Clinical and Experimental Pharmacology and Physiology (2014) 41, 309–316
doi: 10.1111/1440-1681.12216
Melatonin and tadalafil treatment improves erectile dysfunction after spinal cord injury in rats _ Hasan H€ useyin Tavukcßu,* Tarik Emre S Tinay,† Cem Akbal,† Mehmet Ersßahin,‡ ß ener,† Ilker § ¶ € Ozge C ß adırcı, Russel J Reiter** and G€oksel S ß ener¶ ß evik, Selin C ‡
*Department of Urology, Academic Hospital, †Department of Urology, School of Medicine, Marmara University, Department of Neurosurgery, School of Medicine, Istanbul Medeniyet University, Istanbul, §Department of Biochemistry, School of Pharmacy, Cumhuriyet University, Sivas, ¶Department of Pharmacology, School of Pharmacy, Marmara University, Istanbul, Turkey and **Department of Cellular and Structural Biology, The UT Health Science Center, San Antonio, TX, USA
SUMMARY 1. Oxidative stress plays an important role both in spinal cord injury (SCI) and erectile dysfunction (ED). The present study investigated the effects of melatonin and tadalafil treatment alone or in combination on SCI-induced ED. 2. Male Wistar albino rats (n = 40) were divided into five groups: sham-operated control and SCI-injured rats given either vehicle, melatonin (10 mg/kg, i.p.), tadalafil (10 mg/kg, p.o.) or a combination of melatonin and tadalafil. Spinal cord injury was induced using a standard weight-drop method. On Day 7 after SCI, intracavernosal pressure (ICP) was measured and all rats were decapitated. Cavernosal tissues were obtained to examine caspase 3, nitric oxide synthase (NOS), myeloperoxidase (MPO) and superoxide dismutase (SOD) activities, as well as cGMP, nerve growth factor (NGF), malondialdehyde (MDA) and glutathione (GSH) levels. 3. Spinal cord injury caused oxidative damage, as evidenced by increases in MDA and cGMP levels. In addition, MPO and caspase 3 activites were increased after SCI, whereas GSH and NGF levels and SOD activity were reduced. Melatonin effectively reversed these oxidative changes. Furthermore, in rats treated with both melatonin and tadalafil, the recoveries were more pronounced than in rats given either melatonin or tadalafil alone. The ICP/mean arterial pressure value in vehicle-treated SCI rats was significantly higher than in the control group, whereas in the tadalafil- and tadalafil + melatonin-treated groups have returned this value had returned to control levels. 4. As an individual treatment, and especially when combined with tadalafil, a well-known agent in the treatment of ED, melatonin prevented SCI-induced oxidative damage to cavernosal tissues and restored ED, most likely due to its anti-oxidant effects.
Correspondence: G€ oksel Sener, School of Pharmacy, Marmara University, Tibbiye Cad., 34668 Istanbul, Turkey. Email: [email protected] Received 6 October 2013; revision 3 February 2014; accepted 9 February 2014. © 2014 Wiley Publishing Asia Pty Ltd
Key words: apoptosis, erectile spinal cord injury, tadalafil.
dysfunction,
melatonin,
INTRODUCTION Spinal cord injury (SCI), which primarily occurs in young men, results in sexual dysfunction and has a negative impact on physical, social and psychological well being.1,2 Because normal erection depends on a precise balance between psychological, hormonal, neurological, vascular and cavernosal factors, a change in one or a combination of these factors may lead to erectile dysfunction (ED).3,4 The secondary injury resulting from SCI following mechanical compression-induced primary injury involves a cascade of biochemical, molecular and cellular changes, which can lead to even more extensive damage.5 These secondary events include microvascular ischaemia, oxidative stress, excitotoxicity, ion dysregulation and inflammation causing apoptotic cell death, which contributes to numerous medical problems and eventually alters basic organ physiology.6 Although mechanical disruption of the innervating neurons is not amenable to neuroprotective therapy, reductions in secondary injury are susceptible to therapeutic intervention. Because the importance of oxidative stress and inflammation in SCI has been documented in a large number of experimental and clinical studies, the use of anti-oxidants to protect against SCI-induced tissue injury and organ dysfunction has received significant attention.6–8 Similarly, anti-oxidants have been shown to be effective in the treatment of ED induced by oxidative stress.9,10 Melatonin, the major product of the vertebrate pineal gland, functions as a modulator of sleep, sexual behaviour, immune function and circadian rhythms.11–13 Melatonin and its metabolic derivatives are uncommonly effective direct free radical scavengers,14,15 while also stimulating the activities of several anti-oxidative enzymes.16 Melatonin is higly lipid soluble and easily passes the blood–brain barrier and, because of this property, within minutes after its administration it reaches high concentrations in neural tissue.17 In addition to its direct radical-scavenging actions, melatonin is involved in the regulation of many physiological systems, including cardiovascular physiology, through its receptors in the heart and arteries.18,19
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The present study was designed to investigate the protective effects of melatonin against SCI-induced erectile tissue injury and dysfunction. Furthermore, we also examined the effects of tadalafil, a well-known phosphodiesterase inhibitor (PDEI) commonly used to treat ED; this drug was either given alone or in combination with melatonin.
METHODS Animals and experimental design Wistar albino rats (250–300 g) were supplied by the Marmara University (MU) Application and Research Center for Experimental Animals (DEHAMER). All experimental protocols were approved by the MU Animal Care and Use Committee. Rats were randomly divided into five groups of eight rats each: sham-operated control and SCI-damaged rats treated with either vehicle, melatonin (10 mg/kg, i.p.), tadalafil (10 mg/kg, p.o.) or melatonin + tadalafil. Treatments were started following SCI damage and continued for 7 days. At the end of the week-long experimental period after sham surgery or SCI induction, rats were anaesthetized with an i.p. injection of 100 mg/kg ketamine and 1 mg/kg chlorpromazine and, after ICP measurements had been made, cavernosal tissue samples were obtained for biochemical analyses. Induction of SCI Anaesthetized (100 mg/kg, i.p., ketamine and 1 mg/kg, i.p., chlorpromazine) rats were positioned on a thermostat-controlled heating pad in the prone position and a rectal probe was inserted. Under sterile conditions, following a T5–12 midline skin incision and paravertebral muscle dissection, spinous processes and laminar arcs of T7–10 were removed. The dura was left intact. A modified weight-drop model was performed to produce SCI.20 Rats were subjected to an impact of 100 g/cm (10 g weight from a height of 10 cm) to the dorsal surface of the spinal cord. The force was applied via a stainless steel rod (3 mm diameter tip) that was rounded at the surface. The rod was dropped vertically through a 10 cm guide tube that was positioned perpendicular to the centre of the spinal cord. The incision was sutured and rats were placed in a warming chamber, where their body temperature was maintained at approximately 37°C until they were completely awake. Cavernous nerve stimulation and ICP/MAP measurement A week after SCI or sham operation, rats were anaesthetized as described above. An anterior transverse neck incision was made and the muscles were separated. The left internal carotid artery was isolated. The artery was cannulated with a heparinized PE50 tube connected to a pressure transducer and an amplifier unit (COMMAT Pharmacology & Physiology Instruments, Ankara, Turkey). The amplifier was connected to a data-acquisition module (MP35 data-acquisition system; COMMAT, Ankara, Turkey). This allowed MAP to be recorded on a computer using Biopac Student Laboratory PRO recording software (Biopac Systems, Goleta, CA, USA). The penis was circumcised and denuded of skin. At its junction with the pubic arch, the
ischiocavernous muscle was divided and the tunica albuginea was visualized. Intracavernous pressure was measured by inserting a 24 gauge needle into the left crus of the penis. This needle was connected to a transducer, via a heparinized PE-50 tube, for MAP measurement. The original laparotomy incision was extended down to the base of the exposed penis. The cavernosal nerve (CN) was located bilaterally and lying on both sides of the dorsal prostate. Following careful manipulation of the CN with a micromanipulator, a stainless steel bipolar electrode with parallel hooks (1 mm apart) was placed around the nerve, just distal to the ganglion. The electrode cable was attached to a STPTO2-A stimulator (COMMAT Pharmacology & Physiology Instruments); the stimulation parameters were: 1.5 mA, 20 Hz, pulse width 5, 35 ms delay, at 7.5 V for 60 s each. The CN was stimulated and data were recorded individually. The maximum ICP/MAP ratio was calculated by dividing the highest ICP recorded during stimulation by the corresponding MAP and is represented as a percentage.21 Measurement of tissue MDA and GSH levels Corpus cavernosum tissue samples were homogenized with icecold 150 mmol/L KCl for the determination of MDA and GSH levels. The MDA levels were assayed as products of lipid peroxidation by monitoring the formation of thiobarbituric acid-reactive substances, as described previously.22 Results are expressed as nmol MDA/g tissue. The activity of GSH was determined using a modification of the Ellman procedure and results are expressed in lmol GSH/g tissue.23 Measurement of tissue MPO activity Myeloperoxidase activity in cavernosal tissues was measured by a procedure similar to that described by Hillegas et al.24 One unit of enzyme activity was defined as the amount of MPO present that caused a change in absorbance, measured at 460 nm for 3 min, with MPO activity expressed as U/g tissue. Measurement of SOD activity Superoxide dismutase activity in tissue samples was measured according to the method of Mylroie et al.25 A standard curve was prepared with bovine SOD (3000 U; S-2515; Sigma, St. Louis, MO, USA) as a reference. Absorbance readings were taken at 0 and 8 min of illumination and the net absorbance was calculated. Measurement of cGMP levels Tissue levels of cGMP were determined in duplicate using a commercially available ELISA kit according to the manufacturer’s instructions (Enzo Life Sciences, Farmingdale, NY, USA). Total protein was estimated using the Bradford method,49 with cGMP values presented as pmol/mg protein. Measurement of NGF levels Tissue concentrations of NGF were determined by sandwich ELISA using a commercially available kit according to the
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Erectile function in spinal cord injury manufacturer’s instructions (Chemicon International, Temecula, CA, USA). A total protein assay was performed using the Bradford method26 and NGF values are presented as pg/mg protein.
