As a plethora of innovative therapies are now being investigated to treat chronic pain, our protocol provides crucial concepts for the standardization of surgeries required to induce neuropathic pain. Partial sciatic nerve ligation, or PSNL, is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. As PSNL reproduces many symptoms observed in humans with neuropathic pain, this protocol provides crucial concepts to study and develop new pain management therapies.
Demonstrating the procedure will be Kevin Cheng, an MD PhD candidate in my laboratory. To begin, habituate the mice to the Von Frey testing apparatus for one hour prior to testing by placing them in clear plexiglass boxes on a wire mesh in the same room as the testing room. Apply a series of calibrated fine mono filaments perpendicularly to the planter surface of the PSNL ipsilateral hind paw of the animals held in suspended wire mesh cages.
Determine the withdrawal threshold by sequentially increasing or decreasing the stimulus strength corresponding to the size of the filament. Increase the size of the filament when the response is negative and decrease the size of the filament when the animal withdraws its paw. Report negative and positive responses in the data sheet and test the same paw four times with different filaments following the first positive response.
Habituate the mice to the Hargreaves testing apparatus for one hour prior to testing by placing them in clear plexiglass boxes in the same room as the testing room. Establish the baseline paw withdrawal latencies using the Hargreaves apparatus. Focus a radiant heat source onto the planter surface of the hind paw ipsilateral to the PSNL and start stimulation.
When the animal withdraws its paw, wait for a motion detector to halt the stimulus and stop the timer. Adjust the intensity of the heat source to obtain a baseline of paw withdrawal latency of approximately 10 seconds. Next, record the withdrawal time in a table.
Set the hot plate to 52 degrees Celsius. Place the animal in the testing chamber and start a chronometer. Observe for nocifensive behaviors and stop the chronometer as soon as nocifensive behavior is observed.
Then remove the animal from the chamber and record the latency to this behavior. Make sure to clean the apparatus of any urine between each animal tested to avoid affecting the temperature of the heat stimulus. Clean the testing chamber with 70%ethanol between animals to reduce the behavioral impact of odors.
To make a nerve glass hook, turn on the bunsen burner. Hold one end of the glass rod to the fire in one hand. As this glass rod melts, use another glass rod in the other hand to guide and pull at the melting glass on the first rod.
Remove the first glass rod from the fire and allow the end of the melted portion to naturally roll inward to form a small ball shape using the second glass rod. Pinch the toes on a hind paw with tweezers to ensure the absence of paw reflex and check the corneal blink reflex before applying lubricating ophthalmic ointment. Upon choosing which side to perform the surgery on, shape the animal's hind leg around the thigh region, inferiorly toward the patella, superiorly toward the hip and above the femur.
Next, wipe three times with chlorhexidine in one direction with three separate gauzes alternated with warm sterile saline. Then slip the leg through a slit made in a 10 by 10 centimeters sterile drape to create a sterile field around the leg of choice. Using fine surgical scissors make a small two millimeter cut of the skin in the midline of the lateral aspect of the thigh.
Slide the scissors under the skin in a circular motion to break through the fascia and create a clearance, enlarging the incision space. Next, using tying forceps create a sharp one centimeter deep incision vertically at a 90 degree angle in the thigh muscles. Then insert the fine small scissors into the same incision also at a 90 degree angle and spread them open gently to separate the muscles until the sciatic nerve is visualized.
Locate the sciatic nerve running parallel to the vertical thigh in the direction of the hip to the knee. Remove the scissors in the tying forceps from the body before proceeding. Use the extra fine forceps in the nerve glass hook to isolate the nerve from underneath.
Carefully free the nerves from surrounding connective tissues at a site near the trochanter of the femur which is closest to the hip and furthest from the knee. Allow the nerve to rest on the glass rod and ensure that the end of the rod prevents the nerve from rolling off. Place a surgical knot to tie one third of the width of the sciatic nerve using a nine zero nylon suture prior to it dividing into the common peroneal, tibial and sural nerve branches.
Carefully slip the nerve off the glass rod once the knot is complete and tuck it back into the original location at the level below the separated muscles. Next, suture the muscle incision using an absorbable polyglycolic five zero suture. Separately, suture the skin using a non-absorbable polypropylene six zero suture.
Then record the surgery and anesthesia stop time. Allow the mouse to wake up alone in a recovery cage before returning it to a new clean cage. Use the Von Frey, Hargreaves or hot plate test to evaluate both thermal and mechanical hypersensitivity and its potential reversal.
Mice that underwent PSNL surgery demonstrated lower thresholds to mechanical stimuli compared to sham animals over 28 days. Similar results were obtained with thermal hypersensitivity evaluation. Paw withdrawal latencies after exposure to radiant heat stimulus were increased in PSNL animals as well as the withdrawal latencies when animals were placed on a 52 degree Celsius plate.
Groups injected with two different doses of morphine displayed a reversal of hypersensitivity which lasted from one to two hours. Mechanical hypersensitivity returned to baseline four hours after the injection of morphine. When two different doses of ibuprofen were administered intraperitoneally to the mice, the results demonstrated decreased mechanical hypersensitivity compared to saline injected mice.
Limiting additional damage to the nerve is essential as it can affect pain sensory thresholds. The experimenter should pay particular attention and use the glass hook to reduce unintentional damage Following this procedure, this model of chronic neuropathic pain can be used to identify and characterize the antinociceptive effect of drugs leading to the development of new pain management therapies.
