Electrophysiological Methods to Assess Peripheral Pain Block in an Anesthetized Rat

The goal of our research is to develop peripheral electrical and near infrared nerve blocks for the treatment of pain. Technologies such as direct current and high frequency alternating current electrical blocks and near-infrared photobiomodulation offer potential for clinical pain relief. To begin, position the anesthetized rat supine on the surgical table.

Intubate the rat in a supine position using the sheath from a 14 gauge Angiocath intravenous catheter and a laryngoscope with a miller sized 0 or 0.0 blade. Clean the lateral side of the tail vein with lukewarm water, followed by 70%isopropyl alcohol. Then, catheterize the lateral caudal tail vein using a 22 gauge intravenous catheter.

Now, inject approximately one milliliter of sterile saline into the catheterized tail vein. Using polyethylene tubing, connect the inserted tail vein catheter to a syringe containing rocuronium solution at two milligrams per milliliter mounted on a syringe driver. Feel for the caudal most rib on the left flank of the rat, and follow it dorsally to the spine.

Using a scalpel, make a midline incision approximately 60 millimeters long centered on this point. Using the scalpel, scrape the tissue from the spinous processes. Then, use scissors to expose the ribs and locate the caudal most rib.

Confirm the rib location visually and by touch using blunt dissection under a surgical microscope with 3.5 x to 45 x magnification. Mark the T13 spinous process with a permanent marker. Then, perform blunt dissection to create pockets on both sides of T11 and L4 vertebrae.

Slide two vertical posts with base mounts into the T-track of the stereotaxic platform and align them with the T11 and L4 vertebrae without tightening. Insert the cylindrical shafts of the vertebrae clamps into the post clamps, but do not tighten them yet. Starting with the T11 vertebra, hold the spine using toothed forceps.

Insert the jaws of the spine clamps on either side of the vertebra at approximately 45 degrees away from the T13 vertebra and tighten the jaws. Tighten the shaft of the spine clamp in the post clamp. Next, elevate both post clamps to lift the spine, ensuring the torso is supported entirely by the spine clamps.

Then mark the rostral caudal positions of the T13 and L1 spinous processes by placing single sutures into the muscle surface, lateral to the spine. Use toothed forceps, scissors, and a scalpel to clear the muscle and connective tissue from the surface of the vertebrae laminae between T12 and L3.Under a surgical microscope, use Friedman-Pearson rongeurs to remove the vertebrae laminae from L2 to T13. Begin the laminectomy by holding the rongeurs close to horizontal and taking small bites from the caudal part of L2 where it overlaps with L3.Extend the laminectomy rostrally to expose the spinal cord midline, avoiding pressure on the spinal cord and regularly clearing bone fragments from the rongeurs.

Extend the laminectomy two millimeters laterally on each side of the spinal cord midline. Then, use fine forceps and spring scissors to remove the dura mater from the exposed spinal cord. Tint the dura mater before making any incisions.

After the first cut, allow a small amount of cerebral spinal fluid to flow out and gently soak it using a twisted piece of lint-free tissue. Then, remove the remaining portion of the dura mater. Cover the exposed laminectomy site with a piece of tissue dampened with saline until the electrophysiology session begins.

For stimulating the sciatic nerve, expose the nerve and position a custom made bipolar platinum JC style electrode around it with exposed contacts measuring one by three millimeters. Then place the nerve block device adjacent to the exposed nerve. For planter stimulation, insert two 13 millimeter stainless steel needle electrodes, one inside the fifth digit, and the other outside the fourth digit close to the planter surface of the hind paw.

For wide dynamic range neuron recordings, stimulate either the planter surface of the hind paw or directly stimulate the sciatic nerve using a nerve cuff electrode. For a local field potential recordings, apply stimulation directly to the sciatic nerve to avoid diffuse signals. To record the electrophysiological signals, insert 21 gauge 1.5 inch hypodermic needles into the muscle on each side of the spine positioned parallel to the spine.

Digitize the electrophysiological signals continuously and display them in real time. For local field potential recordings, insert the electrode array oriented in the rostral caudal direction, positioning the rostral most electrode level with the suture marking the T13 spinous process as close to the midline as possible without damaging the midline blood vessel. Adjust the stimulation parameters while monitoring the recorded signals.

For the peripheral nerve block, use a carbon separated interface nerve electrode to apply 0.1 to five milliamp cathartic direct current to the sciatic nerve proximal to the side of the stimulating cuff if present Block was induced in both A and C fibers at approximately the same rate as shown by the reduction in local field potential signal area during the direct current application period. Recovery from the block differed between fiber types. Recovery was rapid for C fibers and slow for A fibers.

Longer latency C fibers took longer to block, but recovered earlier than shorter latency C fibers. Using this technique, we can assess the speed of the induction of block and the nerves recovery to normal conduction afterwards. Compared to the behavioral assessments, our electrophysiological techniques allow more precise quantification of the effects of nerve block on the various classes of nerve fiber.

Our future research will focus on the selective block of pain while minimizing the block of the axons involved in movement control.