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Keywords:

  • cavernosal nerve;
  • erectile function;
  • neuropathy;
  • nitric oxide synthetase;
  • electrophysiology;
  • ultrastructure

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

OBJECTIVE

To illustrate the ultrastructural fibre composition of the rat cavernosal nerve at serial levels, from its origin in the main pelvic ganglion to its termination in the corpus cavernosum of the distal penile shaft, and to develop a technique that permits repeated electrophysiological recording from the fibres that form the cavernosal nerve distinct from the axons of the dorsal nerve of the penis (DNP).

MATERIALS AND METHODS

For the light microscope and ultrastructural studies, Sprague–Dawley rats were anaesthetized and the pelvic organs and lower limbs were perfused with glutaraldehyde through the distal aorta. Tissue samples were embedded in epoxy resin and prepared for light and electron microscopy. Frozen tissue was used for the immunohistochemical studies and sections were stained with rabbit anti-nitric oxide synthetase 1 (NOS1). For the electrophysiology, anaesthetized rats were used in sterile conditions. Nerve conduction velocity for the cavernosal nerve was assessed from a point 2 mm below the main (major) pelvic ganglion after stimulating the nerve at the crus penis; multi-unit averaging techniques were used to enhance the recording of slow-conduction activity. Recordings from the DNP were obtained over the proximal shaft after stimulation at the base of the penis.

RESULTS

Step-serial sections of the cavernosal nerve revealed numerous ganglion cells in the initial segments and gradually fewer myelinated fibres at distal levels. At the point of crural entry, the nerve contained almost exclusively unmyelinated axons. As it descended the penile shaft, the nerve separated into small fascicles containing only one to four axons at the level of the distal shaft. In the corpus cavernosum, vesicle-filled presynaptic axon preterminals were close to smooth muscle fibres, but did not seem to be in direct contact. Immunohistochemical evaluation of NOS1 activity showed intense staining of the fibres of the DNP and most of the neurones in the main pelvic ganglion. There was also scattered NOS1 activity in the nerve bundles of the corpus cavernosum. Electrophysiology identified activity in C fibres on the cavernosal nerve and in Aα–Aδ fibres in the DNP.

CONCLUSION

These results show that it is possible to perform integrated cavernosal pressure monitoring and ultrastructural and electrophysiological studies in this model. These yielded accurate data about the erectile status of the penis, and the state of unmyelinated and myelinated fibres in the DNP and cavernosal nerves of the same animal. This study provides a useful template for future studies of experimental diabetic autonomic neuropathy.


Abbreviations
MPG

main (major) pelvic ganglion

DNP

dorsal nerve of the penis

NOS1

nitric oxide synthetase 1

MUA

multiple-unit activity.

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

Erectile and gastrointestinal dysfunction are common and disabling features of human diabetic autonomic neuropathy [1–3]. Studies of autonomic neuropathy in streptozotocin-induced diabetic rats with megacolon indicate that axonal dystrophy is the salient histopathological finding in mesenteric nerves of the ileum [4]. The dystrophic changes are more frequent in the terminal segments of the splanchnic nerves, whereas the proximal nerve and parent ganglion cell are unaffected [4–9]. This pattern of distal axonal degeneration (‘distal axonopathy’) is characteristic of many toxic and metabolic polyneuropathies; it supports the concept that sustained hyperglycaemia causes a perturbation of ganglion cell metabolism, axonal transport, or both, with subsequent loss of integrity of the distal portions of the nerve fibre. The longest nerves are affected first; in time, this metabolic axonal disruption, if uncorrected, will move proximally [10–12]. In humans, distal axonopathy is associated with metabolic neuropathies, including diabetic sensory neuropathy, which gives rise to a ‘stocking-and-glove’ pattern of sensory loss, with sparing of the proximal limbs, trunk and face. Although diabetic somatic neuropathies have been studied extensively, there are serious limitations of experimental studies in autonomic nerves. One is the inability to repeat electrophysiological measurements, because the nerves are relatively inaccessible and animals must be killed at the end of recording; another is the difficulty in measuring the onset and degree of physical changes during activity of the end organ.