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Measurement of tissue caspase 3 and NOS activities A caspase 3 cellular activity assay kit (Calbiochem, San Diego, CA, USA) and NOS activity assay kit (EnzyChrom, BioAssay Systems, Hayward, CA, USA) were used. Caspase 3 and NOS activities are presented as nmol/min per mg protein and U/mg protein, respectively. Western blot analysis of caspase 3 and eNOS
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Caspase 3 and eNOS release were measured directly by western blot analysis, as described previously.8 Protein concentrations in homogenized samples were determined using the Bradford method.26 Briefly, 25 lg protein, resolved by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis, was transferred to a nitrocellulose membrane (sc-3718; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membrane was blocked with 5% non-fat skim milk powder (70166; Sigma) in Tris-buffered saline (TBS) before being washed twice in TBS containing 0.1% Tween-20 and incubated overnight with primary antibody (1 : 500 monoclonal rat anti-caspase 3 (sc-56055; Santa Cruz Biotechnology), anti-eNOS (sc-136977; Santa Cruz Biotechnology) and anti-b-actin (sc-47778; Santa Cruz Biotechnology)). Then, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1 : 1000 goat anti-mouse IgG1-HRP; sc-2060; Santa Cruz Biotechnology) for 2 h at room temperature. The blot was developed with reagents (sc-2048; Santa Cruz Biotechnology). Data were analysed using IMAGEJ Programme OD analysis software (National Institutes of Health, Bethesda, MD, USA). Signals were normalized against b-actin. Statistical analysis Statistical analysis was performed using GRAPHPAD PRISM 4.0 (GraphPad Software, San Diego, CA, USA). All data are expressed as the mean SEM. Groups of data were compared by analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. Two-tailed P < 0.05 was considered significant.
RESULTS Spinal cord injury caused a significant (P < 0.001) increase in myeloperoxidase (MPO) activity in cavernosal tissues compared with activity in sham-operated control rats (Fig. 1a). In SCI-damaged rats that received either melatonin, tadalafil or melatonin + tadalafil, MPO activities were significantly reduced (P < 0.05–0.001), with the levels being near those found in control rats. As a result of SCI, malondialdehyde (MDA) levels in cavernosal tissues increased significantly (P < 0.001). Melatonin alone significantly reduced MDA levels (P < 0.001). In tadalafiltreated SCI rats, tadalafil failed to lower MDA levels, which remained higher than levels in the control rats (P < 0.01). Conversely, with combination treatment, MDA levels decreased
Fig. 1 (a) Myeloperoxidase (MPO) activity and (b) malondialdehyde (MDA) levels in corpus cavernosum tissues of sham-operated control (C) and vehicle (■)-, melatonin ( )-, tadalafil ( ) or melatonin + tadalafil ( )-treated spinal cord injury (SCI) rats. Data are the mean SEM (n = 8 rats in each group). **P < 0.01, ***P < 0.001 compared with control; †P < 0.05, ††P < 0.01, †††P < 0.001 compared with the vehicletreated SCI group.
significantly to levels similar to those in the control group (Fig. 1b). In accordance with the increased oxidative stress, levels of reduced glutathione (GSH), a key anti-oxidant, and the activity of superoxide dismutase (SOD), an anti-oxidant enzyme, were significantly depleted in the cavernosal tissues of vehicle-treated SCI rats (P < 0.05 and P < 0.001, respectively; Fig. 2). Tadalafil alone did not increase GSH levels or SOD activity; in rats given both melatonin and tadalafil there was a significant augmentation of GSH and SOD (Fig. 2), with both GSH levels and SOD activity significantly preserved following melatonin and melatonin + tadalafil treatment (P < 0.05–0.001), but not with tadalafil alone. As shown in Fig. 3, cGMP levels were significantly reduced (P < 0.001) by SCI, whereas these values were increased after all treatments. Nerve growth factor (NGF) levels, which decreased after SCI, were elevated only with combination treatment (P < 0.001). Spinal cord injury caused a significant decrease in nitric oxide synthase (NOS) activity (P < 0.05), whereas melatonin, tadalafil and their combination reversed this decrease in NOS activity (P < 0.01–0.001, Fig. 4a); these levels were approximately the same as those in control rats. In addition, semiquantitative western blot analysis revealed a reduction in endothelial (e) NOS protein expression in tissues of vehicle-treated SCI rats (Fig. 4c).
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Fig. 2 (a) Glutathione (GSH) levels and (b) superoxide dismutase (SOD) activity in corpus cavernosum tissues of sham-operated control (C) and vehicle (■)-, melatonin ( )-, tadalafil ( ) or melatonin + tadalafil ( )-treated spinal cord injury (SCI) rats. Data are the mean SEM (n = 8 rats in each group). *P < 0.05, **P < 0.01, ***P < 0.001 compared with control; †P < 0.05, ††P < 0.01, †††P < 0.001 compared with the vehicle-treated SCI group; ‡P < 0.05 compared with the tadalafil-treated SCI group.
However, eNOS protein expression was increased after treatment with melatonin, tadalafil or melatonin + tadalafil (P < 0.001). Caspase 3 activity of cavernosal tissues, as an index of apoptosis, was significantly elevated in vehicle-treated SCI-damaged rats (P < 0.001; Fig. 4b). Conversely. the SCI-mediated increase in apoptosis was significantly depressed after treatment with melatonin or melatonin + tadalafil (P < 0.001). Despite a slight but significant decrease (P < 0.01), caspase 3 activity remained elevated in tadalafil-treated rats, but in the combination treatment group enzyme activity was lower than that of the tadalafil-treated group (P < 0.05). As described in detail in the Methods, proteins were fixed on the autography film using horseradish peroxidase (HRP)-conjugated secondary antibodies. Different exposure times were used to determine proteins with greater and lesser expression at the same time. When the film was exposed for a sufficiently long enough period of time, in addition to procaspase 3 bands, we obtained cleaved caspase 3, as shown in Fig. 4e. The p20 and p17 subunits of cleaved caspase 3 bands were also apparent; the density of these was especially high in vehicle-treated SCI rats. Similarly, semiquantitative western blot analysis indicated elevated caspase 3 protein expression in cavernosal tissues of vehicle-treated SCI rats, which was significantly attenuated with drug treatment, either alone or in combination (Fig. 4d). As shown in Fig. 5, the ratio of intracavernosal pressure (ICP) to mean arterial pressure (MAP) in SCI rats was significantly
Fig. 3 (a) cGMP and (b) nerve growth factor (NGF) levels in corpus cavernosum tissues of sham-operated control (C) and vehicle (■)-, melatonin ( )-, tadalafil ( ) or melatonin + tadalafil ( )-treated spinal cord injury (SCI) rats. Data are the mean SEM (n = 8 rats in each group). ***P < 0.001 compared with control; †††P < 0.001 compared with the vehicle-treated SCI group; ‡‡P < 0.01 compared with the tadalafil-treated SCI group; §§P < 0.01 compared with the melatonin-treated SCI group.
higher than in control rats (102 11 vs 56 3, respectively; P < 0.01). This increase was reversed by tadalafil and melatonin + tadalafil treatment (P < 0.05). Melatonin alone also decreased the values similar to control levels, but these values were not significantly different from those for SCI rats. The ICP/ MAP ratios in the melatonin-, tadalafil- and melatonin + tadalafil-treated groups were 73 8, 67 8 and 62 10, respectively; these did not differ significantly from control values.
