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Reflexes in sympathetic vasoconstrictor neurones arising from urinary bladder afferents are not amplified early after inflammation in the anaesthetised cat
Pain 101 (2003) 251–257 www.elsevier.com/locate/pain
Reflexes in sympathetic vasoconstrictor neurones arising from urinary bladder afferents are not amplified early after inflammation in the anaesthetised cat H.-J. Ha¨bler*, W. Ja¨nig Physiologisches Institut, Christian-Albrechts-Universita¨t, Olshausenstrasse 40, D-24098 Kiel, Germany Received 19 April 2002; accepted 26 August 2002
Abstract Pathophysiological processes in the viscera can lead to pain and hyperalgesia and exaggerated motility-regulating reflexes. This may be due to sensitisation of visceral afferents (peripheral sensitisation), which has repeatedly been shown to occur as a consequence of e.g. inflammation, and/or to sensitisation of dorsal horn neurones (central sensitisation), which is less well documented in the visceral domain. As an indicator of peripheral sensitisation, we previously analysed the responses of sacral spinal afferents after inflammation of the urinary bladder. Here, we studied reflexes in sympathetic vasoconstrictor neurones supplying skeletal muscle and skin elicited by bladder distension stimuli (vesico-sympathetic reflexes) before and after induction of bladder inflammation. Our aim was to test whether these vesicosympathetic reflexes are amplified after inflammation in a way that would support a major functional role for post-inflammatory central sensitisation processes. Bladder inflammation was induced in anaesthetised cats by instillation of turpentine or mustard oil and vesicosympathetic reflexes were studied 1 and 2 h after induction of the inflammation. Inflammation enhanced on-going activity in vasoconstrictor neurones supplying skeletal muscle (after 1 h to 187:6 ^ 36:8%, mean ^ SEM, P , 0:01, and after 2 h to 139:1 ^ 12:9%, P , 0:05, of baseline activity) and decreased it in most sympathetic neurones supplying skin (to 91:7 ^ 12:5%, P . 0:05, and to 71:6 ^ 11:3%, P , 0:05, respectively, of baseline activity). Relative to the altered baseline activity vesico-sympathetic reflexes to graded distension of the inflamed bladder were quantitatively unchanged with a tendency to be diminished. Thus, the changes in on-going sympathetic vasoconstrictor activity and the distension-evoked reflexes directly mirrored the afferent input from the inflamed urinary bladder into the spinal cord, i.e. no increase of the gain of these reflexes was observed. These results suggest that in the first 2 h of inflammation, peripheral sensitisation processes play the main role for hyperalgesia and hyperreflexia of the urinary bladder. In contrast, central sensitisation appears to be of little importance during this time period. q 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Visceral afferents; Hyperalgesia; Sympathetic reflexes; Central sensitisation; Visceral pain; Urinary bladder
1. Introduction Inflammation leads to allodynia and hyperalgesia in somatic as well as in visceral organs. In somatic tissues, the main underlying pathophysiological mechanisms are well understood: There is a peripheral and a central component both contributing to the hyperalgesic state. At the peripheral level, inflammation sensitises the receptive endings of unmyelinated and thinly myelinated nociceptive afferents to mechanical, chemical or heat stimuli (Schaible and Schmidt, 1985, 1988; Martin et al., 1987; Reeh et al., * Corresponding author. FH Bonn-Rhein-Sieg, Von-Liebig-Str. 20, D53359 Rheinbach, Germany. Tel.: 10049-22-4186-5525; fax: 10049-224186-58525. E-mail address: [email protected] (H.-J. Ha¨bler).
1987; Steen et al., 1992). In addition, there is recruitment of mechanically insensitive (‘silent’) afferents (Schaible and Schmidt, 1988; Meyer et al., 1991; Kress et al., 1992; Schmidt et al., 1995; Xu et al., 2000). As a central component, on-going activity induced in unmyelinated afferents by the inflammatory process is thought to induce sensitisation of dorsal horn neurones (Woolf and Wall, 1986; Cook et al., 1986; Torebjo¨rk et al., 1992). Once sensitised, dorsal horn neurones may show amplified responses to the afferent input from the periphery. In the visceral domain, sensory mechanisms appear to be different from those in somatic tissues. Spinal visceral afferents serve the regulation of autonomic functions in internal organs, evoke viscero-somatic reflexes and are responsible for visceral sensations including discomfort and pain. In many organs, such as urinary bladder and colon, both
0304-3959/02/$20.00 q 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. doi:10.1 016/S0304 -39 59(02)00329-9
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noxious and innocuous stimuli are encoded by thinly myelinated and unmyelinated afferents. It is still a matter of debate whether spinal visceral afferents, innervating a particular organ, consist of subpopulations associated with specific functions or whether these afferents are functionally homogeneous (Ha¨ bler et al., 1993a,b; Sengupta and Gebhart 1994; see Cervero and Ja¨ nig, 1992). Like somatic high threshold afferents, visceral afferents are sensitised by inflammation; i.e. they develop on-going activity and show enhanced responses to natural stimulation (Ha¨ bler et al., 1993b; Su and Gebhart, 1988; Brunsden and Grundy, 1999). In the case of sacral visceral afferents innervating the urinary bladder, this applies to all afferents (Ha¨ bler et al., 1993b). In addition and again similar to somatic tissues, previously silent unmyelinated afferents are recruited and these may acquire novel mechanosensitivity (Ha¨ bler et al., 1990). These changes occurring at the peripheral level of the receptive afferent ending could well explain the sensory and reflex motor disturbances experienced by patients suffering from inflammation of visceral hollow organs. In the visceral domain, evidence for a post-inflammatory central sensitisation of dorsal horn neurones has been presented (McMahon, 1988; Olivar et al., 2000; Sarkar et al., 2000). However, ‘wind-up’, the potentiation of responses in spinal neurones to stimulation of unmyelinated afferents, which is implicated in central sensitisation in the somatic domain, was not observed after stimulating visceral afferents (Laird et al., 1995). In anaesthetised cats, stimulation of spinal visceral afferents evokes powerful vasoconstrictor reflexes which are inhibitory in cutaneous vasoconstrictor (CVC) neurones and excitatory in vasoconstrictor neurones supplying skeletal muscle (MVC neurones) (Ha¨ bler et al., 1992). Thus, distension of the urinary bladder over an intravesical pressure range of 10–90 mmHg elicits graded reflexes in postganglionic sympathetic vasoconstrictor neurones supplying the hindlimb (vesico-sympathetic reflexes) (Ha¨ bler et al., 1992). Previously we analysed, on a quantitative basis, the responses of sacral afferents supplying the inflamed urinary bladder of the cat (Ha¨ bler et al., 1990, 1993b). The aim of the present experiments was to test whether vesico-sympathetic reflexes in response to stimulation of sensitised afferents from the acutely inflamed urinary bladder would be amplified, providing evidence for central sensitisation.
