Journal of Neuroscience Research 00:00–00 (2015)
Role of Oxidative Stress in Surgical Cavernous Nerve Injury in a Rat Model Hui Wang,1 Xie-Gang Ding,1* Shi-Wen Li,1 Hang Zheng,1 Xin-Min Zheng,1 Shrestha Navin,1 Lu Li,2 and Xing-Huan Wang1 1
Department of Urology and Andrology, Zhongnan Hospital, Wuhan University, Wuhan, People’s Republic of China 2 Department of Nutrition and Food Health, School of Public Health, Wuhan University, Wuhan, People’s Republic of China
This study investigates the role of oxidative stress in surgical cavernous nerve (CN) injury in a rat model. Eighty-four male Sprague-Dawley rats were randomly divided into three groups: group 1, sham-operated rats; group 2, bilateral CN-crushed rats; and group 3, bilateral CNtransection-and-sutured-immediately rats. Oxidative stress was evaluated by malondialdehyde levels, super oxide dismutase (SOD) activities, and glutathione peroxidase (GPX) activities in serum. Erectile function was assessed by CN electrostimulation at 3 months with mean maximal intracavernous pressure (ICP) and maximal ICP per mean arterial pressure. Nerve injury was assessed by toluidine blue staining of CNs and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining of penile tissue. GPX protein expression and nitrotyrosine-3 (NT-3) levels in penile tissue were measured. Erectile function and the number of myelinated axons of CNs and NADPH-diaphorase-positive nerve fibers were statistically decreased between groups, from sham to crush to transection. For markers, both nerve-injury groups showed increased oxidative stress markers at early time points, with the transection group showing greater oxidative stress than the crushed group and values normalizing to sham levels by week 12. GPX expression and NT-3 levels in penile tissue were in concordance with the results of SOD and GPX. These results show that oxidative stress plays an important role in injured CNs, and different methods of CN injury can lead to different degrees of oxidative stress in a rat model. VC 2015 Wiley Periodicals, Inc.
Recent findings from experimental studies have suggested a significant association between reactive oxygen species (ROS) and ED, especially in diabetic animal models and in rat models of radiation-induced ED (Suresh and Prakash, 2011; Kimura et al., 2012; Maiorino et al., 2014). In addition, evidence has indicated that neurotomy and manipulation of the CN cause oxidative stress in rat corpus cavernosum and that oxidative stress is more significant in the nerve-resection group (Ozkara et al., 2006). CN injury and transaction ultimately lead to apoptosis of the corporal smooth muscle cells and fibrosis of the corporal tissue (Suresh and Prakash, 2011). Oxidative stress results from an imbalance between the formation of free radicals and the ability of the antioxidants to scavenge ROS (Agarwal et al., 2006; Zhang et al., 2011) and causes accumulation of ROS in the tissue. ROS, which may accumulate in the corpus cavernosum during oxidative stress, has been shown to be a potential cause of ED (Alan et al., 2010). Oxidative stress could be determined in experimental studies by glutathione peroxidase (GPX) activities and nitrotyrosine (NT) levels in the tissue as well as by superoxide dismutase (SOD) activities, GPX activities, and malondialdehyde (MDA) levels in serum. This study investigates the effects of bilateral CNs crushed or bilateral CNs transectioned and sutured immediately to nitric oxide synthase (NOS)containing nerve fibers and pressure after electrostimulation in the rat corpus cavernosum and seeks to determine whether these procedures produce oxidative stress within rat cavernous tissues and peripheral blood.
Key words: erectile dysfunction; cavernous nerve; oxidative stress
It has been reported that the mainstays of treatment for clinically localized prostate cancer (PC) are radical prostatectomy (RP) and radiation therapy (Barazani et al., 2014). Both therapeutic modalities are associated with a high risk of erectile dysfunction (ED). Despite the advent of the cavernous nerve (CN)-sparing surgical technique, ED remains a major problem following RP for PC. The pathophysiological mechanisms of ED, as a complication following nerve-sparing surgical technique, are not completely understood. C 2015 Wiley Periodicals, Inc. V
Contract grant sponsor: National Natural Science Foundation of China; Contract grant number: NSFC303163398 (to X.-G.D.) H. Wang and X.-G. Ding contributed equally to this work. *Correspondence to: Xie-Gang Ding, Department of Urology, Zhongnan Hospital, Wuhan University, 169 Donghu Road, Wuhan 430071, People’s Republic of China. E-Mail: [email protected] Received 8 October 2014; Revised 10 November 2014; Accepted 26 November 2014 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jnr.23545
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MATERIALS AND METHODS Experimental Animals Eight-four white, male Sprague-Dawley rats (weighing 300–350 g) were randomly divided into three groups: group 1, sham-operated rats (n 5 28); group 2, bilateral CN-crushed rats (n 5 28); and group 3, bilateral CN-transection-and-suturedimmediately rats (n 5 28). All rats were obtained from the Experimental Animal Center of Wuhan University, and the Institutional Animal Care and Use Committee of Wuhan University approved all the experimental protocols followed in this study. All surgery was performed with animals under pentobarbital sodium, and all efforts were made to minimize suffering. Surgical Procedures Animals were anesthetized with an i.p. injection of 1% pentobarbital sodium (50 mg/kg), and operations were performed with a surgical microscope (SXP-1C; Medical Optical Instruments Factory of Shanghai Medical Instruments, Shanghai, China). A midline abdominal incision was made to expose the bladder and prostate. After identification of the MPG on the lateral side to bilateral prostates, the CNs, tracking posterolaterally, were identified and isolated. In the sham-operated group, with a midline incision and identification of the bilateral CNs, no further surgical manipulation was done. In the bilateral CN-crushed group, at the point of 3 mm distal to the MPG, the bilateral CNs were crushed with a hemostat clamp (HC-X020; Cheng-He Microsurgical Instruments Factory, Ningbo, China) for 2 min. In the bilateral CN transectionand-sutured-immediately group, CNs were sutured with 10-0 microscopic sutures. In all rats, the wound was closed in two layers. Functional Evaluation and Tissue Procurement The erectile function was calculated in all rats after 3 months by electrostimulation of the CNs and by measuring intracavernous pressure (ICP; n 5 7 in each group). The penis was dissected and the corpus cavernosum and crura of the penis were exposed; the CNs were exposed and isolated via a midline abdominal incision. A 22-G butterfly needle, which was connected to a PE-50 tube and filled with 250 U/ml heparin solution, was inserted into the right penis crura for ICP measurement. Electrostimulation was performed with a stainless-steel bipolar hook electrode (probes 2 mm in diameter and separated by 1 mm). The stimulus parameters were 1.5 mA, frequency 20 Hz, and pulse width 0.2 msec for a duration of 1 min. The maximal amplitude of ICP during cavernous nerve electrostimulation was selected for statistical analysis in each animal. Mean arterial pressure (MAP) was monitored by a PE-50 tube that had been inserted into the carotid artery. The ICPs and MAP were recorded in all rats in a bioinformation acquisition system (BL-420F; Chengdu TME Technology, Chengdu, China). After functional evaluation, the CNs were excised for toluidine blue staining, and the midshaft penis was excised and divided transversally into two pieces, one part for nicotinamide adenine dinucleotide phosphate (NADPH)diaphorase staining and the other part for GPX activities and NT level assays.
