Clinical Neurophysiology xxx (2014) xxx–xxx
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Bladder filling attenuates spinal cord nociceptive reflexes in humans Mariano Serrao a,⇑, Francesca Cortese a, Gaia Fragiotta a, Antonio Luigi Pastore b, Giovanni Palleschi b, Gianluca Coppola c, Antonio Carbone b, Francesco Pierelli d a
Unit of Neurorehabilitation, Department of Medical-Surgical Sciences and Biotechnologies, Sapienza University of Rome, ICOT, Latina, Italy Unit of Urology, Department of Medical-Surgical Sciences and Biotechnologies, Sapienza University of Rome, ICOT, Latina, Italy c G.B. Bietti Eye Foundation-IRCCS, Department of Neurophysiology of Vision and Neurophthalmology, Rome, Italy d IRCCS Neuromed, Pozzilli, IS, Italy b
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Article history: Accepted 12 March 2014 Available online xxxx Keywords: Nociceptive withdrawal reflex Bladder filling Nociception Viscero-somatic interaction Descending control
h i g h l i g h t s The effect of bladder filling on the nociceptive withdrawal reflex (NWR) was evaluated in healthy sub-
jects to examine the viscerosomatic interaction between bladder afferents and somatic nociception. The NWR was reduced in both upper and lower limbs during bladder filling which strongly indicates
that bladder control and nociception share common modulatory descending pathways. The effect of bladder filling on the NWR may represent a useful tool to investigate interactions
between the neural pathways controlling the bladder and pain.
a b s t r a c t Objective: To examine the viscerosomatic interaction between bladder afferents and somatic nociception we evaluated the effect of bladder filling on the nociceptive withdrawal reflex (NWR) in 21 healthy subjects. Methods: NWR was evoked in the lower and upper limbs by stimulating the sural and index finger digital nerves, respectively, while simultaneously recording EMG activity in the biceps femoris and biceps brachialis. NWR pain-related perception was quantified on a 10-point pain scale. Bladder filling was evaluated with suprapubic bladder sonography. Subjects were examined during empty bladder, medium and high level of bladder filling sessions. Results: NWR magnitude in both upper and lower limbs and perceived pain for the upper limb were significantly decreased at higher levels of bladder filling compared to empty bladder sessions. Conclusions: Reduced NWR magnitude in both upper and lower limbs during bladder filling strongly indicates that bladder control and nociception share common modulatory descending pathways. Bladder afferents may activate these pathways to suppress the micturition reflex, but they may also inhibit spinal reflexes to maintain continence during pain stimuli. Significance: The effect of bladder filling on the NWR may represent a useful tool to investigate interactions between the neural pathways controlling the bladder and pain. Ó 2014 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Viscerosomatic convergence of nociceptive information is a neural phenomenon well documented in experimental studies ⇑ Corresponding author. Address: Department of Medical-Surgical Sciences and Biotechnologies, Sapienza University of Rome, ICOT Latina, Via Faggiana 1668, 40100 Latina, Italy. Tel.: +39 07736513337; fax: +39 06 44246965. E-mail address: [email protected] (M. Serrao).
and is considered the primary mechanism underlying referred pain (Sengupta, 2009). Bladder function and pain modulation share peripheral and central neural pathways, including the A-delta and C fibres, spino-thalamic tract, periaqueductal grey (PAG), nucleus raphe magnum (NRM), and nucleus locus coeruleus (LC) (Birder et al., 2010), and use common neurotransmitters such as serotonin and norepinephrine. These common mechanisms suggest a certain degree of overlap between these systems (Mennini and Testa, 2010). A deeper knowledge on the nature of the neural
http://dx.doi.org/10.1016/j.clinph.2014.03.014 1388-2457/Ó 2014 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Serrao M et al. Bladder filling attenuates spinal cord nociceptive reflexes in humans. Clin Neurophysiol (2014), http:// dx.doi.org/10.1016/j.clinph.2014.03.014
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interactions throughout the CNS between pathways from the bladder and pain pathways may shed light on the physiological mechanisms that could be exploited to improve bladder function and pain. Early studies (Evans and McPherson, 1958, 1959) demonstrated that high-pressure bladder distension induced an inhibition of the polysynaptic flexor reflexes in anaesthetized and decerebrated but not in spinalized cats suggesting an activation by bladder nociceptors of the descending inhibitory pathways (e.g. from reticular formation neurons) on the spinal interneurons. Furthermore, the authors found an increase of reflexes excitability in spinalized cats probably due to the unmasking of the segmental spinal excitatory mechanism. Although, so far, no direct evidence has been provided regarding the effect of bladder afferent stimulation on somatic pain in humans, some studies have examined the interaction between gastric and rectal afferents and spinal nociception. Bouhassira et al. (1994) tested the effects of different levels of gastric distension on the lower limb nociceptive flexion reflex in healthy subjects and demonstrated that gastric distension inhibits the reflex. Using a similar approach, these authors (Bouhassira et al., 1998) tested the effects of rectal distension on nociceptive flexion reflex excitability depending on the mode of distension (rapid or slow ramp). They found that rapid distension induced both facilitatory and inhibitory effects on the reflex magnitude, whereas slow-ramp rectal distension produced a volume-dependent depression similar to that observed during gastric distension. To explain these results, the authors hypothesised a convergence of visceral and nociceptive reflex afferents at the same levels of the sacral spinal cord inducing an earlier reflex facilitation in addition to a later supraspinallymediated inhibition. In the present study, we tested the effect of different levels of bladder filling on the nociceptive withdrawal reflex (NWR) in healthy subjects to explore the viscerosomatic interaction between bladder afferents and somatic nociception. The NWR is one of the most reliable tools for assessing treatment efficacy and spinal nociception in humans (Cruccu et al., 2004; Sandrini et al., 2005; Andersen, 2007) and is widely influenced by the descending pathways that modulate pain (Ness and Gebhart, 1990). Thus, we used the NWR to examine how the segmental and suprasegmental descending pathways that receive bladder inputs influence spinal nociceptive processes in humans. In order to better discern descending from the segmental effects, we investigated the NWR changes not only in the lower limb, where a mixed segmental/ descending effect influence might be invoked, but also in the upper limb, where descending modulation is a more likely mechanism although non-segmental propriospinal mechanisms cannot be ruled out (Gerhart et al., 1981; Cadden et al., 1983). 2. Methods 2.1. Participants Twenty-one healthy volunteers (mean age: 28.1 ± 5.9 years; gender: 9 men, 12 women) were enrolled in the study and gave their written informed consent to participate in the study. The study was approved by the local ethics committee and complied with the Helsinki Declaration. All participants reported no previous bladder problems or pain disorders, and were not taking medications on the date of the study enrolment. 2.2. NWR induction To elicit the lower limb NWR, the sural nerve was stimulated percutaneously using a pair of disposable surface Ag/AgCl
