The mechanism underlying CRPS I still remain unclear. Establishing a preclinical animal model mimicking CRPS I will be crucial for exploring the mechanisms underlying CRPS I.The rat CPIP model exhibits many CRPS I like simitance, which include hindlimb edema and hyperemia in the early stage. After model establishment, followed by persistent thermal and mechanical hypersensitivities.
Begin by anesthetizing all rats with sodium phenobarbital. Make sure that the rats are properly anesthetized by pinching their hind paw or tail with forceps. And place vet ointment on the eyes.
Keep the rat on a 37 degree Celsius heated pad during the procedure. Lubricate the right hind paw and ankle with glycerol. Then slide a nitrile 70 durometer O ring into the larger side of a one point five milliliter tube with the snap cap cut off.
Carefully insert the hind paw into the hollow tube until it reaches the bottom. Gradually slide the O ring from the tube to the right hindlimb near the ankle joint. And leave it there for three hours.
Apply the same treatment to the sham group of rats, except with a broken O ring that should not induce ischemia. After three hours, cut off the O ring and monitor the rat until it regains enough consciousness to maintain sternal recumbency. Do not place the rat in the company of other rats until it is fully recovered from anesthesia.
Place the rat in a transparent plexiglass chamber on a mesh floor, and habituate the rat to the environment for 30 minutes before any behavioral testing. Use von frey filaments to test the rat for mechanical allodynia. Begin the test from the middle filament by vertically applying it to the middle plantar surface of the hind paw.
Apply suitable force to bend the filament for up to five seconds. A sudden retraction of the paw is considered a nocifensive behavior. Conduct the mechanical allodynia test on days three, two, one, and every other day until day 13.
Apply the up down testing method to test the threshold and a dickson method for calculating the 50 percent paw withdrawal threshold. To test for thermal hyperalgesia, use hargreaves method by directly aiming the light beam emitted from a 50 watt bulb to the hind paw. Measure the paw withdrawal latency, setting 20 seconds as the cutoff threshold to avoid excessive injury.
Repeat each test three times in five minute intervals for each hind paw, and take the average as the paw withdrawal latency. Conduct the thermal hyperalgesia test on days three, two, one, and every other day until day 13. To test for capsaicin induced acute nocifensive behavior, prepare the 200 millimolar capsaicin stock solution with DMSO, and further dilute it one to 1, 000 in PBS for injection.
Capsaicin is a reagent, which can cause joint irritation to skin, eyes, and respiration. It's important to wear a mask and goggle when preparing capsaicin solution. Then inject 50 microliters of the capsaicin or a vehicle into the hind paw using a 30 gauge needle attached to a one milliliter syringe.
Record nocifensive behavior, such as licking, biting, or flinching of the injected paw using a video camera for 10 minutes after the injection. Evaluate hind paw edema by measuring the increase in paw diameter at 15 minutes, 24 hours, 48 hours, and 72 hours after model establishment. After placing the O ring on the ankle, the ipsilateral hind paw skin showed cyanosis, an indication of tissue hypoxia.
After cutting the O ring, the paw began to fill with blood and showed robust swelling, indicating intense hyperemia. The swelling gradually diminished and returned to normal 48 hours after the procedure. One day after model establishment, the ipsilateral hind paw of the CPIP group exhibited obvious mechanical allodynia that persisted for 13 days of the observation time frame.
The contralateral hind paw of the CPIP group also displayed mechanical hyperalgesia for 13 days. When thermal hyperalgesia was measured, bilateral hind paws of CPIP rats exhibited significantly reduced withdrawal latency in response to noxious thermal stimuli, compared to the sham group rats. The thermal hyperalgesia of the ipsilateral hind paw persisted until the end of the observation time frame, whereas the thermal hyperalgesia of the contralateral hind paw lasted for seven days.
The sham and CPIP groups were tested for nocifensive behavior with the capsaicin injection. When a vehicle was injected, the sham group showed a slight nocifensive response and the CPIP group showed a significantly higher response. More importantly, CPIP rats showed significantly higher responses to capsaicin injection than the sham group, which suggests that the CPIP rats exhibit a phenomenon similar to human patients with CRPS I.By this procedure, the rats are anesthetized before and during model establishment.
The CPIP rat model described in this protocol established by ischemia and Human CRPS I like simitance. Families of these models, more mechanism about CRPS I can be explored.