Erectile dysfunction develops in rats with streptozotocin-induced diabetes, and its treatment was studied widely [13–15]. The murine main (major) pelvic ganglion (MPG), cavernosal nerve (main penile nerve), corpora cavernosum (cavernosum), and dorsal nerve of the penis (DNP) are ideal for studies of autonomic dysfunction for the following reasons: the nerves are very long and are accessible for proximal–distal histopathological studies, the degree of erectile tumescence is measurable readily and accurately, and the paired accessible structures permit electrophysiological studies of the nerves at several time-points in the disease. There are many detailed studies of the light and electron microscopic anatomy of the MPG and cavernosum [14,16–23], but none has focused on the serial composition of the fibre population of the cavernosal nerve or on the ultrastructure of the unmyelinated fibres in the cavernosum. Previous anatomical and immunohistochemical studies of murine streptozotocin neuropathy suggest neuronal loss in the autonomic ganglia; others describe none [24,25]. Although variable changes in DNP and cavernosal nerves are documented, no study has described axonal dystrophy, nor has any systematically analysed the proximal-to-distal gradient of axonal degeneration. Although physiological measures have explored the more proximal segments of the cavernosal nerve [26–28], no previous study has recorded activity from unmyelinated axons in the most distal cavernosal nerve.

The aim of the present study was to provide a histopathological, immunohistochemical and physiological template for further studies of murine autonomic dysfunction in peripheral neuropathies. This report is especially relevant to investigations of erectile dysfunction in diabetic neuropathy. The results are from investigations in control rats aged 2–16 months. The morphological studies highlight features of the MPG and those of serial levels of the cavernosal nerve, starting at the MPG and ending in the distal penile shaft. The electrophysiological studies establish a reliable procedure for repeatedly recording activity from unmyelinated fibres in the cavernosal nerve and from the mixed-fibre population of the DNP in the same animal.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

All experimental studies were conducted according to a protocol approved by the Animal Care and Use Committee of the Albert Einstein College of Medicine. For the histopathological studies, 15 male Sprague–Dawley rats (2 months–2 years) were used, five for the electrophysiological studies, and three for the histochemical studies. Anaesthesia was induced in all rats for anatomical and immunohistochemical study with sodium pentobarbital (35 mg/kg i.p.). Anaesthesia was maintained throughout the experiment, as necessary, by injecting pentobarbital (5–10 mg/kg) every hour as needed. As previously described [13], for intracavernosal pressure monitoring and dissection rats were placed supine, and the bladder, prostate, and abdominal aorta were exposed through a midline abdominal incision. The penis was denuded of skin, followed by removal of the ischiocavernosus muscle to expose both crura (corpora cavernosa). To monitor the intracavernosal pressure, a 23-G needle was filled with 250 U/mL heparin solution, connected to polyethylene-50 tubing, and was inserted into the right corpus cavernosum. The pressure line was connected to a pressure transducer, which in turn was connected via a transducer amplifier to a data acquisition board. A real-time display and pressure measurement was recorded on a Macintosh computer. Intracavernosal pressure was monitored only in five of the rats, which were used for histological studies, and not in rats used for histochemical or physiological studies.

Before dissection, the abdominal aorta was cannulated just below the renal arteries, and the lower body was perfused with cold 4% paraformaldehyde, followed by phosphate-buffered 2.5% glutaraldehyde (pH 7.4). The pelvic ramus was split to enable dissection of the MPG, the entire length of the cavernosal nerve, and the penile crus, shaft and glans. Transverse 1-mm slices were taken from seven levels of the cavernosal nerve and penis (Fig. 1), immersed in cold 1% osmium tetroxide for 1–2 h, dehydrated in a graded series of ethyl alcohol, and embedded in epoxy resin. For light microscopy, 1-µm epoxy resin sections were stained with toluidine blue. For electron microscopy, thin sections were cut on an ultramicrotome, contrasted with lead citrate and uranyl acetate, carbon coated, and examined in a Hitachi H-600 electron microscope (Ibaraki, Japan).

image

Figure 1. The major autonomic components of the cavernosal (penile) nerve. The numbers indicate levels of sections for microscopy (except for the pelvic nerve).