electrodes (Medelec, Oxford, UK; diameter 1 cm, 2 cm inter-electrode distance) positioned immediately below (+) and behind ( ) the lateral malleolus. To elicit the upper limb NWR, the digital nerve was stimulated through a pair of surface electrodes placed on the skin of the index fingertip ( ) and the proximal phalanx (+). The stimulus consisted of 25-ms trains of 5 rectangular pulses (1 ms duration, 200 Hz frequency) delivered through an electrical stimulator (Digitimer DS7A, UK) that were triggered by a data acquisition and analysis interface (CED Power 1401, Cambridge Electronic Design, UK). The EMG signals were amplified with a Digitimer D360 amplifier (band-pass 10-2000 Hz–2000 Hz, Gain 1000). Reflex threshold (RT) was assessed using a staircase method (Willer, 1977) and consisted of 3 series of ascending and descending intensity stimuli. The RT was defined as the minimum stimulation intensity at which it was possible to evoke a stable reflex response in at least 3 out of 5 trials. The intensity of the electrical shocks used for the electrophysiological measurements was then adjusted to 1.5 RT. The subjects were asked to score their pain perception after each stimulus on a 0–10-point numerical rating scale (NRS) anchored by ‘‘no pain’’ (score of 0) and ‘‘worst imaginable pain’’ (score of 10). The electrical stimuli were delivered at varied intervals of at least 40 s to avoid reflex habituation. Electromyographic (EMG) activity was recorded through a pair of Ag/AgCl surface electrodes (Medelec, Oxford, UK; diameter 1 cm, 2 cm inter-electrode distance) positioned over the belly of the biceps femoris in the lower limb and biceps brachialis in the upper limb. 2.3. Bladder filling technique With small sips, participants gradually drank 1 litre of water. They were instructed by the investigator to hold their urine until instructed otherwise. Suprapubic ultrasound (Aloka model: SSD1400 with 3.5 MHz convex probe) evaluation was performed to measure the bladder filling level just after the participants had voided (no voiding desire), and the instances when participants reported a moderate or strong desire of voiding. 2.4. Procedure Before starting formal measurements, the subjects underwent an initial training session to familiarise them with the electrical stimulation procedure and pain intensity ratings, and to reduce any effects due to arousal and/or anxiety. The experimental setup is presented in Fig. 1. In order to avoid possible RT variations due to menstrual cycle or gastric or rectal distensions (Sandrini et al., 2005), the NWR measurements were always performed during the follicular phase in women, at least 2 h after meals and without a desire to evacuate. During electrophysiological recordings, subjects were seated in a comfortable armchair in a quiet room where the temperature was kept at a constant level (23 ± 2 °C). Their lower limbs were positioned to ensure complete muscle relaxation, with the knee flexed at 130° and the ankle at 90°. The upper limb was resting on a horizontal support, slightly flexed at the elbow, the wrist was in line with the forearm, which was situated in the supine position, and the fingers were spontaneously flexed to achieve comfortable muscle relaxation. Neurophysiological data were recorded from subjects in the following 3 sessions: (i) no voiding desire (empty bladder); (ii) moderate voiding desire (medium level of bladder filling); (iii) strong voiding desire (high level of bladder filling). In all subjects, the first session represented the baseline, whereas the conditions for the second and third sessions were chosen randomly.
Please cite this article in press as: Serrao M et al. Bladder filling attenuates spinal cord nociceptive reflexes in humans. Clin Neurophysiol (2014), http:// dx.doi.org/10.1016/j.clinph.2014.03.014
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Fig. 1. Scheme of the experimental design.
Participants were asked to void so that they emptied their bladder completely, and a suprapubic bladder ultrasound was performed to ascertain that the bladder was empty. Afterwards they were invited to sit in the recording position (described above) to undergo baseline measurements (Fig. 1). Thereafter volunteers were invited to drink water until the desire changed from no desire to either moderate or strong voiding desire. At that time, participants underwent bladder ultrasound to measure the bladder filling level before the neurophysiological recordings. 2.5. Data analysis The EMG recordings were rectified, band-pass filtered (10–500 Hz, second order), and sampled at 2 kHz. The root mean square (RMS) amplitude in pre- and post-stimulus time windows (100 ms before and 80–180 ms after) was calculated. The EMG reflex activity was expressed as the difference between post- and pre-stimulus RMS. Reflex magnitude was calculated as the mean RMS of 5 trials. 2.6. Statistical analysis The Statistical Package for the Social Sciences (SPSS) ver. 15 (SPSS, Chicago, IL) was used for all statistical analyses. Two-way repeated measures ANOVA was performed to evaluate the effect of bladder filling (three-level within-subject factor) and gender (two-level between-factor) on the NWR magnitude and NRS scores after having performed the Kolmogorov–Smirnov test for normal distribution. A two-way repeated measures ANOVA was performed to also evaluate the interaction effect of bladder filling/limbs on pain perception (NRS). Greenhouse–Geisser correction was used, when necessary, to circumvent violations of sphericity (i.e. inequalities in the variance of the differences between factors; Greenhouse and Geisser, 1959). The Bonferroni adjustment for multiple comparisons was used for pairwise post hoc analyses. The results for the NWR magnitude are presented as mean values ± standard error of the mean (SEM). 3. Results For all subjects, cystometric capacities were in the normal range. After randomization, 11 subjects performed the strong voiding desire session as first and the moderate desire session as second, and thus 10 subjects performed the moderate session as first and the strong session as second. In 2 subjects, the NWR could not be evoked the upper limb, thus we analysed upper limb NWR data in 19 subjects. Mean values of bladder filling, NWR thresholds and magnitudes, and NRS scores recorded at different voiding desires are reported in Table 1. NWR magnitude changes during bladder filling sessions in a representative subject are shown in Fig. 2.