Oxidative Stress Evaluation Oxidative stress was evaluated by MDA levels, SOD activities, and GPX activities in serum at 1, 2, 4, and 12 weeks. These oxidative stress parameters were determined with assay kits (Nanjing Jiancheng Biology Research Institute, Nanjing, China) according to instructions provided by the manufacturer. Animals were anesthetized with an i.p. injection of 1% pentobarbital sodium (50 mg/kg) and fixed on the stage. After the heartbeat of the site was touched, a syringe with a 4-G needle was used to puncture the most obvious place of heartbeat, and the blood flowed into the syringe; about 6–10 ml of blood was collected from each rat. When the blood had coagulated at room temperature, it was put in the centrifuge at 4,000 rpm for 10 min, and then the serum was obtained. Western Blotting The corpus cavernosum was obtained from each group of rats (n 5 28), and tissues were lysed in triple-detergent radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitor cocktails and phenylmethylsulfonyl fluoride. Tissue protein lysates were centrifuged for 10 min at 14,000 rpm, and then supernatants were harvested for the following experiments. Protein concentrations were estimated with BCA kits. Protein samples (50 lg) were denatured at 95 C for 5 min, and electrophoresis was performed on a 12% discontinuous SDS-polyacrylamide gel and transferred to a PVDF membrane. The membranes were reacted with blocking buffer (5% skim milk in Tris-buffered saline Tween 20 buffer) for 30 min at ambient temperature and incubated with antiGPX antibody (1:1,000; Epitomics, Burlingame, CA). After secondary antibody incubations at ambient temperature for 1 hr, signals were visualized by enhanced chemiluminescence. For NT-3, a mouse monoclonal antibody (1:1,000) was used that detects numerous nitrosylated proteins. We analyzed one immunopositive band at 35 kDa. NT-3 expression levels were quantified for each sample. Densitometry results were normalized by b-actin expression. NADPH-Diaphorase Staining NADPH-diaphorase staining was used to identify NOS in the penile nerve fibers of the proximal penile segment. A midportion of each penile segment was harvested and immediately fixed in 0.1 mol/liter phosphate-buffered saline (PBS; pH 8.0) containing 0.002% picric acid and 2% formaldehyde for 4 hr, then transferred to 30% sucrose and stored overnight at 4 C. The segments were then embedded in optimal cutting temperature compound (Beijing Zhongshan Golden Bridge Biotechnology, Beijing, China). Serial cryosections (8 lm) adhered to the charged slides. After having been air dried for 5 min, the sections were incubated with 0.1 mM NADPH, 0.2 mM nitroblue tetrazolium, and 0.2% Triton X-100 (SigmaAldrich, St. Louis, MO) in 0.1 mol/liter PBS, pH 8.0, for 4 hr at 37 C. The reaction was terminated by washing the sections in buffer. The staining pattern was assessed by counting the number of NADPH-diaphorase-positive nerves in the dorsal nerves (3400 with light microscopy). The endothelium staining was excluded from the count. Journal of Neuroscience Research
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Fig. 1. Changes of maximal ICP and MAP after electrostimulation of CNs at 3 months. A: ICP and MAP in group 1. B: ICP and MAP in group 2. C: ICP and MAP in group 3. D: Maximal ICP in all three groups. E: Maximal ICP/MAP ratio in all three groups. There were seven rats (n 5 7) at week 12 for ICP in each group. *P < 0.05 compared with group 2, #P < 0.05 compared with group 1. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Toluidine Blue Staining Three to four millimeters of CN was obtained from the main trunk of both sides at equivalent points, which were distal to the site of injury. All harvested nerves were fixed in 3% (w/ v) cold glutaraldehyde. The nerve segments were dehydrated with ethanol and postfixed with 1% osmium tetroxide before Journal of Neuroscience Research
being infiltrated with a graded araldite–propylene oxide mixture and embedded in Epon 812 (Serva Feinbiochemica, Heidelberg, NY). An LKB III ultramicrotome (LKB Produkter, Broma, Sweden) was used to obtain 1-lm cross-sections of the embedded nerve, which were then stained with 1% toluidine blue and examined with light microscopy. Images were
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Fig. 2. Serum levels of MDA and serum activities of SOD and GPX in each group. A: Serum activities of SOD (U/ml) in all three groups. B: Serum activities of GPX (U/ml) in all three groups. C: Serum levels of MDA (nmol/ml) in all three groups. *P < 0.05 compared with group 2, # P < 0.05 compared with group 1. There were 28 rats in each group, and each group of different periods (weeks 1, 2, 4, and 12) had seven rats.
captured with an Olympus-DP12 camera (3100 oil immersion objective) and processed in Image Pro-Plus 3.0 (Media Cybernetics, Bethesda, MD). Statistical Analysis Data are expressed as mean 6 SD. The results were analyzed by one-way ANOVA, with significance indicated at P < 0.05. If the difference was significant, a Student-NewmanKeuls test was carried out.