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For the immunohistochemical studies, the MPG, cavernosal nerve and penis were embedded in optimal cooling temperature medium in a dry-ice bath, and cryosectioned (10 µm). The frozen sections were fixed in acetone for 10 min. After blocking with normal sera, sections were incubated with rabbit anti-nitric oxide synthetase 1 (NOS1; Chemicon, Temecula, CA, USA) overnight at 4 °C, followed by incubation with anti-rabbit IgG-fluorescein isothiocyanate (Southern Biotechnology, Birmingham, AL, USA) for 1 h at room temperature. Negative controls (omission of primary antibody) were used to exclude non-specific staining. Sections were viewed under a Zeiss Axioskop (Thornwood, NY, USA).

The electrophysiological studies were performed in five rats. In one rat, recordings were obtained from alternate cavernosal and DNPs on two occasions 5 months apart. The surgery was conducted under general anaesthesia (isoflurane 3.5%/O2 for induction and 2–2.5%/O2 for maintenance). Incisions were made in the midline of the lower abdominal wall to allow access to the proximal part of the cavernosal nerve exiting from the MPG. Distal branches of the cavernosal nerve and DNP were reached through a 1-cm incision in the scrotum that exposed the crura and the proximal ventral penis. The cavernosal nerve was stimulated at the upper level of penile crus or ≈3 mm proximal to it, at the base of the penis (Fig. 1, levels 7–8). The cathode–anode line was perpendicular to the long axis of the penile shaft, and the cathode was located at the site of cavernosal nerve recording. Platinum-needle electrodes were used for recording and stimulation. The activity of the cavernosal nerve was recorded 2–3 mm distal to the MPG; the electrodes were placed over the main branch of the cavernosal nerve, taking care not to damage the prostate capsule. The activity of the DNP was recorded s.c. 20–22 mm distal from the stimulation point along the shaft of the penis. The activity from both nerves was recorded simultaneously in most experiments. Constant current stimulation (0.1–15.0 mA, 0.1–0.5 ms at 1 Hz) was applied (Grass Model 11 stimulator; Grass Model SIU-7 Isolation Unit, Warnick, RI, USA). The neural activity was amplified at a gain of 20 000, digitized at a rate >20 kHz, and averaged using a BioPac MP100 system and dedicated software, AcqKnowledge 3.7.2 (Goleta, CA, USA). Selected data were stored on digital tape for subsequent off-line analysis. Low-amplitude, purely phase-locked responses of slow-conducting fibres were identified by filtering single sweeps (bandpass 450 Hz−5 kHz) and then rectifying and averaging the resultant signal to isolate multiple-unit activity (MUA) [29]. After completing the final electrophysiological measures, rats were killed or were sutured and allowed to recover. After the final recording session, the symphysis pubis was split and the scrotum around the base of the penis was removed to allow tracing of the course of the main cavernosal nerve and measurement of distances from stimulation to recording points.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

HISTOPATHOLOGICAL STUDIES

Longitudinal step-serial sections of the MPG (a 2–3-mm structure adherent to the prostatic fascia) usually showed the ganglion as a curved arc. By light microscopy, the architecture was a compact mixture of variably sized, mostly monopolar neurones and bundles of myelinated and unmyelinated axons. Many of the neurones had large convoluted axon hillocks. In 9-month-old rats there were vacuolar changes (Fig. 2) in scattered neurones (about one per high-power field). There was no evidence of neuronal degeneration. Electron microscopy showed abundant axosomatic synapses, and about a third of the neurones had a dark, granular cytoplasm.

image

Figure 2. A histological section from a 2-year-old rat. This high-power field MPG contains two neurones (arrows) displaying vacuolar changes (1-µm epoxy sections; toluidine blue staining; scale bar, 17 µm).

