ORIGINAL ARTICLE
Effects of intracavernous injection of P2X3 and NK1 receptor antagonists on erectile dysfunction induced by spinal cord transection in rats C.-L. Li1, X.-L. Yang2, J.-J. Wang1, G.-H. Du3, W.-M. Yang3 & H.-P. Zhang1 1 Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China; 2 Department of Urology, Tianyou Hospital, Wuhan University of Science and Technology, Wuhan, Hubei, China; 3 Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
Keywords Erectile dysfunction—NK1 receptor—P2X3 receptor—spinal cord transection Correspondence Hui-Ping Zhang, Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China. Tel./Fax: 0086-27-83692651; E-mail: [email protected]
Accepted: November 11, 2013 doi: 10.1111/and.12217
Summary This study aimed to explore the effects of intracavernous injection (ICI) of P2X3 and NK1 receptor antagonists on erectile dysfunction (ED) induced by spinal cord transection in rats. Sixty male Sprague–Dawley (SD) rats were randomly divided into the following three groups (20 rats each group): sham operation group (C group), thoracic spinal cord transection group (T group) and sacral spinal cord transection group (S group). An ED model was established through complete transection of the thoracic or sacral spinal cord. Intracavernous pressure (ICP) with and without injection of P2X3 (Suramin) or NK1 (GR82334) receptor antagonists was recorded 3 weeks after surgery. Immunohistochemistry was employed to detect the expression of P2X3 and NK1 receptors in the dorsal root ganglion (DRG) and smooth muscle of corpus cavernosum. Data were processed with SPSS 17.0. ICI with Suramin (0.1, 0.3 and 1 mM) or GR82334 (0.1, 0.3 and 1 mM) increased ICP dose dependently in the T and S groups. The expression of P2X3 and NK1 receptors in DRG and smooth muscle of corpus cavernosum was up-regulated in the T and S groups. It is concluded that ICI of P2X3 and NK1 receptor antagonists may improve the recovery of erectile function in a rat model with ED after spinal cord transection.
Introduction Trauma and fall injury are the main causes of spinal cord injury (SCI) (Pickett et al., 2006). Decreased sexual function is a major concern among men with spinal cord injuries (SCIs). Treatment for erectile dysfunction (ED) through oral pharmacotherapy is an effective method of treating this disorder. Phosphodiesterase-5 inhibitors (PDE5) provide a first-line ED therapy for SCI patients (Lombardi et al., 2012). However, PDE5 could worsen the neurological illness of patients with central neurological disorders (CND) (Lombardi et al., 2012). Thus, finding an effective and satisfactory treatment for ED secondary to SCI is necessary. Current methods of establishing an animal model of neurogenic ED involve damaging or cutting the supplying nerves, such as the cavernous, pelvic and dorsal penile nerves (Yamashita et al., 2011; Ying et al., 2013). However, ED after SCI is different from neurogenic ED after © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 25–29
surgery in terms of pathogenesis, pathophysiology and treatment. Thus, a model with ED induced by damaging or cutting the supplying nerve cannot better simulate neurogenic ED after SCI. Vascular smooth muscle relaxation, mediated by nitric oxide (NO), plays an important role in the process of penile erection. Meanwhile, other endogenous neurotransmitters, such as adenosine, ATP, VIP, CGRP and SP, are reported to exist in the corpora cavernosa. However, the role of these neurotransmitters in the erectile process remains unclear. There are seven subtypes of purinergic (P)2X-ATP receptors, and studies have demonstrated that dorsal root ganglion express predominantly homomeric P2X3 and heteromeric P2X2/ 3 receptors (Wirkner et al., 2007). The neurokinin 1 (NK1) receptor, also known as tachykinin receptor 1 (TACR1), is receptor of substance P. The substance P-NK1 receptor system is the most extensively studied neurokinin pathway in the brain and spinal cord 25
P2X3-r and NK1-r antagonists on ED
(Mantyh, 2002). Reports show that the expression of P2X3 and NK1 receptors in DRG is up-regulated in rat models of SCI, and the subtypes of P2X receptors have different levels of expression in the urogenital system. However, the effect of P2X3 and NK1 receptor antagonists on ED induced by spinal cord transection is rarely reported. Thus, complete transection of the spinal cord was performed in this study to establish an ED model that simulates neurogenic ED after SCI in humans. The effects of the intracavernous injection of P2X3 and NK1 receptor antagonists on ED after spinal cord transection in rats were examined. Materials and methods Animals Sixty adult male Sprague–Dawley (SD) rats (10 weeks old, 300–350 g) were purchased from Experimental Animal Center, Tongji Medical College of Huazhong University of Science and Technology. The rats were housed at 24 °C under controlled conditions with free access to standard rat chow and water. All experiments were approved by the Institutional Animal Care and Use Committee of Tongji Medical College and were performed in strict accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (approval no. SCXK 2010-0007).
