Feasibility of Real-Time Conditional Sacral Neuromodulation Using Wireless Bladder Pressure Sensor.

Continuous sacral neuromodulation (SNM) is used to treat overactive bladder, reducing urine leakage and increasing capacity. Conditional SNM applies stimulation in response to changing bladder conditions, and is an opportunity to study neuromodulation effects in various disease states. A key advantage of this approach is saving power consumed by stimulation pulses. This study demonstrated feasibility of automatically applying neuromodulation using a wireless bladder pressure sensor, a real-time control algorithm, and the Medtronic Summit™ RC+S neurostimulation research system. This study tested feasibility of four conditional SNM paradigms over five days in 4 female sheep. Primary outcomes assessed proof of concept of closed-loop system function. While the bladder pressure sensor correlated only weakly to simultaneous catheter-based pressure measurement (correlation 0.26-0.89, median r = 0.52), the sensor and algorithm were accurate enough to automatically trigger SNM appropriately. The neurostimulator executed 98.5% of transmitted stimulation commands with a median latency of 72 ms (n = 1,206), suggesting that rapid decision-making and control is feasible with this platform. On average, bladder capacity increased for continuous SNM and algorithm-controlled paradigms. Some animals responded more strongly to conditional SNM, suggesting that treatment could be individualized. Future research in conditional SNM may elucidate the physiologic underpinnings of differential response and enable clinical translation.

Timing of sacral neurostimulation is important for increasing bladder capacity in the anesthetized rat.

We assessed the effects of limited application of sacral neurostimulation (SNS) during bladder filling on bladder capacity using our previously published SNS model in rats. Female Sprague-Dawley rats (n = 24) were urethane anesthetized (1.2 g/kg sc) and implanted with jugular venous and transvesical bladder catheters. L6/S1 nerve trunks were isolated bilaterally, and two electrodes were placed on each exposed nerve. True bladder capacity (TBC) was determined using stable single-fill cystometrograms. In the first series of experiments, SNS was applied at the onset of bladder filling for 25%, 50%, 75%, and 100% of the previous control filling cycle duration (n = 10). In the second series of experiments, SNS was applied during the first, second, third, and fourth 25% and the first and second 50% of the control fill. In the first series, a significant increase in TBC was observed only when SNS was applied for 75% or 100% of the control fill duration (30% and 35%, respectively, P < 0.05). In the second series, significant increases in TBC only occurred during the fourth 25% period and second 50% period (32% and 43%, respectively, P < 0.001). Results from the second series also revealed an increase in subsequent single-fill bladder capacities (TBC) only when SNS was applied during the second 50% of the prior fill cycle. These data indicate that the application of SNS during the final 50% of the bladder fill cycle is necessary and sufficient for increasing bladder capacity.

Real-Time Changes in Brain Activity during Sacral Neuromodulation for Overactive Bladder.

PURPOSE: We performed functional magnetic resonance imaging to identify changes in brain activity during sacral neuromodulation in women with overactive bladder who were responsive to therapy. MATERIALS AND METHODS: Women recruited into the study had nonneurogenic refractory overactive bladder, responded to sacral neuromodulation and had had a stable program for at least 3 months with no subsequent overactive bladder treatment. Enrolled patients completed validated symptom and quality of life instruments before functional magnetic resonance imaging. Stimulus settings were recorded, devices were switched off for a 5-day washout and instruments were repeated. Three functional magnetic resonance imaging scans with simultaneous sacral neuromodulation stimulation were performed below, at and above stimulus sensory threshold using a block design. This yielded brain activity maps represented by changes in blood oxygenation level dependence. A total of 5 stimulator off and 4 stimulator on cycles of 42 seconds each were imaged. Group analysis was done using a single voxel p value of 0.05 with a false-positive error of 0.05 on cluster analysis. RESULTS: Six of the 13 patients enrolled completed functional magnetic resonance imaging. Median age was 52 years (range 36 to 64). Urinary symptoms and voiding diary data worsened with washout. Overall brain activation generally progressed with increasing stimulation amplitude. However, activation of the right inferior frontal gyrus remained stable while deactivation of the pons and the periacqueductal gray matter only occurred with subsensory stimulation. Sensory stimulation activated the insula but deactivated the medial and superior parietal lobes. Suprasensory stimulation activated multiple structures and the expected S3 somatosensory region. All devices had normal impedance after functional magnetic resonance imaging. CONCLUSIONS: Functional magnetic resonance imaging confirmed that sacral neuromodulation influences brain activity in women with overactive bladder who responded to therapy. These changes varied with stimulus intensity.

