Feasibility of endovascular stimulation of the femoral nerve using a stent-mounted electrode array.

OBJECTIVE: Electrical stimulation of peripheral nerves has long been a treatment option to restore impaired neural functions that cannot be restored by conventional pharmacological therapies. Endovascular neurostimulation with stent-mounted electrode arrays is a promising and less invasive alternative to traditional implanted electrodes, which typically require invasive implantation surgery. In this study, we investigated the feasibility of endovascular stimulation of the femoral nerve using a stent-mounted electrode array and compared its performance to that of a commercially available pacing catheter. Approach: In acute animal experiments, a quadripolar pacing catheter was implanted unilaterally in the femoral artery to stimulate the femoral nerve in a bipolar configuration. Electromyogram (EMG) of the quadriceps and electroneurogram (ENG) of a distal branch of the femoral nerve were recorded. After retrieval of the pacing catheter, a bipolar stent-mounted electrode array was implanted in the same artery and the recording sessions were repeated. Main Results: Stimulation of the femoral nerve was feasible with the stent-electrode array. Although the threshold stimulus intensities required with the stent-mounted electrode array (at 100-500µs increasing pulse width, 2.17 ± 0.87 mA - 1.00 ± 0.11 mA) were more than two times higher than the pacing catheter electrodes (1.05 ± 0.48 mA - 0.57 ± 0.28mA), we demonstrated that, by reducing the stimulus pulse width to 100 µs, the threshold charge per phase and charge density can be reduced to 0.22 ± 0.09 µC and 24.62 ± 9.81 µC/cm2, which were below the tissue-damaging limit, as defined by the Shannon criteria. Significance: The present study is the first to report in vivo feasibility and efficiency of peripheral nerve stimulation using an endovascular stent-mounted electrode array. .

Distribution of smooth muscle actin and collagen in superficial peritoneal endometriotic lesions varies from the surrounding microenvironment.

RESEARCH QUESTION: Do different subtypes of superficial peritoneal endometriotic lesions exist, based on the presence and morphology of smooth muscle, collagen fibres and immune cell populations? DESIGN: A retrospective cohort study of 24 patients, from across the menstrual cycle, with surgically and histologically confirmed endometriosis. Immunofluorescence was used to delineate the CD10 stromal area of lesions (n = 271 lesions from 67 endometriotic biopsies), and then smooth muscle actin (SMA) positive tissue and immune cell populations (CD45+ and CD68+) were quantified within and adjacent to these lesions. Second harmonic generation microscopy was used to evaluate the presence and morphology of type-1 collagen fibres within and surrounding lesions. RESULTS: Overall, immune cell numbers and the area of SMA and collagen within endometriotic lesions tended to be low, but a spectrum of presentations significantly varied, particularly in the adjacent tissue microenvironment, based on lesion locations, the morphology of endometriotic gland profiles, or both. Lesions in which collagen fibres formed well aligned capsules around the CD10+ stromal border were identified compared with lesions in which collagen fibre distribution was random. Considerable inter- and intra-patient variability in the morphology of SMA and collagen was observed within and surrounding lesions. CONCLUSION: These data demonstrate considerable diversity in the presence of immune cells and morphology of SMA and collagen within, but even more so, surrounding endometriotic lesions, even within individual patients. This heterogeneity, especially within individual patients, presents a challenge to incorporating these cell and tissue types into any new endometriosis classification systems or prognostic approaches.

An adeno-associated viral labeling approach to visualize the meso- and microanatomy of mechanosensory afferents and autonomic innervation of the rat urinary bladder.

The urinary bladder is supplied by a rich network of sensory and autonomic axons, commonly visualized by immunolabeling for neural markers. This approach demonstrates overall network patterning but is less suited to understanding the structure of individual motor and sensory terminals within these complex plexuses. There is a further limitation visualizing the lightly myelinated (A-delta) class of sensory axons that provides the primary mechanosensory drive for initiation of voiding. Whereas most unmyelinated sensory axons can be revealed by immunolabeling for specific neuropeptides, to date no unique neural marker has been identified to immunohistochemically label myelinated visceral afferents. We aimed to establish a non-surgical method to visualize and map myelinated afferents in the bladder in rats. We found that in rats, the adeno-associated virus (AAV), AAV-PHP.S, which shows a high tropism for the peripheral nervous system, primarily transduced myelinated dorsal root ganglion neurons, enabling us to identify the structure and regional distribution of myelinated (mechanosensory) axon endings within the muscle and lamina propria of the bladder. We further identified the projection of myelinated afferents within the pelvic nerve and lumbosacral spinal cord. A minority of noradrenergic and cholinergic neurons in pelvic ganglia were transduced, enabling visualization and regional mapping of both autonomic and sensory axon endings within the bladder. Our study identified a sparse labeling approach for investigating myelinated sensory and autonomic axon endings within the bladder and provides new insights into the nerve-bladder interface.

