Multiple Vasodilator Pathways from the Pelvic Plexus to the Penis of the Rat

Multiple vasodilator pathways from the pelvic plexus to the penis of the rat

WG Dail1*, F Harji1, J Gonzales1 and R Galindo1

1Department of Neurosciences, University of New Mexico Health Sciences Center, School of Medicine, Albuquerque, New Mexico, NM 87131-5223, USA

The main penile or cavernous nerve is usually regarded as the most important vasodilator projection in the rat. Although other descending pathways have been described, there is little detailed information on their importance. In this present report, we provide topographic and quantitative information on lateral and ventral penile branches and examine the vasodilator ®bers which join the pudendal neurovascular bundle. Seventeen Sprague-Dawley rats were used. The techniques included injection of dye in the penis to label neurons in the pelvic plexus in combination with transection of the main penile nerve (MPN). NADPH diaphorase (NADPH-d) histochemistry was used to assess the effects of transection of vasodilator pathways on innervation of the penis and for in situ staining of the pelvic plexus. Distinct clusters of penile neurons are aggregated at the origin of several nerve tracts leaving the posterior margin of the major pelvic ganglion (MPG). Multiple NADPH-d ®ber bundles coursed over the anterior surface of the prostate to reach the penis. Branches from these tracts joined the pudendal neurovascular bundle proximal to the hilum of the penis and provided innervation to the artery throughout its course in the pudendal canal. Consistent with the presence of multiple penile pathways, transection of the MPN reduced, but did not eliminate retrograde labeling of penile neurons in the MPG and only modestly decreased NADPH-d ®bers in the penis. This study con®rms that there are multiple pathways by which vasodilator ®bers reach the penis. If a similar allocation of vasodilator output is present in man, preservation of ®ner branches of the pelvic plexus would be important in surgical procedures on the prostate.

Keywords: penis; nitric oxide synthase; pelvic plexus; prostatectomy; cavernous nerve

