The Institutional Animal Studies Committee of the Hines VA Hospital approved these studies. Seven female York-Landrace swine (30±3 Kg) underwent terminal surgeries. Anesthesia was initiated with a pre-anesthetic intramuscular injection of ketamine (25 mg/kg) and xylazine (1-2 mg/kg). After placement of an intravenous catheter, Propofol (3-6 mg/kg) was initiated. After endo-tracheal intubation the swine were maintained at a surgical plane of anesthesia using inhaled isoflurane (0.5-2.0%) and intravenous fentanyl (5-10 µg/kg/hr).¹² Body temperature was maintained at 38°C with an air-blanket heater. Isotonic fluids were administered intravenously at a rate of 10 mg/kg/hr.

Bipolar TECA^R lead electrodes (Fig. 1; 0.36 mm diameter, Carefusion Inc., Middleton WI) were used in initial tests to locate the pudendal nerve and at a distal location in HF blocking protocols. Commercial TECA^R electrodes were modified by removing insulation from the electrode tip to increase the length of the exposed electrode from 0.5 mm to 1.5 mm. Two monopolar TECA^R needle type electrodes were secured together with a separation of 3 mm at the tips to create a bipolar electrode.

Figure 1

Figure 1. Bipolar lead electrodes used in this study: TECA neurological needle; LifeTech barb with 26 gauge insertion needle; Bipolar Permaloc including a polypropylene barb and 16 gauge insertion needle. The needles were not used because the electrode was placed on the exposed pudendal nerve. Arrows highlight the length of stimulating surface of the electrode.

At a proximal location on the pudendal nerve, two LifeTech barb electrodes (EMG type, LifeTech Inc, Houston TX) were inserted adjacent to the nerve. The lead for these electrodes is a single strand of steel wire insulated with Mylar. The electrode tip is 5 mm of exposed wire bent back as a barb. Bipolar stimulation consisted of two electrodes inserted approximately 3 mm apart on the nerve.

At a distal pudendal nerve location, bipolar TECA^R needle electrodes were used in four different HF protocols; the negative electrode was placed distal to the positive electrode. A second electrode used at this location was a bipolar Permaloc^™ electrode (Modified Peterson,⁹ Synapse Biomedical Inc., Oberlin OH). The modified lead electrode had two, helical-wound, multi-stranded, stainless-steel wires insulated with Teflon.^® The electrodes were formed by stripping the insulation from the ends of the lead and using each helix for the two poles of the electrode; the Permaloc^TM electrode was placed on the nerve.

Pulse stimulators (Grass Model S48, Astromed, Houston, TX) isolated from ground were used for stimulation of the proximal pudendal nerve. Stimulation was charge balanced with a 1.2 µF capacitor (www.Digikey.com) in series with the animal and a 4.2 KΩ resistor across the output of the stimulator. A function generator with amplification and electrical isolation was used in the HF nerve blocking protocols. Isolation was with a transformer that had its ground lead disconnected during testing. The function generator produced a balanced square wave, a form shown to be most effective in producing nerve block.^10,12 We modified a biphasic analog amplifier circuit (5 pin, 20 Watt, Pentawatt specification circuit, www.Digikey.com) to provide adequate amplification for the function generator, electrical isolation, and to reduce electrical noise. Stimulating currents were observed on an oscilloscope and were 0.1 to 10 mA. The stimulating current was calculated from the voltage drop across a 100 ohm resistor in series with the stimulating electrodes and by applying Ohm’s law (E = IR).

Following anesthesia and prior to stimulation tests, the urethra was catheterized with a triple-lumen, 7.4Fr balloon-tipped catheter (Model G15540 Urodynamic catheter, Cook Urological Co. Spencer, IN). One of the lumens was used for bladder filling and a second was capped. The third lumen included a balloon and was used for urethral, skeletal-sphincter pressure recording. Techiques for filling the balloon with water and connecting it to a pressure transducer have been detailed else-where.¹³ The urethral meatus in the female swine is recessed 3 to 4 cm within the vaginal opening and catheterization was facilitated by antegrade maneuvers through a 18 gauge needle inserted into the bladder wall. A stylet was advanced through the needle and urethral meatus; the catheter was attached to the stylet and pulled into the bladder. Subsequently, the stylet and needle were removed and the bladder wall closed with a single stitch; the balloon was positioned inside the urethal meatus to record urethral skeletal-sphincter pressures.

