Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

This protocol describes the preparation of rat whole sciatic nerve tissue for ex vivo electrophysiological stimulation and recording in an environmentally-regulated, two-compartment, perfused saline bath.

Transcript

This protocol enables the observation of many neurophysiological phenomena that aren't well understood, such as nerve block carryover after high frequency/high amplitude current injection, while preventing interference from other processes in a living body. This technique yields highly repeatable, robust results and is more practical than in vivo electrophysiology, because complex variables such as depth of anesthesia don't have to be controlled and therefore can't influence measurements. After a euthanization procedure under anesthetic, place a female rat on the dissection table, hold the ankle firmly between the thumb, index finger, and middle finger, and sever the calcaneal tendon using 12-centimeter straight blunt scissors.

Using fine forceps and scissors, make careful incisions through the muscle layers near the middle of the back of the leg until the sciatic nerve is exposed. Once the nerve is visible, moisturize the cavity using ice-cold modified Krebs-Henseleit buffer, or MKHB, to prevent the nerve from drying out. Using hemostats, pull the flaps of skin on each side and maintain the incision open for finer dissection.

Starting from the calcaneal tendon incision location, using fine scissors, interrupt the muscle on the medial side of the leg to free the nerve. Continue to maintain moisture levels in the area with ice-cold MKHB. As the nerve is exposed while moving up the leg, dissect the overlying muscle tissue.

Free the nerve nearer to the spine from connective tissues until reaching the spinal cleft, at which point there is a kink in the nerve. Do not clean the nerve yet, as speed is essential at this stage. To make the dissection easier, using forceps, gently pull the end of the nerve near the ankle.

Then, using fine scissors, severe the nerve close to the spine. Place the dissected nerve in a 15-milliliter centrifuge tube filled with MKHB and place the tube back on the ice until the start of the cleaning procedure. Fill the coated Petri dish halfway with chilled oxygenated MKHB, then place one dissected sciatic nerve in the dish and pin both ends of the nerve to the dish such that the nerve is straight without kinks, torsion, or twists.

Using 6-0 silk sutures or fine thread, tie a double knot around each end of the nerve to prevent cytosol leakage into the buffer. Place the knots just next to the insect pins on the side closer to the center of the nerve to prevent leakage from nerve tissue into the buffer. Under a microscope, using two-millimeter angled spring scissors and fine forceps, remove fat, blood vessels, and muscle tissue from the nerve.

Prune any nerve branches that will not be used in the stimulation and recording. After every five minutes of cleaning, replace the buffer with fresh, chilled, oxygenated MKHB. After cleaning, place the nerve back into the transport tube filled with buffer and rest the tube on ice.

Prepare a clean, dual-chamber nerve bath. Using standard laboratory boss heads and grippers, place the nerve bath below the level of the two-liter bottle placed on the heating stirrer. Connect the drain of the bath to the peristaltic pump inlet.

Connect the outlet of the peristaltic pump to a tube leading back to the buffer bottle. Connect the bath inlet to a tube with an adjustable flow valve and put the tube inside the bottle. Use a three-way valve with a syringe connected to the middle outlet to help with priming the tube as a siphon for gravity-assisted buffer inflow.

Prime the siphon by drawing the syringe until buffer flows into it. Configure the valve such that the buffer flow rate into the bath is five to six millimeters per minute. The flow rate can be increased initially to fill the bath.

Once the bath buffer level has reached the drain, place the nerves in the bath. Using an insect pin, secure the end of the nerve at the corner of the buffer-filled bath chamber. Using 45-degree angled fine forceps and pinching the nerve only at the ends, carefully thread the nerve to be stimulated through the hole in the partition between the two bath chambers.

Using an insect pin, secure the other end of the nerve in the oil chamber of the bath, ensuring that the nerve is straight without being stretched and is free of kinks and twists. Using silicone grease, make a seal to prevent buffer leakage from the buffer chamber into the oil chamber Fill the oil chamber with silicone or mineral oil. Place the silver/silver chloride recording electrode hooks in the oil bath chamber and secure them using boss heads and grippers.

Then, drape the portion of the nerve in the oil bath over the hooks without pulling the nerve taut. Adjust or repair the silicone grease seal if any leakage is observed after moving the nerve. Connect the reference silver/silver chloride electrode to the amplifier ground and place the electrode in the buffer-filled bath chamber by securing it using a laboratory gripper.

After preparing a clean nerve cuff electrode for stimulation, place the electrode in the buffer-filled bath chamber. Using forceps or fine tweezers with blunt or angled tips, open the electrode in the bath to wet the inside of the cuff. If bubbles remain, use a fine syringe to draw buffer from the bath and force the bubbles out of the cuff.

Using a tweezer, gently open the cuff and slide it under the nerve. Close the cuff around the nerve, avoiding any kinking or twisting of the nerve. Connect the stimulation electrode and the current return electrode to the stimulator.

If using a square platinum sheet as the current return electrode, position the sheet away from the nerve in the bath. Secure both the electrodes'lead with tape. Connect the stimulator TTL signal output to channel four of the oscilloscope.

On the oscilloscope screen, press the Trigger channel tab and specify channel four as the triggering channel. Set the trigger level to one volt using the level knob. On the oscilloscope, set time resolution to one millisecond per division and voltage resolution to 10 millivolts per division.

Center the trigger reference in time and set the trigger level to one volt. After connecting the stimulator to the computer, turn on the stimulator by connecting the battery power supply to the power input. Then, start the MATLAB software on the computer.

Execute the custom MATLAB script. Then, open the indicated MATLAB script. Edit the script and set the parameters stimulator pulse amplitude to minus 300 microamperes, stimulator pulse width to 300 microseconds, stimulator number of pulses to 10, and stimulator time between pulses to one second.

Start the stimulation protocol by clicking on Run in the software. The compound action potential, or CAP, had a peak-to-peak amplitude of one millivolt at the electrode and 100 millivolts when amplified. The typical nerve excitability for standard platinum nerve cuffs and custom-made conductive elastomer nerve cuffs are shown here.

The CAP obtained ex vivo over a day of experiments using both sciatic nerves is shown here. Minimal CAP amplitude reduction was observed with the right sciatic nerve. In contrast, the left sciatic nerve CAP amplitude at approximately three millivolts was similar to that of the right sciatic nerve at the start of the experiment more than six hours after nerve extraction.

Emphasize precision and repeatability in dissection, cleaning, and implementation technique. Pay attention to what is happening in the bath. If the nerve signal is lost, it may simply be that the electrodes are not in the good contact or that there is saline in the bath oil partition, and not that the nerve is died.

First time users might be tempted to rush, especially during the dissection phase. This will likely lead to mistakes and damage the nerve. Precision and consistency are key here and will lead to higher quality results.

Special attention should also be given to consistency in buffer preparation.

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Journal (Neuroscience)

Journal (Neuroscience)

Preparation of Rat Sciatic Nerve for Ex Vivo Neurophysiology

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