Name: dissection of pelvic autonomic ganglia and associated nerves in male and female rats
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Description: Watch this Scientific Journal Video about Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats at JoVE.com

Research reported in this publication was supported by the Office of the Director, National Institutes of Health, Stimulating Peripheral Activity to Relieve Conditions (SPARC) Program, Award Number OT2OD023872. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Dr. Bertrand&#39;s fellowship in Dr. Keast&#39;s laboratory was financed by: The University Hospital of N&#238;mes, the faculty of medicine of Montpellier-N&#238;mes, The Association Fran&#231;aise de Chirurgie (AFC), The Soci&#233;t&#233; Interdisciplinaire Francophone d&#39;UroDynamique et de Pelvip&#233;rin&#233;ologie (SIFUD-PP) and the People Programme (Marie Curie Actions) of the European Union&#39;s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement No PCOFUND-GA-2013-609102, through the PRESTIGE programme coordinated by Campus France.

All procedures are to be conducted according the institutional and funding body requirements for animal experimentation. The use of animals for this dissection and the protocol for euthanasia have been approved by the Animal Ethics Committee at the University of Melbourne (Protocol number 1814639).
NOTE: The dissections illustrated here were performed on adult (~10 weeks) male and female Sprague-Dawley rats (Biomedical Sciences Animal Facility, University of Melbourne), weighing 280 g (female) and 350 g (male). Prior to these dissections, the rats were euthanized in a CO2 chamber for 4&#8722;5 min. Immediately following death, MPG were dissected. If dissecting tissue from an animal that has undergone transcardial perfusion with fixative, take precautions to protect the operator from exposure to fixative, i.e., perform dissection in fume cupboard or downdraft cabinet and wear suitable personal protective equipment. A protocol for transcardial perfusion has been published in detail12.
1. Major pelvic ganglion and adjacent nerves: access and resection in a male rat
NOTE: Figure 1 shows anatomical landmarks for MPG visualization in a male rat.
<ol>
	<li>Access to the abdominal cavity and pelvis
	<ol>
		<li>Place the rat in a supine position and access the abdomen and pelvis through a ventral midline incision, taking care to avoid contamination of the surgical field with fur.</li>
		<li>Gently move the abdominal organs to one side using forceps or cotton-tipped applicators. Note the location of the ventral lobes of the prostate gland and the urinary bladder.</li>
		<li>Move the seminal vesicle to the contralateral side.</li>
		<li>Cut the vas deferens to provide better access to the area overlying the ganglion. 
		NOTE: From this point of the dissection, the tissue must not dry out; keep the tissue moist with physiological saline (for fresh tissue dissection) or fixative (for perfusion-fixed animal). Keeping the tissue moist with saline not only benefits tissue structure but also makes the dissection easier as dry nerves are more fragile and tear more easily during handling.</li>
		<li>Identify the dorsolateral lobe of the prostate gland, on the dorsal surface of which is the location of the ganglion; this will not yet be visible.</li>
		<li>To visualize the ganglion, carefully clear away tissues near and overlying the ganglion. If necessary, use a retractor to keep the dissection field clear.</li>
		<li>Remove a nearby aggregate of adipose tissue and open the lateral fascia of the pelvis.</li>
	</ol>
	</li>
	<li>Dissection of the MPG and its associated nerves
	<ol>
		<li>Identify the following locations that provide landmarks for the next steps of the dissection: the dorsolateral lobe of the prostate gland (the ganglion is located on the surface of this lobe, slightly more caudal than the junction between seminal vesicle and prostate) and the seminal vesicles (where they converge at the midline indicates the ganglion location on the animal&#39;s rostrocaudal axis).</li>
		<li>As required from this point, carefully remove any tissue that impedes complete view of the neural structures, avoiding damage to the thin capsule of the prostate gland or major vessels.</li>
		<li>Identify the pelvic nerve by visualizing the following landmarks and features.
		<ol>
			<li>Find the internal iliac vein and its fine branch projecting towards the MPG and the bladder. This vascular branch runs parallel to and is sometimes embedded within the pelvic nerve, then traverses the ganglion.</li>
			<li>Gently place fine-tipped angled forceps under the pelvic nerve and slide the forceps along to free it from surrounding tissue. 
			NOTE: It may also be possible to isolate the pelvic nerve from the small vessel running parallel to it, but for most types of experiments this is not essential. Confirm that the structure is the pelvic nerve by viewing under high magnification to determine that the nerve contains several loosely aggregated fascicles, which are easily distinguished under the dissecting microscope and are characteristic of the pelvic nerve, as none of the other major nerves associated with the ganglion show this clear fasciculation.</li>
		</ol>
		</li>
		<li>Identify the cavernous nerve by visualizing the following landmarks and features.
		<ol>