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Record of intracavernous pressure Three weeks after surgery, the rats were anaesthetised with pentobarbital sodium (50 mg kg 1). Systemic mean arterial pressure (MAP) was monitored by inserting a 22-G cannula into the carotid artery on the left side of the incised neck. The skin overlying the penis was incised, and the crura of the penis were identified. Two 23-G scalp-vein needles filled with 250 U ml 1 of heparin solution were connected with a polyethylene-50 tubing and inserted into the crus; the right one was for measuring the intracavernous pressure (ICP), and the left one was for the intracavernous injection (ICI) of drugs. ICP and MAP were recorded with a bioinformation acquisition system (BL-420F; Chengdu TMA Technology Co. Ltd., Chengdu, China). ICI of drugs After basal ICP (BICP) was recorded, different concentrations (0.1, 0.3 and 1 mM, 100 ll per injection) of P2X3 receptor antagonist, Suramin (Sigma Aldrich, St. Louis, USA), were administered intracavernously. The effects of Suramin were observed for 20 min, and ICP was recorded. After 30 min, different concentrations (0.1, 0.3 and 1 mM, 100 ll per injection) of NK1 receptor antagonist, GR 82334 (Sigma Aldrich, St. Louis, MO, USA), were administered intracavernously. Also, the effects of GR 82334 were observed for 20 min, and ICP was recorded.
Surgical procedures The rats were allowed to adapt to the environment for at least 3 days. Normal sexual function was confirmed prior to surgery by mating the male rats with the female rats. The rats were randomly divided into three equal groups: sham operation group (C group), thoracic spinal cord transection group (T group) and sacral spinal cord transection group (S group). The rats were anaesthetised with intraperitoneal administration of pentobarbital sodium (50 mg kg 1). Laminectomy was performed after midline dorsal incision. Spinal segment T8–T9 was exposed in the C and T groups, and spinal segment L1–L2 was exposed in the S group. The dura and spinal cord were then completely transected in the T and S groups, whereas the spinal cord was exposed and received no further surgical manipulation in the C group. After the surgical procedure, the rats received 10 ml of Ringer’s lactate solution intraperitoneally. All rats were injected intramuscularly with penicillin (120 000 unit kg 1) once a day for 10 days. The bladder was manually emptied three times a day until spinal reflex micturition was recovered. 26
Preparation of tissue sections All rats were sacrificed by decapitation. The DRGs of T10–T12 or S2–S4 and the corpus cavernosum were taken out and transferred immediately into 4% paraformaldehyde. After fixation, the DRGs and corpus cavernosum were processed through dehydration with graded ethanol and subjected to vitrification by dimethylbenzene in paraffin. The tissue paraffin block was sectioned at 4 lm prior to staining. Immunohistochemistry After deparaffinage and dehydration, the sections were incubated with Triton X-100 for 30 min at 37 °C, washed (3 9 5 min) with 0.1 M PBS (pH 7.4) and then blocked with 1% BSA for 1 h. Afterwards, the sections were incubated for 3 h at 37 °C and then for 60 h at 4 °C with rabbit P2X3-R antibody (1 : 200; Santa Cruz Biotechnology) or rabbit NK1-R antibody (1 : 50; Santa Cruz Biotechnology Co. Ltd., Shanghai, China). The sections were washed (3 9 10 min) with 0.1 M PBS (pH 7.4) then incubated with goat anti-rabbit IgG (1 : 100, Boster © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 25–29
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P2X3-r and NK1-r antagonists on ED
Biological Technology, Ltd., Wuhan, China) for 1 h at 37 °C. The sections were washed (3 9 5 min) with 0.1 M PBS (pH 7.4) after incubation and incubated with SABC solution (Boster Biological Technology, Ltd, Wuhan, China) for 1 h at 37 °C. Afterwards, the sections were washed (3 9 5 min) with 0.1 M PBS (pH 7.4) and colorated with fresh DAB solution (Boster Biological Technology, Ltd., Wuhan, China). The sections were then placed on cover slips with mounting medium of buffering glycerol (pH 8.8) and stored at 4 °C until observation. Statistical analysis Data were processed with software SPSS 17.0 (SPSS Inc., Chicago, IL, USA) and expressed as mean values SEM. The results were analysed through one-way ANOVA and Dunnett’s t-test. Averaged P-values < 0.05 were considered statistically significant.