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The cavernosal nerve extended 2.5–3 cm from the base of the MPG before entering the ischiocavernosus muscle; accompanying the deep penile artery, it penetrated the fibrous capsule of the crus penis. The cellular and nerve fibre compositions at descending levels of the cavernosal nerve are shown in Fig. 3.

image

Figure 3. Light microscope sections from three levels of the cavernosal nerve in a 9-month-old rat. 3.1, adjacent to the junction with the MPG (level 2 in Fig. 1); the nerve has three fascicles and contains many neurones, as well as myelinated and unmyelinated fibres. 3.2, at the midpoint (level 3 in Fig. 1) containing a few neurones and fewer fibres. 3.3, proximal to the junction with the crus penis (level 5 in Fig. 1) having only one fascicle and fewer nerve fibres (1-µm epoxy sections; toluidine blue staining; scale bar, 27 µm).

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The proximal third of the cavernosal nerve contained abundant ganglion cells and myelinated and unmyelinated axons, and was virtually indistinguishable from the MPG cytoarchitecture. The number of ganglion cells and myelinated fibres diminished gradually as the nerve descended and entered the crus. At the point of crural entry, the nerve branched into four to six fascicles and became adherent to the adventitia of the anterior branch of the deep penile (cavernosal) artery (Fig. 4.1). There were variable elastic laminae in the anterior branch of this vessel subsequent to its entry into the aperture of the tunica albuginea of the crura (Fig. 4.2). At this point, the cavernosal nerve fascicles were composed almost exclusively of unmyelinated fibres (Fig. 5.1). The fascicles remained closely associated with this artery as it descended the crus into the base of the penis.

image

Figure 4. 4.1, from a 2-year-old rat taken at the point of penetration of the tunica albuginea by the cavernosal nerve (level 6 in Fig. 1). The nerve has divided into several fascicles (arrows) and is embedded in the adventitia of the deep penile (cavernosal) artery. 4.2, from a 9-month-old rat taken at the mid crus (Level 7 in Fig. 1) showing several fascicles (arrows) of the cavernosal nerve adherent to the adventitia of the deep penile artery (1-µm epoxy sections; toluidine blue staining; scale bars (1) 54 µm, (2) 17 µm).

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image

Figure 5. Electron microscope sections showing fascicles of the cavernosal nerve in a 9-month-old rat taken at three levels in the penis: 5.1, from level 7 in Fig. 1, 5.2, from level 8, and 5.3 from level 10. There is steady attrition of nerve fibres, especially myelinated axons during its descent: 5.1 × 14 000, 5.2 × 9000, 5.3 × 7500.

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In the basal third of the penile shaft, the unmyelinated fascicles divided into small bundles of three to 18 axons and were scattered widely among the abundant collagenous septae and smooth muscle fibres (Fig. 5.2). The unmyelinated fibres were no longer associated with vasculature. For the most part, these small bundles were not apparent by light microscopy, but could be seen readily by electron microscopy. Occasional vesicle-filled axonal pre-terminals were associated closely with smooth muscle fibres; we detected no contact between axons and smooth muscle fibres. There were a few small fascicles of three to six thinly myelinated fibres at the outer edge of the dorsal cavernosa, adjacent to the tunica albuginea; some appeared to have penetrated the tunica and were approaching the perineurium of the DNP. Similarly, occasional small clusters of unmyelinated axons were adjacent to helical arterioles, but contacts were not seen. The helical arterioles (Fig. 6.1) had a thin muscularis and, occasionally, plump ‘reactive appearing’ endothelial cells that appeared (Fig. 6.2), on light microscopy, as pads [22]. The spongiosum, by contrast, had bundles of thinly myelinated fibres adjacent to the urethra at all levels of the shaft (Fig. 7).

image

Figure 6. Helical arterioles from a 2-year-old rat. 6.1, light micrograph showing an arteriole with an asymmetric lumen caused by a prominent endothelial pad arrow (1-µm epoxy sections; toluidine blue staining; Original ×630; scale bar, 17 µm). 6.2, electron micrograph of a helical arteriole with a similar asymmetric lumen caused by large, reactive-appearing endothelial cells (original ×12 000).

