A SURGICAL AND FUNCTIONAL APPROACH TO THE PELVIC GROSS NEUROANATOMY OF THE FEMALE YUCATAN MINIPIG

Abstract

The Yucatan minipig is gaining widespread use in studies focused on spinal cord injury. As a large animal model, it offers unique advantages for developing novel and more effective therapies. Successful neuromodulation experiments require precise access to central and peripheral neural structures, which depends on a thorough understanding of topographical anatomy and advanced surgical techniques. The current study describes the topography of the pelvic organs in the female Yucatan minipig, as well as a surgical approach to the principal nerves and branches controlling the pelvic viscera. Eight postmortem specimens, five fixed with 4% paraformaldehyde and three non-fixed, were used to perform dissections under stereoscopy. Muscles that form the lateral and ventrolateral walls of the pelvis were identified. The pudendal nerve, formed by S2 and S3 contributions, includes an extrapelvic component formed by S1 and S2 contributions. The hypogastric nerve converged with the pelvic nerve (formed by the splanchnic branch of the S2 trunk and two anastomotic splanchnic branches from S1 and S3) at the pelvic plexus which supplies the autonomic innervation of the urinary bladder, urethra, vagina, rectum, and cavernous tissue of the clitoris. Together, the current anatomical and neuroanatomical descriptions provide a comprehensive understanding of the structural anatomy of the urogenital and colorectal regions in the female Yucatan minipig.

Graphical Abstract

1. INTRODUCTION

The nervous network involved in the sensory, motor, and autonomic functions of the urogenital and colorectal systems includes a series of nerves arising from the lower thoracic, lumbar, and sacral nerve roots (Hollabaugh et al., 2000; Martinez-Pereira, 2021; Unger et al., 2014). Central or peripheral damage of such neural structures causes dramatic changes in quality of life, including urinary and fecal incontinence, urinary retention, constipation, pelvic pain, and sexual dysfunction, among others (Benevento and Sipski, 2002; Micera et al., 2020; Podnar and Vodušek, 2015).

Management of neurogenic dysfunction of the urogenital and colorectal organs includes a series of procedures based on electrical neuromodulation. The implantation of the stimulators requires detailed knowledge of regional neuroanatomy and highly specialized surgical skills. There is also a desire to search for less invasive surgical methods (Coolen et al., 2019; Rossini et al., 2015; Shepherd et al., 2018; Weber et al., 2004).

Properly designed animal models can contribute to a better understanding of the anatomical pathways involved in the function and dysfunction of the pelvic organs and contribute to developing novel and more effective therapies since current treatments are often inadequate (Gonzalez and Grill, 2019; Keifer and Summers, 2016; Park et al., 2018; Woock et al., 2011). Most neurologic research on animals uses rodent models due to low maintenance cost and reproducibility (Keifer and Summers, 2016). Although rodent research has significantly impacted the physiological and pharmacological knowledge of the pelvic organs, as well as the mechanistic understanding and therapeutic development of the neurourology and colorectal field (Andersson et al., 2011; Mendelsohn, 2009), the opinions of clinicians and scientists about the translational value of murine pre-clinical models remains divided (Keifer and Summers, 2016; Tator et al., 2012; Weber et al., 2004). Larger animal models, such as felines and swine, are perceived to have greater translational potential than rodents due to their clinical relevance (Kwon et al., 2011; Tator et al., 2012). The Yucatan minipig offers a unique advantage due to its manageable size and docile nature, making it an ideal subject for study (Meyerholz et al., 2024; Stricker-Krongrad et al., 2016).

The anatomical and physiological similarities of the lower urinary tract (LUT) and spinal cord between humans and pigs make the Yucatan miniature pig a reliable and valid model for medical research (Andersen et al., 2020; Busscher et al., 2010; Dass et al., 2001; Femi-Akinlosotu et al., 2023; Vicencio et al., 2017; Weber-Levine et al., 2022). Thus, in recent years, the Yucatan miniature pig has gained recognition as a highly promising model for translational research, particularly in the development of novel neuromodulation-based treatments for both intact and spinal cord-injured subjects.

Successful neuromodulation experiments require precise access to central and peripheral neural structures, which depends on a thorough understanding of topographical anatomy and advanced surgical techniques. Accurate targeting and successful experimental outcomes rely on this knowledge and expertise (Guiho et al., 2018b; Keung et al., 2021; Soebadi et al., 2019). The current investigation provides a comprehensive exploration and summary of the topography and surgical approaches to the principal trunk, nerves, and branches innervating the pelvic viscera in the female Yucatan miniature pig. Through detailed gross anatomical dissections, the study identifies key anatomical landmarks, describes the spatial relationships between neural structures and surrounding tissues, and outlines practical surgical strategies to optimize access and minimize invasiveness. The findings aim to enhance the precision and reproducibility of neuromodulation studies in this large animal, contributing to advancements in translational research.

2. METHODS

2.1. Ethical statement

All animal protocols and procedures used in this study were approved by the University of Louisville (UofL) Institutional Animal Care and Use Committee (IACUC) and conducted by the Guide for the Care and Use of Laboratory Animals, 8th edition, and the Animal Welfare Act and Regulations. The UofL animal care program is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

2.2. Subjects

Eight postmortem female Yucatan minipig specimens, weighing 25-28kg, all of them with six lumbar and four sacral vertebrae confirmed by X-ray, and obtained from a swine model of T10 contusion of the spinal cord injury at UofL, were used to perform this study. Changes in the motor cortex and descending motor pathways and reorganization in spinal circuits above and below the spinal cord injury site have been reported (Chen et al., 2002; de Groat and Yoshimura, 2012; Wrigley et al., 2009). However, to our knowledge, no relevant macroscopic changes have been reported in peripheral nerves after spinal cord injury. Therefore, we consider that the macroscopic findings of this study faithfully represent the gross anatomy of the pelvis in the female Yucatan minipig. The protocol used for spinal cord injury has been previously published (Gao et al., 2019; Lee et al., 2013). All specimens were euthanized by anesthetic overdose. Five of them were perfused via trans-cardiac perfusion with 4% paraformaldehyde. One of the perfused animals was used to perform in-block acetylcholinesterase histochemistry (AchE). To visualize, preserve, and describe the vasculature related to the nerves on the pelvis, as well as to use them as surgical landmarks, the pelvis and pelvic organs of three non-perfused specimens were removed in-block and submerged in 4% paraformaldehyde for further dissection.