Table 1 Mean values of bladder filling, NWR thresholds and magnitudes, and NRS scores at different voiding desires. Parameters
Experimental sessions No desire
Moderate desire
Strong desire
Bladder filling volumes
0 ml
202.4 ± 10.6 ml
569.2 ± 12.3 ml
Lower limbs NWR-Th (mA) NWR magnitude (RMS, uV) NRS scores
14.5 ± 1.5 11.7 ± 3.1 6.1 ± 0.5
6.5 ± 2.0 5.8 ± 0.6
4.3 ± 1.2 5.9 ± 0.6
Upper limbs NWR-Th (mA) NWR magnitude (RMS, uV) NRS scores
3.0 ± 0.6 13.4 ± 2.7 6.4 ± 0.4
6.4 ± 1.6 5.8 ± 0.4
2.9 ± 1.5 5.1 ± 0.4
A significant effect of bladder filling on lower limb NWR magnitude was found (main effect, F(1.271,24.153) = 7.501, p = 0.008). Pairwise comparisons revealed higher values in the no desire session than in the moderate and strong voiding desire sessions (Fig. 3). No significant differences were revealed between moderate and strong voiding desire sessions. No significant effect for bladder filling (main effect, F(2,38) = 0.425, p = 0.652) on the lower limb NRS scores was observed. A significant effect of bladder filling on the upper limb NWR magnitude was found (main effect, F(1.356,23.052) = 13.488, p = 0.001). Pairwise comparisons revealed higher values in the no desire session than in both moderate and strong voiding desire sessions, and higher values in the moderate than the strong voiding desire sessions (Fig. 3). A significant effect of bladder filling on the upper limb NRS scores was observed (main effect, F(2,34) = 18.025, p < 0.0005). Pairwise comparisons revealed higher values in the no desire session than in both moderate and strong desires sessions, as well as higher values in the moderate than in the strong voiding desire sessions (Fig. 2). No significant effect for gender and bladder filling/gender interaction was found in either the lower or upper NWR magnitudes, or NRS scores (all, p > 0.05). A significant effect of bladder filling/ limbs interaction was found on NRS scores (main effect, F(2,76) = 8.972, p < 0.0005). Post-hoc analysis revealed significant lower values in upper than in lower limbs during strong desire voiding session (p = 0.043), whereas no other significant differences were found between limbs (all, p > 0.05).
4. Discussion The main result of the present study is that bladder distension inhibited the NWR magnitude in both the upper and lower limbs. In general, the observed reduction in the NWR magnitude during
Please cite this article in press as: Serrao M et al. Bladder filling attenuates spinal cord nociceptive reflexes in humans. Clin Neurophysiol (2014), http:// dx.doi.org/10.1016/j.clinph.2014.03.014
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Fig. 2. NWR magnitude changes during bladder filling sessions in a representative subject.
gradual bladder filling is likely to be the result of a reduced excitability of the spinal cord neurons mediating the NWR. Although a direct segmental reflex-mediated spinal inhibition may potentially occur at the sacral level, the reduction in the NWR magnitude in both upper and lower limbs during bladder filling suggests that the inhibitory effect is mainly mediated by the supraspinal descending pathways. Although our experimental results give no direct indication on which structure might be involved in such inhibition, some indirect evidences may be extrapolated from experiment in animal models and neuroimaging in humans (Blok and Holstege, 1999; Rocha et al., 2001; Fowler and Griffiths, 2010). It is known that when the bladder is storing urine its afferents activate PAG neurons (Holstege, 2010). The ventrolateral PAG triggers micturition in response to bladder afferent inputs through its direct projections to the pontine micturition centre (PMC) located in the caudal pontine tegmentum (Benarroch, 2010). The PMC sends descending projections to the sacral spinal cord via the lateral funiculus. This pathway activates the sacral preganglionic neurons and inhibits, via local interneurons, both the thoracolumbar sympathetic neurons and Onuf nucleus motoneurons innervating the external urethral sphincter (Blok and Holstege, 1999; Sugaya et al., 2005). When the bladder is in storage mode, the PAG receives afferent inputs from the viscera and in turn relays them to the hypothalamus, right insula, dorsal anterior cingulate and lateral prefrontal cortex. These areas then convey this information to the medial prefrontal cortex, which exerts a tonic inhibition on the PAG. If the decision is not to void, the medial prefrontal cortex would maintain a tonic inhibition on PAG; if the decision is to void, withdrawal of this inhibitory control would lead to activation of the PMC (Fowler et al., 2008). Interestingly, PAG has been demonstrated to be involved in spinal nociception. Descending control arises from a number of supraspinal sites, including the midline periaqueductal grey-rostral ventromedial medulla (PAG-RVM) system. Inhibitory control from the PAG-RVM system preferentially suppresses nociceptive inputs mediated by nociceptive A-delta and C fibres (Fields et al., 2006). The PAG projects to the RVM, which in turn sends its outputs to dorsal horn laminae neurons involved in nociception, particularly the WDR neurons which are known to be involved in the polysynaptic nociceptive reflexes (Yang et al., 2001; Sandrini et al., 2005). In this light, the bladder afferents may inhibit spinal reflexes by stimulating the RVM and thus reducing dorsal horn neuron activity. This may be a safety mechanism to maintain continence during painful stimuli and pain-related motor responses that may increase intra-abdominal pressure. In addition to the PAG, other brainstem modulatory descending systems known to produce analgesia, including DNIC (Diffuse Noxious Inhibitory Controls), may be triggered by bladder afferents.
