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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 32  |  Issue : 1  |  Page : 34-39

Can cavernous nerves be spared after radical prostatectomy? Evidence from animal studies


1 Graduate Institute of Biomedical and Pharmaceutical Science, Fu-Jen Catholic University; Department of Urology, Fu Jen Catholic University Hospital, Fu Jen Catholic University, New Taipei City, Taiwan
2 Department of Urology, Fu Jen Catholic University Hospital; School of Medicine, College of Medicine; Graduate Institute of Applied Science and Engineering, Fu Jen Catholic University, New Taipei City, Taiwan
3 School of Medicine, College of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan

Date of Submission24-Jul-2020
Date of Decision15-Oct-2020
Date of Acceptance25-Nov-2020
Date of Web Publication27-Mar-2021

Correspondence Address:
Yi-No Wu
School of Medicine, College of Medicine, Fu Jen Catholic University, New Taipei City
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/UROS.UROS_110_20

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  Abstract 


Purpose: The current study aims at evaluating penile erectile function after nerve-sparing radical prostatectomy (RP) based on evidence from a reliable animal model. The previous model in which the cavernous nerve (CN) is temporarily crushed is reviewed and compared to the present method of prostate lobes removal near the CN to determine which is more relevant to the clinical situation. Materials and Methods: Twenty-four rats were divided into three groups. One group was subjected to temporary CN crushing, one group was subject to prostate lobes removal near the CN, and the last group was preserved (sham group). All rats were re-examined at 4 weeks after the first surgical procedure. The pathological changes of the CNs were evaluated by their gross appearance and immunohistochemistry. Intracavernosal pressure (ICP) was measured as a parameter of male erectile function. Results: The results of the current study demonstrate that the removal of the prostate lobes near the CN led to the degeneration of CN, even with careful sparing techniques. Four weeks after the first surgical procedure, the rats' abdomens were reopened, and CNs were identified in only 60% of the rats with prostate lobes removal near the CN. Furthermore, the remaining CNs in this group were found to be histologically degenerated, with poorer erectile function presented by ICP. In contrast, the CNs after temporary crush were only mildly injured and demyelinated, with evidence of regeneration. The changes in the rats with prostate lobes removal near the CN are much more similar to those in rats with clinical RP with CN sparing. Conclusion: The current study concluded that, in the rat model, CNs will be injured, degenerated, and eventually disappear after prostate lobes removal near the CN; this is very similar to what is observed in clinical RP. Protection of the CNs for erectile function preservation should be investigated further using this animal model.

Keywords: Cavernous nerve, erectile dysfunction, nerve crushing, nerve-sparing, radical prostatectomy


How to cite this article:
Chiang HS, Chang ML, Wu YN. Can cavernous nerves be spared after radical prostatectomy? Evidence from animal studies. Urol Sci 2021;32:34-9

How to cite this URL:
Chiang HS, Chang ML, Wu YN. Can cavernous nerves be spared after radical prostatectomy? Evidence from animal studies. Urol Sci [serial online] 2021 [cited 2021 Apr 19];32:34-9. Available from: https://www.e-urol-sci.com/text.asp?2021/32/1/34/312446




  Introduction Top


Radical prostatectomy (RP) is one of the standard guidelines for the treatment of localized prostatic cancer. Erectile dysfunction (ED) is the major complication of RP, and it has been reported that the incidence of ED after RP ranges from 15% to 80%.[1],[2] ED after RP is believed to be due to the radical procedure that causes injury to the bilateral cavernous nerve (CN). Preservation of the CN during the surgery, the so-called nerve-sparing technique of RP, was developed for postoperative sexual function recovery.[3],[4] However, even after the technical advancement of RP that improved the visibility of the CN, especially the evolution from open surgery to laparoscopic or robotic surgery, ED remains a serious problem after RP.[5],[6]

The CNs are located deep in the bilateral inguinal area, whereby the erection of the male penis results from nerve conduction. Erectile function can be measured and quantized by CN stimulation in animal experiments. In our experience with the rat model, the CN is very thin and difficult to identify. We performed the studies following the previously described model by clamping the CN at different time periods to imitate nerve injury after RP.[7],[8],[9],[10],[11],[12],[13],[14],[15] Eventually, we found that the experimental model using a nerve clamp does not accurately reflect CN injury after RP.

The first important question is, what would happen following CN damage after RP, even if it is preserved using the nerve-sparing technique? There are no reports concerning re-examination of the CN after RP because it is almost impossible to reoperate the patient after RP for this reason. Therefore, it is important to determine the differences in the long-term pathological changes of the CN between the clamping and preserving RP procedures in an animal study. For example, it is important to elucidate whether or not these nerves are still surviving and/or functioning.