2. Materials and methods 2.1. Anaesthesia and animal maintenance Experiments were performed on 16 adult cats (body weight 3.1–4.1 kg) of either sex anaesthetised with a-dgluco-chloralose (50 mg/kg i.p.) after induction with ketamine (Parke-Davis 15 mg/kg, i.m.). Additional doses of chloralose (5–10 mg/kg, i.v.) were given when necessary. Catheters were inserted into the external jugular vein for
drug administration and into the left femoral artery for continuous recording of blood pressure. A sufficient level of anaesthesia was judged from the persistence of miotic pupils and the absence of gross spontaneous fluctuations of blood pressure and heart rate. Additionally, care was taken to make sure that noxious stimulation of skin produced only transient changes in systemic blood pressure. The animals were paralysed with pancuronium bromide (0.2 mg/kg per dose, i.v.) and artificially ventilated with a Starling pump through a tracheal cannula. The respirator was set to a frequency of 18–22 strokes per minute. Tidal volume was adjusted to obtain an end-tidal CO2 concentration between 3.5 and 4.5 vol.%. Arterial pO2, pCO2, pH and plasma bicarbonate were measured at intervals of about 3 h (ABL30, Radiometer Copenhagen) and maintained within the physiological range (pO2 90–100 mmHg, pCO2 close to 30 mmHg, pH close to 7.45, bicarbonate close to 21 mmol/ l). In some experiments left phrenic nerve activity was recorded as an indicator for artificial ventilation and the depth of anaesthesia. Body core temperature was measured intra-oesophageally and kept close to 38.08C by means of a servo-controlled heating blanket. The ECG was recorded conventionally with needle electrodes. At the end of the experiments, the animals were killed by intravenous injection of a saturated potassium chloride solution which was given under deep anaesthesia. All experiments had been approved by the local animal care committee of the state administration and were conducted in accordance with German Federal Law. 2.2. Nerve recording The peroneal nerve was exposed on the left hindlimb. Small filaments containing single or a few post-ganglionic axons with spontaneous activity were split from the superficial peroneal nerve (supplying hairy skin) and from branches of the deep peroneal nerve (supplying skeletal muscle). For identification of sympathetic axons in some experiments the lumbar sympathetic trunk was stimulated electrically with single pulses supramaximal for C fibres. The activity in filaments containing sympathetic units was recorded unipolarly using a pair of platinum wire electrodes with the indifferent electrode connected to nearby tissue. Phrenic nerve activity was recorded bipolarly using platinum hook electrodes. Neural activity was differentially amplified (input resistance 10 MV), filtered (bandpass 120–1200 Hz) and fed into window discriminators. Spike discrimination in single-unit recordings was controlled by means of delay units; the sympathetic action potentials were delayed by 5 ms and displayed on a storage oscilloscope using the output of the window discriminator as a trigger. 2.3. Bladder stimulation and induction of inflammation The urinary bladder was cannulated transurethrally and intravesical pressure was measured continuously. Urinary bladder afferents were stimulated while recording vesico-
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Fig. 1. Reflexes in individual filaments containing MVC neurones to graded distension of the urinary bladder (A) before and (B) 1 and (C) 2 h after intravesical instillation of turpentine or mustard oil ( £ ). Reflexes in MVC neurones were excitatory and similar, although in most cases reduced, 1 and 2 h after inducing an inflammation of the urinary bladder. Reflex amplitudes are expressed as percentages of actual baseline activity.
sympathetic reflexes by a series of graded isotonic distension steps (10–70 mmHg) of 90–120 s duration using a pressure reservoir. Bladder inflammation was induced by the intravesical instillation of 10 ml mustard oil (1–2.5% in low-viscosity paraffin oil, left in the bladder for 10 min) or turpentine oil (25–50% in paraffin oil, left in the bladder for 30 min). Series of isotonic distension stimuli were performed under control conditions and commencing 1 and 2 h, respectively, after bladder inflammation.
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Fig. 3. Mean reflex responses in MVC and CVC neurones before (triangles) and 1 h (filled circles) and 2 h (squares) after bladder inflammation. Overall, reflex magnitudes were similar 1 and 2 h after induction of bladder inflammation, but some responses in MVC neurones 1 h after inflammation were significantly smaller than pre-inflammatory responses (*P , 0:05, Mann–Whitney U-test).
lus and expressed relative to neural activity within a control period of 30–60 s before stimulation. The absolute response of blood pressure to bladder distension was also determined. Data are given as mean ^ SEM. Non-parametric statistical analysis was performed using Mann–Whitney U-test and Wilcoxon test.