2.3. Dissection protocol

Post-perfusion, a surgical microscope (Seiler Instrument Inc., St. Louis, MO) was used to dissect the lumbosacral plexus, focusing on hypogastric, pelvic, and pudendal nerves and branches. The lumbosacral plexus was dissected using four approaches: abdominopelvic, dorsolateral, sagittal, and infrapelvic.

The abdominopelvic approach required the specimen to be in a dorsal recumbency. A skin incision was made from the xiphoid process to the pubic symphysis, extending bilaterally to the iliac crests. The abdominal wall and the parietal peritoneum were removed to expose the urinary bladder, the urethra, the ureter, the round, cardinal, and uterosacral ligaments, and the vaginal vascular pedicle. These organs served as surgical landmarks to identify the hypogastric and obturator nerves. Next, the visceral peritoneum was opened parallel to the external iliac vein to access the paravaginal space. Adipose and lymphatic tissues were bluntly dissected from the promontory to the arcus tendinous. The pelvic fascia and the pubic symphysis were separated. These maneuvers facilitated the dissection of the pudendal nerve branches.

For the dorsolateral approach, the dissection started with proximal to distal reflection of the lumbar paraspinal hindlimb muscles, aimed to expose the spinal nerves of the lumbosacral plexus, complete laminectomies of the last three lumbar vertebrae, and right unilateral laminectomies of S1-S2 were performed. A partial S1-S2 laminectomy was made to spare the sacral foramina as an anatomical reference. The superficial gluteal muscle was reflected from his proximal attachment on the sacrum to expose the sacrotuberous ligaments, the sacro-ischiatic head of the intrapelvic pars of the external obturator, and the pyramid muscles. The pyramid and the obturator muscles were also reflected from the superior edge of the greater sciatic foramen and the lateral edge of the sacrum to expose the origin of S1 to S4 ventral trunks laterally.

The sagittal dissection aimed to explore the sacral paravertebral space and to obtain a clear view of the ventrolateral wall of the internal pelvis. The pubic symphysis and lumbosacral vertebrae were cut through the midline with a surgical bone saw, and all the pelvic viscera were removed.

The infrapelvic approach required the specimen to be in the dorsal recumbency, with hindlimbs fully abducted. A midline skin incision was made from the pubic arch to the clitoris hood and extended bilaterally to the base of the tail. The adductor, gracilis, and semimembranosus muscles were dissected in the midline to access the urogenital diaphragmatic fascia. Next, the nerves were dissected from the subpubic angle to the targeted organs.

2.4. In-Block acetylcholinesterase histochemistry (AchE)

The pelvic viscera were harvested in one of the perfused animals, and AchE histochemistry was performed as previously described. Briefly, the tissue was washed with 0.9% Saline Solution and incubated for 5-6 h at room temperature with a solution consisting of acetylthiocholine-iodide, 0.1 M sodium-hydrogen-maleate (pH 6.0), 0.1M sodium-citrate, 30 mM copper sulphate, 5 mM potassium-ferricyanide, and 0.1% Triton X-100 and distilled water. The tissue was dehydrated with ascending alcohol concentrations and cleared with xylene. After the staining, the pelvic plexus was dissected and photographed under a stereoscope.

3. RESULTS

3.1. Pelvic organs and muscles

In the abdominopelvic approach, removal of the abdominal wall and parietal peritoneum allowed direct visualization of the urinary bladder, uterus, uterine horns, and the broad ligament (Fig. 1). The cranial region of the urethra was also visible. The urethra originated in the bladder neck, ran caudally, attached to the ventral wall of the cranial region of the vagina, passed below the pubic symphysis and merged into the vagina (Fig. 1A, B). At this level, a striated muscle surrounded the urethra and vagina (number 7 in Fig. 1 D, E).

Figure 1.

Ventral view of the main pelvic organs of thefemale Yucatan minipig. (A) Photos showing view the urinary bladder, uterus, and uterine horns. (B) abdominal cavity, the pubic bone, and pelvic and perineal urogenital organs. (C) Uterus folded caudally, allowing the view of the Douglas pouch and the rectum. (D) Extrapelvic urogenital organs after removing the fat and connective tissue, showing the pelvic diaphragmatic hiatus. (E) Pelvic bone and perineal areashowing urogenital organs after removing the pelvic diaphragm. The organs are labeled by name or numbered as follows: (1) urethra, (2) broad ligaments, (3) uterine horns, (4) pubic symphysis, (5) ischiopubic arch, (6) clitoris, (7) urethrovaginal compressor muscle, (8) suspensory ligament of the clitoris, and (9) prepuce of the clitoris.

