Neuromodulation: Technology at the Neural Interface Received: November 25, 2013
Revised: February 22, 2014
Accepted: February 28, 2014
(onlinelibrary.wiley.com) DOI: 10.1111/ner.12189
Effects of Sacral Neuromodulation on Isolated Urinary Bladder Function in a Rat Model of Spinal Cord Injury S¸ükrü Kumsar, MD*; Ulya Keskin, MD†; Alaaddin Akay, MD‡; Uğur Taylan Bilgilisoy, MD§; S¸. Remzi Erdem, MD†; Ç. Levent Pes¸kircioğlu, MD¶; Hakan Özkardes¸, MD¶ Introduction: Sacral neuromodulation has been considered as an effective treatment option for various types of chronic voiding dysfunction, but the mechanism of action has not been well understood. The aim of this study was to evaluate the effect of chronic sacral neuromodulation on isolated bladder functions in a rat model of spinal cord injury. Materials and Methods: Female Sprague-Dawley rats (250–300 g; N = 20) were assigned to four groups as follows: 1) control group (N = 6); 2) spinal cord transection group (SCT; N = 5); 3) spinal cord transection + sacral neuromodulation group (SCT + SNM; N = 5); 4) sham (spinal cord transection + electrode wire implantation without sacral neuromodulation; N = 4). The rats in the SCT, SCT + SNM, and sham groups were anesthetized with ketamine (60 mg/kg, i.p.) and xylazine (7 mg/kg, i.p.). The spinal cord was completely transected at T8–T9 level in SCT and SCT + SNM groups. Electrode wires were implanted into S3 dorsal foramina in both sham and SNM groups, but only the SNM group was subjected to electrical stimulation for four hours a day for three weeks. Twenty-one days later, the rats were sacrificed via anesthetic overdose, and isolated longitudinal bladder strip preparations were placed in organ baths for the investigation of their isometric responses to pharmacological agents. Results: In isometric contraction experiments, SCT was found to increase the contraction responses of the bladder strips to muscarinic stimulation, and SNM could not prevent this increase. In isometric relaxation experiments, SCT caused a decrease in β-adrenergic relaxation responses, and SNM augmented the bladder’s β-adrenergic relaxation responses. Nitric oxide did not affect the relaxation responses. Conclusion: In our rat model of SCT, SNM seemed to alter adrenergic receptor function in the urinary bladder. Further studies are required to clarify the mechanism of these alterations at the level of bladder receptors following sacral neuromodulation. Keywords: Bladder dysfunction, neurostimulation, rats, sacral nerve stimulation, spinal cord injury Conflict of Interest: The authors reported no conflict of interest.
INTRODUCTION Neuromodulation is an innovative treatment of lower urinary tract symptoms and bladder storage dysfunctions secondary to neuromuscular causes. In addition to the application of evolving technologies for sacral neuromodulation (SNM) therapy, it is being used for expanding clinical indications such as neurogenic detrusor overactivity, post-sling-related voiding dysfunction, interstitial cystitis, pelvic pain, pediatric voiding dysfunction, and bowel disorders (1). SNM comprises the electrical stimulation of the sacral nerves that innervate the bladder, urethral sphincter, and pelvic floor muscles. Various theories are suggested to explain the mechanism of action of SNM, such as the following:
www.neuromodulationjournal.com
Address correspondence to: Şükrü Kumsar, MD, Urology Department, Sakarya Training and Research Hospital, Sakarya EAH, Uroloji Kliniği, Sakarya 54100, Turkey. Email: [email protected] * Urology Department, Sakarya Training and Research Hospital, Sakarya, Turkey; † Pharmacology Department, Başkent University Hospital of Medicine, Ankara,Turkey; ‡ Clinic of Urology, Batman State Hospital, Batman, Turkey; § Clinic of Urology, Elbistan State Hospital, Kahramanmaraş, Turkey; and ¶ Urology Department, Başkent University Hospital of Medicine, Ankara,Turkey For more information on author guidelines, an explanation of our peer review process, and conflict of interest informed consent policies, please go to http:// www.wiley.com/bw/submit.asp?ref=1094-7159&site=1 Source(s) of financial support: None.
