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><tbody><tr><td>Resiniferatoxin</td><td>LKT Laboratories</td><td>R1774</td><td>used for s.c./i.c.v. pretreatments and the RTX test</td></tr><tr><td>Acetaminophen</td><td>IWAKI SEIYAKU</td><td></td><td>gifted from IWAKI SEIYAKU</td></tr><tr><td>Pentobarbital sodium salt</td><td>Tokyo Chemical Industry</td><td>P0776</td><td>used for anesthesia</td></tr><tr><td>Ethanol (99.5)</td><td>Wako Pure Chemical Industries</td><td>057-00456</td><td>used for dissolving RTX</td></tr><tr><td>Polyoxyethylene(20) Sorbitan Monooleate</td><td>Wako Pure Chemical Industries</td><td>161-21621</td><td>used for dissolving RTX</td></tr><tr><td>25 &amp;#956;L microsyringe</td><td>Hamilton</td><td>1702LT</td><td>used for i.c.v. injection</td></tr><tr><td>100 &amp;#956;L microsyringe</td><td>Hamilton</td><td>1710LT</td><td>used for intraplantar injection</td></tr><tr><td>26-gauge disposable needle</td><td>TERUMO</td><td>NN-2613S</td><td>used for i.c.v. injection</td></tr><tr><td>30-gauge disposable needle</td><td>NIPRO</td><td>01134</td><td>used for intraplantar injection</td></tr><tr><td>Pressure meter</td><td>Ugo Basile</td><td>Analgesy-Meter Type 7200</td><td>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>

Watch this Scientific Journal Video about Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice at JoVE.com

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

8.1K Views.  Musashino University. Het algemene doel van deze voorbehandeling is het genereren van muizen en dat TRPV1-kanalen worden gedesensibiliseerd in supraspinale regio's. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het neurofarmacie veld. Het belangrijkste voordeel van deze techniek is dat supraspinale selectieve TRPV1 desensibilisatie kan worden veroorzaakt door een vrij eenvoudige manier.Om deze procedure te beginnen, verdoven een muis met pentobarbital natrium intraperitoneally en co...

Video: Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a Xist&#916;:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

Here, we describe a protocol to generate a viable female murine model with non-random X chromosome inactivation, i.e., the maternally-inherited X chromosome is inactive in 100% of the cells. We also describe a protocol to test feasibility, tolerability, and safety of pharmacological reactivation of the inactive X chromosome in vivo.

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all ...

Genetics

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

Medicine

Investigating Migraine-Like Behavior Using Light Aversion in Mice

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as photophobia. Whilst it is impossible to confirm that a mouse is experiencing migraine, light aversion can be used as a behavioral surrogate for the migraine symptom of photophobia. To test for light aversion, we utilize the light/dark assay to measure the time mice freely choose to spend in either a light or dark environment. The assay has been refined by introducing two critical modifications: pre-exposures to the chamber prior to running the test procedure and adjustable chamber lighting, permitting the use of a range of light intensities from 55 lux to 27,000 lux. Because the choice to spend more time in the dark is also indicative of anxiety, we also utilize a light-independent anxiety test, the open field assay, to distinguish anxiety from light-aversive behavior. Here, we describe a modified test paradigm for the light/dark and open field assays. The application of these assays is described for intraperitoneal injection of calcitonin gene-related peptide (CGRP) in two mouse strains and for optogenetic brain stimulation studies.

Rodents are not able to report migraine symptoms. Here, we describe a manageable test paradigm (light/dark and open field assays) to measure light aversion, one of the most common and bothersome symptoms in patients with migraines.

Migraine is a complex neurological disorder characterized by headache and sensory abnormalities, such as hypersensitivity to light, observed as ...

Behavior

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon ...

Establishing a Mouse Model of a Pure Small Fiber Neuropathy with the Ultrapotent Agonist of Transient Receptor Potential Vanilloid Type 1