Extensive heterogeneity in the expression of steroid receptors in superficial peritoneal endometriotic lesions.

RESEARCH QUESTION: Is the expression of steroid hormone receptors (oestrogen receptor-α and progesterone receptor A/B) and proliferative markers (Bcl-2 and Ki67) uniform among superficial peritoneal endometriotic lesions? DESIGN: A retrospective cohort study of 24 patients with surgically and histologically confirmed endometriosis. Immunofluorescence was used to determine the proportion of oestrogen receptor-α (ERα), progesterone receptor A/B, Bcl-2 and Ki67 positive cells in 271 endometriotic lesions (defined as endometriotic gland profile/s within an individual region of CD10 stromal immunostaining from a single biopsy) from 67 endometriotic biopsies from 24 patients. Data were analysed to examine associations related to menstrual cycle stage, lesion location and gland morphology. RESULTS: Oestrogen receptor-α and progesterone receptor A/B expression in superficial peritoneal endometriotic lesions was extremely heterogeneous. Bcl-2 immunostaining in endometriotic lesions was also variable, whereas Ki67 immunostaining was minimal. Menstrual cycle stage associations were limited in steroid hormone receptor and Bcl-2 expression in lesions. Patterns in progesterone receptor A/B and Bcl-2 immunostaining were associated with lesion location. Bcl-2 was differentially expressed, based on lesion gland morphology. CONCLUSIONS: These data demonstrate considerable diversity in the expression of steroid hormone receptors and Bcl-2 between lesions, even within an individual patient.

Dysfunction of the aging female mouse urethra is associated with striated muscle loss and increased fibrosis: an initial report.

The decline of urethral function with advancing age plays a major role in urinary incontinence in women, impairing quality of life and economically burdening the health care system. However, none of the current urinary incontinence treatments address the declining urethral function with aging, and the mechanisms by which aging impacts urethra physiology remain little known or explored. Here, we have compared functional, morphometric, and global gene expression of urethral tissues between young and old female mice. Bladder leak point pressure (LPP) measurement showed that the aged female mice had 26.55% lower LPP compared to younger mice. Vectorized Scale-Invariant Pattern Recognition (VIPR) analysis of the relative abundance of different tissue components revealed that the mid-urethra of old female mice contains less striated muscle, more extracellular matrix/fibrosis, and diminished elastin fibers ratio compared to young mice. Gene expression profiling analysis (bulk RNA-seq of the whole urethra) showed more down-regulated genes in aged than young mice. Immune response and muscle-related (striated and smooth) pathways were predominantly enriched. In contrast, keratinization, skin development, and cell differentiation pathways were significantly downregulated in aged urethral tissues compared to those from young female mice. These results suggest that molecular pathways (i.e., ACVR1/FST signaling and CTGF/TGF-ß signaling) leading to a decreased striated muscle mass and an increase in fibrous extracellular matrix in the process of aging deserve further investigation for their roles in the declined urethral function.

Selective recording of physiologically evoked neural activity in a mixed autonomic nerve using a minimally invasive array.

Real-time closed-loop control of neuromodulation devices requires long-term monitoring of neural activity in the peripheral nervous system. Although many signal extraction methods exist, few are both clinically viable and designed for extracting small signals from fragile peripheral visceral nerves. Here, we report that our minimally invasive recording and analysis technology extracts low to negative signal to noise ratio (SNR) neural activity from a visceral nerve with a high degree of specificity for fiber type and class. Complex activity was recorded from the rat pelvic nerve that was physiologically evoked during controlled bladder filling and voiding, in an extensively characterized in vivo model that provided an excellent test bed to validate our technology. Urethane-anesthetized male rats (n = 12) were implanted with a four-electrode planar array and the bladder instrumented for continuous-flow cystometry, which measures urodynamic function by recording bladder pressure changes during constant infusion of saline. We demonstrated that differential bipolar recordings and cross-correlation analyses extracts afferent and efferent activity, and discriminated between subpopulations of fibers based on conduction velocity. Integrated Aδ afferent fiber activity correlated with bladder pressure during voiding (r2: 0.66 ± 0.06) and was not affected by activating nociceptive afferents with intravesical capsaicin (r2: 0.59 ± 0.14, P = 0.54, and n = 3). Collectively, these results demonstrate our minimally invasive recording and analysis technology is selective in extracting mixed neural activity with low/negative SNR. Furthermore, integrated afferent activity reliably correlates with bladder pressure and is a promising first step in developing closed-loop technology for bladder control.

