Original Paper
Urologia Internationalis
Received: October 13, 2014 Accepted after revision: November 28, 2014 Published online: February 3, 2015
Urol Int DOI: 10.1159/000370248
Calpain Inhibition Improves Erectile Function in a Rat Model of Cavernous Nerve Injury Zhi-Hua Wan Guo-Hao Li Yong-Lian Guo Wen-Zhou Li Lin Chen Department of Urology, the Central Hospital of Wuhan, Wuhan, Hubei, PR China
Abstract Introduction: Erectile dysfunction (ED) after cavernous nerve (CN) injury remains difficult to treat. Calpain plays a critical role in causing neurodegenerative diseases. This study aimed to evaluate whether calpain inhibition preserves erectile function in a rat model of CN injury. Materials and Methods: Rats underwent sham surgery or CN crush injury. The CN-crushed rats were treated with vehicle or MDL28170, a specific calpain inhibitor. At 1, 2, 3, and 7 days postsurgery, major pelvic ganglia (MPG) were harvested, followed by the measurement of erectile function, respectively. At 28 days, penile tissue and distal CN were harvested, followed by the measurement of erectile function in rats. Calpain activity in MPG and corpus cavernosum, as well as TGF-β1/Smad2 and collagen content in corpus cavernosum, were measured by western blot. Neuronal nitric oxide synthase (nNOS) was observed by immunohistochemistry. Results: Increased calpain activity was observed in MPG and corpus cavernosum. CN crush markedly attenuated the erectile responses and nNOS expression in CN, and these were improved by MDL-28170 treatment. Furthermore, treatment prevented increased TGF-β1/Smad2 and collagen expression in corpus cavernosum. Conclusions: Our results suggested that calpain activation plays a role in pathogenesis of
© 2015 S. Karger AG, Basel 0042–1138/15/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/uin
CN injury-associated ED. Calpain inhibition could be a novel approach for preventing the development of ED following © 2015 S. Karger AG, Basel CN injury.
Introduction
Erectile dysfunction (ED) following cavernous nerve (CN) injury frequently accompanies radical prostatectomy. Despite advances in anatomic knowledge and technology, such as nerve sparing and robotic-assisted radical prostatectomy, ED remains the most frequent complication of this surgery [1]. The primary pathophysiological mechanism of postoperative ED is associated with CN damage by surgery. CN can be affected by trauma during surgical manipulation, such as mechanical traction, thermal damage due to electrocautery use, nerve ischemia, apoptosis and local inflammatory reactions [2, 3]. CN damage initiates a series of neuropathological events, termed Wallerian degeneration, which result in the degeneration of the distal axon, as well as reduced or lack of nitric oxide release [4, 5]. Increased reactive oxygen species, cavernous hypoxia, apoptosis, and upregulation of profibrotic factors, such as transforming growth factor-beta1 (TGF-β1), may contribute to structural changes in the corpus cavernosum tissue, such as decreased smooth muscle and endothelial content, and cavernous fibrosis [4–8]. TGF-β1 mediates Guo-Hao Li Department of Urology, the Central Hospital of Wuhan Wuhan, Hubei, 430014 (PR China) E-Mail guohaoli1978 @ 163.com
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Key Words Calpain · Cavernous nerve injury · Erectile dysfunction · Fibrosis · Neuroprotection
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Urol Int DOI: 10.1159/000370248
In optic nerve-crushed animal models, calpain inhibition prevented axonal damage-induced ganglion cell death and cytoskeletal proteolysis in nerve fibers, as well as reduced reactive astrogliosis, avoiding axonal degeneration and maintaining the axon area close to normal values [15, 22]. Furthermore, inhibition of calpain activation can block intracellular the activation of TGF-β1, leading to attenuated Smad2/3 phosphorylation and collagen synthesis [20]. We hypothesize that calpain overactivation is involved in primary nerve degeneration following CN injury, as well as secondary development of penile fibrosis. Because α-spectrin contains a specific calpain cleavage site and releases the 145-kDa spectrin breakdown product (SBDP), we used a primary antibody against α-spectrin to measure the level of 145-kDa SBDP, which has been widely used to quantify calpain activity in vivo [20, 23]. This study used a rat model of bilateral CN crush injury to investigate calpain overactivation after CN injury and the effects of the specific calpain inhibitor MDL-28170 on erectile function, neuroprotection, and corporeal fibrosis. Materials and Methods Animals and Treatment A total of 150 male Sprague-Dawley rats (12 weeks old) were randomly assigned to three equal groups (n = 50 per group): sham control; i.p. injection of phosphate-buffered saline (PBS) vehicle after bilateral CN crush injury; i.p. injection of MDL-28170 dissolved in PBS vehicle at a dose of 20 mg/kg once daily after bilateral CN crush injury. This dose was chosen based on prior experiments [24]. The animals had access to standard rat chow and water ad libitum. Under inhalant anesthesia, the major pelvic ganglion (MPG) was exposed on either side of the prostate via laparotomy. The sham control group underwent laparotomy alone. In the CN crush injury groups, bilateral CN crush injury was performed with a hemostat clamp 5 mm away from the CN origin at the MPG for a duration of 2 min [25]. The abdomen was then sutured. Experimental protocols complied with National Institutes of the Health Guidelines for the Care and Use of Laboratory Animals and were approved by the animal ethics committee of the institution. Tissue Preparation Rats were sacrificed once the erectile functional testing after surgery was completed. At 1 day, 2 days, 3 days, and 7 days after surgery, MPGs were harvested after completing erectile functional testing. At 28 days, penises and distal CN were collected for western blot and histological analyses. All samples were stored at –80 ° C until analyzed. (n = 10 per time point per group).