Cranially, the peritoneum surrounded the urinary bladder and formed a double-layer structure, the lateral vesical ligament. This structure was laterally attached to the broad ligament and the tendinous arch, providing structural support to the urinary bladder. The blood vessels supplying the urinary bladder travelled through this ligament. The broad ligament was formed by a layer of peritoneum that surrounds the uterine horns and is attached to the lateral pelvic wall and the tendinous arch. Dorsal to the bladder, the uterus, cervix, uterovaginal isthmus, and uterine horns were observed (Fig. 1C). Lateral and caudally to the uterine horns, the oviducts and the ovaries were easily identifiable. Blood vessels to the uterus were supplied by arteries coming from branches of the ovarian artery, the internal iliac artery, and the uterine branch of the vaginal artery. The urinary bladder was supplied by a branch of the uterine artery and the caudal branch of the vaginal artery, which arises from the internal iliac artery. The venous drainage was similar. Retroperitoneally and bilaterally to the bladder neck, the ureters and the external iliac vessels were situated. Both structures represented a crucial surgical landmark for the dissection of the sympathetic and parasympathetic innervation of the pelvic organs. Thus, by opening the peritoneum in a sagittal direction, and medially to the external iliac vessels the inguinal ligament and the obturator nerve were found running parallel to these vascular structures (Fig. 2A). At the bladder neck level, there was a vascular bundle containing the vaginal vein and artery, which crossed the paravaginal space from the lateral pelvic wall to the vagina (Fig. 2B). This bundle was the landmark for the hypogastric nerve, the pelvic nerve, the pudendal nerve, the cavernous nerve and the pelvic plexus. The pelvic plexus is a neural structure that provides the main autonomic innervation to urogenital and colorectal organs.

Figure 2.

Pararectal window and landmarks for pelvic innervation. (A) Urogenital organs related to the primary nerves of the pelvis. (B) Zoom of the pararectal space showing primary landmarks and access to the hypogastric nerve (Hgn) and and pelvic nerve; (Pvn).

At the pelvic and perineal regions, the pubic bone, the suspensory ligament of the clitoris, the clitoris, and the perineal musculature were exposed (Fig. 1D). The clitoris was localized distal to the bulbospongiosus muscle and was covered by the prepuce (number 9 in Fig. 1D, E). The suspensory ligament of the clitoris had a “Y” shape. It was attached bilaterally to the ischiatic arch, forming a hiatal gap covered with adipose tissue connecting the intrapelvic cavity to the exterior (Fig. 1C). The muscle constrictor vestibuli could be visualized through the hiatus (Fig. 1E). The ischiocavernosus muscles originated from the ischiopubic rami, about 1cm lateral to the pubic symphysis. They were inserted distally to the midline of the bulbospongiosus muscle, which was attached to the suspensory ligament of the clitoris via a tendon.

With a dorsolateral approach, in craniocaudal order, various muscles were observed in the lateral wall of the pelvis: The intrapelvic pars of the external obturator muscle was made up of three distinguishable heads, the iliac, the sacroischiatic, and the retropubic (numbers 1-3 in Fig. 3A). The iliac head was attached to the body of the ilium; the sacroischiatic head was attached to the superior edge of the greater sciatic notch of the iliac bone, the lateral sacral crest, the sacro-ischiatic ligament and the internal face of the body of the ischium; and the retro-pubic head originates on the ventrolateral wall of the pelvis, the caudal pubic ramus, and ischium. All three heads fused caudally, came out to the pelvis through the caudal portion of the obturator foramen (covered cephalically by the external obturator muscle), and distally ended in a common tendon that attached to the greater trochanter of the femur (Fig. 3A).

Figure 3.

Pelvic muscles of female Yucatan minipig in situ. These photographs show the muscles, associated nerves, and anatomical structures of the pelvis in sagittal (A) and lateral (B) views. The numbers represent: (1) iliac, (2) sacroischiatic, and (3) retropubic heads of the intrapelvic pars of the external obturator muscle, (4) levator ani, (5) coccygeal, gluteus profundus (6) and (7) gemelli muscles. The letters represent: (a) L6–S1 trunk, (b) S2 spinal nerve, (c) intrapelvic trunk of the pudendal nerve, (d) obturator nerve, and (e) ilioinguinal nerve. Yellow arrows point to the nerves of the three heads of the intrapelvic pars of the external obturator muscle. Greater sciatic foramen (blue arrow), obturator foramen (green arrow), sacroiliac fascia (yellow star), and greater trochanter of the proximal femur (red lightning) are also indicated. The orientation labels are the following: (D) dorsal, (V) ventral, (Cau) caudal, and (Cra) cranial.

From the external wall of the pelvis, the intrapelvic pars of the external obturator muscle covered the gap between the sacral and ischium, from the greater sciatic foramen to the lesser sciatic foramen (Fig. 3B). Caudal to the intrapelvic pars of the external obturator muscle, the coccygeal muscle was observed. It originated from the coccyx and inserted on the sacroiliac ligament (number 5 in Fig. 3). Next to it was the levator ani, whose fibers originated from the internal face of the ischium and insert on the lateral coccygeal crest and the arcus tendinous levator ani (Fig. 4). The pubococcygeus muscle did not send fibers to the pelvic viscera. Caudally to the pelvic floor it was localized the external anal sphincter, which had two layers, one deep and another superficial (Fig. 4). There was a gap between the caudal edge of the levator ani muscle and the pars profunda of the external anal sphincter. The latter rounded the dorsal wall of the rectum and was attached to the skin, vagina, and the pelvic fascia. The pars superficial of the external anal sphincter was attached to the pars profunda and surrounded the anus (Fig. 4).

Figure 4.

Pelvic musculature of the female Yucatan minipig. This lateral view of the pelvic bone shows the pelvic muscles and their relationship to the main pelvic organs and fasciae. Muscles and organs are labeled as follows: (1) lateral muscle of the pelvis, (2) coccygeus muscle, (3) iliococcygeus muscle, (4) pubococcygeus muscle, (5) pars profunda of the external anal sphincter, (6) pars superficialis of the external anal sphincter, (7) constrictor vestibuli muscle, (8) sacroiliac fascia, (9) pelvic floor fascia, and (10) rectum. The orientation labels are the following: (D) dorsal, (V) ventral, (Cau) caudal, and (Cra) cranial.

