In vivo characterization of electrodes for selective stimulation of neurovascular bundles during robot-assisted radical prostatectomy

In vivo characterization of electrodes for selective stimulation of neurovascular bundles during robot-assisted radical prostatectomy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In vivo characterization of electrodes for selective stimulation of neurovascular bundles during robot-assisted radical prostatectomy Janez Rozman, Jure Bizjak, Tomaž Smrkolj, Samo Ribarič, Simon Hawlina This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9440557/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The electrical and electrophysiological performance of platinum electrodes within the probe for bipolar selective stimulation of neurovascular bundles (NVB)s during robot-assisted radical prostatectomy was investigated. Quasi-trapezoidal pulse trains (5 min) were delivered to isolated NVBs in 5 patients. The electrical performance of the electrodes was studied by determining the polarization across the electrode–NVB tissue interface using voltage transients. Cavernous EMG (CC-EMG), axial rigidity and computing CC-EMG features were measured in the frequency domain. The most negative E mc and most positive E ma potentials across the electrode–NVB tissue interface reached − 11.49 V and 11.52 V, respectively; the cathodal (σ Q c ) and anodal (σ Q a ) charge densities were 0.963 µA s/mm 2 and 0.435 µA s/mm 2 , respectively. They exceeded tissue injury limits for a short time, but tissue injury did not occur. Impedance | Z | decreased faster with frequency in vitro (50 Hz, 170 Ω) than in vivo (1500 Ω). The median frequencies of the amplitude and power were: CC-EMGr, 26.7 Hz and 50 Hz; CC-EMGl, 28.33 Hz and 43.58 Hz. Both were contaminated with the stimuli and facility mains. Any influence of τ exp preset in the stimuli on electrophysiological performance could not be identified. An increase in penile axial rigidity was not observed in any patient. neurovascular bundle platinum stimulating electrode interface voltage response EMG Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Radical prostatectomy is the surgical treatment for patients with prostate cancer [ 1 – 3 ]. However, it is difficult to remove the gland without affecting prostate neurovascular bundles (NVB)s that innervate the corpora cavernosa (CC) [ 4 – 6 ]. Efferent innervation of the penis comes from parasympathetic, sympathetic and somatic sources. Somatosensory afferents, however, course from the penis to central sites [ 7 , 8 ]. NVBs are composed of thin, wiry post-ganglionic parasympathetic nerves expanding from the pelvic hypogastric plexus. They contain delicate bundles of cavernous nerve (CN) fibres, prostatic branches of the inferior vesical artery, prostatic veins, collagen fibres and adipose tissue, all surrounded by connective tissue. They travel posterolaterally to the prostate, eventually reaching the spongy erectile tissues of the CC [ 9 ]. Parasympathetic CN fibres that arise from the prostatic plexus innervate the helicine arteries of the erectile tissue in the cavernous spaces. They mediate vasodilation and stimulate the erection of the penis. They are extremely susceptible to stretch. Rodrigues et al. [ 10 ] assumed that numerous nerve fibres are located in the anterior and anterolateral positions and that CNs are probably formed by the posterior nerve fibres located at a safer distance from the prostate. Despite recent advances in surgical techniques, erectile dysfunction still occurs as a consequence of injury caused during radical prostatectomy, mainly to the CNs and less to the arteries within the NVB. A surgical technique called nerve-sparing prostatectomy was developed to preserve the NVBs [ 1 , 2 ]. When appropriately accomplished during robot-assisted radical prostatectomy (RARP), nerve sparing largely improves recovery of erectile function (EF). Robotic assistance offers excellent visualization of the NVBs and high surgical precision [ 1 – 3 ]. However, despite advancements in NVB preservation, potency rates have remained unsatisfactory. Tode et al. [ 11 ] showed that regeneration after injury is limited because afferent axons regenerate into the peripheral nerve stump. Thus, even when spared, the recovery of EF may take months after the procedure [ 3 , 10 ]. Therefore, the focus is on how to improve RARP techniques to preserve the functionality of the NVBs. Intraoperative nerve monitoring was introduced as a technique to assist the surgeon in minimizing the risk of harm to the NVBs. The technique was based on investigations on the distribution of CN fibres around the prostate using nerve stimulation during radical prostatectomy [ 12 ]. In recent decades, the concepts for electrical nerve stimulation involved non-fibre-selective stimulation, which in turn frequently causes undesirable effects. Accordingly, various models of selective nerve fibre stimulation and electrodes that selectively stimulate certain features have been developed [ 13 , 14 ]. Peripheral nerve fibres are classified into 4 subclasses according to their diameter, conduction velocity, and extent of myelination [ 15 , 16 ]. One subclass is a large group of unmyelinated slowly conducting C-fibres with axon diameters from 0.2 µm to 1.2 µm and up to 3 µm in some cases. They are widely distributed in hairy and glabrous skin, transmitting cutaneous and visceral peripheral signals to the central nervous system. C-fibres are also present in the glabrous skin of the glans penis. They play a crucial role in the nervous transmission of peripheral sexual stimuli to higher cortical sexual arousal areas [ 16 , 17 ]. Generally, unmyelinated C-fibres that have higher stimulation thresholds (between 1 and 3 mA) are activated less reliably than myelinated ones (about 0.15 mA) and stay activated for higher currents. The exact threshold depends on the stimulation frequency, pulse duration and electrode placement. C-fibres typically require higher stimulation intensities or longer duration than Aβ- and Aδ-fibres [ 15 , 16 ]. Therefore, the intensity, frequency and pattern of electrical stimuli play a critical role in determining which fibre types are activated. Other fibre types, such as thinly myelinated Aδ-fibres, can also be activated at certain frequencies; 5 Hz is often cited for C-fibre activation, but the optimal range for C-fibre stimulation is broader. Frequencies between 20 and 50 Hz have been identified as the blocking frequency for C-fibre afferents [ 18 ]. In CN stimulation, a frequency range of 10–20 Hz, pulse duration 0.5–1 ms, stimulation duration 30–60 s and voltage 2.5–8 V are commonly used to induce erectile responses [ 19 ]. NVBs require high charge densities for stimulation due to a high content of non-myelinated nerve C-fibres [ 20 , 21 ]. This may result in a high energy transfer and the occurrence of reversible and potentially irreversible chemical reactions. The use of current, biphasic stimulating pulses in neural tissue stimulation, with cathodal and anodal phases of equal shape and charge per phase, is a common practice [ 22 , 23 ]. Charge delivery has been shown to be highly influenced by the electrode–NVB interface, where transduction of electrons in the metallic electrode to ions in the tissue occurs [ 24 , 25 ]. Accordingly, impedance | Z | that occurs at the electrode–NVB interface is crucially affected by the electrode geometry. The efficiency of neural excitation, however, is highly dependent on the current density on the electrode surface and the spatial distribution of the electric field generated in the tissue [ 26 ]. Although efforts have been made to find optimum positions at the NVB, little work has been done to test the efficiency of NVB stimulation using analytically driven configurations of the electrodes and stimulus waveforms. Warman et al. [ 27 ] showed that nerve fibre excitation could be predicted using the driving function of a neuron, which is proportional to the second spatial derivative of the extracellular potential and thus, the spatial derivative of the current density in the tissue. Accordingly, an electrode with greater roughness would increase the efficiency of the nerve fibre stimulation [ 26 , 27 ]. Every material appropriate for the production of electrodes has a charge that delimits reversibility and irreversibility of the electrochemical processes at the interface. Pure platinum is frequently used because it can inject charge by both double-layer charging and Faradaic processes [ 28 , 29 ]. However, they can be either reversible or irreversible. It was shown that all irreversible electrochemical processes are harmful. The platinum charge injection limits within which the electrolysis of water is avoided in chronic stimulation with biphasic cathode first pulses were determined to be between 50 and 150 µC cm − 2 (geometric area) [ 29 – 31 ]. Platinum as a stimulating electrode material has the advantage of decreasing |Z| with increasing frequency. In vitro electrochemical techniques such as cyclic voltammetry, |Z| and voltage transient (SVT) measurements can be used to characterize an electrode for nerve tissue stimulation [ 32 , 33 ]. Their results enable the selection and design of the electrode material, and selection of stimulation pulse waveform and parameters for efficient and safe nerve tissue stimulation [ 24 , 34 ]. SVTs are frequently used to estimate the charge injection limit Q inj , which defines the charge that can be injected by the electrode in a current stimulation pulse using only reversible processes. SVTs determine both negative ( E mc ) and positive ( E ma ) potential extremes across the electrode–electrolyte interface. These potential extremes are then compared with the established maximum potentials beyond which it is considered unsafe to polarize the electrode (typically the potential window of water electrolysis). In most in vivo testing, however, the electrode is placed onto or into the neural tissue to develop the electrode–NVB interface [ 32 , 33 ]. Corpus cavernous EMG (CC-EMG) is a measure of the electrical activity of the smooth corpus cavernosum muscle (CCM), which is a prerequisite for erection. According to Wagner et al. [ 35 ], CC-EMG is the most direct electrophysiological indicator of any malfunction in the integrity circuits between the CNS and CCM related to EF. Klotz and Herschorn [ 36 ] described how to successfully capture CC-EMG during prostatectomy. Some authors claimed that CC-EMG reflects the summation of the membrane current of a group of CCM muscle cells and is the source of the electrical activity that may be measured [ 37 – 40 ]. Much later, Vírseda-Chamorro et al. [ 41 ] declared that CC-EMG of the erectile tissue recorded during erection could be used as a diagnostic technique in patients with erectile dysfunction. In addition, Leddy et al. [ 42 ] have identified the origin of the low-frequency CC-EMG complexes in volunteers with normal EF using penile surface electrodes. They concluded that the CCM tissue was the origin of the CC-EMG complex and not the penile skin or surrounding tissue. Recently, Yildiz et al. [ 43 ] aimed to evaluate the changes in penile sensation using electrophysiological tests in patients who underwent RARP. The results showed decreased penile sensation due to CN damage and a possible dorsal penile nerve injury. However, the CC-EMG generating source is still not clearly known [ 44 , 45 ]. Monopolar needle electrodes and/or non-invasive surface electrodes placed on the penis could be used for CC-EMG recording. Amplification of CC-EMG and attenuation of environmental noise can be accomplished using a differential amplifier [ 45 , 46 ]. However, a low-pass and high-pass filter should be used to acquire the most optimal frequency range of the CC-EMG (0.1–5 Hz), and a notch filter should be used to remove the noise from the power lines [ 47 ]. Jiang et al. [ 45 ] showed that peripheral autonomic injuries could be suspected if the amplitude and duration of CC-EMG signals diminished. Thus, careful and thorough CC-EMG monitoring during RARP may provide further information useful for preserving EF. The present work focuses on modelling and in vivo testing of the geometry of the cathode and waveform of a stimulating pulse based on the electrochemical and neurophysiological mechanisms to provide efficient, safe and potentially selective fibre-type NVB stimulation. A concept capable of preferential activation of a population of C-fibres in an NVB to potentially effect faster, stronger and more durable erections was proposed. We tested our hypothesis that such stimulating electrodes could activate a population of nerve fibres within the NVB in a more physiological manner in vivo during RARP. The ultimate intention was to estimate the usability of the concept in further development of an intraoperative measuring procedure aimed at improving recovery of EF after prostatectomy. Methods Design of stimulating pulse and electrodes The NVB is composed of myelinated Aδ-fibres, also designated as B-fibres (diameter [ D ] = 1–5 µm, CV Aδ =3–15 m/s), and unmyelinated C-fibres ( D = 0.5–2 µm, CV c =0.5–2 m/s) [ 10 , 16 ]. Most are C-fibres; B-fibres are in the minority. The differing properties of each individual fibre result in different action potential (AP) thresholds, refractory periods, and the duration of the APs [ 48 , 49 ]. Consequently, any change in the amplitude and waveform of the CC-EMG is due to a change in the number of fibres that are firing. With increasing stimulation current i c , however, the number of axons firing is equivalent to the sum of all those whose thresholds are met by a given input. The latency between the application of the stimulus and the onset of the CC-EMG is a function of the events during the depolarization and an autonomic signal path between the recording site and the site of the stimulation. It was assumed that the i c , required for activation of the AP in Aδ-fibres, is significantly lower than that required for C-fibre activation [ 50 , 51 ], that Aδ- and C-fibres could be activated at any site on the cathode and that the APs elicited would propagate simultaneously in both directions. According to the paradigm, the AP in the C-fibres should reach the inner edge of the circular