The overall goal of this pretreatment is to generate mice and that TRPV1 channels are desensitized at supraspinal regions. This method can help answer key questions in the neuropharmalogical field. The main advantage of this technique is that supraspinal selective TRPV1 desensitization can be induced by a quite simple way.
To begin this procedure, anesthetize a mouse with pentobarbital sodium intraperitoneally and check for loss of righting reflex. For subcutaneous treatment, inject RTX at 20 micrograms per milliliter into the back of the neck at a volume of 0.1 milliliter per 10 grams body weight. For the control group, inject the vehicle in the same way.
Pass a disposable 27 gauge needle through a metal tube to expose 3 to 3.5 millimeter tip of the needle. Then hold the squamosal bones of the mouse firmly with the fingers. Move the needle laterally on the scalp and find the sagittal suture as the needle tip is hooked on the suture.
Move the tip about one millimeter to the right, then move the tip rostrally to find the coronal suture. Afterward, insert the needle slowly and vertically. Inject the RTX solution over 10 seconds and hold it for another 10 seconds afterward.
Subsequently, withdraw the needle slowly and return the mouse to its home cage. One week after RTX injection, at least 60 minutes prior to the test, transfer the mouse to the testing room. At least 30 minutes prior to the test, place the mouse in a plexiglass cage in order to allow it to acclimate to the environment.
Then 20 minutes before the test, administer acetaminophen at 300 milligrams per kilogram to the mouse intraperitoneally. 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 pad and inject 20 microliters of RTX solution at 0.05 micrograms per milliliter.
Subsequently, measure the period of licking and biting behavior in the glabrous region of the affected paw in each five-minute block. One week after RTX injection, transfer the mouse to the testing room. Place the mouse in a plexiglass cage.
Mark the spots at 1.5 and 2.5 centimeters from the base of the tail. Next, hold the mouse loosely in a small cloth bag and apply pressure to the spots with a blunt probe. Please note that cutoff pressure of 250 gram is imposed to avoid tissue damage.
Determine the pressure required to elicit escape behavior and calculate the nociceptive threshold by averaging the pressure determined at the two spots. Repeat the procedures every 15 minutes. After obtaining the baseline, administer acetaminophen at 300 micrograms per kilogram to the mouse intraperitoneally, then repeat the tail pressure test every 15 minutes until it reaches 90 minutes.
These two figures show the responsiveness of SC or ICV treated mice to the intraplantar injection of RTX. The licking and biting behavior of vehicle-treated mice was remarkable in the first 10 minutes. Although the SC pretreated mice did not show licking and biting behavior at all, the ICV pretreated mice normally responded to the plantar injection of RTX.
Moreover, intraperitoneal administration of acetaminophen reduced the licking and biting behavior of vehicle ICV treated mice, but not in RTX ICV treated mice. This graph shows the analgesic effects of acetaminophen at 300 micrograms per kilogram 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 ICV with RTX.
Once mastered, this ICV injection technique can be applied for other drugs. Don't forget that working with Resiniferatoxin can be extremely hazardous and make sure to use rubber gloves and glasses for protection when handling.