Western Blot Analysis Total proteins were prepared and protein concentrations were measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad Laboratories, Hercules, Calif., USA). A total of 40 μg total protein was separated on 10% SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane (Pierce, Rockford, Ill., USA). Before im-
Wan/Li/Guo/Li/Chen
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fibrotic effects by activating receptor-associated Smad2 and Smad3. Following phosphorylated, Smad2 translocates to the nucleus and regulates the transcription of TGF-β target genes. It has been reported that upregulation of the TGF-β1/Smad signaling pathway might play an important role in ED-associated cavernosal fibrosis [7, 9]. As a result of this denervation and structural changes in erectile tissue, often severe and difficult-to-treat ED develops. Calpains are a family of calcium-dependent, non-lysosomal neutral cysteine endopeptidases present in the majority of mammalian cells [10]. To date, at least 15 additional mammalian calpains have been identified [10, 11]. Calpain-1 and calpain-2 are the two major typical calpains. Under physiological conditions, calpain possesses low levels of activity and plays an important role in cellular events, such as cytoskeletal turnover, proliferation, migration, and differentiation of endothelial cells, fibroblasts, and myoblasts [10–13]. Excessively activated calpain has been associated with acute or chronic neurodegenerative diseases such as crush lesion, ischemia, and Alzheimer’s disease [14, 15]. Excessive accumulation of intracellular calcium plays a key role in initiating and mediating numerous events in the nerve secondary damage cascade, including calpain overactivation [14, 16]. In cerebrospinal fluid with traumatic brain injury, enhanced calpain activity levels were found at 24 and 48 h post-injury, and the levels remained significantly elevated at days 5–7 post-injury [17]. After an optic nerve crush injury, calpain content increased as early as 12 h post-lesion, peaking at 48 h [18]. Overactivation of calpains results in the proteolysis of cytoskeletal proteins αII spectrin, βII spectrin, and ankyrinB. This cleavage of cytoskeletal proteins leads to axonal transport disruption and structural collapse, culminating in secondary axonal injury and possibly cell death [15, 19]. Proteolysis of the axonal cytoskeletal proteins by calpain has been proposed to be a major contributor to axonal degeneration after nervous system injury [14, 15, 19]. Additionally, a chronic hypoxia state can cause calpain overactivation, which leads to fibrosis through the activation of TGF-β1 and the Smad signal pathway [20]. MDL-28170 is a highly selective cysteine peptidase inhibitor with a Ki for calpain of 8 nM; it is among the best inhibitors at crossing cell membranes [21]. MDL-28170 has been reported to have neuroprotective effects in numerous rodent neurotrauma models, including spinal cord injury, optic nerve injury, neonatal hypoxia-ischemia and focal cerebral ischemia, and also to reduce neuronal loss and improve locomotor functions in a mouse model of Parkinson’s disease [14, 15, 19].
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Fig. 1. a 145-kDa spectrin break down product (SBDP) protein levels in major pelvic ganglion (MPG) at 1, 2, 3 and 7 days after bilateral cavernous nerve (CN) crush injury. b 145-kDa SBDP protein levels in corpus cavernosum 28 days after CN injury. 145-kDa
SBDP protein levels were quantified by Western blot. β-actin was used as a loading control. The bar graphs show the relative levels of SBDP protein normalized to β-actin. * p < 0.05 vs. sham control.
munostaining, the membranes were blocked with 5% non-fat milk overnight at 4 ° C, followed by incubation with appropriate dilutions of the primary specific antibody in PBS Tween-20 at 4 ° C for 16 h. The primary antibodies included anti-αII-spectrin (1:1,000; Enzo Life Sciences, Plymouth Meeting, Pa., USA), Smad2 and phosphoSmad2 (p-Smad2) (1:1,000, Cell Signaling, Beverly, Mass., USA), collagen I, collagen IV, and TGF-β1 (1:1,000; Abcam, Cambridge, Mass., USA), and anti-β-actin (1: 1,000; Santa Cruz Biotechnology, Santa Cruz, Calif., USA). The horseradish peroxidase-conjugated secondary antibody incubation was performed at room temperature for 1 h. The membranes were then treated with an enhanced chemiluminescence reagent (Amersham, Arlington Heights, Ill., USA), and the signals were detected by the exposure of the membranes to X-ray films (Kodak, Rochester, N.Y., USA). The signal intensity was quantified by densitometry with Gel pro3.0 image software (Media Cybernetics, Silver Spring, Md., USA) and normalized to β-actin expression. Samples were run in duplicate, and the ratios for each sample were averaged and reported as means ± SD (n = 10 per time point per group).
ronal nitric oxide synthase (nNOS) antibody (1:150; Santa Cruz Biotechnology, Santa Cruz, Calif., USA), followed by 1-h immersion in 1:100 dilution of a secondary antibody conjugated with fluorescein isothiocyanate. Nuclear staining was performed with 4’,6-diamidino-2-phenylindole (DAPI; Abcam, Cambridge, Mass., USA). Digital images were acquired using an Olympus FV500 fluorescence microscope equipped with a Nikon camera (n = 10 per group).
Measurement of Erectile Responses Erectile function was measured as previously described [26]. The ratio between the intracavernosal pressure (ICP) and mean systemic arterial blood pressure (MAP) obtained at the peak of erectile response, as well as total ICP determined by the area under the curve (mm Hg/s), were calculated.
