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Traction on the mesentery as a model of visceral nociception
Pain 80 (1999) 319–328
Traction on the mesentery as a model of visceral nociception Ulrike Holzer-Petsche*, Birgit Brodacz Department of Experimental and Clinical Pharmacology, Karl-Franzens-University, Universita¨tsplatz 4, A-8010 Graz, Austria Received 2 March 1998; received in revised form 25 September 1998; accepted 27 October 1998
Abstract Traction of the mesentery is known to induce strong autonomic reactions in patients undergoing abdominal surgery. An experimental model using this stimulus in anaesthetized rats has been developed, which allows the comparison of noxious mechanical and chemical stimulation of the mesentery. Graded traction on a bundle of jejunal vessels with 2–30 g led to reflex changes in blood pressure and intragastric pressure, the size of which correlated with the strength of the stimulus. Comparable responses were elicited by clamping the same vessels either at their distal or proximal end or by applying 100 ml 0.6% acetic acid or 0.1 mM bradykinin. These reflexes are fairly insensitive towards impairment of the autonomic system. Only the combination of phentolamine and propranolol reduced the cardiovascular responses to all stimuli but at the same time, significantly lowered basal blood pressure. The adrenoceptor antagonists affected the gastric response to acid only. Atropine on its own was ineffective. Administered together with the combination of adrenoceptor blockers it had no further influence on the cardiovascular reflexes but significantly reduced the gastric responses to stretch, proximal clamping and acid. Acute desensitization of small diameter afferents with capsaicin almost abolished the reflex responses to acid. The cardiovascular, but not the gastric, response to traction was reduced by capsaicin. Morphine led to dose-dependent reductions of the reflex responses in a naloxone-reversible manner, whereas indomethacin was inactive. The bradykinin B2-antagonist icatibant abolished the reflex in response to the application of bradykinin but not to acid or traction. It is concluded that the measurement of the cardiovascular and gastric responses of anaesthetized rats to traction on the mesentery is a suitable method to investigate acute visceral nociception. Chemical stimuli to the mesentery are transmitted by capsaicin-sensitive afferents, but there is a dichotomy regarding capsaicin’s influence on visceral mechanonociception. Opioid mechanisms are always involved, whereas prostaglandins or bradykinin have no role in the reflexes evoked by acid or traction. Intact a- or b-adrenergic (as tested with unselective receptor antagonists) or muscarinic mechanisms are required for the reaction of the end organs in the reflex but they have no role in the afferent or central processing. 1999 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Visceral pain; Mechanonociception; Chemonociception
1. Introduction The method most frequently used to study visceral mechanonociception is distension of a viscus. In the case of the intestine, however, this procedure leads not only to the stimulation of stretch receptors in the intestinal wall but often also to a displacement of the gut with concomitant
* Corresponding author. Tel.: +43-316-380-4510; fax: +43-316-3809645; e-mail: [email protected]
excitation of mesenteric mechanoreceptors (Morrison, 1973). Thus, experiments using distension most likely measure the effects of a mixture of visceral stimuli. Here, another model to study visceral nociception in anaesthetized rats is characterized, which allows to study the effects of traction on the mesentery. Such a stimulus has already been described in 1942 by Lewis (Lewis, 1942) to induce pain in humans and is well known from the clinical setting to induce strong autonomic reactions in patients undergoing abdominal surgery. The mechanoreceptors that sense these stimuli are situated near the jejunal arteries, in particular close to their branching points, and the fibres travel with
0304-3959/99/$ - see front matter 1999 International Association for the Study of Pain. Published by Elsevier Science B.V. PII: S03 04-3959(98)002 33-4
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the splanchnic nerves (Bessou and Perl, 1966; Morrison, 1973). The experiments were done in rats under barbiturate anaesthesia. In these animals pseudaffective reflexes, i.e. changes in blood pressure and intragastric pressure, served as indicators of nociception. Since the animals’ ability to react in such ways strongly depends on the general status of the autonomic nervous system, the influence of drugs inhibiting a- or b-adrenergic receptors or muscarinic receptors was investigated. By these means the reliability of the responses could be judged under different baseline states of the cardiovascular system and the stomach. Only then was the experimental model tested for its sensitivity towards drugs known to inhibit nociceptive mechanisms. Preliminary results have been published in abstract form (Holzer-Petsche and Brodacz, 1995, 1997).