Results Observation of the rats The rats in the T and S groups lost their hindlimb movement and hindlimb reflex ability after surgery. After 3 weeks, 13 and 15 rats in the T and S groups were alive respectively; all the 20 rats in the C group were alive. Two rats died of anaesthesia, three of infection, three of bladder rupture and four of unknown reasons.
Table 1 Effect of Suramin on ICP in C, T and S groups
Effects of Suramin and GR 82334 on ICP of rats ICI of Suramin (0.1, 0.3 and 1 mM) had no effect on ICP in the C group (P = 0.9491, Table 1); however, it increased ICP in the T and S groups (P < 0.001, Table 1) except for the 0.1 mM dose (P = 0.5937, Table 1). Compared with the 0.1 mM dose of Suramin, ICP was also significantly increased with 0.3 mM dose and 1 mM dose in the T and S groups (P < 0.0001). Thirty minutes after recording the effect of Suramin, GR 82334 were intracavernously injected. GR 82334 (0.1, 0.3 and 1 mM) had no effect on ICP in the C group (P = 0.8164, Table 2); however, it increased ICP dose dependently in the T and S groups (P < 0.0001, Table 2). Expression of P2X3 and NK1 receptors in DRG The P2X3 receptor were expressed weakly in the DRG in the C group but were strongly and extensively expressed in the T and S groups. The NK1 receptor was up-regulated in the DRG in T and S groups, compared with the C group. Furthermore, the P2X3 and NK1 receptors were predominantly located in the parvicellular neuron (Fig. 1). Expression of P2X3 and NK1 receptors in corpus cavernosum Compared with the C group, the expression of P2X3 and NK1 receptors was up-regulated in the smooth muscle of the corpus cavernosum in the T and S groups. (Fig. 2).
Dose of Suramin ICP (mmHg)
0.1 mM
0.3 mM
1 mM
C group (n = 20) T group (n = 13) S group (n = 15)
12.61 2.71 (n = 6) 14.23 3.21 (n = 4) 13.02 2.45 (n = 4)
12.33 3.12 (n = 6) 19.35 2.45a,c (n = 5) 18.25 3.25a,c (n = 5)
12.82 2.65 (n = 7) 26.24 3.16b,c (n = 4) 24.24 1.24b,c (n = 6)
a P = 0.0006, bP < 0.0001: compared with the C group, ICP was significantly increased in the T and S groups after ICI of 0.1, 0.3 and 1 mM dose of Suramin. c P < 0.0001. Compared with the 0.1 mM dose of Suramin, ICP was significantly increased with 0.3 mM dose and 1 mM dose in the T and S groups.
Table 2 Effect of GR 82334 after Suramin on ICP in C, T and S groups
Dose of GR 82334 ICP (mmHg)
0.1 mM
0.3 mM
1 mM
C group (n = 20) T group (n = 13) S group (n = 15)
13.11 4.10 (n = 6) 30.36 2.34a (n = 4) 28.21 3.26a (n = 4)
14.10 3.23 (n = 7) 36.36 2.50a,b (n = 5) 34.37 4.23a,b (n = 5)
14.34 3.56 (n = 7) 41.32 2.35a,b (n = 4) 39.24 2.41a,b (n = 6)
a P < 0.0001: compared with the C group, ICP was significantly increased in the T and S groups after ICI of 0.1, 0.3 and 1 mM dose of GR82334. b P < 0.0001: compared with 0.1 mM dose of GR82334, ICP were significantly increased with 0.3 mM dose and 1 mM dose in the T and S groups.
© 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 25–29
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P2X3-r and NK1-r antagonists on ED
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(a)
(b)
(c)
(d)
(e)
(f)
Fig. 1 Expression of P2X3 and NK1 receptors in the DRG neurons of rats in C, T and S groups. (a–c) Expression of P2X3 receptor in the DRG neurons of rats in C, T and S groups respectively. (d–f) Expression of NK1 receptor in the DRG neurons of rats in C, T and S groups respectively. Compared with the C group, the expression of P2X3 and NK1 receptors was up-regulated in the DRG neurons in the T and S groups.
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 2 Expression of P2X3 and NK1 receptors in the corpus cavernosum of rats in C, T and S groups. (a–c) Expression of P2X3 receptor in the corpus cavernosum of rats in C, T and S groups respectively. (d–f) Expression of NK1 receptor in the corpus cavernosum of rats in C, T and S groups respectively. Compared with the C group, the expression of P2X3 and NK1 receptors was up-regulated in the corpus cavernosum in the T and S groups.