anode (A) after i c has exponentially decayed to a point that is inefficient at blocking their conduction [ 52 ]. It was assumed that the latest AP of the Aδ-fibres, however, arrives at the inner edge of the anode and passes away as the hyperpolarizing effect of the anode is weak. For this purpose, the time separations between the AP of Aδ- and C-fibres to reach the inner edge of an anode were calculated. The intensity of the anodal phase of the stimulus had to be lower than the threshold of the anodal activation of the Aδ-fibres to prevent excitation due to reversed polarity on the cathode during the anodal phase. On the anode, hyperpolarization caused by inward current should be decayed exponentially to prevent any anodal break excitation when APs arrive from the cathode. Activation of fibres with different conduction velocity (CV) could therefore be achieved by changing the time constant τ exp of exponential i c decay and by adjusting the intensity of i c . The resulting stimulus shown in Fig. 1 b was a current, charge-balanced pulse composed of a quasi-trapezoidal cathodal phase with a square leading edge of intensity i c , a plateau of the cathodal phase with the width of t c , followed by an exponentially decaying phase with the width of t exp and the time constant τ exp , and ended with a wide, rectangular, anodic phase with width t a and intensity i a . The electrodes for NVB stimulation were designed considering the published results of studies modelling the selective stimulation of peripheral nerves [ 27 , 50 , 52 ], the available structural topography of the NVBs, and the paradigm developed. The distributions of the fibre diameters and physical dimensions were taken from the literature to define the relationship between the structural topography and the physical model [ 7 , 15 , 16 ]. For the NVB simulation, the aforementioned quasi-trapezoidal biphasic rectangular and cathodal first current stimulating pulse pairs with a frequency of 10 Hz were intended to be delivered to the NVB via specifically designed stimulation electrodes. The quantities that were considered for the design of the stimulation pulse and stimulation electrodes are presented in Table 1 . Table 1 Quantities considered for the design of the stimulating probe Parameter Acronym Value Type of nerve fibre NA A δ , C Diameter of A δ -fibres d Aδ 1–5 µm Diameter of C-fibres d c 0.5–2 µm Conduction velocity of A δ -fibres CV Aδ 3–15 m/s Conduction velocity of C-fibres CV c 0.5–2 m/s Average conduction velocity of C- and A δ -fibres CV av 8 m/s Distance from the centre if the cathode to the circular edge D ce 3.7 mm Average travel time of the action potential from the centre of the cathode to the anode t ce 465 µs Manufacture of the stimulating probe The material used to craft the stimulation electrodes with a specific shape and arrangement was chosen based on the mechanical and electrochemical characteristics [ 32 , 33 ]. A spiral-shaped stimulation cathode and a ring-shaped anode were crafted from a 0.25-mm-thick cold-rolled platinum ribbon (purity, 99.99 wt %) (Zlatarna Celje d. d., Kersnikova 19, 3000 Celje, Republic of Slovenia). The first step in crafting the electrodes involved the design and computer-supported drawings of the arrangement at dimensions that matched the proposed paradigm. A file of the drawings was then transferred to the laser cutting machine to cut out an electrode arrangement from the platinum ribbon. Once cut out, the geometric surface of the cathode obtained was 27 mm 2 and that of the anode was 23 mm 2 . In the third step, a piece of platinum wire was welded onto the back of each electrode using a capacitive discharge spot welder. In addition, a few additional bent pieces were welded on both electrodes as anchors within an encapsulant to prevent the electrodes from separating from the probe. Lead wires to the electrodes made of fluorinated ethylene propylene (FEP) insulated stranded wire (AS 637, Cooner Wire, Chatsworth, CA) were welded onto the 2 pieces of platinum wire. Self-curing denture material (ProBase Cold Professional PMMA denture base material, Ivoclar, Schaan, Liechtenstein) was used to encapsulate the electrodes. An arrangement of electrodes was then inserted into the model made of high-quality RTV mould-making silicone rubber (Extreme Silicone) and filled with denture material. After the material had cured, the model was removed from the casting material, inserted into the titanium body of the probe and adhered using medical-grade silicone adhesive (ASC, Applied Silicone Corporation, Part No: 40064, MED RTV adhesive, implant grade, Santa Paula, CA). When finalized, the area where the effect of NVB stimulation could potentially be produced by the cathode was around 50 mm 2 . The geometric surface of both electrodes was enlarged by mechanical grinding with sandpaper (Waterproof Silica Carbide Paper FEPA P#500, Struers ApS, Denmark) to increase irregularity on the cathode surface and thus to decrease the current density at the interface with the NVB tissue. As a result, the | Z | of both electrodes was lowered so they became more suitable for NVB stimulation. Figure 1 d shows a stimulation probe with the spiral cathode at the centre of the probe and the ring-shaped anode close to the circumference. Among the sterilization methods that could be applied with the FEP insulation material, low-temperature hydrogen peroxide gas plasma sterilization was selected. Assessment of the electrical properties The | Z | of the electrodes was investigated under simulated physiological conditions in phosphate-buffered saline (PBS) (NaCl, 7.36 g/L; Na 2 HPO 4 , 11,5 g/L; NaH 2 PO 4 ·H 2 O, 3.04 g/L) as a reference. |Z| values at the interface of the cathode and the anode were measured in a chamber filled with PBS using an LCR meter (AT2816A Precision Digital LCR Meter, Changzhou Applent Instruments, Ltd., Jiangsu, China). In both in vitro and in vivo testing, the stimuli proposed in the paradigm were delivered from the custom-designed nerve stimulator shown in Fig. 2 a, which was connected to the voltage booster amplifier shown in Fig. 2 c. The corresponding | Z | at the interface of the cathode and the anode in PBS solution versus frequency is shown in Fig. 2 d. The relevant parameters of the stimuli are shown in Table 2 . When testing the stimuli, the intensity of the cathodal and anodal parts of the stimulus pulse pair i c and i a was identified by measuring the voltage drop across the precision serial resistor at the stimulator output (10 Ω). Table 2 Stimulation parameters and physical quantities used during testing of the electrical properties of the stimulating probe Stimulation parameter/physical quantity Acronym Value Intensity of the cathodal phase (stimulating current) i c 10–70 mA Width of the cathodal phase t c 500 µs Delay between the phases d NA Width of cathodal exponential decay t exp 300 µs Time constant of cathodal exponential decay τ exp Arbitrary set µs Intensity of the anodic phase i a 10 mA Width of the anodic phase t a 1000 µs Frequency f 10 Hz Cathodal geometric surface A c 27 mm 2 Anodal geometric surface A a 23 mm 2 Table 3 Electrical quantities and elements that contributed to the voltage drop across the electrode–NVB tissue interface ∆ V Stimulation parameter/physical quantity Acronym Value Stimulating/cathodal charge Q c 26 µA s Anodal charge Q a 10 µA s Cathodal charge density σQ c 0.963 µA s/mm 2 Anodal charge density σQ a 0.435 µA s/mm 2 Entire voltage drop across the electrode–NVB tissue interface ∆ V 40.4 V Polarization across the electrode–NVB tissue interface Δ E p 24.0149 V Potential of the cathode at the onset of the stimulating pulse E ipp 12.53 V Access voltage (drop across the NVB resistance R i plus over-potential terms) V a 16.38 V Maximum positive polarization across the electrode–NVB tissue interface E ma 11.52 V Maximum negative polarization across the electrode–NVB tissue interface E mc −11.49 V The stimulation/cathodal charge Q c was calculated as an integral of the surface under the cathodal current i c between the start and the end of the cathodal part of the stimulus, and nodal charge Q a was calculated as an integral of the surface under the anodal current i c between the start and the end of the anodal part of the stimulus. To identify the potential harm that could occur at the NVB tissue below the cathode during pulsing, cathodal charge density σQ c was calculated considering the charge at the cathodal part and the geometric surface of the cathode. Similarly, anodal charge density σQ a was calculated considering the charge at the anodal part and the geometric surface of the anode. The electrical properties of both the cathode and the anode were studied in vivo using the simple technique of stimulating SVT measurements between the anode and the cathode during RARP while they were in firm contact with the NVB. However, the electrical properties were analysed in one particular stimulating pulse selected from the 5-min train of identical stimulation pulses. Furthermore, | Z | across both the cathode-NVB tissue and the anode-NVB tissue was determined to investigate the electrical properties of the cathode and anode under in vivo physiological conditions. | Z | was calculated from the current i c flowing between the cathode and anode through the NVB tissue and the SVT that developed between the cathode and anode during NVB stimulation [ 44 ]. E mc and E ma were defined across the electrode–NVB tissue interface. These potentials were then tested to establish if any exceeded the values confining the water electrolysis window defined by the cyclic voltammetry CV values [− 0.60 V, + 0.85 V] measured in PBS [ 53 ]. The stimulation parameters, physical quantities, their values and acronyms that were used/delivered during testing of the electrical properties of the stimulating probe are presented in Table 2 . Assessment of the physiological properties Five patients (mean age, 57 years) scheduled for RARP were enrolled in the study. All were confirmed to have clinically localized prostate cancer (stage T1 or T2). After being informed about the purpose and procedures of the study, they provided written informed consent. Before the surgery, the probe was introduced into the operating field through the fifth lateral port (AirSeal), and lead wires remained in part within the port and in part outside the port to allow connection to the stimulator output. During the sparing procedure, the NVBs were carefully removed and isolated from the prostate and freed from excessive fat tissue in preparation for testing the physiological performance. The probe was then placed in the middle of the NVB for 5 min, once on the left side and once on the right side [ 19 ]. NVB stimulation was delivered via stimulation electrodes connected to the custom-designed nerve stimulator when the parameters identified in Table 1 were predefined via dials by the principal investigator. Based on the above-mentioned paradigm, i c was adjusted to 50 mA after firm contact of the probe was established by the surgeon. It was necessary to activate predominant non-myelinated NVB fibres located at a certain distance from the stimulating electrodes, therefore firm contact of the probe is crucial to deliver a stimulating charge onto the NVB at a density not harmful to the NVB tissue. A multi-sensorial device combining commercial and custom-crafted sensors was developed to monitor and evaluate eventual penile erectile events. The main parts of the device to measure penile axial rigidity involve a set of 4 different-sized custom-developed bell-shaped probes and a tensile/compressive force transducer (Type S2, Hottinger Brüel & Kjær GmbH, Darmstadt, Germany). Before the measurements, the appropriate size of the bell-shaped probe was selected for each patient and mounted onto the multisensorial device. For the study of smooth muscle activity within the penile shaft, dual-channel CC-EMG measurements were performed using a high-performance general-purpose amplifier (ETH-256 2-Channel Bridge/ECG/EMG/EEG/Bio-Amplifier, iWorx/CB Sciences, Dover, DE) with the following settings: gain, ×100; low-pass filter, 50 Hz; high-pass filter, 0.03 Hz. A 3-lead isolated biopotential recording preamplifier (C-ISO-256, iWorx/CB Sciences, Dover, DE) was used to provide galvanical isolation between the patient and the ETH-256 with the following settings: gain, ×400; high-pass filter, 0.05 Hz; low-pass filter, 2500 Hz. CC-EMG was measured between the base and the tip region of the penis using surface and subdermal needle electrodes [ 19 ]. Intracavernosal CC-EMG was measured with platinum-iridium disposable needle electrodes (TE/S46-638; Technomed Europe, Maastricht-Airport, The Netherlands) connected to positive inputs of the recording preamplifier. They were inserted at the 3 o'clock position into the middle of the left CC penile shaft and at the 9 o’clock position into the middle of the right CC penile shaft. Their tips were positioned around the centre of the CCs. For surface CC-EMG measurements, however, 2 pre-gelled 20-mm disposable silver and silver chloride electrodes (Ag/AgCl) (part number 019-400400; Natus, Middleton, WI) were placed on the left CC shaft at the 5 o’clock position and on the right CC shaft at the 7 o’clock position as close as possible to the proximal part of the penis [ 19 ]. One pair of surface and subdermal needle electrodes was placed on the left and another on the right pubis to serve as a ground for the CC-EMG signals. Data acquisition and offline signal analysis The data were gathered at a sampling rate f s =200.00 kHz/channel with 24-bit resolution using a high-performance I/O data acquisition system (DEWE-43a; Dewesoft d. o. o., Trbovlje, Republic of Slovenia) and data acquisition software (DewesoftX). The stimulation intensity i c was assessed by measuring the voltage drop across the precision serial 10 Ω resistor connected to the stimulator output. During the acquisition, the CC-EMGr and CC-EMGr signals were filtered and stored on a portable computer (Lenovo W541, Lenovo, Beijing, China). Offline signal analysis was performed using MATLAB R2024b software (Mathworks Inc., Natick, MA). Recordings of i c and SVT were analysed after a reconstructed NVB stimulation duration (NVB sd ) of 305.77 s. The DC