			<li>After following the pelvic nerve to its junction with the ganglion, follow the cavernous nerve as it travels across the prostate and then caudally towards the cavernous bodies of the penis.</li>
			<li>If microscope magnification permits, note that there is a small group of delicate nerves emerging from the ganglion between the pelvic and cavernous nerves; these are the rectal nerves that travel to the lower bowel.</li>
		</ol>
		</li>
		<li>Identify the hypogastric nerve by visualizing the following landmarks and features.
		<ol>
			<li>Identify where the hypogastric nerve joins the ganglion at its cranial edge, after travelling alongside the ureter.</li>
			<li>Confirm that the hypogastric nerve is much thinner than either the pelvic or cavernous nerves and is not accompanied by large vessels.</li>
		</ol>
		</li>
		<li>Identify the MPG by visualizing the following features.
		<ol>
			<li>Visualize the ventral, dorsal and cranial edges of the ganglion, forming a triangular shape.</li>
			<li>Confirm the location of each major nerve: the pelvic nerves emerging from the ganglion&#39;s dorsal edge, the cavernous nerve at the most caudal corner of the ganglion, the hypogastric nerve from its cranial edge, and the accessory nerves emerging from the ganglion&#39;s ventral edge.</li>
		</ol>
		</li>
		<li>Identify the accessory nerves by visualizing the following landmarks and features.
		<ol>
			<li>After clearing tissue to enable visualization of the ventral edge of the ganglion, identify a cluster of nerves that project towards the urinary and reproductive tracts.</li>
			<li>If microscope magnification permits, identify one caudal group of nerves that enter between the prostate lobes and one rostral group between the seminal vesicle and the bladder.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Removal of the MPG with its associated nerves
	<ol>
		<li>Gently slide forceps between the ganglion and the underlying prostate gland, being careful not to puncture the thin capsule of the prostate. Disrupt any connections between the ganglion and the prostate.</li>
		<li>Clear any final connections with surrounding tissues for the lengths of nerves required for the experiment, then cut each nerve.</li>
		<li>Using fine forceps, move the ganglion with its nerves to the appropriate solution for the experiment and confirm that each of the main nerves are intact.</li>
	</ol>
	</li>
</ol>
2. Major pelvic ganglion and adjacent nerves: access and resection in a female rat
NOTE: Figure 2 shows anatomical landmarks for MPG visualization in a female rat.
<ol>
	<li>Access to the abdominal cavity and pelvis
	<ol>
		<li>Place the rat in a supine position and access the abdomen and pelvis through a ventral midline incision, taking care to avoid contamination of the surgical field with fur. 
		NOTE: From this point of the dissection, the tissue must not dry out; keep the tissue moist with physiological saline (for fresh tissue dissection) or fixative (for perfusion-fixed animal).</li>
		<li>Gently move the abdominal organs to one side using forceps or cotton-tipped applicators. Note the location of the uterine horn, urinary bladder and rectum.</li>
		<li>Cut the ovarian and uterine vessels and retract the uterine horn.</li>
		<li>Enter the peritoneal space and gently clear away an aggregate of adipose tissue located near the uterine cervix.</li>
	</ol>
	</li>
	<li>Dissection of the MPG and its associated nerves
	<ol>
		<li>Identify the lateral wall of the uterine cervix, just caudal to its junction with the uterine horns; this region is the primary landmark for defining the MPG location on the animal&#39;s rostrocaudal axis.</li>
		<li>As required from this point, carefully remove any tissue that impedes complete view of the neural structures, avoiding damage to major vessels.</li>
		<li>Identify the pelvic nerve by visualizing the following landmarks and features.
		<ol>
			<li>Find the internal iliac vein and its fine branch projecting towards the MPG and the bladder. This branch runs parallel to and is sometimes embedded within the pelvic nerve, then traverses the ganglion.</li>
			<li>Confirm that the structure is the pelvic nerve by viewing under high magnification to determine that the nerve contains several loosely aggregated fascicles, which are easily distinguished under the dissecting microscope and are characteristic of the pelvic nerve, as none of the other major nerves associated with the ganglion show this clear fasciculation.</li>
		</ol>
		</li>
		<li>Identify the hypogastric nerve by visualizing the following landmarks and features.
		<ol>
			<li>Identify where the hypogastric nerve joins the ganglion at its cranial edge, after travelling alongside the ureter.</li>
			<li>Confirm that the hypogastric nerve is much thinner than either the pelvic or cavernous nerves and is not accompanied by large vessels.</li>
		</ol>
		</li>
		<li>Identify the cavernous nerve by visualizing the following landmarks and features.
		<ol>
			<li>After following the pelvic nerve to its junction with the ganglion, follow the cavernous nerve as it travels caudally along the lateral wall of the cervix towards the vagina.</li>
			<li>If microscope magnification permits, note that there is a small group of delicate nerves emerging from the ganglion between the pelvic and cavernous nerves; these are the rectal nerves that travel to the lower bowel.</li>
		</ol>
		</li>
		<li>Identify the accessory nerves by visualizing the following landmarks and features. 
