2.1.

Animal Preparation

Unmyelinated nerves of Aplysia were used. Aplysia can be maintained for many hours and have previously been used to define appropriate parameters for optical block in myelinated rat sciatic nerve.^10,11 Animals 300 to 400 g were used, as their nerves are 4 to 7 cm, with a diameter of 0.5 to 1.5 mm, comparable to rat sciatic nerve. Animals were anesthetized with isotonic magnesium chloride. The pleural-abdominal nerves were maintained in Aplysia saline¹² at room temperature after dissection. All experiments were performed in vitro.

2.2.

Experimental Setup

Suction electrodes and optical fibers were placed on the nerve (Fig. 1): a monopolar electrode for stimulation (a); 2 en passant electrodes for bipolar proximal recording (b); 2 optical fibers for IR block (c); 2 en passant electrodes for KHFAC block (d); and 2 electrodes for bipolar distal recording (e). En passant electrodes were chosen for convenience; cuff electrodes have previously been used successfully. Electrodes were filled with Aplysia saline before suctioning the nerve into the electrode to preserve nerve viability. An Ag/AgCl wire was inserted in each electrode. Electrical stimulation to generate nerve action potentials was controlled by a pulse generator (A310 Accupulser, WPI Instruments, Sarasota, Florida) via a stimulus isolator (WPI A360, WPI Instruments). KHFAC block was delivered by a controlled constant-current function generator (Model 6221, Keithley Instruments, Cleveland, Ohio) using a sinusoidal waveform. An inductor (8.2 H) was placed across the function generator outputs to minimize DC leakage. The nerve compound action potential (CAP) was monitored using AxoGraph X (AxoGraph Scientific).

Fig. 1

Nerve preparation incorporating kilohertz high-frequency alternating current (KHFAC) and IR lasers. From left to right: stimulating electrode (a), proximal recording electrode (b), KHFAC blocking electrode (d), and distal recording electrode (e). Position of optical fibers between the proximal and blocking electrodes (c) are shown schematically (see text).

For IR block, 600-µm multimode optical fibers (P600-2-VIS-NIR, Ocean Optics, Dunedin, Florida) were placed on either side of the same nerve cross section to produce more uniform IR exposure. They were coupled to 2 Capella lasers (Lockheed Martin Aculight, Bothell, Washington) centered at 1860 and 1863 nm due to different wavelength tuning ranges, but with similar water absorption coefficients. The optical fibers gently touched the nerve sheath, producing a 600-µm spot at the nerve surface. The fibers had a numerical aperture of 0.22 (i.e., a beam divergence of 25.4 deg in air), so the spot was slightly larger at the axons due to divergence and scattering through the typically 100-µm thick sheath. Since the onset response travels both anterogradely and retrogradely from the KHFAC block electrodes, applying the lasers to block the onset response near the proximal electrodes allowed the distal electrodes to serve as a control for the same KHFAC block. Varying placement of the optical fibers between the KHFAC and the distal or proximal recording electrodes had no effect on the results.

2.3.

Experimental Protocol

Three experiments were performed on three different nerves, using an A-B-A protocol. During protocol A, a train of action potentials was blocked by KHFAC; protocol B added IR inhibition to generate onset response block. Protocol A was repeated as a control. A current just above the stimulation threshold produced CAPs of sufficient amplitude to assess block effectiveness. The minimum amplitude KHFAC waveform at which block was observed was consistently at a frequency of 10 kHz and amplitude ranging from 10 to 15 mA (peak-to-peak). For the two lasers, radiant exposures per pulse ranged from 0.177 to $0.254\mathrm{J}/{\mathrm{cm}}^2$. Both lasers were switched on at the same time and emitted laser light for 30 s before the KHFAC waveform was applied, using 200-μs pulses at 200 Hz, to allow the temperature to reach a higher value.¹¹ Nerve health was assessed before and after every experiment by comparing the propagating CAPs traveling down the length of the nerve.