The overall goal of this procedure is to measure rodent antinociceptive behaviors with the operant or facial pain assessment device. This is accomplished by first training the rodents on the oad for a few sessions until stable baselines are obtained. The next stage of the procedure is to remove the rodent's buckle hair and fast them the day before the experiment.
Next, baseline measurements at normal and painful temperatures are made in the oped temperature variable bars that stimulate the facial derma. If the rodent wants to gain the reward. The final stage is to introduce a pain or analgesic stimulus under the same conditions and measure the licked contact frequencies.
For example, capsaicin reduces lick frequency under painful conditions. Ultimately, results can show altered pain behaviors by analysis of the frequency of licks in the total contacts. The main advantage of this technique over existing methods like the P withdrawal or von fry, or the tail flick assay, is that instead of using reflex based measures of nociception, the orofacial pain assessment device or oped uses an operant reward conflict assay The night before an oped training session.
The rodents should not have any food to eat for this demonstration. The fast time is 15 hours, give or take an hour. The rodents must first be trained to a consistent behavior at non aversive temperatures.
This takes about six sessions. At three sessions per week. Mice can lick 600 to a thousand times per session, whereas rats can lick up to 2000 times per session.
This demonstration shows how to program a simple experiment using the Orofacial software. It has many other temperature ramping protocols and pain models available. Also, the design can be saved in any state of completion.
After turning on op pads and opening the software, turn on white noise to control for ambient noise. Next, under file, select a new experiment. Then under the subheading protocol, choose a name for the protocol and select whether it is to be blinded or not.
Under the OPED cages menu, select new oped cage and then add all connected oped cages. Under outputs, select temperature controllers. Then thermal element, adjust starting temperature to a neutral temperature if needed, adjust the ramping temperatures under the oped temperature cycle tab to change the temperature from neutral to hot to cold.
Just make the adjustments here. For step one, set the temperature to 45 degrees Celsius. Set the ramp duration to 30 seconds and set that temperature to remain for three minutes.
For step two, make the settings 33 degrees, 30 seconds, and three minutes. For step three, make the temperature seven degrees the ramp, 60 seconds and remain time. Three minutes.
Make step four, like step two, but with a 62nd ramp. Then check the box for after the following period of time and set that value to three minutes. Now under the fields tab, add any extra notes needed about the subjects.
For example, make an area for animal ID by selecting new field and changing the name to animal id. Then choose the animals tab, then text, and then use this field as the animal ID look under the stage tab. The OPPA automatically sets a first stage, name the stage, and set the duration of test period.
The behavioral session shouldn't last for longer than 10 to 20 minutes. cause after that time, the rows become satiated. Also, when you're doing experiments for several days, additional stages can be added to make the data analysis simpler.
Next, under the calculations, select new calculation and name it L over F to represent the L to face ratio. In the section labeled, enter the calculation in the area below. Adjust it to read L.Number of activations over contact.
Number of activations to create time periods for a simpler data analysis, select analysis and new time period. Name 1 33 degrees C and select the box for this period is the same in all stages. For the starts at field, enter zero.
And for the NZ field, put three minutes. Now repeat this labeling process for each time period. Finally, save and name the protocol.
It can now be used for new experiments because hair is an insulator, hairless, rodents are best for all operant procedures. Otherwise, a day or two before the animals are tested, remove just their buccal facial hair, leaving the whiskers to do this. First anesthetize the rodent.
Next, shave the cheek hair with clippers. Once the hair is clipped, apply eye ointment followed by hair removal cream, and wait a few minutes. Then wash the hair removal cream and hair away with water.
On the day of testing, anesthetize the animal with, for example, 3%isof fluorine inhalation. Next, apply ophthalmic fat ointment to both eyes. Later, avoid getting any topical drug treatments in the eyes.
Now apply a 0.1%capsaicin cream bilaterally on the exposed cheeks with a sterile cotton swab, and wait five minutes. Meanwhile, soak some gauze pads in warm 40 degrees Celsius water. After five minutes, wipe the cream off with the wet pads, followed by an alcohol swab.
Now return the rodent to its cage and set a timer for 30 minutes. First, prepare a room temperature reward mixture of one part sweetened condensed milk, two parts water. Load the mixture into the oped bottles.
Second, set up the liquid catch tray, the plexiglass cage, and the metal flooring grate on the oped machine. Once that is assembled, attach the wiring to connect the cage to the rest of the oped system. Place a reward bottle on the stand with its spout accessible to the rodent.
Initially, position the bottle a little deep so it can be withdrawn if better contacts are needed. Now, load the experiment in the software. Enter the animals being tested under the subheading.
Experiment at a title, and then add the treatment groups such as capsaicin and control. Then enter the number of animals in each group. Under the subheading tests, select animal treatments and data.
Add the letter of the treatment and the animal id. The boxes should now have the designated animal ID on their screens. Now, press the button on the box to adjust the thermos to the right temperature.
When the light goes green, place a rodent inside. For this experiment, rats are started 30 minutes after the capsaicin cream was wiped off. Now press the button again and the orange light will turn on.
Adjust the distance. The bottle is from the box to be able to lick. The rat must make contact with the thermos on its buccal region, not the vibra region.
Proper adjustment will result in a solid red box for licking above an outlined orange box for contacts. An outlined red box indicates a problem. Once the testing session is over, the oped will alert the experimenter with a tone.
Now return the rodent to its cage. If another rodent is to be tested, the box will indicate its animal ID and repeat the procedure. Many rats can be run simultaneously on the op pad.
In one protocol, up to 16 boxes can be run simultaneously. Thus, 40 to 80 rats can be tested per hour. When the experiment is complete, shut off the machine and remove the cage wiring by hand or machine wash and sanitize the great box bottle and liquid tray.
In the oped software, data analysis tools are available. Under the subheading.Results. Select whether to see a text graph or statistical analysis report.
In the graph report settings box, select a variable to examine. For instance, under calculation results, check the L over F box. Now specify the on the X Axi show field as the time period and show different series four field as the treatment.
Then select view the report. Saving, printing, copying, or emailing the report can be done at this time. Certain data points may be excluded in the box below the grouping step if needed.
If needed. The raw data in a spreadsheet form is accessible under the data subheading. The OPED was programmed for a ramping protocol for the test sessions.
Analysis of a single rodents test session shows that the lick number is high for every segment of the session at the neutral temperature, but low at the aversive temperatures. As expected, long bouts of contact are made at the neutral temperatures. Those durations decrease and the number of contacts increase during periods where the temperatures are painful.
The total amount of reward ingested is similar to the number of licks. Animals prefer feeding at the neutral temperatures to the painful temperatures. The lick to face ratio for the baseline session was calculated by the OPPA for all the animals without caps a s an application, this ratio is much higher during the three non-painful sessions than at the painful sessions.
One-way, Inova and Bonferroni tests bear out the significance. On a subsequent day. The group was then divided into capsaicin treated rodents and naive rodents.
Eight in each group at neutral temperatures. The groups did not behave significantly differently. Capase and treated rodents had a significantly lower LTA F ratio at 45 Celsius and an insignificant higher LTA F ratio at seven.
Once mastered, this technique can be done on hundreds of animals in a single day using several ePad boxes. This allows for high throughput pain testing of nociceptive or analgesic conditions if performed properly.
