Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

Summary

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.

Abstract

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.

Introduction

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 sensors^1,2, 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 nociception

Protocol

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

1. 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.
   1. Add 500 µL of ethanol to 1 mg of RTX.
   2. Add 5....

Results

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 ± 0.3 °C, n = 6; RTX-treated group, 38.7 ± 0.2 °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.......

Discussion

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 need.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Resiniferatoxin	LKT Laboratories	R1774	used for s.c./i.c.v. pretreatments and the RTX test
Acetaminophen	IWAKI SEIYAKU		gifted from IWAKI SEIYAKU
Pentobarbital sodium salt	Tokyo Chemical Industry	P0776	used for anesthesia
Ethanol (99.5)	Wako Pure Chemical Industries	057-00456	used for dissolving RTX
Polyoxyethylene(20) Sorbitan Monooleate	Wako Pure Chemical Industries	161-21621	used for dissolving RTX
25 μL microsyringe	Hamilton	1702LT	used for i.c.v. injection
100 μL microsyringe	Hamilton	1710LT	used for intraplantar injection
26-gauge disposable needle	TERUMO	NN-2613S	used for i.c.v. injection
30-gauge disposable needle	NIPRO	01134	used for intraplantar injection
Pressure meter	Ugo Basile	Analgesy-Meter Type 7200	used for tail pressure test