Introduction An initial study by Langworthy15 used methylene blue stained preparations to describe a prominent postganglionic ®ber bundle which projected to the Much of the recent interest in the neural control of penis (descending branch to the penis) and rectum penile erection have focused on intriguing questions in the rat. A cluster of nerve cell somata, presumed regarding vasodilator neurotransmitters,1,2 the cen- to be penile, were said to be related to the origin of tral control of erection3±6 and mechanical factors this nerve. Purinton et al 16 con®rmed these ob- which promote ®lling of the penis.7 A thorough servations and identi®ed two other ®ber tracts from knowledge of peripheral nerve pathways is also a the rat major pelvic ganglion to the penis; lateral and necessary element in experiments designed to study ventral penile nerves. The main branch (descending autonomic control of erection. Information of a branch of Langworthy) reaches the penis by cour- general nature is available on the organization of sing between the rectum and urethra, while the the pelvic plexus in large species such as the rabbit lateral and ventral branches run over the prostate and cat;8 however, size considerations and the gland to travel distally on the urethra.16 complexity of the pelvic plexus in these species Since the main penile nerve (of Purinton et al;16 have prompted the move to a smaller animal model. often called the cavernous nerve) is an easily located Much relevant information now exists on the and sizable efferent nerve tract, it is often the neuroanatomy of the pelvic portion of the auto- only one stimulated or lesioned in functio- nomic nervous system of the rat,8,9,10 consequently, nal studies.2,11,12,17 Indeed, some recent studies in this species is now one of the more useful models the rat further emphasize the dominance of for studies of erectile physiology.2,11 ± 14 this pathway. For example, nitric oxide synthase immunoreactive (NOS-IR) nerve ®bers in the *Correspondence: Dr WG Dail E-mail: [email protected] cavernous spaces were completely eliminated one Received December 28, 1998; accepted in revised form March week after bilateral lesion of the rat cavernous 23, 1999 nerves; although some faint staining remained in Nitric oxide pathways to the penis WG Dail et al 278 the adventitia of major arterial branches of the containing nerves (n 2). Additional observations penile crura.2 Similarly, NADPH-d ®bers in the were made on the presence and origin of NADPH-d penile shaft were either completely eliminated or ®bers in the DNP. greatly reduced at three weeks following bilateral transection of the cavernous nerves.17 These data suggest that the ancillary pathways identi®ed by Retrograde tracer studies Purinton et al,16 or that the ganglion cells located distally along efferent nerve tracts18 which would also escape axotomy, contribute only a small Animals were anesthetized with Nembutal (30 mg= fraction of the NADPH-d ®bers in rat erectile tissue. kg, i.p.; Abbot Laboratories, Chicago, IL). Procedures A recent investigation however, has again called for injecting the penile crura have been described attention to the importance of multiple ®ber tracts previously.18 Brie¯y, 4 ml of Fluorogold (FG; Fluor- which convey vasodilator messages to the penis.19 ochrome, Inglewood, CO) were injected into each Transection of these ®bers on the prostatic capsule penile crus. A previous study has shown that a (sparing the MPNs) reduced electrically induced small volume of dye in the penile crus does not label cavernosal pressures in the rat by about 50%.19 In neurons which innervate the corpus spongiosum.18 possible agreement with this ®nding and in contrast The scrotal incision was closed by sutures and the to previous reports, our preliminary observations animals were allowed to recover. Animals were found that transection of the MPN of the rat did anesthetized at 7 d after dye placement and perfused not noticeably affect NADPH-d staining in the rat with 0.5% methylene blue in buffer, followed by 4% penile crura. paraformaldehyde. Methylene blue enhanced the As a thorough knowledge of all pathways taken outline of the MPG and its efferent nerve tracts and by vasodilator ®bers to the penis is of clinical importance, this present study sought to provide more detailed information on this issue. The approach included neurectomies in various combi- nations with retrograde tracer studies and staining for NADPH-d ®bers. In addition, information was sought on a possible NADPH-d innervation of the internal pudendal artery, since this vessel may serve as an additional route by which vasodilator ®bers reach erectile tissues.2

Materials and methods

Animals and experimental groups

Seventeen male rats (Sprague-Dawley, 200±300 g) were used in these studies. Animals were kept at 23C with a 12=12 h light=dark cycle and were allowed free access to food and water. The use of animals conformed to the rules outlined in NIH Publication 80±23 and all operative procedures employed sterile surgical techniques. Four groups of animals were studied: (1) Fluorogold-labeled pro®les of penile neurons were counted in the MPG in animals receiving a transection of the right MPN (n 5) and in animals in which all ®bers along the caudal margin of the MPG were severed (n 2) (Figure 1); (2) NADPH-d nerve ®bers were quanti®ed Figure 1 Major features of the genitalia of the male rat. The in the penile crura 7 d after removal of the ®rst 5 mm major pelvic ganglion lies on the posteriolateral lobe of the of the right MPN (n 3); (3) NADPH-d innervation prostate. At least three distinct branches may be traced to the of the internal pudendal artery and its distal branch, region of the penis. The largest of these is the main penile or the dorsal artery of the penis, was assessed in cavernous nerve. Other branches lie more lateral and ventral along the urethra. Distally, all three penile branches may form a control animals (n 3) and in those in which the plexus with the dorsal artery and dorsal nerve of the penis. The MPG had been removed 7 d earlier (n 2); and [4] arrow indicates where the penile branches were severed. the internal viscera were stained in situ for NADPH-d mpg major pelvic ganglion; mpn main penile nerve. Nitric oxide pathways to the penis WG Dail et al 279 In the second type of neurectomy, scissors were used to cut close to the ventral margin of the MPG to interrupt the MPN and all ancillary penile nerves (lateral and ventral branches of Purinton). This cut extended ventrally, but did not include direct branches of the MPG to the urinary bladder. Finally, in the third type of denervation, the MPG was excised taking care to control bleeding from vessels coursing nearby and through the ganglion.