The ventrolateral wall of the pelvis consisted of the perineal membrane, which was attached to the ischiopubic ramus, the lateral wall of the vagina, and the coccyx. The perineal muscles, bulbospongiosus and ischiocavernosus, were vestigial structures found in this fascia. The sacrococcygeal muscle was found medially and attached to the lateral sacral crest and the lateral coccygeal crest.

3.2. Innervation

3.2.1. Lumbosacral spinal segments, foramina, and sacral sympathetic trunk

In the dorsal and dorsolateral approach, upon conducting a laminectomy and spinal cord dissection at the lumbosacral segments, a discrepancy was found between the spinal segments (right side in Fig. 5), vertebrae (left side in Fig. 5), and nerve foramina (Figs. 5 and 6A). As a surgical reference, there is a gap between sacral vertebrae S1 and S2. By removing the ligamentum flavum on the lateral side of the intervertebral space, the S2 nerve root could consistently be identified (Fig. 6A). Dorsal to the foramina, the lumbosacral sympathetic trunks were revealed (Fig. 6B). They ran along either side of the vertebral column. After the second segment of the sacrum, it lay closer to the median sacral artery. This proximity continues as the trunks descend along the sacrum length, maintaining a close relationship with the median sacral artery. A pocket of adipose tissue is associated with each sacral ventral foramen.

Figure 5.

Spatial correspondence between vertebral and spinal segments and the spinal nerves in the female Yucatan minipig. The yellow lines mark the vertebral levels. Arrows point to the apparent origin of L6 (green), S1 (yellow), S2 (red), and S3 (blue) spinal nerves. Black lines mark the spinal segments.

Figure 6.

Source, travel, and branches of the principal lumbosacral nerves of the pelvis in the female Yucatan minipig. (A) Anatomical position of S1–S3 nerve roots relative to the sacral vertebrae and sacral dorsal foramina. (B) Components of the sympathetic trunk and adjacent anatomical structures. Green arrows show the rami communicants, yellow arrows point to the pelvic sympathetic ganglia, and red arrows point to the sacral sympathetic trunk. (C) Transgluteal access to the main nerves of the pelvis. (E) Lateral dissection of the pelvis showing the main pelvic innervation in a living specimen, highlighting the size and proportion of different pelvic nerves. (F) Terminal branches of the pudendal nerve on the extrapelvic face of the pelvic diaphragm. Labeled structures: (1) pelvic nerve, (2) S1–S2 anastomotic nerve, (3) pudendal nerve, (4) communicating branch of the sciatic nerve to the pudendal nerve, (5) sciatic nerve, (6) common trunk for perineal nerves, (7) dorsal nerve of clitoris (DNC), (8) rectalis and external anal sphincter nerve, (9) rectal nerves, (10) perineal superficial, (11) external urethral sphincter, (12) deep perineal nerve, (13) mammarious caudalis, and (14) bulbospongiosus nerve.

Each trunk extended from the lumbar region down to the coccyx, running along either side of the vertebral column. Four pairs of interconnected sympathetic ganglia were identified, positioned slightly cranial to the corresponding ventral sacral foramina. The first two ganglia were notably large and triangular, while the last two were smaller and more challenging to observe. The sympathetic ganglia were connected to the spinal nerves via the communicantes rami (see arrows in Fig. 6B), which originated from the medial angle of each ganglion and traveled obliquely in a caudal direction towards the sacral foramina. Only one small sympathetic splanchnic nerve was confirmed by dissection. It came from the S3 sympathetic ganglia and joined the pelvic plexus. The sympathetic trunk continues caudally to the coccyx. The coccygeal ganglia were not identified due to the medio-sagittal sectioning of the sacral and coccygeal vertebrae; thus, the ganglia impar and the pars coccygealis could not be confirmed.

3.2.2. Spinal nerve branching

In a dorsolateral approach, reflection of the gluteal and lumbar muscles gave access to the sacral spinal nerves. By reflecting the intrapelvic pars of the external obturator muscle from the upper edge of the greater sciatic foramen, it was possible to reach into the pelvic cavity. This surgical window allows access to the S1-S4 spinal nerves (Fig. 6C). The S1 fascicles importantly contributed to the sciatic nerve, which gave of an anastomotic branch (number 4 in Fig. 6C). Fibers coming from the S1 trunk and the anastomotic branch of S2 seem to be the primary source of the communicating branch of the sciatic to the pudendal nerve. However, since the sciatic nerve is made up of fibers coming from S1, L5-L6 trunk, the presence of fibers coming from L5-L6 segments on the communicating branch of the sciatic nerve, the pudendal nerve cannot be ruled out. The sciatic nerve emerged through the greater sciatic foramen along with the pudendal vessels and ran between the intrapelvic pars of the external obturator muscle and the gluteus medius muscle.

The spinal S2 nerve was divided into three branches; the first one, the S2 splanchnic nerve, joined the S3 splanchnic nerve to form the pelvic nerve (number 1 in Fig. 6C-D). A second one, the thickest trunk, joined the S3 anastomotic branch and formed the pudendal nerve (number 3 in Fig. 6C-D). A third branch anastomoses with the S1 nerve (Number 2 in Fig. 6C, D).

As mentioned above, there was an anastomotic branch of S3 spinal nerve to the pudendal nerve, and S3 splanchnic nerve joined S2 splanchnic nerve to form the pelvic nerve. In addition to these fibers, two other nerves emerged from S3, an accessory splanchnic nerve, which joins the paracervical ganglion, and a branch traveling in caudal direction to join a branch of S4, which innervates the coccygeus muscle, the levator ani, and the pars profunda of the external anal sphincter. The S4 spinal nerve also had a small splanchnic nerve that supplied the rectum and the vagina, as well as the cavernous nerve.