A novel statistical methodology for quantifying the spatial arrangements of axons in peripheral nerves.

A thorough understanding of the neuroanatomy of peripheral nerves is required for a better insight into their function and the development of neuromodulation tools and strategies. In biophysical modeling, it is commonly assumed that the complex spatial arrangement of myelinated and unmyelinated axons in peripheral nerves is random, however, in reality the axonal organization is inhomogeneous and anisotropic. Present quantitative neuroanatomy methods analyze peripheral nerves in terms of the number of axons and the morphometric characteristics of the axons, such as area and diameter. In this study, we employed spatial statistics and point process models to describe the spatial arrangement of axons and Sinkhorn distances to compute the similarities between these arrangements (in terms of first- and second-order statistics) in various vagus and pelvic nerve cross-sections. We utilized high-resolution transmission electron microscopy (TEM) images that have been segmented using a custom-built high-throughput deep learning system based on a highly modified U-Net architecture. Our findings show a novel and innovative approach to quantifying similarities between spatial point patterns using metrics derived from the solution to the optimal transport problem. We also present a generalizable pipeline for quantitative analysis of peripheral nerve architecture. Our data demonstrate differences between male- and female-originating samples and similarities between the pelvic and abdominal vagus nerves.

Computational modeling of endovascular peripheral nerve stimulation using a stent-mounted electrode array.

Objective.Endovascular neuromodulation has attracted substantial interest in recent years as a minimally invasive approach to treat neurological disorders. In this study, we investigated with a computational model the feasibility of stimulating peripheral nerves with an endovascular stent-mounted electrode array.Approach.Anatomically realistic FEM models were constructed for the pudendal and vagal neurovascular bundles. The electromagnetic fields generated from electrical stimuli were computed using Sim4Life NEURON models to predict dynamic axonal responses.Main results.The models predict that the stimulation thresholds of the endovascular stent-electrode array configurations tested are comparable to that of ring electrodes and are dependent on the inter-electrode distance and orientation of the device. Arranging multiple electrodes along the longitudinal axis of the nerve lowers surface charge density without sacrificing axon recruitment, whereas arranging electrodes along the circumference of the blood vessel reduces the risk of misalignment but lowers axon recruitment.Significance.Overall, this study predicts that the endovascular stent-electrode array is a feasible stimulation option for peripheral nerves, and the electrode array can be flexibly optimized to achieve the lowest stimulation threshold.

Engineered AAVs for non-invasive gene delivery to rodent and non-human primate nervous systems.

Gene therapy offers great promise in addressing neuropathologies associated with the central and peripheral nervous systems (CNS and PNS). However, genetic access remains difficult, reflecting the critical need for the development of effective and non-invasive gene delivery vectors across species. To that end, we evolved adeno-associated virus serotype 9 (AAV9) capsid in mice and validated two capsids, AAV-MaCPNS1 and AAV-MaCPNS2, across rodent species (mice and rats) and non-human primate (NHP) species (marmosets and rhesus macaques). Intravenous administration of either AAV efficiently transduced the PNS in rodents and both the PNS and CNS in NHPs. Furthermore, we used AAV-MaCPNS1 in mice to systemically deliver the following: (1) the neuronal sensor jGCaMP8s to record calcium signal dynamics in nodose ganglia and (2) the neuronal actuator DREADD to dorsal root ganglia to mediate pain. This conclusively demonstrates the translatability of these two systemic AAVs across four species and their functional utility through proof-of-concept studies in mice.

High-throughput segmentation of unmyelinated axons by deep learning.