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to degeneration and injury of small-diameter sensory neurons, referred to as small fiber neuropathy. Present animal models of small fiber neuropathy affect both large- and small-diameter sensory fibers and thus create a neuropathology too complex to properly assess the effects of injured small-diameter sensory fibers. Therefore, it is necessary to develop an experimental model of pure small fiber neuropathy to adequately examine these issues. This protocol describes an experimental model of small fiber neuropathy specifically affecting small-diameter sensory nerves with resiniferatoxin (RTX), an ultrapotent agonist of transient receptor potential vanilloid type 1 (TRPV1), through a single dose of intraperitoneal injection, referred to as RTX neuropathy. This RTX neuropathy showed pathological manifestations and behavioral abnormalities that mimic the clinical characteristics of patients with small fiber neuropathy, including intraepidermal nerve fiber (IENF) degeneration, specifically injury in small-diameter neurons, and induction of thermal hypoalgesia and mechanical allodynia. This protocol tested three doses of RTX (200, 50, and 10 &#181;g/kg, respectively) and concluded that a critical dose of RTX (50 &#181;g/kg) is required for the development of typical small fiber neuropathy manifestations, and prepared a modified immunostaining procedure to investigate IENF degeneration and neuronal soma injury. The modified procedure is fast, systematic, and economic. Behavioral evaluation of neuropathic pain is critical to reveal the function of small-diameter sensory nerves. The evaluation of mechanical thresholds in experimental rodents is particularly challenging and this protocol describes a customized metal mesh that is suitable for this type of assessment in rodents. In summary, RTX neuropathy is a new and easily established experimental model to evaluate the molecular significance and intervention underlying neuropathic pain for the development of therapeutic agents.

This study establishes an experimental model of pure small fiber neuropathy with resiniferatoxin (RTX). A unique dose of RTX (50 &#181;g/kg) is optimal for developing a small fiber neuropathy model that mimics patient characteristics and could help investigate the nociceptive molecular significance underlying neuropathic pain.

Patients with diabetes mellitus (DM) or those experiencing the neurotoxic effects of chemotherapeutic agents may develop sensation disorders due to ...

Intracranial Pharmacotherapy and Pain Assays in Rodents

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the development of effective therapeutics. Intracranial pharmacology presents an important tool for understanding the molecular and cellular mechanisms of pain in the brain, as well as for novel treatments. Here we present a protocol that integrates intracranial pharmacology with pain behavior testing. Specifically, we show how to infuse analgesic drugs into a select brain region, which may be responsible for pain modulation. Furthermore, to determine the effect of the candidate drug in the central nerve system, pain assays are performed after intracranial treatment. Our results demonstrate that intracranial administration of analgesic drugs in a targeted region can provide relief of pain in rodents. Thus, our protocol successfully demonstrates that intracranial pharmacology, combined with pain behavior testing, can be a powerful tool for the study of pain mechanisms in the brain.

Here we present a protocol to perform intracranial pharmacological experiments followed by pain behavior assays in rodents. This protocol allows researchers to deliver molecular and cellular targets in the brain, for pharmacologic agents in the treatment of pain.

Pain is a salient sensory experience with affective and cognitive dimensions. However, central mechanisms for pain remain poorly understood, hindering the ...

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

Elimination of Serotonergic Neurons by Stereotaxic Injection of 5,7-Dihydroxytryptamine in the Dorsal Raphe Nuclei of Mice

Stereotaxic injection has been widely used for direct delivery of compounds or viruses to targeted brain areas in rodents. Direct targeting of serotonergic neurons in the dorsal raphe nucleus (DRN) can cause excessive bleeding and animal death, due to its location below the superior sagittal sinus (SSS). This protocol describes the generation of a DRN serotonergic neuron-lesioned mouse model (&#62;90% survival rate) with stable loss of &#62;70% 5-HT-positive cells in the DRN. The lesion is induced by stereotaxic injection of a selective serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) into the DRN using an angled approach (30&#176; in the anterior/posterior direction) to avoid injury to the SSS. DRN serotonergic neuron-lesioned mice display anxiety-associated behavior alterations, which helps to confirm success of the DRN lesion surgery. This method is used here for DRN lesions, but it can also be used for other stereotaxic injections that require angular injections to avoid midline structures. This DRN serotonergic neuron-lesioned mouse model provides a valuable tool for understanding the role of serotonergic neurons in the pathogenesis of psychiatric disorders, such as generalized anxiety disorder and major depressive disorder.

This protocol describes a dorsal raphe nucleus (DRN)-lesioned mouse model (&#62;90% survival rate in experimental mice) with stable loss of dorsal raphe serotonergic neurons by stereotaxic injection of 5,7-dihydroxytryptamine into the DRN using an angled approach to prevent injury to the superior sagittal sinus.

Stereotaxic injection has been widely used for direct delivery of compounds or viruses to targeted brain areas in rodents. Direct targeting of ...

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

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

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

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

Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice

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

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

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

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

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