Discussion An ED model was developed through complete transection of the spinal cord in rats. This procedure was seldom 28
performed in previous studies on neurogenic ED. The present study demonstrates that P2X3 and NK1 receptors (i) have low basal expression in the DRG and corpus cavernosum; (ii) increase in the DRG and corpus © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 25–29
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cavernosum after the transection of the spinal cord; (iii) are related to ED after spinal cord transection; and (iv) ICI of P2X3 and NK1 receptor antagonists can improve the recovery of the erectile function in a rat model with ED. The male reproductive function depends largely on the neurological integrity of the pelvic floor and spinal cord. Erectile function diminished in patients with neurological damage from SCI (Fode et al., 2012). Neurological problems can cause impairment in sperm quality, which is most often seen in men with SCI. Only about 9% of men with SCI can ejaculate through masturbation or sexual intercourse (Brackett et al., 2010). Thus, studying the related mechanism involved in ED after SCI is important. In present study, the expression of P2X3 and NK1 receptors was up-regulated in the DRGs of model rats with ED, which is consistent with the findings of other researchers (Burnstock, 2000; Weissner et al., 2006; Xiang et al., 2008). Furthermore, P2X3 and NK1 receptors were predominantly located in the parvicellular neuron of DRGs. When ICI of P2X3 and NK1 receptor antagonists was implemented, ICP increased, which was highly related to erection. Thus, the P2X3 and NK1 receptors are involved in the regulation of erectile function. The present study has several limitations. Only the receptors and not the expression of P2X3 and NK1 were detected in this study. The gene and protein levels of the receptors were also not quantified with real-time PCR and Western blot analysis. Exposing spinal segment T8– T9 in rats was set as control group to both T and S groups, which was a little inappropriate. Another control group that exposing spinal segment L1–L2 should have been set. Also, model groups without treatment of Suramin and GR 82334 were not set, so the expression of P2X3 and NK1 receptors could not be detected for comparison before and after the treatment. The results of ICP and immunohistochemistry indicate that the P2X3 and NK1 receptors play an important role in ED induced by spinal cord transection. ICI together with the P2X3 and NK1 receptors can increase ICP and could serve as potential methods of therapeutic intervention for ED after spinal cord transection. The gene and protein levels of P2X3 and NK1 receptors must be detected in the future, and their mechanism in ED after spinal cord transection must be explored further.
© 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 25–29
P2X3-r and NK1-r antagonists on ED
Conclusions These studies demonstrate a functional role for P2X3 and NK1 receptors signal ED in normal rats and in those with ED induced by spinal cord transection. P2X3 and NK1 receptor signalling could serve as potential methods of therapeutic intervention for ED induced by spinal cord transection. References Brackett NL, Ibrahim E, Iremashvili V, Aballa TC, Lynne CM (2010) Treatment for ejaculatory dysfunction in men with spinal cord injury: an 18-year single center experience. J Urol 183:2304–2308. Burnstock G (2000) P2X receptors in sensory neurones. Br J Anaesth 84:476–488. Fode M, Krogh-Jespersen S, Brackett NL, Ohl DA, Lynne CM, Sonksen J (2012) Male sexual dysfunction and infertility associated with neurological disorders. Asian J Androl 14:61–68. Lombardi G, Nelli F, Celso M, Mencarini M, Del Popolo G (2012) Treating erectile dysfunction and central neurological diseases with oral phosphodiesterase type 5 inhibitors. Review of the literature. J Sex Med 9:970–985. Mantyh PW (2002) Neurobiology of substance P and the NK1 receptor. J Clin Psychiatry 63(Suppl 11):6–10. Pickett GE, Campos-Benitez M, Keller JL, Duggal N (2006) Epidemiology of traumatic spinal cord injury in Canada. Spine, 31:799–805. Weissner W, Winterson BJ, Stuart-Tilley A, Devor M, Bove GM (2006) Time course of substance P expression in dorsal root ganglia following complete spinal nerve transection. J Comp Neurol 497:78–87. Wirkner K, Sperlagh B, Illes P (2007) P2X3 receptor involvement in pain states. Mol Neurobiol 36:165–183. Xiang Z, Xiong Y, Yan N, Li X, Mao Y, Ni X, He C, Lamotte RH, Burnstock G, Sun J (2008) Functional up-regulation of P2X 3 receptors in the chronically compressed dorsal root ganglion. Pain 140:23–34. Yamashita S, Kato R, Kobayashi K, Hisasue S, Arai Y, Tsukamoto T (2011) Inhibition of interleukin-6 attenuates erectile dysfunction in a rat model of nerve-sparing radical prostatectomy. J Sex Med 8:1957–1964. Ying C, Yang M, Zheng X, Hu W, Wang X (2013) Effects of intracavernous injection of adipose-derived stem cells on cavernous nerve regeneration in a rat model. Cell Mol Neurobiol 33:233–420.