Axonal characterizations of connectomes in healthy and disease phenotypes are surprisingly incomplete and biased because unmyelinated axons, the most prevalent type of fibers in the nervous system, have largely been ignored as their quantitative assessment quickly becomes unmanageable as the number of axons increases. Herein, we introduce the first prototype of a high-throughput processing pipeline for automated segmentation of unmyelinated fibers. Our team has used transmission electron microscopy images of vagus and pelvic nerves in rats. All unmyelinated axons in these images are individually annotated and used as labeled data to train and validate a deep instance segmentation network. We investigate the effect of different training strategies on the overall segmentation accuracy of the network. We extensively validate the segmentation algorithm as a stand-alone segmentation tool as well as in an expert-in-the-loop hybrid segmentation setting with preliminary, albeit remarkably encouraging results. Our algorithm achieves an instance-level [Formula: see text] score of between 0.7 and 0.9 on various test images in the stand-alone mode and reduces expert annotation labor by 80% in the hybrid setting. We hope that this new high-throughput segmentation pipeline will enable quick and accurate characterization of unmyelinated fibers at scale and become instrumental in significantly advancing our understanding of connectomes in both the peripheral and the central nervous systems.

Computational Modeling of an Endovascular Peripheral Nerve Interface.

Implantable neuromodulation devices that interface with the peripheral nervous system are a promising approach to restore functions lost to nerve damage. Existing nerve stimulation electrodes require direct contact with the target nerve and are associated with mechanical nerve damage and fibrous tissue encapsulation. Endovascularly delivered electrode arrays may provide a less invasive solution. Using a hybrid tissue conductor-neuron model and computational simulations, this study demonstrates the feasibility of delivering electrical stimulation of a peripheral nerve from a blood vessel in the vicinity of the target and predicts that the stimulation intensity required strongly depends on nerve-vessel distance and relative orientation, which are important factors to consider when screening candidate blood vessels for electrode implantation.

Computational modelling of nerve stimulation and recording with peripheral visceral neural interfaces.

Objective.Neuromodulation of visceral nerves is being intensively studied for treating a wide range of conditions, but effective translation requires increasing the efficacy and predictability of neural interface performance. Here we use computational models of rat visceral nerve to predict how neuroanatomical variability could affect both electrical stimulation and recording with an experimental planar neural interface.Approach.We developed a hybrid computational pipeline,VisceralNerveEnsembleRecording andStimulation (ViNERS), to couple finite-element modelling of extracellular electrical fields with biophysical simulations of individual axons. Anatomical properties of fascicles and axons in rat pelvic and vagus nerves were measured or obtained from public datasets. To validate ViNERS, we simulated pelvic nerve stimulation and recording with an experimental four-electrode planar array.Main results.Axon diameters measured from pelvic nerve were used to model a population of myelinated and unmyelinated axons and simulate recordings of electrically evoked single-unit field potentials (SUFPs). Across visceral nerve fascicles of increasing size, our simulations predicted an increase in stimulation threshold and a decrease in SUFP amplitude. Simulated threshold changes were dominated by changes in perineurium thickness, which correlates with fascicle diameter. We also demonstrated that ViNERS could simulate recordings of electrically-evoked compound action potentials (ECAPs) that were qualitatively similar to pelvic nerve recording made with the array used for simulation.Significance.We introduce ViNERS as a new open-source computational tool for modelling large-scale stimulation and recording from visceral nerves. ViNERS predicts how neuroanatomical variation in rat pelvic nerve affects stimulation and recording with an experimental planar electrode array. We show ViNERS can simulate ECAPS that capture features of our recordings, but our results suggest the underlying NEURON models need to be further refined and specifically adapted to accurately simulate visceral nerve axons.

Comparing endometriotic lesions with eutopic endometrium: time to shift focus?

Endometriosis is a heterogeneous disease in terms of patient symptoms, treatment responsiveness and the presentation of endometriotic lesions. This article explores the histological features of endometriotic lesions, highlighting their sometimes underappreciated heterogeneity. We note the variability in evidence for and against the menstrual cycle responsiveness of lesions and consider the utility of drawing parallels between endometriotic lesions and eutopic endometrium. We ask whether histopathologic features beyond just the presence/absence of endometrial-like glands and/or stroma could help improve disease stratification. At the same time, we acknowledge the desire of many clinicians and patients to avoid invasive surgery thereby limiting the ability to histologically phenotype lesions. The ability to derive clinically useful histological information from endometriotic lesions, in association with patient data, would be invaluable to clinicians to help improve treatment options in such a diverse group of patients. However, in suggesting that a shift in focus may enable the development of a better patient stratification system, we recognise that our wish for a single comprehensive stratification system may be beyond reach for a disease of such diverse presentation.

