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Keywords:

  • Impotence;
  • nitric oxide synthase;
  • denervation;
  • erectile dysfunction

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. References

Objective

To investigate changes in histology and nitric oxide synthase (NOS) activity in cavernosal tissues from rats with neurogenic erectile dysfunction induced experimentally.

Materials and methods

Twenty-four adult male Sprague-Dawley rats were divided equally into three groups and underwent a sham operation (control, group 1), unilateral (group 2) or bilateral (group 3) cavernosal nerve resection. Three months later they were killed and the cavernosal tissues analysed histologically by light and transmission electron microscopy, with NOS activity detected using an NADPH-diaphorase staining technique.

Results

On light and electron microscopy, while penile nerves and cavernosal smooth muscle cells had a normal morphological appearance in the eight control rats, there were degenerative changes of the myelinated penile nerves and axonal fibrosis in groups 2 and 3. However, these changes were not significant. Using NADPH-diaphorase staining, NOS activity was detected in all three groups in endothelial cells and cavernosal structures. However, the staining was more intense in endothelial cells and cavernosal muscles of rats in group 2 than in the other groups.

Conclusion

NOS activity was increased in the cavernosal tissue after cavernosal denervation, but the pharmacological action of nitric oxide may be impaired.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. References

Penile erection is governed by the relaxation of smooth muscle which surrounds the arterioles and vascular sinusoids of the corpus cavernosum [1]. The relaxation of penile smooth muscle is controlled locally by neurotransmitters and by the endothelium, which lines the lacunar spaces and releases endothelium-derived relaxing factor [2,3]. Although their identity remains uncertain, recent evidence points to a few possible neurotransmitters. VIP was proposed as the principal neurotransmitter causing dilatation of penile smooth muscle and immunoreactive VIP was detected within the penile nerves by Andersson et al. [4]. Recently, although VIP has been used in the treatment of erectile dysfunction, some authors have reported that direct administration of VIP does not elicit a physiological erection [5,6]. Therefore other neurotransmitters were investigated; nitric oxide (NO) has been detected at high concentration in penile neurons and ganglion cells, and VIP was found at the same location [7]. NO is a potent relaxant of vascular smooth muscle, its action being mediated through activation of cGMP [8,9]. Ignaro et al. [10] showed that the release of NO, the accumulation of cGMP and the relaxation of corpus cavernosum muscle result from the stimulation of nonadrenergic, noncholinergic (NANC) nerves. Kim et al. [11] also showed NANC relaxation of human corpus cavernosum and the attenuation of relaxation by substances that interfere with the synthesis or effects of NO in in vitro experiments. Moreover, in vivo experiments have shown that inhibitors of NO synthesis and cGMP formation prevent the erectile response to pelvic nerve stimulation, suggesting that the NO/cGMP pathway acts as the predominant mechanism of erectogenic neurotransmission [12,13].

In the present study, we investigated changes of NO synthase (NOS) activity in the cavernosal tissues of rats in which neurogenic erectile dysfunction was induced experimentally by denervation, and evaluated the histopathological changes in these denervated rats using light and electron microscopy.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. References

Twenty-four adult male Sprague-Dawley rats were divided into three equal groups that underwent a sham operation (group 1, controls), or unilateral (group 2) or bilateral (group 3) cavernosal nerve resection. The surgical procedure was performed under anaesthesia with ketamine (0.5 mL/kg). Through a suprapubic incision, the urinary bladder was retracted laterally to locate the major pelvic plexus on the lateral surface of the prostate. The major branch of the cavernosal nerve, running caudally from the pelvic plexus, was isolated and excised, using an operative microscope. The cavernosal tissue was then inverted and all nervous structures (superficial and deep cavernosal nerves) on the cavernosal tissue (identified using frozen sections) were totally resected along the corpus cavernosum. This procedure was also performed under the operative microscope to avoid resecting vascular structures. The pelvic plexus and penile cavernosal nerves were resected only on the right side in group 2, but were resected on both sides in group 3 (Fig. 1). In the controls, the nerves were exposed but not resected. All three groups were then maintained separately for 3 months.

image

Figure 1. Anatomy of the major pelvic ganglion and cavernosal nerve in the rat, and the incisions for denervation.