RESULTS Evaluation of Erectile Function Erectile function was evaluated by the maximal ICP (Fig. 1). No erectile dysfunction was identified in group 1, with a pressure of 102.5 6 9.0 cmH2O. Group 2 showed a significant reduction in maximal ICP of 52.4 6 11.2 cmH2O, consistent with a state of ED. In group 3, the mean maximal ICP was 34.1 6 9.7 cmH2O,
which was significantly lower than that in other groups (P < 0.05). The change of maximal ICP/MAP ratio was consistent with the maximal ICP in the three groups. Oxidative Stress Parameters in Serum Figure 2A,B shows the mean activities of SOD and GPX in serum. At weeks 1, 2, and 4, the mean activities of SOD and GPX were significantly increased in group 3 compared with those in the other two groups (P < 0.05). At weeks 1 and 2, the mean activities of SOD and GPX were significantly increased in group 2 compared with those of group 1 (P < 0.05). At week 4, there was no appreciable difference between group 1 and group 2 (P > 0.05). At week 12, there were no appreciable differences in the mean activities of SOD and GPX for the three groups (P >0.05). Figure 2C shows the mean levels of MDA in the serum. At weeks 1, 2, and 4, the serum level in group 3 Journal of Neuroscience Research
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Fig. 3. NADPH-diaphorase staining of dorsal nerves. A: In group 1, there was predominant blue staining of nerve fibers. B: In group 2, there was a paucity of blue-stained fibers. C: In group 3, there was a significant decrease in the number of blue-stained nerve fibers relative to groups 1 and 2. D: Results of NADPH-diaphorase-positive nerve fibers of dorsal nerves for each group. Arrows in A–C indicate
NADPH-diaphorase-positive nerve fibers. There were seven rats (n 5 7) at week 12 for NADPH-diaphorase staining in each group. *P < 0.05 compared with group 2, #P < 0.05 compared with group 1. Scale bars 5 600 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
was significantly increased compared with that of the other two groups (P < 0.05). At weeks 2 and 4, there was no appreciable difference between group 1 and group 2 (P > 0.05). At week 12, there were no appreciable differences among the three groups (P > 0.05).
(P < 0.05) compared with group 1, and group 3 GPX protein expression was much higher than that in group 2 (P < 0.05). However, it had decreased to normal levels in the two groups at weeks 4 and 12 (Fig. 5). Western blot analysis of NT-3 showed one immunopositive band at 35 kDa. At weeks 1, 2, and 4, the NT-3 level was significantly increased in group 2 (P < 0.05) and group 3 (P < 0.05) compared with group 1, and the group 3 level was much higher than that in group 2 (P < 0.05). However, it had decreased to normal level in the two groups at week 12 (Fig. 6).
NADPH-Diaphorase Staining There were significantly fewer NADPHdiaphorase-positive nerve fibers in the dorsal nerves of groups 2 and 3 than in those of group 1 (P < 0.05), and group 3 had a significant decrease compared with group 2 in the number of NADPH-diaphorase-positive nerve fibers (P < 0.05; Fig. 3). Toluidine Blue Staining of CNs The numbers of myelinated axons in groups 2 and 3 were significantly less than those in group 1 (P < 0.05), and group 3 showed a significant decrease compared with group 2 in the number of myelinated axons (P < 0.05; Fig. 4). Western Blot Analysis At weeks 1 and 2, GPX protein expression was significantly increased in group 2 (P < 0.05) and group 3 Journal of Neuroscience Research
DISCUSSION Despite the fact that CNs are preserved in nerve-sparing surgical procedures performed in radical prostatectomies to maintain erectile function, erectile capacity cannot always be maintained. Many previous studies have suggested that oxidative stress and apoptotic changes occur in cavernous body smooth muscle and endothelial cells (Elcioglu et al., 2011). One team of researchers found that oxidative stress and apoptosis may reach their peak levels at 2–7 days when the cavernous body was cross-sectioned (Walsh et al., 1994). Because ED in diabetic rats has been attributed to free radicals resulting from oxidative stress, we hypothesized that oxidative stress could be a potential
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Fig. 4. Myelinated axons of CNs were assessed by toluidine blue staining of histological specimens. A: In group 1, there were abundant myelinated axons of CNs that had a normal morphological appearance. B: In group 2, the number of myelinated axons decreased substantially and displayed the phenomenon of atrophy. C: In group 3, there was a significant decrease in the regeneration of well-orientated
myelinated axons relative to groups 1 and 2. D: The number of myelinated axons in all three groups. Arrows in A–C indicate myelinated axons of CNs. There were seven rats (n 5 7) at week 12 for toluidine blue staining in each group. *P < 0.05 compared with group 2, #P < 0.05 compared with group 1. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
cause of ED after surgical trauma. This study investigated histomorphological and functional changes and markers of oxidative stress after CN crush or CN transection with immediate suturing in different time periods. In the current study, functional assessments confirmed that group 2 (bilateral CNs crushed) and group 3 (bilateral CNs transectioned and sutured immediately) had significantly lower ICP than group 1, and group 2 had higher ICP than group 3. This observation is consistent with the clinical consequence of ED after prostate surgery with bilateral nerve preservation. In clinical practice, bilateral nerve-sparing prostatectomy patients suffer ED less than do unilateral nerve-sparing patients. In animal experiments, neurotomy-group animals also had lower ICP than the manipulation group during the functional evaluation. NO is the main mediator of penile erection and the most important neurotransmitter that mediates the relaxation of smooth muscle cell in the corpus cavernosum (Becker et al., 2002; Burnett, 2004). In the penis, NO is formed by both endothelial NOS and neuronal NOS.