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Effects of intracavernous injection of P2X3 and NK1 receptor antagonists on erectile dysfunction induced by spinal cord transection in rats C.-L. Li1, X.-L. Yang2, J.-J. Wang1, G.-H. Du3, W.-M. Yang3 & H.-P. Zhang1 1 Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China; 2 Department of Urology, Tianyou Hospital, Wuhan University of Science and Technology, Wuhan, Hubei, China; 3 Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
Keywords Erectile dysfunction—NK1 receptor—P2X3 receptor—spinal cord transection Correspondence Hui-Ping Zhang, Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China. Tel./Fax: 0086-27-83692651; E-mail: [email protected]
Accepted: November 11, 2013 doi: 10.1111/and.12217
Summary This study aimed to explore the effects of intracavernous injection (ICI) of P2X3 and NK1 receptor antagonists on erectile dysfunction (ED) induced by spinal cord transection in rats. Sixty male Sprague–Dawley (SD) rats were randomly divided into the following three groups (20 rats each group): sham operation group (C group), thoracic spinal cord transection group (T group) and sacral spinal cord transection group (S group). An ED model was established through complete transection of the thoracic or sacral spinal cord. Intracavernous pressure (ICP) with and without injection of P2X3 (Suramin) or NK1 (GR82334) receptor antagonists was recorded 3 weeks after surgery. Immunohistochemistry was employed to detect the expression of P2X3 and NK1 receptors in the dorsal root ganglion (DRG) and smooth muscle of corpus cavernosum. Data were processed with SPSS 17.0. ICI with Suramin (0.1, 0.3 and 1 mM) or GR82334 (0.1, 0.3 and 1 mM) increased ICP dose dependently in the T and S groups. The expression of P2X3 and NK1 receptors in DRG and smooth muscle of corpus cavernosum was up-regulated in the T and S groups. It is concluded that ICI of P2X3 and NK1 receptor antagonists may improve the recovery of erectile function in a rat model with ED after spinal cord transection.
Introduction Trauma and fall injury are the main causes of spinal cord injury (SCI) (Pickett et al., 2006). Decreased sexual function is a major concern among men with spinal cord injuries (SCIs). Treatment for erectile dysfunction (ED) through oral pharmacotherapy is an effective method of treating this disorder. Phosphodiesterase-5 inhibitors (PDE5) provide a first-line ED therapy for SCI patients (Lombardi et al., 2012). However, PDE5 could worsen the neurological illness of patients with central neurological disorders (CND) (Lombardi et al., 2012). Thus, finding an effective and satisfactory treatment for ED secondary to SCI is necessary. Current methods of establishing an animal model of neurogenic ED involve damaging or cutting the supplying nerves, such as the cavernous, pelvic and dorsal penile nerves (Yamashita et al., 2011; Ying et al., 2013). However, ED after SCI is different from neurogenic ED after © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 25–29
surgery in terms of pathogenesis, pathophysiology and treatment. Thus, a model with ED induced by damaging or cutting the supplying nerve cannot better simulate neurogenic ED after SCI. Vascular smooth muscle relaxation, mediated by nitric oxide (NO), plays an important role in the process of penile erection. Meanwhile, other endogenous neurotransmitters, such as adenosine, ATP, VIP, CGRP and SP, are reported to exist in the corpora cavernosa. However, the role of these neurotransmitters in the erectile process remains unclear. There are seven subtypes of purinergic (P)2X-ATP receptors, and studies have demonstrated that dorsal root ganglion express predominantly homomeric P2X3 and heteromeric P2X2/ 3 receptors (Wirkner et al., 2007). The neurokinin 1 (NK1) receptor, also known as tachykinin receptor 1 (TACR1), is receptor of substance P. The substance P-NK1 receptor system is the most extensively studied neurokinin pathway in the brain and spinal cord 25
P2X3-r and NK1-r antagonists on ED
(Mantyh, 2002). Reports show that the expression of P2X3 and NK1 receptors in DRG is up-regulated in rat models of SCI, and the subtypes of P2X receptors have different levels of expression in the urogenital system. However, the effect of P2X3 and NK1 receptor antagonists on ED induced by spinal cord transection is rarely reported. Thus, complete transection of the spinal cord was performed in this study to establish an ED model that simulates neurogenic ED after SCI in humans. The effects of the intracavernous injection of P2X3 and NK1 receptor antagonists on ED after spinal cord transection in rats were examined. Materials and methods Animals Sixty adult male Sprague–Dawley (SD) rats (10 weeks old, 300–350 g) were purchased from Experimental Animal Center, Tongji Medical College of Huazhong University of Science and Technology. The rats were housed at 24 °C under controlled conditions with free access to standard rat chow and water. All experiments were approved by the Institutional Animal Care and Use Committee of Tongji Medical College and were performed in strict accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (approval no. SCXK 2010-0007).
