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 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 action^9,10.

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 agonists^11,12. Combined with the local application (intrathecal^13,14, intracisternal^15,16,17, 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 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 stimuli^11,13,22. 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.

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 500 µL of polyoxyethylene (20) sorbitan monooleate to the solution above and vortex well.
   3. Add 4 mL of physiological saline to the mixture and vortex well.
   4. Aliquot 40 µL of the solution into 1.5-mL screw cap tubes, and store them at -40 °C.
2. Acetaminophen
   1. 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.

2. Subcutaneous or Intracerebroventricular Injection of RTX

1. Thaw the stocked solution prepared in 1.1. above and dilute it to 20 µg/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).
2. Anesthetize mice with pentobarbital sodium salt (60 mg/kg, intraperitoneally), and check for loss of the righting reflex.
3. For s.c. treatment, inject RTX (20 µg/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.
4. For i.c.v. treatment, inject 5 µL of RTX (20 µg/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.
   1. 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).
   2. 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.
   3. Move the needle laterally on the scalp, and find the sagittal suture as the needle tip is hooked on the suture.
   4. 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).
   5. Insert the needle slowly and vertically, inject the RTX solution in about 10 seconds, and hold it for about 10 seconds.
   6. 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.
5. Assign the pretreated mice as subjects for the RTX test or the tail pressure test (Step 3 and 4, respectively).

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 °C.

1. One week after pretreatments with RTX (Step 2.), transfer mice to the testing room at least 60 min prior to starting the test.
2. Weigh and place each mouse individually in a plexiglass cage (29.5 × 17.5 × 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.
3. Administer acetaminophen (300 mg/kg) to the mouse intraperitoneally 20 min before the test.
4. 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 µL of RTX solution (0.05 µg/mL).
5. Measure the period of licking/biting behavior in theglabrous region of the affected paw in each 5-min block.

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 °C.

1. 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.
2. Mark the spots at 1.5 and 2.5 cm from the base of the tail.
3. 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.
4. 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.
5. Repeat steps 4.3. to 4.4. every 15 min.
6. 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.

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