DISCUSSION The data of the present study demonstrate that SCI causes inflammatory and oxidative damage in erectile tissue because cavernosal MDA levels and MPO and caspase 3 activities were elevated. In association with the elevated oxidative injury, there was a depletion of the tissue anti-oxidants GSH and SOD. Furthermore, cGMP and eNOS, which are important parameters of erectile function, were depressed. Furthermore, the findings of the present study clearly show that melatonin and tadalafil, when given in combination, reverse these changes and protect cavernosal tissues against SCI-mediated tissue damage. Spinal cord injury causes a series of pathophysiological changes that compromise organ function; these negative changes have a tremendous impact on the patient and his family. A major complication that influences quality of life in men is ED.27,28 It is believed that reductions in nitric oxide (NO) bioavailability
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Fig. 4 (a) Nitric oxide synthase (NOS) and (b) caspase 3 activity and (c) endothelial (e) NOS and (d) caspase 3 protein expression in corpus cavernosum tissues of sham-operated control (C) and vehicle (■)-, melatonin ( )-, tadalafil ( ) or melatonin + tadalafil ( )-treated spinal cord injury (SCI) rats. Data are the mean SEM (n = 8 rats in each group). *P < 0.05, **P < 0.01, ***P < 0.001 compared with control; †P < 0.05, †††P < 0.001, ††† P < 0.001 compared with the vehicle-treated SCI group; ‡P < 0.05, ‡‡P < 0.01 compared with the tadalafil-treated SCI group; §§P < 0.01 compared with the melatonin-treated SCI group. (e) Western blot analysis of eNOS and procaspase-3. Mel, melatonin; TD, tadalafil.
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Fig. 5 Intracavernosal pressure (ICP)/mean arterial pressure (MAP) ratio in sham-operated control (C) and vehicle (■)-, melatonin ( )-, tadalafil ( ) or melatonin + tadalafil ( )-treated spinal cord injury (SCI) rats. Data are the mean SEM (n = 8 rats in each group). **P < 0.01 compared with control; †P < 0.05 compared with the vehicle-treated SCI group.
due to increased generation of reactive oxygen species (ROS) is a major determinant of ED.29 In the present study, we monitored ROS-induced tissue damage via the end-product of lipid peroxidation, namely MDA, and found that SCI caused a significant increase in MDA levels in corpus cavernosum tissues. In addition to ROS-induced damage, an inflammatory response has been suggested as an important factor in the development of secondary injury following SCI. Activated neutrophils exacerbate tissue injury through the production of oxygen metabolites and activation of cytotoxic enzymes, including MPO.30 Herein, the presence of increased neutrophil accumulation, as evidenced by elevated MPO activity in cavernosal tissues, indicates that neutrophil accumulation contributes to SCI-induced oxidative injury in cavernosal tissue. The ability of melatonin to inhibit neutrophil accumulation and the associated MPO activity is well known.31,32 Because oxidizing processes trigger the activation of caspases, changes in the redox status elevate oxidative stress; under these conditions, cells are killed either by necrosis or apoptosis.33 Apoptosis is a major complicating process related to oxidative events in the pathogenesis of secondary injury after SCI.34 In a previous study, we demonstrated that SCI increases caspase 3 activity in bladder tissue, in which MDA levels and MPO activity were also increased and bladder contractility impaired.8 In agreement with these observations, the results of the present study demonstrate that, in the cavernosal tissues of rats with SCI, caspase 3 activation is increased as a result of oxidative stress. Reduced glutathione and SOD are critical components of the anti-oxidant system.35 In the present study, both GSH levels and SOD activity were found to be depressed in cavernosal tissues of rats with SCI; these reductions likely elevated oxidative damage and contributed to ED. We aslo investigated NOS activity and cGMP levels, major indicators in the NO/cGMP signalling pathway, in erectile tissue. Spinal cord injury significantly reduced NOS activity and cGMP levels. The drop in NOS activity was associated with depressed eNOS, as determined by western blot analysis. It is well known that NO is an important mediator for the relaxation of the corpus cavernosum smooth muscle and vasculature. Nitric oxide generated by neuronal (n) NOS is considered a key factor responsible for the immediate relaxation of the corpus cavernosum, whereas
NO from eNOS is essential for maintaining relaxation.36,37 Kim et al.38 suggested that the reductions in nNOS and eNOS levels can cause circulatory and structural changes in penile tissues, resulting in ED. Nerve growth factor, a member of the neurotrophin family, is important for the growth, maintenance and survival of peripheral sensory and autonomic neurons.39 It functions as a signalling molecule.40 In a previous study, we demonstrated that SCI reduced NGF levels in bladder tissue in which contractility of bladder strips was impaired.41 There are limited published data regarding NGF in penile tissues or its relationship with ED. Burgers et al.,42 after ablating the cavernous nerve in rats, used NGF alone or in combination with interposed nerve grafts and demonstrated their potential for reducing the loss of surgically induced nerve injuries. Herein we demonstrated for the first time that NGF levels were significantly decreased after SCI, which may contribute to the development of ED. In animal models of SCI, melatonin has proven highly effective in reducing lipid peroxidation, stimulating several anti-oxidant enzymes and inhibiting apoptosis.43,44 Conversely, tadalafil, a PDEI, is a widely used agent in the treatment of ED because it blocks the degredation of cGMP and thereby augments the local relaxant effects of NO.45,46 Looking at the ICP/MAP ratio, the elevated values in SCI rats were lowered to control levels by tadalafil or tadalafil + melatonin treatment, whereas melatonin alone did not significantly change these values. Rivas et al.47 previously claimed that SCI rats exhibited a supersensitive response to pharmacological erectile therapy on ICP cavernosometry; unlike in the present study, those authors measured only ICP values. Temeltas et al.48 reported higher ICP/MAP values in SCI; however, these authors cautioned that these higher values should not be interpreted as erectile improvement. Furthermore, Temeltas et al.48 evaluated sexual function using the method of penile reflexes described earlier by Vargas et al.49 and reported that penile erections after nerve stimulation were significantly depressed after SCI despite the higher ICP/MAP values. In accordance with those results, we found that ICP/MAP values were higher in the SCI compared with control group. Conversely, melatonin alone decreased this significant increase in ICP/MAP values in SCI rats close to control levels, but these levels did not differ significantly from values in either the control or SCI rats. However, the ICP/MAP values in the tadalafil and tadalafil + melatonin groups were significantly lower than those of SCI rats. Our biochemical analysis also supported the results in predicting treatment responses to melatonin and tadalafil in ED after SCI damage. In conclusion, the findings of the present study show the critical pathogenic contribution of increased oxidative stress in SCIinduced ED. Furthermore, our results suggest that, when used in combination, melatonin and tadalafil induce similar beneficial effects through different mechanisms of action. Thus, when these agents are given in combination, the prevention of cavernosal damage and consequent ED may be possible.
REFERENCES 1. Giuliano F, Sanchez-Ramos A, L€ochner-Ernst D et al. Efficacy and safety of tadalafil in men with erectile dysfunction following spinal cord injury. Arch. Neurol. 2007; 64: 1584–92.