Image and Statistical Analysis Three mid-penile tissue sections per animal were included for statistical analysis. Computerized histomorphometric analysis was performed using image-pro plus 6.0 image processing software (Media Cybernetics). For the analysis of nNOS content, the ratio of the number of nNOS-positive fibers per nerve over the total area of the nerve was calculated at ×400 magnification. All results were expressed as means ± SD. The data were analyzed by analysis of variance with Student-Newman-Keuls posthoc analysis using SPSS 12.0 software (SPSS Inc., Chicago, Ill., USA). Differences were considered statistically significant when p < 0.05.
Results
Immunohistochemistry Adjacent sections (5 μm) were cut at the middle region of the penis shaft. After blocking with 3% goat serum/PBS/0.3% tritonX-100, the sections were incubated overnight at 4 ° C with a rabbit anti-neu-
Calpain Activity as Determined by α-Spectrin Degradation Figure 1a shows that compared with the sham control, CN crush injury led to a 2.4-fold (35.7 ± 3.4% for CN in-
Effect of Calpain Inhibition after CN Injury
Urol Int DOI: 10.1159/000370248
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tile response to electrical stimulation of the cavernous nerve measured by intracavernosal pressure/mean systemic arterial blood pressure (ICP/MAP) and total ICP (the area under curve) in sham
jury vs. 14.7 ± 3.7% for sham control, p < 0.05), 2.2-fold (33.8 ± 3.6% vs. 15.7 ± 3.8%, p < 0.05), and 1.8-fold (25.8 ± 3.5% vs. 14.1 ± 3.4%, p < 0.05) increase in the SBDP protein at 1, 2, and 3 days post-surgery, respectively, indicating that calpain activity in MPG increased as a result of CN crush injury. At 7 days, SBDP levels in CN-crushed rats decreased, but were not significantly different compared with the sham control. We also detected a significant increase in the amount of SBDP protein from penile tissue of CN-crushed rats at 28 days (36.3 ± 2.4 vs. 16.5 ± 2.8%, p < 0.05) (fig. 1b). MDL-28170 treatment significantly blocked these increases (p < 0.05). Assessment of Erectile Function The effect of cavernous nerve stimulation on erectile function in vivo was measured to evaluate the functional consequence of calpain inhibition (fig. 2). The ratios of maximal ICP to MAP and total ICP were markedly lower in vehicle-treated rats than in sham control rats. The decrease in erectile function was noted at the earliest time Urol Int DOI: 10.1159/000370248
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Fig. 2. Effect of calpain inhibitor MDL-28170 treatment on neurogenic-mediated erectile responses. a Bar graph summary of erec-
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control and CN-crushed rats treated with vehicle only or MDL28170 at 1, 2, 3, 7 and 28 days after cavernous nerve crushing. b Representative ICP and MAP in response to electrostimulation of cavernous nerve in each group. * p < 0.05 vs. sham control; # p < 0.05 vs. CN-crushed rats treated with vehicle.
point assayed (day 1) and persisted up to 4 weeks after cavernous nerve crushing. When compared with vehicle-treated rats, CN-crushed rats treated with MDL28170 had significantly greater increases in ICP/MAP and total ICP in response to cavernous nerve stimulation in all time points assayed (p < 0.05). However, the erectile responses remaining reduced compared with the sham controls. nNOS-Positive Nerve Protection nNOS-positive nerves were quantified by histomorphometric analysis of the percentage of dorsal nerve area that stained positive for neuronal-specific NOS. Representative images of each group are shown in figure 3. There was a significant decrease in nNOS content of dorsal penile nerves at 28 days after bilateral CN crush injury (p < 0.05). Following MDL-28170 treatment, nNOS content in the dorsal nerves was significantly greater compared with vehicle-treated CN-crushed controls (p < 0.05). Wan/Li/Guo/Li/Chen
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nal nitric oxide synthase (nNOS)-positive nerve protection. Sections were stained for nNOS (green) and DAPI (blue) in the dorsal penile nerve 28 days after operation. Quantitative analysis of
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Fig. 4. Effect of calpain inhibitor MDL-28170 treatment on trans-
forming growth factor-beta1 (TGF-β1)/Smad2 expression in corpus cavernosum. a The protein expression of TGF-β1, phosphoSmad2 (p-Smad2) and total Smad2 in rat corpus cavernosum tissues were measured by Western blot. β-actin was used as a loading
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nNOS expression was quantified as the relative percentage of nNOS expression in area stain for the entire dorsal penile nerve cross-section. Magnification is ×400. * p < 0.05 vs. sham control.
Fig. 3. Effect of calpain inhibitor MDL-28170 treatment on neuro-
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control. b Relative optical density changes of TGF-β1 normalized to β-actin. c Data are presented as the ratio of phosphorylated protein to total protein. * p < 0.05 vs. sham control; # p < 0.05 vs. CNcrushed rats treated with vehicle.
Activation of TGF-β1/Smad2 in the Corpus Cavernosum TGF-β1 and p-Smad2/Smad2 protein levels were measured in cavernous tissue by western blot analysis. The ratio of p-Smad2 to total Smad2 was quantified to determine Smad2 activity. In CN-crushed rats, TGF-β1 protein levels and the ratio of p-Smad2 to total Smad2 were significantly increased compared with the sham controls (85.3 ± 3.4% for CN injury vs. 37.5 ± 5.6% for sham control and 0.93 ± 0.06 vs. 0.48 ± 0.04, respectively, p < 0.05, fig. 4). In response to MDL-28170 treatment, TGF-β1 protein and the ratio of p-Smad2 to total Smad2 were significantly decreased (85.3 ± 3.4% for CN injury vs. 54.3 ± 5.3% for MDL-28170 and 0.93 ± 0.06 vs. 0.68 ± 0.04, respectively, p < 0.05).
