Patients affected by lower urinary tract dysfunctions (LUTDs), such as overactive bladder, lose control of micturition with invalidating consequences on their physical, social and emotional well-being. 
Pudendal neuromodulation has been successfully used to treat LUTDs symptoms by injecting current through a tined lead electrode [1]. However, the epineural electrode migration compromised its efficacy and reliability [2]. Furthermore, to our knowledge, neuromodulation techniques can yet not restore the sensory perception of bladder fullness. 
Intraneural electrodes instead are inserted through the nerve enhancing electrode stability and providing a highly selective stimulation of nerves [3]. Intraneural stimulation has been proven to restore touch sensations in upper-limb amputees. 
Therefore, the selective stimulation of pudendal afferents could restore bladder fullness perception while the stimulation of efferents could restore the micturition process control. To investigate this novel approach to pudendal neuromodulation based on the use of intraneural electrodes, here we present a first explorative study on pig pudendal nerve surgical access and stimulation.

All the experimental procedures, performed under general anesthesia, ended with animal suppression. 
Two surgical procedures to expose the pudendal nerve were tested in five pigs. Two animals were subjected to abdominal surgery while a transgluteal approach was performed on the others. 
Four samples of pudendal nerves were explanted proximally to the branching point from two animals to perform a histological analysis. Samples were fixed in 4% paraformaldehyde (PFA) in 0.1M phosphate buffer saline (PBS), dehydrated, and embedded in paraffin. 13 sections of 4µm were cut by a microtome (Reichert-Jung, DE) and stained with Hematoxylin and Eosin (H&E). The nerve dimension and fascicles distribution were estimated. 
The pudendal nerve was bilaterally exposed in four animals using a transgluteal surgery and implanted with a 1mm inner diameter cuff electrode (Microprobes, US). Electromyographic (EMG) microneedles electrodes were positioned on the external anal sphincter (EAS) and the external urethral sphincter (EUS). The pudendal nerves were stimulated with a biphasic square current pulse with incremental amplitude from 10μA to 1000μA, 200μs pulse width, and 3Hz frequency. The EMG signal of the sphincters was recorded with a sampling frequency of 750Hz, amplified (×1000) and bandpass filtered at 10–250Hz. For every stimulus, three EMG maximum peak responses were averaged. The experimental setup is shown in Figure 1.

The pudendal nerves were successfully exposed using both surgical approaches. 
The EAS contraction visually observed during the stimulation confirmed the identification of the pudendal nerve. 
The histological analysis (Figure 2) allowed to estimate a nerve mean diameter of (947±28)μm and fascicles mean diameter of (166±67)μm. 
The EMG signal confirmed the activation of EAS and EUS during the pudendal nerve stimulation. The current threshold that induced the contraction of the sphincters was 200μA, resulting in a 40nC charge. The EMG signal amplitude increased with the current pulse up to a plateau value of 50mV.

Both the surgical approaches showed to be reliable for pudendal nerve exposure. However, the abdominal approach may cause damage to the visceral organs, making difficult the clinical translation. The transgluteal approach preserved the abdomen integrity, resulting in less invasiveness. 
The histology allowed to calculate the optimal dimension of the cuff electrode used during this study. This information will allow to develop an intraneural electrode customized for the pudendal nerve. 
The cuff electrode showed to successfully stimulate the pudendal nerve but it still does not prove the selectivity in afferent and efferent fibers recruitment. Therefore future experiments will involve the use of intraneural electrodes. 
The current limitation of this study is the lack of long-term evaluations, proving the efficacy and suitability of the system for clinical applications.

The less invasive surgical procedure for the pudendal nerve stimulation in pigs was defined. 
The contraction of the EAS and EUS sphincters confirmed the identification of the pudendal nerve. The threshold to activate the muscles was determined. 
The nerve features were estimated and the implanted electrode dimensions were established. 
Future steps will involve the use of intraneural electrodes for the selective activation of the sphincters and the sensorial pathways using a biomimetic stimulation pattern. Cortical activity recordings will confirm the restoration of bladder fullness sensory feedback.

Hassouna T., Banakhar M., Hassouna M., Pudendal Nerve Stimulation to Treat Lower Urinary Tract Dysfunction, Nerves and Nerve Injuries, Chapter 27, 2015
Peters K., Feber K., Bennett R., Sacral Versus Pudendal Nerve Stimulation for Voiding Dysfunction: A Prospective, Single-Blinded, Randomized, Crossover Trial, Neurourology and Urodynamics, 2005
Larson C., Meng E., A review for the peripheral nerve interface designer, Journal of Neuroscience Methods, 2020