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Erectile function in spinal cord injury 2. Biering-Sørensen I, Hansen RB, Biering-Sørensen F. Sexual function in a traumatic spinal cord injured population 10–45 years after injury. J. Rehabil. Med. 2012; 44: 926–31. 3. Agarwal A, Nandipati KC, Sharma RK, Zippe CD, Raina R. Role of oxidative stress in the pathophysiological mechanism of erectile dysfunction. J. Androl. 2006; 27: 335–47. 4. Silva FH, M onica FZ, Bau FR et al. Superoxide anion production by NADPH oxidase plays a major role in erectile dysfunction in middle-aged rats: Prevention by antioxidant therapy. J. Sex. Med. 2013; 10: 960–71. 5. Azbill RD, Mu X, Bruce-Keller AJ, Mattson MP, Springer JE. Impaired mitochondrial function, oxidative stress and altered antioxidant enzyme activities following traumatic spinal cord injury. Brain Res. 1997; 765: 283–90. 6. Hall ED. Antioxidant therapies for acute spinal cord injury. Neurotherapeutics 2011; 8: 152–67. 7. Bastani NE, Kostovski E, Sakhi AK et al. Reduced antioxidant defense and increased oxidative stress in spinal cord injured patients. Arch. Phys. Med. Rehabil. 2012; 93: 2223–8. 8. Cevik O, Ersßahin M, Sener TE et al. Beneficial effects of quercetin on rat urinary bladder after spinal cord injury. J. Surg. Res. 2013; 183: 695–703. 9. Qiu XF, Li XX, Chen Y et al. Mobilisation of endothelial progenitor cells: One of the possible mechanisms involved in the chronic administration of melatonin preventing erectile dysfunction in diabetic rats. Asian J. Androl. 2012; 14: 481–6. 10. Zhang W, Wang Y, Yang Z et al. Antioxidant treatment with quercetin ameliorates erectile dysfunction in streptozotocin-induced diabetic rats. J. Biosci. Bioeng. 2011; 112: 215–8. 11. Cardinali DP, Srinivasan V, Brzezinski A, Brown GM. Melatonin and its analogs in insomnia and depression. J. Pineal Res. 2012; 52: 365–74. 12. Mauriz JL, Collado PS, Veneroso C, Reiter RJ, Gonzalez-Gallego J. A review of the molecular aspects of melatonin’s antiinflammatory actions: Recent insights and new perspectives. J. Pineal Res. 2013; 54: 245–57. 13. Hardeland R, Madrid JA, Tan DX, Reiter RJ. Melatonin, the circadian multioscillator system and health: The need for detailed analyses of peripheral melatonin signalling. J. Pineal Res. 2012; 52: 139– 66. 14. Galano A, Tan DX, Reiter RJ. Melatonin as a natural ally against oxidative stress: A physicochemical examination. J. Pineal Res. 2011; 51: 1–16. 15. Galano A, Tan DX, Reiter RJ. On the free radical scavenging activities of melatonin’s metabolites, AFMK and AMK. J. Pineal Res. 2013; 54: 245–57. 16. Rodriguez C, Mayo JC, Sainz RM et al. Regulation of antioxidant enzymes: A significant role for melatonin. J. Pineal Res. 2004; 36: 1–9. 17. Reiter RJ, Tan DX, Osuna C, Gitto E. Actions of melatonin in the reduction of oxidative stress: A review. J. Biomed. Sci. 2000; 7: 444–58. 18. Tengattini S, Reiter RJ, Tan DX, Terron MP, Rodella LF, Rezzani R. Cardiovascular diseases: Protective effects of melatonin. J. Pineal Res. 2008; 44: 16–25. 19. Er(ahin M, Sehirli O, Toklu HZ et al. Melatonin improves cardiovascular function and ameliorates renal, cardiac and cerebral damage in rats with renovascular hypertension. J. Pineal Res. 2009; 47: 97–106. 20. Allen AR. Surgery of experimental lesion of spinal cord equivalent to crush injury of fracture dislocation of spinal column. A preliminary report. JAMA 1911; 57: 878–80. 21. Mullerad M, Donohue JF, Li PS, Scardino PT, Mulhall JP. Functional sequelae of cavernous nerve injury in the rat: Is there model dependency. J. Sex. Med. 2006; 3: 77–83. 22. Beuge JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978; 52: 302.
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23. Beutler E. Glutathione in red blood cell metabolism. In: Beutler E (ed.). A Manuel of Biochemical Methods. Grune & Stratton, NewYork, NY, 1975; 112–4. 24. Hillegas LM, Griswold DE, Brickson B, Albrightson-Winslow C. Assesment of myeloperoxidase activity in whole rat kidney. J. Pharmacol. Methods 1990; 24: 285–95. 25. Mylroie AA, Collins H, Umbles C, Kyle J. Erythrocyte superoxide dismutase activity and other parameters of copper status in rats ingesting lead acetate. Toxicol. Appl. Pharmacol. 1986; 82: 512–20. 26. Bradford MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976; 72: 248–54. 27. Barbonetti A, Cavallo F, Felzani G, Francavilla S, Francavilla F. Erectile dysfunction is the main determinant of psychological distress in men with spinal cord injury. J. Sex. Med. 2012; 9: 830–6. 28. Rizio N, Tran C, Sorenson M. Efficacy and satisfaction rates of oral PDE5s in the treatment of erectile dysfunction secondary to spinal cord injury: A review of literature. J. Spinal Cord Med. 2012; 35: 219–28. 29. Maas R, Schwedhelm E, Albsmeier J, B€oger RH. The pathophysiology of erectile dysfunction related to endothelial dysfunction and mediators of vascular function. Vasc. Med. 2002; 7: 213–25. 30. Khan MA, Thompson CS, Jeremy JY, Mumtaz FH, Mikhailidis P, Morgan RJ. The effect of superoxide dismutase on nitric oxide-mediated and electrical field-stimulated diabetic rabbit cavernosal smooth muscle relaxation. BJU Int. 2001; 87: 98–103. 31. Kettle AJ, Winterbourn CC. Myeloperoxidase: A key regulator of neutrophil oxidant production. Redox Rep. 1997; 3: 3–15. 32. Sewerynek E, Reiter RJ, Melchiorri D, Ortiz GG, Lewinski A. Oxidative damage in the liver induced by ischemia–reperfusion: Protection by melatonin. Hepatogastroenterology 1996; 43: 898–905. € Koyun D, Tetik S et al. Melatonin protects against 33. Sehirli AO, ischemic heart failure in rats. J. Pineal Res. 2013; 55: 138–48. 34. Kannan K, Jain SK. Oxidative stress and apoptosis. Pathophysiology 2000; 7: 153–63. 35. Rong W, Wang J, Liu X et al. Naringin treatment improves functional recovery by increasing BDNF and VEGF expression, inhibiting neuronal apoptosis after spinal cord injury. Neurochem. Res. 2012; 37: 1615–23. 36. Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL. Glutathione dysregulation and the etiology and progression of human diseases. Biol. Chem. 2009; 390: 191–214. 37. Andersson KE. Erectile physiological and pathophysiological pathway involved in erectile dysfunction. J. Urol. 2003; 170(Suppl.): S6–14. 38. Kim YW, Park SY, Kim JY et al. Metformin restores the penile expression of nitric oxide synthase in high-fat-fed obese rats. J. Androl. 2007; 28: 555–60. 39. Barde YA. The nerve growth factor family. Prog. Growth Factor Res. 1990; 2: 237–48. 40. Fiore M, Chaldakov GN, Aloe L. Nerve growth factor as a signaling molecule for nerve cells and also for the neuroendocrine–immune systems. Rev. Neurosci. 2009; 20: 133–45. € € 41. Ersßahin M, Ozdemir Z, Ozsavcı D et al. Melatonin treatment protects against spinal cord injury induced functional and biochemical changes in rat urinary bladder. J. Pineal Res. 2012; 52: 340–8. 42. Burgers JK, Nelson RJ, Quinlan DM, Walsh PC. Nerve growth factor, nerve grafts and amniotic membrane grafts restore erectile function in rats. J. Urol. 1991; 146: 463–8. 43. Samantaray S, Sribnick EA, Das A et al. Melatonin attenuates calpain upregulation, axonal damage and neuronal death in spinal cord injury in rats. J. Pineal Res. 2008; 44: 348–57. 44. Schiaveto-de-Souza A, da-Silva CA, Defino HL, Del Bel EA. Effect of melatonin on the functional recovery from experimental traumatic compression of the spinal cord. Braz. J. Med. Biol. Res. 2013; 46: 348–58.
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45. Lombardi G, Nelli F, Celso M, Mencarini M, Del Popolo G. Treating erectile dysfunction and central neurological diseases with oral phosphodiesterase type 5 inhibitors. Review of the literature. J. Sex. Med. 2012; 9: 970–85. 46. Hamidi Madani A, Asadolahzade A, Mokhtari G et al. Assessment of the efficacy of combination therapy with folic acid and tadalafil for the management of erectile dysfunction in men with type 2 diabetes mellitus. J. Sex. Med. 2013; 10: 1146–50. 47. Rivas DA, Chancellor MB, Huang B, Salzman SK. Erectile response to topical, intraurethral and intracorporal pharmacotherapy
in a rat model of spinal injury. J. Spinal Cord Med. 1995; 18: 245–50. 48. Temeltas G, Dagci T, Evren V, Lekili M. Effects of neuronal and glial precursor cells transplantation on erectile function after experimentally induced spinal cord injury. J. Sex. Med. 2009; 6: 3265–73. 49. Vargas VM, Torres D, Corona F et al. Cholinergic facilitation of erection and ejaculation in spinal cord-transected rats. Int. J. Impot. Res. 2004; 16: 86–90.