A Chronic, Conscious Large Animal Platform to Quantify Therapeutic Effects of Sacral Neuromodulation on Bladder Function.

PURPOSE: Sacral neuromodulation is a Food and Drug Administration approved therapy for urinary urge incontinence, urgency-frequency and fecal incontinence. Most preclinical studies have used anesthetized preparations in small animals. To expand the testing capabilities of sacral neuromodulation stimulation parameters and novel concepts we created a large animal model in fully conscious sheep. MATERIALS AND METHODS: Six adult female sheep were tested weekly using 10 trials of single fill cystometry, similar to clinical urodynamics. Maximal bladder capacity was measured without (trials 1 to 5) and with (trials 6 to 10) sacral neuromodulation. A mixed effects regression model was used to analyze the effect of sacral neuromodulation on bladder capacity. RESULTS: Acute sacral neuromodulation significantly increased bladder capacity in conscious female sheep from 75.2 to 118.7 ml, an almost 60% increase. This was not simply an effect of repeat cystometric trials since testing without sacral neuromodulation was not associated with an increase in bladder capacity. CONCLUSIONS: These data demonstrate the effects of acute sacral neuromodulation on bladder capacity in the conscious sheep. This model represents a useful testing platform for novel sacral neuromodulation concepts such as alternate methods and parameters of therapy delivery.

Differential modulation of neurons in the rostral ventromedial medulla by neurokinin-1 receptors.

The rostral ventromedial medulla (RVM) is part of descending circuitry that modulates nociceptive processing at the level of the spinal cord. RVM output can facilitate pain transmission under certain conditions such as inflammation, and thereby contribute to hyperalgesia. Evidence suggests that substance P and activation of neurokinin-1 (NK-1) receptors in the RVM are involved in descending facilitation of nociception. We showed previously that injection of NK-1 receptor antagonists into the RVM attenuated mechanical and heat hyperalgesia produced by intraplantar injection of capsaicin. Furthermore, intraplantar injection of capsaicin excited ON cells in the RVM and inhibited ongoing activity of OFF cells. In the present studies, we therefore examined changes in responses of RVM neurons to mechanical and heat stimuli after intraplantar injection of capsaicin and determined the role of NK-1 receptors by injecting a NK-1 receptor antagonist into the RVM prior to capsaicin. After capsaicin injection, excitatory responses of ON cells and inhibitory responses of OFF cells evoked by mechanical and heat stimuli applied to the injected, but not contralateral, paw were increased. Injection of the NK-1 antagonist L-733,060 did not alter evoked responses of ON or OFF cells but attenuated the capsaicin-evoked enhanced responses of ON cells to mechanical and heat stimuli with less of an effect on the enhanced inhibitory responses of OFF cells. These data support the notion that descending facilitation from RVM contributes to hyperalgesia and that NK-1 receptors, presumably located on ON cells, play an important role in initiating descending facilitation of nociceptive transmission.

Excitation of cutaneous C nociceptors by intraplantar administration of anandamide.