Once the pudendal nerve and the communicating branch of the sciatic nerve nerve joined outside of the pelvis, the mixed nerve branched out all terminal nerves before reentering the pelvic cavity (Fig 6 D, E). From these nerves emerge the innervation to distal regions of the urogenital organs and perineal muscles. The dorsal nerve of the clitoris was a branch of the pudendal nerve emerging from the pelvic cavity through the angle formed between the ischiocavernosus muscle and the ischiopubic ramus. At that level, branched out to form the nerves for the bulbospongiosus and ischiocavernosus muscles, the dorsal nerve of the clitoris, and the mammarius caudalis nerve (Fig. 6F).

3.2.3. Main nerves and the pelvic ganglionated plexus

Pudendal nerve

The pudendal nerve was made up of branches of S2 and S3 and a communicant branch between S1-S2 (Fig. 7). This nerve travelled parallel to the rectum and the intrapelvic pars of the external obturator muscle (branching in some cases an accessory trunk), passing through the intrapelvic pars of the external obturator muscle to exit the pelvis. Outside of the pelvis, the communicating branch of the sciatic nerve joins the pudendal nerve. In three animals, the pudendal nerve branched into a nerve that exits the pelvis by perforating the intrapelvic pars of the external obturator muscle and joining the pudendal nerve and the communicating branch of the sciatic nerve. This branch was referred to as the accessory pudendal trunk. The pudendal nerve and the communicating branch of the sciatic nerve to the pudendal nerve (as well as the accessory pudendal nerve when present) joined together before reentering the pelvis through the lesser sciatic foramen, accompanied by the internal pudendal artery and vein. After reentering the pelvis, the pudendal nerve gave rise to branches such as the rectal nerve, the perineal nerve, and a branch innervating the external anal sphincter (in some animals, the perineal nerve emerges from this nerve), a nerve to the constrictor vestibuli muscle, and a nerve to the external urethral sphincter. The latter anastomoses with the urethral nerve coming from the pelvic plexus. The sensory components of the pudendal nerve leave the pelvis laterally to the ischial arch and branch into three nerves: one innervates the skin of the pubic area, including the mammarius caudalis, and the other innervates the dorsal nerve of the clitoris. The third branch innervates the bulbospongiosus and ischiocavernosus muscles (Fig. 7).

Figure 7.

Innervation of the pelvis in situ. This photograph shows the principal nerves of the pelvis in a lateral view. The nerves are labeled as follows: (1) hypogastric nerve, (2) pelvic nerve, (3) pudendal nerve, (4) communicating branch of the sciatic nerve to the pudendal nerve, (5) common trunk of the perineal nerves, (6) external urethral sphincter nerve, (7) constrictor vestibuli nerve, (8) dorsal nerve of the clitoris, (9) deep perineal nerve, (10) external anal sphincter nerve, (11) coccygeus nerve, and (12) rectalis caudal nerve. The orientation labels are the following: (D) dorsal, (V) ventral, (Cau) caudal, and (Cra) cranial.

Obturator Nerve

The L3-L4 spinal nerves formed the obturator nerve. It ran caudally under the psoas muscle and entered the pelvic space parallel to the inguinal ligaments and the internal iliac vessels. It branched into small nerves for each of the three heads of the intrapelvic pars of the external obturator muscle, leaving the pelvis through the obturator foramen, and was subdivided into the terminal nerves outside of the pelvic cavity (Fig. 3A).

Hypogastric nerve

The hypogastric nerves originated from the caudal mesenteric plexus, which originated from the splanchnic branches of the lumbar spinal nerves. It descended bilaterally into the pelvis attached to the visceral peritoneum that surrounds the lateral wall of the vagina, branching small nerves to the rectum, the cranial region of the vagina, and the ureters, and mixed with the pelvic nerve to form the pelvic plexus (number 1 in Figs. 7, 8, 9).

Figure 8:

The pelvic ganglionated plexus in female Yucatan minipig in situ. This photograph shows the source, branches, and principal ganglia of the pelvic ganglionated plexus stained with acetylcholinesterase. The neural structures are labeled as follows: (1) hypogastric nerve, (2) pelvic nerve, (3) S3 splanchnic nerve, (4) paracervical ganglion, (5) accessory paracervical ganglion, (6) anastomotic nerve, (7) urethral sphincter nerve, (8) vaginal nerve, (9) rectal nerve, and (10) cavernous nerve, 11 vesical nerves The orientation labels are the following: (D) dorsal, (V) ventral, (Cau) caudal, and (Cra) cranial.

Figure 9.

Innervation of the pelvic organs in the female Yucatan minipig. Lateral view of the pelvic organs and nerves labeled as follows: (1) S1, (2) S2, (3) S3, (4) S4, (5) communicating branch of the sciatic nerve to the pudendal nerve, (6) pudendal nerve, (7) anastomotic branch S1–S2, (8) pelvic nerve, (9) hypogastric nerve, (10) cavernous nerve, (11) paracervical ganglion, (12) accessory paracervical ganglion, (13) dorsal nerve of clitoris (DNC), (14) bladder neck nerve, (15) external urethral sphincter nerve, (16) constrictor vestibuli muscle, (17) mammarious caudal nerve, (18) bulbospongiosus nerve, (19) DNC clitoral branch, (20) deep perineal branches, (21) nerve to the pars profunda, (22) nerve to the pars superficialis of the external anal sphincter, (23) rectal nerve, (24) coccygeus nerve, (25) rectalis caudal nerve, (26) urinary bladder nerves and (27) sacral sympathetic chain. Labeled structures: (A) urinary bladder, (B) urethra, (C) external urethral sphincter, (D) constrictor vestibuli muscle, (E) clitoris, (F) pars interna of the external anal sphincter, (G) pars superficialis of the external anal sphincter, (H) rectum, and (I) pubic symphysis. The orientation labels are the following: (D) dorsal, (V) ventral, (Cau) caudal, and (Cra) cranial.