3. Results 2.4. Data analysis Window discriminated nerve impulses, electrocardiogram (ECG), mean arterial blood pressure and intravesical pressure were simultaneously fed into an IBM-compatible computer with ADC and counter interface. Original signals were also stored on magnetic tape for off-line analysis. Because sympathetic reflexes needed some time to build up to maximum during bladder distension, reflexes were quantified using the period 31–90 s after onset of the stimu-
As described previously (Ha¨ bler et al., 1992), isotonic distension and active contractions of the uninflamed urinary bladder evoked graded excitatory responses in post-ganglionic MVC neurones (Figs. 1A and 3A) and in a minority of CVC neurones, while the majority of CVC neurones were inhibited (Figs. 2A and 3B). Arterial blood pressure also showed a graded increase to bladder distension (Fig. 4). Instillation of inflammatory agents elicited pronounced isovolumetric bladder contractions, each being mirrored
Fig. 2. Reflexes in individual filaments containing CVC neurones to graded distension of the urinary bladder (A) before and (B) 1 and (C) 2 h after intravesical instillation of turpentine or mustard oil ( £ ).The majority of CVC neurones exhibited inhibitory reflexes.
Fig. 4. Responses of mean arterial blood pressure (BP) to graded distension of the urinary bladder before (triangles) and 1 h (filled circles) and 2 h (squares) after intravesical instillation of turpentine or mustard oil. BP responses were slightly enhanced after inflammation. *P , 0:05, response 2 h post-inflammation significantly different from pre-inflammatory response (Mann–Whitney U-test).
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by reciprocal reflexes in MVC and CVC neurones and by increases in blood pressure (Fig. 5). Between bladder contractions, on-going activity remained elevated in MVC neurones and reduced in CVC neurones. After removing the irritant by draining the bladder, sympathetic activity also did not return to baseline, but remained altered in most MVC and CVC neurones throughout the post-inflammatory observation period of 2 h. On-going activity was increased in 10/ 12 MVC filaments 1 h after inflammation (on an average to 187:6 ^ 36:8% of baseline activity, P , 0:01, n ¼ 12) and in 6/8 MVC filaments 2 h after inflammation (to 139:1 ^ 12:9% of baseline activity, P , 0:05) (Fig. 6A). Responses in CVC neurones were more inhomogeneous; compared to the control period on-going activity decreased in 8/13 CVC filaments and increased in 5/13 CVC filaments 1 h after inflammation (on an average to 91:7 ^ 12:5% of baseline activity, P . 0:05), while 8/9 CVC filaments showed reduced on-going activity 2 h post-inflammation (to 71:6 ^ 11:3% of baseline activity, P , 0:05)(Fig. 6B). After, but not before bladder inflammation, short irregular bursts of MVC or CVC activity were frequently observed which were related neither to intravesical pressure nor to any other obvious events (see CVC in Fig. 5). Distension-evoked vesico-sympathetic reflexes in individual MVC and CVC filaments were similar before and from 1 to 2 h following bladder inflammation (Figs. 1 and 2), and the average curves relating the responses of MVC neurones (Fig. 3A) and CVC neurones (Fig. 3B), respectively, to bladder pressure were also similar. However, there was a tendency
Fig. 6. Changes of on-going activity in individual filaments containing MVC (A1) and CVC (B1) fibres and mean changes (A2, B2) 1 and 2 h after induction of bladder inflammation. Most MVC neurones, but also five multifibre preparations containing CVC axons, showed increased ongoing activity after inflammation, whereas on-going activity in most CVC neurones decreased. *P , 0:05, **P , 0:01, significantly different from pre-inflammatory on-going activity (Wilcoxon test).
towards decreased responses after inflammation (Fig. 3). Only a few individual sympathetic neurones showed enhanced reflexes after inflammation, the enhancement, however, in relation to on-going activity being modest (Fig. 7). The possibility that after inflammation the relatively high level of on-going activity in MVC neurones precluded stronger reflex responses to distension was ruled out, as there was almost no correlation between the two variables (Fig. 8). Consistent with the enhanced level of on-going activity in MVC neurones, which are involved in the regulation of vascular resistance, baseline arterial blood pressure was higher after inflammation than before (Fig. 7). The phasic responses of arterial pressure to bladder distension were also slightly, in part significantly, enhanced after inflammation, particularly in the middle pressure range (Fig. 4).
4. Discussion Fig. 5. Changes of on-going activity in MVC and CVC neurones after induction of bladder inflammation. Instillation of turpentine oil ( " ) acutely induced large micturition contractions each of them being accompanied by phasic activation of MVC, phasic inhibition of CVC neurones and phasic increases of blood pressure. Twenty minutes later (20 0 ) bladder contractions were more frequent and smaller in amplitude leading to tonically increased MVC and tonically decreased CVC activity. After removing the irritant ( # ) the elevated and diminished level, respectively, of vasoconstrictor activity did not return to the original baseline within 90 min. With some latency high-frequency bursts occurred in CVC activity which were unrelated to bladder pressure.
The present study was designed to test whether an inflammation of the urinary bladder amplifies the central transmission of afferent input arising from the inflamed viscus. In analogy to measuring wind-up, which is a potentiation of motor reflexes upon electrical high intensity, low frequency simulation of the corresponding afferent input, we studied vasoconstrictor reflexes elicited by adequately stimulating primary afferents in the urinary bladder before and within 1– 2 h after inflaming the bladder with either turpentine or mustard oil. We expected that we would be able to distin-
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Fig. 7. MVC multifibre preparation showing increased responses to bladder distension after inflammation. However, in relation to baseline activity, the increased neural responses after inflammation to a 50 mmHg distension step were only modest. Blood pressure responses were also increased, but baseline blood pressure was higher after inflammation.