Collagen Content in the Corpus Cavernosum Western blot analysis was performed to evaluate expression of collagen I and collagen IV in the corpus cavernosum (fig. 5). The relative protein level of collagen I significantly increased in CN-crushed rats (78.7 ± 6.4% for CN injury vs. 27.9 ± 5.2% for sham control, p < 0.05). The expression significantly reduced in response to MDL28170 treatment (p < 0.05) and was indistinguishable from the sham. Western blot analysis also revealed that collagen IV protein expression was significantly increased in CN-crushed rats (98.7 ± 6.8% for CN injury vs. 33.1 ± 5.6% for sham control, p < 0.05). Treatment with MDL28170 significantly decreased the expression (98.7 ± 6.8% for CN injury vs. 67.1 ± 5.2% for sham control, p < 0.05), although the expression remained increased compared with that of the sham control rats.
Effect of Calpain Inhibition after CN Injury
Urol Int DOI: 10.1159/000370248
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Discussion
As penile neuropathy is a major pathogenic basis for ED in patients after radical prostatectomy, there has been great interest in finding therapeutic approaches that promote neuroprotection and nerve regeneration for facilitating functional recovery. In the present study, we used a rat model of bilateral CN crush injury to investigate the role of calpain in CN injury-induced ED. Our results indicated that impaired erectile function in CN-crushed rats was associated with increased levels of calpain activity, decreased nNOS expression in the dorsal penile nerve and increased cavernous expression of TGF-β1/Smad2 and collagen deposition. Results also demonstrated that pharmacological inhibition of calpain by MDL-28170 at least partially improved these deficits, indicating that calpain inhibition could attenuate the development of CN injury-induced ED. Following initial mechanical injury to the CN, additional tissue damage resulted from secondary processes, including inflammation, oxidative stress, ischemia, excitotoxicity, free radical production, and apoptosis [3, 27]. Excessively activated calpain has been associated with acute or chronic neurodegenerative diseases. Consistent with previous reports using other nervous system models [17, 18], results from the present study, using a model of bilateral CN crush injury, showed enhanced levels of calpain activity in MPG at the early postoperative period, as well as calpain activity gradually decreased to control levels within 7 days. Proteolysis of the axonal cytoskeletal proteins αII spectrin, βII spectrin, and ankyrinB by calpain has been proposed to be a major contributor to axonal degeneration after injury in central or peripheral nervous systems [14, 15, 19]. Thus, calpain represents a rational and feasible 6
Urol Int DOI: 10.1159/000370248
b
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target for neuroprotective therapeutic interventions. In this study, we found that the content of nNOS-positive fiber content in the dorsal nerves of CN-crushed rats improved after MDL-28170 treatment compared with vehicle-treated rats, indicating the neuroprotective effects of calpain inhibition after bilateral CN crush injury. Compared with sham control rats, electrical stimulation of the cavernous nerve resulted in markedly decreased erectile function in CN-crushed rats treated with vehicle. Partial, but significant, enhancement of erectile response was observed in the MDL-28170-treated group. This enhancement occurred at the earliest time point assayed and persisted for the duration of the experiment (28 days) after cavernous nerve crushing. The MDL-28170-treated group exhibited improved erectile function, although the improvement did not reach control levels. It has been hypothesized that cavernosal nerve damage, such as that after radical prostatectomy, is associated with hypoxia-induced activation of the TGF-β1/Smad signaling pathway in the corpus cavernosum, which further leads to fibrosis and loss of smooth muscle content in erectile tissue [6, 7]. Results from the current study confirmed previous reports showing activation of the TGF-β1/Smad2 pathway and increased collagen content in CN-crushed rats. Inhibition of the TGF-β pathway resulted in a significant decrease of collagen deposition and apoptosis in the corpus cavernosum following bilateral CN injury [28]. Indeed, the TGF-β1/Smad pathway is important in the pathogenesis of ED following cavernosal nerve damage. Interestingly, calpain has been suggested to be one of the main suspected perpetrators in chronic hypoxic/ischemic injury. Calpain-mediated proteolysis and apoptotic processes are involved in the pathogenesis of many tissues as a result of hypoxic conditions, such as the brain, Wan/Li/Guo/Li/Chen
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28170 treatment on collagen I and IV expression in corpus cavernosum. a The protein expression of collagen I and IV in rat corpus cavernosum tissues were measured by Western blot. β-actin was used as a loading control. b Relative optical density changes of collagen I and IV normalized to β-actin. * p < 0.05 vs. sham control; # p < 0.05 vs. CN-crushed rats treated with vehicle.
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liver, heart, skeletal muscles, pulmonary artery, retina, and kidney [19, 20, 29, 30]. Moreover, calpain can induce TGF-β activation through an unknown mechanism [20, 30]. The present study demonstrated enhanced calpain activity in cavernous tissue after bilateral CN crush injury, and calpain inhibition by MDL-28170 resulted in decreased TGF-β1/Smad2 expression and collagen I and IV content. These results implicated that calpain inhibition had protective effects on cavernous tissue fibrosis after CN injury. In conclusion, this study provided evidence for the role of calpain activation in the pathogenesis of CN inju-
ry-induced ED. In addition, treatment with a calpain inhibitor provided neuroprotection against axonal degeneration produced by a crush lesion and amelioration of penile fibrosis, thereby preserving erectile function. These data suggested that calpain inhibition could be a promising strategy for preventing the development of diabetic ED.
Conflicts of Interest The authors declare no competing financial interests.