Pelvic nerve

The pelvic nerve was formed by the splanchnic nerves S1 to S3. The pelvic nerve travels in a dorsoventral direction, perpendicular to the rectum and vagina, and merges with the hypogastric nerve to reach the pelvic plexus (Figs. 7, 8, 9).

The pelvic ganglionated plexus

Dissection of the paravaginal-pararectal space on the animal stained with AchE revealed a paired and multi-ganglionated autonomic nerve plexus, the pelvic ganglionated plexus (PGP, Fig. 8). This plexus was supplied by the hypogastric nerve, the pelvic nerve, the S3-S4 splanchnic pelvic nerves, and the sacral splanchnic nerves emerging from the sacral sympathetic trunk (Fig. 8). The PGP included multiple small ganglia, with the most noticeable being the paracervical ganglion (also known as Frankenhäuser’s ganglion). This ganglion (number 4 in Fig. 8) has a cylindrical shape with branches coming from the hypogastric nerve in the cranial region, and fibers from the pelvic nerve (number 2 in Fig. 8) and the S3 splanchnic nerve on the dorsolateral side (number 3 in Fig. 8). On the ventral edge of the PGP, there was an anastomotic nerve to the accessory paracervical ganglion and multiple branches to the urethra and the EUS (Figs. 8,9). Nerves arising from the PGP anastomoses with a branch of the pudendal nerve and innervates the EUS. From the caudal edge of the PGP emerged branches innervating the vagina, rectum, and the erectile tissue of the clitoris, the cavernous nerve (Figs. 8, 9). The cavernous nerve runs parallel to the rectum in between the vaginal and the rectal nerves. The accessory paracervical ganglion was circular, anatomically related to branches of the hypogastric and the pelvic nerves on the dorsal side. On the ventral side, there was a nerve going to the urinary bladder (number 11 in Fig. 8), and on the caudal side, branches to the urethral and vaginal nerves. The urethral, vaginal, cavernous, and rectal nerves arose from the ganglionated plexus.

The source, course and topography of the above-described pelvic innervation in the female Yucatan minipig is summarized in Figure 9.

4. DISCUSSION

Targeting the peripheral nerves of the pelvis holds significant implications in restorative neurosciences for neurogenic conditions affecting the urogenital tract, both in translational research and clinical applications. This highlights the importance of precise anatomical knowledge and innovative surgical techniques. The Yucatan Minipig has been gaining prominence as a model for studying the function and neurogenic dysfunction of the lower urinary tract and for new therapies based on central and peripheral neuromodulation. For it, it is crucial to know the porcine pelvic urogenital organ topography and its innervation. The existing literature on porcine pelvic nerve anatomy primarily relies on removal of surrounding organs and tissues to expose the gross anatomical features of the nerves and other structures of interest, which do not provide a realistic representation of the anatomy within a practical surgical context (Bosa, 1965; Gandhi, 1966; Goshal and Getty, 1969; Kauffman and St Clair, 1965; Martinez-Pereira, 2021; Popesko, 1977; Sack et al., 1982; Tsaknakis, 1971). Moreover, studies using retrograde labeling methods often omit descriptions of the peripheral nerves (Botti et al., 2009; Kozłowska et al., 2018a, 2018b; Panu et al., 2001; Pidsudko, 2014; Ragionieri et al., 2013), and the reports about neurophysiological experiments involving electrical neuromodulation frequently lack detailed descriptions of the surgical approaches and the regional anatomy (Andersen et al., 2006; Dalmose et al., 2002; Guiho et al., 2018a; Guiho et al., 2019; Guiho et al., 2018b; Jezernik and Sinkjaer, 1998; Jezernik et al., 1997; Kneist et al., 2010; Møller et al., 2008; Soebadi et al., 2019; Wang et al., 2019). Furthermore, studies focusing on surgical approaches in the pelvic region of pigs are limited in scope, often concentrating on a single nerve, and frequently, the nomenclature provided does not align with established anatomical references for pigs (Foditsch et al., 2014; Giannotti et al., 2021; Wen et al., 1999). These limitations may hinder accurate access to the neural structures targeted during neuromodulation and reduce the reproducibility of the experiments. This study presents an integrative and comprehensive information on the pelvic neuroanatomy of female Yucatan minipigs, emphasizing surgical access to the primary neural structures that innervate the pelvic organs. It details the origins, pathways, targets, and anatomical landmarks of these nerves, providing valuable insights for surgical procedures and anatomical understanding. The photos and schematics showing all the structures will be a reference resource for researchers interested in studying the urogenital system in swine models.

Regarding our findings in surgical approaches, a previous study described a laparoscopic approach to dissect and cuff the pudendal nerve (Foditsch et al., 2014). Although their results match our findings in the abdominal approach dissections, they did not explore the plausibility of reaching the hypogastric, pelvic, and cavernous nerves using the same approach. It is possible since we have demonstrated that all these structures are located in the same area. We have referred to landmarks to identify these nerves, complementing and broadening the horizon in the laparoscopic implantation of neuroprosthesis in subjects with neurourologic conditions (Possover, 2022).

