To begin, wear appropriate personal protection equipment. Before the procedure, weigh the rat to determine the correct dose for weight-based medications and ventilator settings. After anesthetizing the rat, place the rat in a prone position.
To confirm the depth of anesthesia, use forceps to gently press the hind limb footpad and check for the absence of a withdrawal response. Now apply a veterinarian approved petroleum-based ointment to the rat's eyes to prevent dryness. Observe the respiratory rate and assess for withdrawal responses as shown earlier.
Then using clippers, shave the hind limb of the rat. Use adhesive tape to position the limb with the ankle fixed at 90 degrees dorsiflexion, the knee in extension, and the hip in abduction. Afterwards, insert a pair of insulated 28 gauge monopolar needles for stimulating the sciatic nerve with the cathode placed in the region of the proximal hind limb and the anode positioned more proximally within the subcutaneous tissue, overlaying the sacrum.
Then position a disposable ground electrode on the contralateral hind limb or tail. Carefully insert a 27 gauge, 25 millimeter autoclave needle electrode, specifically designed for single fiber electromyography into the right gastrocnemius muscle. Insert the single fiber electromyography needle electrode in parallel with the gastrocnemius muscle fibers to capture single fiber action potential.
Apply constant current stimulation to the right sciatic nerve at 10 hertz frequency, using an intensity range of 0.3 to 10 milliamperes and a pulse duration of 0.1 milliseconds. Adjust the gain from 200 microvolts to 1000 microvolts per division to enhance the visualization of potential. Configure the sweep speed to be 500 microseconds per division.
To classify a response as an SFAP, it must exhibit a rise time shorter than 500 microseconds, a minimum amplitude of 200 microvolts, and demonstrate all or none behavior. Calculate the jitter or variability of SFAP latency between consecutive discharges after at least 50 stimulations using the given equation. Assess and quantify blocking.
After evaluating 10 synapses from each animal, calculate the jitter and determine the average values per animal. When recording potentials that show intermittent blocking, increase the stimulus intensity to confirm that SFAP failure is not due to submaximal stimulation. During rocuronium administration, single fiber EMG showed increased variability of transmission and neuromuscular junction blocking as compared to healthy individuals.