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Rats were tested for copulatory behaviour, using the mating test described by Walsh et al. [14], 3 months after the operation. The test was terminated at the first intromission or after 45 min. After this examination, all the rats were killed and the corpus cavernosum removed for histopathological examination using light and electron microscopy, and to evaluate NOS activity.

For light and electron microscopy, tissue specimens were fixed in buffered 2.5% glutaraldehyde for 24 h (Sorenson phosphate buffer solution, SPB, a mixture of 20% 0.07 mol/L dibasic Na2HPO4 and 80% 0.2 mol/L KH2PO4 , pH 7.38). After 24 h, the specimens were washed with SPB three times for 15 min. The tissues were post-fixed with equal volumes of SPB and OsO4 2% for 1 h. After re-washing in SPB for 15 min, the specimens were dehydrated in increasing concentrations of alcohol (50–100%). The tissue was then embedded in epoxy resin and half-thickness sections cut using an ultratome (LKB Nova, Uppsala, Sweden). Specimens were stained with toluidine blue and examined by light microscopy. For electron microscopy, 60–90 nm sections were cut using the same microtome, and contrasted with uranyl acetate and lead citrate. The sections were examined using a JOEL-JEM 1200 EM (JEOL, Tokyo, Japan).

For the histopathological study, 2–3 mm thick transverse sections from the shaft of the penis were immediately snap-frozen in liquid nitrogen and stored at −70°C. Cryostat sections (10 μm) were mounted on slides and briefly air-dried at room temperature. The sections were fixed in 4% paraformaldehyde in 0.05 mol/L phosphate buffer, pH 8.0, for 5 min. After rinsing with buffer the sections were incubated in a mixture of β-NADPH (1 mg/mL, Sigma Chem. Co. St Louis, MO, USA), nitroblue tetrazolium (0.1 mg/mL, Sigma) and 0.2% Triton-X in 0.1 mol/L phosphate buffer, pH 8.0, for one hour at 37°C. Washed sections were mounted with buffered glycerol. Sections were not counterstained but the blue staining pattern of the endothelium and cavernosal smooth muscle cells was compared with surgical models treated with PBS and covered with glycerol. The presence of β-NADPH-positive structures was obvious as dense blue regions. The tissues were examined at high magnification and evaluated semiquantitatively as: 0, no staining; +, poor staining, particularly of endothelial cells; ++, moderate staining of the endothelial cells, nerves and smooth muscles of cavernosal tissue and arterioles; +++, strong staining of endothelial cells, nerves and smooth muscles of cavernosal tissue and arterioles.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. References

All rats in groups 1 and 2 achieved intromission, but none in group 3 were capable of erection. Although there were no significant differences among the groups on light microscopy in the corpus cavernosum and vascular systems, there was a prominent atrophy of the penile nerves in groups 2 and 3 (Fig. 2). On electron microscopy, in the control group the axonal structures of the penile nerves, the myelinated and unmyelinated nerve fibres, the endothelial cell structure of the corpus cavernosum and the connective tissue cells like histiocytes, macrophages and the endothelial cells were intact (Fig. 3a). However, ≈1% of the connective tissue cells showed apoptotic figures. In groups 2 and 3, there were degenerative changes of the penile nerves and axonal fibrosis (Fig. 3b); apoptosis was observed in ≈2% of cells. However, these changes were not marked and not statistically different among the groups.

image

Figure 2. Light micrograph of a cavernosal body. a, The cavernosal bodies, arteries and the nerves are apparently intact in the control group. (×200). b, The nerve section shown is apparently degenerated and most of the axons have no or a thinner myelin sheath in the denervated rats. The cavernosal bodies and arteries appear normal. Toluidine blue. ×400.

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image

Figure 3. Electron micrograph of the penile nerves. a, Both myelinated and unmyelinated axons have a normal structure in the control group. b, Degenerated axons, both myelinated and unmyelinated, have been replaced with connective tissue elements, although a few intact myelinated axons were observed in the denervated groups.