References
Effect of Calpain Inhibition after CN Injury
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Urologia Internationalis
Received: October 13, 2014 Accepted after revision: November 28, 2014 Published online: February 3, 2015
Urol Int DOI: 10.1159/000370248
Calpain Inhibition Improves Erectile Function in a Rat Model of Cavernous Nerve Injury Zhi-Hua Wan Guo-Hao Li Yong-Lian Guo Wen-Zhou Li Lin Chen Department of Urology, the Central Hospital of Wuhan, Wuhan, Hubei, PR China
Abstract Introduction: Erectile dysfunction (ED) after cavernous nerve (CN) injury remains difficult to treat. Calpain plays a critical role in causing neurodegenerative diseases. This study aimed to evaluate whether calpain inhibition preserves erectile function in a rat model of CN injury. Materials and Methods: Rats underwent sham surgery or CN crush injury. The CN-crushed rats were treated with vehicle or MDL28170, a specific calpain inhibitor. At 1, 2, 3, and 7 days postsurgery, major pelvic ganglia (MPG) were harvested, followed by the measurement of erectile function, respectively. At 28 days, penile tissue and distal CN were harvested, followed by the measurement of erectile function in rats. Calpain activity in MPG and corpus cavernosum, as well as TGF-β1/Smad2 and collagen content in corpus cavernosum, were measured by western blot. Neuronal nitric oxide synthase (nNOS) was observed by immunohistochemistry. Results: Increased calpain activity was observed in MPG and corpus cavernosum. CN crush markedly attenuated the erectile responses and nNOS expression in CN, and these were improved by MDL-28170 treatment. Furthermore, treatment prevented increased TGF-β1/Smad2 and collagen expression in corpus cavernosum. Conclusions: Our results suggested that calpain activation plays a role in pathogenesis of
© 2015 S. Karger AG, Basel 0042–1138/15/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/uin
CN injury-associated ED. Calpain inhibition could be a novel approach for preventing the development of ED following © 2015 S. Karger AG, Basel CN injury.
Introduction
Erectile dysfunction (ED) following cavernous nerve (CN) injury frequently accompanies radical prostatectomy. Despite advances in anatomic knowledge and technology, such as nerve sparing and robotic-assisted radical prostatectomy, ED remains the most frequent complication of this surgery [1]. The primary pathophysiological mechanism of postoperative ED is associated with CN damage by surgery. CN can be affected by trauma during surgical manipulation, such as mechanical traction, thermal damage due to electrocautery use, nerve ischemia, apoptosis and local inflammatory reactions [2, 3]. CN damage initiates a series of neuropathological events, termed Wallerian degeneration, which result in the degeneration of the distal axon, as well as reduced or lack of nitric oxide release [4, 5]. Increased reactive oxygen species, cavernous hypoxia, apoptosis, and upregulation of profibrotic factors, such as transforming growth factor-beta1 (TGF-β1), may contribute to structural changes in the corpus cavernosum tissue, such as decreased smooth muscle and endothelial content, and cavernous fibrosis [4–8]. TGF-β1 mediates Guo-Hao Li Department of Urology, the Central Hospital of Wuhan Wuhan, Hubei, 430014 (PR China) E-Mail guohaoli1978 @ 163.com
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Key Words Calpain · Cavernous nerve injury · Erectile dysfunction · Fibrosis · Neuroprotection
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Urol Int DOI: 10.1159/000370248
In optic nerve-crushed animal models, calpain inhibition prevented axonal damage-induced ganglion cell death and cytoskeletal proteolysis in nerve fibers, as well as reduced reactive astrogliosis, avoiding axonal degeneration and maintaining the axon area close to normal values [15, 22]. Furthermore, inhibition of calpain activation can block intracellular the activation of TGF-β1, leading to attenuated Smad2/3 phosphorylation and collagen synthesis [20]. We hypothesize that calpain overactivation is involved in primary nerve degeneration following CN injury, as well as secondary development of penile fibrosis. Because α-spectrin contains a specific calpain cleavage site and releases the 145-kDa spectrin breakdown product (SBDP), we used a primary antibody against α-spectrin to measure the level of 145-kDa SBDP, which has been widely used to quantify calpain activity in vivo [20, 23]. This study used a rat model of bilateral CN crush injury to investigate calpain overactivation after CN injury and the effects of the specific calpain inhibitor MDL-28170 on erectile function, neuroprotection, and corporeal fibrosis. Materials and Methods Animals and Treatment A total of 150 male Sprague-Dawley rats (12 weeks old) were randomly assigned to three equal groups (n = 50 per group): sham control; i.p. injection of phosphate-buffered saline (PBS) vehicle after bilateral CN crush injury; i.p. injection of MDL-28170 dissolved in PBS vehicle at a dose of 20 mg/kg once daily after bilateral CN crush injury. This dose was chosen based on prior experiments [24]. The animals had access to standard rat chow and water ad libitum. Under inhalant anesthesia, the major pelvic ganglion (MPG) was exposed on either side of the prostate via laparotomy. The sham control group underwent laparotomy alone. In the CN crush injury groups, bilateral CN crush injury was performed with a hemostat clamp 5 mm away from the CN origin at the MPG for a duration of 2 min [25]. The abdomen was then sutured. Experimental protocols complied with National Institutes of the Health Guidelines for the Care and Use of Laboratory Animals and were approved by the animal ethics committee of the institution. Tissue Preparation Rats were sacrificed once the erectile functional testing after surgery was completed. At 1 day, 2 days, 3 days, and 7 days after surgery, MPGs were harvested after completing erectile functional testing. At 28 days, penises and distal CN were collected for western blot and histological analyses. All samples were stored at –80 ° C until analyzed. (n = 10 per time point per group).