© 2014 International Neuromodulation Society
Neuromodulation 2014; ••: ••–••
1
1. SNM affects the “neuroaxis” at various levels and restores the balance between excitatory and inhibitory regulation at various locations within the peripheral and central nervous systems (2). 2. SNM may activate the afferent bladder somatosensors that run to the micturition center in the brain stem and/or may activate the hypogastric sympathic nerves (3).
3. The bladder tends to respond to neural stimulation initially with rapid contraction followed by slow, longer-lasting relaxation; however, recurrent, repetitive electrical stimulation causes a decay and down-regulation in the bladder’s response and reduces detrusor muscle activity (4). 4. Stimulation of the afferent sacral nerves in either the pelvis or lower extremities increases the inhibitory stimuli to
KUMSAR ET AL. the efferent pelvic nerve and reduces detrusor contractility (5). 5. The hypogastric nerve is stimulated through the activation of sympathetic fibers at low bladder volumes, and pudendal nerve nuclei in the spinal cord are directly stimulated at maximal bladder volume (6). However, the precise mechanism of action of SNM is still not entirely clear. Animal experiments investigating the mechanism of SNM action have been generally focused on experimental urinary bladder inflammation formation and spinalization (6–9). Even though there have been various studies on spinalized rat neuromodulation models in the literature, to the best of our knowledge, this is the first study to investigate the effect of neuromodulation at tissue level together with the functional findings (isometric contraction-relaxation responses) from isolated bladder strips (10,11). The objective of the present study was to investigate the ex vivo effect of long-term SNM on the isometric responses of isolated bladder strips obtained from rats that previously underwent spinal cord transection to various pharmacological agents in isolated organ baths.
MATERIALS AND METHODS The present study was started after having the project proposal approved by the Başkent University Local Ethical Committee on Research in Experimental Animals (DA 08/30). The study was performed in 20 adult (12–14 months) female Sprague-Dawley rats (250–300 g) at Başkent University Experimental Research Center. The animals were fed with standard rat chow and tap water ad libitum, and they were kept in the animal facility with constant environmental conditions (room temperature: 20 ± 2°C, relative humidity: 50 ± 10%, light/dark cycle: 12 h/12 h). The experiments were conducted in accordance with the Declaration of Helsinki and the Guide for the Use and Care of Laboratory Animals published by the American National Institutes of Health. The animals were randomly assigned to four groups, as follows: 1. Control group (N = 6) 2. Spinal cord transection group (SCT; N = 5) 3. Spinal cord transection + sacral neuromodulation group (SCT + SNM; N = 5) 4. Sham (spinal cord transection + electrode wire implantation without sacral neuromodulation; N = 4)
2
For antimicrobial prophylaxis, enrofloxacin (10 mg/kg, i.m.) was administered to the rats for three days starting before the surgery. After providing appropriate conditions (disinfection and sterilization) for surgery, the experimental animals to be subjected to SCT procedure were anesthetized by ketamine (60 mg/kg, i.p.) and xylazine (7 mg/kg, i.p.). The rats were placed in prone position pursuant to back shaving and field disinfection. A dorsal midline skin incision between the levels of T7 and T10 was applied. The spinal processes and lamina of T8 and T9 were removed; then, a complete anatomic transection of the medulla spinalis was applied by a scalpel at T8–T9 level. For electrode implantation to the sacral foramina, a midline incision was applied at the sacral region, and paravertebral muscles were pulled to the laterals. The third sacral foramen was found, and the response (ipsilateral hind limb movement) of the underlying www.neuromodulationjournal.com
nerve was tested by a stimulation needle. Then, one end of a copper wire subcutaneously placed through a tunnel on the back of the rat was inserted into the S3 foramen. The other end of the wire was pulled out and affixed at the interscapular region to be connected to the stimulator device (Medtronic Model 3625, Medtronic, Inc., Minneapolis, MN, USA) for neuromodulation. The Medtronic stimulator was not implanted. One end of the wire was connected to the stimulator device for stimulation. Neuromodulation (monopolar current up to 80% [0.5–2 mA] of the stimulation amplitude to develop hind limb movement at 2 V and 25 Hz frequency) was applied for 21 days, beginning on the day after surgery, for four hours a day. All the rats with transected spinal cords were induced to micturate by manual urinary bladder massage three