| Urine collection, storage and voiding periodically are mediated by the coordination of the bladder and urethra, which is controlled by complicated neural circuit. The urine storage and voiding are completely dependent on central neural control. There are two bladder functional modes:storage and voiding. So the neural control for lower urinary tract often has on-off switch-like activity. In addition, urine voiding is also under voluntary control by cerebral cortex.Due to the complicated neural control in the LUT, many injury, irritation and disease can cause urinary dysfunction including bladder overactivity, urinary retention, neuronergic bladder and detrusor-sphincter dyssynergia. Modulating certain peripheral nerve, stimulating or blocking nerve conduction, would get some theraputic effect. This study consists of two parts:(1) Role of μ, κ, and δ opioid receptors in tibial inhibition of bladder overactivity in cats. (2) Conduction Block of Pudendal Nerve by Changing Local Temperature.Part 1:Role of μ,κ, and δ opioid receptors in tibial inhibition of bladder overactivity in cats.Introduction:Overactive bladder syndrome (OAB), characterized by urinary urgencyusually accompanied by frequencywith/without incontinence, which greatly impacted the quality of patient’s life and significantly affected work productivity. Medication such as anticholinergic drugs is often un-satisfactory for OAB treatment due to its limited efficacy and/or undesirable side effects. Therefore, neuromodulation therapy becomes an attractive option for drug-refractory patients.Neuromodulation by stimulating peripheral nerves with different modes of electric current was reported to alleviate OAB symtoms. Currently, the US Food and Drug Administration approved sacral nerve stimulation and tibial nerve stimulation for OAB treatment, while pudendal nerve stimulation was also reported in OAB treatment but not approved now. Plenty of references supported neuromodulation as an effective, safe and minimally invasive option for OAB treatment. And the American Urological Association Guideline recommended neuromodulation as the third line treatment for OAB patients. However, the mechanisms such as the neural circuit and transmitters underlying neuromodulation therapy is currently not fully understood.Our previous study in cats has indicated that opioid receptors play a major role in OAB inhibition induced by tibial nerve stimulation (TNS), because intravenous administration of naloxone (an opioid receptor antagonist) can completely remove the TNS inhibition. Consequently TNS inhibition can be greatly enhanced when combined with a low dose of tramadol (an opioid receptor agonist).Opioid receptors play a key role in neuromodulatory system which inhibit neuron activity with numerous transmitters or control transmitter release. Opioid receptors was also report modulate micturation reflex both in spinal and supraspinal levels. Naloxone suppressed the enkephalinergic inhibitory control to disinhibit the parasympathetic outflow to bladder at spinal level. Intracerebroventricular morphine or endorphin inhibit bladder activity which was reversed by naloxone. And administrationfentanyl in the dorsal pontine tegmentum also inhibited bladder motility.However, it is known that opioid drugs such as tramadol can produce significant adverse effects. Currently three opioid receptorsubtypes(μ, κ, and δ) have been identified and different subtypes may modulate opioid functions differentially. The exact role of different opioid subtypes in TNS inhibition remains unclear.Therefore, determining the role of different opioid receptor subtypes in TNS inhibition may further refine the effective combinations between TNS and opioid drugs, so that the bladder overactivity can be suppressed more effectively and at the same time the adverse effects of opioid drugsmay be reduced by targeting a subtype of opioid receptors.Objective:This study in cats is designed to determine which subtype of opioid receptorsis involved in TNS inhibition of the bladder overactivity induced by 0.25% acetic acid (AA) irritation.Method:1. Experimental SetupA total of 22 cats were used in this study, which were divided into 3 groups for pharmacological studies with cumulative dose of cyprodime (a selective μ opioid receptor antagonist), nor-Binaltorphimine(a selective κ opioid receptor antagonist) or naltrindole(a selective δ opioid receptor antagonist).The animals were anesthetized by isoflurane during surgery and maintained with a-chloralose anesthesia during data collection. A pulse oximeter was attached on the tongue to monitor the heart rate and blood oxygen level. A tracheotomywas performed and a tube was inserted to maintain the airway open. A catheter was inserted into right carotid artery to monitor systemic blood pressure. Another catheter was inserted into the left cephalic vein for saline and drug administration. Through an abdominal incision, the ureters were isolated, cut and drained externally. A double-lumen catheter was inserted into the bladder