Diffuse noxious inhibitory controls (DNIC) represents a phenomenon in which, via spino-bulbo-spinal loops, the activity of wide dynamic range neurons in the spinal dorsal horn and in trigeminal nuclei is attenuated in response to noxious stimuli applied to a remote area of the body (Villanueva and Le Bars, 1995). Interestingly, it was demonstrated in animal models that PAG can modulate DNIC to produce analgesic-like effects (Dickenson and Le Bars, 1983; Bouhassira et al., 1992; Bajic and Proudfit, 1999). In rats, distension of abdominal viscera (the colon and urinary bladder) depressed activity of somatic lumbar dorsal horn neurons possibly by DNIC activation (Cadden and Morrison, 1991). Bouhassira and colleagues (Bouhassira et al., 1992, 1998) speculated DNIC-induced NWR depression to be a consequence of rectal and gastric distension in humans. It has been suggested that DNIC may modulate NWR and pain in several cognitive and physiological states (i.e. stress, anxiety, sleep, respiration, menstrual cycle, etc.) (reviewed by Sandrini et al. (2005)). More recently, the term ‘‘conditioned pain modulation’’ (CPM) has been coined for the psychophysical protocols that explore DNIC in humans (Yarnitsky et al., 2010). In this light, the CPM system may represent the final common output pathway to brainstem and spinal cord nociceptive neurons, which serves to regulate and adapt pain thresholds in cognitive and homeostatic contexts. In the present study, we found a different inhibitory effect of bladder distension on pain perception in the upper vs lower limbs. Particularly, while a significant decrease in the upper limb NRS score was observed, no significant changes were observed in the lower limb NRS scores. One plausible explanation for such a finding may be a convergence of excitatory and inhibitory effects in the somatic sacral spinal cord neurons mediating the flexion reflexes as demonstrated in previous animal studies (Ness and Gebhart, 1987; Shefchyk, 2001; Peles et al., 2004). It is known that afferents from the bladder travel via the pelvic and hypogastric nerves and terminate in the superficial dorsal horn (Birder et al., 2010) from which inputs are conveyed to the supraspinal structures via the spino-thalamic tract. Wide dynamic range neurons, located in laminae II and V, modulate the NWR and receive afferents conveying multimodal painful and non-painful information from both somatic and visceral tissues and organs (Cervero, 1995; Laird et al., 1995; Drewes et al., 2003). A spatio-temporal summation of bladder inputs at this level, during gradual bladder filling, may explain the different voiding sensations related to the different levels of bladder distension, which range from normal voiding sensation to a clear pain perception (Xu and Gebhart, 2007; De Groat and Yoshimura, 2009; Kanai and Andersson, 2010). We speculated that the mechanosensitive inputs related to bladder distension might interfere with somatic pain processing mechanisms at both sacral spinal and supraspinal
Please cite this article in press as: Serrao M et al. Bladder filling attenuates spinal cord nociceptive reflexes in humans. Clin Neurophysiol (2014), http:// dx.doi.org/10.1016/j.clinph.2014.03.014
M. Serrao et al. / Clinical Neurophysiology xxx (2014) xxx–xxx
Fig. 3. NWR magnitude and NRS scores during bladder filling sessions. ⁄p < 0.05,
levels, inducing a segmental-mediated facilitation followed by a supraspinal-mediated inhibition of the NWR pain-related perception. This effect would also explain the lack of significant differences in the lower limb NWR magnitude between moderate and strong voiding desire sessions (Fig. 3). A potential bias induced by the order sequence of the experimental sessions may be present and limits the results of our study. Although the moderate and strong voiding desire sessions were randomly performed, the no desire session was always performed first. However, the trend for a progressive reflex magnitude reduction from an empty to high level of bladder filling trials (Fig. 3) gives us a reasonable degree of certainty that the NWR inhibition effect was related to the bladder filling. Our experimental design did not include a ‘‘post-control’’ session which could have been done at the end of the experiment after the subjects had voided. However, control session should have done at different time intervals from the bladder emptying due to the descending inhibitory after effect on the spinal neurons. Indeed, in previous studies it has been demonstrated that the NWR remains inhibited for a variable time period after the end of the cold pressor test (Piché et al., 2009). In animal models, a long lasting post-effects directly related to the duration of conditioning painful stimulus has been reported (Le Bars et al., 1979). In this regard, an ‘‘ad hoc’’ experiment, investigating on the after-effect recovery curve of the NWR should be performed. Further studies are warranted to investigate on this aspect. In summary, our study indicates that bladder filling activates descending pathways that inhibit nociception at the spinal level.
5
⁄⁄
p < 0.01 at post hoc analysis.
A central role for the PAG as the main centre of functional connectivity between bladder afferents during storage and nociception control may be hypothesised. The effect of bladder filling on NWR may represent a useful tool to investigate the interaction between bladder and pain pathways. It would be interesting to evaluate the bladder-triggered NWR inhibition in either somatic (i.e. low back pain) or visceral (i.e. bladder pain and irritable bowel pain) chronic pain syndromes and/or their pharmacological effect.