		NOTE: The accessory nerves are difficult to see but project from the medial aspect of the MPG. After clearing tissue to enable visualization of the ventral edge of the ganglion, identify a cluster of very delicate nerves that project towards the urinary and reproductive tracts.</li>
		<li>Identify the MPG by visualizing the following features.
		<ol>
			<li>Visualize the ventral, dorsal, and cranial edges of the ganglion, which form a triangular shape.</li>
			<li>Confirm the location of each major nerve: the pelvic nerves emerging from the ganglion&#39;s dorsal edge, the cavernous nerve at the most caudal corner of the ganglion, the hypogastric nerve from its cranial edge, and the accessory nerves emerging from the ganglion&#39;s ventral edge.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Removal of the MPG with its associated nerves
	<ol>
		<li>Gently place fine-tipped angled forceps under the pelvic nerve and slide the forceps along to free it from the underlying uterine cervix and surrounding tissue. 
		NOTE: It may also be possible to isolate the pelvic nerve from the small vessel running parallel to it, but for most types of experiments this is not essential. If demonstrating the dissection, place a suture under the pelvic nerve, to facilitate its visualization.</li>
		<li>Repeat the process for the cavernous nerve, then the hypogastric nerve, and finally the accessory nerves.</li>
		<li>Gently slide forceps between the ganglion and the underlying uterine cervix. Disrupt any connections between the ganglion and the cervix.</li>
		<li>Clear any final connections with surrounding tissues for the lengths of nerves required for the experiment, then cut each nerve.</li>
		<li>Using fine forceps, move the ganglion with its nerves to the appropriate solution for the experiment and confirm that each of the main nerves are intact.</li>
	</ol>
	</li>
</ol>
3. Confirmation of ganglion components (optional)
<ol>
	<li>After removal of the ganglion, immerse ganglion in a conventional histological fixative (e.g., 4% buffered formalin) for a minimum of 1 h, wash out fixative with 0.1 M phosphate buffer and process tissue for cryosectioning and fluorescence immunohistochemistry, as previously described13. 
	NOTE: Many high-quality antibodies that specifically recognize these three neural markers are commercially available. See Table of Materials for the reagents used for the labeling shown in Figure 3.</li>
	<li>Alternatively, process ganglia intact (wholemounts) for immunohistochemistry using a similar method as above but increasing the incubation times for the antibodies to 4 days (primary antibody) and 2 days (secondary antibody).</li>
	<li>To demonstrate a major population of sensory axons, use antibodies against calcitonin gene-related peptide (CGRP). 
	NOTE: The recommended dilution of the antibody used in this study is 1:5,000.</li>
	<li>To demonstrate noradrenergic sympathetic neurons, use antibodies against tyrosine hydroxylase (TH). 
	NOTE: The recommended dilution of the antibody used in this study is 1:5,000.</li>
	<li>To demonstrate a major population of cholinergic neurons, use antibodies against neuronal nitric oxide synthase (NOS). 
	NOTE: The recommended dilution of the antibody used in this study is 1:500.</li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+injury+of+pelvic+visceral+sensory+nerves+alters+GFR&#945;+(GDNF+family+receptor+alpha)+localization+in+sensory+and+autonomic+pathways+of+the+sacral+spinal+cord.">Peripheral injury of pelvic visceral sensory nerves alters GFR&#945; (GDNF family receptor alpha) localization in sensory and autonomic pathways of the sacral spinal cord.</a> Frontiers in Neuroanatomy. 9, 43 (2015).</li><li>Keast, J. R., Luckensmeyer, G. B., Schemann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=All+pelvic+neurons+in+male+rats+contain+immunoreactivity+for+the+synthetic+enzymes+of+either+noradrenaline+or+acetylcholine.">All pelvic neurons in male rats contain immunoreactivity for the synthetic enzymes of either noradrenaline or acetylcholine.</a> Neuroscience Letters. 196 (3), 209-212 (1995).</li><li>Keast, J. R., de Groat, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+characterization+of+pelvic+neurons+which+project+to+the+bladder,+colon,+or+penis+in+rats.">Immunohistochemical characterization of pelvic neurons which project to the bladder, colon, or penis in rats.</a> Journal of Comparative Neurology. 288 (3), 387-400 (1989).</li><li>Dail, W. G., Moll, M. A., Weber, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+vasoactive+intestinal+polypeptide+in+penile+erectile+tissue+and+in+the+major+pelvic+ganglion+of+the+rat.">Localization of vasoactive intestinal polypeptide in penile erectile tissue and in the major pelvic ganglion of the rat.</a> Neuroscience. 10 (4), 1379-1386 (1983).</li><li>Keast, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+possible+neural+source+of+nitric+oxide+in+the+rat+penis.">A possible neural source of nitric oxide in the rat penis.</a> Neuroscience Letters. 143 (1-2), 69-73 (1992).</li><li>Kepper, M. E., Keast, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmitter+profile+and+spinal+inputs+of+pelvic+ganglion+cells+projecting+with+preganglionic+axons+along+the+hypogastric+and+pelvic+nerves+of+the+male+rat.">Transmitter profile and spinal inputs of pelvic ganglion cells projecting with preganglionic axons along the hypogastric and pelvic nerves of the male rat.</a> Neuroscience Letters. 280 (2), 123-126 (2000).</li></ol>