The penis of NOS fibers originates from the dorsal part of CNs, and NADPH-diaphorase staining is able to evaluate regeneration of CNs. The current study shows that NADPH-diaphorase-positive nerve fibers were statistically decreased among the groups, from sham to crush to transection. Oxidative stress will be produced when the balance between formation of free radicals in the body and the elements that remove them is broken, and this leads to ROS accumulation in tissue (Strzyzewski et al., 2013). It will result in functional tissue damage and cell death if ROS are not effectively neutralized by an antioxidant defense system (Wu et al., 2014). The possible mechanisms of ED related to oxidative stress may have two aspects, one being the accumulation of NO in the penis and smooth muscle relaxation (Li et al., 2012; Bivalacqua et al., 2013) and the other being structural and functional impairment of the corporal smooth muscle and endothelium. To investigate whether a surgical trauma to the CN, which was simulated as bilateral CNs crushed or bilateral Journal of Neuroscience Research
Oxidative Stress in Surgical CN Injury
Fig. 5. GPX expression in the corpus cavernosum for each group. A: Representative immunoblot of GPX and b-actin protein expression in rat penes in each group. B: Graph shows relative levels of GPX protein normalized to b-actin. There were 28 rats (n 5 28) in each group, and each group of different periods (weeks 1, 2, 4, and 12) had seven rats. #P < 0.05 compared with group 1, *P < 0.05 compared with group 2. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
CNs transectioned and sutured immediately, could cause oxidative stress in the corpus cavernosum, Western blotting was performed to detect the expression of GPX and NT-3 in penis tissue. GPX, one of the antioxidant enzymes, plays an important role in maintaining the peroxidation balance by facilitating the breakdown of hydrogen peroxide to oxygen and H2O by and reverting lipid peroxidation, converting peroxides into nonreactive products (Doktorovova et al., 2014; Oyeyipo et al., 2014). NT-3 not only is an indicator of protein oxidative damage but also is a marker of oxidative stress. In our experiment, GPX and NT-3 (35 kDa) levels in penis tissue significantly increased in groups 2 and 3 compared with group 1 at weeks 1 and 2, and group 3 had levels that were increased further. This study also measured MDA levels, SOD activities, and GPX activities in serum. SOD, which is well known to be involved in the tissue scavenging system during oxidative stress and in protecting tissue against free radicals, is another indirect indicator for oxidative stress. MDA, which is recognized as a biomarker of peroxidation and oxidative stress, has been widely used to assess the extent of lipid peroxidation. In this study, oxidative stress caused an increase in MDA in groups 2 and 3. At the same time, SOD and GPX activities were also increased to antagonize the oxidative stress effects by reducing superoxide to form hydrogen peroxide and hydrogen peroxide to water, respectively. In addition, Journal of Neuroscience Research
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Fig. 6. NT-3 levels in the corpus cavernosum for each group; one immunopositive band (35 kDa) was analyzed. A: Representative immunoblot of NT-3 and b-actin expression in rat penes for each group. B: Graph shows the relative levels of NT-3 normalized to bactin. There were 28 (n 5 28) rats in each group, and each group of different periods (weeks 1, 2, 4, and 12) had seven rats. *P < 0.05 compared with group 2, #P < 0.05 compared with group 1. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
GPX can reduce lipid peroxides directly. These results demonstrate that crush or transection and sutured immediately of cavernous nerves causes oxidative stress in the erectile tissue and serum. The transection and suture procedure caused more prominent oxidative stress than crush. CONCLUSION This study confirms that bilateral CN crush or bilateral CN transection and suturing causes a decrease of ICP, myelinated CN axons, and NOS fibers. Oxidative stress, which occurred after both bilateral crush and bilateral transection and suture of CNs, was more prominent in the sham-operated group. From the data presented here, we can conclude that surgical manipulation of the CN will result in oxidative stress in rat corpus cavernosum. These data provide evidence in support of the hypothesis that ED associated with CN manipulation is related, in part, to oxidative stress in the corpus cavernosum. Although antioxidant treatment improved erectile function in aged or diabetic rats, further investigation is required to determine whether antioxidant treatment can also prevent ED after surgical trauma. ACKNOWLEDGMENTS S.-W.L., X.-M.Z., and H.Z. were responsible for the integrity of the entire study. X.-H.W. was responsible for study concepts. H.W. and S.N. performed research. X.G.D. and H.W. analyzed data. L.L. was responsible for
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statistical analysis. H.W. was responsible for article preparation. H.W. wrote the article. X.-G.D. and H.W. approved the final version of the article. All authors read and approved the final article and have no competing financial interests. REFERENCES Agarwal A, Nandipati KC, Sharma RK, Zippe CD, Raina R. 2006. Role of oxidative stress in the pathophysiological mechanism of erectile dysfunction. J Androl 27:335–347. Alan C, Kocoglu H, Resit Ersay A, Anil Kurt H, Ertung Y, Alan H. 2010. [Biochemical changes in cavernosal tissue caused by single sided cavernosal nerve resection and the effects of alpha lipoic acid on these changes]. Actas Urol Esp 34:874–881. Barazani Y, Stahl PJ, Nagler HM, Stember DS. 2014. Is there a rationale for penile rehabilitation following radical prostatectomy? Am J Mens Health 9:35–43. Becker AJ, Uckert S, Stief CG, Scheller F, Knapp WH, Machtens SA, Kuczyk MA, Jonas U. 2002. Systemic and cavernous plasma levels of vasoactive intestinal polypeptide during sexual arousal in healthy males. World J Urol 20:59–63. Bivalacqua TJ, Musicki B, Hsu LL, Berkowitz DE, Champion HC, Burnett AL. 2013. Sildenafil citrate-restored eNOS and PDE5 regulation in sickle cell mouse penis prevents priapism via control of oxidative/nitrosative stress. PLoS One 8:e68028. Burnett AL. 2004. Novel nitric oxide signaling mechanisms regulate the erectile response. Int J Impot Res 16(suppl 1):S15–S19. Doktorovova S, Santos DL, Costa I, Andreani T, Souto EB, Silva AM. 2014. Cationic solid lipid nanoparticles interfere with the activity of antioxidant enzymes in hepatocellular carcinoma cells. Int J Pharm 471: 18–27. Elcioglu HK, Kabasakal L, Ozkan N, Celikel C, Ayanoglu-Dulger G. 2011. A study comparing the effects of rosiglitazone and/or insulin
treatments on streptozotocin induced diabetic (type I diabetes) rat aorta and cavernous tissues. Eur J Pharmacol 660:476–484. Kimura M, Rabbani ZN, Zodda AR, Yan H, Jackson IL, Polascik TJ, Donatucci CF, Moul JW, Vujaskovic Z, Koontz BF. 2012. Role of oxidative stress in a rat model of radiation-induced erectile dysfunction. J Sex Med 9:1535–1549. Li M, Zhuan L, Wang T, Rao K, Yang J, Quan W, Liu J, Ye Z. 2012. Apocynin improves erectile function in diabetic rats through regulation of NADPH oxidase expression. J Sex Med 9:3041–3050. Maiorino MI, Bellastella G, Esposito K. 2014. Diabetes and sexual dysfunction: current perspectives. Diabetes Metab Syndr Obes 7:95– 105. Oyeyipo IP, Raji Y, Bolarinwa AF. 2014. Nicotine alters serum antioxidant profile in male albino rats. North Am J Med Sci 6:168–171. Ozkara H, Alan C, Atukeren P, Uyaner I, Demirci C, Gumustas MK, Alici B. 2006. Changes of nitric oxide synthase-containing nerve fibers and parameters for oxidative stress after unilateral cavernous nerve resection or manuplation in rat penis. Chin J Physiol 49:160–166. Strzyzewski KW, Piorunska-Stolzmann M, Majewski W, Kasprzak M, Strzyzewski W. 2013. Effect of surgical treatment on lipid peroxidation parameters and antioxidant status in the serum of patients with peripheral arterial disease. Dis Markers 35:647–652. Suresh S, Prakash S. 2011. Effect of Mucuna pruriens (Linn.) on oxidative stress-induced structural alteration of corpus cavernosum in streptozotocin-induced diabetic rat. J Sex Med 8:1943–1956. Walsh PC, Partin AW, Epstein JI. 1994. Cancer control and quality of life following anatomical radical retropubic prostatectomy: results at 10 years. J Urol 152:1831–1836. Wu YT, Wu SB, Wei YH. 2014. Roles of sirtuins in the regulation of antioxidant defense and bioenergetic function of mitochondria under oxidative stress. Free Radic Res:1–15. Zhang W, Wang Y, Yang Z, Qiu J, Ma J, Zhao Z, Bao T. 2011. Antioxidant treatment with quercetin ameliorates erectile dysfunction in streptozotocin-induced diabetic rats. J Biosci Bioeng 112:215–218.