A mechanism linking perinatal 2,3,7,8 tetrachlorodibenzo-p-dioxin exposure to lower urinary tract dysfunction in adulthood.

Benign prostatic hyperplasia/lower urinary tract dysfunction (LUTD) affects nearly all men. Symptoms typically present in the fifth or sixth decade and progressively worsen over the remainder of life. Here, we identify a surprising origin of this disease that traces back to the intrauterine environment of the developing male, challenging paradigms about when this disease process begins. We delivered a single dose of a widespread environmental contaminant present in the serum of most Americans [2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), 1 µg/kg], and representative of a broader class of environmental contaminants, to pregnant mice and observed an increase in the abundance of a neurotrophic factor, artemin, in the developing mouse prostate. Artemin is required for noradrenergic axon recruitment across multiple tissues, and TCDD rapidly increases prostatic noradrenergic axon density in the male fetus. The hyperinnervation persists into adulthood, when it is coupled to autonomic hyperactivity of prostatic smooth muscle and abnormal urinary function, including increased urinary frequency. We offer new evidence that prostate neuroanatomical development is malleable and that intrauterine chemical exposures can permanently reprogram prostate neuromuscular function to cause male LUTD in adulthood.

Spatiotemporal mapping of sensory and motor innervation of the embryonic and postnatal mouse urinary bladder.

The primary function of the urinary bladder is to store urine (continence) until a suitable time for voiding (micturition). These distinct processes are determined by the coordinated activation of sensory and motor components of the nervous system, which matures to enable voluntary control at the time of weaning. Our aim was to define the development and maturation of the nerve-organ interface of the mouse urinary bladder by mapping the organ and tissue distribution of major classes of autonomic (motor) and sensory axons. Innervation of the bladder was evident from E13 and progressed dorsoventrally. Increasing defasciculation of axon bundles to single axons within the muscle occurred through the prenatal period, and in several classes of axons underwent further maturation until P7. Urothelial innervation occurred more slowly than muscle innervation and showed a clear regional difference, from E18 the bladder neck having the highest density of urothelial nerves. These features of innervation were similar in males and females but varied in timing and tissue density between different axon classes. We also analysed the pelvic ganglion, the major source of motor axons that innervate the lower urinary tract and other pelvic organs. Cholinergic, nitrergic (subset of cholinergic) and noradrenergic neuronal cell bodies were present prior to visualization of these axon classes within the bladder. Examination of cholinergic structures within the pelvic ganglion indicated that connections from spinal preganglionic neurons to pelvic ganglion neurons were already present by E12, a time at which these autonomic ganglion neurons had not yet innervated the bladder. These putative preganglionic inputs increased in density prior to birth as axon terminal fields continued to expand within the bladder tissues. Our studies also revealed in numerous pelvic ganglion neurons an unexpected transient expression of calcitonin gene-related peptide, a peptide commonly used to visualise the peptidergic class of visceral sensory axons. Together, our outcomes enhance our understanding of neural regulatory elements in the lower urinary tract during development and provide a foundation for studies of plasticity and regenerative capacity in the adult system.

Sex differences in c-Fos and EGR-1/Zif268 activity maps of rat sacral spinal cord following cystometry-induced micturition.

Storage and voiding of urine from the lower urinary tract (LUT) must be timed precisely to occur in appropriate behavioral contexts. A major part of the CNS circuit that coordinates this activity is found in the lumbosacral spinal cord. Immediate early gene (IEG) activity mapping has been widely used to investigate the lumbosacral LUT-related circuit, but most reports focus on the effects of noxious stimulation in anesthetized female rats. Here we use c-Fos and EGR-1 (Zif268) activity mapping of lumbosacral spinal cord to investigate cystometry-induced micturition in awake female and male rats. In females, after cystometry c-Fos neurons in spinal cord segments L5-S2 were concentrated in the sacral parasympathetic nucleus (SPN), dorsal horn laminae II-IV, and dorsal commissural nucleus (SDCom). Comparisons of cystometry and control groups in male and female revealed sex differences. Activity mapping suggested dorsal horn laminae II-IV was activated in females but showed net inhibition in males. However, inhibition in male rats was not detected by EGR-1 activity mapping, which showed low coexpression with c-Fos. A class of catecholamine neurons in SPN and SDCom neurons were also more strongly activated by micturition in females. In both sexes, most c-Fos neurons were identified as excitatory by their absence of Pax2 expression. In conclusion, IEG mapping in awake male and female rats has extended our understanding of the functional molecular anatomy of the LUT-related circuit in spinal cord. Using this approach, we have identified sex differences that were not detected by previous studies in anesthetized rats.