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Record of intracavernous pressure Three weeks after surgery, the rats were anaesthetised with pentobarbital sodium (50 mg kg 1). Systemic mean arterial pressure (MAP) was monitored by inserting a 22-G cannula into the carotid artery on the left side of the incised neck. The skin overlying the penis was incised, and the crura of the penis were identified. Two 23-G scalp-vein needles filled with 250 U ml 1 of heparin solution were connected with a polyethylene-50 tubing and inserted into the crus; the right one was for measuring the intracavernous pressure (ICP), and the left one was for the intracavernous injection (ICI) of drugs. ICP and MAP were recorded with a bioinformation acquisition system (BL-420F; Chengdu TMA Technology Co. Ltd., Chengdu, China). ICI of drugs After basal ICP (BICP) was recorded, different concentrations (0.1, 0.3 and 1 mM, 100 ll per injection) of P2X3 receptor antagonist, Suramin (Sigma Aldrich, St. Louis, USA), were administered intracavernously. The effects of Suramin were observed for 20 min, and ICP was recorded. After 30 min, different concentrations (0.1, 0.3 and 1 mM, 100 ll per injection) of NK1 receptor antagonist, GR 82334 (Sigma Aldrich, St. Louis, MO, USA), were administered intracavernously. Also, the effects of GR 82334 were observed for 20 min, and ICP was recorded.
Surgical procedures The rats were allowed to adapt to the environment for at least 3 days. Normal sexual function was confirmed prior to surgery by mating the male rats with the female rats. The rats were randomly divided into three equal groups: sham operation group (C group), thoracic spinal cord transection group (T group) and sacral spinal cord transection group (S group). The rats were anaesthetised with intraperitoneal administration of pentobarbital sodium (50 mg kg 1). Laminectomy was performed after midline dorsal incision. Spinal segment T8–T9 was exposed in the C and T groups, and spinal segment L1–L2 was exposed in the S group. The dura and spinal cord were then completely transected in the T and S groups, whereas the spinal cord was exposed and received no further surgical manipulation in the C group. After the surgical procedure, the rats received 10 ml of Ringer’s lactate solution intraperitoneally. All rats were injected intramuscularly with penicillin (120 000 unit kg 1) once a day for 10 days. The bladder was manually emptied three times a day until spinal reflex micturition was recovered. 26
Preparation of tissue sections All rats were sacrificed by decapitation. The DRGs of T10–T12 or S2–S4 and the corpus cavernosum were taken out and transferred immediately into 4% paraformaldehyde. After fixation, the DRGs and corpus cavernosum were processed through dehydration with graded ethanol and subjected to vitrification by dimethylbenzene in paraffin. The tissue paraffin block was sectioned at 4 lm prior to staining. Immunohistochemistry After deparaffinage and dehydration, the sections were incubated with Triton X-100 for 30 min at 37 °C, washed (3 9 5 min) with 0.1 M PBS (pH 7.4) and then blocked with 1% BSA for 1 h. Afterwards, the sections were incubated for 3 h at 37 °C and then for 60 h at 4 °C with rabbit P2X3-R antibody (1 : 200; Santa Cruz Biotechnology) or rabbit NK1-R antibody (1 : 50; Santa Cruz Biotechnology Co. Ltd., Shanghai, China). The sections were washed (3 9 10 min) with 0.1 M PBS (pH 7.4) then incubated with goat anti-rabbit IgG (1 : 100, Boster © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 25–29
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P2X3-r and NK1-r antagonists on ED
Biological Technology, Ltd., Wuhan, China) for 1 h at 37 °C. The sections were washed (3 9 5 min) with 0.1 M PBS (pH 7.4) after incubation and incubated with SABC solution (Boster Biological Technology, Ltd, Wuhan, China) for 1 h at 37 °C. Afterwards, the sections were washed (3 9 5 min) with 0.1 M PBS (pH 7.4) and colorated with fresh DAB solution (Boster Biological Technology, Ltd., Wuhan, China). The sections were then placed on cover slips with mounting medium of buffering glycerol (pH 8.8) and stored at 4 °C until observation. Statistical analysis Data were processed with software SPSS 17.0 (SPSS Inc., Chicago, IL, USA) and expressed as mean values SEM. The results were analysed through one-way ANOVA and Dunnett’s t-test. Averaged P-values < 0.05 were considered statistically significant.
