Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Our data indicate that algogens reduce scratching behaviors, but the effect of capsaicin on scratching behaviors remain unclear. Therefore a simultaneous measurement of each amount of pain are important. Traditional method provide information for only itch or pain sensations, but the cheek injection model provide information for itch and pain, separately and simultaneously.

The cheek injection can be difficult at first, as it takes practice to discriminate the scratching, wiping, and grooming behaviors. Video demonstration of the injection and behavioral analysis can be helpful for mastering the techniques. For a cheek injection experiment, on the day before evaluation, shave the fur on the cheek of each mouse with hair clippers, and mount a video camera with a 30 to 60 frames per second frame rate onto a tripod, in a temperature, humidity, and sound-controlled environment.

Place four cages under the camera in the indicated orientation, and place screens between each cage to prevent the mice from seeing each other. One day after shaving, place one mouse into each recording cage for one hour to acclimatize the animals to the recording conditions. If a topical treatment is to be used, apply it at this time.

While the animals are acclimating, load the test substance of interest into one 0.5 milliliter syringe equipped with a 29-or 30-gauge needle per mouse. At the end of the acclimation period, start the video recording prior to injection. Then inject a 10 microliter solution of the test substance intradermally into the shaved cheek of the awake animal.

Then return the injected mice to their respective cages and continue the video recording for 30 minutes. At the end of the experiment, load the video into a suitable video editing software program. Count each time a mouse raises a hind leg toward the cheek and scratches several times over one or a few seconds before putting their leg down as one scratch bout.

Mice use a single foreleg for wiping, and both forelegs for grooming, but only wiping behaviors are counted as pain-related behaviors. For a neck injection experiment, one day before the experiment, shave the fur on the nape of each mouse’s neck with hair clippers. One day after shaving, acclimate the mice in the recording cages for one hour before injecting 10 microliters of the test substance of interest intradermally into the shaved neck, under awake conditions.

Return the injected animals to their recording cages and record the mouse behaviors for 30 minutes. Then count the scratching bouts on the neck and any face wiping behaviors as just demonstrated. In the neck injection model, histamine induces strong scratching behaviors, while vehicle induces mild scratching.

In the cheek injection model, capsaicin does not induce scratching behaviors, but induces wiping behaviors, which are thought to be related to pain. Following injection into the cheek, histamine mainly induces scratching behaviors with weak wiping behaviors. The application of an anti-histamine reagent inhibits scratching, suggesting that histamine-induced scratching behaviors are caused by the reaction of histamine on histamine receptors.

In this experiment, the application of an itch mediator induced scratching, but not wiping behaviors, indicating that the tested mediator acts as a pruritogen, but not an algogen in mice, similar to its effects in humans. This method allows itch and pain sensations to be easily and clearly distinguished with one procedure. A detailed behavior analysis can be done by focusing on either itch or pain sensation.

Itch and pain behaviors accord different responses. Also these techniques are not difficult. The cheek injection and observation procedure may let various of our training peers to master.

We appreciate introduction of the cheek injection model by Professor LaMotte in Yale University.