NADPH diaphorase histochemistry

Nitric oxide synthase was localized by the NADPH- d histochemical method, using a modi®cation20 of the technique of Hope and Vincent.21 Animals were perfused with physiological saline and then with 4% phosphate buffered paraformaldehye. The penile crura and shaft were removed by sharp dissec- tion, further subdivided, and placed in ®xative for 4 h. Tissues were sectioned on a cryostat at 16 mm, af®xed to coated slides and subsequently stained for NADPH-d. In other similarly perfused animals, the pubic rami were removed by rongeurs to expose the DNP and accompanying internal pudendal vessels. Figure 2 Penile ganglion cells in the MPG labeled by Fluorogold injection of the penile crura. (a) Penile neurons occur throughout The neurovascular bundle, dissected free from its the dorso-caudal region of the MPG. Neurons cluster in several entry into the pudendal canal to its penile branches, lobes of the ganglion which then give rise to penile nerves: was studied in whole mounts and after cryostat cavernous lobe (1), lobe for lateral penile nerves (2), and a lobe for sectioning. For in situ NADPH-d preparations the ventral penile nerves (3). (b) Prior transection of the MPN (bilaterally in 2 animals), pelvic fascia overlying reduces labeling in the cavernous lobe, but neurons are abundant in the more anterior region of the ganglion. (Calibration: 400 mm). the MPG and its efferent branches and their connections to the pudendal nerve was carefully teased away. A block of tissue consisting of the permitted their removal from the surface of the internal genitalia, urinary bladder, urethra and base prostate gland. A layer of tissue including the MPG of the penis was removed and stained for NADPH-d. and its efferent branches were removed as a single sheet down to and including the junction of efferent branches with the DNP. After washout of ®xative, Quantitation tissues were examined as whole mounts using the appropriate ®lters to visualize FG. After washout of ®xative, all Fluorogold-®lled penile neuronal pro®les were counted in the MPG of animals receiving a prior transection of the MPN Procedure for neurectomy (counts were not made in intact MPG since published data is available). Superimposed labeled neurons could not always be reliably distinguished Three types of neurectomies were performed on in the MPG, therefore counts are semiquantitative animals deeply anesthetized with Nembutal: (1) and data is expressed as average number ( Æ s.d.) of axotomy of the MPN; (2) axotomy of the MPN along neurons per ganglion. Photographs were taken with all ventrally-directed branches of the MPG; and depicting the position of labeled neurons in each (3) unilateral removal of the MPG (retrograde experiment. labeling and neurectomy were performed at the An image analysis program (Bioquant=TRW, same time). The MPN is the largest branch of the Nashville, TN) was used to measure the NADPH- MPG and was easily identi®ed as it arose from the d ®ber population in sections of the penile crura. dorsal portion of the ganglion. Transection of the Taking care to avoid quantitation of blood vessel MPN was performed at a point where the triangular innervation, random ®elds of cavernosal smooth lobe of the ganglion noticeably tapers to become the muscle were digitally captured with a color video much thinner efferent nerve (Figure 1). The ®rst camera. The left crus served as an internal control in 5 mm of the nerve was then surgically excised in each animal in order to minimize problems with accordance with a previously described method.17 variation in histochemical staining. The computer Nitric oxide pathways to the penis WG Dail et al 280 was used to determine the area of the frame of the ganglion (Figure 2a). Penile neurons were occupied by stained ®bers (each frame measured highly concentrated in the lobe of the ganglion 0.9 mm2). An average areal measurement (nerve giving rise to the MPN (Figure 2a). Preservation of ®bers=mm2) was taken from the analysis of 20 efferent bundles in whole mount preparations of the frames in each crus. Results were calculated as the MPG indicated that many penile neurons cluster percent difference in ®ber density in the two sides. along the caudal margin of the ganglion near the Representative photographs of NADPH-d ®bers in origin of several nerve tracts (Figure 2a). A small the penile crura and shaft were taken with Kodak number of FG-labeled ganglion cells were found T-Max ®lm. along the proximal portion of some of these nerve tracts, but their incidence is higher distally, near the penis. This pattern was also true of the MPN; Results neuronal somata were scarce in the proximal portion, but were present in large clusters distally (data not shown). The pattern of localization of penile neurons was A large number of penile neurons in the MPG consistent with published data8; labeled ganglion were ®lled with FG even when the MPN was cells were found in the dorsal and the caudal region severed (Figure 2b). There were virtually no labeled