guish changes in these reflexes due to inflammation-induced sensitisation of bladder afferents from changes due to a potential central sensitisation of neurones involved in the integration of noxious input from the inflamed urinary bladder. Consistent with previous results (Ha¨ bler et al., 1992), we saw reciprocal responses in MVC and CVC neurones. After inflammation, there was a sustained increase of baseline on-going activity in MVC neurones while a decrease of on-going activity was observed in most CVC neurones. However, the gain of the vesico-sympathetic reflexes, indicated by the changes of activity relative to baseline activity in response to graded distension of the inflamed bladder, was not enhanced. The possibility that strong increases of on-going activity in MVC neurones precluded stronger excitatory responses seems unlikely, as there was almost no inverse correlation between the two variables. These results suggest that in the early phase after inducing inflammation the efficacy of the afferent input from the bladder is not amplified in central neurones. Therefore, central sensitisation processes may not play a major role, at least in the generation of vesico-sympathetic reflexes, in this time period. Earlier studies showed that bladder inflammation excited previously silent and mechanically unresponsive unmyelinated sacral afferents which, thereafter, exhibited on-going activity and some of them acquired novel mechanosensitivity (Ha¨ bler et al., 1990). More than half of the mechanosensitive sacral afferents, most of them being thinly myelinated and some unmyelinated, which normally show no on-going activity when the bladder is empty, were activated by mustard or turpentine oil at short latency (Ha¨ bler et al., 1990, 1993b) and then continuously exhibited irregular on-going activity. The responses to bladder distension of these spinal afferents were greatly enhanced for at least
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2 h after turpentine oil. In contrast, the mechanical responsiveness of bladder afferents changed only little within 1– 2 h after mustard oil which, thereafter, tended to induce desensitisation (Ha¨ bler et al., 1993b). From these results it follows that during inflammation there is a continuous substantial afferent inflow from the bladder to the spinal cord and that the afferent input to the spinal cord is enhanced during bladder distension, at least after turpentine oil. The increased and decreased on-going activity of MVC and CVC neurones, respectively, after bladder inflammation is consistent with a continuous reflex mediated by the novel on-going activity in both thinly myelinated and unmyelinated sacral bladder afferents. However, from the increased responses of bladder afferents to distension stimuli, in particular after turpentine oil, it would have been expected that vesico-sympathetic reflexes would be considerably enhanced which was not the case. An obvious possibility would be that the changed sympathetic on-going activity obscured the enhanced reflexes after inflammation. Indeed, when the magnitude of distension-evoked reflexes was related to the sympathetic on-going activity present before inflammation, the excitatory responses were considerably greater 1 h after inflammation. However, an hour after post-inflammation the absolute magnitude of distensionevoked reflexes had again diminished, suggesting that the gain of these reflexes decreased rather than increased with time after inflammation. Thus, the sympathetic vasoconstrictor reflexes kept a similar, if not identical, close quantitative relationship to the activity in sacral bladder afferents before and after inflammation. It has been speculated that mechanically insensitive silent C-fibre afferents might serve a function different from that of their mechanosensitive counterparts (Michaelis et al., 1996). From the present study it can be concluded that at least with respect to eliciting reflexes in sympathetic neurones they appear to have effects similar to those of other sacral afferents supplying the bladder. Therefore,
Fig. 8. There is almost no correlation between relative changes in on-going activity following bladder inflammation and normalised excitatory reflexes to bladder distension in MVC neurones. Correlation coefficient 0.17, P ¼ 0:47.
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these mechanically insensitive afferent fibres may differ from the other bladder afferents in their encoding properties, but not in their central connectivity. Several studies present evidence that dorsal horn neurones may show enhanced responses after noxious stimulation or inflammation of visceral organs. Conditioning stimuli applied to visceral afferents in the absence of inflammation increased the somatic receptive fields of dorsal horn neurones receiving convergent viscero-somatic input (Cervero et al., 1992; Laird et al., 1996). After bladder inflammation with turpentine oil, rat dorsal horn neurones responsive to bladder stimulation to some extent increased their responses to bladder distension and to electrical stimulation of the pelvic nerve (McMahon, 1988). Neurones of the post-synaptic dorsal column system excited by colon distension increased their responses after colon inflammation with mustard oil (Al-Chaer et al., 1997), and rat dorsal horn neurones sensitive to electrical stimulation of the pelvic nerve responded more strongly to colorectal distension after colon inflammation with acetic acid (Olivar et al., 2000). Roza et al. (1998) found a more complex behaviour of dorsal horn neurones responsive to ureter input 1–4 days after implantation of a ureteric stone. The prevalence and frequency of on-going activity was enhanced in these neurones, but otherwise there were signs of increased as well as decreased excitability. In these studies, the apparently increased responses to distension of the inflamed viscus could be explained by a higher level of convergent input from sensitised visceral afferents and does not necesssarily reflect an increase in the gain of transmission of this particular afferent input. It was argued that electrical stimulation of the pelvic nerve would avoid the problem of adequately stimulating sensitised afferents, and therefore, reveal central sensitisation, but the afferent impulses evoked electrically still impinge on neurones receiving continuously increased afferent input from the inflamed viscus. Thus, these studies may be reconciled with the present results which indicate that the increased afferent input into the spinal cord due to peripheral sensitisation of visceral afferents after inflammation is sufficient to explain the magnitude of vesico-sympathetic reflexes early after inflammation. Wind-up represents a true increase in the gain of motor reflexes upon low frequency stimulation at C fibre strength of the corresponding dorsal root, and it is widely assumed to indicate sensitisation of dorsal horn neurones (Doubell et al., 1999; see Herrero et al., 2000; but see Woolf, 1996). However, wind-up could not be elicited by electrical stimulation of visceral afferents in the splanchnic nerve (Cervero, 1995; Laird et al., 1995) which appears to be consistent with our failure to observe amplified vesico-sympathetic reflexes after bladder inflammation. At present, there is no experimental basis to refute the assumption that spinal somatomotor or sympathetic reflexes are valid and representative indicators of the excitability of dorsal horn neurones involved in the processing of corresponding afferent input.