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doi: 10.1111/1440-1681.12216
Melatonin and tadalafil treatment improves erectile dysfunction after spinal cord injury in rats _ Hasan H€ useyin Tavukcßu,* Tarik Emre S Tinay,† Cem Akbal,† Mehmet Ersßahin,‡ ß ener,† Ilker § ¶ € Ozge C ß adırcı, Russel J Reiter** and G€oksel S ß ener¶ ß evik, Selin C ‡
*Department of Urology, Academic Hospital, †Department of Urology, School of Medicine, Marmara University, Department of Neurosurgery, School of Medicine, Istanbul Medeniyet University, Istanbul, §Department of Biochemistry, School of Pharmacy, Cumhuriyet University, Sivas, ¶Department of Pharmacology, School of Pharmacy, Marmara University, Istanbul, Turkey and **Department of Cellular and Structural Biology, The UT Health Science Center, San Antonio, TX, USA
SUMMARY 1. Oxidative stress plays an important role both in spinal cord injury (SCI) and erectile dysfunction (ED). The present study investigated the effects of melatonin and tadalafil treatment alone or in combination on SCI-induced ED. 2. Male Wistar albino rats (n = 40) were divided into five groups: sham-operated control and SCI-injured rats given either vehicle, melatonin (10 mg/kg, i.p.), tadalafil (10 mg/kg, p.o.) or a combination of melatonin and tadalafil. Spinal cord injury was induced using a standard weight-drop method. On Day 7 after SCI, intracavernosal pressure (ICP) was measured and all rats were decapitated. Cavernosal tissues were obtained to examine caspase 3, nitric oxide synthase (NOS), myeloperoxidase (MPO) and superoxide dismutase (SOD) activities, as well as cGMP, nerve growth factor (NGF), malondialdehyde (MDA) and glutathione (GSH) levels. 3. Spinal cord injury caused oxidative damage, as evidenced by increases in MDA and cGMP levels. In addition, MPO and caspase 3 activites were increased after SCI, whereas GSH and NGF levels and SOD activity were reduced. Melatonin effectively reversed these oxidative changes. Furthermore, in rats treated with both melatonin and tadalafil, the recoveries were more pronounced than in rats given either melatonin or tadalafil alone. The ICP/mean arterial pressure value in vehicle-treated SCI rats was significantly higher than in the control group, whereas in the tadalafil- and tadalafil + melatonin-treated groups have returned this value had returned to control levels. 4. As an individual treatment, and especially when combined with tadalafil, a well-known agent in the treatment of ED, melatonin prevented SCI-induced oxidative damage to cavernosal tissues and restored ED, most likely due to its anti-oxidant effects.
Correspondence: G€ oksel Sener, School of Pharmacy, Marmara University, Tibbiye Cad., 34668 Istanbul, Turkey. Email: [email protected] Received 6 October 2013; revision 3 February 2014; accepted 9 February 2014. © 2014 Wiley Publishing Asia Pty Ltd
Key words: apoptosis, erectile spinal cord injury, tadalafil.
dysfunction,
melatonin,
INTRODUCTION Spinal cord injury (SCI), which primarily occurs in young men, results in sexual dysfunction and has a negative impact on physical, social and psychological well being.1,2 Because normal erection depends on a precise balance between psychological, hormonal, neurological, vascular and cavernosal factors, a change in one or a combination of these factors may lead to erectile dysfunction (ED).3,4 The secondary injury resulting from SCI following mechanical compression-induced primary injury involves a cascade of biochemical, molecular and cellular changes, which can lead to even more extensive damage.5 These secondary events include microvascular ischaemia, oxidative stress, excitotoxicity, ion dysregulation and inflammation causing apoptotic cell death, which contributes to numerous medical problems and eventually alters basic organ physiology.6 Although mechanical disruption of the innervating neurons is not amenable to neuroprotective therapy, reductions in secondary injury are susceptible to therapeutic intervention. Because the importance of oxidative stress and inflammation in SCI has been documented in a large number of experimental and clinical studies, the use of anti-oxidants to protect against SCI-induced tissue injury and organ dysfunction has received significant attention.6–8 Similarly, anti-oxidants have been shown to be effective in the treatment of ED induced by oxidative stress.9,10 Melatonin, the major product of the vertebrate pineal gland, functions as a modulator of sleep, sexual behaviour, immune function and circadian rhythms.11–13 Melatonin and its metabolic derivatives are uncommonly effective direct free radical scavengers,14,15 while also stimulating the activities of several anti-oxidative enzymes.16 Melatonin is higly lipid soluble and easily passes the blood–brain barrier and, because of this property, within minutes after its administration it reaches high concentrations in neural tissue.17 In addition to its direct radical-scavenging actions, melatonin is involved in the regulation of many physiological systems, including cardiovascular physiology, through its receptors in the heart and arteries.18,19
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The present study was designed to investigate the protective effects of melatonin against SCI-induced erectile tissue injury and dysfunction. Furthermore, we also examined the effects of tadalafil, a well-known phosphodiesterase inhibitor (PDEI) commonly used to treat ED; this drug was either given alone or in combination with melatonin.
METHODS Animals and experimental design Wistar albino rats (250–300 g) were supplied by the Marmara University (MU) Application and Research Center for Experimental Animals (DEHAMER). All experimental protocols were approved by the MU Animal Care and Use Committee. Rats were randomly divided into five groups of eight rats each: sham-operated control and SCI-damaged rats treated with either vehicle, melatonin (10 mg/kg, i.p.), tadalafil (10 mg/kg, p.o.) or melatonin + tadalafil. Treatments were started following SCI damage and continued for 7 days. At the end of the week-long experimental period after sham surgery or SCI induction, rats were anaesthetized with an i.p. injection of 100 mg/kg ketamine and 1 mg/kg chlorpromazine and, after ICP measurements had been made, cavernosal tissue samples were obtained for biochemical analyses. Induction of SCI Anaesthetized (100 mg/kg, i.p., ketamine and 1 mg/kg, i.p., chlorpromazine) rats were positioned on a thermostat-controlled heating pad in the prone position and a rectal probe was inserted. Under sterile conditions, following a T5–12 midline skin incision and paravertebral muscle dissection, spinous processes and laminar arcs of T7–10 were removed. The dura was left intact. A modified weight-drop model was performed to produce SCI.20 Rats were subjected to an impact of 100 g/cm (10 g weight from a height of 10 cm) to the dorsal surface of the spinal cord. The force was applied via a stainless steel rod (3 mm diameter tip) that was rounded at the surface. The rod was dropped vertically through a 10 cm guide tube that was positioned perpendicular to the centre of the spinal cord. The incision was sutured and rats were placed in a warming chamber, where their body temperature was maintained at approximately 37°C until they were completely awake. Cavernous nerve stimulation and ICP/MAP measurement A week after SCI or sham operation, rats were anaesthetized as described above. An anterior transverse neck incision was made and the muscles were separated. The left internal carotid artery was isolated. The artery was cannulated with a heparinized PE50 tube connected to a pressure transducer and an amplifier unit (COMMAT Pharmacology & Physiology Instruments, Ankara, Turkey). The amplifier was connected to a data-acquisition module (MP35 data-acquisition system; COMMAT, Ankara, Turkey). This allowed MAP to be recorded on a computer using Biopac Student Laboratory PRO recording software (Biopac Systems, Goleta, CA, USA). The penis was circumcised and denuded of skin. At its junction with the pubic arch, the
ischiocavernous muscle was divided and the tunica albuginea was visualized. Intracavernous pressure was measured by inserting a 24 gauge needle into the left crus of the penis. This needle was connected to a transducer, via a heparinized PE-50 tube, for MAP measurement. The original laparotomy incision was extended down to the base of the exposed penis. The cavernosal nerve (CN) was located bilaterally and lying on both sides of the dorsal prostate. Following careful manipulation of the CN with a micromanipulator, a stainless steel bipolar electrode with parallel hooks (1 mm apart) was placed around the nerve, just distal to the ganglion. The electrode cable was attached to a STPTO2-A stimulator (COMMAT Pharmacology & Physiology Instruments); the stimulation parameters were: 1.5 mA, 20 Hz, pulse width 5, 35 ms delay, at 7.5 V for 60 s each. The CN was stimulated and data were recorded individually. The maximum ICP/MAP ratio was calculated by dividing the highest ICP recorded during stimulation by the corresponding MAP and is represented as a percentage.21 Measurement of tissue MDA and GSH levels Corpus cavernosum tissue samples were homogenized with icecold 150 mmol/L KCl for the determination of MDA and GSH levels. The MDA levels were assayed as products of lipid peroxidation by monitoring the formation of thiobarbituric acid-reactive substances, as described previously.22 Results are expressed as nmol MDA/g tissue. The activity of GSH was determined using a modification of the Ellman procedure and results are expressed in lmol GSH/g tissue.23 Measurement of tissue MPO activity Myeloperoxidase activity in cavernosal tissues was measured by a procedure similar to that described by Hillegas et al.24 One unit of enzyme activity was defined as the amount of MPO present that caused a change in absorbance, measured at 460 nm for 3 min, with MPO activity expressed as U/g tissue. Measurement of SOD activity Superoxide dismutase activity in tissue samples was measured according to the method of Mylroie et al.25 A standard curve was prepared with bovine SOD (3000 U; S-2515; Sigma, St. Louis, MO, USA) as a reference. Absorbance readings were taken at 0 and 8 min of illumination and the net absorbance was calculated. Measurement of cGMP levels Tissue levels of cGMP were determined in duplicate using a commercially available ELISA kit according to the manufacturer’s instructions (Enzo Life Sciences, Farmingdale, NY, USA). Total protein was estimated using the Bradford method,49 with cGMP values presented as pmol/mg protein. Measurement of NGF levels Tissue concentrations of NGF were determined by sandwich ELISA using a commercially available kit according to the
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Erectile function in spinal cord injury manufacturer’s instructions (Chemicon International, Temecula, CA, USA). A total protein assay was performed using the Bradford method26 and NGF values are presented as pg/mg protein.