times a day. After 21 days, the urinary bladder was completely removed under ketamine (60 mg/kg, i.p.) and xylazine (7 mg/kg, i.p.) anesthesia, vertically opened at the midline in a Petri dish containing ice-cold Krebs-Henseleit solution, and longitudinal urinary bladder strip preparations (10 × 0.3 mm) were isolated. The isolated bladder strips were mounted in 10 mL organ baths containing Krebs-Henseleit solution (NaCl, 118.2 mmol; KCl, 4.7 mmol; MgSO4, 12 mmol; CaCl2, 2.5 mmol; KH2PO4, 1.2 mmol; NaHCO3, 25 mmol; glucose, 11.1 mmol). The solution was maintained at 37°C and aerated with 95% O2 and 5% CO2 gas mixture. An initial resting tension of 1 g was applied to the tissues, which were allowed to equilibrate for 60 min without adding any test agents into the baths. Isometric contractions were displayed and recorded on a computerized physiological data acquisition system (BioPac, MP100, Commat, Ankara, Turkey) through a force-displacement transducer (FTO3, Commat, Ankara, Turkey). The functional data (isometric responses) from the bladder preparations were obtained by adding contracting and relaxing pharmacological agents at increasing concentrations into the organ baths. The pharmacological agents applied and their final concentrations in the isolated organ baths were as follows: carbachol (cholinergic agonist, 1 nM–300 μM; Sigma, St. Louis, MO, USA), isoproterenol (beta adrenergic agonist, 1 nM–100 μM; Sigma), sodium nitroprusside (SNP; exogenous NO donor, 1 nM–300 μM; Sigma), atropine (muscarinic receptor antagonist, 1 μM; Sigma), propranolol (beta adrenergic receptor antagonist, 1 μM; Sigma). Each contractile response was normalized to the initial submaximal KCl (60 mM) contraction. All of the agents were dissolved in distilled water. The agents were cumulatively added into the isolated organ baths. Inhibitors and antagonists were added into the organ baths 20 min before the agonists for incubation. The baths were washed out with fresh Krebs-Henseleit solution three times with 5-min intervals between different procedures. Statistical Analysis Data, expressed as mean and SEM, were statistically analyzed by two-way ANOVA (GraphPad Prism Version 4.0, GraphPad Software Inc., San Diego, CA, USA). Any statistical differences among the groups were further evaluated by post hoc Bonferroni test. A p value
Revised: February 22, 2014
Accepted: February 28, 2014
(onlinelibrary.wiley.com) DOI: 10.1111/ner.12189
Effects of Sacral Neuromodulation on Isolated Urinary Bladder Function in a Rat Model of Spinal Cord Injury S¸ükrü Kumsar, MD*; Ulya Keskin, MD†; Alaaddin Akay, MD‡; Uğur Taylan Bilgilisoy, MD§; S¸. Remzi Erdem, MD†; Ç. Levent Pes¸kircioğlu, MD¶; Hakan Özkardes¸, MD¶ Introduction: Sacral neuromodulation has been considered as an effective treatment option for various types of chronic voiding dysfunction, but the mechanism of action has not been well understood. The aim of this study was to evaluate the effect of chronic sacral neuromodulation on isolated bladder functions in a rat model of spinal cord injury. Materials and Methods: Female Sprague-Dawley rats (250–300 g; N = 20) were assigned to four groups as follows: 1) control group (N = 6); 2) spinal cord transection group (SCT; N = 5); 3) spinal cord transection + sacral neuromodulation group (SCT + SNM; N = 5); 4) sham (spinal cord transection + electrode wire implantation without sacral neuromodulation; N = 4). The rats in the SCT, SCT + SNM, and sham groups were anesthetized with ketamine (60 mg/kg, i.p.) and xylazine (7 mg/kg, i.p.). The spinal cord was completely transected at T8–T9 level in SCT and SCT + SNM groups. Electrode wires were implanted into S3 dorsal foramina in both sham and SNM groups, but only the SNM group was subjected to electrical stimulation for four hours a day for three weeks. Twenty-one days later, the rats were sacrificed via anesthetic overdose, and isolated longitudinal bladder strip preparations were placed in organ baths for the investigation of their isometric responses to pharmacological agents. Results: In isometric contraction experiments, SCT was found to increase the contraction responses of the bladder strips to muscarinic stimulation, and SNM could not prevent this increase. In isometric relaxation experiments, SCT caused a decrease in β-adrenergic relaxation responses, and SNM augmented the bladder’s β-adrenergic relaxation responses. Nitric oxide did not affect the relaxation responses. Conclusion: In our rat model of SCT, SNM seemed to alter adrenergic receptor function in the urinary bladder. Further studies are required to clarify the mechanism of these alterations at the level of bladder receptors following sacral neuromodulation. Keywords: Bladder dysfunction, neurostimulation, rats, sacral nerve stimulation, spinal cord injury Conflict of Interest: The authors reported no conflict of interest.