Concerning the dorsolateral approach, the study of Wen and collaborators reporting the dorsal surgical approach to access the pelvic and pudendal nerves in pigs is one of the most referenced studies in the field (Wen et al., 1999). However, this study did not provide landmarks to reach the nerves or a topographical description to recognize each spinal nerve of the sacral trunks specifically, which can be an essential factor for researchers with little experience in neurosurgery. In addition, the description of the pudendal nerve during the subgluteal dissection did not differentiate between the pudendal nerve arising from sacral spinal nerves or the communicating branch of the sciatic nerve to the pudendal nerve, leaving a space for misinterpretation and mistakes. The study performed by Gianotti et al. (2021) also named the exposed nerve generically as the pudendal nerve. Based on the description and figures presented in our study, we interpreted this nerve as the communicating branch of the sciatic nerve to the pudendal nerve. The anatomical references described in this study (sacral intervertebral spaces as landmarks to differentiate S1 from S2 spinal nerves, anatomy of the pelvic and pudendal nerves and the communicating branch of the sciatic nerve to the pudendal nerve, as well as the greater sciatic foramen and the sciatic nerve itself) allows to consider the greater sciatic foramen as a potential site to perform a laparoscopic approach to target those nerves (Knudsen et al., 2015).

Regarding the infrapubic approach, to the best of our knowledge, this is the first study describing the regional neuroanatomy of the perineal area in the female pig. The results obtained represent a valuable tool for further investigation of the neuromodulation of the dorsal nerve of the clitoris, which is one of the targets for the treatment of urological and sexual neurogenic dysfunctions (Bottorff et al., 2024; Martens et al., 2011; Possover, 2023; Qvist et al., 2023; Sakr et al., 2023). Additionally, this approach can be used to permanently catheterize the urethra for research purposes (Taylor and Smeak, 2021).

Regarding nerve organization, our findings corroborated previous anatomical descriptions of the lumbosacral innervation in different breeds of domestic pigs and further information about the autonomic innervation arising from the PGP and the surgical approaches. The fact that there were not many discrepancies in the peripheral nerve distribution and targets demonstrated that the anatomical configuration of the lumbosacral plexus remains consistent in Sus scrofa species and suggests that experimental outcomes obtained from pigs can be extrapolated, regardless of breed. However, some differences exist between our findings and other studies (Tsaknakis,1971), for example, about the origin of the pelvic and pudendal nerves, Tsaknakis described S3 as the primary source, while we consistently identified S2 as the main trunk for these nerves, with contribution of the precede and following spinal nerves. In line with our description, these peripheral nerves have been described to originate from more than one spinal nerve in other species, including humans (Cruz et al., 2017; Cruz et al., 2004; Pastelín et al., 2012).

It is interesting to note that the cavernous nerve was not mentioned in Tsaknakis' description (1971). Although a nerve emerging from the pelvic plexus running caudally to the middle part of the retractor penis, presumably the cavernous nerve, was previously described (Bosa, 1965), a specific nomenclature for this nerve was not assigned. In our study, a nerve emerging from the PGP that ran in caudal direction was recognized as the cavernous nerve and was consistently identified in all specimens dissected in the current investigation. Whether this nerve innervates the cavernous corpus of the clitoris or the rectum requires further studies, as well as its potential as target for modulation therapies.

Even if the Veterinary Anatomical Nomenclature summarizes the consensus regarding the nomenclature to be used in veterinary sciences to ensure clarity, accuracy, and comparability across research efforts in neuroanatomy, there are multiple examples of different terms used to name the same structure between studies. For example, the pudendal nerve is described consistently as formed by two different spinal trunks; these trunks are named upper and lower cords of the pudendal nerve in humans (Shafik et al., 1995), the motor and sensory branches of the pudendal nerve in rats and rabbit (Cruz et al., 2004, 2014 & 2017; Pastelín et al., 2012), and the anastomotic branch of the sciatic nerve for the pudendal nerve (Tsaknakis, 1971) or the communicating branch from nerve ischiadicus to nerve pudendus (Bosa, 1965). This discrepancy underscores a scientific and physiological issue beyond mere terminology controversy. Both components of the nerve (lower lumbar travelling through the sciatic nerve and sacral) appear to have been targeted for neuromodulation purposes in pigs, with the term 'pudendal nerve' being used interchangeably (Foditsch et al., 2014; Giannotti et al., 2023). Cruz et al., 2014) (Cruz et al., 2017). ). However, it is known that in rats, the pudendal nerve components are the motor and sensory branches arising from the L6- S1 trunk (Cruz et al., 2004; Pastelín et al., 2012), and it is recognized that the motor components also originate from rostral segments, travelling through the lumbosacral trunk, together with the sciatic nerve fibers, while in rabbits, 50% of the animals the pudendal nerve arises from S2, with components of S3 and S1 (Pastelín et al. 2012; Cruz et al. 2017; Arellano et al. 2019). Additionally, the existence and probable role of the communicating branch of the sciatic nerve to the pudendal nerve has not been discussed. Unpublished results of our lab showing strong anal contractions in response to the stimulation of the communicating branch of the sciatic nerve to the pudendal nerve and the anal contraction due to the stimulation of the L6-S1 spinal cord segment (Guiho et al., 2018b) (sacral segments) and the lack of a clear anal contractile response to the direct stimulation of the pudendal nerve (Andersen et al., 2006), suggest the possibility of a functional difference between the two components of the pudendal nerve, making it mandatory to perform electrophysiological studies to investigate this possibility further. Regarding the nomenclature, we decided to use the term “the communicating branch of the sciatic nerve to the pudendal nerve” to refer to the extrapelvic component of the pudendal nerve, using the suggestion of the Veterinary Anatomical Nomenclature regarding this kind of nerves.