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Measurement of tissue caspase 3 and NOS activities A caspase 3 cellular activity assay kit (Calbiochem, San Diego, CA, USA) and NOS activity assay kit (EnzyChrom, BioAssay Systems, Hayward, CA, USA) were used. Caspase 3 and NOS activities are presented as nmol/min per mg protein and U/mg protein, respectively. Western blot analysis of caspase 3 and eNOS
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Caspase 3 and eNOS release were measured directly by western blot analysis, as described previously.8 Protein concentrations in homogenized samples were determined using the Bradford method.26 Briefly, 25 lg protein, resolved by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis, was transferred to a nitrocellulose membrane (sc-3718; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membrane was blocked with 5% non-fat skim milk powder (70166; Sigma) in Tris-buffered saline (TBS) before being washed twice in TBS containing 0.1% Tween-20 and incubated overnight with primary antibody (1 : 500 monoclonal rat anti-caspase 3 (sc-56055; Santa Cruz Biotechnology), anti-eNOS (sc-136977; Santa Cruz Biotechnology) and anti-b-actin (sc-47778; Santa Cruz Biotechnology)). Then, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1 : 1000 goat anti-mouse IgG1-HRP; sc-2060; Santa Cruz Biotechnology) for 2 h at room temperature. The blot was developed with reagents (sc-2048; Santa Cruz Biotechnology). Data were analysed using IMAGEJ Programme OD analysis software (National Institutes of Health, Bethesda, MD, USA). Signals were normalized against b-actin. Statistical analysis Statistical analysis was performed using GRAPHPAD PRISM 4.0 (GraphPad Software, San Diego, CA, USA). All data are expressed as the mean SEM. Groups of data were compared by analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests. Two-tailed P < 0.05 was considered significant.
RESULTS Spinal cord injury caused a significant (P < 0.001) increase in myeloperoxidase (MPO) activity in cavernosal tissues compared with activity in sham-operated control rats (Fig. 1a). In SCI-damaged rats that received either melatonin, tadalafil or melatonin + tadalafil, MPO activities were significantly reduced (P < 0.05–0.001), with the levels being near those found in control rats. As a result of SCI, malondialdehyde (MDA) levels in cavernosal tissues increased significantly (P < 0.001). Melatonin alone significantly reduced MDA levels (P < 0.001). In tadalafiltreated SCI rats, tadalafil failed to lower MDA levels, which remained higher than levels in the control rats (P < 0.01). Conversely, with combination treatment, MDA levels decreased
Fig. 1 (a) Myeloperoxidase (MPO) activity and (b) malondialdehyde (MDA) levels in corpus cavernosum tissues of sham-operated control (C) and vehicle (■)-, melatonin ( )-, tadalafil ( ) or melatonin + tadalafil ( )-treated spinal cord injury (SCI) rats. Data are the mean SEM (n = 8 rats in each group). **P < 0.01, ***P < 0.001 compared with control; †P < 0.05, ††P < 0.01, †††P < 0.001 compared with the vehicletreated SCI group.
significantly to levels similar to those in the control group (Fig. 1b). In accordance with the increased oxidative stress, levels of reduced glutathione (GSH), a key anti-oxidant, and the activity of superoxide dismutase (SOD), an anti-oxidant enzyme, were significantly depleted in the cavernosal tissues of vehicle-treated SCI rats (P < 0.05 and P < 0.001, respectively; Fig. 2). Tadalafil alone did not increase GSH levels or SOD activity; in rats given both melatonin and tadalafil there was a significant augmentation of GSH and SOD (Fig. 2), with both GSH levels and SOD activity significantly preserved following melatonin and melatonin + tadalafil treatment (P < 0.05–0.001), but not with tadalafil alone. As shown in Fig. 3, cGMP levels were significantly reduced (P < 0.001) by SCI, whereas these values were increased after all treatments. Nerve growth factor (NGF) levels, which decreased after SCI, were elevated only with combination treatment (P < 0.001). Spinal cord injury caused a significant decrease in nitric oxide synthase (NOS) activity (P < 0.05), whereas melatonin, tadalafil and their combination reversed this decrease in NOS activity (P < 0.01–0.001, Fig. 4a); these levels were approximately the same as those in control rats. In addition, semiquantitative western blot analysis revealed a reduction in endothelial (e) NOS protein expression in tissues of vehicle-treated SCI rats (Fig. 4c).
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Fig. 2 (a) Glutathione (GSH) levels and (b) superoxide dismutase (SOD) activity in corpus cavernosum tissues of sham-operated control (C) and vehicle (■)-, melatonin ( )-, tadalafil ( ) or melatonin + tadalafil ( )-treated spinal cord injury (SCI) rats. Data are the mean SEM (n = 8 rats in each group). *P < 0.05, **P < 0.01, ***P < 0.001 compared with control; †P < 0.05, ††P < 0.01, †††P < 0.001 compared with the vehicle-treated SCI group; ‡P < 0.05 compared with the tadalafil-treated SCI group.
However, eNOS protein expression was increased after treatment with melatonin, tadalafil or melatonin + tadalafil (P < 0.001). Caspase 3 activity of cavernosal tissues, as an index of apoptosis, was significantly elevated in vehicle-treated SCI-damaged rats (P < 0.001; Fig. 4b). Conversely. the SCI-mediated increase in apoptosis was significantly depressed after treatment with melatonin or melatonin + tadalafil (P < 0.001). Despite a slight but significant decrease (P < 0.01), caspase 3 activity remained elevated in tadalafil-treated rats, but in the combination treatment group enzyme activity was lower than that of the tadalafil-treated group (P < 0.05). As described in detail in the Methods, proteins were fixed on the autography film using horseradish peroxidase (HRP)-conjugated secondary antibodies. Different exposure times were used to determine proteins with greater and lesser expression at the same time. When the film was exposed for a sufficiently long enough period of time, in addition to procaspase 3 bands, we obtained cleaved caspase 3, as shown in Fig. 4e. The p20 and p17 subunits of cleaved caspase 3 bands were also apparent; the density of these was especially high in vehicle-treated SCI rats. Similarly, semiquantitative western blot analysis indicated elevated caspase 3 protein expression in cavernosal tissues of vehicle-treated SCI rats, which was significantly attenuated with drug treatment, either alone or in combination (Fig. 4d). As shown in Fig. 5, the ratio of intracavernosal pressure (ICP) to mean arterial pressure (MAP) in SCI rats was significantly
Fig. 3 (a) cGMP and (b) nerve growth factor (NGF) levels in corpus cavernosum tissues of sham-operated control (C) and vehicle (■)-, melatonin ( )-, tadalafil ( ) or melatonin + tadalafil ( )-treated spinal cord injury (SCI) rats. Data are the mean SEM (n = 8 rats in each group). ***P < 0.001 compared with control; †††P < 0.001 compared with the vehicle-treated SCI group; ‡‡P < 0.01 compared with the tadalafil-treated SCI group; §§P < 0.01 compared with the melatonin-treated SCI group.
higher than in control rats (102 11 vs 56 3, respectively; P < 0.01). This increase was reversed by tadalafil and melatonin + tadalafil treatment (P < 0.05). Melatonin alone also decreased the values similar to control levels, but these values were not significantly different from those for SCI rats. The ICP/ MAP ratios in the melatonin-, tadalafil- and melatonin + tadalafil-treated groups were 73 8, 67 8 and 62 10, respectively; these did not differ significantly from control values.
