repeated administration of drugs is required, cannulation is necessary.
The RTX test presented here is an easy-to-use approach for assessing the function of peripheral TRPV13,19. Nociceptive behavior can be observed most prominently at a dose of 1-10 ng RTX and inhibited by co-injection of capsazepine, a TRPV1 antagonist19,25. In the formalin test some groups video-tape the experiments, but the post-hoc observation is often difficult because mice tend to cover the affected paw with the head and body. Therefore, experimenters in our laboratory observe and measure the licking/biting behavior directly. In this scenario, care should be taken not to disturb the mice. In addition, in pain tests, it is very important to sufficiently calm the mouse. Excessively strong gripping and a noisy environment could produce stress-induced analgesia and delay the nociceptive response.
Mice that are i.c.v.-pretreated with RTX show a normal nociceptive response in the RTX test and the tail pressure test. However, these mice are insensitive to the analgesic effects of acetaminophen, which has been suggested to mediate central TRPV19,10. These results suggest that supraspinal-selective TRPV1 desensitization can be induced in RTX-i.c.v. mice. Although TRPV1 desensitization has been performed with local application of agonists13,14,15,16,17,18, supraspinal-selective desensitization has not yet been achieved. The RTX-i.c.v. injection protocols presented here will provide a convenient experimental model for studying the role of TRPV1 in supraspinal function.

Animals receive various physical and chemical stimuli from their environment through sensors on the peripheral nerves. The transient receptor potential vanilloid type 1 (TRPV1) is one of the thermosensitive, nonselective cation channels that act as heat sensors1,2, and activation and/or modulation of TRPV1 is known to be a key step for nociception in both normal and inflammatory contexts3. Although the overall expression pattern is controversial, expression of TRPV1 has also been suggested in supraspinal regions, being involved in various brain activities (including nociception4, thermoregulation5, anxiety6, attention deficit hyperactivity disorder7, and epilepsy8). Moreover, it has recently been suggested that acetaminophen, a widely used painkiller, mediates the activation of central TRPV1 to elicit its analgesic action9,10.
Administration of excess TRPV1 agonist including capsaicin and resiniferatoxin (RTX) to animals leads to the death of TRPV1-positive neurons and long-lasting desensitization to TRPV1 agonists11,12. Combined with the local application (intrathecal13,14, intracisternal15,16,17, and intraganglional18), this chemical ablation approach has provided an alternative way to investigate the physiological functions of TRPV1. We have recently reported that intracerebroventricular (i.c.v.) injection of RTX inhibits the analgesic effect of acetaminophen in mice, suggesting supraspinal-selective TRPV1 desensitization19. In this manuscript, we present the precise protocol for i.c.v. injection and subsequent pain tests.
Direct injection of drugs into the ventricles of the brain makes it possible to study their central effects while minimalizing any peripheral effects. The i.c.v. injection procedure presented here is a modification of the method reported by Haley and McCormick20. This method is&#160;simple involving insertion of an injection needle into the lateral ventricles through the coronal suture and does not require any special equipment or surgical procedures for cannulation.
Peripheral local application of TRPV1 agonists evokes a burning pain sensation and neurogenic inflammation. Mice that are systemically treated with RTX, and TRPV1-KO mice, are insensitive to this stimulation13. We have performed intraplantar injection of RTX (RTX test) to confirm the preservation of peripheral TRPV1 in RTX-i.c.v. mice. This method is a modification of the conventional formalin test21.
It has been reported that mice systemically treated with RTX and TRPV1-KO mice show a normal threshold to mechanical stimuli11,13,22. Here we present a procedure for the tail pressure test for testing changes in the analgesic effect of acetaminophen.
All of these procedures are orthodox and versatile, and can be applied to studies of other drugs.