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Clinical Neurophysiology journal homepage: www.elsevier.com/locate/clinph
Bladder filling attenuates spinal cord nociceptive reflexes in humans Mariano Serrao a,⇑, Francesca Cortese a, Gaia Fragiotta a, Antonio Luigi Pastore b, Giovanni Palleschi b, Gianluca Coppola c, Antonio Carbone b, Francesco Pierelli d a
Unit of Neurorehabilitation, Department of Medical-Surgical Sciences and Biotechnologies, Sapienza University of Rome, ICOT, Latina, Italy Unit of Urology, Department of Medical-Surgical Sciences and Biotechnologies, Sapienza University of Rome, ICOT, Latina, Italy c G.B. Bietti Eye Foundation-IRCCS, Department of Neurophysiology of Vision and Neurophthalmology, Rome, Italy d IRCCS Neuromed, Pozzilli, IS, Italy b
a r t i c l e
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Article history: Accepted 12 March 2014 Available online xxxx Keywords: Nociceptive withdrawal reflex Bladder filling Nociception Viscero-somatic interaction Descending control
h i g h l i g h t s The effect of bladder filling on the nociceptive withdrawal reflex (NWR) was evaluated in healthy sub-
jects to examine the viscerosomatic interaction between bladder afferents and somatic nociception. The NWR was reduced in both upper and lower limbs during bladder filling which strongly indicates
that bladder control and nociception share common modulatory descending pathways. The effect of bladder filling on the NWR may represent a useful tool to investigate interactions
between the neural pathways controlling the bladder and pain.
a b s t r a c t Objective: To examine the viscerosomatic interaction between bladder afferents and somatic nociception we evaluated the effect of bladder filling on the nociceptive withdrawal reflex (NWR) in 21 healthy subjects. Methods: NWR was evoked in the lower and upper limbs by stimulating the sural and index finger digital nerves, respectively, while simultaneously recording EMG activity in the biceps femoris and biceps brachialis. NWR pain-related perception was quantified on a 10-point pain scale. Bladder filling was evaluated with suprapubic bladder sonography. Subjects were examined during empty bladder, medium and high level of bladder filling sessions. Results: NWR magnitude in both upper and lower limbs and perceived pain for the upper limb were significantly decreased at higher levels of bladder filling compared to empty bladder sessions. Conclusions: Reduced NWR magnitude in both upper and lower limbs during bladder filling strongly indicates that bladder control and nociception share common modulatory descending pathways. Bladder afferents may activate these pathways to suppress the micturition reflex, but they may also inhibit spinal reflexes to maintain continence during pain stimuli. Significance: The effect of bladder filling on the NWR may represent a useful tool to investigate interactions between the neural pathways controlling the bladder and pain. Ó 2014 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
1. Introduction Viscerosomatic convergence of nociceptive information is a neural phenomenon well documented in experimental studies ⇑ Corresponding author. Address: Department of Medical-Surgical Sciences and Biotechnologies, Sapienza University of Rome, ICOT Latina, Via Faggiana 1668, 40100 Latina, Italy. Tel.: +39 07736513337; fax: +39 06 44246965. E-mail address: [email protected] (M. Serrao).
and is considered the primary mechanism underlying referred pain (Sengupta, 2009). Bladder function and pain modulation share peripheral and central neural pathways, including the A-delta and C fibres, spino-thalamic tract, periaqueductal grey (PAG), nucleus raphe magnum (NRM), and nucleus locus coeruleus (LC) (Birder et al., 2010), and use common neurotransmitters such as serotonin and norepinephrine. These common mechanisms suggest a certain degree of overlap between these systems (Mennini and Testa, 2010). A deeper knowledge on the nature of the neural
http://dx.doi.org/10.1016/j.clinph.2014.03.014 1388-2457/Ó 2014 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Serrao M et al. Bladder filling attenuates spinal cord nociceptive reflexes in humans. Clin Neurophysiol (2014), http:// dx.doi.org/10.1016/j.clinph.2014.03.014
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interactions throughout the CNS between pathways from the bladder and pain pathways may shed light on the physiological mechanisms that could be exploited to improve bladder function and pain. Early studies (Evans and McPherson, 1958, 1959) demonstrated that high-pressure bladder distension induced an inhibition of the polysynaptic flexor reflexes in anaesthetized and decerebrated but not in spinalized cats suggesting an activation by bladder nociceptors of the descending inhibitory pathways (e.g. from reticular formation neurons) on the spinal interneurons. Furthermore, the authors found an increase of reflexes excitability in spinalized cats probably due to the unmasking of the segmental spinal excitatory mechanism. Although, so far, no direct evidence has been provided regarding the effect of bladder afferent stimulation on somatic pain in humans, some studies have examined the interaction between gastric and rectal afferents and spinal nociception. Bouhassira et al. (1994) tested the effects of different levels of gastric distension on the lower limb nociceptive flexion reflex in healthy subjects and demonstrated that gastric distension inhibits the reflex. Using a similar approach, these authors (Bouhassira et al., 1998) tested the effects of rectal distension on nociceptive flexion reflex excitability depending on the mode of distension (rapid or slow ramp). They found that rapid distension induced both facilitatory and inhibitory effects on the reflex magnitude, whereas slow-ramp rectal distension produced a volume-dependent