INTRODUCTION Neuromodulation is an innovative treatment of lower urinary tract symptoms and bladder storage dysfunctions secondary to neuromuscular causes. In addition to the application of evolving technologies for sacral neuromodulation (SNM) therapy, it is being used for expanding clinical indications such as neurogenic detrusor overactivity, post-sling-related voiding dysfunction, interstitial cystitis, pelvic pain, pediatric voiding dysfunction, and bowel disorders (1). SNM comprises the electrical stimulation of the sacral nerves that innervate the bladder, urethral sphincter, and pelvic floor muscles. Various theories are suggested to explain the mechanism of action of SNM, such as the following:
www.neuromodulationjournal.com
Address correspondence to: Şükrü Kumsar, MD, Urology Department, Sakarya Training and Research Hospital, Sakarya EAH, Uroloji Kliniği, Sakarya 54100, Turkey. Email: [email protected] * Urology Department, Sakarya Training and Research Hospital, Sakarya, Turkey; † Pharmacology Department, Başkent University Hospital of Medicine, Ankara,Turkey; ‡ Clinic of Urology, Batman State Hospital, Batman, Turkey; § Clinic of Urology, Elbistan State Hospital, Kahramanmaraş, Turkey; and ¶ Urology Department, Başkent University Hospital of Medicine, Ankara,Turkey For more information on author guidelines, an explanation of our peer review process, and conflict of interest informed consent policies, please go to http:// www.wiley.com/bw/submit.asp?ref=1094-7159&site=1 Source(s) of financial support: None.
© 2014 International Neuromodulation Society
Neuromodulation 2014; ••: ••–••
1
1. SNM affects the “neuroaxis” at various levels and restores the balance between excitatory and inhibitory regulation at various locations within the peripheral and central nervous systems (2). 2. SNM may activate the afferent bladder somatosensors that run to the micturition center in the brain stem and/or may activate the hypogastric sympathic nerves (3).
3. The bladder tends to respond to neural stimulation initially with rapid contraction followed by slow, longer-lasting relaxation; however, recurrent, repetitive electrical stimulation causes a decay and down-regulation in the bladder’s response and reduces detrusor muscle activity (4). 4. Stimulation of the afferent sacral nerves in either the pelvis or lower extremities increases the inhibitory stimuli to
KUMSAR ET AL. the efferent pelvic nerve and reduces detrusor contractility (5). 5. The hypogastric nerve is stimulated through the activation of sympathetic fibers at low bladder volumes, and pudendal nerve nuclei in the spinal cord are directly stimulated at maximal bladder volume (6). However, the precise mechanism of action of SNM is still not entirely clear. Animal experiments investigating the mechanism of SNM action have been generally focused on experimental urinary bladder inflammation formation and spinalization (6–9). Even though there have been various studies on spinalized rat neuromodulation models in the literature, to the best of our knowledge, this is the first study to investigate the effect of neuromodulation at tissue level together with the functional findings (isometric contraction-relaxation responses) from isolated bladder strips (10,11). The objective of the present study was to investigate the ex vivo effect of long-term SNM on the isometric responses of isolated bladder strips obtained from rats that previously underwent spinal cord transection to various pharmacological agents in isolated organ baths.