The authors have nothing to disclose.

Neural control of the pelvic organs is mediated by complex pathways including somatic, parasympathetic, sympathetic and visceral sensory components14,15,16,17. Most of these pathways originate in or pass through the MPG. The dissection protocols outlined here provide an introduction to MPG anatomy, the related associated nerves and nearby macroscopic anatomical landmarks; the latter are illustrated by anatomical schematics. Other approaches to MPG dissection may also be successful, but we find the one described here to be robust and suitable for a researcher new to this area of the nervous system.
The most critical aspects of the protocol are correct identification of each major nerve and the complete removal of MPG tissue. With careful viewing and handling of the tissues, MPG tissues can be removed for anatomical, molecular and electrophysiological in vitro studies18,19,20,21,22. The protocol can also be adapted for in vivo experimental manipulation23,24,25, noting that in this case, great care must be taken to minimise contact with the primary nerves associated with the ganglion or to damage nearby vasculature. If the experiment requires selective denervation by interruption of one or more nerves, it is recommended to ligate the severed nerve to prevent reinnervation and confounding of analyses. This dissection protocol could also be utilized for the mouse, where there is also an MPG with comparable function26,27,28.
For neuroanatomical studies, the best preservation of antigens and tissue structure is obtained by dissecting the MPG from an anesthetized animal that has been perfused transcardially with histological fixative appropriate to the experiment29; however, identification of the ganglion and nerve structures are more difficult after this process, as the tissue coloration is lost. It is recommended to become proficient in identifying and dissecting the ganglion from nonperfused animals before attempting this dissection after perfusion. Likewise, it is recommended to first become proficient in the dissection in males because for animals of equivalent age and body size, the MPG and its associated nerves are much smaller in females.
To validate that the tissue removed is indeed the MPG, the researcher is first advised to check the location and features of each primary nerve. Many dissectors find the pelvic nerve and cavernous nerve the easiest to identify in situ; the hypogastric and accessory nerves are more delicate and more difficult to distinguish from the surrounding tissue. If these nerves are no longer available because of problems during dissection, or if there is uncertainty regarding their structure, it is recommended that initial MPG dissections are characterized with conventional histology (to confirm presence of neuronal cell bodies8) and secondly with immunohistochemistry (to identify that both cholinergic and noradrenergic neurons are present30,31) (Figure 3). To validate correct identification of the major nerves, the cavernous nerves are readily identified by their high density of neuronal cell bodies in their initial portion close to the MPG; most of these neurons express markers of cholinergic, nitrergic neurons32,33. The pelvic, hypogastric and accessory nerves have very few neuronal cell bodies34.
There are several common pitfalls in performing this dissection. If novice dissectors have problems finding any of the major nerves or the MPG, they are encouraged to return to the steps that describe the key landmarks. It is very common to become so focused on finding the microstructures that one loses track of the macroscopic context. Most commonly, novice dissectors either move too far rostral in their dissection site or remain too &#39;superficial&#39;-i.e., too close to the ventral opening of the abdomen, rather than examining deeper (i.e., more dorsal) structures. A common problem during dissection is damage to the vasculature during dissection. If bleeding starts, gently hold a cotton-tipped applicator over the source until bleeding stops, then flush the area liberally with saline before recommencing dissection. It is possible the MPG will not be usable for experiments if contaminated with too much blood or if the dissection is delayed too long while waiting for bleeding to stop. Another common dissection error is damage to the capsule of the prostate gland which significantly impairs the MPG visualization and removal. This capsule is a very delicate structure that is easily punctured while removing the fat from the lateral wall of the prostate, even if using only a cotton-tipped applicator. Finally, the main nerves associated with the MPG are easily damaged during the process of identifying each one and then during removal of the MPG. Dissectors are encouraged to develop a routine whereby each nerve is isolated in turn, in a particular order, so that there is less opportunity for confusion during the final steps of ganglion removal.
This dissection did not seek to trace each of the components of the accessory nerves to specific organs, or to identify each of the many microganglia that lie at various points between the pelvic ganglia and the pelvic organs8. These are quite difficult to visualize in vivo without using specific stains; however, they can be removed by following each of the nerve tracts towards the organs, and utilizing specific neural stains post hoc to determine ganglion location. These microganglia, even though comprising only small fraction of the neuronal population compared to the MPG, may provide specific types of input to the organs to which they are most closely located. We note here a limitation in the field that neither these microganglia nor many of the small nerve tracts exiting the MPG to travel to pelvic organs yet have broadly accepted names. Moreover, a similarly detailed study of microganglia has not yet been conducted in female rats.
In summary, the protocol and schematics provided here provide researchers with tools to study the primary structures providing the autonomic nerve supply to the pelvic organs, as well as the major peripheral conduits of sensory nerves from lumbosacral dorsal root ganglia that travel via the MPG to pelvic organs.