depression similar to that observed during gastric distension. To explain these results, the authors hypothesised a convergence of visceral and nociceptive reflex afferents at the same levels of the sacral spinal cord inducing an earlier reflex facilitation in addition to a later supraspinallymediated inhibition. In the present study, we tested the effect of different levels of bladder filling on the nociceptive withdrawal reflex (NWR) in healthy subjects to explore the viscerosomatic interaction between bladder afferents and somatic nociception. The NWR is one of the most reliable tools for assessing treatment efficacy and spinal nociception in humans (Cruccu et al., 2004; Sandrini et al., 2005; Andersen, 2007) and is widely influenced by the descending pathways that modulate pain (Ness and Gebhart, 1990). Thus, we used the NWR to examine how the segmental and suprasegmental descending pathways that receive bladder inputs influence spinal nociceptive processes in humans. In order to better discern descending from the segmental effects, we investigated the NWR changes not only in the lower limb, where a mixed segmental/ descending effect influence might be invoked, but also in the upper limb, where descending modulation is a more likely mechanism although non-segmental propriospinal mechanisms cannot be ruled out (Gerhart et al., 1981; Cadden et al., 1983). 2. Methods 2.1. Participants Twenty-one healthy volunteers (mean age: 28.1 ± 5.9 years; gender: 9 men, 12 women) were enrolled in the study and gave their written informed consent to participate in the study. The study was approved by the local ethics committee and complied with the Helsinki Declaration. All participants reported no previous bladder problems or pain disorders, and were not taking medications on the date of the study enrolment. 2.2. NWR induction To elicit the lower limb NWR, the sural nerve was stimulated percutaneously using a pair of disposable surface Ag/AgCl
electrodes (Medelec, Oxford, UK; diameter 1 cm, 2 cm inter-electrode distance) positioned immediately below (+) and behind ( ) the lateral malleolus. To elicit the upper limb NWR, the digital nerve was stimulated through a pair of surface electrodes placed on the skin of the index fingertip ( ) and the proximal phalanx (+). The stimulus consisted of 25-ms trains of 5 rectangular pulses (1 ms duration, 200 Hz frequency) delivered through an electrical stimulator (Digitimer DS7A, UK) that were triggered by a data acquisition and analysis interface (CED Power 1401, Cambridge Electronic Design, UK). The EMG signals were amplified with a Digitimer D360 amplifier (band-pass 10-2000 Hz–2000 Hz, Gain 1000). Reflex threshold (RT) was assessed using a staircase method (Willer, 1977) and consisted of 3 series of ascending and descending intensity stimuli. The RT was defined as the minimum stimulation intensity at which it was possible to evoke a stable reflex response in at least 3 out of 5 trials. The intensity of the electrical shocks used for the electrophysiological measurements was then adjusted to 1.5 RT. The subjects were asked to score their pain perception after each stimulus on a 0–10-point numerical rating scale (NRS) anchored by ‘‘no pain’’ (score of 0) and ‘‘worst imaginable pain’’ (score of 10). The electrical stimuli were delivered at varied intervals of at least 40 s to avoid reflex habituation. Electromyographic (EMG) activity was recorded through a pair of Ag/AgCl surface electrodes (Medelec, Oxford, UK; diameter 1 cm, 2 cm inter-electrode distance) positioned over the belly of the biceps femoris in the lower limb and biceps brachialis in the upper limb. 2.3. Bladder filling technique With small sips, participants gradually drank 1 litre of water. They were instructed by the investigator to hold their urine until instructed otherwise. Suprapubic ultrasound (Aloka model: SSD1400 with 3.5 MHz convex probe) evaluation was performed to measure the bladder filling level just after the participants had voided (no voiding desire), and the instances when participants reported a moderate or strong desire of voiding. 2.4. Procedure Before starting formal measurements, the subjects underwent an initial training session to familiarise them with the electrical stimulation procedure and pain intensity ratings, and to reduce any effects due to arousal and/or anxiety. The experimental setup is presented in Fig. 1. In order to avoid possible RT variations due to menstrual cycle or gastric or rectal distensions (Sandrini et al., 2005), the NWR measurements were always performed during the follicular phase in women, at least 2 h after meals and without a desire to evacuate. During electrophysiological recordings, subjects were seated in a comfortable armchair in a quiet room where the temperature was kept at a constant level (23 ± 2 °C). Their lower limbs were positioned to ensure complete muscle relaxation, with the knee flexed at 130° and the ankle at 90°. The upper limb was resting on a horizontal support, slightly flexed at the elbow, the wrist was in line with the forearm, which was situated in the supine position, and the fingers were spontaneously flexed to achieve comfortable muscle relaxation. Neurophysiological data were recorded from subjects in the following 3 sessions: (i) no voiding desire (empty bladder); (ii) moderate voiding desire (medium level of bladder filling); (iii) strong voiding desire (high level of bladder filling). In all subjects, the first session represented the baseline, whereas the conditions for the second and third sessions were chosen randomly.
Please cite this article in press as: Serrao M et al. Bladder filling attenuates spinal cord nociceptive reflexes in humans. Clin Neurophysiol (2014), http:// dx.doi.org/10.1016/j.clinph.2014.03.014
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Fig. 1. Scheme of the experimental design.