component was then removed from i c and SVT, and the corresponding spectral density was calculated using MATLAB Welch’s power spectral density estimate. The absolute impedance | Z | was calculated simply by dividing the spectral density of i c and SVT. With regard to the analysis of the CC-EMG recordings, frequency spectral cumulative curves (amplitude and power spectrum curves) were calculated for the right (CC-EMG r ) and left (CC-EMG l ). An amplitude cumulative curve represents the sum of the amplitudes of spectral components, and a power spectrum cumulative curve represents the sum of the powers of spectral components. Results Assessment of electrical properties A stimulation current pulse with a cathodal intensity of i c = − 50 mA and the resulting SVT are shown in Fig. 3 . The SVT trace represents the waveform of the voltage difference between the cathode and anode during injection of a stimulating current i c . There were also several elements shown in Fig. 3 that contributed to the entire voltage drop (∆ V ), and they were accounted for in the calculation of E mc and E ma : Δ E p , polarization across the electrode–electrolyte interface; E ipp , potential of the cathode at the onset of the pulse; and V a , access voltage (drop across the electrolyte resistance R i plus over-potential terms). The values of these elements read from the SVT trace are presented numerically in Table 4. The table also shows Q c and Q a, as well as the corresponding cathodal ( σQ c ) and anodal ( σQ a ) charge density. E mc is defined by Eq. (1) [ 53 , 54 ]; Δ E p was obtained by subtraction of V a from the measured voltage drop ∆ V . E mc = E ipp + Δ E p = E ipp + (Δ V − V a ) = 12.53 V − (40.4 − 16.38) V = − 11.49 V (1) The value of E ma , also read from the SVT trace shown in Fig. 4 , was 11.52 V. Figure 4 shows the absolute | Z | calculated using the frequency domain Fourier analysis method. This method was used because it provided a more accurate estimation of | Z | than the time domain method, especially when recordings contain both pulses and noise. Assessment of physiological properties Figure 5 . shows the CC-EMG records in the left and right CC and the amplitude and power spectrum cumulative curves for both CC-EMGs in one of the 5 patients (GM) while the left and right NVB were stimulated. Figure 5 a shows i c (top trace), CC-EMGr (intermediate trace) and CC-EMGl while the left NVB was stimulated. Figure 5 b shows the amplitude and power spectrum cumulative curves for CC-EMGr. Figure 5 c shows i c (top trace), CC-EMGr (intermediate trace) and CC-EMGl while the right NVB was stimulated. Figure 5 d shows the amplitude and power spectrum cumulative curves for CC-EMGl. Discussion The present article reviews the electrical and physiological properties of the electrode–NVB tissue interface for stimulation electrodes within the probe to be used in NVB stimulation during RARP. The goal of the study was to develop and test the paradigm and stimulation system for efficient and selective NVB stimulation to elicit erectile events during RARP. The study focused on testing the hypothesis that stimulating electrodes within the probe and predefined stimulating pulses are appropriate for effective NVB stimulation. A quantitative description of the stimulating electrode–NVB tissue interface from the in vivo measurements has been documented. The ultimate goal of the study was to assess the status of NVB preservation by the surgeon’s judgement during the operation and the changes in CC-EMG related to NVB stimulation. The surgeon should correctly identify the NVB to ensure exact preservation and avoid injury to the NVB during RARP [ 1 – 4 ]. For this purpose, bilateral nerve-sparing procedures were performed in the 5 patients enrolled in the study. Assessment of electrical properties The most significant results on testing the electrical properties of the probe are shown graphically in Fig. 3 , as well as the relation between the stimulation pulse and elicited SVT and elements that contributed to the entire voltage drop ∆ V across the interface between the cathode and the anode and NVB tissue. The numerical values of the electrical quantities and elements from Fig. 3 are presented in Table 4. A practical issue in determining E mc was the difficulty in accurately measuring V a . As shown in Fig. 3 , the onset of i c elicited near-instantaneous voltage, so that V a could be easily determined. However, when i c was terminated, the behaviour of the potential in the exponential decay region, where i c approached the lowest value, meant that V a could not be determined easily [ 53 , 54 ] because exponential decay plays a specific role. However, E ma was determined with relative ease. As seen in the SVT, E mc and E ma reached − 11.49 V and 11.52 V, respectively. It was also noticed that V a and Δ E p were not the same value (Fig. 3 ). The in vivo impedance |Z| of the stimulation electrodes was determined mainly by the actual area of the cathode and the anode that was obtained by mechanical grinding during fabrication [ 26 ]. Thus, a surface of the cathode modified with rough sandpaper could deliver more current to the NVB tissue and provide more activation for a fixed input voltage. As the total i c delivered was reduced, activation was obtained at a reduced input power. | Z | decreased exponentially from high values at low frequencies to low values at high frequencies (Fig. 4 ), thus indicating the pure capacitive nature of the electrode–NVB tissue interface. It was also shown that coherence between i c and SVT was near 1, indicating that they were strongly connected. The original question was how to develop stimulation electrodes and a stimulation pulse to enable safe and efficient NVB stimulation at the same time. The greatest concern in deciding on the dimensions of the cathode and the anode was to avoid an electrochemical regimen such that charge injection during NVB stimulation would not remain within reversible limits. The results show that there was a circumstance when the stimulation electrodes operated in a regimen whereby potentials were pushed outside the safe region. Electrochemical reactions that occurred at the cathode owing to the charge Q c injected via the cathodal phase were not completely reversed by the charge Q a injected via the anodic phase [ 14 , 24 , 28 ]. Accordingly, the data on the electrical properties of the probe only partly support our hypothesis. Namely, E mc and E ma largely exceed the safe potential limits of water electrolysis [0.60 V, + 0.85 V]. This is not a desirable property of clinical stimulation electrodes because, in conditions where Faradaic charge transfer predominates over capacitive charge transfer, chemical reactions may occur [ 24 , 28 , 29 ]. Fortunately, the NVB stimulations were not permanent and lasted only 5 min for the left and right NVB. As more becomes known about RARP, it is necessary to provide a basis for the choice of materials and the design of stimulating electrodes for NVB stimulation. To reveal certain quantitative information about the anode– and cathode–NVB tissue interface and limits of the stimulation pulses, the electrochemical techniques deployed were selected based on their ability to enable short-term selective and safe NVB stimulation. The results on the electrical performance of the electrode–NVB tissue interface were consistent with reports from other investigators [ 22 , 24 – 26 , 29 ]. To stimulate a certain group of NVB fibres and avoid injury associated with a high charge density, NVB stimulation started only when firm contact of the stimulation electrodes and the location was made so that the i c was delivered at the lowest density and lowest impedance | Z |. Such conditions were expected because the parameters of the stimulation pulses that were applied to the NVB tissue to depolarize non-myelinated nerve fibres were relatively high. The results show that both in vitro and in vivo, low frequencies during pulsing resulted in significant differences in measured | Z |. Due to the different conditions at the 2 interfaces, | Z | at 50 Hz was 170 Ω in vitro and 1500 Ω in vivo. However, aiming to obtain | Z | of the interface only between stimulation electrodes and NVB tissue, a resistivity of 22.26 Ω (12.26 Ω leading wires, 10 Ω measurement resistor) must be deducted from the measured valus of | Z |. Furthermore, | Z | during pulsing in vitro lowered faster with increasing frequency than during pulsing in vivo due to a much larger number and greater mobility of ionic carriers available in the in vitro environment. Our further work could test different stimulation waveforms and parameters to answer the questions raised by our results with regard to the electrical properties of the probe. Assessment of physiological properties As shown in Fig. 5 a and c, NVB stimulation elicited high-quality CC-EMG waveforms in all our patients. In the upper trace in Fig. 5 a, the amplitude of CC-EMGr, which was recorded from the right CC at the start of an 85-s-long left NVB stimulation, can be seen to be lower than that of CC-EMGl, which was recorded from the left CC. Within the next 80 s, when the left NVB was stimulated with intensity i c =70 mA, the CC-EMGr amplitude recorded from the right CC was also lower than the CC-EMGl recorded from the left CC. Within the remaining 135 s, when the left NVB was stimulated with the same intensity i c =70 mA, the CC-EMG amplitude recorded from the right and left CC was the highest and almost the same. Within this time period, the appearance of both CC-EMG waves was synchronized. However, Fig. 5 c (upper trace) shows that with right NVB stimulation with intensity i c =60 mA for the entire time period of 300 s, the CC-EMGr amplitude recorded from the right CC was roughly the same as the CC-EMGl recorded from the left CC. Within this time period, both CC-EMG waves appeared almost synchronized. The results do not confirm the original question of whether it is possible to selectively stimulate fibre populations of different types and different diameters within the NVB. Any effect indicating faster, stronger and more durable erections based on activation of a preferential population of C-fibres in an NVB could not be identified. Therefore, the hypothesis proposing fibre-type selective NVB stimulation tested by exploiting the difference in the threshold between different nerve fibre diameters has not been confirmed. However, the ability of the electrodes to activate a certain volume of NVB tissue with as low as possible voltage and power requirements, an important desire [ 26 , 28 ], was confirmed. The results also show that NVB stimulation is strongly dependent on the physical proximity of the electrodes deployed and the distance between the electrodes and the NVB tissue [ 29 , 48 , 52 ]. The results on the physiological properties of the electrodes were consistent with reports from other investigators [ 31 , 55 , 56 ]. This may be seen in Fig. 5 , which shows CC-EMG signals recorded in the left and right CC just after RARP while the left and right NVBs were stimulated. All 4 CC-EMG traces were of high quality and, as such, they confirmed the functionality of the isolated NVB tissue [ 41 , 44 , 45 ]. However, the measurements were accomplished in patients with satisfactory EF pre-operatively. With regard to the assessment of other physiological properties of the probe, no increase in axial rigidity was obtained in any of the assessments. This outcome is in contrast to outcomes published in the literature [ 3 , 5 , 36 , 44 ]. One possible reason for the absence of axial rigidity could be constriction of blood vessels due to NVB stimulation [ 57 ]. Another possible reason for the lack of erectile responses may include the limited time allocated for NVB stimulation. Nevertheless, the surface area of the stimulation cathode may not have been able to establish effective electrical contact with a sufficient number of NVB neural fibres. The extent and neurological outcome of these effects on EF are not known and should be assessed in further research [ 58 ]. One parameter for a quasi-trapezoidal pulse that might be important for confirmation of the paradigm is the arbitrarily preset τ exp . Its precise influence on EF was not identified. It could only be speculated that the AP of the Aδ-fibres were not blocked, whereas the AP of the C-fibres passed through. One method that might be used to confirm or discredit the proposed paradigm is CC-EMG [42, 59]. CC-EMG features in the frequency domain were computed from an NVB stimulation duration (NVB sd ) of 305.77 s. The amplitude and power spectrum cumulative curve computed is shown in Fig. 5 b for CC-EMGr and in Fig. 5 d for CC-EMGl. The median frequency (MF) is an indicator of the CCM status under NVB stimulation conditions. It is obtained from the amplitude and power spectrum cumulative curves of a recorded CC-EMG [ 47 ]. Reasons for changes in the CC-EMG are modulation of the recruitment firing rate, grouping and slowing of the CV and synchronization of the signal. The MF in the amplitude cumulative curve in Fig. 5 b was 26.7 Hz, and the MF in the power spectrum cumulative curve was 50 Hz. Furthermore, the MF in the amplitude cumulative curve shown in Fig. 5 d was 28.23 Hz and the MF in the power spectrum cumulative curve was 43.58 Hz. It may be seen in the cumulative curves of both CC-EMGs that they were contaminated with harmonics that came from the NVB stimulation pulses and with frequency that came from the facility mains. Results for the physiological properties from the assessment probe also showed that no measurable increases in penile axial rigidity were observed in response to NVB stimulation in any of the patients. Further research should consider even longer NVB stimulation periods to obtain a more consistent picture on the status of the NVB tissue just after RARP. Further improvement in the NVB stimulation scheme, including different patterns of stimulation pulses, will be implemented to elicit measurable axial rigidity during NVB stimulation. In addition, a probe with a larger and modified area of stimulating electrodes will be crafted. Conclusion The results suggest that CC-EMG may provide valuable intraoperative feedback regarding the functional integrity of NVBs during RARP. However, no immediate measurable changes in EF parameters, such as axial rigidity, were observed after NVB stimulation, highlighting the complexity of real-time intraoperative assessment of EF. The results show that the design of the stimulating electrodes, based on the electrical properties of the real area of the cathode and properties of NVB nerve fibres, could be useful for the development of probes for NVB stimulation. If successfully developed further, the paradigm would enable significant improvement of current NVB sparing during RARP. A preference towards nerve fibre-type NVB stimulation may be advantageous for eliciting adequate EF during RARP and for preserving sexual function after RARP. However, more detailed investigations of neural control systems, considering the realistic structural topography of the NVB and the presence of a spatio-temporal constraint based on the electrophysiology of unmyelinated C-fibres, should be carried out based on this work. Declarations Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the National Medical Ethics Committee, Ministry of Health, Republic of Slovenia (2 July, 2024/no. 0120–138/2024-2711-6). Consent to participate Informed consent was obtained from all participants in the study. Funding This work was supported in part by Slovenian Research and Innovation Agency (grant number P3-0171) and in part by University Medical Centre Ljubljana (grant number 292401). Competing interests The authors have no relevant financial or non-financial interests to disclose. Author Contribution J.R. Developed the model for selective NVB stimulation, methods, hardware and wrote the manuscript. J.B. Performed surgery and CC-EMG measurements.T.S. Revised the research methods.S.R. Assisted with interpreting the data, discussed the results and commented on the manuscript.S.H. Perfomed surgery and NVB stimulation. Acknowledgements The authors would like to thank members of the team in the surgical facility for their assistance with the experiments. 