Second, if the animal's prostate is not removed, the CNs do not seem to be injured by a procedure similar to clinical RP. Does the way in which the CN is temporarily clamped cause ED of the animal, similar to what is observed in removal of the animal's prostate?

This study aims at exploring the severity of the damage to the CNs of animals after prostate removal. A re-open procedure was performed after animal prostate removal, erectile function was measured by stimulating the CNs, and the results were compared to those of the previous model, in which CN injury was induced after a period of clamping. The differences in pathophysiological changes of the erectile tissues, as well as the resulting ED, were compared between the two models.


  Materials and Methods Top


Experimental animals

In total, 24 male Sprague-Dawley rats (8 weeks old) were used in this study. The rats were supplied by BioLasco Taiwan Co., Ltd (Taipei, Taiwan), and the study was approved by the Fu Jen Catholic University Animal Care and Use Committee (IACUC approval no: A10730). All study procedures and methods were performed in accordance with the approved guidelines.

Experimental design

The animals were randomly assigned to three groups, including a sham group, and each group was designed to undergo laparotomy. In the bilateral CN crush (BCNC) group, the CNs were identified and then clamped for 2 min, as in our previous procedure, to destroy the erectile function. In the prostate removal (cavernous nerve-sparing in prostate remove [CNSP]) group, the prostate lobes near the CN were completely removed with CN sparing. For the sham group, no further surgical manipulation was performed.

Re-laparotomy was performed 4 weeks after the surgical procedure for each group of rats, and erectile function was assessed by intracavernosal pressure (ICP). Erectile tissues of every rat were subject to immunohistochemistry and transmission electron microscopy (TEM). The erectile tissues of the rats belonging to each group were compared in relation to erectile function.

Surgical procedures

The animals were first anesthetized with an intraperitoneal injection of sodium pentobarbital (40 mg/kg). A lower abdominal incision was made after shaving, cleaning the skin, and then completing the sterilization with an iodine-based solution. The prostate gland was exposed, and the posterolateral CNs and the major pelvic ganglion (MPG) were identified. In the group of rats undergoing CN crushing, the nerve was isolated and crushed for 2 min per side, 5 mm from their origin in the MPG, using a hemostat clamp (Roboz Surgical Instrument Co Inc., Gaithersburg, MD, USA). In the group of rats subjected to prostate removal, the prostate was meticulously dissected and detached, with the CNs remaining intact in the previous site. In the sham group, no further surgical procedures were performed. The abdomens were then closed with one-layer sutures and sterilized with gauze protection.

Measurement of erectile responses and arterial blood pressure

After the CNs were exposed, the crura of the penis was identified and a 24G needle containing 50 U/mL heparin solution was inserted into one side of the crus and then connected to polyester-50 tubing for ICP measurement with an MP 36 pressure transducer (Biopac System Inc., Goleta, CA, USA) and BSL software, version 3.7.3. The CNs were stimulated using a bipolar stainless steel electrode. A monophasic rectangular pulse was rested by a computer with a DS3 constant-current isolated stimulator (AutoMate Scientific Inc., CA, USA). The stimulus parameters included a 7.5-mA amplitude, 20-Hz frequency, 0.2-ms pulse width, and 60-s duration. Arterial blood pressure was assessed concurrently with ICP. The real-time response of the erectile tissue was determined based on the maximal ICP, minimum ICP, changes in ICP (ΔICP), area under the ICP curve, and mean arterial blood pressure.

Histology and immunohistochemistry

Rats were sacrificed by the administration of a high dose of pentobarbital sodium solution, and the tissues from 24 rats were collected from the middle portion of the penis. The tissues were then fixed with 10% formaldehyde (w/v) for 24 h, followed by dehydration, postfixation, and embedding in paraffin blocks. The embedded tissues were sliced into five slices, fixed, and dewaxed in xylene for 10 min (total, 3 times). The hydration of tissues was processed by graded alcohol (100%, 95%, 80%, and 70%) and ddH2O for 5 min. Subsequently, slides were soaked in a blocking solution containing 10% goat serum, 2% bovine serum albumin, and 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature. For further analysis of α-smooth muscle actin (α-SMA), mouse anti-α-SMA (Abcam Inc., Cambridge, MA, USA) was added and incubated at 4°C overnight. Next, the tissues were incubated with a secondary antibody (1:400 diluted) conjugated to Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) for 1 h. Nuclear co-localization was performed using 4',6-diamidino-2-phenylindole (DAPI). The immunohistochemistry results of the samples were collected by fluorescence microscopy, and ImageJ (National Institutes of Health, Bethesda, MD, USA) was employed for the computerized histomorphometric analyses.