via a small cut in the proximal urethra. One lumen of the catheter was connected to a pump to slowly infuse saline or 0.25% acetic acid (AA) into the bladder and the other lumen was connected with a pressure transducer to measure intravesical pressure.The tibial nerve was exposed on the left leg above the ankle and a tripolar cuff electrode was implanted for stimulation. All incisions were closed by sutures at the end of surgery.2. Stimulation parametersUniphasic rectangular pulses (5-Hz frequency,0.2-ms pulse width) were used to stimulate the tibial nerve via the cuff electrode. The intensity threshold (T) for inducing observable toe movement was determined by gradually increasing the stimulation intensity. Based on our previous studies, intensities of 2T or 4T were used in this study to suppress the bladder overactivity induced by 0.25% AA irritation.3. Stimulation protocols and drug administrationAt the beginning of each experiment, multiple cystometrograms (CMGs) were performed with saline infusion to determine the bladder capacity. Once stable bladder capacity was obtained,0.25% AA was infused into the bladder to irritate nociceptive C-fiber bladder afferents and induce overactive bladder reflex. Repeated CMGs were performed with AA infusion until the bladder capacity was stabilized, which was followed by additional four AA CMGs:(1) control CMG without TNS, (2) CMG during 2T TNS, (3) CMG during 4T TNS, (4) control CMG without TNS. Then, the animals were divided into 3 groups for pharmacological studies.In the first group, cumulative doses (0.003,0.01,0.03,0.1,0.3 and 1 mg/kg) of cyprodime (a selective μ opioid receptor antagonist) were administrated intravenously. Ten minutes after administering each dose, four AA CMGs were performed:(1) control CMG without TNS, (2) CMG during 2T TNS, (3) CMG during 4T TNS, (4) control CMG without TNS. Similar drug testing protocol was also used in the second and third groups of cats, but cumulative doses (0.03,0.1,0.3,1.0,3.0 and 10.0 mg/kg) of nor-Binaltorphimine(a selective κ opioid receptor antagonist) were given in the second group and cumulative doses (0.03,0.1,0.3,1.0,3.0 and 10.0 mg/kg) of naltrindole(a selective 8 opioid receptor antagonist)were given in the third group.At the end ofexperiment naloxone (1 mg/kg) was administrated, which was followed by the four repeated CMGs (control,2T,4T and control).4. Data analysisBladder capacity was measured during each CMG and normalized to the saline control CMG in each experiment so that the results from different animals could be compared. Repeated measurements in the same animal under the same experimental conditions were averaged. The results from different animals are reported as mean±SD. Statistical significance (P<0.05) was detected by a paired t-test or ANOVA followed by Dunnett or Bonferroni multiple comparison.Result:1. TNS Inhibition of Bladder Overactivity:The bladder irritated by 0.25% AA became overactive and significantly (P<0.01) reduced its capacity to 21.15±12.21% of saline control capacity. TNS at 2T or 4T intensity suppressed the bladder overactivity and significantly (P<0.01)increased bladder capacity to 52.92±17.76% or57.39±21.62% of the saline control capacity, respectively. After TNS the bladder capacity returned to pre-stimulation volume, indicating that there was no post-stimulation inhibition.2. Effects of Selective Opioid Receptor AntagonistsonTNS Inhibition and Bladder OveractivityCyprodime did not change the control bladder capacity during repeated AA CMGs but significantly (P<0.05) reduced TNS inhibition starting from 0.1 mg/kg dose and completely eliminated the inhibition induced by both 2T and 4T TNS at doses of 0.3-1 mg/kg.Nor-Binaltorphimine significantly (P<0.05) increased the control bladder capacity at doses of 1-10 mg/kg and also significantly (P<0.05) reduced TNS inhibition starting from 1 mg/kg dose and completely eliminated the inhibition induced by both 2T and 4T TNS at doses of 3-10 mg/kg.Naltrindole significantly (P<0.05) increased AA control capacity and reduced TNS inhibition at doses of 1-10 mg/kg, but significant amount of TNS inhibition was still remained even after the largest dose.3. Effect of Naloxone after Blocking a Subtype of Opioid ReceptorNaloxone had no effect on bladder capacity with/without TNS in cyprodime pre-treated cats. However, it significantly (P<0.05) reduced bladder capacity in nor-Binaltorphimine pre-treated cats and completely eliminated the inhibitory effect of nor-Binaltorphimine on bladder overactivity. In addition, naloxone also eliminated the remaining TNS inhibition in naltrindole pre-treated cats without changing the control bladder capacity.Conclusion:This study in cats revealed several important roles of different opioid receptor subtypes (μ, κ, and δ) in TNS inhibition and AA irritation-induced bladder overactivity. Our results indicate a major role of μ and κ opioid receptors in TNS inhibition while the 8 opioid receptor plays a minor role. Meanwhile, κ and δ opioid receptors play an excitatory role in