The rat is the one of the best characterized species used in the study of the pelvic organ physiology and anatomy. While excellent resources exist for descriptions of these organs1,2, they generally do not provide information on the related neural structures or do so at insufficient resolution to guide an experimental study. As detailed further below, the organization of the autonomic ganglia that regulate pelvic organ function is quite different to the rest of the autonomic nervous system, making it difficult to accurately infer pelvic innervation features from neuroanatomical information available for other autonomic ganglia. This deficiency in resources to guide researchers entering this area may have slowed research on neural regulation of pelvic organs. Here we describe protocols for accessing this region of the nervous system for further in vitro studies or experimental intervention.
The bilateral major pelvic ganglia (MPG; synonyms: pelvic ganglia; paracervical ganglia [female]; Frankenh&#228;user&#39;s ganglion [female]) are the primary source of postganglionic sympathetic and parasympathetic neurons innervating pelvic organs of rodents; the inferior hypogastric plexus comprises the equivalent neuronal structure in humans3,4,5,6. Sensory projections from lumbar and sacral dorsal root ganglia also travel via the MPG to reach the pelvic organs. Therefore, understanding the neural circuitry and biology of the MPG is critical for preclinical studies on a myriad of clinical conditions relating to the development and adult function of pelvic organs. Several excellent descriptions of rodent MPG have been published7,8, but our experience is that in general these descriptions do not always provide sufficient guidance to practically inform an experimental dissection or manipulation of these structures when recovery of the animal is required. Moreover, the majority of MPG studies focus on male rats. In female rats, the MPG are smaller9 and have distinct anatomical landmarks, and therefore require a distinctly tailored guide to visualization and dissection.
Sympathetic and parasympathetic pathways are distinguished by their anatomy, specifically the location of their preganglionic neurons, with sympathetic pathways having preganglionic neurons in the thoraco-lumbar spinal cord and the parasympathetic preganglionic neurons located in the brainstem (cranial nerve projections) and sacral spinal cord. In most other regions of the autonomic system, their target ganglion neurons are located in distinct sympathetic or parasympathetic ganglia. However, the MPG are unusual in being mixed sympathetic-parasympathetic ganglia, and therefore at a macroscopic scale are sites of convergence from preganglionic axons of both thoraco-lumbar and sacral spinal regions. We have therefore included in our protocols the location and description of these primary nerve tracts that connect each spinal region with the MPG, facilitating experimental analysis or separate manipulation of these neural components. We also note for readers specifically comparing these ganglia across species, that in rodents the spinal preganglionic neurons that are &#39;functionally sacral&#39;, e.g., are active and required during micturition, defecation and penile erection, are located at spinal levels L6-S1 rather than exclusively in sacral segments10; likewise L6 and S1 dorsal root ganglia provide the major &#39;sacral&#39; sensory input to pelvic organs. In rodents, sensory and preganglionic input from more rostral neural circuits is concentrated in spinal levels L1 and L210.
Here we describe a protocol to access the MPG and their associated nerve tracts in male and female rats, and support this with schematics to illustrate specific landmarks. This protocol guides surgical access to these structures in an experimental context of removing the tissue for in vitro studies, e.g., isolating MPG neurons for molecular characterization or primary culture. It can also be adapted to MPG removal after intracardiac perfusion with fixative, although this is a more difficult dissection because the neural tissue becomes more difficult to visualize when the adjacent tissues are devoid of blood. This protocol can also be integrated into a surgical setting for experimental intervention of these nerve pathways (e.g., nerve resection, microinjection of neural tracers). These types of dissections are increasingly important for the growing field of bioelectronic medicine, where new targets and approaches for neuromodulation to treat clinical conditions of the pelvic viscera are being developed11. We present the complete protocol first for male rats, then a replicate of the protocol tailored specifically for female rats.

<table><tbody><tr><td>Anti-calcitonin gene-related peptide; RRID AB_259091</td><td>Merck</td><td>C8198</td><td></td></tr><tr><td>Anti-nitric oxide synthase, RRID AB_2533937</td><td>Invitrogen</td><td>61-7000</td><td></td></tr><tr><td>Anti-rabbit IgG, Cy3 tag, RRID AB_2307443</td><td>Jackson</td><td>711-165-152</td><td></td></tr><tr><td>Anti-tyrosine hydroxylase, RRID AB_390204</td><td>Millipore</td><td>AB152</td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus</td><td>SZ40, SC</td><td></td></tr><tr><td>Dumont AA epoxy coated forceps</td><td>Fine Science Tools</td><td>11210-10</td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Dumont #5/45 curved forceps</td><td>Fine Science Tools</td><td>11251-35</td><td></td></tr><tr><td>LED light source</td><td>Schott</td><td>KL 1600</td><td></td></tr><tr><td>Micro-Adson forceps</td><td>Fine Science Tools</td><td>11019-12</td><td></td></tr><tr><td>Student Vannas spring scissors</td><td>Fine Science Tools</td><td>91500-09</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools</td><td>14054-13</td><td></td></tr></tbody></table>

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.