DISCUSSION The data of the present study demonstrate that SCI causes inflammatory and oxidative damage in erectile tissue because cavernosal MDA levels and MPO and caspase 3 activities were elevated. In association with the elevated oxidative injury, there was a depletion of the tissue anti-oxidants GSH and SOD. Furthermore, cGMP and eNOS, which are important parameters of erectile function, were depressed. Furthermore, the findings of the present study clearly show that melatonin and tadalafil, when given in combination, reverse these changes and protect cavernosal tissues against SCI-mediated tissue damage. Spinal cord injury causes a series of pathophysiological changes that compromise organ function; these negative changes have a tremendous impact on the patient and his family. A major complication that influences quality of life in men is ED.27,28 It is believed that reductions in nitric oxide (NO) bioavailability
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Fig. 4 (a) Nitric oxide synthase (NOS) and (b) caspase 3 activity and (c) endothelial (e) NOS and (d) caspase 3 protein expression in corpus cavernosum tissues of sham-operated control (C) and vehicle (■)-, melatonin ( )-, tadalafil ( ) or melatonin + tadalafil ( )-treated spinal cord injury (SCI) rats. Data are the mean SEM (n = 8 rats in each group). *P < 0.05, **P < 0.01, ***P < 0.001 compared with control; †P < 0.05, †††P < 0.001, ††† P < 0.001 compared with the vehicle-treated SCI group; ‡P < 0.05, ‡‡P < 0.01 compared with the tadalafil-treated SCI group; §§P < 0.01 compared with the melatonin-treated SCI group. (e) Western blot analysis of eNOS and procaspase-3. Mel, melatonin; TD, tadalafil.
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Fig. 5 Intracavernosal pressure (ICP)/mean arterial pressure (MAP) ratio in sham-operated control (C) and vehicle (■)-, melatonin ( )-, tadalafil ( ) or melatonin + tadalafil ( )-treated spinal cord injury (SCI) rats. Data are the mean SEM (n = 8 rats in each group). **P < 0.01 compared with control; †P < 0.05 compared with the vehicle-treated SCI group.
due to increased generation of reactive oxygen species (ROS) is a major determinant of ED.29 In the present study, we monitored ROS-induced tissue damage via the end-product of lipid peroxidation, namely MDA, and found that SCI caused a significant increase in MDA levels in corpus cavernosum tissues. In addition to ROS-induced damage, an inflammatory response has been suggested as an important factor in the development of secondary injury following SCI. Activated neutrophils exacerbate tissue injury through the production of oxygen metabolites and activation of cytotoxic enzymes, including MPO.30 Herein, the presence of increased neutrophil accumulation, as evidenced by elevated MPO activity in cavernosal tissues, indicates that neutrophil accumulation contributes to SCI-induced oxidative injury in cavernosal tissue. The ability of melatonin to inhibit neutrophil accumulation and the associated MPO activity is well known.31,32 Because oxidizing processes trigger the activation of caspases, changes in the redox status elevate oxidative stress; under these conditions, cells are killed either by necrosis or apoptosis.33 Apoptosis is a major complicating process related to oxidative events in the pathogenesis of secondary injury after SCI.34 In a previous study, we demonstrated that SCI increases caspase 3 activity in bladder tissue, in which MDA levels and MPO activity were also increased and bladder contractility impaired.8 In agreement with these observations, the results of the present study demonstrate that, in the cavernosal tissues of rats with SCI, caspase 3 activation is increased as a result of oxidative stress. Reduced glutathione and SOD are critical components of the anti-oxidant system.35 In the present study, both GSH levels and SOD activity were found to be depressed in cavernosal tissues of rats with SCI; these reductions likely elevated oxidative damage and contributed to ED. We aslo investigated NOS activity and cGMP levels, major indicators in the NO/cGMP signalling pathway, in erectile tissue. Spinal cord injury significantly reduced NOS activity and cGMP levels. The drop in NOS activity was associated with depressed eNOS, as determined by western blot analysis. It is well known that NO is an important mediator for the relaxation of the corpus cavernosum smooth muscle and vasculature. Nitric oxide generated by neuronal (n) NOS is considered a key factor responsible for the immediate relaxation of the corpus cavernosum, whereas
NO from eNOS is essential for maintaining relaxation.36,37 Kim et al.38 suggested that the reductions in nNOS and eNOS levels can cause circulatory and structural changes in penile tissues, resulting in ED. Nerve growth factor, a member of the neurotrophin family, is important for the growth, maintenance and survival of peripheral sensory and autonomic neurons.39 It functions as a signalling molecule.40 In a previous study, we demonstrated that SCI reduced NGF levels in bladder tissue in which contractility of bladder strips was impaired.41 There are limited published data regarding NGF in penile tissues or its relationship with ED. Burgers et al.,42 after ablating the cavernous nerve in rats, used NGF alone or in combination with interposed nerve grafts and demonstrated their potential for reducing the loss of surgically induced nerve injuries. Herein we demonstrated for the first time that NGF levels were significantly decreased after SCI, which may contribute to the development of ED. In animal models of SCI, melatonin has proven highly effective in reducing lipid peroxidation, stimulating several anti-oxidant enzymes and inhibiting apoptosis.43,44 Conversely, tadalafil, a PDEI, is a widely used agent in the treatment of ED because it blocks the degredation of cGMP and thereby augments the local relaxant effects of NO.45,46 Looking at the ICP/MAP ratio, the elevated values in SCI rats were lowered to control levels by tadalafil or tadalafil + melatonin treatment, whereas melatonin alone did not significantly change these values. Rivas et al.47 previously claimed that SCI rats exhibited a supersensitive response to pharmacological erectile therapy on ICP cavernosometry; unlike in the present study, those authors measured only ICP values. Temeltas et al.48 reported higher ICP/MAP values in SCI; however, these authors cautioned that these higher values should not be interpreted as erectile improvement. Furthermore, Temeltas et al.48 evaluated sexual function using the method of penile reflexes described earlier by Vargas et al.49 and reported that penile erections after nerve stimulation were significantly depressed after SCI despite the higher ICP/MAP values. In accordance with those results, we found that ICP/MAP values were higher in the SCI compared with control group. Conversely, melatonin alone decreased this significant increase in ICP/MAP values in SCI rats close to control levels, but these levels did not differ significantly from values in either the control or SCI rats. However, the ICP/MAP values in the tadalafil and tadalafil + melatonin groups were significantly lower than those of SCI rats. Our biochemical analysis also supported the results in predicting treatment responses to melatonin and tadalafil in ED after SCI damage. In conclusion, the findings of the present study show the critical pathogenic contribution of increased oxidative stress in SCIinduced ED. Furthermore, our results suggest that, when used in combination, melatonin and tadalafil induce similar beneficial effects through different mechanisms of action. Thus, when these agents are given in combination, the prevention of cavernosal damage and consequent ED may be possible.
REFERENCES 1. Giuliano F, Sanchez-Ramos A, L€ochner-Ernst D et al. Efficacy and safety of tadalafil in men with erectile dysfunction following spinal cord injury. Arch. Neurol. 2007; 64: 1584–92.