Participants were asked to void so that they emptied their bladder completely, and a suprapubic bladder ultrasound was performed to ascertain that the bladder was empty. Afterwards they were invited to sit in the recording position (described above) to undergo baseline measurements (Fig. 1). Thereafter volunteers were invited to drink water until the desire changed from no desire to either moderate or strong voiding desire. At that time, participants underwent bladder ultrasound to measure the bladder filling level before the neurophysiological recordings. 2.5. Data analysis The EMG recordings were rectified, band-pass filtered (10–500 Hz, second order), and sampled at 2 kHz. The root mean square (RMS) amplitude in pre- and post-stimulus time windows (100 ms before and 80–180 ms after) was calculated. The EMG reflex activity was expressed as the difference between post- and pre-stimulus RMS. Reflex magnitude was calculated as the mean RMS of 5 trials. 2.6. Statistical analysis The Statistical Package for the Social Sciences (SPSS) ver. 15 (SPSS, Chicago, IL) was used for all statistical analyses. Two-way repeated measures ANOVA was performed to evaluate the effect of bladder filling (three-level within-subject factor) and gender (two-level between-factor) on the NWR magnitude and NRS scores after having performed the Kolmogorov–Smirnov test for normal distribution. A two-way repeated measures ANOVA was performed to also evaluate the interaction effect of bladder filling/limbs on pain perception (NRS). Greenhouse–Geisser correction was used, when necessary, to circumvent violations of sphericity (i.e. inequalities in the variance of the differences between factors; Greenhouse and Geisser, 1959). The Bonferroni adjustment for multiple comparisons was used for pairwise post hoc analyses. The results for the NWR magnitude are presented as mean values ± standard error of the mean (SEM). 3. Results For all subjects, cystometric capacities were in the normal range. After randomization, 11 subjects performed the strong voiding desire session as first and the moderate desire session as second, and thus 10 subjects performed the moderate session as first and the strong session as second. In 2 subjects, the NWR could not be evoked the upper limb, thus we analysed upper limb NWR data in 19 subjects. Mean values of bladder filling, NWR thresholds and magnitudes, and NRS scores recorded at different voiding desires are reported in Table 1. NWR magnitude changes during bladder filling sessions in a representative subject are shown in Fig. 2.
Table 1 Mean values of bladder filling, NWR thresholds and magnitudes, and NRS scores at different voiding desires. Parameters
Experimental sessions No desire
Moderate desire
Strong desire
Bladder filling volumes
0 ml
202.4 ± 10.6 ml
569.2 ± 12.3 ml
Lower limbs NWR-Th (mA) NWR magnitude (RMS, uV) NRS scores
14.5 ± 1.5 11.7 ± 3.1 6.1 ± 0.5
6.5 ± 2.0 5.8 ± 0.6
4.3 ± 1.2 5.9 ± 0.6
Upper limbs NWR-Th (mA) NWR magnitude (RMS, uV) NRS scores
3.0 ± 0.6 13.4 ± 2.7 6.4 ± 0.4
6.4 ± 1.6 5.8 ± 0.4
2.9 ± 1.5 5.1 ± 0.4
A significant effect of bladder filling on lower limb NWR magnitude was found (main effect, F(1.271,24.153) = 7.501, p = 0.008). Pairwise comparisons revealed higher values in the no desire session than in the moderate and strong voiding desire sessions (Fig. 3). No significant differences were revealed between moderate and strong voiding desire sessions. No significant effect for bladder filling (main effect, F(2,38) = 0.425, p = 0.652) on the lower limb NRS scores was observed. A significant effect of bladder filling on the upper limb NWR magnitude was found (main effect, F(1.356,23.052) = 13.488, p = 0.001). Pairwise comparisons revealed higher values in the no desire session than in both moderate and strong voiding desire sessions, and higher values in the moderate than the strong voiding desire sessions (Fig. 3). A significant effect of bladder filling on the upper limb NRS scores was observed (main effect, F(2,34) = 18.025, p < 0.0005). Pairwise comparisons revealed higher values in the no desire session than in both moderate and strong desires sessions, as well as higher values in the moderate than in the strong voiding desire sessions (Fig. 2). No significant effect for gender and bladder filling/gender interaction was found in either the lower or upper NWR magnitudes, or NRS scores (all, p > 0.05). A significant effect of bladder filling/ limbs interaction was found on NRS scores (main effect, F(2,76) = 8.972, p < 0.0005). Post-hoc analysis revealed significant lower values in upper than in lower limbs during strong desire voiding session (p = 0.043), whereas no other significant differences were found between limbs (all, p > 0.05).
4. Discussion The main result of the present study is that bladder distension inhibited the NWR magnitude in both the upper and lower limbs. In general, the observed reduction in the NWR magnitude during
Please cite this article in press as: Serrao M et al. Bladder filling attenuates spinal cord nociceptive reflexes in humans. Clin Neurophysiol (2014), http:// dx.doi.org/10.1016/j.clinph.2014.03.014
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Fig. 2. NWR magnitude changes during bladder filling sessions in a representative subject.
gradual bladder filling is likely to be the result of a reduced excitability of the spinal cord neurons mediating the NWR. Although a direct segmental reflex-mediated spinal inhibition may potentially occur at the sacral level, the reduction in the NWR magnitude in both upper and lower limbs during bladder filling suggests that the inhibitory effect is mainly mediated by the supraspinal descending pathways. Although our experimental results give no direct indication on which structure might be involved in such inhibition, some indirect evidences may be extrapolated from experiment in animal models and neuroimaging in humans (Blok and Holstege, 1999; Rocha et al., 2001; Fowler and Griffiths, 2010). It is known that when the bladder is storing urine its afferents activate PAG neurons (Holstege, 2010). The ventrolateral PAG triggers micturition in response to bladder afferent inputs through its direct projections to the pontine micturition centre (PMC) located in the caudal pontine tegmentum (Benarroch, 2010). The PMC sends descending projections to the sacral spinal cord via the lateral funiculus. This pathway activates the sacral preganglionic neurons and inhibits, via local interneurons, both the thoracolumbar sympathetic neurons and Onuf nucleus motoneurons innervating the external urethral sphincter (Blok and Holstege, 1999; Sugaya et al., 2005). When the bladder is in storage mode, the PAG receives afferent inputs from the viscera and in turn relays them to the hypothalamus, right insula, dorsal anterior cingulate and lateral prefrontal cortex. These areas then convey this information to the medial prefrontal cortex, which exerts a tonic inhibition on the PAG. If the decision is not to void, the medial prefrontal cortex would maintain a tonic inhibition on PAG; if the decision is to void, withdrawal of this inhibitory control would lead to activation of the PMC (Fowler et al., 2008). Interestingly, PAG has been demonstrated to be involved in spinal nociception. Descending control arises from a number of supraspinal sites, including the midline periaqueductal grey-rostral ventromedial medulla (PAG-RVM) system. Inhibitory control from the PAG-RVM system preferentially suppresses nociceptive inputs mediated by nociceptive A-delta and C fibres (Fields et al., 2006). The PAG projects to the RVM, which in turn sends its outputs to dorsal horn laminae neurons involved in nociception, particularly the WDR neurons which are known to be involved in the polysynaptic nociceptive reflexes (Yang et al., 2001; Sandrini et al., 2005). In this light, the bladder afferents may inhibit spinal reflexes by stimulating the RVM and thus reducing dorsal horn neuron activity. This may be a safety mechanism to maintain continence during painful stimuli and pain-related motor responses that may increase intra-abdominal pressure. In addition to the PAG, other brainstem modulatory descending systems known to produce analgesia, including DNIC (Diffuse Noxious Inhibitory Controls), may be triggered by bladder afferents.