A successful dissection will not only remove the complete body of the MPG intact, but also retain the first segment of each of the major nerves still attached. These nerves are valuable indicators of ganglion orientation in vivo and therefore provide essential information for many types of anatomical studies (e.g., mapping expression patterns or cellular changes after an experimental perturbation). Although preserving the associated nerves can be of less importance for some experiment types (e.g., ganglion dissociation for culture of isolated neurons), the presence of nerves also provides a way of handling the ganglion without touching (and potentially damaging) the neuronal cell bodies.
An unsuccessful dissection will have an incomplete or damaged ganglion, or where the primary nerves are no longer attached. It is also possible that ganglia or nerves are unknowingly damaged during dissection, either because the physical damage is too subtle to detect under the dissecting microscope or because the damage only becomes apparent during certain types of assays. For example, if the ganglion tissue becomes dry during dissection, the tissue may appear normal during later handling, but will show high levels of non-specific fluorescence on the surface.
Examples of dissected MPG are shown in Figure 3, which provides examples of the entire MPG visualized as a whole thickness complete ganglion (Figure 3A) and an MPG that has been cryosectioned for performing immunofluorescence to demonstrate noradrenergic and cholinergic neurons (Figure 3B,C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60904/60904fig1.jpg" /> 
Figure 1: Anatomical landmarks for MPG visualization in a male rat. 1, seminal vesicle; 2, urinary bladder; 3, coagulating gland; 4 &#38; 5, accessory nerves; 6, prostate (ventral lobe); 7, cavernous nerve; 8, vas deferens; 9, urethra; 10, bulbocavernosus muscle; 11, ischiocavernosus muscle; 12, rectal nerves; 13, abductor caudae externus; 14, major pelvic ganglion; 15, pelvic nerve; 16, abductor caudae internus; 17, hypogastric nerve; 18, internal iliac vein; 19, flexor caudae brevis; 20, flexor caudae longus; 21, ureter; 22, psoas major; 23, abdominal aorta; 24, inferior vena cava. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60904/60904fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60904/60904fig2.jpg" /> 
Figure 2: Anatomical landmarks for MPG visualization in a female rat. 1, distal colon; 2, urinary bladder; 3, uterine body; 4, hypogastric nerve; 5, accessory nerves; 6, major pelvic ganglion; 7, cavernous nerve; 8, vagina; 9, urethra; 10, rectum; 11, abductor caudae externus; 12, rectal nerves; 13, flexor caudae brevis; 14, pelvic nerve; 15, internal iliac vein; 16, abductor caudae internus; 17, flexor caudae longus; 18, external iliac artery; 19, ureter; 20, psoas major; 21, uterine horn; 22, abdominal aorta. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60904/60904fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60904/60904fig3.jpg" /> 
Figure 3: Immunohistochemically labelled MPG from adult male rats. All preparations have been visualized with a conventional widefield fluorescence microscope equipped with a monochrome camera, then digitally colorized. (A) Wholemount (complete thickness), fixed MPG with associated nerves, immunohistochemically labelled for the sensory nerves that express calcitonin gene-related peptide (CGRP); 1, pelvic nerve (showing the multiple fascicles); 2, cavernous nerve; 3, hypogastric nerve; 4, accessory nerves; 5, rectal nerves; 6, major pelvic ganglion (MPG). (B,C) Cryosections (14 &#181;m) of fixed MPG, immunohistochemically labelled to demonstrate the mixed noradrenergic-cholinergic nature of the ganglion; (B) noradrenergic neurons demonstrated by antibody for tyrosine hydroxylase and (C) a major population of cholinergic neurons by antibody for neuronal nitric oxide synthase. Calibration bar represents (A) 1,000 &#181;m, (B,C) 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60904/60904fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Dissection of Pelvic Autonomic Ganglia and Associated Nerves in Male and Female Rats at JoVE.com

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

The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.

The bilateral major pelvic ganglia (MPG; synonym, pelvic ganglia) are the primary source of postganglionic sympathetic and parasympathetic neurons ...

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Unit 1

The Autonomic Nervous System

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14.6K Views.  University of Melbourne. The major pelvic ganglia contain parasympathetic and sympathetic neurons that innervate pelvic organs. Here we describe a dissection method and provide schematics for identification of these ganglia and their associated nerves. These methods can be applied to experimental manipulation of these ganglia in vivo or removal post-mortem for further study.

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

Patch Clamp Recordings on Intact Dorsal Root Ganglia from Adult Rats

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of recordings are conducted on dissociated DRG neurons, which is a standard preparation for most laboratories. Neuronal properties, however, can be altered by axonal injury resulting from enzyme digestion used in acquiring dissociated neurons. Further, dissociated neuron preparations cannot fully represent the microenvironment of the DRG since loss of contact with satellite glial cells that surround the primary sensory neurons is an unavoidable consequence of this method. To overcome the limitations in using conventional dissociated DRG neurons for patch clamp recordings, in this report we describe a method to prepare intact DRGs and conduct patch clamp recordings on individual primary sensory neurons ex vivo. This approach permits the fast and straightforward preparation of intact DRGs, mimicking in vivo conditions by keeping DRG neurons associated with their surrounding satellite glial cells and basement membrane. Furthermore, the method avoids axonal injury from manipulation and enzyme digestion such as when dissociating DRGs. This ex vivo preparation can additionally be used to study the interaction between primary sensory neurons and satellite glial cells.

This manuscript describes how to prepare intact dorsal root ganglia for patch clamp recordings. This preparation maintains the microenvironment for neurons and satellite glial cells, thus avoiding the phenotypic and functional changes seen using dissociated DRG neurons.

Patch clamp studies from dorsal root ganglia (DRGs) neurons have increased our understanding of the peripheral nervous system. Currently, the majority of ...

Biochemistry

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Biology

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation.
We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass.
We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Nerve-sparing Mid-urethral Obstruction NeMO in Female Small Rodents

Traditional modeling of partial bladder outlet obstruction in rodents is fraught with animal mortality. A denervation injury from dissection around the proximal urethra and bladder neck is also of major concern. We developed and evaluated a safe and reliable mid-urethral obstruction model, avoiding the shortcomings of the traditional model.

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal ...

