The overall goal of this procedure is to perform chronic constriction, injury of the sciatic nerve, a commonly used rodent model of neuropathic pain, and quantitatively measure the resulting pain behaviors. This is accomplished by first placing four chromic gut ligatures around the exposed sciatic nerve at mid thigh level to create a constriction injury to the sciatic nerve. Next, monitor pain behaviors in the hind paw at desired time points by quantitatively measuring paw withdrawal threshold to mechanical stimulus using a von fray anthes and paw withdrawal latency to thermal stimulus using a plantar analgesia meter.
Ultimately results can be obtained that show the existence of mechanical and thermal pain, hypersensitivity of the injured hind paw through the measurement of evoked withdrawal responses to both mechanical and thermal stimuli. This method of peripheral nerve injury first developed by bene and Z can help us answer key questions in the field of chronic pain, such as what are the peripheral and central mechanisms by which neuropathic pain is generated. The implications of this technique extend toward therapy of neuropathic pain because this model provides the basis to examine novel strategies to reduce pain hypersensitivity.
Generally, individuals new to this method will struggle because placing the cmic gut sutures around the static nerve and an appropriate tightness to cause sufficient but not excessive nerve injury is particularly challenging. To begin this procedure, disinfect the surgical work surface with 70%ethanol and prepare the sterile instruments gauze staples and swabs, which have been previously autoclaved. Next, anesthetize the rat in an induction chamber using 5%ISO fluorine.
Deliver 2%ISO fluorine to it through a custom made face mask during the surgery. After that, shave the left hind leg of the rat, then place the animal onto a thermo regulated heating mat at 37 degrees Celsius. Apply lubricating ophthalmic ointment to the eyes.
Then prepare the chromic gut suture by cutting it into small pieces of about three centimeters, immerse them in sterile saline. Next, sterilize the shaved area with three alternate applications of alcohol and iodine solution. With the animal lying on its thorax, elevate its left hind leg and hold the femur at 90 degrees to the spine using masking tape on the foot.
Then make an incision to the skin about three to four millimeters below the femur. Separate the skin from the muscle surrounding the incision by cutting through the connective tissue. Use a pair of blunt scissors to cut through the connective tissue between the gluteus superficialis and the biceps fems muscles.
Next, use a retractor to widen the gap between the two muscles in order to allow clear visualization of the sciatic nerve, then use the forceps and micro scissors to gently free approximately 10 millimeters of the sciatic nerve from the surrounding connective tissue. Tie the first ligature proximal to the trifurcation of the sciatic nerve for each ligature. Start with a single loose loop.
Then grasp the two ends close to the loop and pull until the loop is just barely snug and the ligature does not slide along the nerve. Then hold the loop in its proper position and place a second loop on top of the first loop to complete the knot. Next, cut the loose ends of the ligature to a round one millimeter constriction of the nerve should be minimal.
Repeat the steps for the remaining three ligatures by tying each additional ligature one millimeter more proximal than the previous. Also with minimal constriction at the end of the surgery, use sutures to close the muscle layer and staples to fasten the skin. Next, use iodine solution to sterilize the wound.
Observe the animal closely during the anesthesia recovery period and allow it to recover in a separate cage with flat paper bedding. Before starting the experiment. Handle the animal daily for several days.
Then prior to the behavioral testing, habituate the rat in the enclosure. The testing environment should be kept quiet and well controlled with constant temperature and humidity levels, and each testing session should be carried out at a similar time of the day. To study the mechanical withdrawal threshold on the day of testing, place the rat into the elevated mesh floor testing cage, 15 to 30 minutes before the experiment using a dynamic plantar von fray anter position the touch stimulator unit with the filament directly beneath the mid planter surface of the hind paw and press the start key.
This lifts the filament to mechanically stimulate the planter surface of the hind paw with an increasing force. Next, record the mechanical withdrawal threshold from both injured and uninjured hind paws. This device automatically records and displays the maximum for supplied upon withdrawal reaction.
Repeat the mechanical stimulation three times with an interval of about five minutes between stimuli testing and uninjured hind paws in a counterbalanced order, and then calculate the mean of the paw withdrawal thresholds. To study thermal withdrawal latency on the day of testing, place the rat into the glass floor. Testing cage 15 to 30 minutes before the experiment using a thermal planter analgesia instrument, position the infrared source directly beneath the mid planter surface of the hind paw and press the start key.
This exposes the planter surface of the hind paw to a beam of radiant heat through a transparent glass surface. Then record the withdrawal latency from both injured and uninjured hind paws. This device automatically records the time taken from the onset of the thermal stimulus to the withdrawal of the paw from the heat source.
Repeat the heat stimulation at least three times with an interval of about five minutes between stimuli testing injured and uninjured hind paws in a counterbalanced order, and then calculate the mean of paw withdrawal Latencies here shows the mechanical withdrawal threshold in both injured and uninjured paws of wistar rats before and up to 12 days after chronic constriction of the sciatic nerve. There is a significant reduction in paw withdrawal threshold to mechanical stimuli in the injured ipsilateral hind paw compared to the uninjured contralateral hind paw. Shown here is the thermal withdrawal latency in both injured and uninjured paws before and up to 12 days after chronic constriction of the sciatic nerve.
Similarly, the thermal withdrawal latency is significantly reduced in the injured ipsilateral hind paw compared to the uninjured contralateral hind paw. Once mastered the CCI surgery can be properly performed in as little as 20 minutes per animal, resulting in long-term pain hypersensitivity of the hind paw. While attempting this procedure, it's important to remember to loosely tie the ligatures as it is the self strangulation beneath the ligatures following edema, which causes a constriction After its development.
This technique of peripheral nerve injury combined with testing of pain hypersensitivity P wave for researchers in the field of neuropathic pain to explore the mechanisms underlying chronic pain due to nerve injury.