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Int J Impot Res 22(3):171–178. https://doi.org/10.1038/ijir.2010.5 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9440557","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":625377013,"identity":"71eb8149-94ae-4782-a8ba-7a423287bdb8","order_by":0,"name":"Janez Rozman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYBACxgYILccPIhMKSNBiLAliJBiQYFuiwQEQRYwW5tntzz7+zLmTYHx+deKHBwYM8vxiBwg4bM6B5BmS257lmd14u1kC6DDDmbMTCGiZkXCYwXDb4WKzG2c3gLQkGNwmqCWxmSFx2+HEzTPObv5BpJZkZoaDQC0b+Hu3EWtLGjNj47bDxhI3eLdZJBhIEPaL4Yz0x4w/tx2W4+8/u/nmjwobeX5pQloaYCwJsEoJ/MpBQB7O4j9AWPUoGAWjYBSMTAAA5kpHhaPGP/oAAAAASUVORK5CYII=","orcid":"","institution":"ITIS d. o. o. Ljubljana","correspondingAuthor":true,"prefix":"","firstName":"Janez","middleName":"","lastName":"Rozman","suffix":""},{"id":625377014,"identity":"f59f0e0d-f146-4fd2-9360-b6d694979f57","order_by":1,"name":"Jure Bizjak","email":"","orcid":"","institution":"University Medical Centre Ljubljana","correspondingAuthor":false,"prefix":"","firstName":"Jure","middleName":"","lastName":"Bizjak","suffix":""},{"id":625377015,"identity":"c0c35f30-d504-4559-a0d1-839faff5172a","order_by":2,"name":"Tomaž Smrkolj","email":"","orcid":"","institution":"University Medical Centre Ljubljana","correspondingAuthor":false,"prefix":"","firstName":"Tomaž","middleName":"","lastName":"Smrkolj","suffix":""},{"id":625377016,"identity":"351a9bba-b19d-4082-9ede-f47f15812e3d","order_by":3,"name":"Samo Ribarič","email":"","orcid":"","institution":"Faculty of Medicine, University of Ljubljana","correspondingAuthor":false,"prefix":"","firstName":"Samo","middleName":"","lastName":"Ribarič","suffix":""},{"id":625377017,"identity":"c4abad20-869d-4d59-b056-b4843ad08554","order_by":4,"name":"Simon Hawlina","email":"","orcid":"","institution":"University Medical Centre Ljubljana","correspondingAuthor":false,"prefix":"","firstName":"Simon","middleName":"","lastName":"Hawlina","suffix":""}],"badges":[],"createdAt":"2026-04-16 16:24:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9440557/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9440557/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108012529,"identity":"8582e298-2bd7-4fce-bb09-9a0463a6c6cb","added_by":"auto","created_at":"2026-04-28 13:15:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":451652,"visible":true,"origin":"","legend":"\u003cp\u003eComponents of the paradigm for selective NVB stimulation. (a) Schematic diagram of the cathode and the anode above the NVB nerves and blood vessels. (b) Waveform of the quasi-trapezoidal stimulating pulse. (c) Arrangement, shape and dimensions of the cathode and the anode. (d) Probe\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9440557/v1/7ed7f828e6804f0f68abe37d.png"},{"id":108012714,"identity":"1489ff75-464e-4353-a1b7-43dadf148550","added_by":"auto","created_at":"2026-04-28 13:16:02","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":121689,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Nerve stimulator. (b) Foot switch. (c) Voltage booster amplifier. (d) |\u003cem\u003eZ\u003c/em\u003e| at the interface of the cathode and the anode in PBS\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9440557/v1/08df32c02249e23d366cf4ad.jpeg"},{"id":108012689,"identity":"44e1f0a4-06bb-45cc-aa81-73ca7e74edf1","added_by":"auto","created_at":"2026-04-28 13:15:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":54459,"visible":true,"origin":"","legend":"\u003cp\u003eStimulation pulse and elicited SVT in the cathode with indicated elements that contributed to the entire voltage drop ∆\u003cem\u003eV\u003c/em\u003e\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9440557/v1/fa832db085c84d4c5ac4d4ab.png"},{"id":108012401,"identity":"7881022c-360c-4858-876d-a4a6ad3c6c0f","added_by":"auto","created_at":"2026-04-28 13:15:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":43911,"visible":true,"origin":"","legend":"\u003cp\u003eAbsolute |\u003cem\u003eZ\u003c/em\u003e| versus frequency in vivo at coherence ³0.5 (MATLAB R2024b)\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9440557/v1/3417797b4af89b6f233b64c1.png"},{"id":108012399,"identity":"4049a229-1d42-4caf-ba51-129f13009081","added_by":"auto","created_at":"2026-04-28 13:15:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":261833,"visible":true,"origin":"","legend":"\u003cp\u003eRecords of CC-EMG in the left and right CC-EMG, and the amplitude and corresponding power spectrum cumulative curves while the left and right NVB were stimulated. (a) \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e (top trace), CC-EMGr (intermediate trace) and CC-EMGl while the left NVB was stimulated. (b) Amplitude and power spectrum cumulative curve for CC-EMGr. (c) \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e (top trace), CC-EMGr (intermediate trace) and CC-EMGl while the right NVB was stimulated. (d) Amplitude and power spectrum cumulative curve for CC-EMGl\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9440557/v1/d681112b2763798ef47a5f04.png"},{"id":108181270,"identity":"e4ecd39b-5407-4e1a-a42b-a67674b02112","added_by":"auto","created_at":"2026-04-30 08:58:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1429872,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9440557/v1/2149015e-1aff-47c0-92e3-9e33b715dbd2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"In vivo characterization of electrodes for selective stimulation of neurovascular bundles during robot-assisted radical prostatectomy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRadical prostatectomy is the surgical treatment for patients with prostate cancer [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, it is difficult to remove the gland without affecting prostate neurovascular bundles (NVB)s that innervate the corpora cavernosa (CC) [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEfferent innervation of the penis comes from parasympathetic, sympathetic and somatic sources. Somatosensory afferents, however, course from the penis to central sites [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. NVBs are composed of thin, wiry post-ganglionic parasympathetic nerves expanding from the pelvic hypogastric plexus. They contain delicate bundles of cavernous nerve (CN) fibres, prostatic branches of the inferior vesical artery, prostatic veins, collagen fibres and adipose tissue, all surrounded by connective tissue. They travel posterolaterally to the prostate, eventually reaching the spongy erectile tissues of the CC [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Parasympathetic CN fibres that arise from the prostatic plexus innervate the helicine arteries of the erectile tissue in the cavernous spaces. They mediate vasodilation and stimulate the erection of the penis. They are extremely susceptible to stretch. Rodrigues et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] assumed that numerous nerve fibres are located in the anterior and anterolateral positions and that CNs are probably formed by the posterior nerve fibres located at a safer distance from the prostate.\u003c/p\u003e \u003cp\u003eDespite recent advances in surgical techniques, erectile dysfunction still occurs as a consequence of injury caused during radical prostatectomy, mainly to the CNs and less to the arteries within the NVB. A surgical technique called nerve-sparing prostatectomy was developed to preserve the NVBs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. When appropriately accomplished during robot-assisted radical prostatectomy (RARP), nerve sparing largely improves recovery of erectile function (EF). Robotic assistance offers excellent visualization of the NVBs and high surgical precision [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, despite advancements in NVB preservation, potency rates have remained unsatisfactory. Tode et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] showed that regeneration after injury is limited because afferent axons regenerate into the peripheral nerve stump. Thus, even when spared, the recovery of EF may take months after the procedure [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, the focus is on how to improve RARP techniques to preserve the functionality of the NVBs. Intraoperative nerve monitoring was introduced as a technique to assist the surgeon in minimizing the risk of harm to the NVBs. The technique was based on investigations on the distribution of CN fibres around the prostate using nerve stimulation during radical prostatectomy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent decades, the concepts for electrical nerve stimulation involved non-fibre-selective stimulation, which in turn frequently causes undesirable effects. Accordingly, various models of selective nerve fibre stimulation and electrodes that selectively stimulate certain features have been developed [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePeripheral nerve fibres are classified into 4 subclasses according to their diameter, conduction velocity, and extent of myelination [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. One subclass is a large group of unmyelinated slowly conducting C-fibres with axon diameters from 0.2 \u0026micro;m to 1.2 \u0026micro;m and up to 3 \u0026micro;m in some cases. They are widely distributed in hairy and glabrous skin, transmitting cutaneous and visceral peripheral signals to the central nervous system. C-fibres are also present in the glabrous skin of the glans penis. They play a crucial role in the nervous transmission of peripheral sexual stimuli to higher cortical sexual arousal areas [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Generally, unmyelinated C-fibres that have higher stimulation thresholds (between 1 and 3 mA) are activated less reliably than myelinated ones (about 0.15 mA) and stay activated for higher currents. The exact threshold depends on the stimulation frequency, pulse duration and electrode placement. C-fibres typically require higher stimulation intensities or longer duration than Aβ- and Aδ-fibres [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, the intensity, frequency and pattern of electrical stimuli play a critical role in determining which fibre types are activated. Other fibre types, such as thinly myelinated Aδ-fibres, can also be activated at certain frequencies; 5 Hz is often cited for C-fibre activation, but the optimal range for C-fibre stimulation is broader. Frequencies between 20 and 50 Hz have been identified as the blocking frequency for C-fibre afferents [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn CN stimulation, a frequency range of 10\u0026ndash;20 Hz, pulse duration 0.5\u0026ndash;1 ms, stimulation duration 30\u0026ndash;60 s and voltage 2.5\u0026ndash;8 V are commonly used to induce erectile responses [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. NVBs require high charge densities for stimulation due to a high content of non-myelinated nerve C-fibres [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This may result in a high energy transfer and the occurrence of reversible and potentially irreversible chemical reactions.