<table border="0" cellpadding="0" cellspacing="0" width="872">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Resiniferatoxin</td>
			<td style="width:191px;">LKT Laboratories</td>
			<td style="width:117px;">R1774</td>
			<td style="width:319px;">used for s.c./i.c.v. pretreatments and the RTX test</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Acetaminophen</td>
			<td style="width:191px;">IWAKI SEIYAKU</td>
			<td style="width:117px;"></td>
			<td style="width:319px;">gifted from IWAKI SEIYAKU</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">Pentobarbital sodium salt</td>
			<td style="width:191px;">Tokyo Chemical Industry</td>
			<td style="width:117px;">P0776</td>
			<td style="width:319px;">used for anesthesia</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Ethanol (99.5)</td>
			<td style="width:191px;">Wako Pure Chemical Industries</td>
			<td style="width:117px;">057-00456</td>
			<td style="width:319px;">used for dissolving RTX</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Polyoxyethylene(20) Sorbitan Monooleate</td>
			<td style="width:191px;">Wako Pure Chemical Industries</td>
			<td style="width:117px;">161-21621</td>
			<td style="width:319px;">used for dissolving RTX</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">25 mL microsyringe</td>
			<td style="width:191px;">Hamilton</td>
			<td style="width:117px;">1702LT</td>
			<td style="width:319px;">used for i.c.v. injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">100 mL microsyringe</td>
			<td style="width:191px;">Hamilton</td>
			<td style="width:117px;">1710LT</td>
			<td style="width:319px;">used for intraplantar injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">26-gauge disposable needle</td>
			<td style="width:191px;">TERUMO</td>
			<td style="width:117px;">NN-2613S</td>
			<td style="width:319px;">used for i.c.v. injection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:247px;">30-gauge disposable needle</td>
			<td style="width:191px;">NIPRO</td>
			<td style="width:117px;">01134</td>
			<td style="width:319px;">used for intraplantar injection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:247px;">Pressure meter</td>
			<td style="width:191px;">Ugo Basile</td>
			<td style="width:117px;">Analgesy-Meter Type 7200</td>
			<td style="width:319px;">used for tail pressure test</td>
		</tr>
	</tbody>
</table>

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 i.c.v.-treated mice show no apparent abnormalities in their appearance, spontaneous activities, body weight19 and core body temperature (Vehicle-treated group, 38.4 &#177; 0.3 &#176;C, n = 6; RTX-treated group, 38.7 &#177; 0.2 &#176;C, n = 6).
Figure 2A-B show the responsiveness of s.c.- or i.c.v.-treated mice to the intraplantar injection of RTX. The licking/biting behavior of vehicle-treated mice was remarkable in the first 10 min19. Although the s.c.-pretreated mice did not show licking/biting behavior at all, the i.c.v.-pretreated mice normally responded to the plantar injection of RTX. Moreover, as shown in Figure 2B, intraperitoneal administration of acetaminophen (300 mg/kg) reduced the licking/biting behavior of vehicle-i.c.v.-treated mice but not that of RTX-i.c.v.-treated mice.
Figure 2C shows the analgesic effects of acetaminophen (300 mg/kg) in the tail pressure test. Acetaminophen reduced the nociceptive response of vehicle-pretreated mice in both tests, but the analgesic effects of acetaminophen were inhibited in mice that were pretreated i.c.v. with RTX.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57570/57570fig1.jpg" /> 
Figure 1: Photographic and schematic views of i.c.v. injection. (A) Needle used for i.c.v. injection. (B) Schema of the mouse skull and the movement of the needle tip. Squamosal bones are shown in blue. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57570/57570fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57570/57570fig2.jpg" /> 
Figure 2: Nociceptive responses of mice that were pretreated s.c. or i.c.v. with RTX. (A) Time course (left panel) and total time of licking/biting behavior (right panel) of s.c.-pretreated mice. RTX was injected into the plantar area at time zero (indicated by arrow head). (B) Time course (left panel) and total time of licking/biting behavior (right panel) of i.c.v.-pretreated mice. Either acetaminophen (300 mg/kg) or its vehicle (20% propyleneglycol) was administered intraperitoneally 20 min before intraplantar injection of RTX (indicated by arrow head). (C) Mechanical pain threshold in the tail of i.c.v.-pretreated mice and the analgesic effect of acetaminophen.All data were expressed as mean &#177; SEM. The number of mice in each group is shown in parentheses. The two-tailed Mann-Whitney U-test was used to compare the data for two groups. Differences at P&#60;0.05 were considered to be significant. AcAP, acetaminophen; PG, propyleneglycol; n.s., not significant; i.pl., intraplantar injection. These figures have been modified from Fukushima et al19. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57570/57570fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