Results Observation of the rats The rats in the T and S groups lost their hindlimb movement and hindlimb reflex ability after surgery. After 3 weeks, 13 and 15 rats in the T and S groups were alive respectively; all the 20 rats in the C group were alive. Two rats died of anaesthesia, three of infection, three of bladder rupture and four of unknown reasons.
Table 1 Effect of Suramin on ICP in C, T and S groups
Effects of Suramin and GR 82334 on ICP of rats ICI of Suramin (0.1, 0.3 and 1 mM) had no effect on ICP in the C group (P = 0.9491, Table 1); however, it increased ICP in the T and S groups (P < 0.001, Table 1) except for the 0.1 mM dose (P = 0.5937, Table 1). Compared with the 0.1 mM dose of Suramin, ICP was also significantly increased with 0.3 mM dose and 1 mM dose in the T and S groups (P < 0.0001). Thirty minutes after recording the effect of Suramin, GR 82334 were intracavernously injected. GR 82334 (0.1, 0.3 and 1 mM) had no effect on ICP in the C group (P = 0.8164, Table 2); however, it increased ICP dose dependently in the T and S groups (P < 0.0001, Table 2). Expression of P2X3 and NK1 receptors in DRG The P2X3 receptor were expressed weakly in the DRG in the C group but were strongly and extensively expressed in the T and S groups. The NK1 receptor was up-regulated in the DRG in T and S groups, compared with the C group. Furthermore, the P2X3 and NK1 receptors were predominantly located in the parvicellular neuron (Fig. 1). Expression of P2X3 and NK1 receptors in corpus cavernosum Compared with the C group, the expression of P2X3 and NK1 receptors was up-regulated in the smooth muscle of the corpus cavernosum in the T and S groups. (Fig. 2).
Dose of Suramin ICP (mmHg)
0.1 mM
0.3 mM
1 mM
C group (n = 20) T group (n = 13) S group (n = 15)
12.61 2.71 (n = 6) 14.23 3.21 (n = 4) 13.02 2.45 (n = 4)
12.33 3.12 (n = 6) 19.35 2.45a,c (n = 5) 18.25 3.25a,c (n = 5)
12.82 2.65 (n = 7) 26.24 3.16b,c (n = 4) 24.24 1.24b,c (n = 6)
a P = 0.0006, bP < 0.0001: compared with the C group, ICP was significantly increased in the T and S groups after ICI of 0.1, 0.3 and 1 mM dose of Suramin. c P < 0.0001. Compared with the 0.1 mM dose of Suramin, ICP was significantly increased with 0.3 mM dose and 1 mM dose in the T and S groups.
Table 2 Effect of GR 82334 after Suramin on ICP in C, T and S groups
Dose of GR 82334 ICP (mmHg)
0.1 mM
0.3 mM
1 mM
C group (n = 20) T group (n = 13) S group (n = 15)
13.11 4.10 (n = 6) 30.36 2.34a (n = 4) 28.21 3.26a (n = 4)
14.10 3.23 (n = 7) 36.36 2.50a,b (n = 5) 34.37 4.23a,b (n = 5)
14.34 3.56 (n = 7) 41.32 2.35a,b (n = 4) 39.24 2.41a,b (n = 6)
a P < 0.0001: compared with the C group, ICP was significantly increased in the T and S groups after ICI of 0.1, 0.3 and 1 mM dose of GR82334. b P < 0.0001: compared with 0.1 mM dose of GR82334, ICP were significantly increased with 0.3 mM dose and 1 mM dose in the T and S groups.
© 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 25–29
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P2X3-r and NK1-r antagonists on ED
C.-L. Li et al.
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 1 Expression of P2X3 and NK1 receptors in the DRG neurons of rats in C, T and S groups. (a–c) Expression of P2X3 receptor in the DRG neurons of rats in C, T and S groups respectively. (d–f) Expression of NK1 receptor in the DRG neurons of rats in C, T and S groups respectively. Compared with the C group, the expression of P2X3 and NK1 receptors was up-regulated in the DRG neurons in the T and S groups.
(a)
(b)
(c)
(d)
(e)
(f)
Fig. 2 Expression of P2X3 and NK1 receptors in the corpus cavernosum of rats in C, T and S groups. (a–c) Expression of P2X3 receptor in the corpus cavernosum of rats in C, T and S groups respectively. (d–f) Expression of NK1 receptor in the corpus cavernosum of rats in C, T and S groups respectively. Compared with the C group, the expression of P2X3 and NK1 receptors was up-regulated in the corpus cavernosum in the T and S groups.