An anal balloon for pressure recording was fashioned from silastic tubing (15 mm outside diameter). The ends of the tube were sealed with silicone (Med-1037, NuSil Inc, Carpinteria, CA) and were further enlarged to maintain its position in the sphincter. Pressure transducers (World Precision Instruments Inc, Sarasota, FL) were used for urodynamic measures and recorded with a digital system (PowerLab, AD Instruments Inc, Colorado Springs, CO).

An 8 cm incision was made lateral to the sacral spine; hemostasis was maintained with electro-cautery. Blunt dissection and separtion of paraspinal muscles from their fascial plane was carried down approximately 3 cm to the spinal level and the pudendal nerve identified; the nerve is medial to the sciatic nerve.¹⁴ Identification of the nerve was confirmed with low current stimulation using single pulses and observing urethral and anal sphincter pressure spikes without leg movement. Two LifeTech barb electrodes (Fig. 2) were implanted at the proximal end of the nerve. Intermittent urethral and anal sphincter contraction were induced with stimulus trains applied every 2.5 s. Trains consisted of 3 pulses applied at 40 Hz with 50 µs pulse durations. Stimulation currents were increased from 0.25 to 7 mA to induce maximal sphincter contractions without leg muscle contractions.

At the distal pudendal nerve location (Fig. 2), bipolar TECA^R needle electrodes were placed on the nerve. Sphincter pressure responses were recorded to simultaneous 10 kHz stimulation ^{at 0.25 to 10 mA for 15 s and intermittent stimulation at the proximal nerve location. To limit the total number of stimulations at this frequency, the number of animals tested at the lowest stimulation currents was limited to only those animals that demonstrated nerve blocking at higher currents; thus, three animals were not tested at 0.25 mA and one animal was not tested at 0.5 mA. Also, the number of animals tested at the highest stimulating currents was limited to animals that did not demonstrate leg muscle contractions have baseline pressures over 30 cm H₂0 at the end of 15 s during lower stimulating currents; thus, two animals were not tested at both 5 and 10 mA (See Table 1 legend).}

Three different HFs were compared; 1.2 kHz was tested because this was the frequency used by Passover et. al. in their clinical study of pudendal nerve blocking⁸ and 10 kHz was tested because it has been reported to be the most effective nerve blocking frequency in animal studies.^6,10 We also tested 40 kHz because during initial studies unwanted stimulatory effects were encountered.

Three additional tests were conducted at 10 kHz: one, the TECA^R electrodes was tested 2.5 mm away from the nerve; two, the bipolar Permaloc^R electrode was placed next to the pudendal nerve; and three, effects of cutting the pudendal nerve proximal to the proximal electrode was evaluated. Only effects on intermittent peak sphincter pressures were assessed.

One final test was conducted to assess a problem observed during initial testing which was gas evolution seen as bubbles from the TECA ^R needle electrode.. For further assessment, the electrodes were placed against muscle so that the electrodes and bubbles could be seen and the current was increased until bubbles were observed.

Animals were sacrificed by administering intravenously 50 ml of saturated KCl while under surgical anesthesia. During autopsy the implanted electrodes were photographed. Summary results are presented as mean±SEM. Student’s t-tests with paired data was used for statistical analyses and a statistical significance was set at p < 0.05.

Once the pudendal nerve was identified, proximal LifeTech barb electrodes on the pudendal nerve were stimulated with trains of pulses every 2.5 s. These induced intermittent urethral and anal sphincter contractions with high pressures and without leg movements; these trains were used during HF blocking protocols.

High-frequency, 10 kHz, stimulation at 0.25 to 10 mA was tested at a location distal to the proximal electrodes (Fig. 2). As shown for one animal in Figure 3, increasing the stimulating current from 0.25 to 5 mA induced greater increases in the baseline urethral and anal pressures at the beginning of stimulation. At the end of 15 s of stimulation the baseline pressures continued to be elevated and were higher at higher stimulating currents. Elevated baseline pressures at the end of the 15 s are unwanted as they show nerve stimulatory effects.

Figure 2

Figure 2. Pudendal nerve with proximal electrodes for intermittent stimulation and distal electrodes for HF nerve-blocking protocols. Proximal electrodes were bipolar LifeTech barb and distal electrodes were bipolar TECA. Gluteus and paraspinal muscles were pulled lateral.

Similar results were observed in all seven animals (Table 1). Initial baseline urethral sphincter pressures increased with increasing currents whereas at the end of 15 s of stimulation baseline pressures tended to vary. These end pressures tended to increase from 11±6 cm H₂0 at 0.25 mA to 17±3 cm H₂0 at 1 mA (P = 0.26; n = 4, see Table 1 legend). Stimulation at 10 mA, however, tended to have lower end baseline pressures of 11±3 compared to the response at 1 mA (P = 0.57; n = 5). This trend toward lower pressures occurred even though initial baseline pressures at these higher currents were greater. A maximal initial baseline pressure of 45±4 cm H₂0 was observed at 10 mA showing strong sphincter stimulation. Similar baseline sphincter pressure results were observed for the anal sphincter although all of the pressures were lower than urethral pressures.