Western Blot Analysis Total proteins were prepared and protein concentrations were measured using a Bio-Rad colorimetric protein assay kit (Bio-Rad Laboratories, Hercules, Calif., USA). A total of 40 μg total protein was separated on 10% SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane (Pierce, Rockford, Ill., USA). Before im-
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fibrotic effects by activating receptor-associated Smad2 and Smad3. Following phosphorylated, Smad2 translocates to the nucleus and regulates the transcription of TGF-β target genes. It has been reported that upregulation of the TGF-β1/Smad signaling pathway might play an important role in ED-associated cavernosal fibrosis [7, 9]. As a result of this denervation and structural changes in erectile tissue, often severe and difficult-to-treat ED develops. Calpains are a family of calcium-dependent, non-lysosomal neutral cysteine endopeptidases present in the majority of mammalian cells [10]. To date, at least 15 additional mammalian calpains have been identified [10, 11]. Calpain-1 and calpain-2 are the two major typical calpains. Under physiological conditions, calpain possesses low levels of activity and plays an important role in cellular events, such as cytoskeletal turnover, proliferation, migration, and differentiation of endothelial cells, fibroblasts, and myoblasts [10–13]. Excessively activated calpain has been associated with acute or chronic neurodegenerative diseases such as crush lesion, ischemia, and Alzheimer’s disease [14, 15]. Excessive accumulation of intracellular calcium plays a key role in initiating and mediating numerous events in the nerve secondary damage cascade, including calpain overactivation [14, 16]. In cerebrospinal fluid with traumatic brain injury, enhanced calpain activity levels were found at 24 and 48 h post-injury, and the levels remained significantly elevated at days 5–7 post-injury [17]. After an optic nerve crush injury, calpain content increased as early as 12 h post-lesion, peaking at 48 h [18]. Overactivation of calpains results in the proteolysis of cytoskeletal proteins αII spectrin, βII spectrin, and ankyrinB. This cleavage of cytoskeletal proteins leads to axonal transport disruption and structural collapse, culminating in secondary axonal injury and possibly cell death [15, 19]. Proteolysis of the axonal cytoskeletal proteins by calpain has been proposed to be a major contributor to axonal degeneration after nervous system injury [14, 15, 19]. Additionally, a chronic hypoxia state can cause calpain overactivation, which leads to fibrosis through the activation of TGF-β1 and the Smad signal pathway [20]. MDL-28170 is a highly selective cysteine peptidase inhibitor with a Ki for calpain of 8 nM; it is among the best inhibitors at crossing cell membranes [21]. MDL-28170 has been reported to have neuroprotective effects in numerous rodent neurotrauma models, including spinal cord injury, optic nerve injury, neonatal hypoxia-ischemia and focal cerebral ischemia, and also to reduce neuronal loss and improve locomotor functions in a mouse model of Parkinson’s disease [14, 15, 19].
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Fig. 1. a 145-kDa spectrin break down product (SBDP) protein levels in major pelvic ganglion (MPG) at 1, 2, 3 and 7 days after bilateral cavernous nerve (CN) crush injury. b 145-kDa SBDP protein levels in corpus cavernosum 28 days after CN injury. 145-kDa
SBDP protein levels were quantified by Western blot. β-actin was used as a loading control. The bar graphs show the relative levels of SBDP protein normalized to β-actin. * p < 0.05 vs. sham control.
munostaining, the membranes were blocked with 5% non-fat milk overnight at 4 ° C, followed by incubation with appropriate dilutions of the primary specific antibody in PBS Tween-20 at 4 ° C for 16 h. The primary antibodies included anti-αII-spectrin (1:1,000; Enzo Life Sciences, Plymouth Meeting, Pa., USA), Smad2 and phosphoSmad2 (p-Smad2) (1:1,000, Cell Signaling, Beverly, Mass., USA), collagen I, collagen IV, and TGF-β1 (1:1,000; Abcam, Cambridge, Mass., USA), and anti-β-actin (1: 1,000; Santa Cruz Biotechnology, Santa Cruz, Calif., USA). The horseradish peroxidase-conjugated secondary antibody incubation was performed at room temperature for 1 h. The membranes were then treated with an enhanced chemiluminescence reagent (Amersham, Arlington Heights, Ill., USA), and the signals were detected by the exposure of the membranes to X-ray films (Kodak, Rochester, N.Y., USA). The signal intensity was quantified by densitometry with Gel pro3.0 image software (Media Cybernetics, Silver Spring, Md., USA) and normalized to β-actin expression. Samples were run in duplicate, and the ratios for each sample were averaged and reported as means ± SD (n = 10 per time point per group).
ronal nitric oxide synthase (nNOS) antibody (1:150; Santa Cruz Biotechnology, Santa Cruz, Calif., USA), followed by 1-h immersion in 1:100 dilution of a secondary antibody conjugated with fluorescein isothiocyanate. Nuclear staining was performed with 4’,6-diamidino-2-phenylindole (DAPI; Abcam, Cambridge, Mass., USA). Digital images were acquired using an Olympus FV500 fluorescence microscope equipped with a Nikon camera (n = 10 per group).
Measurement of Erectile Responses Erectile function was measured as previously described [26]. The ratio between the intracavernosal pressure (ICP) and mean systemic arterial blood pressure (MAP) obtained at the peak of erectile response, as well as total ICP determined by the area under the curve (mm Hg/s), were calculated.