Journal of Neuroscience Research
Role of Oxidative Stress in Surgical Cavernous Nerve Injury in a Rat Model Hui Wang,1 Xie-Gang Ding,1* Shi-Wen Li,1 Hang Zheng,1 Xin-Min Zheng,1 Shrestha Navin,1 Lu Li,2 and Xing-Huan Wang1 1
Department of Urology and Andrology, Zhongnan Hospital, Wuhan University, Wuhan, People’s Republic of China 2 Department of Nutrition and Food Health, School of Public Health, Wuhan University, Wuhan, People’s Republic of China
This study investigates the role of oxidative stress in surgical cavernous nerve (CN) injury in a rat model. Eighty-four male Sprague-Dawley rats were randomly divided into three groups: group 1, sham-operated rats; group 2, bilateral CN-crushed rats; and group 3, bilateral CNtransection-and-sutured-immediately rats. Oxidative stress was evaluated by malondialdehyde levels, super oxide dismutase (SOD) activities, and glutathione peroxidase (GPX) activities in serum. Erectile function was assessed by CN electrostimulation at 3 months with mean maximal intracavernous pressure (ICP) and maximal ICP per mean arterial pressure. Nerve injury was assessed by toluidine blue staining of CNs and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining of penile tissue. GPX protein expression and nitrotyrosine-3 (NT-3) levels in penile tissue were measured. Erectile function and the number of myelinated axons of CNs and NADPH-diaphorase-positive nerve fibers were statistically decreased between groups, from sham to crush to transection. For markers, both nerve-injury groups showed increased oxidative stress markers at early time points, with the transection group showing greater oxidative stress than the crushed group and values normalizing to sham levels by week 12. GPX expression and NT-3 levels in penile tissue were in concordance with the results of SOD and GPX. These results show that oxidative stress plays an important role in injured CNs, and different methods of CN injury can lead to different degrees of oxidative stress in a rat model. VC 2015 Wiley Periodicals, Inc.
Recent findings from experimental studies have suggested a significant association between reactive oxygen species (ROS) and ED, especially in diabetic animal models and in rat models of radiation-induced ED (Suresh and Prakash, 2011; Kimura et al., 2012; Maiorino et al., 2014). In addition, evidence has indicated that neurotomy and manipulation of the CN cause oxidative stress in rat corpus cavernosum and that oxidative stress is more significant in the nerve-resection group (Ozkara et al., 2006). CN injury and transaction ultimately lead to apoptosis of the corporal smooth muscle cells and fibrosis of the corporal tissue (Suresh and Prakash, 2011). Oxidative stress results from an imbalance between the formation of free radicals and the ability of the antioxidants to scavenge ROS (Agarwal et al., 2006; Zhang et al., 2011) and causes accumulation of ROS in the tissue. ROS, which may accumulate in the corpus cavernosum during oxidative stress, has been shown to be a potential cause of ED (Alan et al., 2010). Oxidative stress could be determined in experimental studies by glutathione peroxidase (GPX) activities and nitrotyrosine (NT) levels in the tissue as well as by superoxide dismutase (SOD) activities, GPX activities, and malondialdehyde (MDA) levels in serum. This study investigates the effects of bilateral CNs crushed or bilateral CNs transectioned and sutured immediately to nitric oxide synthase (NOS)containing nerve fibers and pressure after electrostimulation in the rat corpus cavernosum and seeks to determine whether these procedures produce oxidative stress within rat cavernous tissues and peripheral blood.