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image

Figure 7. Two sections from the corpus spongiosum of a 2-year-old rat. 7.1, light micrograph of a 1-µm, epoxy resin toluidine blue-stained section containing one fascicle with many myelinated fibres. There are also two isolated myelinated axons adjacent to the urethra (arrows; scale bar, 17 µm). 7.2, electron micrograph of a small fascicle in the spongiosum containing a mixture of myelinated and unmyelinated fibres (original ×12 000).

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In the middle and distal thirds of the penile shaft cavernosa, unmyelinated fibre fascicles could not be identified reliably by light microscopy. Electron microscopy detected a few bundles of three to eight unmyelinated fibres in the mid-shaft level. In the distal third, only rare isolated fibres were seen (Fig. 5.3). On electron microscopy, small clusters of unmyelinated axons deployed in the walls of the cavernosal sinusoids and vesicle-filled presynaptic axonal processes were occasionally evident adjacent to smooth muscle fibres (Fig. 8).

image

Figure 8. Electron micrograph of a section from the corpus cavernosum of a 9-month-old rat. There are two vesicle-containing presynaptic unmyelinated axons (arrows) in close proximity to smooth muscle fibres (original ×12 000).

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IMMUNOHISTOCHEMICAL STUDIES

Immunostaining for neuronal NOS revealed many intensely stained neuronal cell bodies and nerve fibres distributed throughout the MPG. In the penis, there was strong NOS reactivity in nerves surrounding the deep penile and dorsal arteries, as well as in the DNP and scattered small nerve bundles in the cavernosal bodies (Fig. 9).

image

Figure 9. Representative immunohistochemical localization of nNOS in the rat penis cavernosum. There is reactivity in myelinated nerve fibre bundles (arrows) at the outer edge and faint reactivity among the smooth muscle fibres in the centre (C) (fluorescein isothiocyanate immunofluorescence; original ×250).

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ELECTROPHYSIOLOGICAL STUDIES

As expected, the fibre types activated in the distal cavernosal nerve were a function of stimulation intensity. The initial response was elicited at intensities as low as 0.5 mA and reflected a conduction velocity of ≈13.5 m/s (Fig. 10.1). This activity presumably represented thinly myelinated afferents in cavernosal nerve. The paucity of the signal amplitude, the simplicity of the response, and its lack of growth with increased stimulation intensity was consistent with the presence of only limited axons in this category in the cavernosal nerve, as suggested by the histological studies above.

image

Figure 10. Responses in the distal cavernosal nerve at different intensities of stimulation. 10.1, Averaged (n = 50) evoked potentials (EP; thick line traces) and MUA (thin line traces) are visible at four intensities of stimulation. The parameters of stimulation (duration and constant current of a square pulse) are presented on the right. Open arrow points to the response of the fastest fibres in the cavernosal nerve, closed arrows depict appearance of the responses in different pools of slow-conducting fibres in the same nerve. Dashed lines indicate the range of nerve conduction velocity in responses of slow-conducting fibres written at the bottom. Black dot indicates the location of stimulus artifact. Amplitude calibrations are given by vertical lines: thick lines for EP are 5 µV, thin lines for MUA are 3 µV; the time scale is the same for all traces and is given at the bottom (in ms). 10.2, Evaluation of the changes in MUA of the responses in distal cavernosal nerve shown above over pre-stimulus period and different ranges of slow nerve conduction velocity (x-axis) with increasing intensity of stimulation (y-axis). The magnitude of the MUA is in V/time bin (z-axis).