Anandamide has been characterized as both an endocannabinoid and endovanilloid. Consistent with its actions as an endovanilloid, previous studies have demonstrated that anandamide can excite primary sensory neurons in vitro via transient receptor potential vanilloid type one (TRPV1) receptors. In the present study, we sought to determine if anandamide excited cutaneous C nociceptors in vivo and if this excitation correlated with nocifensive behaviors. Using teased-fiber electrophysiological methods in the rat, C nociceptors isolated from the tibial nerve with receptive fields (RFs) on the plantar surface of the hindpaw were studied. Injection of anandamide into the RF dose-dependently excited nociceptors at doses of 10 and 100 microg. The TRPV1 receptor antagonists, capsazepine or SB 366791, were applied to the RF to determine if excitation by anandamide was mediated through TRPV1 receptors. Intraplantar injection of either capsazepine (10 microg) or SB 366791 (3 microg) attenuated the excitation produced by 100 microg anandamide. We also determined whether excitation of C nociceptors by anandamide was associated with nocifensive behaviors. Intraplantar injection of 100 microg anandamide produced nocifensive behaviors that were attenuated by pre-treatment with either capsazepine or SB 366791. Furthermore, we determined if intraplantar injection of anandamide altered withdrawal responses to radiant heat. Neither intraplantar injection of anandamide nor vehicle produced antinociception or hyperalgesia to radiant heat. Our results indicate that anandamide excited cutaneous C nociceptors and produced nocifensive behaviors via activation of TRPV1 receptors.

Cannabinoid modulation of cutaneous Adelta nociceptors during inflammation.

Previous studies have demonstrated that locally administered cannabinoids attenuate allodynia and hyperalgesia through activation of peripheral cannabinoid receptors (CB(1) and CB(2)). However, it is currently unknown if cannabinoids alter the response properties of nociceptors. In the present study, correlative behavioral and in vivo electrophysiological studies were conducted to determine if peripheral administration of the cannabinoid receptor agonists arachidonyl-2'-chloroethylamide (ACEA) or (R)-(+)-methanandamide (methAEA) could attenuate mechanical allodynia and hyperalgesia, and decrease mechanically evoked responses of Adelta nociceptors. Twenty-four hours after intraplantar injection of complete Freund's adjuvant (CFA), rats exhibited allodynia (decrease in paw withdrawal threshold) and hyperalgesia (increase in paw withdrawal frequency), which were attenuated by both ACEA and methAEA. The antinociceptive effects of these cannabinoids were blocked by co-administration with the CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophen yl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) but not with the CB(2) receptor antagonist 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-y l](4-methoxyphenyl)methanone (AM630). ACEA and methAEA did not produce antinociception under control, non-inflamed conditions 24 h after intraplantar injection of saline. In parallel studies, recordings were made from cutaneous Adelta nociceptors from inflamed or control, non-inflamed skin. Both ACEA and methAEA decreased responses evoked by mechanical stimulation of Adelta nociceptors from inflamed skin but not from non-inflamed skin, and this decrease was blocked by administration of the CB(1) receptor antagonist AM251. These results suggest that attenuation of mechanically evoked responses of Adelta nociceptors contributes to the behavioral antinociception produced by activation of peripheral CB(1) receptors during inflammation.

Activity of murine raphe magnus cells predicts tachypnea and on-going nociceptive responsiveness.

In rats, opioids produce analgesia in large part by their effects on two cell populations in the medullary raphe magnus (RM). To extend our mechanistic understanding of opioid analgesia to the genetically tractable mouse, we characterized behavioral reactions and RM neural responses to opioid administration. d-Ala(2), N-Me-Phe(4)-Gly(5)ol-enkephalin, a mu-opioid receptor agonist, microinjected into the murine RM produced cardiorespiratory depression and reduced slow wave electroencephalographic activity as well as increased the noxious heat-evoked withdrawal latencies. As in rat, RM cell types that were excited and inhibited by noxious stimuli, termed on and off cells, respectively, were observed in mice. However, in contrast to findings in rat, opioid doses that suppressed withdrawals did not alter the background discharge rate of murine on and off cells, suggesting that the cellular mechanisms by which the murine RM generates opioid analgesia are substantially different from those in rats. Murine on cell discharge did not predict the latency or magnitude of an ensuing withdrawal but did correlate to the magnitude and latency of concurrent withdrawals. Although opioids failed to alter the background discharge of on and off cells, they reduced the responses of RM neurons to noxious stimulation, further evidence that RM modulates on-going withdrawals. In characterizing the role of RM in respiratory modulation, we found that on cells burst and off cells paused during tachypneic events. The effects of opioids in the murine RM on homeostasis and the association of on and off cell discharge with tachypnea corroborate roles for opioid signaling in RM beyond analgesia.