\u003c/p\u003e \u003cp\u003eThe use of current, biphasic stimulating pulses in neural tissue stimulation, with cathodal and anodal phases of equal shape and charge per phase, is a common practice [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Charge delivery has been shown to be highly influenced by the electrode\u0026ndash;NVB interface, where transduction of electrons in the metallic electrode to ions in the tissue occurs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Accordingly, impedance |\u003cem\u003eZ\u003c/em\u003e| that occurs at the electrode\u0026ndash;NVB interface is crucially affected by the electrode geometry. The efficiency of neural excitation, however, is highly dependent on the current density on the electrode surface and the spatial distribution of the electric field generated in the tissue [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough efforts have been made to find optimum positions at the NVB, little work has been done to test the efficiency of NVB stimulation using analytically driven configurations of the electrodes and stimulus waveforms. Warman et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] showed that nerve fibre excitation could be predicted using the driving function of a neuron, which is proportional to the second spatial derivative of the extracellular potential and thus, the spatial derivative of the current density in the tissue. Accordingly, an electrode with greater roughness would increase the efficiency of the nerve fibre stimulation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEvery material appropriate for the production of electrodes has a charge that delimits reversibility and irreversibility of the electrochemical processes at the interface. Pure platinum is frequently used because it can inject charge by both double-layer charging and Faradaic processes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, they can be either reversible or irreversible. It was shown that all irreversible electrochemical processes are harmful. The platinum charge injection limits within which the electrolysis of water is avoided in chronic stimulation with biphasic cathode first pulses were determined to be between 50 and 150 \u0026micro;C cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e (geometric area) [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Platinum as a stimulating electrode material has the advantage of decreasing |Z| with increasing frequency. In vitro electrochemical techniques such as cyclic voltammetry, |Z| and voltage transient (SVT) measurements can be used to characterize an electrode for nerve tissue stimulation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Their results enable the selection and design of the electrode material, and selection of stimulation pulse waveform and parameters for efficient and safe nerve tissue stimulation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSVTs are frequently used to estimate the charge injection limit \u003cem\u003eQ\u003c/em\u003e\u003csub\u003einj\u003c/sub\u003e, which defines the charge that can be injected by the electrode in a current stimulation pulse using only reversible processes. SVTs determine both negative (\u003cem\u003eE\u003c/em\u003e\u003csub\u003emc\u003c/sub\u003e) and positive (\u003cem\u003eE\u003c/em\u003e\u003csub\u003ema\u003c/sub\u003e) potential extremes across the electrode\u0026ndash;electrolyte interface. These potential extremes are then compared with the established maximum potentials beyond which it is considered unsafe to polarize the electrode (typically the potential window of water electrolysis). In most in vivo testing, however, the electrode is placed onto or into the neural tissue to develop the electrode\u0026ndash;NVB interface [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCorpus cavernous EMG (CC-EMG) is a measure of the electrical activity of the smooth corpus cavernosum muscle (CCM), which is a prerequisite for erection. According to Wagner et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], CC-EMG is the most direct electrophysiological indicator of any malfunction in the integrity circuits between the CNS and CCM related to EF. Klotz and Herschorn [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] described how to successfully capture CC-EMG during prostatectomy. Some authors claimed that CC-EMG reflects the summation of the membrane current of a group of CCM muscle cells and is the source of the electrical activity that may be measured [\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Much later, V\u0026iacute;rseda-Chamorro et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] declared that CC-EMG of the erectile tissue recorded during erection could be used as a diagnostic technique in patients with erectile dysfunction. In addition, Leddy et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] have identified the origin of the low-frequency CC-EMG complexes in volunteers with normal EF using penile surface electrodes. They concluded that the CCM tissue was the origin of the CC-EMG complex and not the penile skin or surrounding tissue.\u003c/p\u003e \u003cp\u003eRecently, Yildiz et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] aimed to evaluate the changes in penile sensation using electrophysiological tests in patients who underwent RARP. The results showed decreased penile sensation due to CN damage and a possible dorsal penile nerve injury. However, the CC-EMG generating source is still not clearly known [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Monopolar needle electrodes and/or non-invasive surface electrodes placed on the penis could be used for CC-EMG recording. Amplification of CC-EMG and attenuation of environmental noise can be accomplished using a differential amplifier [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, a low-pass and high-pass filter should be used to acquire the most optimal frequency range of the CC-EMG (0.1\u0026ndash;5 Hz), and a notch filter should be used to remove the noise from the power lines [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eJiang et al. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] showed that peripheral autonomic injuries could be suspected if the amplitude and duration of CC-EMG signals diminished. Thus, careful and thorough CC-EMG monitoring during RARP may provide further information useful for preserving EF.\u003c/p\u003e \u003cp\u003eThe present work focuses on modelling and in vivo testing of the geometry of the cathode and waveform of a stimulating pulse based on the electrochemical and neurophysiological mechanisms to provide efficient, safe and potentially selective fibre-type NVB stimulation. A concept capable of preferential activation of a population of C-fibres in an NVB to potentially effect faster, stronger and more durable erections was proposed. We tested our hypothesis that such stimulating electrodes could activate a population of nerve fibres within the NVB in a more physiological manner in vivo during RARP. The ultimate intention was to estimate the usability of the concept in further development of an intraoperative measuring procedure aimed at improving recovery of EF after prostatectomy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDesign of stimulating pulse and electrodes\u003c/h2\u003e \u003cp\u003eThe NVB is composed of myelinated Aδ-fibres, also designated as B-fibres (diameter [\u003cem\u003eD\u003c/em\u003e]\u0026thinsp;=\u0026thinsp;1\u0026ndash;5 \u0026micro;m, CV\u003csub\u003eAδ\u003c/sub\u003e=3\u0026ndash;15 m/s), and unmyelinated C-fibres (\u003cem\u003eD\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.5\u0026ndash;2 \u0026micro;m, CV\u003csub\u003ec\u003c/sub\u003e=0.5\u0026ndash;2 m/s) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Most are C-fibres; B-fibres are in the minority. The differing properties of each individual fibre result in different action potential (AP) thresholds, refractory periods, and the duration of the APs [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Consequently, any change in the amplitude and waveform of the CC-EMG is due to a change in the number of fibres that are firing. With increasing stimulation current \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e, however, the number of axons firing is equivalent to the sum of all those whose thresholds are met by a given input. The latency between the application of the stimulus and the onset of the CC-EMG is a function of the events during the depolarization and an autonomic signal path between the recording site and the site of the stimulation. It was assumed that the \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e, required for activation of the AP in Aδ-fibres, is significantly lower than that required for C-fibre activation [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], that Aδ- and C-fibres could be activated at any site on the cathode and that the APs elicited would propagate simultaneously in both directions. According to the paradigm, the AP in the C-fibres should reach the inner edge of the circular anode (A) after \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e has exponentially decayed to a point that is inefficient at blocking their conduction [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. It was assumed that the latest AP of the Aδ-fibres, however, arrives at the inner edge of the anode and passes away as the hyperpolarizing effect of the anode is weak. For this purpose, the time separations between the AP of Aδ- and C-fibres to reach the inner edge of an anode were calculated. The intensity of the anodal phase of the stimulus had to be lower than the threshold of the anodal activation of the Aδ-fibres to prevent excitation due to reversed polarity on the cathode during the anodal phase. On the anode, hyperpolarization caused by inward current should be decayed exponentially to prevent any anodal break excitation when APs arrive from the cathode. Activation of fibres with different conduction velocity (CV) could therefore be achieved by changing the time constant \u003cem\u003eτ\u003c/em\u003e\u003csub\u003eexp\u003c/sub\u003e of exponential \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e decay and by adjusting the intensity of \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e. The resulting stimulus shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb was a current, charge-balanced pulse composed of a quasi-trapezoidal cathodal phase with a square leading edge of intensity \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e, a plateau of the cathodal phase with the width of \u003cem\u003et\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e, followed by an exponentially decaying phase with the width of \u003cem\u003et\u003c/em\u003e\u003csub\u003eexp\u003c/sub\u003e and the time constant \u003cem\u003eτ\u003c/em\u003e\u003csub\u003eexp\u003c/sub\u003e, and ended with a wide, rectangular, anodic phase with width \u003cem\u003et\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e and intensity \u003cem\u003ei\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe electrodes for NVB stimulation were designed considering the published results of studies modelling the selective stimulation of peripheral nerves [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], the available structural topography of the NVBs, and the paradigm developed. The distributions of the fibre diameters and physical dimensions were taken from the literature to define the relationship between the structural topography and the physical model [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For the NVB simulation, the aforementioned quasi-trapezoidal biphasic rectangular and cathodal first current stimulating pulse pairs with a frequency of 10 Hz were intended to be delivered to the NVB via specifically designed stimulation electrodes. The quantities that were considered for the design of the stimulation pulse and stimulation electrodes are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eQuantities considered for the design of the stimulating probe\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcronym\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eValue\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType of nerve fibre\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA\u003csub\u003eδ\u003c/sub\u003e, C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiameter of A\u003csub\u003eδ\u003c/sub\u003e-fibres\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ed\u003c/em\u003e\u003csub\u003eAδ\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u0026ndash;5 \u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDiameter of C-fibres\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ed\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u0026ndash;2 \u0026micro;m\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConduction velocity of A\u003csub\u003eδ\u003c/sub\u003e-fibres\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCV\u003csub\u003eAδ\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u0026ndash;15 m/s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConduction velocity of C-fibres\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCV\u003csub\u003ec\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.5\u0026ndash;2 m/s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage conduction velocity of C- and A\u003csub\u003eδ\u003c/sub\u003e-fibres\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCV\u003csub\u003eav\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8 m/s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDistance from the centre if the cathode to the circular edge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eD\u003c/em\u003e\u003csub\u003ece\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.7 mm\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage travel time of the action potential from the centre of the cathode to the anode\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003et\u003c/em\u003e\u003csub\u003ece\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e465 \u0026micro;s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eManufacture of the stimulating probe\u003c/h3\u003e\n\u003cp\u003eThe material used to craft the stimulation electrodes with a specific shape and arrangement was chosen based on the mechanical and electrochemical characteristics [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. A spiral-shaped stimulation cathode and a ring-shaped anode were crafted from a 0.25-mm-thick cold-rolled platinum ribbon (purity, 99.99 wt %) (Zlatarna Celje d. d., Kersnikova 19, 3000 Celje, Republic of Slovenia). The first step in crafting the electrodes involved the design and computer-supported drawings of the arrangement at dimensions that matched the proposed paradigm. A file of the drawings was then transferred to the laser cutting machine to cut out an electrode arrangement from the platinum ribbon. Once cut out, the geometric surface of the cathode obtained was 27 mm\u003csup\u003e2\u003c/sup\u003e and that of the anode was 23 mm\u003csup\u003e2\u003c/sup\u003e. In the third step, a piece of platinum wire was welded onto the back of each electrode using a capacitive discharge spot welder. In addition, a few additional bent pieces were welded on both electrodes as anchors within an encapsulant to prevent the electrodes from separating from the probe. Lead wires to the electrodes made of fluorinated ethylene propylene (FEP) insulated stranded wire (AS 637, Cooner Wire, Chatsworth, CA) were welded onto the 2 pieces of platinum wire.