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However, the longer duration of activity recorded over the cavernosal nerve, elicited only by high-intensity stimulation, was complex and multiphasic. Stimulation intensities of >6 mA elicited MUA with conduction velocities of 1–2.5 m/s, consistent with C-fibre activation (Fig. 10.1). Increasing the intensity to 14 mA activated fibres with an even higher threshold and increased the amplitude of the slower portion of the C-fibre response, i.e. velocities of 1–1.5 m/s (Fig. 10.1). The application of MUA techniques to peripheral nerve assessment provided outstanding resolution of the subcomponents of C-fibre activation of the distal cavernosal nerve. Findings consistent with C-fibre activity were detected in all five rats examined. Velocities of <1.0 m/s were also detected in two of the five rats (data not shown).

By contrast to the findings in the distal cavernosal nerve, physiological responses overlying the proximal DNP were larger, much shorter in latency, and monotonic in morphological features (Fig. 11). This pattern was consistent with activation of axons with conduction velocities of 36–44 m/s (i.e. consistent with activity in Aα–Aδ afferents). In accord with the thinning of axonal cross-sectional diameter and the decrease in internodal distances, recording at more distal sites along the nerve revealed a relative slowing of maximal conduction velocities and a reduction in compound sensory amplitude (Fig. 11).

image

Figure 11. Averaged (n = 50) responses in the DNP at two distances along the penile shaft, with the calculated nerve conduction velocity for each point and for the segment between them. Dashed lines mark the onset of responses. Black dots indicate the location of stimulus artefact. Amplitude calibration (thick vertical lines) is 50 µV. The time scale is at the bottom of the figure is in ms.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

The present report describes ultrastructural and light microscope data from perfusion-fixed tissue designed to permit serial analysis of the fibre population of the cavernosal nerve from its origin in the MPG to its termination in the penile cavernosum. Previous reports have depicted, in a series of meticulous studies, the gross anatomical, light microscopic and histochemical features of the autonomic innervation of the rat penis [14,16–24].

The MPG is a tightly packed peripheral ganglion and the only such mammalian structure that contains both sympathetic and parasympathetic neurones. The neurones were almost all unipolar, with surfaces studded with abundant axosomatic synaptic boutons. In the 9-month-old rats, occasional cells showed vacuolar changes superficially similar to degenerative changes associated with senescence and some toxic neuropathies (Fig. 2) [30,31]. The oldest rats were aged 9 months, and neither axonal nor neuronal degenerative changes were prominent. For these reasons it seems improbable that it reflected a degenerative process. This type of vacuolar change is also a well-described normal finding in sympathetic ganglia of younger animals; it is probably related to a secretory function, not a degenerative feature [32]. In senescent animals, this phenomenon should be an important consideration for studies that assess cellular degeneration in the MPG of older rats as a component of autonomic neuropathies [24,33].

The cavernosal nerve extended ≈3 cm from its origin in the MPG before penetrating the ischiocavernosus muscle and the crus penis; in its brief course, it gave rise to several branches and changed dramatically in composition (Fig. 3). In the initial 1 cm from its origin, the nerve resembled an extension of the MPG, with an abundant population of neurones and both myelinated and unmyelinated fibres. At the point of penetration of the fibrous capsule of the crus, the nerve was almost exclusively composed of unmyelinated axons (Fig. 4); there were few remaining myelinated fibres and no neurones. Descending the crus, the nerve branched frequently, and at the level of the base of the penis, there was a barely discernible nerve; there were small fascicles of three to 12 unmyelinated axons enveloped by Schwann cells and rare myelinated fibres (Fig. 5). These fascicles were less apparent in the middle third of the penile shaft. In the distal shaft, there were only a few widely scattered small fascicles and isolated axons; these isolated axons were often filled with clear and dense-core vesicles, were devoid of enveloping Schwann cell processes, and were usually close to cavernosal smooth muscle cells (Fig. 8). Physical contacts between these presumably presynaptic axons and smooth muscle fibres were not detected; there were no defined synaptic structures. Most of the smooth muscle fibres in the mid and distal shaft appeared to be remote from the nearest unmyelinated axon. Gap junctional complexes between the smooth muscle cells were reported in previous studies on immunohistochemical stains targeted at connexin 43, but have been rarely seen by electron microscopy [34]. The present immunohistochemical data showed abundant NOS staining in DNP fibres and MPG neurones, but only scattered reaction in the cavernosum (Fig. 9). Previous reports described similar findings [35,36]. Our anatomical and histochemical findings correlated closely with studies that indicate that impulse and chemo-transmission in cavernosal smooth muscle, in contrast to striate muscle, is not totally dependent on individual nerve–muscle synaptic contact; gap junction channels considerably facilitate impulse transmission [34,37].