Raphe magnus neurons help protect reactions to visceral pain from interruption by cutaneous pain.

Suppression of reactions to one noxious stimulus by a spatially distant noxious stimulus is termed heterotopic antinociception. In lightly anesthetized rats, a noxious visceral stimulus, colorectal distension (CRD), suppressed motor withdrawals but not blood pressure or heart rate changes evoked by noxious hindpaw heat. Microinjection of muscimol, a GABA(A) receptor agonist, into raphe magnus (RM) reduced CRD-evoked suppression of withdrawals, evidence that RM neurons contribute to this heterotopic antinociception. To understand how brain stem neurons contribute to heterotopic antinociception, RM neurons were recorded during CRD-elicited suppression of hindpaw withdrawals. Although subsets of RM neurons that were excited (on cells) or inhibited (off cells) by noxious cutaneous stimulation were either excited or inhibited by CRD, on cells were inhibited and off cells excited by an intracerebroventricularly administered opioid, evidence that the nociception-facilitating and -inhibiting functions of on and off cells, respectively, are predicted by the cellular response to noxious cutaneous stimulation alone and not by the response to CRD. When recorded during CRD-elicited antinociception, RM cell discharge resembled the pattern observed in response to CRD stimulation alone. However, when hindpaw withdrawal suppression was incomplete, RM cell discharge resembled the pattern observed in response to heat alone. We propose that on cells excited by CRD facilitate responses to CRD itself, which in turn augments excitation of off cells that then act to suppress cutaneous nociception. RM cells may thereby contribute to the dominance of quiet recuperative reactions evoked by potentially life-threatening visceral stimuli over transient somatomotor activity elicited by less-injurious noxious cutaneous stimuli.

Use-dependent inhibition of P2X3 receptors by nanomolar agonist.

P2X3 receptors desensitize within 100 ms of channel activation, yet recovery from desensitization requires several minutes. The molecular basis for this slow rate of recovery is unknown. We designed experiments to test the hypothesis that this slow recovery is attributable to the high affinity (< 1 nM) of desensitized P2X3 receptors for agonist. We found that agonist binding to the desensitized state provided a mechanism for potent inhibition of P2X3 current. Sustained applications of 0.5 nM ATP inhibited > 50% of current to repetitive applications of P2X3 agonist. Inhibition occurred at 1000-fold lower agonist concentrations than required for channel activation and showed strong use dependence. No inhibition occurred without previous activation and desensitization. Our data are consistent with a model whereby inhibition of P2X3 by nanomolar [agonist] occurs by the rebinding of agonist to desensitized channels before recovery from desensitization. For several ATP analogs, the concentration required to inhibit P2X3 current inversely correlated with the rate of recovery from desensitization. This indicates that the affinity of the desensitized state and recovery rate primarily depend on the rate of agonist unbinding. Consistent with this hypothesis, unbinding of [32P]ATP from desensitized P2X3 receptors mirrored the rate of recovery from desensitization. As expected, disruption of agonist binding by site-directed mutagenesis increased the IC50 for inhibition and increased the rate of recovery.

Roles for pain modulatory cells during micturition and continence.