\u003c/p\u003e \u003cp\u003eSelf-curing denture material (ProBase Cold Professional PMMA denture base material, Ivoclar, Schaan, Liechtenstein) was used to encapsulate the electrodes. An arrangement of electrodes was then inserted into the model made of high-quality RTV mould-making silicone rubber (Extreme Silicone) and filled with denture material. After the material had cured, the model was removed from the casting material, inserted into the titanium body of the probe and adhered using medical-grade silicone adhesive (ASC, Applied Silicone Corporation, Part No: 40064, MED RTV adhesive, implant grade, Santa Paula, CA).\u003c/p\u003e \u003cp\u003eWhen finalized, the area where the effect of NVB stimulation could potentially be produced by the cathode was around 50 mm\u003csup\u003e2\u003c/sup\u003e. The geometric surface of both electrodes was enlarged by mechanical grinding with sandpaper (Waterproof Silica Carbide Paper FEPA P#500, Struers ApS, Denmark) to increase irregularity on the cathode surface and thus to decrease the current density at the interface with the NVB tissue. As a result, the |\u003cem\u003eZ\u003c/em\u003e| of both electrodes was lowered so they became more suitable for NVB stimulation. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed shows a stimulation probe with the spiral cathode at the centre of the probe and the ring-shaped anode close to the circumference.\u003c/p\u003e \u003cp\u003eAmong the sterilization methods that could be applied with the FEP insulation material, low-temperature hydrogen peroxide gas plasma sterilization was selected.\u003c/p\u003e\n\u003ch3\u003eAssessment of the electrical properties\u003c/h3\u003e\n\u003cp\u003eThe |\u003cem\u003eZ\u003c/em\u003e| of the electrodes was investigated under simulated physiological conditions in phosphate-buffered saline (PBS) (NaCl, 7.36 g/L; Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 11,5 g/L; NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO, 3.04 g/L) as a reference. |Z| values at the interface of the cathode and the anode were measured in a chamber filled with PBS using an LCR meter (AT2816A Precision Digital LCR Meter, Changzhou Applent Instruments, Ltd., Jiangsu, China).\u003c/p\u003e \u003cp\u003eIn both in vitro and in vivo testing, the stimuli proposed in the paradigm were delivered from the custom-designed nerve stimulator shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, which was connected to the voltage booster amplifier shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec. The corresponding |\u003cem\u003eZ\u003c/em\u003e| at the interface of the cathode and the anode in PBS solution versus frequency is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe relevant parameters of the stimuli are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. When testing the stimuli, the intensity of the cathodal and anodal parts of the stimulus pulse pair \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e and \u003cem\u003ei\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e was identified by measuring the voltage drop across the precision serial resistor at the stimulator output (10 Ω).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStimulation parameters and physical quantities used during testing of the electrical properties of the stimulating probe\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStimulation parameter/physical quantity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcronym\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eValue\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntensity of the cathodal phase (stimulating current)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u0026ndash;70 mA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWidth of the cathodal phase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003et\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500 \u0026micro;s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDelay between the phases\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ed\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWidth of cathodal exponential decay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003et\u003c/em\u003e\u003csub\u003eexp\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e300 \u0026micro;s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime constant of cathodal exponential decay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eτ\u003c/em\u003e\u003csub\u003eexp\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eArbitrary set \u0026micro;s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntensity of the anodic phase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ei\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 mA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWidth of the anodic phase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003et\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1000 \u0026micro;s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFrequency\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ef\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 Hz\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCathodal geometric surface\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27 mm\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnodal geometric surface\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eA\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23 mm\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eElectrical quantities and elements that contributed to the voltage drop across the electrode\u0026ndash;NVB tissue interface ∆\u003cem\u003eV\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStimulation parameter/physical quantity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAcronym\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eValue\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStimulating/cathodal charge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eQ\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e26 \u0026micro;A s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnodal charge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eQ\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 \u0026micro;A s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCathodal charge density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eσQ\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.963 \u0026micro;A s/mm\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnodal charge density\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eσQ\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.435 \u0026micro;A s/mm\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEntire voltage drop across the electrode\u0026ndash;NVB tissue interface\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e∆\u003cem\u003eV\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.4 V\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePolarization across the electrode\u0026ndash;NVB tissue interface\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eΔ\u003cem\u003eE\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.0149 V\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePotential of the cathode at the onset of the stimulating pulse\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003csub\u003eipp\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.53 V\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAccess voltage (drop across the NVB resistance \u003cem\u003eR\u003c/em\u003e\u003csub\u003ei\u003c/sub\u003e plus over-potential terms)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eV\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.38 V\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaximum positive polarization across the electrode\u0026ndash;NVB tissue interface\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003csub\u003ema\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.52 V\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaximum negative polarization across the electrode\u0026ndash;NVB tissue interface\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003csub\u003emc\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;11.49 V\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe stimulation/cathodal charge \u003cem\u003eQ\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e was calculated as an integral of the surface under the cathodal current \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e between the start and the end of the cathodal part of the stimulus, and nodal charge \u003cem\u003eQ\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e was calculated as an integral of the surface under the anodal current \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e between the start and the end of the anodal part of the stimulus.\u003c/p\u003e \u003cp\u003eTo identify the potential harm that could occur at the NVB tissue below the cathode during pulsing, cathodal charge density \u003cem\u003eσQ\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e was calculated considering the charge at the cathodal part and the geometric surface of the cathode. Similarly, anodal charge density \u003cem\u003eσQ\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e was calculated considering the charge at the anodal part and the geometric surface of the anode.\u003c/p\u003e \u003cp\u003eThe electrical properties of both the cathode and the anode were studied in vivo using the simple technique of stimulating SVT measurements between the anode and the cathode during RARP while they were in firm contact with the NVB. However, the electrical properties were analysed in one particular stimulating pulse selected from the 5-min train of identical stimulation pulses. Furthermore, |\u003cem\u003eZ\u003c/em\u003e| across both the cathode-NVB tissue and the anode-NVB tissue was determined to investigate the electrical properties of the cathode and anode under in vivo physiological conditions. |\u003cem\u003eZ\u003c/em\u003e| was calculated from the current \u003cem\u003ei\u003c/em\u003e\u003csub\u003e\u003cb\u003ec\u003c/b\u003e\u003c/sub\u003e flowing between the cathode and anode through the NVB tissue and the SVT that developed between the cathode and anode during NVB stimulation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. \u003cem\u003eE\u003c/em\u003e\u003csub\u003emc\u003c/sub\u003e and \u003cem\u003eE\u003c/em\u003e\u003csub\u003ema\u003c/sub\u003e were defined across the electrode\u0026ndash;NVB tissue interface. These potentials were then tested to establish if any exceeded the values confining the water electrolysis window defined by the cyclic voltammetry CV values [\u0026minus;\u0026thinsp;0.60 V, +\u0026thinsp;0.85 V] measured in PBS [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The stimulation parameters, physical quantities, their values and acronyms that were used/delivered during testing of the electrical properties of the stimulating probe are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eAssessment of the physiological properties\u003c/h3\u003e\n\u003cp\u003eFive patients (mean age, 57 years) scheduled for RARP were enrolled in the study. All were confirmed to have clinically localized prostate cancer (stage T1 or T2). After being informed about the purpose and procedures of the study, they provided written informed consent.\u003c/p\u003e \u003cp\u003eBefore the surgery, the probe was introduced into the operating field through the fifth lateral port (AirSeal), and lead wires remained in part within the port and in part outside the port to allow connection to the stimulator output. During the sparing procedure, the NVBs were carefully removed and isolated from the prostate and freed from excessive fat tissue in preparation for testing the physiological performance.\u003c/p\u003e \u003cp\u003eThe probe was then placed in the middle of the NVB for 5 min, once on the left side and once on the right side [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. NVB stimulation was delivered via stimulation electrodes connected to the custom-designed nerve stimulator when the parameters identified in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e were predefined via dials by the principal investigator. Based on the above-mentioned paradigm, \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e was adjusted to 50 mA after firm contact of the probe was established by the surgeon. It was necessary to activate predominant non-myelinated NVB fibres located at a certain distance from the stimulating electrodes, therefore firm contact of the probe is crucial to deliver a stimulating charge onto the NVB at a density not harmful to the NVB tissue.\u003c/p\u003e \u003cp\u003eA multi-sensorial device combining commercial and custom-crafted sensors was developed to monitor and evaluate eventual penile erectile events. The main parts of the device to measure penile axial rigidity involve a set of 4 different-sized custom-developed bell-shaped probes and a tensile/compressive force transducer (Type S2, Hottinger Br\u0026uuml;el \u0026amp; Kj\u0026aelig;r GmbH, Darmstadt, Germany). Before the measurements, the appropriate size of the bell-shaped probe was selected for each patient and mounted onto the multisensorial device.