Transmission electron microscopy

Segments of CN tissue were obtained from each group of rats and sliced into small pieces for TEM analysis. The segments were treated overnight with 2.5% phosphate-buffered glutaraldehyde (0.1 M, pH 7.2) to fix the tissue, and the postfix process used 1% phosphate-buffered osmium tetroxide (0.1 M, pH 7.2). The segments were then dehydrated in graded concentrations of ethanol alcohol and embedded in Epon-812. Subsequently, 1-μm sections (semi-thin) were stained with toluidine blue, and ultra-thin sections from selected blocks were stained with uranyl acetate and lead citrate. All images of the segments were observed using a JEOL JEM-1400 transmission electron microscope (JEOL, Japan).

Statistical analysis

Statistical analyses were performed using SPSS software, version 18.0 (SPSS Inc. Chicago, IL, USA). Data are presented as mean ± standard deviation. One-way ANOVA was used to compare the difference in each component among groups. The discrepancy in erectile function was measured by ICP, which was evaluated by Pearson's correlation analysis. A two-sided P < 0.05 was considered statistically significant.


  Results Top


Gross findings

During the surgical procedure, the CNs were carefully separated from the surface of the prostate between the MPG and apex of the prostate. The abdomens were reopened 4 weeks after the surgery, and all of the CNs could be identified in the BCNC group and sham group. In contrast, only 60% of the CNs could be identified in the CNSP group [Figure 1] and [Figure 2], and only 20% of the normal ICP could be measured [Figure 2].
Figure 1: Anatomical pathology of the major pelvic ganglion and the cavernous nerve in rats

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Figure 2: Percentage of nerve preserved animal number (a) and intracavernosal pressure response (b)

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Inracavernonosal pressure change

We measured the arterial pressure and the ICP of the CNs in the different groups after reopening of the abdomens and identified the existing CNs. The ICP represents the erectile function of the penis, which is responded to by the CNs. We found that the ICP of the rats in the BCNC and CNSP groups was significantly lower than that in the sham group [Table 1]. More remarkably, the ICP of the CNSP group was significantly lower than that of the BCNC group, indicating that CN injury is more severe with prostate lobes removal near the CN than with temporary CN crush. Furthermore, 4 weeks after surgery, the presence of CNs degeneration and subsequent ED (ICP decrease) in the CNSP group clearly demonstrated that clinical RP could not preserve erectile function, even when accompanied by CN sparing.
Table 1: Results of the sham, bilateral cavernous nerve crush, and cavernous nerve-sparing in prostate remove groups

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Immunocytochemistry staining

Immunofluorescence staining for α-SMA expression in the corpus cavernosum demonstrated that the volume of smooth muscle was reduced in the BCNC and CNSP groups compared to the sham group. In particular, in the CNSP group, considerably, more of the smooth muscle in the corpus cavernosum is replaced by fibrotic tissues, and this irreversible change may cause ED [Figure 3].
Figure 3: (a) Immunofluorescence staining for α-smooth muscle actin expression in the corpus cavernosum of sham, bilateral cavernous nerve crush, and cavernous nerve sparing in prostate remove groups. (b) α-smooth muscle actin expression in the corpus cavernosum. *Indicates a significant difference with the sham group (P < 0.05)

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Scanning transmission electron microscopy

Scanning TEM was performed to investigate the pathological changes in the CNs and examine the seriousness of the nerve degeneration. The CNs degeneration was more prominent in the CNSP group than the sham group, and demyelination of the CNs could be identified over a large area. Furthermore, the number of demyelination nerves in this group was much higher than that in the BCNC group. Remyelination occurred in the BCNC group but was never observed in the CNSP group [Figure 4]. The ultrastructure of the pathological change in the CNs provided further evidence for the use of CNSP in an animal model that has a similar outcome to that of clinical RP.
Figure 4: Transmission electron microscope images of cavernous nerve in the sham, bilateral cavernous nerve crush, and cavernous nerve sparing in prostate remove groups. The red box is magnified in the upper right corner. The scale bar indicates 5 μm

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  Discussion Top


In the generation of the model of CN injury, it became clear that it was very difficult to identify the nerve and may have been easier through the MPG.[16] The CN is very thin, and we followed the previously described procedure to crush the nerve temporarily to imitate injury of the nerve after prostatectomy. Electrical stimulation of the CN to measure the ICP was also performed as a standard by which to determine erectile function. However, we questioned what would happen to the CN if the prostate was removed. After a serial trial to preserve the bilateral CNs, we successfully removed the prostate. This procedure can be considered similar to the clinical “nerve-sparing technique” for RP in prostatic cancer.