Medicine

Cavernous Nerve Stimulation and Recording of Intracavernous Pressure in a Rat

The stimulation of the cavernous nerve (CN) and measurement of intracavernous pressure (ICP) have been used extensively to test and evaluate therapies for erectile dysfunction. However, the methods used vary between laboratories, and pitfalls still exist. The goal of this study was to describe a surgical technique that would provide a reliable and reproducible model. By exposing the ischiocavernosus muscle at its point of insertion on the ischial tuberosity, the penile crus could be cannulated with minimal dissection and injury to the structures involved in erectile function. Repeated stimulation of the CN, without the need for lifting and drying, was achieved by using a 125 &#181;m bipolar silver electrode and biocompatible silicon glue to isolate the electrode-nerve complex. This method prevents neuropraxia by reducing stretching and drying the nerve and provides complete isolation of the nerve, negating electrical leakage and preventing stimulation of alternative pathways.

This study describes a simplified surgical procedure and technique for performing cavernous nerve stimulation with the isolation of the nerve-electrode complex using silicone glue and intracavernous pressure measurement.

The stimulation of the cavernous nerve (CN) and measurement of intracavernous pressure (ICP) have been used extensively to test and evaluate therapies for ...

Spinal Cord Lateral Hemisection and Asymmetric Behavioral Assessments in Adult Rats

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human Brown-S&#233;quard syndrome&#160;is a common type of incomplete SCI caused by a lesion to one half of the spinal cord which results in paralysis and loss of&#160;proprioception&#160;on the same (or ipsilesional) side as the injury, and loss of pain and temperature sensation on the opposite (or contralesional) side. Adequate methodologies for producing a spinal cord lateral hemisection (HX) and assessing neurological impairments are essential to establish a reliable animal model of Brown-S&#233;quard syndrome. Although lateral hemisection model plays a pivotal role in basic and translational research, standardized protocols for creating such a hemisection and assessing unilateralized function are lacking. The goal of this study is to describe step-by-step procedures to produce a rat spinal lateral HX at the 9th thoracic (T9) vertebral level. We, then, describe a combined behavior scale for HX (CBS-HX) that provides a simple and sensitive assessment of asymmetric neurological performance for unilateral SCI. The CBS-HX, ranging from 0 to 18, is composed of 4 individual assessments which include unilateral hindlimb stepping (UHS), coupling, contact placing, and grid walking. For CBS-HX, the ipsilateral and contralateral hindlimbs are assessed separately. We found that, after a T9 HX, the ipsilateral hindlimb showed impaired behavior function whereas the contralateral hindlimb showed substantial recovery. The CBS-HX effectively discriminated behavioral functions between ipsilateral and contralateral hindlimbs and detected temporal progression of recovery of the ipsilateral hindlimb. The CBS-HX components can be analyzed separately or in combination with other measures when needed. Although we only provided visual descriptions of the surgical procedures and behavioral assessments of a thoracic HX, the principle may be applied to other incomplete SCIs and at other levels of the injury.

Here we describe surgical procedures to produce a reliable spinal cord lateral hemisection (HX) at the 9th thoracic level in adult rats and neurobehavioral assessments designed for detecting asymmetric deficits after such a unilateral injury.

Incomplete spinal cord injury (SCI) often leads to impairments of sensorimotor functions and is clinically the most frequent type of SCI. Human ...

Detrusor Underactivity Model in Rats by Conus Medullaris Transection

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was performed in a total of 40 female Wistar rats (control group: 10 rats; test group: 30 rats) weighing 200&#8211;220 g, and the conus medullaris was transected at the L4&#8210;L5 level in the test group. All the rats were housed and fed under the same environmental conditions for six weeks. In the test group, urine voiding was performed twice daily for six weeks, and mean residual urine volume was recorded. A cystometrogram was performed in both groups. Maximum cystometric capacity (MCC), detrusor opening pressure (DOP), and compliance of the bladder were recorded and calculated. The test group showed significant urinary retention after the surgery, both during and after the spinal shock. However, no abnormality was observed in the control group. When compared to the control group, the MCC and compliance of bladder in the test group was significantly higher than that of the test group (3.24 &#177; 2.261 mL versus 1.04 &#177; 0.571 mL; 0.43 &#177; 0.578 mL/cmH2O versus 0.032 &#177; 0.016 mL/cmH2O), whereas DOP in the test group was lower than control (20.28 &#177; 14.022 cmH2O versus 35 &#177; 13.258 cmH2O). This method of establishing an animal model of DU by the conus medullaris transection offers an excellent opportunity to understand DU&#8217;s pathophysiology in a better manner.

We present a method for establishing a detrusor underactivity model by conus medullaris transection in rats. Detrusor underactivity was successfully stimulated in these animals. The model can be used for studying urinary tract function.

The goal of the presented protocol was to establish a detrusor underactivity (DU) model in the rat through conus medullaris transection. Laminectomy was ...

Whole Central and Peripheral Nervous System Mice Dissection

Animal models represent the workhorse of the neuroscience field. Despite this, today, there is still no step-by-step protocol to dissect a complete rodent nervous system, nor is there a complete schematic representing it that is freely available. Only methods to harvest the brain, the spinal cord, a specific dorsal root ganglion, and the sciatic nerve (separately) are available. Here, we provide detailed pictures and a schematic of the central and peripheral murine nervous system. More importantly, we outline a robust procedure to perform its dissection. The 30 min pre-dissection step allows isolating the intact nervous system within the vertebra with muscles free of viscera and skin. A 2-4 h dissection follows it under a micro-dissection microscope to expose the spinal cord and the thoracic nerves, and finally peel the whole central and peripheral nervous system off the carcass. This protocol represents a significant step forward in studying the anatomy and pathophysiology of the nervous system globally. For example, the dissected dorsal root ganglions from a neurofibromatosis type I mice model can be further processed for histology to unravel changes in tumor progression.