Simulating bidirectional peripheral neural interfaces in EIDORS.

Bioelectronic neural interfaces that deliver adaptive therapeutic stimulation in an intelligent manner must be able to sense and stimulate activity within the same nerve. Existing minimally-invasive peripheral neural interfaces can provide a read-out of the aggregate level of activity via electrical recordings of nerve activity, but these recordings are limited in terms of their specificity. Computational simulations can provide fine-grained insight into the contributions of different neural populations to the extracellular recording, but integration of the signals from individual nerve fibers requires knowledge of spread of current in the complex (heterogenous, anisotropic) extracellular space. We have developed a model which uses the open-source EIDORS package for extracellular stimulation and recording in the pelvic nerve. The pelvic nerve is the primary source of autonomic innervation to the pelvic organs, and a prime target for electrical stimulation to treat a variety of voiding disorders. We simulated recordings of spontaneous and electrically-evoked activity using biophysical models for myelinated and unmyelinated axons. As expected, stimulus thresholds depended strongly on both fibre type and electrode-fibre distance. In conclusion, EIDORS can be used to accurately simulate extracellular recording in complex, heterogenous neural geometries.

Functional segregation within the pelvic nerve of male rats: a meso- and microscopic analysis.

The pelvic splanchnic nerves are essential for pelvic organ function and have been proposed as targets for neuromodulation. We have focused on the rodent homologue of these nerves, the pelvic nerves. Our goal was to define within the pelvic nerve the projections of organ-specific sensory axons labelled by microinjection of neural tracer (cholera toxin, subunit B) into the bladder, urethra or rectum. We also examined the location of peptidergic sensory axons within the pelvic nerves to determine whether they aggregated separately from sacral preganglionic and paravertebral sympathetic postganglionic axons travelling in the same nerve. To address these aims, microscopy was performed on the major pelvic ganglion (MPG) with attached pelvic nerves, microdissected from young adult male Sprague-Dawley rats (6-8 weeks old) and processed as whole mounts for fluorescence immunohistochemistry. The pelvic nerves were typically composed of five discrete fascicles. Each fascicle contained peptidergic sensory, cholinergic preganglionic and noradrenergic postganglionic axons. Sensory axons innervating the lower urinary tract (LUT) consistently projected in specific fascicles within the pelvic nerves, whereas sensory axons innervating the rectum projected in a complementary group of fascicles. These discrete aggregations of organ-specific sensory projections could be followed along the full length of the pelvic nerves. From the junction of the pelvic nerve with the MPG, sensory axons immunoreactive for calcitonin gene-related peptide (CGRP) showed several distinct patterns of projection: some projected directly to the cavernous nerve, others projected directly across the surface of the MPG to the accessory nerves and a third class entered the MPG, encircling specific cholinergic neurons projecting to the LUT. A subpopulation of preganglionic inputs to noradrenergic MPG neurons also showed CGRP immunoreactivity. Together, these studies reveal new molecular and structural features of the pelvic nerves and suggest functional targets of sensory nerves in the MPG. These anatomical data will facilitate the design of experimental bioengineering strategies to specifically modulate each axon class.

Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats.

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the functionally equivalent structure in humans is the inferior hypogastric plexus. The major pelvic ganglia also provide the route by which lumbar and sacral sensory axons reach the pelvic organs. These complex, mixed ganglia can prove challenging to identify and dissect for further experimental study of normal autonomic mechanisms or to establish preclinical models of disease, injury or visceral pain. Here we describe a protocol to access and visualize these ganglia and their associated nerve tracts. We provide this protocol with schematics for both male and female rats, as the ganglion size and landmarks for identification differ between sexes. The protocol describes removal of the ganglion for in vitro studies, but this method can be integrated into a surgical recovery protocol for experimental interventions (e.g., nerve crush, nerve resection) or for mapping neuronal circuits (e.g., by microinjection of neural tracers). We also demonstrate the primary structures of the ganglion and its associated nerves immediately following dissection and following immunohistochemical staining.