There were small fascicles of myelinated fibres in the cavernosal penis located circumferentially under the tunica; some appeared to pierce the tunica and join the DNP. Taken in concert with the virtual absence of myelinated axons in the cavernosal nerve at its entry into the crus, this strongly suggests that large-fibre sensory activity from the penile shaft reaches the spinal cord via the DNP. It also seems likely that most myelinated fibres in the cavernosal nerve are not sensory; they were mostly preganglionic parasympathetic axons. Communication between branches of pudendal and cavernosal nerves has been described in humans [38].

By contrast to the corpora cavernosa, the corpus spongiosum contained clusters of myelinated fibres, as well as unmyelinated bundles. Both fibre types heavily innervated the glans. The abundant myelinated fibres are probably the anatomical substrate for the lower threshold and sensitivity of these structures compared with the cavernosum.

The reliable identification of subsets of physiological activity in unmyelinated axons of the distal cavernosal nerve represented a unique and critical finding of the present study. These axons play a key role in erectile function, and their pathophysiology is a cardinal feature of the onset and progression of a wide variety of polyneuropathies. Although many studies have monitored changes in somatic nerves associated with disease and with putative therapies, the lack of objective and direct measures of autonomic nerve physiology has hampered research into autonomic neuropathies. Previous studies recording myelinated and unmyelinated fibre activity of the cavernosal nerve used classical techniques that required large surgical exposures, nerve dissection before recording, and termination by killing the rat [27,28]. A salient technical advantage of the present experiments was the ability to isolate low-amplitude, poorly phase-locked responses using the MUA technique (Fig. 10) originally developed to measure activity across cortical laminae and afferent fibre tracts of the CNS [29].

Based on the reports of others [4–9], it is very likely that the unmyelinated fibres will be especially vulnerable in autonomic neuropathies. Therefore, the ability to evaluate accurately their waveforms, amplitudes, and velocities at different temporal stages is critical. Recent human studies reported that loss of thermal sensitivity, a method dependent on small and largely unmyelinated axons, was clearly associated with erectile dysfunction [39]. Our ability to differentiate slow and fast conduction activity in autonomic nerves of the penis, and to repeat these measures over time, should strengthen the value of animal models of autonomic neuropathy and erectile dysfunction.

The present study is the first to integrate electrophysiology with ultrastructural anatomy of the mammalian cavernosal nerve. It affords the ability to integrate the measured erectile status of the penis, the anatomical state of the axonal population at various levels of the cavernosal and DNPs, and the physiological activities of these fibres. It will be a useful template for future experimental investigations of autonomic neuropathy in diabetes.

ACKNOWLEDGEMENTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

Supported by the National Institute of Digestive, Diabetes and Kidney Diseases (grant no. P01-DK060037) and the National Institute of Neurological Disease and Stroke (grant nos. NS041194, NS11920, and NS08952), National Institutes of Health, Bethesda, Maryland. We thank Miriam Pakingan and Yvonne Kress for expert technical assistance.

CONFLICT OF INTEREST

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES

None declared. Source of funding: National Institutes of Health.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGEMENTS
  8. CONFLICT OF INTEREST
  9. REFERENCES