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Reflexes in sympathetic vasoconstrictor neurones arising from urinary bladder afferents are not amplified early after inflammation in the anaesthetised cat H.-J. Ha¨bler*, W. Ja¨nig Physiologisches Institut, Christian-Albrechts-Universita¨t, Olshausenstrasse 40, D-24098 Kiel, Germany Received 19 April 2002; accepted 26 August 2002
Abstract Pathophysiological processes in the viscera can lead to pain and hyperalgesia and exaggerated motility-regulating reflexes. This may be due to sensitisation of visceral afferents (peripheral sensitisation), which has repeatedly been shown to occur as a consequence of e.g. inflammation, and/or to sensitisation of dorsal horn neurones (central sensitisation), which is less well documented in the visceral domain. As an indicator of peripheral sensitisation, we previously analysed the responses of sacral spinal afferents after inflammation of the urinary bladder. Here, we studied reflexes in sympathetic vasoconstrictor neurones supplying skeletal muscle and skin elicited by bladder distension stimuli (vesico-sympathetic reflexes) before and after induction of bladder inflammation. Our aim was to test whether these vesicosympathetic reflexes are amplified after inflammation in a way that would support a major functional role for post-inflammatory central sensitisation processes. Bladder inflammation was induced in anaesthetised cats by instillation of turpentine or mustard oil and vesicosympathetic reflexes were studied 1 and 2 h after induction of the inflammation. Inflammation enhanced on-going activity in vasoconstrictor neurones supplying skeletal muscle (after 1 h to 187:6 ^ 36:8%, mean ^ SEM, P , 0:01, and after 2 h to 139:1 ^ 12:9%, P , 0:05, of baseline activity) and decreased it in most sympathetic neurones supplying skin (to 91:7 ^ 12:5%, P . 0:05, and to 71:6 ^ 11:3%, P , 0:05, respectively, of baseline activity). Relative to the altered baseline activity vesico-sympathetic reflexes to graded distension of the inflamed bladder were quantitatively unchanged with a tendency to be diminished. Thus, the changes in on-going sympathetic vasoconstrictor activity and the distension-evoked reflexes directly mirrored the afferent input from the inflamed urinary bladder into the spinal cord, i.e. no increase of the gain of these reflexes was observed. These results suggest that in the first 2 h of inflammation, peripheral sensitisation processes play the main role for hyperalgesia and hyperreflexia of the urinary bladder. In contrast, central sensitisation appears to be of little importance during this time period. q 2002 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Visceral afferents; Hyperalgesia; Sympathetic reflexes; Central sensitisation; Visceral pain; Urinary bladder
1. Introduction Inflammation leads to allodynia and hyperalgesia in somatic as well as in visceral organs. In somatic tissues, the main underlying pathophysiological mechanisms are well understood: There is a peripheral and a central component both contributing to the hyperalgesic state. At the peripheral level, inflammation sensitises the receptive endings of unmyelinated and thinly myelinated nociceptive afferents to mechanical, chemical or heat stimuli (Schaible and Schmidt, 1985, 1988; Martin et al., 1987; Reeh et al., * Corresponding author. FH Bonn-Rhein-Sieg, Von-Liebig-Str. 20, D53359 Rheinbach, Germany. Tel.: 10049-22-4186-5525; fax: 10049-224186-58525. E-mail address: [email protected] (H.-J. Ha¨bler).
1987; Steen et al., 1992). In addition, there is recruitment of mechanically insensitive (‘silent’) afferents (Schaible and Schmidt, 1988; Meyer et al., 1991; Kress et al., 1992; Schmidt et al., 1995; Xu et al., 2000). As a central component, on-going activity induced in unmyelinated afferents by the inflammatory process is thought to induce sensitisation of dorsal horn neurones (Woolf and Wall, 1986; Cook et al., 1986; Torebjo¨rk et al., 1992). Once sensitised, dorsal horn neurones may show amplified responses to the afferent input from the periphery. In the visceral domain, sensory mechanisms appear to be different from those in somatic tissues. Spinal visceral afferents serve the regulation of autonomic functions in internal organs, evoke viscero-somatic reflexes and are responsible for visceral sensations including discomfort and pain. In many organs, such as urinary bladder and colon, both
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noxious and innocuous stimuli are encoded by thinly myelinated and unmyelinated afferents. It is still a matter of debate whether spinal visceral afferents, innervating a particular organ, consist of subpopulations associated with specific functions or whether these afferents are functionally homogeneous (Ha¨ bler et al., 1993a,b; Sengupta and Gebhart 1994; see Cervero and Ja¨ nig, 1992). Like somatic high threshold afferents, visceral afferents are sensitised by inflammation; i.e. they develop on-going activity and show enhanced responses to natural stimulation (Ha¨ bler et al., 1993b; Su and Gebhart, 1988; Brunsden and Grundy, 1999). In the case of sacral visceral afferents innervating the urinary bladder, this applies to all afferents (Ha¨ bler et al., 1993b). In addition and again similar to somatic tissues, previously silent unmyelinated afferents are recruited and these may acquire novel mechanosensitivity (Ha¨ bler et al., 1990). These changes occurring at the peripheral level of the receptive afferent ending could well explain the sensory and reflex motor disturbances experienced by patients suffering from inflammation of visceral hollow organs. In the visceral domain, evidence for a post-inflammatory central sensitisation of dorsal horn neurones has been presented (McMahon, 1988; Olivar et al., 2000; Sarkar et al., 2000). However, ‘wind-up’, the potentiation of responses in spinal neurones to stimulation of unmyelinated afferents, which is implicated in central sensitisation in the somatic domain, was not observed after stimulating visceral afferents (Laird et al., 1995). In anaesthetised cats, stimulation of spinal visceral afferents evokes powerful vasoconstrictor reflexes which are inhibitory in cutaneous vasoconstrictor (CVC) neurones and excitatory in vasoconstrictor neurones supplying skeletal muscle (MVC neurones) (Ha¨ bler et al., 1992). Thus, distension of the urinary bladder over an intravesical pressure range of 10–90 mmHg elicits graded reflexes in postganglionic sympathetic vasoconstrictor neurones supplying the hindlimb (vesico-sympathetic reflexes) (Ha¨ bler et al., 1992). Previously we analysed, on a quantitative basis, the responses of sacral afferents supplying the inflamed urinary bladder of the cat (Ha¨ bler et al., 1990, 1993b). The aim of the present experiments was to test whether vesico-sympathetic reflexes in response to stimulation of sensitised afferents from the acutely inflamed urinary bladder would be amplified, providing evidence for central sensitisation.