Discussion An ED model was developed through complete transection of the spinal cord in rats. This procedure was seldom 28
performed in previous studies on neurogenic ED. The present study demonstrates that P2X3 and NK1 receptors (i) have low basal expression in the DRG and corpus cavernosum; (ii) increase in the DRG and corpus © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 25–29
C.-L. Li et al.
cavernosum after the transection of the spinal cord; (iii) are related to ED after spinal cord transection; and (iv) ICI of P2X3 and NK1 receptor antagonists can improve the recovery of the erectile function in a rat model with ED. The male reproductive function depends largely on the neurological integrity of the pelvic floor and spinal cord. Erectile function diminished in patients with neurological damage from SCI (Fode et al., 2012). Neurological problems can cause impairment in sperm quality, which is most often seen in men with SCI. Only about 9% of men with SCI can ejaculate through masturbation or sexual intercourse (Brackett et al., 2010). Thus, studying the related mechanism involved in ED after SCI is important. In present study, the expression of P2X3 and NK1 receptors was up-regulated in the DRGs of model rats with ED, which is consistent with the findings of other researchers (Burnstock, 2000; Weissner et al., 2006; Xiang et al., 2008). Furthermore, P2X3 and NK1 receptors were predominantly located in the parvicellular neuron of DRGs. When ICI of P2X3 and NK1 receptor antagonists was implemented, ICP increased, which was highly related to erection. Thus, the P2X3 and NK1 receptors are involved in the regulation of erectile function. The present study has several limitations. Only the receptors and not the expression of P2X3 and NK1 were detected in this study. The gene and protein levels of the receptors were also not quantified with real-time PCR and Western blot analysis. Exposing spinal segment T8– T9 in rats was set as control group to both T and S groups, which was a little inappropriate. Another control group that exposing spinal segment L1–L2 should have been set. Also, model groups without treatment of Suramin and GR 82334 were not set, so the expression of P2X3 and NK1 receptors could not be detected for comparison before and after the treatment. The results of ICP and immunohistochemistry indicate that the P2X3 and NK1 receptors play an important role in ED induced by spinal cord transection. ICI together with the P2X3 and NK1 receptors can increase ICP and could serve as potential methods of therapeutic intervention for ED after spinal cord transection. The gene and protein levels of P2X3 and NK1 receptors must be detected in the future, and their mechanism in ED after spinal cord transection must be explored further.
© 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 25–29
P2X3-r and NK1-r antagonists on ED
Conclusions These studies demonstrate a functional role for P2X3 and NK1 receptors signal ED in normal rats and in those with ED induced by spinal cord transection. P2X3 and NK1 receptor signalling could serve as potential methods of therapeutic intervention for ED induced by spinal cord transection. References Brackett NL, Ibrahim E, Iremashvili V, Aballa TC, Lynne CM (2010) Treatment for ejaculatory dysfunction in men with spinal cord injury: an 18-year single center experience. J Urol 183:2304–2308. Burnstock G (2000) P2X receptors in sensory neurones. Br J Anaesth 84:476–488. Fode M, Krogh-Jespersen S, Brackett NL, Ohl DA, Lynne CM, Sonksen J (2012) Male sexual dysfunction and infertility associated with neurological disorders. Asian J Androl 14:61–68. Lombardi G, Nelli F, Celso M, Mencarini M, Del Popolo G (2012) Treating erectile dysfunction and central neurological diseases with oral phosphodiesterase type 5 inhibitors. Review of the literature. J Sex Med 9:970–985. Mantyh PW (2002) Neurobiology of substance P and the NK1 receptor. J Clin Psychiatry 63(Suppl 11):6–10. Pickett GE, Campos-Benitez M, Keller JL, Duggal N (2006) Epidemiology of traumatic spinal cord injury in Canada. Spine, 31:799–805. Weissner W, Winterson BJ, Stuart-Tilley A, Devor M, Bove GM (2006) Time course of substance P expression in dorsal root ganglia following complete spinal nerve transection. J Comp Neurol 497:78–87. Wirkner K, Sperlagh B, Illes P (2007) P2X3 receptor involvement in pain states. Mol Neurobiol 36:165–183. Xiang Z, Xiong Y, Yan N, Li X, Mao Y, Ni X, He C, Lamotte RH, Burnstock G, Sun J (2008) Functional up-regulation of P2X 3 receptors in the chronically compressed dorsal root ganglion. Pain 140:23–34. Yamashita S, Kato R, Kobayashi K, Hisasue S, Arai Y, Tsukamoto T (2011) Inhibition of interleukin-6 attenuates erectile dysfunction in a rat model of nerve-sparing radical prostatectomy. J Sex Med 8:1957–1964. Ying C, Yang M, Zheng X, Hu W, Wang X (2013) Effects of intracavernous injection of adipose-derived stem cells on cavernous nerve regeneration in a rat model. Cell Mol Neurobiol 33:233–420.
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