We studied how the nervous system selects between noxious stimulus-evoked withdrawals and micturition, movements that are necessary for survival but use overlapping muscles and therefore cannot occur simultaneously. In lightly anesthetized rats, micturition was favored, because noxious stimulation never interrupted micturition, whereas withdrawals were suppressed during voiding. Neurons in the ventromedial medulla (VMM) are a major source of descending antinociceptive signals. To test whether VMM neurons support withdrawal suppression during micturition, the discharge of VMM neurons was recorded during continence and micturition. VMM cells that were inhibited (M-inh) or excited (M-exc) during micturition were observed. M-inh cells were excited by noxious cutaneous stimulation and thus are likely nociception facilitating, whereas M-exc cells were inhibited by noxious heat and are likely nociception inhibiting. The excitation of nociception-inhibiting M-exc and inhibition of nociception-facilitating M-inh cells predicts suppression of withdrawals during micturition. M-exc cells were typically silent before micturition, whereas most M-inh cells fired before micturition, suggesting that these cells may also play a preparatory role for micturition. To test this idea, we examined manipulations that either advanced or delayed the onset of micturition. Hypothalamic stimulation and noxious paw heat advanced micturition while exciting M-inh cells and inhibiting M-exc cells. In contrast, colorectal distension, a stimulus that delays micturition, inhibited M-inh cells and excited M-exc cells. These results suggest a model in which, during continence, VMM M-inh cells facilitate and M-exc cells inhibit bladder afferents, advancing micturition onset when M-inh cells are activated and delaying onset when M-exc cells are activated.

Role for raphe magnus neuronal responses in the behavioral reactions to colorectal distension.

The brain stem is necessary for the expression of behavioral reactions to noxious visceral inputs. Neurons in raphe magnus (RM) and the adjacent nucleus reticularis magnocellularis (NRMC) respond to visceral stimuli and can facilitate the behavioral reaction to visceral stimulation. To determine which RM and NRMC cells could play a role in generating the reaction to colorectal distension (CRD), the responses of RM and NRMC cells to multiple intensities of CRD were compared with simultaneously evoked cardiovascular and visceromotor reactions in halothane-anesthetized rats. Most neurons (89%) responded to CRD with one of three basic response patterns. For cells with a graded response pattern, the response magnitude increased with increasing stimulation intensity. For flat responding cells, the response magnitude was not different across suprathreshold stimulation intensities. Finally, neurons with a switch response pattern responded to low- and high-intensity CRD in opposing directions. Cells were either inhibited or excited by CRD in each of these categories. Responses of cells with both graded and switch response patterns were significantly correlated with CRD-evoked tachycardia, pressor reaction, and hunching. The activity of graded-responding cells have the greatest predictive value for CRD-evoked reactions. Flat-responding cells have nonlinear responses that may augment reactions to stimuli above the noxious threshold. Cells with switch type response patterns may contribute to differential reactions evoked by CRD stimuli within the noxious range. In sum, RM and NRMC neurons respond to CRD with a variety of patterns, each of which may contribute to the sculpting of CRD reactions in different ways.

Raphe magnus neurons respond to noxious colorectal distension.

Physiological studies of neurons in raphe magnus (RM) and the adjacent nucleus reticularis magnocellularis (NRMC) have demonstrated that the response to noxious cutaneous stimulation predicts the response to opioid administration and therefore a cell's functional role in nociceptive modulation. Although visceral stimulation, like opioids, elicits antinociception, little is known about how RM and NRMC cells respond to visceral stimulation. Therefore RM and NRMC cells were tested for their responses to both colorectal distension (CRD) and noxious cutaneous heat in halothane-anesthetized rats. Less than a third of serotonergic cells responded to CRD with small increases or decreases in discharge rate. In contrast, almost two-thirds of nonserotonergic cells responded to CRD stimulation with either excitatory (35%) or inhibitory (30%) responses to CRD. The response to heat did not predict the response to CRD with nearly equal proportions of heat-excited, -inhibited, and -unaffected cells being excited, inhibited, or unaffected by CRD. The dissociation between the responses to cutaneous heat and CRD demonstrates that cell classes based on the response to noxious heat are not homogeneous and may play multiple functional roles.