MATERIALS AND METHODS The present study was started after having the project proposal approved by the Başkent University Local Ethical Committee on Research in Experimental Animals (DA 08/30). The study was performed in 20 adult (12–14 months) female Sprague-Dawley rats (250–300 g) at Başkent University Experimental Research Center. The animals were fed with standard rat chow and tap water ad libitum, and they were kept in the animal facility with constant environmental conditions (room temperature: 20 ± 2°C, relative humidity: 50 ± 10%, light/dark cycle: 12 h/12 h). The experiments were conducted in accordance with the Declaration of Helsinki and the Guide for the Use and Care of Laboratory Animals published by the American National Institutes of Health. The animals were randomly assigned to four groups, as follows: 1. Control group (N = 6) 2. Spinal cord transection group (SCT; N = 5) 3. Spinal cord transection + sacral neuromodulation group (SCT + SNM; N = 5) 4. Sham (spinal cord transection + electrode wire implantation without sacral neuromodulation; N = 4)
2
For antimicrobial prophylaxis, enrofloxacin (10 mg/kg, i.m.) was administered to the rats for three days starting before the surgery. After providing appropriate conditions (disinfection and sterilization) for surgery, the experimental animals to be subjected to SCT procedure were anesthetized by ketamine (60 mg/kg, i.p.) and xylazine (7 mg/kg, i.p.). The rats were placed in prone position pursuant to back shaving and field disinfection. A dorsal midline skin incision between the levels of T7 and T10 was applied. The spinal processes and lamina of T8 and T9 were removed; then, a complete anatomic transection of the medulla spinalis was applied by a scalpel at T8–T9 level. For electrode implantation to the sacral foramina, a midline incision was applied at the sacral region, and paravertebral muscles were pulled to the laterals. The third sacral foramen was found, and the response (ipsilateral hind limb movement) of the underlying www.neuromodulationjournal.com
nerve was tested by a stimulation needle. Then, one end of a copper wire subcutaneously placed through a tunnel on the back of the rat was inserted into the S3 foramen. The other end of the wire was pulled out and affixed at the interscapular region to be connected to the stimulator device (Medtronic Model 3625, Medtronic, Inc., Minneapolis, MN, USA) for neuromodulation. The Medtronic stimulator was not implanted. One end of the wire was connected to the stimulator device for stimulation. Neuromodulation (monopolar current up to 80% [0.5–2 mA] of the stimulation amplitude to develop hind limb movement at 2 V and 25 Hz frequency) was applied for 21 days, beginning on the day after surgery, for four hours a day. All the rats with transected spinal cords were induced to micturate by manual urinary bladder massage three times a day. After 21 days, the urinary bladder was completely removed under ketamine (60 mg/kg, i.p.) and xylazine (7 mg/kg, i.p.) anesthesia, vertically opened at the midline in a Petri dish containing ice-cold Krebs-Henseleit solution, and longitudinal urinary bladder strip preparations (10 × 0.3 mm) were isolated. The isolated bladder strips were mounted in 10 mL organ baths containing Krebs-Henseleit solution (NaCl, 118.2 mmol; KCl, 4.7 mmol; MgSO4, 12 mmol; CaCl2, 2.5 mmol; KH2PO4, 1.2 mmol; NaHCO3, 25 mmol; glucose, 11.1 mmol). The solution was maintained at 37°C and aerated with 95% O2 and 5% CO2 gas mixture. An initial resting tension of 1 g was applied to the tissues, which were allowed to equilibrate for 60 min without adding any test agents into the baths. Isometric contractions were displayed and recorded on a computerized physiological data acquisition system (BioPac, MP100, Commat, Ankara, Turkey) through a force-displacement transducer (FTO3, Commat, Ankara, Turkey). The functional data (isometric responses) from the bladder preparations were obtained by adding contracting and relaxing pharmacological agents at increasing concentrations into the organ baths. The pharmacological agents applied and their final concentrations in the isolated organ baths were as follows: carbachol (cholinergic agonist, 1 nM–300 μM; Sigma, St. Louis, MO, USA), isoproterenol (beta adrenergic agonist, 1 nM–100 μM; Sigma), sodium nitroprusside (SNP; exogenous NO donor, 1 nM–300 μM; Sigma), atropine (muscarinic receptor antagonist, 1 μM; Sigma), propranolol (beta adrenergic receptor antagonist, 1 μM; Sigma). Each contractile response was normalized to the initial submaximal KCl (60 mM) contraction. All of the agents were dissolved in distilled water. The agents were cumulatively added into the isolated organ baths. Inhibitors and antagonists were added into the organ baths 20 min before the agonists for incubation. The baths were washed out with fresh Krebs-Henseleit solution three times with 5-min intervals between different procedures. Statistical Analysis Data, expressed as mean and SEM, were statistically analyzed by two-way ANOVA (GraphPad Prism Version 4.0, GraphPad Software Inc., San Diego, CA, USA). Any statistical differences among the groups were further evaluated by post hoc Bonferroni test. A p value