Figure 3 Representative photographs of NADPH-d nerve ®bers in the penis of a rat in which the right MPN was transected 7 d earlier. (a) intact left crus. (b) `denervated' right crus. (c) proximal portion of penile shaft. Note that the density of NADPH-d ®bers in the control and denervated tissues appear similar. Right dorsal penile nerve (R); Left dorsal penile nerve (L); dorsal vein of penis (dv). (Calibration: 200 mm). Nitric oxide pathways to the penis WG Dail et al 281 neurons in the lobe of the ganglion giving rise to the efferent tracts in this location and occasionally, MPN, however, there was a large concentration of some of them were closely associated with the DNP. penile neurons in the caudal=anterior portions of the The proximal portion of the DNP had only a modest MPG (average of 88.8 Æ 16.3; six ganglia from ®ve component of NADPH-d ®bers (Figure 5b) compared animals). Fewer than three neurons were labeled in to that seen in the penile shaft (Figure 3c). As the the two rats (four ganglia) in which the MPN and all DNP passed distal to the ischiourethralis muscle, tracts along the caudal margin of the MPG were many of the NADPH-d bundles diverged and transected. distributed around the shaft of the penis (Figure There was no marked visual difference in the 5c). The bundles were closely related to a rich density of NADPH-d ®bers between right and left plexus of vessels in the subcutaneous tissue of the penile crus seven days after removal of the ®rst penis. Many of the vessels were accompanied by 5 mm of the right MPN (Figure 3a,b); a dense ground NADPH-d ®bers and penetrated the tunica albuginea plexus of ®bers was distributed over the cavernous to reach cavernous spaces. smooth muscle as well as in the adventitia of penile In situ staining of the pelvic viscera revealed a arteries. However, quantitative analysis revealed concentration of NADPH-d neurons in the dorso- that there was some loss of ®bers in two of the three caudal region of the MPG (Figure 6 A & B). Four to ®ve animals when the left intact crus was compared to deeply stained nerve bundles extended caudally over the right `denervated' crus (0,21 and 24% decrease). the prostate (posterolateral lobe) and onto the urethra. Finally, there was no striking visual difference in The lateral penile nerve (one or more bundles) and the the density of NADPH-d ®bers between the right and thinner ventral penile nerve (usually single) commu- left DNP after chronic interruption of the MPN nicated with the pudendal neurovascular bundle (Figure 3c). near the base of the penis (Figure 6A & B). Smaller The internal pudendal artery has a NADPH-d branches of both nerves formed a plexus and rami®ed ground plexus all along its length from its entry into on the DNP and on the dorsal artery of the penis and the pudendal canal to the origin of branches to the its tributaries. The MPN, the most prominent branch penis (Figure 4a,b). Preterminal NADPH-d ®ber of the MPG, was directed dorsally and assumed a bundles joined the vessel at branch points (bulb position in a groove between the urethra and rectum. and crural arteries) and rami®ed proximally (Figure The two MPNs formed a ladder-like plexus on the 4b). Although not quantitated, removal of the posterior surface of the urethra. The branching ipsilateral MPG for seven days decreased, but did pattern of the MPN to the penis varied even in the not eliminate NADPH-d innervation of the internal same animal; in two of the four specimens, one or pudendal artery. more branches of the MPN arose distal to the prostate A NADPH-d nerve plexus was also distributed to and joined the pudendal plexus. The main trunk of the DNP, joining it at a site just proximal to the the MPN maintained its location in the groove ischiourethralis muscle (Figure 5a). There were between urethra and rectum and gave rise to small numerous NADPH-d neuronal pro®les along the branches to penile vessels.