Key words: erectile dysfunction; cavernous nerve; oxidative stress
It has been reported that the mainstays of treatment for clinically localized prostate cancer (PC) are radical prostatectomy (RP) and radiation therapy (Barazani et al., 2014). Both therapeutic modalities are associated with a high risk of erectile dysfunction (ED). Despite the advent of the cavernous nerve (CN)-sparing surgical technique, ED remains a major problem following RP for PC. The pathophysiological mechanisms of ED, as a complication following nerve-sparing surgical technique, are not completely understood. C 2015 Wiley Periodicals, Inc. V
Contract grant sponsor: National Natural Science Foundation of China; Contract grant number: NSFC303163398 (to X.-G.D.) H. Wang and X.-G. Ding contributed equally to this work. *Correspondence to: Xie-Gang Ding, Department of Urology, Zhongnan Hospital, Wuhan University, 169 Donghu Road, Wuhan 430071, People’s Republic of China. E-Mail: [email protected] Received 8 October 2014; Revised 10 November 2014; Accepted 26 November 2014 Published online 00 Month 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jnr.23545
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MATERIALS AND METHODS Experimental Animals Eight-four white, male Sprague-Dawley rats (weighing 300–350 g) were randomly divided into three groups: group 1, sham-operated rats (n 5 28); group 2, bilateral CN-crushed rats (n 5 28); and group 3, bilateral CN-transection-and-suturedimmediately rats (n 5 28). All rats were obtained from the Experimental Animal Center of Wuhan University, and the Institutional Animal Care and Use Committee of Wuhan University approved all the experimental protocols followed in this study. All surgery was performed with animals under pentobarbital sodium, and all efforts were made to minimize suffering. Surgical Procedures Animals were anesthetized with an i.p. injection of 1% pentobarbital sodium (50 mg/kg), and operations were performed with a surgical microscope (SXP-1C; Medical Optical Instruments Factory of Shanghai Medical Instruments, Shanghai, China). A midline abdominal incision was made to expose the bladder and prostate. After identification of the MPG on the lateral side to bilateral prostates, the CNs, tracking posterolaterally, were identified and isolated. In the sham-operated group, with a midline incision and identification of the bilateral CNs, no further surgical manipulation was done. In the bilateral CN-crushed group, at the point of 3 mm distal to the MPG, the bilateral CNs were crushed with a hemostat clamp (HC-X020; Cheng-He Microsurgical Instruments Factory, Ningbo, China) for 2 min. In the bilateral CN transectionand-sutured-immediately group, CNs were sutured with 10-0 microscopic sutures. In all rats, the wound was closed in two layers. Functional Evaluation and Tissue Procurement The erectile function was calculated in all rats after 3 months by electrostimulation of the CNs and by measuring intracavernous pressure (ICP; n 5 7 in each group). The penis was dissected and the corpus cavernosum and crura of the penis were exposed; the CNs were exposed and isolated via a midline abdominal incision. A 22-G butterfly needle, which was connected to a PE-50 tube and filled with 250 U/ml heparin solution, was inserted into the right penis crura for ICP measurement. Electrostimulation was performed with a stainless-steel bipolar hook electrode (probes 2 mm in diameter and separated by 1 mm). The stimulus parameters were 1.5 mA, frequency 20 Hz, and pulse width 0.2 msec for a duration of 1 min. The maximal amplitude of ICP during cavernous nerve electrostimulation was selected for statistical analysis in each animal. Mean arterial pressure (MAP) was monitored by a PE-50 tube that had been inserted into the carotid artery. The ICPs and MAP were recorded in all rats in a bioinformation acquisition system (BL-420F; Chengdu TME Technology, Chengdu, China). After functional evaluation, the CNs were excised for toluidine blue staining, and the midshaft penis was excised and divided transversally into two pieces, one part for nicotinamide adenine dinucleotide phosphate (NADPH)diaphorase staining and the other part for GPX activities and NT level assays.
Oxidative Stress Evaluation Oxidative stress was evaluated by MDA levels, SOD activities, and GPX activities in serum at 1, 2, 4, and 12 weeks. These oxidative stress parameters were determined with assay kits (Nanjing Jiancheng Biology Research Institute, Nanjing, China) according to instructions provided by the manufacturer. Animals were anesthetized with an i.p. injection of 1% pentobarbital sodium (50 mg/kg) and fixed on the stage. After the heartbeat of the site was touched, a syringe with a 4-G needle was used to puncture the most obvious place of heartbeat, and the blood flowed into the syringe; about 6–10 ml of blood was collected from each rat. When the blood had coagulated at room temperature, it was put in the centrifuge at 4,000 rpm for 10 min, and then the serum was obtained. Western Blotting The corpus cavernosum was obtained from each group of rats (n 5 28), and tissues were lysed in triple-detergent radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitor cocktails and phenylmethylsulfonyl fluoride. Tissue protein lysates were centrifuged for 10 min at 14,000 rpm, and then supernatants were harvested for the following experiments. Protein concentrations were estimated with BCA kits. Protein samples (50 lg) were denatured at 95 C for 5 min, and electrophoresis was performed on a 12% discontinuous SDS-polyacrylamide gel and transferred to a PVDF membrane. The membranes were reacted with blocking buffer (5% skim milk in Tris-buffered saline Tween 20 buffer) for 30 min at ambient temperature and incubated with antiGPX antibody (1:1,000; Epitomics, Burlingame, CA). After secondary antibody incubations at ambient temperature for 1 hr, signals were visualized by enhanced chemiluminescence. For NT-3, a mouse monoclonal antibody (1:1,000) was used that detects numerous nitrosylated proteins. We analyzed one immunopositive band at 35 kDa. NT-3 expression levels were quantified for each sample. Densitometry results were normalized by b-actin expression. NADPH-Diaphorase Staining NADPH-diaphorase staining was used to identify NOS in the penile nerve fibers of the proximal penile segment. A midportion of each penile segment was harvested and immediately fixed in 0.1 mol/liter phosphate-buffered saline (PBS; pH 8.0) containing 0.002% picric acid and 2% formaldehyde for 4 hr, then transferred to 30% sucrose and stored overnight at 4 C. The segments were then embedded in optimal cutting temperature compound (Beijing Zhongshan Golden Bridge Biotechnology, Beijing, China). Serial cryosections (8 lm) adhered to the charged slides. After having been air dried for 5 min, the sections were incubated with 0.1 mM NADPH, 0.2 mM nitroblue tetrazolium, and 0.2% Triton X-100 (SigmaAldrich, St. Louis, MO) in 0.1 mol/liter PBS, pH 8.0, for 4 hr at 37 C. The reaction was terminated by washing the sections in buffer. The staining pattern was assessed by counting the number of NADPH-diaphorase-positive nerves in the dorsal nerves (3400 with light microscopy). The endothelium staining was excluded from the count. Journal of Neuroscience Research
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Fig. 1. Changes of maximal ICP and MAP after electrostimulation of CNs at 3 months. A: ICP and MAP in group 1. B: ICP and MAP in group 2. C: ICP and MAP in group 3. D: Maximal ICP in all three groups. E: Maximal ICP/MAP ratio in all three groups. There were seven rats (n 5 7) at week 12 for ICP in each group. *P < 0.05 compared with group 2, #P < 0.05 compared with group 1. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Toluidine Blue Staining Three to four millimeters of CN was obtained from the main trunk of both sides at equivalent points, which were distal to the site of injury. All harvested nerves were fixed in 3% (w/ v) cold glutaraldehyde. The nerve segments were dehydrated with ethanol and postfixed with 1% osmium tetroxide before Journal of Neuroscience Research
being infiltrated with a graded araldite–propylene oxide mixture and embedded in Epon 812 (Serva Feinbiochemica, Heidelberg, NY). An LKB III ultramicrotome (LKB Produkter, Broma, Sweden) was used to obtain 1-lm cross-sections of the embedded nerve, which were then stained with 1% toluidine blue and examined with light microscopy. Images were
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Fig. 2. Serum levels of MDA and serum activities of SOD and GPX in each group. A: Serum activities of SOD (U/ml) in all three groups. B: Serum activities of GPX (U/ml) in all three groups. C: Serum levels of MDA (nmol/ml) in all three groups. *P < 0.05 compared with group 2, # P < 0.05 compared with group 1. There were 28 rats in each group, and each group of different periods (weeks 1, 2, 4, and 12) had seven rats.
captured with an Olympus-DP12 camera (3100 oil immersion objective) and processed in Image Pro-Plus 3.0 (Media Cybernetics, Bethesda, MD). Statistical Analysis Data are expressed as mean 6 SD. The results were analyzed by one-way ANOVA, with significance indicated at P < 0.05. If the difference was significant, a Student-NewmanKeuls test was carried out.