\u003c/p\u003e \u003cp\u003eFor the study of smooth muscle activity within the penile shaft, dual-channel CC-EMG measurements were performed using a high-performance general-purpose amplifier (ETH-256 2-Channel Bridge/ECG/EMG/EEG/Bio-Amplifier, iWorx/CB Sciences, Dover, DE) with the following settings: gain, \u0026times;100; low-pass filter, 50 Hz; high-pass filter, 0.03 Hz. A 3-lead isolated biopotential recording preamplifier (C-ISO-256, iWorx/CB Sciences, Dover, DE) was used to provide galvanical isolation between the patient and the ETH-256 with the following settings: gain, \u0026times;400; high-pass filter, 0.05 Hz; low-pass filter, 2500 Hz. CC-EMG was measured between the base and the tip region of the penis using surface and subdermal needle electrodes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Intracavernosal CC-EMG was measured with platinum-iridium disposable needle electrodes (TE/S46-638; Technomed Europe, Maastricht-Airport, The Netherlands) connected to positive inputs of the recording preamplifier. They were inserted at the 3 o'clock position into the middle of the left CC penile shaft and at the 9 o\u0026rsquo;clock position into the middle of the right CC penile shaft. Their tips were positioned around the centre of the CCs. For surface CC-EMG measurements, however, 2 pre-gelled 20-mm disposable silver and silver chloride electrodes (Ag/AgCl) (part number 019-400400; Natus, Middleton, WI) were placed on the left CC shaft at the 5 o\u0026rsquo;clock position and on the right CC shaft at the 7 o\u0026rsquo;clock position as close as possible to the proximal part of the penis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. One pair of surface and subdermal needle electrodes was placed on the left and another on the right pubis to serve as a ground for the CC-EMG signals.\u003c/p\u003e\n\u003ch3\u003eData acquisition and offline signal analysis\u003c/h3\u003e\n\u003cp\u003eThe data were gathered at a sampling rate \u003cem\u003ef\u003c/em\u003e\u003csub\u003es\u003c/sub\u003e=200.00 kHz/channel with 24-bit resolution using a high-performance I/O data acquisition system (DEWE-43a; Dewesoft d. o. o., Trbovlje, Republic of Slovenia) and data acquisition software (DewesoftX). The stimulation intensity \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e was assessed by measuring the voltage drop across the precision serial 10 Ω resistor connected to the stimulator output. During the acquisition, the CC-EMGr and CC-EMGr signals were filtered and stored on a portable computer (Lenovo W541, Lenovo, Beijing, China). Offline signal analysis was performed using MATLAB R2024b software (Mathworks Inc., Natick, MA).\u003c/p\u003e \u003cp\u003eRecordings of \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e and SVT were analysed after a reconstructed NVB stimulation duration (NVB\u003csub\u003esd\u003c/sub\u003e) of 305.77 s. The DC component was then removed from \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e and SVT, and the corresponding spectral density was calculated using MATLAB Welch\u0026rsquo;s power spectral density estimate. The absolute impedance |\u003cem\u003eZ\u003c/em\u003e| was calculated simply by dividing the spectral density of \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e and SVT. With regard to the analysis of the CC-EMG recordings, frequency spectral cumulative curves (amplitude and power spectrum curves) were calculated for the right (CC-EMG\u003csub\u003er\u003c/sub\u003e) and left (CC-EMG\u003csub\u003el\u003c/sub\u003e). An amplitude cumulative curve represents the sum of the amplitudes of spectral components, and a power spectrum cumulative curve represents the sum of the powers of spectral components.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of electrical properties\u003c/h2\u003e \u003cp\u003eA stimulation current pulse with a cathodal intensity of \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;50 mA and the resulting SVT are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The SVT trace represents the waveform of the voltage difference between the cathode and anode during injection of a stimulating current \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e. There were also several elements shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e that contributed to the entire voltage drop (∆\u003cem\u003eV\u003c/em\u003e), and they were accounted for in the calculation of \u003cem\u003eE\u003c/em\u003e\u003csub\u003emc\u003c/sub\u003e and \u003cem\u003eE\u003c/em\u003e\u003csub\u003ema\u003c/sub\u003e: Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e, polarization across the electrode\u0026ndash;electrolyte interface; \u003cem\u003eE\u003c/em\u003e\u003csub\u003eipp\u003c/sub\u003e, potential of the cathode at the onset of the pulse; and \u003cem\u003eV\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e, access voltage (drop across the electrolyte resistance R\u003csub\u003ei\u003c/sub\u003e plus over-potential terms). The values of these elements read from the SVT trace are presented numerically in Table\u0026nbsp;4. The table also shows \u003cem\u003eQ\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e and \u003cem\u003eQ\u003c/em\u003e\u003csub\u003ea,\u003c/sub\u003e as well as the corresponding cathodal (\u003cem\u003eσQ\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e) and anodal (\u003cem\u003eσQ\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e) charge density. \u003cem\u003eE\u003c/em\u003e\u003csub\u003emc\u003c/sub\u003e is defined by Eq.\u0026nbsp;(1) [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]; Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e was obtained by subtraction of \u003cem\u003eV\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e from the measured voltage drop ∆\u003cem\u003eV\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eE\u003c/em\u003e \u003csub\u003emc\u003c/sub\u003e = \u003cem\u003eE\u003c/em\u003e\u003csub\u003eipp\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e = \u003cem\u003eE\u003c/em\u003e\u003csub\u003eipp\u003c/sub\u003e + (Δ\u003cem\u003eV\u003c/em\u003e\u0026thinsp;\u0026minus;\u0026thinsp;\u003cem\u003eV\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e )\u0026thinsp;=\u0026thinsp;12.53 V \u0026minus; (40.4\u0026thinsp;\u0026minus;\u0026thinsp;16.38) V\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;11.49 V (1)\u003c/p\u003e \u003cp\u003eThe value of \u003cem\u003eE\u003c/em\u003e\u003csub\u003ema\u003c/sub\u003e, also read from the SVT trace shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, was 11.52 V.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the absolute |\u003cem\u003eZ\u003c/em\u003e| calculated using the frequency domain Fourier analysis method. This method was used because it provided a more accurate estimation of |\u003cem\u003eZ\u003c/em\u003e| than the time domain method, especially when recordings contain both pulses and noise.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAssessment of physiological properties\u003c/h3\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. shows the CC-EMG records in the left and right CC and the amplitude and power spectrum cumulative curves for both CC-EMGs in one of the 5 patients (GM) while the left and right NVB were stimulated. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea shows \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e (top trace), CC-EMGr (intermediate trace) and CC-EMGl while the left NVB was stimulated. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb shows the amplitude and power spectrum cumulative curves for CC-EMGr. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec shows \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e (top trace), CC-EMGr (intermediate trace) and CC-EMGl while the right NVB was stimulated. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed shows the amplitude and power spectrum cumulative curves for CC-EMGl.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present article reviews the electrical and physiological properties of the electrode\u0026ndash;NVB tissue interface for stimulation electrodes within the probe to be used in NVB stimulation during RARP. The goal of the study was to develop and test the paradigm and stimulation system for efficient and selective NVB stimulation to elicit erectile events during RARP. The study focused on testing the hypothesis that stimulating electrodes within the probe and predefined stimulating pulses are appropriate for effective NVB stimulation. A quantitative description of the stimulating electrode\u0026ndash;NVB tissue interface from the in vivo measurements has been documented. The ultimate goal of the study was to assess the status of NVB preservation by the surgeon\u0026rsquo;s judgement during the operation and the changes in CC-EMG related to NVB stimulation. The surgeon should correctly identify the NVB to ensure exact preservation and avoid injury to the NVB during RARP [\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. For this purpose, bilateral nerve-sparing procedures were performed in the 5 patients enrolled in the study.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of electrical properties\u003c/h2\u003e \u003cp\u003eThe most significant results on testing the electrical properties of the probe are shown graphically in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, as well as the relation between the stimulation pulse and elicited SVT and elements that contributed to the entire voltage drop ∆\u003cem\u003eV\u003c/em\u003e across the interface between the cathode and the anode and NVB tissue. The numerical values of the electrical quantities and elements from Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e are presented in Table\u0026nbsp;4. A practical issue in determining \u003cem\u003eE\u003c/em\u003e\u003csub\u003emc\u003c/sub\u003e was the difficulty in accurately measuring \u003cem\u003eV\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the onset of \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e elicited near-instantaneous voltage, so that \u003cem\u003eV\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e could be easily determined. However, when \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e was terminated, the behaviour of the potential in the exponential decay region, where \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e approached the lowest value, meant that \u003cem\u003eV\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e could not be determined easily [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] because exponential decay plays a specific role. However, \u003cem\u003eE\u003c/em\u003e\u003csub\u003ema\u003c/sub\u003e was determined with relative ease. As seen in the SVT, \u003cem\u003eE\u003c/em\u003e\u003csub\u003emc\u003c/sub\u003e and \u003cem\u003eE\u003c/em\u003e\u003csub\u003ema\u003c/sub\u003e reached\u0026thinsp;\u0026minus;\u0026thinsp;11.49 V and 11.52 V, respectively. It was also noticed that \u003cem\u003eV\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e and Δ\u003cem\u003eE\u003c/em\u003e\u003csub\u003ep\u003c/sub\u003e were not the same value (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe in vivo impedance \u003cem\u003e|Z|\u003c/em\u003e of the stimulation electrodes was determined mainly by the actual area of the cathode and the anode that was obtained by mechanical grinding during fabrication [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Thus, a surface of the cathode modified with rough sandpaper could deliver more current to the NVB tissue and provide more activation for a fixed input voltage. As the total \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e delivered was reduced, activation was obtained at a reduced input power. |\u003cem\u003eZ\u003c/em\u003e| decreased exponentially from high values at low frequencies to low values at high frequencies (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), thus indicating the pure capacitive nature of the electrode\u0026ndash;NVB tissue interface. It was also shown that coherence between \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e and SVT was near 1, indicating that they were strongly connected.\u003c/p\u003e \u003cp\u003eThe original question was how to develop stimulation electrodes and a stimulation pulse to enable safe and efficient NVB stimulation at the same time. The greatest concern in deciding on the dimensions of the cathode and the anode was to avoid an electrochemical regimen such that charge injection during NVB stimulation would not remain within reversible limits. The results show that there was a circumstance when the stimulation electrodes operated in a regimen whereby potentials were pushed outside the safe region. Electrochemical reactions that occurred at the cathode owing to the charge \u003cem\u003eQ\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e injected via the cathodal phase were not completely reversed by the charge \u003cem\u003eQ\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e injected via the anodic phase [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccordingly, the data on the electrical properties of the probe only partly support our hypothesis. Namely, \u003cem\u003eE\u003c/em\u003e\u003csub\u003emc\u003c/sub\u003e and \u003cem\u003eE\u003c/em\u003e\u003csub\u003ema\u003c/sub\u003e largely exceed the safe potential limits of water electrolysis [0.60 V, +\u0026thinsp;0.85 V]. This is not a desirable property of clinical stimulation electrodes because, in conditions where Faradaic charge transfer predominates over capacitive charge transfer, chemical reactions may occur [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Fortunately, the NVB stimulations were not permanent and lasted only 5 min for the left and right NVB.\u003c/p\u003e \u003cp\u003eAs more becomes known about RARP, it is necessary to provide a basis for the choice of materials and the design of stimulating electrodes for NVB stimulation. To reveal certain quantitative information about the anode\u0026ndash; and cathode\u0026ndash;NVB tissue interface and limits of the stimulation pulses, the electrochemical techniques deployed were selected based on their ability to enable short-term selective and safe NVB stimulation. The results on the electrical performance of the electrode\u0026ndash;NVB tissue interface were consistent with reports from other investigators [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. To stimulate a certain group of NVB fibres and avoid injury associated with a high charge density, NVB stimulation started only when firm contact of the stimulation electrodes and the location was made so that the \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e was delivered at the lowest density and lowest impedance |\u003cem\u003eZ\u003c/em\u003e|. Such conditions were expected because the parameters of the stimulation pulses that were applied to the NVB tissue to depolarize non-myelinated nerve fibres were relatively high.