Here, we present a protocol to complete the dissection of central and peripheral nervous systems of mice. The dissected tissues are further analyzed in downstream applications, such as taking gross pictures or histology.

Animal models represent the workhorse of the neuroscience field. Despite this, today, there is still no step-by-step protocol to dissect a complete rodent ...

Targeting Mouse Sympathetic Neurons: A Lumbar Sympathectomy Approach

Surgical Lumbar Sympathectomy in Mice

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays many critical roles in maintaining bodily homeostasis, but it has rarely been studied in the context of peripheral nerve injury. The extent of postganglionic sympathetic neuronal functions in distal targets in the periphery is currently unclear. To better explore the role of sympathetic innervation of peripheral targets, a surgical &#34;knock-out&#34; model provides an alternative approach. Although this can be achieved chemically, chemical destruction of postganglionic sympathetic neurons can be nonspecific and dose-dependent. The use of a surgical lumbar sympathectomy in mice, once thought to be &#34;virtually not practicable&#34; in small animals, allows for specific targeting of postganglionic sympathetic neurons that innervate the hind limbs. This manuscript describes how to surgically remove the L2-L5 lumbar sympathetic ganglia from a mouse as a survival surgery, which reliably decreases the hind paw sweat response and the number of sympathetic axons in the sciatic nerve.

Author Spotlight: Exploring Plasticity of Sympathetic Neurons

This manuscript presents a protocol for surgically removing the postganglionic lumbar sympathetic neurons from a mouse. This procedure will facilitate a multitude of studies aimed at investigating the role of sympathetic innervation in distal tissue targets.

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays ...

Isolation and Characterization of the Murine Uterosacral Ligaments and Pelvic Floor Organs

Pelvic organ prolapse (POP) is a condition that affects the integrity, structure, and mechanical support of the pelvic floor. The organs in the pelvic floor are supported by different anatomical structures, including muscles, ligaments, and pelvic fascia. The uterosacral ligament (USL) is a critical load-bearing structure, and injury to the USL results in a higher risk of developing POP. The present protocol describes the dissection of murine USLs and the pelvic floor organs alongside the acquisition of unique data on the USL biochemical composition and function using Raman spectroscopy and the evaluation of mechanical behavior. Mice are an invaluable model for preclinical research, but dissecting the murine USL is a difficult and intricate process. This procedure presents an approach to guide the dissection of murine pelvic floor tissues, including the USL, to enable multiple assessments and characterization. This work aims to aid the dissection of pelvic floor tissues by basic scientists and engineers, thus expanding the accessibility of research on the USL and pelvic floor conditions and the preclinical study of women's health using mouse models.

This article presents a detailed protocol for dissecting uterosacral ligaments and other pelvic floor tissues, including the cervix, rectum, and bladder in mice, to expand the study of female reproductive tissues.

Pelvic organ prolapse (POP) is a condition that affects the integrity, structure, and mechanical support of the pelvic floor. The organs in the pelvic ...

Standardized 5/6 Nephrectomy in Rats for Renal Insufficiency and Malperfusion

5/6 Nephrectomy Using Sharp Bipolectomy Via Midline Laparotomy in Rats

Chronic kidney disease (CKD) affects over 10% of the global population, amounting to more than 800 million individuals worldwide. The advances in the treatment of CKD have had a significant impact on patient outcomes. While in the past, CKD was often considered a death sentence, with many patients succumbing to the complications of end-stage renal disease, it is now increasingly being managed as a chronic condition with the availability of dialysis and kidney transplantation, as well as new pharmaceutical developments such as SGLT2 inhibitors or nonsteroidal mineralocorticoid receptor antagonists. 
Yet, there continues to be a growing demand for further exploration into the pathophysiological processes and potential therapeutic interventions. Reliable biological models play a crucial role in facilitating this research. Given the multifaceted nature of renal disease, which encompasses not only cell biology but also vascular microanatomy and endocrine signaling, an appropriate model must possess a level of biological complexity that only an animal model can offer, rendering rodents an obvious choice.
This manuscript, therefore, provides an intricate, systematic protocol for surgically reducing renal parenchyma through midline laparotomy and combined total and partial nephrectomy in rats for survival as well as non-survival applications. It emphasizes the critical role of precise surgical techniques in ensuring consistent and reliable outcomes. Prime examples of potential applications for this model include biomolecular and pharmaceutical studies as well as the development of innovative intraoperative imaging modalities, such as hyperspectral imaging, to objectively visualize and differentiate renal malperfusion.

This manuscript introduces a standardized protocol for a 5/6 nephrectomy by sharp bipolectomy via midline laparotomy in a rat model, aiming to induce a state of renal insufficiency via renal parenchyma reduction with a great degree of methodical precision and low risk of technical error.

Chronic kidney disease (CKD) affects over 10% of the global population, amounting to more than 800 million individuals worldwide. The advances in the ...