RESULTS Evaluation of Erectile Function Erectile function was evaluated by the maximal ICP (Fig. 1). No erectile dysfunction was identified in group 1, with a pressure of 102.5 6 9.0 cmH2O. Group 2 showed a significant reduction in maximal ICP of 52.4 6 11.2 cmH2O, consistent with a state of ED. In group 3, the mean maximal ICP was 34.1 6 9.7 cmH2O,
which was significantly lower than that in other groups (P < 0.05). The change of maximal ICP/MAP ratio was consistent with the maximal ICP in the three groups. Oxidative Stress Parameters in Serum Figure 2A,B shows the mean activities of SOD and GPX in serum. At weeks 1, 2, and 4, the mean activities of SOD and GPX were significantly increased in group 3 compared with those in the other two groups (P < 0.05). At weeks 1 and 2, the mean activities of SOD and GPX were significantly increased in group 2 compared with those of group 1 (P < 0.05). At week 4, there was no appreciable difference between group 1 and group 2 (P > 0.05). At week 12, there were no appreciable differences in the mean activities of SOD and GPX for the three groups (P >0.05). Figure 2C shows the mean levels of MDA in the serum. At weeks 1, 2, and 4, the serum level in group 3 Journal of Neuroscience Research
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Fig. 3. NADPH-diaphorase staining of dorsal nerves. A: In group 1, there was predominant blue staining of nerve fibers. B: In group 2, there was a paucity of blue-stained fibers. C: In group 3, there was a significant decrease in the number of blue-stained nerve fibers relative to groups 1 and 2. D: Results of NADPH-diaphorase-positive nerve fibers of dorsal nerves for each group. Arrows in A–C indicate
NADPH-diaphorase-positive nerve fibers. There were seven rats (n 5 7) at week 12 for NADPH-diaphorase staining in each group. *P < 0.05 compared with group 2, #P < 0.05 compared with group 1. Scale bars 5 600 lm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
was significantly increased compared with that of the other two groups (P < 0.05). At weeks 2 and 4, there was no appreciable difference between group 1 and group 2 (P > 0.05). At week 12, there were no appreciable differences among the three groups (P > 0.05).
(P < 0.05) compared with group 1, and group 3 GPX protein expression was much higher than that in group 2 (P < 0.05). However, it had decreased to normal levels in the two groups at weeks 4 and 12 (Fig. 5). Western blot analysis of NT-3 showed one immunopositive band at 35 kDa. At weeks 1, 2, and 4, the NT-3 level was significantly increased in group 2 (P < 0.05) and group 3 (P < 0.05) compared with group 1, and the group 3 level was much higher than that in group 2 (P < 0.05). However, it had decreased to normal level in the two groups at week 12 (Fig. 6).
NADPH-Diaphorase Staining There were significantly fewer NADPHdiaphorase-positive nerve fibers in the dorsal nerves of groups 2 and 3 than in those of group 1 (P < 0.05), and group 3 had a significant decrease compared with group 2 in the number of NADPH-diaphorase-positive nerve fibers (P < 0.05; Fig. 3). Toluidine Blue Staining of CNs The numbers of myelinated axons in groups 2 and 3 were significantly less than those in group 1 (P < 0.05), and group 3 showed a significant decrease compared with group 2 in the number of myelinated axons (P < 0.05; Fig. 4). Western Blot Analysis At weeks 1 and 2, GPX protein expression was significantly increased in group 2 (P < 0.05) and group 3 Journal of Neuroscience Research
DISCUSSION Despite the fact that CNs are preserved in nerve-sparing surgical procedures performed in radical prostatectomies to maintain erectile function, erectile capacity cannot always be maintained. Many previous studies have suggested that oxidative stress and apoptotic changes occur in cavernous body smooth muscle and endothelial cells (Elcioglu et al., 2011). One team of researchers found that oxidative stress and apoptosis may reach their peak levels at 2–7 days when the cavernous body was cross-sectioned (Walsh et al., 1994). Because ED in diabetic rats has been attributed to free radicals resulting from oxidative stress, we hypothesized that oxidative stress could be a potential
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Fig. 4. Myelinated axons of CNs were assessed by toluidine blue staining of histological specimens. A: In group 1, there were abundant myelinated axons of CNs that had a normal morphological appearance. B: In group 2, the number of myelinated axons decreased substantially and displayed the phenomenon of atrophy. C: In group 3, there was a significant decrease in the regeneration of well-orientated
myelinated axons relative to groups 1 and 2. D: The number of myelinated axons in all three groups. Arrows in A–C indicate myelinated axons of CNs. There were seven rats (n 5 7) at week 12 for toluidine blue staining in each group. *P < 0.05 compared with group 2, #P < 0.05 compared with group 1. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
cause of ED after surgical trauma. This study investigated histomorphological and functional changes and markers of oxidative stress after CN crush or CN transection with immediate suturing in different time periods. In the current study, functional assessments confirmed that group 2 (bilateral CNs crushed) and group 3 (bilateral CNs transectioned and sutured immediately) had significantly lower ICP than group 1, and group 2 had higher ICP than group 3. This observation is consistent with the clinical consequence of ED after prostate surgery with bilateral nerve preservation. In clinical practice, bilateral nerve-sparing prostatectomy patients suffer ED less than do unilateral nerve-sparing patients. In animal experiments, neurotomy-group animals also had lower ICP than the manipulation group during the functional evaluation. NO is the main mediator of penile erection and the most important neurotransmitter that mediates the relaxation of smooth muscle cell in the corpus cavernosum (Becker et al., 2002; Burnett, 2004). In the penis, NO is formed by both endothelial NOS and neuronal NOS.