2. Methods 2.1. Experimental setup The experiments were approved by the Commission for Animal Experiments of the Austrian Ministry of Science. Female Sprague–Dawley rats (200–300 g body weight; Forschungsinstitut fu¨r Versuchstierkunde, Himberg, Austria) were fasted overnight with free access to tap water. The experimental setup is shown in Fig. 1. Under phenobarbitone anaesthesia (250 mg/kg i.p.) the trachea was cannulated to keep the airways patent and the oesophagus ligated in the neck. Mean arterial pressure (MAP) was monitored from a carotid artery. A jugular vein was cannulated for continuous i.v. infusion of isotonic saline (1.5 ml/h) and for drug injections. After laparotomy, a cannula was introduced into the stomach through an incision in the duodenum and fixed by a ligation around the pylorus for measurement of intragastric pressure (IGP) at a constant volume of 5 ml isotonic saline. The body temperature was kept at 37–38°C by means of a heating pad. A hook was positioned around the superior mesenteric
artery just before its division into the jejunal arteries to prevent displacement of the major vessels. A thread was tied loosely around a jejunal artery and vein just proximal to their branching to the gut. The other end of the thread was led over a pulley in order to attach weights of 2–30 g for stretching the vessels. The intestinal loop was placed onto the abdominal wall, on a piece of cotton soaked in saline, and covered with parafilm. As judged by inspection, this procedure did not compress the blood vessels, neither did it seem it to induce any mechanical irritation, since there were no alterations to the cardiovascular or gastric parameters. The effect of traction on the jejunal vessels for 1 min was compared with the effect of clamping the same vessels with a bulldog clamp right before their branching into the arcades (‘distal clamp’) for 1 min or at their leaving the mesenteric artery (‘proximal clamp’) for 3 min. The effect of the ‘distal clamp’ corresponds with a pure mechanical stimulus comparable with pinching the skin, whereas the ‘proximal clamp’ also leads to a transient hypoxia in a short gut segment as judged by the darkening and reddening of the supplied tissue. A further stimulus consisted in dropping 100 ml of 0.6% (v/v) acetic acid onto the same bundle of jejunal vessels (‘acid’). In one set of experiments, 10 pmol bradykinin was applied in a volume of 100 ml. Chemicals applied in such a way did not get into contact with the parietal peritoneum, thus the stimulus was purely visceral. After a first series of stimuli at 5 min-intervals (2, 5, 10, 20 and 30 g, distal and proximal clamp and acid) the experimental animals were injected i.v. with a single drug or a drug combination (1 ml/kg) and 10 min later (30 min in the case of indomethacin) the series of stimuli were repeated. Changes in MAP and IGP were taken as signs of pseudaffective reflexes (Cervero and McRitchie, 1982; HolzerPetsche, 1992). The reflex size after drug injection was then compared with the value obtained during the first series of stimuli, thereby compensating for interindividual variations. In the experiments with capsaicin the test stimuli were traction with 20 g and acetic acid, each applied once as a control stimulus. At time 0 capsaicin was injected s.c. (50 mg/kg). After capsaicin administration, traction was repeated every 10 min up to 60 min after capsaicin. The chemical stimulus was repeated 35 and 65 min after capsaicin. Both types of stimuli were therefore tested in the same rats. 2.2. Statistics
Fig. 1. Diagram of the experimental setup. A detailed description is given in Section 2.
The results are presented as mean ± SEM. If there was no response to a particular stimulus, the value was entered into the calculation as 0. The software SigmaStat was used to calculate Student’s t-test, paired t-test, one-way ANOVA, or one-way repeated measure ANOVA (followed by Dunnett’s test), as appropriate. Relationships between the strength of traction and the size of the reflex responses were determined
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chloride, Aldrich (Steinheim, Germany); atropine sulfate, Merck (Darmstadt, Germany); capsaicin, Serva (Heidelberg, Germany); morphine hydrochloride, Diosynth (Apeldoorn, The Netherlands); naloxone hydrochloride, DuPont (Geneva, Switzerland); indomethacin, Merck Sharp and Dohme (Rahway, NJ, USA); bradykinin, Bachem (Bubendorf, Switzerland); icatibant, Hoechst (Frankfurt, Germany). Capsaicin was dissolved in 10% ethanol, 10% Tween 80 and 80% isotonic NaCl at 25 mg/ml and injected s.c. at a volume of 2 ml/kg. Indomethacin was dissolved in 0.66% Na2CO3. All other drugs were dissolved in, and diluted with, isotonic saline and injected i.v. at 1 ml/kg.
3. Results 3.1. Characterization of the experimental model
Fig. 2. Tracings of mean arterial pressure (MAP) and intragastric pressure (IGP) during mesenteric stimulation. The duration of the mechanical stimuli is indicated by black bars, application of acetic acid by arrowhead. Asterisks indicate spontaneous contractions. The evaluation of the response is shown for the 30 g stimulus.
by a first order regression using the least squares method, and the Pearson product moment correlation coefficient was calculated as an indicator for the strength of the association. 2.3. Materials The chemicals were obtained from the following sources: phentolamine methane-sulfonate and propranolol hydro-
Graded traction on the jejunal vessels with weights of 5– 30 g led to prompt and transient changes in MAP and IGP (Fig. 2). Traction with 2 g was subthreshold in all cases but one. The changes in MAP usually consisted of an immediate fall followed by a rise, whereas the stomach reacted only with relaxations reflected in a transient lowering of the IGP, similar to those observed in a previous study (HolzerPetsche, 1992). Between animals it was unpredictable whether an increase or a decrease of MAP would prevail, in some animals there was even only a monophasic response. However, there was no correlation between the basal blood pressure at the time of the stimulus and the quality of the response. Only when MAP fell below 50 mmHg, did the responses to noxious stimulation cease consistently. Therefore, the total deviation of MAP from baseline (absolute value of rise plus absolute value of fall) rather than the isolated falls or rises was evaluated (see Fig. 2). By this method, a highly significant correlation was obtained between the magnitude of the responses and the weight
Fig. 3. Pseudaffective reflex responses to mesenteric traction with weights of 2–30 g. Individual values are plotted (n = 9 for MAP n = 6 for IGP). The line shows the first order regression calculated by the least squares method. The Pearson product–moment correlation coefficient (r) is inserted in each graph. All correlations are statistically significant (P , 0.001).