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Erectile function in spinal cord injury 2. Biering-Sørensen I, Hansen RB, Biering-Sørensen F. Sexual function in a traumatic spinal cord injured population 10–45 years after injury. J. Rehabil. Med. 2012; 44: 926–31. 3. Agarwal A, Nandipati KC, Sharma RK, Zippe CD, Raina R. Role of oxidative stress in the pathophysiological mechanism of erectile dysfunction. J. Androl. 2006; 27: 335–47. 4. Silva FH, M onica FZ, Bau FR et al. Superoxide anion production by NADPH oxidase plays a major role in erectile dysfunction in middle-aged rats: Prevention by antioxidant therapy. J. Sex. Med. 2013; 10: 960–71. 5. Azbill RD, Mu X, Bruce-Keller AJ, Mattson MP, Springer JE. Impaired mitochondrial function, oxidative stress and altered antioxidant enzyme activities following traumatic spinal cord injury. Brain Res. 1997; 765: 283–90. 6. Hall ED. Antioxidant therapies for acute spinal cord injury. Neurotherapeutics 2011; 8: 152–67. 7. Bastani NE, Kostovski E, Sakhi AK et al. Reduced antioxidant defense and increased oxidative stress in spinal cord injured patients. Arch. Phys. Med. Rehabil. 2012; 93: 2223–8. 8. Cevik O, Ersßahin M, Sener TE et al. Beneficial effects of quercetin on rat urinary bladder after spinal cord injury. J. Surg. Res. 2013; 183: 695–703. 9. Qiu XF, Li XX, Chen Y et al. Mobilisation of endothelial progenitor cells: One of the possible mechanisms involved in the chronic administration of melatonin preventing erectile dysfunction in diabetic rats. Asian J. Androl. 2012; 14: 481–6. 10. Zhang W, Wang Y, Yang Z et al. Antioxidant treatment with quercetin ameliorates erectile dysfunction in streptozotocin-induced diabetic rats. J. Biosci. Bioeng. 2011; 112: 215–8. 11. Cardinali DP, Srinivasan V, Brzezinski A, Brown GM. Melatonin and its analogs in insomnia and depression. J. Pineal Res. 2012; 52: 365–74. 12. Mauriz JL, Collado PS, Veneroso C, Reiter RJ, Gonzalez-Gallego J. A review of the molecular aspects of melatonin’s antiinflammatory actions: Recent insights and new perspectives. J. Pineal Res. 2013; 54: 245–57. 13. Hardeland R, Madrid JA, Tan DX, Reiter RJ. Melatonin, the circadian multioscillator system and health: The need for detailed analyses of peripheral melatonin signalling. J. Pineal Res. 2012; 52: 139– 66. 14. Galano A, Tan DX, Reiter RJ. Melatonin as a natural ally against oxidative stress: A physicochemical examination. J. Pineal Res. 2011; 51: 1–16. 15. Galano A, Tan DX, Reiter RJ. On the free radical scavenging activities of melatonin’s metabolites, AFMK and AMK. J. Pineal Res. 2013; 54: 245–57. 16. Rodriguez C, Mayo JC, Sainz RM et al. Regulation of antioxidant enzymes: A significant role for melatonin. J. Pineal Res. 2004; 36: 1–9. 17. Reiter RJ, Tan DX, Osuna C, Gitto E. Actions of melatonin in the reduction of oxidative stress: A review. J. Biomed. Sci. 2000; 7: 444–58. 18. Tengattini S, Reiter RJ, Tan DX, Terron MP, Rodella LF, Rezzani R. Cardiovascular diseases: Protective effects of melatonin. J. Pineal Res. 2008; 44: 16–25. 19. Er(ahin M, Sehirli O, Toklu HZ et al. Melatonin improves cardiovascular function and ameliorates renal, cardiac and cerebral damage in rats with renovascular hypertension. J. Pineal Res. 2009; 47: 97–106. 20. Allen AR. Surgery of experimental lesion of spinal cord equivalent to crush injury of fracture dislocation of spinal column. A preliminary report. JAMA 1911; 57: 878–80. 21. Mullerad M, Donohue JF, Li PS, Scardino PT, Mulhall JP. Functional sequelae of cavernous nerve injury in the rat: Is there model dependency. J. Sex. Med. 2006; 3: 77–83. 22. Beuge JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978; 52: 302.
315
23. Beutler E. Glutathione in red blood cell metabolism. In: Beutler E (ed.). A Manuel of Biochemical Methods. Grune & Stratton, NewYork, NY, 1975; 112–4. 24. Hillegas LM, Griswold DE, Brickson B, Albrightson-Winslow C. Assesment of myeloperoxidase activity in whole rat kidney. J. Pharmacol. Methods 1990; 24: 285–95. 25. Mylroie AA, Collins H, Umbles C, Kyle J. Erythrocyte superoxide dismutase activity and other parameters of copper status in rats ingesting lead acetate. Toxicol. Appl. Pharmacol. 1986; 82: 512–20. 26. Bradford MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976; 72: 248–54. 27. Barbonetti A, Cavallo F, Felzani G, Francavilla S, Francavilla F. Erectile dysfunction is the main determinant of psychological distress in men with spinal cord injury. J. Sex. Med. 2012; 9: 830–6. 28. Rizio N, Tran C, Sorenson M. Efficacy and satisfaction rates of oral PDE5s in the treatment of erectile dysfunction secondary to spinal cord injury: A review of literature. J. Spinal Cord Med. 2012; 35: 219–28. 29. Maas R, Schwedhelm E, Albsmeier J, B€oger RH. The pathophysiology of erectile dysfunction related to endothelial dysfunction and mediators of vascular function. Vasc. Med. 2002; 7: 213–25. 30. Khan MA, Thompson CS, Jeremy JY, Mumtaz FH, Mikhailidis P, Morgan RJ. The effect of superoxide dismutase on nitric oxide-mediated and electrical field-stimulated diabetic rabbit cavernosal smooth muscle relaxation. BJU Int. 2001; 87: 98–103. 31. Kettle AJ, Winterbourn CC. Myeloperoxidase: A key regulator of neutrophil oxidant production. Redox Rep. 1997; 3: 3–15. 32. Sewerynek E, Reiter RJ, Melchiorri D, Ortiz GG, Lewinski A. Oxidative damage in the liver induced by ischemia–reperfusion: Protection by melatonin. Hepatogastroenterology 1996; 43: 898–905. € Koyun D, Tetik S et al. Melatonin protects against 33. Sehirli AO, ischemic heart failure in rats. J. Pineal Res. 2013; 55: 138–48. 34. Kannan K, Jain SK. Oxidative stress and apoptosis. Pathophysiology 2000; 7: 153–63. 35. Rong W, Wang J, Liu X et al. Naringin treatment improves functional recovery by increasing BDNF and VEGF expression, inhibiting neuronal apoptosis after spinal cord injury. Neurochem. Res. 2012; 37: 1615–23. 36. Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL. Glutathione dysregulation and the etiology and progression of human diseases. Biol. Chem. 2009; 390: 191–214. 37. Andersson KE. Erectile physiological and pathophysiological pathway involved in erectile dysfunction. J. Urol. 2003; 170(Suppl.): S6–14. 38. Kim YW, Park SY, Kim JY et al. Metformin restores the penile expression of nitric oxide synthase in high-fat-fed obese rats. J. Androl. 2007; 28: 555–60. 39. Barde YA. The nerve growth factor family. Prog. Growth Factor Res. 1990; 2: 237–48. 40. Fiore M, Chaldakov GN, Aloe L. Nerve growth factor as a signaling molecule for nerve cells and also for the neuroendocrine–immune systems. Rev. Neurosci. 2009; 20: 133–45. € € 41. Ersßahin M, Ozdemir Z, Ozsavcı D et al. Melatonin treatment protects against spinal cord injury induced functional and biochemical changes in rat urinary bladder. J. Pineal Res. 2012; 52: 340–8. 42. Burgers JK, Nelson RJ, Quinlan DM, Walsh PC. Nerve growth factor, nerve grafts and amniotic membrane grafts restore erectile function in rats. J. Urol. 1991; 146: 463–8. 43. Samantaray S, Sribnick EA, Das A et al. Melatonin attenuates calpain upregulation, axonal damage and neuronal death in spinal cord injury in rats. J. Pineal Res. 2008; 44: 348–57. 44. Schiaveto-de-Souza A, da-Silva CA, Defino HL, Del Bel EA. Effect of melatonin on the functional recovery from experimental traumatic compression of the spinal cord. Braz. J. Med. Biol. Res. 2013; 46: 348–58.
© 2014 Wiley Publishing Asia Pty Ltd
316
HH Tavukcßu et al.
45. Lombardi G, Nelli F, Celso M, Mencarini M, Del Popolo G. Treating erectile dysfunction and central neurological diseases with oral phosphodiesterase type 5 inhibitors. Review of the literature. J. Sex. Med. 2012; 9: 970–85. 46. Hamidi Madani A, Asadolahzade A, Mokhtari G et al. Assessment of the efficacy of combination therapy with folic acid and tadalafil for the management of erectile dysfunction in men with type 2 diabetes mellitus. J. Sex. Med. 2013; 10: 1146–50. 47. Rivas DA, Chancellor MB, Huang B, Salzman SK. Erectile response to topical, intraurethral and intracorporal pharmacotherapy
in a rat model of spinal injury. J. Spinal Cord Med. 1995; 18: 245–50. 48. Temeltas G, Dagci T, Evren V, Lekili M. Effects of neuronal and glial precursor cells transplantation on erectile function after experimentally induced spinal cord injury. J. Sex. Med. 2009; 6: 3265–73. 49. Vargas VM, Torres D, Corona F et al. Cholinergic facilitation of erection and ejaculation in spinal cord-transected rats. Int. J. Impot. Res. 2004; 16: 86–90.
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