Figure 4 NADPH-d innervation of the internal pudendal artery (dorsal artery of the penis). (a), A segment of the vessel in the proximal pudendal canal; (b) the common penile artery (cpa) giving rise to the artery of the bulb (ab). (Calibration: 125 mm). Nitric oxide pathways to the penis WG Dail et al 282

Figure 5 NADPH-d contributions to the dorsal nerve of the penis. (a) Several bundles join the DNP at the base of the penis. (b) A section of the DNP in the proximal portion of Alcock's canal. (c) NADPH-d ®bers (arrows), derived from branches of the DNP, pierce the tunica albuginea (ta) to enter the cavernous spaces of the shaft. Note the NADPH-d innervation of the numerous small arteries in the fascia super®cial to the tunica. Calibration in (a), 200 mm; in (b), 100 mm; in (c), 200 mm).

Discussion reported a 50% drop in cavernosal pressure (induced by pelvic nerve stimulation) after cutting ®bers running on the lateral and anterior capsule of This study has provided evidence that penile the rat prostate. Data from this present study suggest neurons located in the MPG of the rat take multiple that the vasodilator projection interrupted by Zvara pathways to the penis, in addition to the main et al 19 consists of the lateral and ventral penile penile or cavernous nerve. These alternate pathways nerves. Assuming that all NADPH-d ganglion cells (broadly de®ned as lateral and ventral penile subserve the same function in the penis (their branches) are only brie¯y treated in previous processes release nitric oxide which relaxes smooth reports, but account for a signi®cant proportion of muscle) then a large portion of the penile vasodi- the penile vasodilator out¯ow. Evidence is also lator out¯ow follows these alternate pathways (the presented which con®rms, explains and extends average of 88 neurons which project through these previous studies which show that the pelvic plexus nerves may be about one half of the total of penile (the MPG and more distal ganglion cells) exerts wide neurons in the MPG18). In our whole mount control over penile vasculature. preparations, many ®bers arise directly from the Our observations of multiple pathways to the MPG, rather than from the cavernous nerve as penis are in agreement with Zvara et al 19 who depicted by Zvara et al. 19 That the MPG gives rise Nitric oxide pathways to the penis WG Dail et al 283

