This protocol outlines a non-invasive method to induce chronic back pain in mice for up to 10 weeks, which can be performed quickly and reliably with minimal training. The most difficult aspect of this technique is to recognize how injecting ligament feels versus injecting muscle. Feeling resistance upon contact indicates ligament injection.
No resistance means the needle could be in the muscle or abdominal cavity. To begin, take a sterile flat surface to place the mouse. Then, place a heating pad recovery station next to the surgery setup to immediately transfer mice after injection.
Next, prepare the urokinase and a Hamilton syringe. Draw the solution beforehand to minimize the time the mouse spends under anesthesia. After anesthetizing the mouse, use two fingers to locate the lumbar spinal segments at L2 and L3 below the rib cage and verify the injection site.
Place the tip of the Hamilton syringe next to the spine. Position the syringe at approximately 45-degree angle into the ligament immediately adjacent to the bone. Insert the needle tip gently, but firmly, into the ligament, and inject slowly until all five microliters have been injected.
Remove the needle gently and slowly. Then, place the mouse in the heat recovery station until it wakes up and becomes mobile. Check the mice for one hour after surgery to ensure all normal motor function continues.
After the surgery, check the weight and injection site of the mice daily for a week to prevent any infection or complications. Before testing, move each animal into a separate clear cubicle on the testing table with a screen top. Then, stimulate the foot pad with the 3.61 Von Frey filament that elicits four grams of force.
A foot withdrawal of three to five stimuli is considered a positive response. Now, apply the next weaker filament in the series until the animal stops responding to the mechanical stimulation. Use the higher filament until a response is elicited, then switch back to the lower filament.
Using a curve fitting algorithm, calculate the mechanical withdrawal threshold, which is the minimal amount of force needed to elicit a response 50%of the time. To perform the Hargreaves Test, place the mice in cubicles on a glass surface heated from below by an infrared emitter. Measure the time from applying infrared light stimulus on the mouse's hind paw to withdrawal as foot latency.
For the Cold Plate Test, place the mice on the cold plate apparatus cooled to minus nine degrees Celsius. Record the time the mouse takes to lift its foot after being placed on the apparatus as the latency to withdraw the foot. To perform the Anxiety Test, position each animal into the place preference test box with a passageway between two chambers.
One apparatus chamber should be brightly lit, while the opposite side should remain dark. Using a computer, monitor the animal's light and dark occupancy times and transitions during a 10-minute test. Next, place the model rodents or naive animals on the zero maze, or use the two-chamber light or dark place preference test.
Assess the time spent in closed and open areas of the maze using a stopwatch. For the Sucrose Splash Depression Test, spray a 10 to 30%sucrose solution on the dorsal code and score the frequency, duration, and latency of grooming for 10 minutes. To perform novel object tests, acclimate mice individually to a clear plastic cage with an open top for one hour.
Add two identical toy mini figures to opposite corners of the cage for five minutes. Acclimatize the animals on the testing day for one hour, and place the two identical mini figures in the same positions of the cage for five minutes before returning them to the home cage. Then, replace one of the original figures with a distinctly different novel object.
After four hours, return the mice to the test cage and record the time spent exploring the objects. For motor function assessment, construct a tunnel from a paper towel tube cut lengthwise on one edge. Spread open the tunnel on a clean piece of printer paper.
Hold the mice gently until they are calm. Apply non-toxic India ink on a mouse's paws by placing them into an ink well, stamp pad, or using a cotton swab. Release the mice at the tunnel entrance.
Allow them to run through the tunnel and capture them at the end. Score paw prints based on stride length, stride width, and toe spread. Mechanical and thermal hypersensitivity assays demonstrated that the chronic back pain mice had significantly increased mechanical, heat, and cold sensitivity compared to the naive controls.
Anxiety analysis showed that the total time spent in the light chamber of the light and dark box and the number of rearing events were significantly lower in mice with chronic back pain, indicating anxiety. During the Sucrose Splash Test, mice with chronic back pain exhibited lower grooming time and the number of grooming. However, the amount of time before grooming starts was significantly increased.
Stride length between mice with chronic back pain and naive mice was significantly different. The most important thing to remember when attempting this procedure is that you are aiming for the ligaments around the spine, not the muscle. The chronic back pain model provides a reliable tool to assess a variety of novel research, pharmacologic, and other potential therapeutics.
