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Regeneration
of neuronal nitric oxide synthase (nNOS)-containing nerve fibers in rat
corpus cavernosum
Xin-Hua
ZHANG, Li-Quan HU, Xin-Min ZHENG, Shi-Wen LI
Research
Center of Urology and Andrology, Hubei Medical University, Wuhan 430071,
China
Asian J Androl 1999 Sep; 1: 135-138
Keywords:
nitric-oxide
synthase; nerve fibers; cavernous nerve; regeneration
Abstract
Aim: To investigate the effect of cavernous nerve injury on the nNOS-containing nerve fibers in rat corpus cavernosum. Methods: Thirty-three male SD rats were randomized into 3 groups: 5 rats underwent pelvic exploration without transection of cavernous nerve as the sham-operated controls, the unilateral injury group (14 rats) had the cavernous nerve cut on one side, and the bilateral injury group (14 rats) had the nerves cut on both sides. Corpora cavernosa were harvested at the 3rd week and 6th month after surgery. nNOS-positive nerve fibers were examined with strepavidin peroxidase immunohistochemistry techniques (SP method). Results: After bilateral ablation, the nNOS-positive nerve fibers were significantly decreased at both the 3rd week (174) and the 6th month (164). For the unilateral injury group, the nNOS-positive nerve fibers were similarly decreased on the side of the neurotomy at the 3rd week (186), but by the 6th month, the number increased significantly (619) and approximated the level on the contralateral side (8113). Conclusion: In rats after unilateral cavernous nerve ablation, nNOS-containing nerve fibers might regenerate 6 months after operation, but regeneration did not occur in animals with bilateral cavernous nerve injury. Results suggest that during pelvic radical surgery, the cavernous nerve should be preserved at least on one side in order to accomplish adequate regeneration.1 Introduction
Previous
studies indicated that after radical prostatectomy, the erectile function
might recover in 41%-69% of patients in whom at least one neurovascular
bundle is preserved[1,2], but many experienced a recovery time
of 6 to 18 months.
2 Materials and methods
2.1
Materials
Adult
male Sprague Dawley rats (60-90 d, 150-250 g) were purchased from the
Experimental Animal Center, Hubei Medical University. Polyclonal antibody
(prepared in rabbit) to
nNOS was purchased from Santa Cruz (USA), SP kit from Zymend (USA), operating
microscope from Olympus (Japan), and cryoultramicrotomy from AO (USA).
2.2
Animal management
Thirty-three
rats were randomized into 3 groups: sham-operated controls (n=5),
unilateral cavernous nerve ablation group (n=14), and bilateral
cavernous nerve ablation group (n=14).
Under
general anesthesia, a lower abdominal midline incision was made and the
cavernous nerves were identified in the area posterolateral to the prostate[3]
with the assistance of a surgical microscope (10). In the control group,
the cavernous nerves were just identified but not cut. In the bilateral
group, cavernous nerves on both sides were transected and 5-mm segments
removed. For the unilateral group, only the left cavernous nerve was ablated.
One rat in the bilateral group
died from peritonitis on the first day postoperation. Three weeks after
surgery, 5 rats in each group were sacrificed and the middle segment of
corpus cavernosum
was harvested. The remaining rats underwent the same procedure 6 months
after surgery. At that time, only 6 rats in the bilateral group were available
(2 died from upper respiratory infection).
2.3
Strepavidin-peroxidase immunohistochemistry staining (SP method)
Tissue
specimens were embedded in OCT compound and fast frozen at -30 in cryoultramicrotomy.
Cryostat tissue sections were cut at 5 m thickness. After air-dried,
sections were fixed for 10 min in pure acetone. The main steps of SP
method involved: 0.3% H2O2 inactivating endogenous
peroxidaseblocked by normal
goat serumtreated with 1:50 dilution of antibody to nNOS30-min incubation at
37followed by an overnight incubation at 4 in refrigeratorbiotin-anti-antibody
to nNOSstrepavidin peroxidasestained with DAB substrateobserve 5
random fields under light microscope (magnification400) and count the
number of brown-stained positive nerve fibers present in the fields.