2. Materials and methods 2.1. Anaesthesia and animal maintenance Experiments were performed on 16 adult cats (body weight 3.1–4.1 kg) of either sex anaesthetised with a-dgluco-chloralose (50 mg/kg i.p.) after induction with ketamine (Parke-Davis 15 mg/kg, i.m.). Additional doses of chloralose (5–10 mg/kg, i.v.) were given when necessary. Catheters were inserted into the external jugular vein for
drug administration and into the left femoral artery for continuous recording of blood pressure. A sufficient level of anaesthesia was judged from the persistence of miotic pupils and the absence of gross spontaneous fluctuations of blood pressure and heart rate. Additionally, care was taken to make sure that noxious stimulation of skin produced only transient changes in systemic blood pressure. The animals were paralysed with pancuronium bromide (0.2 mg/kg per dose, i.v.) and artificially ventilated with a Starling pump through a tracheal cannula. The respirator was set to a frequency of 18–22 strokes per minute. Tidal volume was adjusted to obtain an end-tidal CO2 concentration between 3.5 and 4.5 vol.%. Arterial pO2, pCO2, pH and plasma bicarbonate were measured at intervals of about 3 h (ABL30, Radiometer Copenhagen) and maintained within the physiological range (pO2 90–100 mmHg, pCO2 close to 30 mmHg, pH close to 7.45, bicarbonate close to 21 mmol/ l). In some experiments left phrenic nerve activity was recorded as an indicator for artificial ventilation and the depth of anaesthesia. Body core temperature was measured intra-oesophageally and kept close to 38.08C by means of a servo-controlled heating blanket. The ECG was recorded conventionally with needle electrodes. At the end of the experiments, the animals were killed by intravenous injection of a saturated potassium chloride solution which was given under deep anaesthesia. All experiments had been approved by the local animal care committee of the state administration and were conducted in accordance with German Federal Law. 2.2. Nerve recording The peroneal nerve was exposed on the left hindlimb. Small filaments containing single or a few post-ganglionic axons with spontaneous activity were split from the superficial peroneal nerve (supplying hairy skin) and from branches of the deep peroneal nerve (supplying skeletal muscle). For identification of sympathetic axons in some experiments the lumbar sympathetic trunk was stimulated electrically with single pulses supramaximal for C fibres. The activity in filaments containing sympathetic units was recorded unipolarly using a pair of platinum wire electrodes with the indifferent electrode connected to nearby tissue. Phrenic nerve activity was recorded bipolarly using platinum hook electrodes. Neural activity was differentially amplified (input resistance 10 MV), filtered (bandpass 120–1200 Hz) and fed into window discriminators. Spike discrimination in single-unit recordings was controlled by means of delay units; the sympathetic action potentials were delayed by 5 ms and displayed on a storage oscilloscope using the output of the window discriminator as a trigger. 2.3. Bladder stimulation and induction of inflammation The urinary bladder was cannulated transurethrally and intravesical pressure was measured continuously. Urinary bladder afferents were stimulated while recording vesico-
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Fig. 1. Reflexes in individual filaments containing MVC neurones to graded distension of the urinary bladder (A) before and (B) 1 and (C) 2 h after intravesical instillation of turpentine or mustard oil ( £ ). Reflexes in MVC neurones were excitatory and similar, although in most cases reduced, 1 and 2 h after inducing an inflammation of the urinary bladder. Reflex amplitudes are expressed as percentages of actual baseline activity.
sympathetic reflexes by a series of graded isotonic distension steps (10–70 mmHg) of 90–120 s duration using a pressure reservoir. Bladder inflammation was induced by the intravesical instillation of 10 ml mustard oil (1–2.5% in low-viscosity paraffin oil, left in the bladder for 10 min) or turpentine oil (25–50% in paraffin oil, left in the bladder for 30 min). Series of isotonic distension stimuli were performed under control conditions and commencing 1 and 2 h, respectively, after bladder inflammation.
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Fig. 3. Mean reflex responses in MVC and CVC neurones before (triangles) and 1 h (filled circles) and 2 h (squares) after bladder inflammation. Overall, reflex magnitudes were similar 1 and 2 h after induction of bladder inflammation, but some responses in MVC neurones 1 h after inflammation were significantly smaller than pre-inflammatory responses (*P , 0:05, Mann–Whitney U-test).
lus and expressed relative to neural activity within a control period of 30–60 s before stimulation. The absolute response of blood pressure to bladder distension was also determined. Data are given as mean ^ SEM. Non-parametric statistical analysis was performed using Mann–Whitney U-test and Wilcoxon test.
