The overall goal of this procedure is to observe the development of Osteoarthritis pain like behavior in the mouse knee joint, by intra-articularly injecting Monoiodoacetate and measuring mechanical sensitivity and weight bearing deficits. This method can help answer key questions in the area of Osteoarthritis pain, such as which mechanisms underlying pain in Osteoarthritis, and which pharmacological targets are most promising for future therapy. The main advantage of this technique is that it's easy to perform and allows adjustment of disease verity by altering the dose of MIA.
Thus, the model provides constant pain like responses throughout the test period. The implication of this technique extend towards therapy overweight pain because the MIA model responds to conventional pain relieving therapies, and may be useful for drug discovery. For this protocol it is customary to house 8 to 10 week old mice in groups of five and let them acclimate to the housing room.
In setting up the experiment, randomize the mice keeping the average body weight of each group the same. Label the animals with numbers to blind the experimenter to their treatments. On the day of injection prepare the MIA solution in sterile saline at the desired concentrations.
Always prepare the solution wearing a mask and gloves. The highest recommend dose is one milligram per ten microliters. Use sterile saline for the control mice.
Now, anesthetize a mouse using an Anesthetic Trolley. First use an isoflurane chamber and continue delivering the same gas via a nose cone during the injection. Treat the eyes with Ophthalmic ointment, so they do not dry out.
Once the anesthesia is confirmed using a toe pinch place the mouse on its back, and trim and wipe the area surrounding the knee joint with alcohol. The patellar tendon will become visible as a white line below the patella. To stabilize the injection site secure the knee in a bent position.
Put an index finger beneath the knee joint, and a thumb above the anterior surface of the ankle joint. To find the precise site of injection, run a 26 gage needle horizontally along the knee until it finds the gap beneath the patella. Then, apply gentle pressure to mark the area and then lift the needle and the syringe vertically for the injection.
Guiding the needle with a thumb, insert the patellar tendon, perpendicular to the tibia. No resistance should be felt. Make the injection superficial to the site of entry.
The depth of the injection is critical to this model. Great care must be given to ensure that the needle has not gone too deep and is hence passed through the ankle joint. After the injection massage the knee to ensure an even distribution of the solution.
Then, place the mouse back into a clean recovery cage on a heated pad and allow it to recover. Observe the mouse until it regains sternal recumbency. Then, return it to its home cage.
Before beginning behavioral experiments always put on a gown, gloves, and a mask. On the two days prior to testing bring the mice to the testing room and let them each acclimate to being in acrylic cubicles atop a wire mesh grid in two hour sessions. On the test days habituate the animals for up to 60 minutes before testing.
Then, apply the calibrated von Frey hairs to the plantar surface of the hind paw. Start with a 0.07 gram hair. Hold the hair in the position for three seconds, or until the paw in withdrawn.
After ten seconds apply the next hair following the up-down method, until a largest force where it responds is found. Do not exceed one gram of force. A response occurring at 0.1 gram or less is considered allodynic.
Once a withdrawal is detected retest the animal. Begin with a filament one notch below the filament that produced the withdrawal. Then, continue downward through the range of forces until no response is observed followed by ascending through the forces until the response force is found again.
The goal is to collect a sequence of 6 responses in order to obtain a k'value. Repeat the process on the paws inpsilateral and contralateral to the injected limb. This procedure should be used before the injection to get a baseline, and for several days after the injection to ascertain the development of mechanical allodynia.
This test utilizes a weight and capacitance tester calibrated with a 100 gram weight before each use. Like the von Frey test, perform this test before and after the injections. To begin, train each mouse to walk into a Plexiglass chamber on the apparatus and sit in the holding box.
To do so, place the mouse in front of the holding box, lift the entrance 45 degrees and allow the mouse to freely walk in. Then, close the box. Allow the animals to move freely until they adopt a sitting posture.
Repeat this training for at least two days until the mouse stays still and does not lean on either side of the chamber. Each hind paw must be positioned on the appropriate recording pad for measurment. The measurement takes one second to collect.
Make three separate measurements of the weight born on each hind paw every session. Use the mean values to calculate the difference in weight borne by the ipsilateral and contralateral paws. Animals are considered hypersensitive if they display a weight bearing imbalance of 45 percent or less on one paw.
A time course of MIA induced mechanical hypersensitivity in the ipsilateral hind paws was made using a range of doses. The lowest dose of MIA induced a 50%decrease of thresholds on day ten, and a 70%decrease by day 28. The highest dose resulted in a significant drop of thresholds by day five.
Weight bearing changes were also calculated. The lowest MIA dose had no measured effect on weight bearing. But the next largest dose resulted in a significant reduction in the weight borne on the ipsilateral paw ten days post injection.
After watching this video you should have a good understanding of how to perform intra-articular injections of MIA into the knee of the mouse. and conduct suitable tests to assess the development of pain like behavior. Once mastered, this technique can be done in five minutes per mouse for injection if it is performed properly.
While attempting this procedure it is important to remember to keep the animals calm at all times. Don't forget that working with Monoiodoacetate can be extremely hazardous, and precautions such as wearing gloves and masks should always be taken when performing this procedure. Following this procedure other methods like histology and immuno is the chemistry of knee joint can be performed in order to answer additional questions.
After it's development this technique paved the way for researchers in the field of Osteoarthritis pain to explore the pharmacology of the disease in rodents.