Figure 3

Table 1. HF (10 kHz) pudendal nerve blocking with bipolar TECA electrodes during intermittent proximal pudendal nerve stimulation

^a intermittent peak urethral pressure near the end of the 15 s of 10 kHz stimulation was measured above baseline urethral pressures induced by the HF.

^bdetermination of complete block of intermittent urethral sphincter contractions shown by no changes in urethral pressure or no observable movement of the perineum to proximal nerve stimulation and near the end of the 15 s of HF stimulation. Note, evaluation of blocking confounded by high baseline pressures at the end of the 15 s of HF stimulation.

^c as detailed in methods, not all animals were tested with HF stimulation and low currents because higher currents did not show any blocking effects; for 0.25 mA: n = 4 for statistical comparisons of recorded pressures; n = 7 for assessment of complete block; for 0.5 mA: n = 6 for recorded pressures; n = 7 for complete block.

^d not all animals tested at HF and high currents because lower currents showed blocking with baseline pressures of 30 cm H₂0 at the end of 15 s or strong leg contractions: for 2 mA: n = 6 for recorded pressures; n = 7 for complete block; for 5 mA: n = 5 for recorded pressures; n = 6 for complete block; for 10 mA: n = 5 for recorded pressures; n = 6 for complete block.

^s comparison of urethral baseline pressure at the end of the 15 s of HF: 1 mA vs 0.25 mA (p = 0.26, not significant); 1 mA vs 10 mA (p = 0.57, not significant).

^t comparison of peak intermittent urethral pressure above baseline at the end of the 15 s of HF: 1 mA vs 0.25 mA (p = 0.14, not significant); 1 mA vs 10 mA (p = 0.37, not significant).

In contrast to these unwanted stimulatory effects on the baseline sphincter pressures, the HF stimulation at 10 kHz also produced blocking of intermittent urethral and anal sphincter contractions induced from the proximal electrode. At 0.25 mA (Fig 30-A) there was no blocking of the urethral intermittent contractions whereas the anal contractions were slightly reduced. At 0.5 mA (Fig 3-B) the urethral sphincter contractions were partially reduced whereas the anal contractions were blocked. At 1 and 5 mA (C, D) the intermittent contractions were completely blocked in both sphincters. These blocking effects to the HF stimulation, in contrast to the stimulatory effects seen as increased sphincter baseline pressures, were further confirmed by observation and palpation of the anal sphincter and perineal region. When intermittent sphincter pressures were absent or greatly reduced, there was an abatement of contractions as determined by visual observation and palpation.

Figure 4

Figure 3. Urethral and anal sphincter pressures recorded during intermittent stimulation of the proximal electrodes and 10 kHz stimulation of the distal electrode. Paradoxical effects of the HF stimulation include blocking of intermittent sphincter contractions and unwanted stimulatory affects with increased baseline sphincter pressures. Bipolar Life-Tech barb electrodes were used at the proximal location and bipolar TECA needle electrodes were used for HF blocking at the distal location. Phasic contractions to intermittent stimulation induced with trains of pulses every 2.5 s (3 pulses at 40 Hz, 50 µs,

Similar blocking effects of intermittent sphincter contractions with 10 kHz stimulation were observed in all seven animals (Table 1). Increasing the HF blocking current from 0.25 mA to 1 mA caused complete block of intermittent contractions in six animals whereas 0.25 mA only produced complete block in one animal. At even higher stimulating currents from 2 to 10 mA results were similar showing complete block of the intermittent contractions. Importantly, in none of the seven animals were we able to show blocking of the intermittent urethral contractions without some unwanted stimulatory effect as evidenced by lack of increase in baseline urethral pressures. Similar intermittent sphincter pressure responses to the HF stimulation were also observed for the anal sphincter. Peak pressures recorded in the anal sphincter, however, were lower than for the urethral sphincter.