With respect to the pelvic ganglionated plexus (PGP), we observed multiple ganglia scattered throughout the plexus and on the serosal surface of the pelvic organs, similar to the configurations found in humans, rabbits, dogs, and cats (Andersen et al., 2020; Dail, 1996; Landa-García et al., 2024; Li and Masuko, 2001; Woźniak and Skowrońska, 1967). However, it is important to notice in our results that one ganglion was much larger than the others, similar to that described in rats and guinea pigs, species with a single distinct macroscopic ganglion and several small accessory ganglia (Arellano et al., 2019; Mirto-Aguilar et al., 2021; Mirto-Aguilar et al., 2019).

It has been reported in pig that the anterior pelvic ganglion -component of the pelvic plexus- innervates the bladder, and that its neurons contain both adrenergic and cholinergic neurons (tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP), substance P (SP), choline acetyltransferase (ChAT) and vesicular acetylcholine transporter, (VAChT) (Listowska and Pidsudko, 2021; Pidsudko, 2014). According to the position, this ganglion could be the accessory paracervical ganglion described in our study.

The preganglionic innervation of the PGP originates from thoracolumbar and sacral segments. Parasympathetic and sensory neurons from the vagina, uterine cervix, urinary bladder, and urethra are located at S2-S4 (Kozłowska et al., 2018a, 2018b; Wasowicz et al., 1998) while the sympathetic preganglionic neurons are found from T10-S4(Botti et al., 2009; Lepiarczyk et al., 2015; Panu et al., 2001; Ragionieri et al., 2013; Wasowicz et al., 1998).

In relation to the somatomotor innervation, it has been described that the motoneurons innervating the bulbospongiosus, cremaster, and external anal sphincter muscles are located dorsolateral to the central canal of the spinal cord lumbosacral segments,L2-L4 for the cremaster and S1-S3 for the others (Blok et al., 1996; Botti et al., 2006; Botti et al., 2009). Presence of sympathetic neurons innervating these muscles has also been described (Botti et al., 2006; Botti et al., 2009; Crowe and Burnstock, 1989). The anastomosis of the urethral branch of the pudendal nerve and the urethral branch of the pelvic plexus found in this study, and previously described by Bosa (1965), could explain this close relationship between the striated urogenital musculature and the sympathetic system, which raises the question about the autonomic control of the sphincters, their roll on the urogenital neurogenic dysfunction, and the potential therapeutic targets of the sympathetic system for the treatment of the sequelae of neural injuries.

Concerning the musculature covering the ventrolateral wall of the pelvis, this musculature has been named the internal obturator muscle (Bassett, 1965; Bassett, 1971; Bosa, 1965; Goshal and Getty, 1969; Kauffman and St Clair, 1965), the intrapelvic pars of the external obturator muscle (Macdonald and Kneepkens, 1995; Sack et al., 1982; Urbankova et al., 2017), or the pyramid muscle (Melgaard and Rijkhoff, 2011). In pigs, cows, goats, lambs, babirusa, and hippopotami, this muscle covers virtually the entire inner aspect of the ventral and lateral wall of the pelvic canal and passes the pelvis through the obturator foramen. In all other species, including human beings, the muscle appears to pass through the lesser sciatic foramen (Bassett, 1965; Bassett, 1971; Fisher et al., 2010; Leal et al., 2021; Urbankova et al., 2017). The uncommon anatomical disposition of this muscle could be the origin of differences in the nomenclature. Basset concludes that the pyramid muscle does not exist in the pig, and the anatomical characteristics of the muscle described in this study are potentially exclusive of the Artiodactyla (Bassett, 1965; Bassett, 1971). According to the Veterinary Anatomical Nomenclature, the differentiation of the nomenclature of this structure remains based on the source of the innervation. The term obturator muscle should be used to refer only to the muscle in Carnivora and the horse, which is innervated by a branch of the ischiadicus nerve, and which passes through the lesser sciatic foramen. While the term intrapelvic pars of the external obturator muscle is applied to the muscle in Artiodactyla because it is innervated by branches of the obturator nerve, and it passes through the obturator foramen. Since the Veterinary Anatomical Nomenclature represents the consensus of the anatomical research community, the term 'intrapelvic pars of the external obturator muscle' is the appropriate designation for this structure in pigs, reflecting its anatomical characteristics and innervation pattern.

Finally, it has been claimed that larger animal models, such as swine, are perceived to have greater translational potential than rodents due to anatomical similarities of LUT. Indeed, our study showed that the general organization of the lumbosacral spinal nerves in pigs does not significantly differ from those described in rats, for example, in both species there is a contribution of sciatic fibers to the pudendal nerve, and a major ganglion in the pelvic plexus. It is also important to notice that in female pigs the urethra ends into the vagina, instead of having an independent urinary meatus, as occur in women and rodents. Thus, it is important to consider that both animal models have advantages and disadvantages.

In conclusion, this study provides a detailed and comprehensive analysis of the pelvic neuroanatomy of female Yucatan minipigs, highlighting the importance of precise anatomical knowledge for surgical procedures and neuromodulation therapies. By addressing the limitations of previous studies and offering new insights into the origins, pathways, and targets of pelvic nerves, this research enhances our understanding of the urogenital system in swine models. The findings underscore the value of standardized terminology and further physiological studies to improve the reproducibility and accuracy of neuromodulation experiments. Ultimately, this work contributes valuable information for translational research and clinical applications to treat neurogenic urogenital dysfunctions.

Highlights.

The urethra of the Female minipig is not in direct relation to the pelvic floor

The pudendal nerve roots are surgically accessible ventrally and laterally

The hypogastric and pelvic nerves are surgically accessible ventrally

The multiganglionated inferior pelvic plexus includes two paravaginal major ganglia

The subgluteal space is a potential laparoscopic approach to pelvic innervation