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 ...

intracerebroventricular-treatment-with-resiniferatoxin-pain-tests

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.0K Views.  Musashino University. 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.

Video: Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

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 ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Comprehensive Autopsy Program for Individuals with Multiple Sclerosis

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 days a year. Participants consent to donate their brain and spinal cord. Most patients were followed by neurologists at the Cleveland Clinic Mellen Center for MS Treatment and Research. Their clinical courses and neurological disabilities are well-characterized. Soon after death, the body is transported to the MS Imaging Center, where the brain is scanned in situ by 3 T magnetic resonance imaging (MRI). The body is then transferred to the autopsy room, where the brain and spinal cord are removed. The brain is divided into two hemispheres. One hemisphere is immediately placed in a slicing box and alternate 1 cm-thick slices are either fixed in 4% paraformaldehyde for two days or rapidly frozen in dry ice and 2-methylbutane. The short-fixed brain slices are stored in a cryopreservation solution and used for histological analyses and immunocytochemical detection of sensitive antigens. Frozen slices are stored at -80 &#176;C and used for molecular, immunocytochemical, and in situ hybridization/RNA scope studies. The other hemisphere is placed in 4% paraformaldehyde for several months, placed in the slicing box, re-scanned in the 3 T magnetic resonance (MR) scanner and sliced into centimeter-thick slices. Postmortem in situ MR images (MRIs) are co-registered with 1 cm-thick brain slices to facilitate MRI-pathology correlations. All brain slices are photographed and brain white-matter lesions are identified. The spinal cord is cut into 2 cm segments. Alternate segments are fixed in 4% paraformaldehyde or rapidly frozen. The rapid procurement of postmortem MS tissues allows pathological and molecular analyses of MS brains and spinal cords and pathological correlations of brain MRI abnormalities. The quality of these rapidly-processed postmortem tissues (usually within 6 h of death) is of great value to MS research and has resulted in many high-impact discoveries.

Multiple sclerosis is an inflammatory demyelinating disease with no cure. Analysis of brain tissue provides important clues to understanding the pathogenesis of the disease. Here we discuss the methodology and downstream analysis of MS brain tissue collected through a unique rapid autopsy program in operation at the Cleveland Clinic.

We describe a rapid tissue donation program for individuals with multiple sclerosis (MS) that requires scientists and technicians to be on-call 24/7, 365 ...

Burn Injury-Induced Pain and Depression-Like Behavior in Mice

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the high mortality and sequelae in low- and middle-income countries. Burns frequently induce intense spontaneous pain and persistent allodynia as well as life-threatening problem. More importantly, excessive pain is often accompanied by depression, which may significantly decrease the quality of life. This article shows how to develop an animal model for the study of burn-induced pain and depression-like behavior. After anesthesia, burn injury was induced by dipping one hind paw of the mouse into hot water (65 &#176;C &plusmn; 0.5 &#176;C) for 3 s. The von Frey test and automated gait analysis were performed every 2 days after burn injury. In addition, depression-like behavior was examined using the forced swimming test, and the rota-rod test was performed to differentiate the abnormal motor function after burn injury. The main purpose of this study is to describe the development of an animal model for the study of burn injury-induced pain and depression-like behavior in mice.

A transient scald injury (65 &#176;C &plusmn; 0.5 &#176;C, 3 s) of one hind paw decreases the threshold (g) to von Frey filament stimulation of the ipsilateral side and alters gait pattern. Besides, burn injury induces depression-like behavior in the forced swimming test.

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of 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 ...

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

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

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

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