Figure 6 NADPH-d staining of the rat pelvic plexus; left (a) and right (b) sides of the same animal. Major pelvic ganglion (mpg), main penile nerve (mpn), lateral penile nerve (lpn), ventral penile nerve (vpn), branches of main penile nerve to the pudendal neurovascular bundle (arrow heads), dorsal nerve of the penis (dnp). (Calibration: 2 mm). to lateral and ventral penile nerves is supported by penis.29 For example, if visual observation of the obvious clustering of penile neurons in the tumescence is the sole measure, it is conceivable ventral margin of the ganglion at the origin of the that an increase in blood ¯ow may not result in ancillary nerves and secondly, by the ability to detectable swelling. Moreover, denervation may not retrogradely label many neurons even when the have an equal effect on all penile tissues. Sachs and MPN was transected. Therefore, the original naming Liu30 found that transection of the MPN and all ®ne of these branches as lateral and ventral penile nerves branches coursing toward the corpora cavernosa appears justi®ed.16 penis and urethra (presumably, the `®ne branches' The neuroanatomy described herein, which is correspond to lateral and ventral penile nerves) likely similar to that in most mammals,8 may help to weakens, but does not prevent moderate to strong explain why surgery of the prostate has had a erection of the glans penis. variable effect on potency. Although favorable Consistent with observations on retrograde label- results have been reported when care is taken to ing, transection of the MPN did not eliminate preserve the more laterally placed neurovascular NADPH-d ®bers in the penile crura and in the bundles,22 ± 25 other studies have reported less DNP. It is presumed that ganglion cells whose axons success in retaining erectile ability.26,27 In animal project through the lateral and ventral branches of models, cutting the cavernous nerve (main penile the MPG could account for this result. In addition, nerve) usually results in erectile failure;2 however, the abundant penile ganglion cells along the MPN care must be taken in interpreting results, since the distal to the site of transection may contribute to the manner in which erection is measured varies residual ®bers, since the NADPH-d staining in their widely, ranging from gross observation of tumes- processes would not be expected to change at one cence,12 to direct measurement of intracavernosal week following simple decentralization. The possi- pressure2,11,13,28 and plethysmography of the entire bility that ®ber crossing from the opposite uninjured Nitric oxide pathways to the penis WG Dail et al 284 side could account for the residual innervation the tunica albuginea to reach the cavernous spaces cannot be eliminated; however, this is not deemed of the penile shaft along with perforating branches likely due to the separate course of the NADPH-d of the dorsal arteries of the penis (perforating bundles along the dorsolateral and anterior urethra en arteries have been previously described in the route to the widely separate crura. The present results rat).37 If perforating neurovascular bundles are a of a modest reduction of NADPH-d ®bers in two of the common occurrence, the DNP may provide an three animals examined are in contrast to earlier important route for vasodilator ®bers to erectile reports that interruption of the cavernous nerves in tissue. Preservation of this nerve in surgical the rat greatly reduced or eliminated NADPH-d procedures involving the penile shaft should take staining in the crura2 and in the penile shaft.17 The this putative vasodilator function into account. reason for the variance in results is unclear, but may include differences in denervation procedures (the Conclusions extra care taken for preservation of lateral and ventral branches in the present report), histochemical technique, post-lesion interval or sampling site in the In summary, ancillary vasodilator pathways, penis (the crura or the penile shaft). although somewhat variable in number compared The NADPH-d ground plexus of the internal to the singular main penile nerve, comprise a pudendal artery is consistent with the pelvic constant and substantial projection of the MPG to nerve-induced dilatation of this vessel.31,32 Recur- the penis. The data presented here has both rent nerves from the pelvic plexus seem mandated functional and clinical implications as it is clear by the intervening pelvic ¯oor musculature which that stimulation of the MPN activates only a portion separates the pelvic plexus from the pudendal of the vasodilator out¯ow to the penis; moreover, neurovascular bundle. Therefore, the recurrent in- the course of the ancillary nerves over the prostate nervation of the internal pudendal artery is a neural renders them at risk in surgical resections. Finally, substrate by which the pelvic plexus can control in addition to direct branches to the penis (efferent relaxation of this vessel concomitant with that of penile nerves), the pelvic plexus coordinates penile penile resistance vessels and cavernous smooth blood ¯ow through a substantial input to the muscle. That there was only a slight loss of the internal pudendal artery and, by virtue of the DNP, NADPH-d nerve net of the internal pudendal artery to perforating branches of the dorsal artery along the after removal of the ipsilateral MPG further suggests shaft of the penis. that ganglion cells, located distally along the pelvic plexus, provide innervation to the artery as they do to the erectile tissue of the penis. Acknowledgements The substantial contribution of NADPH-d ®bers from the pelvic plexus to the DNP con®rms and Research in this manuscript was supported by NIH extends observations which detail an autonomic RO1 NS19839-13. contribution to this sensory nerve.9,20,33 ± 36 Our data also indicates that the MPN may or may not make a direct contribution to the DNP; however, References even when no contribution was noted, the MPN always gave rise to a ®ne plexus to penile vessels 1 Andersson KE, Wagner G. 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