Diffuse noxious inhibitory controls (DNIC) represents a phenomenon in which, via spino-bulbo-spinal loops, the activity of wide dynamic range neurons in the spinal dorsal horn and in trigeminal nuclei is attenuated in response to noxious stimuli applied to a remote area of the body (Villanueva and Le Bars, 1995). Interestingly, it was demonstrated in animal models that PAG can modulate DNIC to produce analgesic-like effects (Dickenson and Le Bars, 1983; Bouhassira et al., 1992; Bajic and Proudfit, 1999). In rats, distension of abdominal viscera (the colon and urinary bladder) depressed activity of somatic lumbar dorsal horn neurons possibly by DNIC activation (Cadden and Morrison, 1991). Bouhassira and colleagues (Bouhassira et al., 1992, 1998) speculated DNIC-induced NWR depression to be a consequence of rectal and gastric distension in humans. It has been suggested that DNIC may modulate NWR and pain in several cognitive and physiological states (i.e. stress, anxiety, sleep, respiration, menstrual cycle, etc.) (reviewed by Sandrini et al. (2005)). More recently, the term ‘‘conditioned pain modulation’’ (CPM) has been coined for the psychophysical protocols that explore DNIC in humans (Yarnitsky et al., 2010). In this light, the CPM system may represent the final common output pathway to brainstem and spinal cord nociceptive neurons, which serves to regulate and adapt pain thresholds in cognitive and homeostatic contexts. In the present study, we found a different inhibitory effect of bladder distension on pain perception in the upper vs lower limbs. Particularly, while a significant decrease in the upper limb NRS score was observed, no significant changes were observed in the lower limb NRS scores. One plausible explanation for such a finding may be a convergence of excitatory and inhibitory effects in the somatic sacral spinal cord neurons mediating the flexion reflexes as demonstrated in previous animal studies (Ness and Gebhart, 1987; Shefchyk, 2001; Peles et al., 2004). It is known that afferents from the bladder travel via the pelvic and hypogastric nerves and terminate in the superficial dorsal horn (Birder et al., 2010) from which inputs are conveyed to the supraspinal structures via the spino-thalamic tract. Wide dynamic range neurons, located in laminae II and V, modulate the NWR and receive afferents conveying multimodal painful and non-painful information from both somatic and visceral tissues and organs (Cervero, 1995; Laird et al., 1995; Drewes et al., 2003). A spatio-temporal summation of bladder inputs at this level, during gradual bladder filling, may explain the different voiding sensations related to the different levels of bladder distension, which range from normal voiding sensation to a clear pain perception (Xu and Gebhart, 2007; De Groat and Yoshimura, 2009; Kanai and Andersson, 2010). We speculated that the mechanosensitive inputs related to bladder distension might interfere with somatic pain processing mechanisms at both sacral spinal and supraspinal
Please cite this article in press as: Serrao M et al. Bladder filling attenuates spinal cord nociceptive reflexes in humans. Clin Neurophysiol (2014), http:// dx.doi.org/10.1016/j.clinph.2014.03.014
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Fig. 3. NWR magnitude and NRS scores during bladder filling sessions. ⁄p < 0.05,
levels, inducing a segmental-mediated facilitation followed by a supraspinal-mediated inhibition of the NWR pain-related perception. This effect would also explain the lack of significant differences in the lower limb NWR magnitude between moderate and strong voiding desire sessions (Fig. 3). A potential bias induced by the order sequence of the experimental sessions may be present and limits the results of our study. Although the moderate and strong voiding desire sessions were randomly performed, the no desire session was always performed first. However, the trend for a progressive reflex magnitude reduction from an empty to high level of bladder filling trials (Fig. 3) gives us a reasonable degree of certainty that the NWR inhibition effect was related to the bladder filling. Our experimental design did not include a ‘‘post-control’’ session which could have been done at the end of the experiment after the subjects had voided. However, control session should have done at different time intervals from the bladder emptying due to the descending inhibitory after effect on the spinal neurons. Indeed, in previous studies it has been demonstrated that the NWR remains inhibited for a variable time period after the end of the cold pressor test (Piché et al., 2009). In animal models, a long lasting post-effects directly related to the duration of conditioning painful stimulus has been reported (Le Bars et al., 1979). In this regard, an ‘‘ad hoc’’ experiment, investigating on the after-effect recovery curve of the NWR should be performed. Further studies are warranted to investigate on this aspect. In summary, our study indicates that bladder filling activates descending pathways that inhibit nociception at the spinal level.
5
⁄⁄
p < 0.01 at post hoc analysis.
A central role for the PAG as the main centre of functional connectivity between bladder afferents during storage and nociception control may be hypothesised. The effect of bladder filling on NWR may represent a useful tool to investigate the interaction between bladder and pain pathways. It would be interesting to evaluate the bladder-triggered NWR inhibition in either somatic (i.e. low back pain) or visceral (i.e. bladder pain and irritable bowel pain) chronic pain syndromes and/or their pharmacological effect.
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