3. Results 2.4. Data analysis Window discriminated nerve impulses, electrocardiogram (ECG), mean arterial blood pressure and intravesical pressure were simultaneously fed into an IBM-compatible computer with ADC and counter interface. Original signals were also stored on magnetic tape for off-line analysis. Because sympathetic reflexes needed some time to build up to maximum during bladder distension, reflexes were quantified using the period 31–90 s after onset of the stimu-
As described previously (Ha¨ bler et al., 1992), isotonic distension and active contractions of the uninflamed urinary bladder evoked graded excitatory responses in post-ganglionic MVC neurones (Figs. 1A and 3A) and in a minority of CVC neurones, while the majority of CVC neurones were inhibited (Figs. 2A and 3B). Arterial blood pressure also showed a graded increase to bladder distension (Fig. 4). Instillation of inflammatory agents elicited pronounced isovolumetric bladder contractions, each being mirrored
Fig. 2. Reflexes in individual filaments containing CVC neurones to graded distension of the urinary bladder (A) before and (B) 1 and (C) 2 h after intravesical instillation of turpentine or mustard oil ( £ ).The majority of CVC neurones exhibited inhibitory reflexes.
Fig. 4. Responses of mean arterial blood pressure (BP) to graded distension of the urinary bladder before (triangles) and 1 h (filled circles) and 2 h (squares) after intravesical instillation of turpentine or mustard oil. BP responses were slightly enhanced after inflammation. *P , 0:05, response 2 h post-inflammation significantly different from pre-inflammatory response (Mann–Whitney U-test).
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by reciprocal reflexes in MVC and CVC neurones and by increases in blood pressure (Fig. 5). Between bladder contractions, on-going activity remained elevated in MVC neurones and reduced in CVC neurones. After removing the irritant by draining the bladder, sympathetic activity also did not return to baseline, but remained altered in most MVC and CVC neurones throughout the post-inflammatory observation period of 2 h. On-going activity was increased in 10/ 12 MVC filaments 1 h after inflammation (on an average to 187:6 ^ 36:8% of baseline activity, P , 0:01, n ¼ 12) and in 6/8 MVC filaments 2 h after inflammation (to 139:1 ^ 12:9% of baseline activity, P , 0:05) (Fig. 6A). Responses in CVC neurones were more inhomogeneous; compared to the control period on-going activity decreased in 8/13 CVC filaments and increased in 5/13 CVC filaments 1 h after inflammation (on an average to 91:7 ^ 12:5% of baseline activity, P . 0:05), while 8/9 CVC filaments showed reduced on-going activity 2 h post-inflammation (to 71:6 ^ 11:3% of baseline activity, P , 0:05)(Fig. 6B). After, but not before bladder inflammation, short irregular bursts of MVC or CVC activity were frequently observed which were related neither to intravesical pressure nor to any other obvious events (see CVC in Fig. 5). Distension-evoked vesico-sympathetic reflexes in individual MVC and CVC filaments were similar before and from 1 to 2 h following bladder inflammation (Figs. 1 and 2), and the average curves relating the responses of MVC neurones (Fig. 3A) and CVC neurones (Fig. 3B), respectively, to bladder pressure were also similar. However, there was a tendency
Fig. 6. Changes of on-going activity in individual filaments containing MVC (A1) and CVC (B1) fibres and mean changes (A2, B2) 1 and 2 h after induction of bladder inflammation. Most MVC neurones, but also five multifibre preparations containing CVC axons, showed increased ongoing activity after inflammation, whereas on-going activity in most CVC neurones decreased. *P , 0:05, **P , 0:01, significantly different from pre-inflammatory on-going activity (Wilcoxon test).
towards decreased responses after inflammation (Fig. 3). Only a few individual sympathetic neurones showed enhanced reflexes after inflammation, the enhancement, however, in relation to on-going activity being modest (Fig. 7). The possibility that after inflammation the relatively high level of on-going activity in MVC neurones precluded stronger reflex responses to distension was ruled out, as there was almost no correlation between the two variables (Fig. 8). Consistent with the enhanced level of on-going activity in MVC neurones, which are involved in the regulation of vascular resistance, baseline arterial blood pressure was higher after inflammation than before (Fig. 7). The phasic responses of arterial pressure to bladder distension were also slightly, in part significantly, enhanced after inflammation, particularly in the middle pressure range (Fig. 4).
4. Discussion Fig. 5. Changes of on-going activity in MVC and CVC neurones after induction of bladder inflammation. Instillation of turpentine oil ( " ) acutely induced large micturition contractions each of them being accompanied by phasic activation of MVC, phasic inhibition of CVC neurones and phasic increases of blood pressure. Twenty minutes later (20 0 ) bladder contractions were more frequent and smaller in amplitude leading to tonically increased MVC and tonically decreased CVC activity. After removing the irritant ( # ) the elevated and diminished level, respectively, of vasoconstrictor activity did not return to the original baseline within 90 min. With some latency high-frequency bursts occurred in CVC activity which were unrelated to bladder pressure.
The present study was designed to test whether an inflammation of the urinary bladder amplifies the central transmission of afferent input arising from the inflamed viscus. In analogy to measuring wind-up, which is a potentiation of motor reflexes upon electrical high intensity, low frequency simulation of the corresponding afferent input, we studied vasoconstrictor reflexes elicited by adequately stimulating primary afferents in the urinary bladder before and within 1– 2 h after inflaming the bladder with either turpentine or mustard oil. We expected that we would be able to distin-
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Fig. 7. MVC multifibre preparation showing increased responses to bladder distension after inflammation. However, in relation to baseline activity, the increased neural responses after inflammation to a 50 mmHg distension step were only modest. Blood pressure responses were also increased, but baseline blood pressure was higher after inflammation.