irritation-induced bladder overactivity.Part 2 Conduction Block of Pudendal Nerve by Changing Local TemperatureIntroductionIn normal conditions, coordination and transition between bladder detrusor and urethral sphincter managed urine storage and voiding which was controlled by the central and peripheral nervous systems. In some pathological conditions, this coordination was disturbed and the urethral sphincter contracted synchronous with the bladder detrusor which was defined as detrusor sphincter dyssynergia (DSD). DSD caused high bladder pressure and dysuria. Long-term DSD leaded to urinary infection, vesicoureteric reflux and even renal failure. For DSD treatment, intermittent catheterization remained as a mainstay in DSD treatment and other treatment included transurethral sphincterotomy, botulinum A toxin injection and intraurethral stent, which were usually unsatisfactory due to limited efficacy and risk of permanent incontinence. Local blocking pudendal nerve using phenol solution get similar effect to botulinum A toxin injection. If the pudendal nerve is reversibly blocked during voiding and restored after voiding, the dysuria is alleviated without incontinenece.Nerve block was widely applied in basic and clinical studies. Many techniques were raised to achieve a selective nerve block which was categorized as chemical drugs, surgery, electronic stimulation and temperature regulation. However, surgeries have side effect and even cause irreversible damage. Injection of local anesthetics has been used for many years to produce brief nerve block because it is difficult to deliver these drugs chronically. Recently, high-frequency (kHz) electrical stimulation generated by implantable stimulators is being used clinically to chronically block the vagus nerve for obesity treatment or to block the spinal roots for treatments of chronic pain. High-frequency stimulation has also been proposed to block the pudendal nerve to restorelower urinary tract function after spinal cord injury. However, high-frequency stimulation will always generate an initial nerve firing before it can block nerve conduction. The initial nerve firing is problematic for many clinical applications such as suppressing pain, because initial painful sensationcould be induced before nerve block occurs. Local nerve temperature regulation were much attractive because extremely heating and cold both induced nerve block.Nerve block by modifying temperature on local nerve has been raised for a century. Heating can block nerve conductivity in squid, bullfrog and cats. But heating results are difficult to control and overheating often cause irreversible damage on nerve. While nerve block by cooling is safe and reversible. But Schumacher reported pudendal nerve was blocked until cooling down to 6.2℃, which is easy to low the nerve to such low degree for treatment in vivo. Therefore, a thermal block technology which is effective, safe and easy to manage is in demand, and it could potentially be used for many clinical applications to treat chronic diseases such as obesity, pain, heart failure, and bladder dysfunction after spinal cord injury.ObjectiveTo evaluate the effect of local temperature to pudendal nerve conductivity, and find a safe convenient method to achieve reversible pudendal nerve block by modifying local temperature.Method1. Experimental SetupThe animals were anesthetized by isoflurane during surgery and maintained with a-chloralose anesthesia during data collection. A pulse oximeter was attached on the tongue to monitor the heart rate and blood oxygen level. Atracheotomywas performed and a tube was inserted to maintain the airway open. A catheter was inserted into right carotid artery to monitor systemic blood pressure. Another catheter was inserted into the left cephalic vein for saline administration. Through an abdominal incision, the ureters were isolated, cut and drained externally. A double-lumen catheter was inserted into the urethral via a small cut in the proximal urethra. One lumen of the catheter was connected to a pump to slowly infuse saline into the urethra and the other lumen was connected with a pressure transducer to measure intraurethral pressure.The urethral branches of pudendal nerves were exposed via 3-4 cm incisions between the tail and sciatic notch and then cut bilaterally at the distal ends. The right or left pudendal nerve was studiedindividually. The separated branch was put through a small (9 mm long) coil (2 mm inner coil diameter) of copper tubing, through which water at different degrees ran to modify the local temperature at the covered nerve. A thermometer probe was inserted adjacent to the coil and the pudendal nerve to monitor the temperature at the local nerve. The size of the coil just fit the nerve and the thermocouple, so that the thermocouple tip was in contact with both the nerve and the coil. A hook electrode for electrical stimulation was placed on the nerve central to the copper coil to test whether local temperature change could block the urethral response