\u003c/p\u003e \u003cp\u003eThe results show that both in vitro and in vivo, low frequencies during pulsing resulted in significant differences in measured |\u003cem\u003eZ\u003c/em\u003e|. Due to the different conditions at the 2 interfaces, |\u003cem\u003eZ\u003c/em\u003e| at 50 Hz was 170 Ω in vitro and 1500 Ω in vivo. However, aiming to obtain |\u003cem\u003eZ\u003c/em\u003e| of the interface only between stimulation electrodes and NVB tissue, a resistivity of 22.26 Ω (12.26 Ω leading wires, 10 Ω measurement resistor) must be deducted from the measured valus of |\u003cem\u003eZ\u003c/em\u003e|. Furthermore, |\u003cem\u003eZ\u003c/em\u003e| during pulsing in vitro lowered faster with increasing frequency than during pulsing in vivo due to a much larger number and greater mobility of ionic carriers available in the in vitro environment.\u003c/p\u003e \u003cp\u003eOur further work could test different stimulation waveforms and parameters to answer the questions raised by our results with regard to the electrical properties of the probe.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAssessment of physiological properties\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and c, NVB stimulation elicited high-quality CC-EMG waveforms in all our patients. In the upper trace in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, the amplitude of CC-EMGr, which was recorded from the right CC at the start of an 85-s-long left NVB stimulation, can be seen to be lower than that of CC-EMGl, which was recorded from the left CC. Within the next 80 s, when the left NVB was stimulated with intensity \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e=70 mA, the CC-EMGr amplitude recorded from the right CC was also lower than the CC-EMGl recorded from the left CC. Within the remaining 135 s, when the left NVB was stimulated with the same intensity \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e=70 mA, the CC-EMG amplitude recorded from the right and left CC was the highest and almost the same. Within this time period, the appearance of both CC-EMG waves was synchronized.\u003c/p\u003e \u003cp\u003eHowever, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec (upper trace) shows that with right NVB stimulation with intensity \u003cem\u003ei\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e=60 mA for the entire time period of 300 s, the CC-EMGr amplitude recorded from the right CC was roughly the same as the CC-EMGl recorded from the left CC. Within this time period, both CC-EMG waves appeared almost synchronized.\u003c/p\u003e \u003cp\u003eThe results do not confirm the original question of whether it is possible to selectively stimulate fibre populations of different types and different diameters within the NVB. Any effect indicating faster, stronger and more durable erections based on activation of a preferential population of C-fibres in an NVB could not be identified.\u003c/p\u003e \u003cp\u003eTherefore, the hypothesis proposing fibre-type selective NVB stimulation tested by exploiting the difference in the threshold between different nerve fibre diameters has not been confirmed. However, the ability of the electrodes to activate a certain volume of NVB tissue with as low as possible voltage and power requirements, an important desire [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], was confirmed. The results also show that NVB stimulation is strongly dependent on the physical proximity of the electrodes deployed and the distance between the electrodes and the NVB tissue [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe results on the physiological properties of the electrodes were consistent with reports from other investigators [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. This may be seen in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, which shows CC-EMG signals recorded in the left and right CC just after RARP while the left and right NVBs were stimulated. All 4 CC-EMG traces were of high quality and, as such, they confirmed the functionality of the isolated NVB tissue [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. However, the measurements were accomplished in patients with satisfactory EF pre-operatively. With regard to the assessment of other physiological properties of the probe, no increase in axial rigidity was obtained in any of the assessments. This outcome is in contrast to outcomes published in the literature [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. One possible reason for the absence of axial rigidity could be constriction of blood vessels due to NVB stimulation [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Another possible reason for the lack of erectile responses may include the limited time allocated for NVB stimulation.\u003c/p\u003e \u003cp\u003eNevertheless, the surface area of the stimulation cathode may not have been able to establish effective electrical contact with a sufficient number of NVB neural fibres. The extent and neurological outcome of these effects on EF are not known and should be assessed in further research [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne parameter for a quasi-trapezoidal pulse that might be important for confirmation of the paradigm is the arbitrarily preset \u003cem\u003eτ\u003c/em\u003e\u003csub\u003eexp\u003c/sub\u003e. Its precise influence on EF was not identified. It could only be speculated that the AP of the Aδ-fibres were not blocked, whereas the AP of the C-fibres passed through.\u003c/p\u003e \u003cp\u003eOne method that might be used to confirm or discredit the proposed paradigm is CC-EMG [42, 59]. CC-EMG features in the frequency domain were computed from an NVB stimulation duration (NVB\u003csub\u003esd\u003c/sub\u003e) of 305.77 s. The amplitude and power spectrum cumulative curve computed is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb for CC-EMGr and in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed for CC-EMGl. The median frequency (MF) is an indicator of the CCM status under NVB stimulation conditions. It is obtained from the amplitude and power spectrum cumulative curves of a recorded CC-EMG [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Reasons for changes in the CC-EMG are modulation of the recruitment firing rate, grouping and slowing of the CV and synchronization of the signal. The MF in the amplitude cumulative curve in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb was 26.7 Hz, and the MF in the power spectrum cumulative curve was 50 Hz. Furthermore, the MF in the amplitude cumulative curve shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed was 28.23 Hz and the MF in the power spectrum cumulative curve was 43.58 Hz. It may be seen in the cumulative curves of both CC-EMGs that they were contaminated with harmonics that came from the NVB stimulation pulses and with frequency that came from the facility mains.\u003c/p\u003e \u003cp\u003eResults for the physiological properties from the assessment probe also showed that no measurable increases in penile axial rigidity were observed in response to NVB stimulation in any of the patients.\u003c/p\u003e \u003cp\u003eFurther research should consider even longer NVB stimulation periods to obtain a more consistent picture on the status of the NVB tissue just after RARP. Further improvement in the NVB stimulation scheme, including different patterns of stimulation pulses, will be implemented to elicit measurable axial rigidity during NVB stimulation. In addition, a probe with a larger and modified area of stimulating electrodes will be crafted.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results suggest that CC-EMG may provide valuable intraoperative feedback regarding the functional integrity of NVBs during RARP. However, no immediate measurable changes in EF parameters, such as axial rigidity, were observed after NVB stimulation, highlighting the complexity of real-time intraoperative assessment of EF. The results show that the design of the stimulating electrodes, based on the electrical properties of the real area of the cathode and properties of NVB nerve fibres, could be useful for the development of probes for NVB stimulation.\u003c/p\u003e \u003cp\u003eIf successfully developed further, the paradigm would enable significant improvement of current NVB sparing during RARP. A preference towards nerve fibre-type NVB stimulation may be advantageous for eliciting adequate EF during RARP and for preserving sexual function after RARP. However, more detailed investigations of neural control systems, considering the realistic structural topography of the NVB and the presence of a spatio-temporal constraint based on the electrophysiology of unmyelinated C-fibres, should be carried out based on this work.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003e This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the National Medical Ethics Committee, Ministry of Health, Republic of Slovenia (2 July, 2024/no. 0120\u0026ndash;138/2024-2711-6).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent to participate\u003c/strong\u003e \u003cp\u003e Informed consent was obtained from all participants in the study.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported in part by Slovenian Research and Innovation Agency (grant number P3-0171) and in part by University Medical Centre Ljubljana (grant number 292401).\u003c/p\u003e \u003cp\u003eCompeting interests\u003c/p\u003e \u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.R. Developed the model for selective NVB stimulation, methods, hardware and wrote the manuscript. J.B. Performed surgery and CC-EMG measurements.T.S. Revised the research methods.S.R. Assisted with interpreting the data, discussed the results and commented on the manuscript.S.H. Perfomed surgery and NVB stimulation.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors would like to thank members of the team in the surgical facility for their assistance with the experiments.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eUniversity of Florida Health Robotic nerve-sparing radical prostatectomy. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://urology.ufl.edu/patient-care/robotic-laparoscopic-urologic-surgery/procedures/robotic-nerve-sparing-radical-prostatectomy/\u003c/span\u003e\u003cspan address=\"https://urology.ufl.edu/patient-care/robotic-laparoscopic-urologic-surgery/procedures/robotic-nerve-sparing-radical-prostatectomy/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiology Insights Cavernous nerve function damage and rehabilitation. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://biologyinsights.com/cavernous-nerve-function-damage-and-rehabilitation/\u003c/span\u003e\u003cspan address=\"https://biologyinsights.com/cavernous-nerve-function-damage-and-rehabilitation/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNguyen LN, Head L, Witiuk K, Punjani N, Mallick R, Cnossen S, Fergusson DA, Cagiannos I, Lavall\u0026eacute;e LT, Morash C, Breau RH (2017) The risks and benefits of cavernous neurovascular bundle sparing during radical prostatectomy: a systematic review and meta-analysis. 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Int J Impot Res 22(3):171\u0026ndash;178. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ijir.2010.5\u003c/span\u003e\u003cspan address=\"10.1038/ijir.2010.5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"neurovascular bundle, platinum, stimulating electrode, interface, voltage response, EMG","lastPublishedDoi":"10.21203/rs.3.rs-9440557/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9440557/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe electrical and electrophysiological performance of platinum electrodes within the probe for bipolar selective stimulation of neurovascular bundles (NVB)s during robot-assisted radical prostatectomy was investigated. Quasi-trapezoidal pulse trains (5 min) were delivered to isolated NVBs in 5 patients. The electrical performance of the electrodes was studied by determining the polarization across the electrode\u0026ndash;NVB tissue interface using voltage transients. Cavernous EMG (CC-EMG), axial rigidity and computing CC-EMG features were measured in the frequency domain. The most negative \u003cem\u003eE\u003c/em\u003e\u003csub\u003emc\u003c/sub\u003e and most positive \u003cem\u003eE\u003c/em\u003e\u003csub\u003ema\u003c/sub\u003e potentials across the electrode\u0026ndash;NVB tissue interface reached\u0026thinsp;\u0026minus;\u0026thinsp;11.49 V and 11.52 V, respectively; the cathodal (σ\u003cem\u003eQ\u003c/em\u003e\u003csub\u003ec\u003c/sub\u003e) and anodal (σ\u003cem\u003eQ\u003c/em\u003e\u003csub\u003ea\u003c/sub\u003e) charge densities were 0.963 \u0026micro;A s/mm\u003csup\u003e2\u003c/sup\u003e and 0.435 \u0026micro;A s/mm\u003csup\u003e2\u003c/sup\u003e, respectively. They exceeded tissue injury limits for a short time, but tissue injury did not occur. Impedance |\u003cem\u003eZ\u003c/em\u003e| decreased faster with frequency in vitro (50 Hz, 170 Ω) than in vivo (1500 Ω). The median frequencies of the amplitude and power were: CC-EMGr, 26.7 Hz and 50 Hz; CC-EMGl, 28.33 Hz and 43.58 Hz. Both were contaminated with the stimuli and facility mains. Any influence of \u003cem\u003eτ\u003c/em\u003e\u003csub\u003eexp\u003c/sub\u003e preset in the stimuli on electrophysiological performance could not be identified. An increase in penile axial rigidity was not observed in any patient.\u003c/p\u003e","manuscriptTitle":"In vivo characterization of electrodes for selective stimulation of neurovascular bundles during robot-assisted radical prostatectomy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-28 13:11:53","doi":"10.21203/rs.3.rs-9440557/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2653ac8a-d710-464b-a998-861c10d5c8fb","owner":[],"postedDate":"April 28th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T13:11:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-28 13:11:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9440557","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9440557","identity":"rs-9440557","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}