guish changes in these reflexes due to inflammation-induced sensitisation of bladder afferents from changes due to a potential central sensitisation of neurones involved in the integration of noxious input from the inflamed urinary bladder. Consistent with previous results (Ha¨ bler et al., 1992), we saw reciprocal responses in MVC and CVC neurones. After inflammation, there was a sustained increase of baseline on-going activity in MVC neurones while a decrease of on-going activity was observed in most CVC neurones. However, the gain of the vesico-sympathetic reflexes, indicated by the changes of activity relative to baseline activity in response to graded distension of the inflamed bladder, was not enhanced. The possibility that strong increases of on-going activity in MVC neurones precluded stronger excitatory responses seems unlikely, as there was almost no inverse correlation between the two variables. These results suggest that in the early phase after inducing inflammation the efficacy of the afferent input from the bladder is not amplified in central neurones. Therefore, central sensitisation processes may not play a major role, at least in the generation of vesico-sympathetic reflexes, in this time period. Earlier studies showed that bladder inflammation excited previously silent and mechanically unresponsive unmyelinated sacral afferents which, thereafter, exhibited on-going activity and some of them acquired novel mechanosensitivity (Ha¨ bler et al., 1990). More than half of the mechanosensitive sacral afferents, most of them being thinly myelinated and some unmyelinated, which normally show no on-going activity when the bladder is empty, were activated by mustard or turpentine oil at short latency (Ha¨ bler et al., 1990, 1993b) and then continuously exhibited irregular on-going activity. The responses to bladder distension of these spinal afferents were greatly enhanced for at least
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2 h after turpentine oil. In contrast, the mechanical responsiveness of bladder afferents changed only little within 1– 2 h after mustard oil which, thereafter, tended to induce desensitisation (Ha¨ bler et al., 1993b). From these results it follows that during inflammation there is a continuous substantial afferent inflow from the bladder to the spinal cord and that the afferent input to the spinal cord is enhanced during bladder distension, at least after turpentine oil. The increased and decreased on-going activity of MVC and CVC neurones, respectively, after bladder inflammation is consistent with a continuous reflex mediated by the novel on-going activity in both thinly myelinated and unmyelinated sacral bladder afferents. However, from the increased responses of bladder afferents to distension stimuli, in particular after turpentine oil, it would have been expected that vesico-sympathetic reflexes would be considerably enhanced which was not the case. An obvious possibility would be that the changed sympathetic on-going activity obscured the enhanced reflexes after inflammation. Indeed, when the magnitude of distension-evoked reflexes was related to the sympathetic on-going activity present before inflammation, the excitatory responses were considerably greater 1 h after inflammation. However, an hour after post-inflammation the absolute magnitude of distensionevoked reflexes had again diminished, suggesting that the gain of these reflexes decreased rather than increased with time after inflammation. Thus, the sympathetic vasoconstrictor reflexes kept a similar, if not identical, close quantitative relationship to the activity in sacral bladder afferents before and after inflammation. It has been speculated that mechanically insensitive silent C-fibre afferents might serve a function different from that of their mechanosensitive counterparts (Michaelis et al., 1996). From the present study it can be concluded that at least with respect to eliciting reflexes in sympathetic neurones they appear to have effects similar to those of other sacral afferents supplying the bladder. Therefore,
Fig. 8. There is almost no correlation between relative changes in on-going activity following bladder inflammation and normalised excitatory reflexes to bladder distension in MVC neurones. Correlation coefficient 0.17, P ¼ 0:47.
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these mechanically insensitive afferent fibres may differ from the other bladder afferents in their encoding properties, but not in their central connectivity. Several studies present evidence that dorsal horn neurones may show enhanced responses after noxious stimulation or inflammation of visceral organs. Conditioning stimuli applied to visceral afferents in the absence of inflammation increased the somatic receptive fields of dorsal horn neurones receiving convergent viscero-somatic input (Cervero et al., 1992; Laird et al., 1996). After bladder inflammation with turpentine oil, rat dorsal horn neurones responsive to bladder stimulation to some extent increased their responses to bladder distension and to electrical stimulation of the pelvic nerve (McMahon, 1988). Neurones of the post-synaptic dorsal column system excited by colon distension increased their responses after colon inflammation with mustard oil (Al-Chaer et al., 1997), and rat dorsal horn neurones sensitive to electrical stimulation of the pelvic nerve responded more strongly to colorectal distension after colon inflammation with acetic acid (Olivar et al., 2000). Roza et al. (1998) found a more complex behaviour of dorsal horn neurones responsive to ureter input 1–4 days after implantation of a ureteric stone. The prevalence and frequency of on-going activity was enhanced in these neurones, but otherwise there were signs of increased as well as decreased excitability. In these studies, the apparently increased responses to distension of the inflamed viscus could be explained by a higher level of convergent input from sensitised visceral afferents and does not necesssarily reflect an increase in the gain of transmission of this particular afferent input. It was argued that electrical stimulation of the pelvic nerve would avoid the problem of adequately stimulating sensitised afferents, and therefore, reveal central sensitisation, but the afferent impulses evoked electrically still impinge on neurones receiving continuously increased afferent input from the inflamed viscus. Thus, these studies may be reconciled with the present results which indicate that the increased afferent input into the spinal cord due to peripheral sensitisation of visceral afferents after inflammation is sufficient to explain the magnitude of vesico-sympathetic reflexes early after inflammation. Wind-up represents a true increase in the gain of motor reflexes upon low frequency stimulation at C fibre strength of the corresponding dorsal root, and it is widely assumed to indicate sensitisation of dorsal horn neurones (Doubell et al., 1999; see Herrero et al., 2000; but see Woolf, 1996). However, wind-up could not be elicited by electrical stimulation of visceral afferents in the splanchnic nerve (Cervero, 1995; Laird et al., 1995) which appears to be consistent with our failure to observe amplified vesico-sympathetic reflexes after bladder inflammation. At present, there is no experimental basis to refute the assumption that spinal somatomotor or sympathetic reflexes are valid and representative indicators of the excitability of dorsal horn neurones involved in the processing of corresponding afferent input.
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