induced by electrical stimulation. Adjacent skin flaps were sutured to form a small organ pool filled with warm saline(35-37℃) to imitate a vivo homeostasis.2. A total of 21 pudendal nerves were involved in this experiment. Temperature modifications on pudendal nerves were managed as follows:2.1 The pudendal nerves were briefly (50-60 second duration) cooled sequentially to temperatures of 30,25,20,15,10,5 and 2℃.2.2 The pudendal nerves were briefly (50-60 second duration) heated sequentially to temperatures of 42,44,46,48,50,52, and54℃ in+2℃ steps. Once a reversible complete heat block was observed, the temperature was not further increased.2.3 The brief cooling protocol was repeated to examine the changes in cold block temperatures; andthe duration of change was then monitored by repeatedly (50-150 second interval) and briefly (50-60 seconds) coolingthe nerve until the cold block temperature returned to control level,which was confirmed by repeating the brief cooling protocol.2.4 Different heating durations (0.6-3.8 minutes) were tested at the reversible block temperature (50-54℃) to determine the heating duration for a non-reversible block.2.5 The repeated cooling protocol as described above was performed initially to determine the cold block temperature. Then, the nerve was heated 3 times for a period of 5 minutes to 46℃ or 48℃, which are temperatures just below the heat block temperature (50-54℃). After each heating the cold block temperature was measured by the repeated cooling protocol.3. Data analysisAmplitude of urethral pressure response to stimulation was measured during temperature modification and normalized to control amplitude just before temperature modification in each experiment so that the results from different animals could be compared. Repeated measurements in the same animal under the same experimental conditions were averaged. The results from different animals are reported as Mean±SD. Statistical significance (P<0.05) was detected by a paired t-test or ANOVA followed by Dunnett or Bonferroni multiple comparison.ResultShort trains (5 seconds on and 20 seconds off) of pudendal nerve stimulation induced short duration EUS contractions that generated relatively consistent urethral pressure increases of amplitude greater than 40 cmH2O. Manual perfusion of cold/hot water through the copper coil quickly (5-10 seconds) decreased/increased the local temperature that was maintained for 50-60 seconds. Once the perfusion was stopped, the temperature quickly (5-10 seconds) returned to the saline pool temperature of 35-37℃.1.When the temperature was gradually decreased by local cooling, a partial block of pudendal nerve conduction occurred starting at 15℃. In the 20 tested nerves, a complete block was firstly achieved at 15℃ in 2 nerves, at 10℃ in 4 nerves, at 5℃ in 11 nerves and at 2℃ in 3 nerves. The mean cold block temperature was 6.55±3.83℃. The urethral contraction responses fully recovered once the temperature was returned to the warm saline pool temperature after both the brief (50-60 seconds) and long-lasting (4.5-10 minutes) complete cold block.2.When the temperature was gradually increased by local heating, a partial block of nerve conduction occurred starting at 50℃. In the 14 tested nerves, a complete block was firstly achieved at 50℃ in 2 nerves, at 52℃ in 6 nerves, and at 54℃ in 6 nerves. The mean hot block temperature was 52.57±1.45℃. Although heat block of short duration (1.27℃0.44 min) was fully reversible, a longer duration (2.74℃0.54 min) produced a non-reversible block.3.Reversible complete heat block increased the temperature for cold block. The temperature for cold block after brief heating was significantly higher than that before heating (17.92±1.30℃ VS 7.17±1.36℃, respectively). On average a brief (0.5-1.0 minute) reversible complete heat block at 50-54℃ shifted the cold block response curve to a temperature about 10℃ higher than the control.The increased temperature for cold block decreased with time and fully recovered to innitial conditions in 42.84±7.00 min.4.The temperature for cold block could also be increased by non-block heating at 46-48℃. However, in the same nerve repeated (3 times) heating at 46-48℃ for 5 minutes each time, which had no effect on nerve conductivity, gradually increased the temperature for cold block from 6.43±0.92℃ to 14.29±1.70℃.ConclusionThis study in cats showed that the pudendal nerve can be reversibly blocked by locally cooling the nerve below 15℃ or by a brief local heating above 50℃. However, the cold block temperature could be increased to 10-25℃ after a reversible complete heat block at 50-54℃ or after repeated non-block heating at 46-48℃. This study discovered a novel method to block mammalian myelinated nerves at room temperatures (10-25℃), providing the possibility to develop an implantable device to block axonal conduction and treat many chronic diseases such as obesity, pain, heart failure, and bladder dysfunction after spinal cord injury. |
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