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The staining of β-NADPH was recorded as + only in the endothelial cells in the control group (Fig. 4). In group 3, NOS activity was slightly increased and staining was assessed as + in the endothelial cells and sinusoids (Fig. 5). The endothelium and cavernosal smooth muscle cells were stained more obviously in group 2 than in group 3 or the controls (Fig. 6).

image

Figure 4. Nitric oxide synthase activity in the control group. a, Stained endothelial cells. b, Cavernosal body (β-NADPH, ×40).

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image

Figure 5. . Moderately increased nitric oxide synthase activity in the rats from group 3. a, An arterial endothelial section. b, Cavernosal sinusoids (β-NADPH, ×40).

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image

Figure 6. . Nitric oxide synthase activity in rats from group 2; staining was obviously more intense in the endothelial cells (a) and cavernosal structures (b). β-NADPH. ×40.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. References

Nitric oxide was described in 1979 as a potent relaxant of peripheral vascular smooth muscle, with an action mediated by cGMP [15]. NO is synthesized from endogenous l-arginine by NOS, located in the vascular endothelium, and N-substituted analogues of l-arginine inhibit NOS and endothelium-dependent relaxation [16[17]–18]. NO, which acts as a physiological mediator of penile erection, has many functions, e.g. bactericidal, tumoricidal and a smooth muscle relaxant, and has been extensively studied [19]. The release of NO is modulated by several local factors, e.g. oxygen tension, circulating prolactin level and castration [20].

It is unclear whether NO is released directly from nerves or indirectly from the endothelium after activation by a yet unknown neurotransmitter [21,22]. Ehmke et al. showed that the density of NOS/VIP-containing cavernosal nerve fibres in patients with diabetes mellitus (DM) was halved [23]. Therefore, reduced nerve-evoked NO formation may be responsible for impaired corpus cavernosum relaxation in men with cavernosal dysfunction and DM [9]. On the contrary, a recent study on diabetic rats showed that DM did not affect NOS activity. The increase in penile NOS activity was reported to be related mainly to penile NOS of non-neuronal origin [24]. It has also been shown that potential sources of NO within the corpus cavernosum are neurons (Type I), smooth muscle (Type II) and the endothelium (Type III) [20].

Brock et al. claimed that inactivity or impairment of NOS may be related to neurogenic erectile dysfunction, despite increased tissue staining of NO [8]. The present study is amongst the first to examine NOS activity in denervated rats; there was significantly more NOS activity in cavernosal tissues in rats in group 2 and 3 than in the control rats. Thus, the increased activity of NOS may be explained by another neurotransmitter as yet unknown, except possibly cGMP.

Short-term studies in dogs have shown ultrastructural changes in cavernosal smooth muscle after cavernosal nerve ablation [25]. Later studies emphasized the importance of the involvement of neurotransmitters and the CNS in erectile control [26], but Martinez et al. showed that cavernosal nerve resection in rats resulted in insignificant changes to penile erectile tissue. They also observed no decrease in the acetylcholinesterase and catecholamine-positive fibres [27]. It was proposed that the earlier findings may be related to the nerves running along the cavernosal blood vessels or accessory genital nerves, present in previous studies [27,28]. In the present study, all nerves around the vascular bundle were resected in group 3; no electrical activity was detected, although some was present in rats in group 2 (data not shown). There were no significant differences on light and electron microscopy in groups 2 and 3; this may be explained by cross innervation of one corpus cavernosum from the other, as suggested by Walsh et al. [29].

In conclusion, NO is the most important relaxant neurotransmitter in cavernosal tissue and is released from various sources. Although there were no significant histopathological changes in rats with neurogenic erectile dysfunction, NOS activity was increased. Even though functionally impaired, particularly after nerve ablation, NOS activity can continue, induced by unknown neurotransmitters.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. References
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Footnotes
  1. Presented at the meeting of the European Society for Impotence Research, Madrid, October 1–4, 1997.