2.4
Statistical analysis
Data were compared with the non-parametric kruskal-Wallis followed by Nemenyi test. Statistical significance was set at P<0.05.
3 Results
The
number of nNOS-positive nerve fibers in each group stained by immunohistochemistry
is shown in Table 1. It could be seen that three weeks after bilateral
ablation, only a minimal number of nNOS-positive nerve fibers were present
(Figure 1A). A mean of 174 fibers
were found in 5 random fields. Six months later, the immunohistochemical
staining pattern was similar to those seen in
Figure 1A, and the number
of nNOS nerve fibers was not significantly different (164 in 5 random
fields, P>0.05). For the sham-operated group, plenty of nNOS-containing
nerve fibers were seen (Figure 1B)
with 9017 fibers in 5 visual fields, being significantly higher than
that of the bilateral ablation group (P<0.002). In the unilateral
ablation group, the number of nNOS-positive fibers on the side of neurotomy
at the 3rd week was 186, which was similar to that in the bilateral
ablation group (P<0.05).
Table
1. nNOS-Positive nerve fibers in corpus cavernosum (5 random fields).
|
Time |
Control |
Unilateral |
Bilateral |
|
|
Intact |
Neurotomy |
|||
|
3
weeks |
9017 |
186 |
8810 |
174 |
|
6
months |
|
619 |
8113 |
164 |
At
the 6th month, however, a significant increase of nNOS-positive fibers
was found (Figure 1C) with 619
fibers in 5 random fields. When compared with the 3rd week finding and
the results of the bilateral ablation group, the differences were both
significant (P<0.05). The data of the contralateral side were
similar to that of the control group (P>0.05) , both at the
3rd week and 6th month.
4 Discussion
Recent
in vitro and in vivo studies have strongly suggested that
nitric oxide (NO)
is the main neurotransmitter of the NANC nerves involved in penile erection[4-6].
The synthesis of NO from L-arginine is catalyzed by nitric oxide
synthase (NOS). There are mainly 2 types of NOS in the penis, the endothelial
NOS (eNOS) and the nNOS, of which the nNOS may play the principal role
in penile erection and is more interesting to researchers[7-8].
Within
the past decade, cadaveric dissection and animal studies have identified
the nerve responsible for penile erection, now known as the cavernous
nerve[9]. For the study of penile erection, many animal models
of neuronal erectile dysfunction (ED) have been established, among which
the rat is the excellent animal as demonstrated
by Quinlan and associates[3].
In
urology practice, a number of patients experienced ED after radical pelvic
surgery, such as radical prostatectomy, radical cystectomy, and abdominoperineal
resection of rectum. Neurogenic ED arising from these surgeries is thought
to arise from
complication of cavernernous nerve injury. Among these ED patients, some
regained potency after 6 to 18 months. We believe that regeneration of
nerve fibers has been involved in this recovering process.
In
our unilateral neurotomy group, the nNOS-positive nerve fibers decreased
to a minimum at the
side of neurotomy at the 3rd week. However, by the 6th month, the fibers
increased almost to the level of the intact side. We thought that regeneration
of nNOS nerve fibers had taken place in the neurotomy side. The present finding
also provided a good explanation to the result of Walsh et al[5],
who preserved the cavernous nerve unilaterally during radical pelvic surgery
and erectile function did recovered in a number of cases.
In
our bilateral ablation group, both at the 3rd week and 6th month after
bilateral cavernous nerve neurotomy, only a minimal number of nNOS-positive
nerve fibers could be found. Apparently, regeneration did not occur. Permanent ED
following pelvic surgery can be explained by injury to bilateral cavernous
nerves. The regeneration mechanism of nNOS nerve fibers in the corpus
cavernosum is not clear. One possible explanation is the sprouting of
intact nerves from the contralateral corpus cavernosum.
References
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Correspondence
to Dr. Xin-Hua ZHANG.
Tel: +86-27-8731 7743 Fax: +86-27-8731 7743
Received
1999-08-09 Accepted 1999-09-13