The penis of NOS fibers originates from the dorsal part of CNs, and NADPH-diaphorase staining is able to evaluate regeneration of CNs. The current study shows that NADPH-diaphorase-positive nerve fibers were statistically decreased among the groups, from sham to crush to transection. Oxidative stress will be produced when the balance between formation of free radicals in the body and the elements that remove them is broken, and this leads to ROS accumulation in tissue (Strzyzewski et al., 2013). It will result in functional tissue damage and cell death if ROS are not effectively neutralized by an antioxidant defense system (Wu et al., 2014). The possible mechanisms of ED related to oxidative stress may have two aspects, one being the accumulation of NO in the penis and smooth muscle relaxation (Li et al., 2012; Bivalacqua et al., 2013) and the other being structural and functional impairment of the corporal smooth muscle and endothelium. To investigate whether a surgical trauma to the CN, which was simulated as bilateral CNs crushed or bilateral Journal of Neuroscience Research
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Fig. 5. GPX expression in the corpus cavernosum for each group. A: Representative immunoblot of GPX and b-actin protein expression in rat penes in each group. B: Graph shows relative levels of GPX protein normalized to b-actin. There were 28 rats (n 5 28) in each group, and each group of different periods (weeks 1, 2, 4, and 12) had seven rats. #P < 0.05 compared with group 1, *P < 0.05 compared with group 2. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
CNs transectioned and sutured immediately, could cause oxidative stress in the corpus cavernosum, Western blotting was performed to detect the expression of GPX and NT-3 in penis tissue. GPX, one of the antioxidant enzymes, plays an important role in maintaining the peroxidation balance by facilitating the breakdown of hydrogen peroxide to oxygen and H2O by and reverting lipid peroxidation, converting peroxides into nonreactive products (Doktorovova et al., 2014; Oyeyipo et al., 2014). NT-3 not only is an indicator of protein oxidative damage but also is a marker of oxidative stress. In our experiment, GPX and NT-3 (35 kDa) levels in penis tissue significantly increased in groups 2 and 3 compared with group 1 at weeks 1 and 2, and group 3 had levels that were increased further. This study also measured MDA levels, SOD activities, and GPX activities in serum. SOD, which is well known to be involved in the tissue scavenging system during oxidative stress and in protecting tissue against free radicals, is another indirect indicator for oxidative stress. MDA, which is recognized as a biomarker of peroxidation and oxidative stress, has been widely used to assess the extent of lipid peroxidation. In this study, oxidative stress caused an increase in MDA in groups 2 and 3. At the same time, SOD and GPX activities were also increased to antagonize the oxidative stress effects by reducing superoxide to form hydrogen peroxide and hydrogen peroxide to water, respectively. In addition, Journal of Neuroscience Research
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Fig. 6. NT-3 levels in the corpus cavernosum for each group; one immunopositive band (35 kDa) was analyzed. A: Representative immunoblot of NT-3 and b-actin expression in rat penes for each group. B: Graph shows the relative levels of NT-3 normalized to bactin. There were 28 (n 5 28) rats in each group, and each group of different periods (weeks 1, 2, 4, and 12) had seven rats. *P < 0.05 compared with group 2, #P < 0.05 compared with group 1. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
GPX can reduce lipid peroxides directly. These results demonstrate that crush or transection and sutured immediately of cavernous nerves causes oxidative stress in the erectile tissue and serum. The transection and suture procedure caused more prominent oxidative stress than crush. CONCLUSION This study confirms that bilateral CN crush or bilateral CN transection and suturing causes a decrease of ICP, myelinated CN axons, and NOS fibers. Oxidative stress, which occurred after both bilateral crush and bilateral transection and suture of CNs, was more prominent in the sham-operated group. From the data presented here, we can conclude that surgical manipulation of the CN will result in oxidative stress in rat corpus cavernosum. These data provide evidence in support of the hypothesis that ED associated with CN manipulation is related, in part, to oxidative stress in the corpus cavernosum. Although antioxidant treatment improved erectile function in aged or diabetic rats, further investigation is required to determine whether antioxidant treatment can also prevent ED after surgical trauma. ACKNOWLEDGMENTS S.-W.L., X.-M.Z., and H.Z. were responsible for the integrity of the entire study. X.-H.W. was responsible for study concepts. H.W. and S.N. performed research. X.G.D. and H.W. analyzed data. L.L. was responsible for
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statistical analysis. H.W. was responsible for article preparation. H.W. wrote the article. X.-G.D. and H.W. approved the final version of the article. All authors read and approved the final article and have no competing financial interests. REFERENCES Agarwal A, Nandipati KC, Sharma RK, Zippe CD, Raina R. 2006. Role of oxidative stress in the pathophysiological mechanism of erectile dysfunction. J Androl 27:335–347. Alan C, Kocoglu H, Resit Ersay A, Anil Kurt H, Ertung Y, Alan H. 2010. [Biochemical changes in cavernosal tissue caused by single sided cavernosal nerve resection and the effects of alpha lipoic acid on these changes]. Actas Urol Esp 34:874–881. Barazani Y, Stahl PJ, Nagler HM, Stember DS. 2014. Is there a rationale for penile rehabilitation following radical prostatectomy? Am J Mens Health 9:35–43. Becker AJ, Uckert S, Stief CG, Scheller F, Knapp WH, Machtens SA, Kuczyk MA, Jonas U. 2002. Systemic and cavernous plasma levels of vasoactive intestinal polypeptide during sexual arousal in healthy males. World J Urol 20:59–63. Bivalacqua TJ, Musicki B, Hsu LL, Berkowitz DE, Champion HC, Burnett AL. 2013. Sildenafil citrate-restored eNOS and PDE5 regulation in sickle cell mouse penis prevents priapism via control of oxidative/nitrosative stress. PLoS One 8:e68028. Burnett AL. 2004. Novel nitric oxide signaling mechanisms regulate the erectile response. Int J Impot Res 16(suppl 1):S15–S19. Doktorovova S, Santos DL, Costa I, Andreani T, Souto EB, Silva AM. 2014. Cationic solid lipid nanoparticles interfere with the activity of antioxidant enzymes in hepatocellular carcinoma cells. Int J Pharm 471: 18–27. Elcioglu HK, Kabasakal L, Ozkan N, Celikel C, Ayanoglu-Dulger G. 2011. A study comparing the effects of rosiglitazone and/or insulin
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