We performed CN stimulation to induce penile erection to prove that the nerve was functioning well. The ICP is markedly reduced if it is measured immediately after the cavernous is injured, either by clamping or performing prostatectomy. In comparison to the ICP of the penile erection after clamping the CN, prostate removal led to greater damage to erectile function (sham operation as normal control).

In an experiment to observe the long-term pathological changes in the CN after prostate removal, we reopened the animal at different times, from 4 weeks to 6 weeks, after prostatectomy. Following prostatectomy, only 60% of the CN was still able to be identified; therefore, even after nerve sparing during prostatectomy, some of the CNs may be completely degenerated postoperatively within a certain period. Furthermore, if the CN remained, the erectile function was remarkably impaired, as determined by the ICP measured after electrical stimulation among the different groups. The ICP of rats whose CN was injured by clamping was higher than that of the rats, in which the prostate lobes near the CN had been removed. Thus, after prostatectomy, even with CN preservation, the nerve becomes degenerated or dysfunctional and eventually leads to erectile failure.

Similarly, pathological changes were noticed in the cavernous tissue and CN of the rats 4 weeks later, either after clamping the CN or after removal of the prostate lobes near the CN. Electron microscopy examination showed that demyelination of the CN after removal is very prominent compared to that after clamping. The demyelination of the CN represents an irreversible change in the erectile tissue, which may reflect irreversible ED after RP. Clinically, ED after RP is frequently unresponsive to medical treatment, such as oral phosphodiesterase 5 inhibitors or intracavernosal injection; this type of ED cannot be spontaneously resolved and is one of the frequent indications of the implantation of the penile prosthesis for young patients. In contrast, if we clamped the CN for 2 min, demyelination phenomena were not so remarkable; this is one reason why the ED of the rats (ICP decreased) is not as serious as that observed following prostatectomy. The abdomens of the animals were reopened 4 weeks after clamping of the CN, and all of the CNs were identified and remained intact, with no degeneration or gross abnormalities observed. This might be related to the clinical observation that a rather mild CN injury could be self-repaired by remyelination and lead to spontaneous recovery of erectile function in some patients.

Based on the results of this study, we concluded that temporary clamping of the CN can only induce mild, temporary injury of the CN and may provide the opportunity for spontaneous recovery as a result of nerve remyelination. This type of CN injury is different from the complications experiences with prostate lobes removal near the CN, especially clinical, in the case of RP. For the RP, even combined with the CN-sparing technique, associated injury of the CNs, such as tearing, cauterization, and adjacent vessel ablation, was inevitable and may ultimately lead to the degeneration of CNs.[17],[18] Based on the measurement of ICP in rats 4 weeks after surgery, the ED caused by prostate lobes removal near the CN is more serious than that caused by CN clamping. This may be one of the reasons that our previous animal studies of ED using CN clampnig had a better recovery rate with different treatments. The results of these previous studies did not accurately represent definitive therapies for ED patients after RP.

Future studies of cavernous injury after RP should be conducted using an animal model of prostate lobes removal near the CN, in which ED caused by CN injury could also be predicted more precisely. Since ED is an inevitably high-risk complication in RP combined with CN sparing,[19] it is necessary to develop other innovations for nerve protection. In the era of minimally invasive surgery, especially with the rise in popularity of robotic prostatectomy, it may be more feasible to administer medication or a device to enhance the viability of the CNs in addition to the surgical sparing technique.[20]


  Conclusion Top


The current study concluded that, in the rat model, CNs will be injured, degenerated, and eventually disappear after prostate lobes removal near the CN; this is very similar to what is observed in clinical RP. Prospective research on an animal model of prostate removal is ongoing in our laboratory. We believe that this model represents the most effective way to provide reliable evidence before clinical trials.

Acknowledgment

The authors would like to thank Mr. Yen-Sheng Wu of the electron microscope laboratory of Tzong Jwo Jang, Fu Jen Catholic University for technical assistance.

Financial support and sponsorship

This work was supported by the Cardinal Tien Hospital and Fu Jen Catholic University Education and Development Cooperation Project (108-CTH-FJU-04).

Conflicts of interest

Prof. Han-Sun Chiang, an editorial board member at Urological Science, had no role in the peer review process of or decision to publish this article. The other authors declared that they have no conflicts of interest.



 
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