Effects of 10 kHz stimulation on baseline pressures near the end of the 15 s stimulation was compared for 1.2 kHz. As shown in Figure 4, 1.2 kHz and 2 mA (A) induced higher urethral baseline pressures than did 10 kHz and 2 mA (B). Lower baseline pressures at 10 kHz compared to 1.2 kHz were observed in four out of the five animals tested at these two frequencies. Slight to moderate leg movements occurred during 1.2 kHz and 10 kHz in two of the five animals tested. The unwanted leg muscle contractions are shown in Figure 4-A by the thick line in the urethral pressure recording. In contrast, baseline pressures were often reduced with 40 kHz stimulation compared to 10 or 1.2 kHz. The second of the two recordings at 40 kHz and 5mA (Fig-D) show this with a pressure that changes little when the HF is turned off. The lowest baseline pressures were all recorded at the end of 15 s with 40 kHz compared to 10 kHz or 1.2 kHz for the five animals tested; also, leg muscle contractions were not observed in any of the animals at this higher stimulating frequency.

Figure 5

Figure 4. Urethral sphincter pressures recorded during intermittent stimulation of the proximal pudendal nerve electrodes and 1.2, 10 and 40 kHz HF stimulation at distal electrodes. Both nerve blocking and stimulation are shown to the HF stimulation. Stimulation at 40 kHz and 5 mA demonstrated the most reduction in unwanted increases in baseline urethral sphincter pressures while still partially blocking intermittent sphincter contractions. Electrodes and methods used are described in Figure 3.

Effects of 10 kHz stimulation on intermittent urethral contractions were also compared to 1.2 kHz and 40 kHz stimulation. As shown in Figure 4, at 10 kHz and 1.2 kHz (2 mA, A and B) intermittent contractions were blocked. For four of the five animals tested with these two stimulation frequencies, similar blocking of intermittent contractions occurred; whereas in the fifth animal 10 mA was required to demonstrate a complete block of intermittent contractions at 10 kHz compared to only 1 mA at 1.2 kHz. As shown in Figure 4, at 40 kHz with 2 and 5 mA (Fig 4-C, D) only partial blocking of the intermittent contractions occurred; however, 5 mA (D) was more effective than 2 mA (C). In four of the five animals tested with 40 kHz, no complete blocking of the intermittent contractions were observed whereas in the fifth animal a complete block was observed only at high currents of 10 mA.

TECA^R bipolar lead electrodes were tested 2.5 mm away from the pudendal nerve at 10 kHz. In two of the three animals, there was no effect of stimulation as evidenced by the lack of change in baseline pressures or intermittent urethral contractions at 5, 8, or 10 mA. In the third animal, there were small baseline changes (initial pressure of 10 cm H₂0) in the urethral sphincter pressures at 10 mA but not 5 mA. At both 5 and 10 mA, however, there were no changes in the intermittent urethral contractions for any of the three animals. With stimulations 2.5 mm from the nerve, anal sphincter pressures also showed little or no change.

Bipolar Permaloc^TM lead electrodes were tested on the nerve at 10 kHz. Using 5 mA, of current changes in baseline sphincter pressures were much less than those observed with the TECA^R bipolar needle electrodes on the nerve. In addition, Permaloc^TM electrodes produced no blocking of the intermittent urethral sphincter contractions at 5 mA in any of the five animals tested. Higher currents of 10 mA (four animals) and 14 mA (one animal) induced higher baseline pressures and complete blocking of the intermittent urethral sphincter contractions in two animals. Another concern was greater spread of electrical fields with the Permaloc electrodes. In four of the five animals tested, current spread was indicated by slight or moderate leg muscle contractions at 10 or 14 mA; at 5 mA current spread occurred in only one animal.

The pudendal nerve was transected proximal to the stimulation electrodes to assess spinal reflex effects on pudendal nerve stimulation. Nerve transection had no effect on peak intermittent contractions, 26±6 cm H₂0 (n = 4), both before and after cutting. In one of the remaining three animals, effects of pudendal nerve transection could not be assessed because of intermittent sphincter peak pressure changes that may have been associated with slight changes in position of the proximal electrodes on the pudendal nerve; in the other two the tests were not conducted due to limited surgical time. Similar to the urethral sphincter, Intermittent contractions for the anal sphincter were 4±1 (n = 4) before and after transection of the nerve. Thus, spinal reflexes do not play a role in stimulation induced intermittent sphincter contractions.

Gas released from the TECA ^R electrodes due to electrolysis was observed at higher stimulating currents and particularly at 1.2 kHz. Gas production started at 10 mA of current at 1.2 kHz and 20 mA of current at 40 kHz; higher currents led to more gas production. Permaloc^TM electrodes, which have a larger surface area, did not produce gas at the same stimulating currents and frequencies. Gas production was not due to heating of the tissue based on two observations: one, there was no visible signs of tissue damage such as tissue reddening; and, two, when electrodes were held between the investigators fingers with isotonic saline to produce the same electrode resistance as in animals, there was no perceptible increase in temperature with prolonged stimulation.