Image and Statistical Analysis Three mid-penile tissue sections per animal were included for statistical analysis. Computerized histomorphometric analysis was performed using image-pro plus 6.0 image processing software (Media Cybernetics). For the analysis of nNOS content, the ratio of the number of nNOS-positive fibers per nerve over the total area of the nerve was calculated at ×400 magnification. All results were expressed as means ± SD. The data were analyzed by analysis of variance with Student-Newman-Keuls posthoc analysis using SPSS 12.0 software (SPSS Inc., Chicago, Ill., USA). Differences were considered statistically significant when p < 0.05.
Results
Immunohistochemistry Adjacent sections (5 μm) were cut at the middle region of the penis shaft. After blocking with 3% goat serum/PBS/0.3% tritonX-100, the sections were incubated overnight at 4 ° C with a rabbit anti-neu-
Calpain Activity as Determined by α-Spectrin Degradation Figure 1a shows that compared with the sham control, CN crush injury led to a 2.4-fold (35.7 ± 3.4% for CN in-
Effect of Calpain Inhibition after CN Injury
Urol Int DOI: 10.1159/000370248
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tile response to electrical stimulation of the cavernous nerve measured by intracavernosal pressure/mean systemic arterial blood pressure (ICP/MAP) and total ICP (the area under curve) in sham
jury vs. 14.7 ± 3.7% for sham control, p < 0.05), 2.2-fold (33.8 ± 3.6% vs. 15.7 ± 3.8%, p < 0.05), and 1.8-fold (25.8 ± 3.5% vs. 14.1 ± 3.4%, p < 0.05) increase in the SBDP protein at 1, 2, and 3 days post-surgery, respectively, indicating that calpain activity in MPG increased as a result of CN crush injury. At 7 days, SBDP levels in CN-crushed rats decreased, but were not significantly different compared with the sham control. We also detected a significant increase in the amount of SBDP protein from penile tissue of CN-crushed rats at 28 days (36.3 ± 2.4 vs. 16.5 ± 2.8%, p < 0.05) (fig. 1b). MDL-28170 treatment significantly blocked these increases (p < 0.05). Assessment of Erectile Function The effect of cavernous nerve stimulation on erectile function in vivo was measured to evaluate the functional consequence of calpain inhibition (fig. 2). The ratios of maximal ICP to MAP and total ICP were markedly lower in vehicle-treated rats than in sham control rats. The decrease in erectile function was noted at the earliest time Urol Int DOI: 10.1159/000370248
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Fig. 2. Effect of calpain inhibitor MDL-28170 treatment on neurogenic-mediated erectile responses. a Bar graph summary of erec-
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control and CN-crushed rats treated with vehicle only or MDL28170 at 1, 2, 3, 7 and 28 days after cavernous nerve crushing. b Representative ICP and MAP in response to electrostimulation of cavernous nerve in each group. * p < 0.05 vs. sham control; # p < 0.05 vs. CN-crushed rats treated with vehicle.
point assayed (day 1) and persisted up to 4 weeks after cavernous nerve crushing. When compared with vehicle-treated rats, CN-crushed rats treated with MDL28170 had significantly greater increases in ICP/MAP and total ICP in response to cavernous nerve stimulation in all time points assayed (p < 0.05). However, the erectile responses remaining reduced compared with the sham controls. nNOS-Positive Nerve Protection nNOS-positive nerves were quantified by histomorphometric analysis of the percentage of dorsal nerve area that stained positive for neuronal-specific NOS. Representative images of each group are shown in figure 3. There was a significant decrease in nNOS content of dorsal penile nerves at 28 days after bilateral CN crush injury (p < 0.05). Following MDL-28170 treatment, nNOS content in the dorsal nerves was significantly greater compared with vehicle-treated CN-crushed controls (p < 0.05). Wan/Li/Guo/Li/Chen
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nal nitric oxide synthase (nNOS)-positive nerve protection. Sections were stained for nNOS (green) and DAPI (blue) in the dorsal penile nerve 28 days after operation. Quantitative analysis of
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Fig. 4. Effect of calpain inhibitor MDL-28170 treatment on trans-
forming growth factor-beta1 (TGF-β1)/Smad2 expression in corpus cavernosum. a The protein expression of TGF-β1, phosphoSmad2 (p-Smad2) and total Smad2 in rat corpus cavernosum tissues were measured by Western blot. β-actin was used as a loading
Crush + vehicle
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nNOS expression was quantified as the relative percentage of nNOS expression in area stain for the entire dorsal penile nerve cross-section. Magnification is ×400. * p < 0.05 vs. sham control.
Fig. 3. Effect of calpain inhibitor MDL-28170 treatment on neuro-
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control. b Relative optical density changes of TGF-β1 normalized to β-actin. c Data are presented as the ratio of phosphorylated protein to total protein. * p < 0.05 vs. sham control; # p < 0.05 vs. CNcrushed rats treated with vehicle.
Activation of TGF-β1/Smad2 in the Corpus Cavernosum TGF-β1 and p-Smad2/Smad2 protein levels were measured in cavernous tissue by western blot analysis. The ratio of p-Smad2 to total Smad2 was quantified to determine Smad2 activity. In CN-crushed rats, TGF-β1 protein levels and the ratio of p-Smad2 to total Smad2 were significantly increased compared with the sham controls (85.3 ± 3.4% for CN injury vs. 37.5 ± 5.6% for sham control and 0.93 ± 0.06 vs. 0.48 ± 0.04, respectively, p < 0.05, fig. 4). In response to MDL-28170 treatment, TGF-β1 protein and the ratio of p-Smad2 to total Smad2 were significantly decreased (85.3 ± 3.4% for CN injury vs. 54.3 ± 5.3% for MDL-28170 and 0.93 ± 0.06 vs. 0.68 ± 0.04, respectively, p < 0.05).
Collagen Content in the Corpus Cavernosum Western blot analysis was performed to evaluate expression of collagen I and collagen IV in the corpus cavernosum (fig. 5). The relative protein level of collagen I significantly increased in CN-crushed rats (78.7 ± 6.4% for CN injury vs. 27.9 ± 5.2% for sham control, p < 0.05). The expression significantly reduced in response to MDL28170 treatment (p < 0.05) and was indistinguishable from the sham. Western blot analysis also revealed that collagen IV protein expression was significantly increased in CN-crushed rats (98.7 ± 6.8% for CN injury vs. 33.1 ± 5.6% for sham control, p < 0.05). Treatment with MDL28170 significantly decreased the expression (98.7 ± 6.8% for CN injury vs. 67.1 ± 5.2% for sham control, p < 0.05), although the expression remained increased compared with that of the sham control rats.
Effect of Calpain Inhibition after CN Injury
Urol Int DOI: 10.1159/000370248
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Discussion
As penile neuropathy is a major pathogenic basis for ED in patients after radical prostatectomy, there has been great interest in finding therapeutic approaches that promote neuroprotection and nerve regeneration for facilitating functional recovery. In the present study, we used a rat model of bilateral CN crush injury to investigate the role of calpain in CN injury-induced ED. Our results indicated that impaired erectile function in CN-crushed rats was associated with increased levels of calpain activity, decreased nNOS expression in the dorsal penile nerve and increased cavernous expression of TGF-β1/Smad2 and collagen deposition. Results also demonstrated that pharmacological inhibition of calpain by MDL-28170 at least partially improved these deficits, indicating that calpain inhibition could attenuate the development of CN injury-induced ED. Following initial mechanical injury to the CN, additional tissue damage resulted from secondary processes, including inflammation, oxidative stress, ischemia, excitotoxicity, free radical production, and apoptosis [3, 27]. Excessively activated calpain has been associated with acute or chronic neurodegenerative diseases. Consistent with previous reports using other nervous system models [17, 18], results from the present study, using a model of bilateral CN crush injury, showed enhanced levels of calpain activity in MPG at the early postoperative period, as well as calpain activity gradually decreased to control levels within 7 days. Proteolysis of the axonal cytoskeletal proteins αII spectrin, βII spectrin, and ankyrinB by calpain has been proposed to be a major contributor to axonal degeneration after injury in central or peripheral nervous systems [14, 15, 19]. Thus, calpain represents a rational and feasible 6
Urol Int DOI: 10.1159/000370248
b
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target for neuroprotective therapeutic interventions. In this study, we found that the content of nNOS-positive fiber content in the dorsal nerves of CN-crushed rats improved after MDL-28170 treatment compared with vehicle-treated rats, indicating the neuroprotective effects of calpain inhibition after bilateral CN crush injury. Compared with sham control rats, electrical stimulation of the cavernous nerve resulted in markedly decreased erectile function in CN-crushed rats treated with vehicle. Partial, but significant, enhancement of erectile response was observed in the MDL-28170-treated group. This enhancement occurred at the earliest time point assayed and persisted for the duration of the experiment (28 days) after cavernous nerve crushing. The MDL-28170-treated group exhibited improved erectile function, although the improvement did not reach control levels. It has been hypothesized that cavernosal nerve damage, such as that after radical prostatectomy, is associated with hypoxia-induced activation of the TGF-β1/Smad signaling pathway in the corpus cavernosum, which further leads to fibrosis and loss of smooth muscle content in erectile tissue [6, 7]. Results from the current study confirmed previous reports showing activation of the TGF-β1/Smad2 pathway and increased collagen content in CN-crushed rats. Inhibition of the TGF-β pathway resulted in a significant decrease of collagen deposition and apoptosis in the corpus cavernosum following bilateral CN injury [28]. Indeed, the TGF-β1/Smad pathway is important in the pathogenesis of ED following cavernosal nerve damage. Interestingly, calpain has been suggested to be one of the main suspected perpetrators in chronic hypoxic/ischemic injury. Calpain-mediated proteolysis and apoptotic processes are involved in the pathogenesis of many tissues as a result of hypoxic conditions, such as the brain, Wan/Li/Guo/Li/Chen
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28170 treatment on collagen I and IV expression in corpus cavernosum. a The protein expression of collagen I and IV in rat corpus cavernosum tissues were measured by Western blot. β-actin was used as a loading control. b Relative optical density changes of collagen I and IV normalized to β-actin. * p < 0.05 vs. sham control; # p < 0.05 vs. CN-crushed rats treated with vehicle.
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liver, heart, skeletal muscles, pulmonary artery, retina, and kidney [19, 20, 29, 30]. Moreover, calpain can induce TGF-β activation through an unknown mechanism [20, 30]. The present study demonstrated enhanced calpain activity in cavernous tissue after bilateral CN crush injury, and calpain inhibition by MDL-28170 resulted in decreased TGF-β1/Smad2 expression and collagen I and IV content. These results implicated that calpain inhibition had protective effects on cavernous tissue fibrosis after CN injury. In conclusion, this study provided evidence for the role of calpain activation in the pathogenesis of CN inju-
ry-induced ED. In addition, treatment with a calpain inhibitor provided neuroprotection against axonal degeneration produced by a crush lesion and amelioration of penile fibrosis, thereby preserving erectile function. These data suggested that calpain inhibition could be a promising strategy for preventing the development of diabetic ED.
Conflicts of Interest The authors declare no competing financial interests.
References
Effect of Calpain Inhibition after CN Injury
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