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applied (Fig. 3). However, after traction with 5 g or distal clamping responses often seemed random, although basal values were not significantly altered. Therefore, in the experiments testing drug effects, only stimuli .5 g were evaluated. When a clamp was put on the bundle of vessels, the reflex responses to the ‘distal clamp’ were generally smaller than those to the ‘proximal clamp’, probably as an indication of the different number of nociceptors stimulated at the two clamping sites. Furthermore, the time course of the responses differed from those to traction: during clamping MAP and IGP, after the peak, remained at a plateau value for the duration of the stimulus. After application of a chemical stimulus, a prompt change of MAP and IGP was seen, after which both parameters gradually returned to baseline, most likely reflecting the time course of elimination of the chemical agent. Although respiration was not recorded, the rats breathed faster and deeper during a stimulus (see Ness and Gebhart, 1990). At the end of the first experiments, after euthanasia, the maximum weight tolerated by a bundle of mesenteric vessels before rupture was evaluated. In 11 rats, mesenteric vessels tolerated traction with at least 75 g, in one rat, however, they ruptured at 40 g. Therefore no weight higher than 30 g was applied. After i.v. injection of isotonic saline (1 ml/kg) there was a tendency for greater responses to most stimuli (Fig. 4 top panels). These differences reached statistical significance for the IGP response to traction with 30 g. Since, however, the anticipated effect of the test drugs was to reduce rather than enhance nociception, the responses of the animals to mesenteric noxious stimuli were evaluated before and after systemic administration of drugs.
Injection of atropine (1 mg/kg i.v.) alone had no effect on basal measurements, in combination with the adrenoceptor blockers it lowered basal MAP but did not change basal IGP (Table 1). At the end of the experiment 2/8 rats in the latter group had a basal MAP ,50 mmHg. On its own, atropine did not influence the responses to the noxious stimuli. If given in combination with the a- and badrenoceptor blockers it led to a reduction of all reflex responses, which was statistically significant except for the response to distal clamping (Fig. 4 bottom panels). Quantitatively, the MAP responses did not differ from those observed with the adrenoceptor blockers alone, whereas the gastric responses became smaller than in the former group. These observations also show that reliable reflex responses could be obtained over a wide range of basal MAP and that lowering of basal MAP per se did not necessarily abolish the reflex response. In individual rats it was noted that the MAP responses ceased consistently only when basal MAP fell well below 50 mmHg, regardless of the type of drug injected. For this reason, rats with MAP
3.2. Efferent mechanisms Phentolamine (1 mg/kg i.v. as bolus followed by an infusion of 1 mg/kg × h) significantly reduced basal MAP while propranolol (0.3 mg/kg i.v. followed by 0.3 mg/kg × h) reduced basal MAP and increased IGP (Table 1). As expected, administration of phentolamine plus propranolol strongly modified basal MAP as detailed in Table 1. In this group, 4/10 rats had a basal MAP ,50 mmHg by the end of the experiment, while in none of the control rats or the rats injected with only one compound such a profound alteration of basal MAP was observed. Basal intragastric pressure was not significantly altered after phentolamine plus propranolol. Administration of either phentolamine or propranolol alone had no influence on the reflexes induced by the various noxious stimuli. The combination of the a- and badrenoceptor antagonists reduced the changes in MAP after all stimuli, but the reduction of the responses to clamping did not reach statistical significance (Fig. 4 middle panel). The gastric response was significantly reduced by the antagonists only when acetic acid was used as a stimulus (Fig. 4 middle panel).
Fig. 4. Reflex changes of mean arterial pressure (MAP, left panels) and intragastric pressure (IGP, right panels) in response to traction of the mesentery with weights of 10, 20 and 30 g, clamping of the vessels under investigation close to the gut for 1 min (‘D’ distal), clamping close to the branching from the superior mesenteric artery for 3 min (‘P’ proximal) and to application of 100 ml 0.6% acetic acid (‘Ac’). White columns, 1st series of stimuli before drug; hatched columns, 2nd series of stimuli started 10 min after i.v. injection of drugs as indicated. NaCl, 1 ml/kg isotonic saline (n = 6); Phe + Pro, 1 mg/kg phentolamine as bolus followed by 1 mg/kg × h together with 0.3 mg/kg propranolol as bolus followed by 0.3 mg/kg × h (n = 10); Atr + Phe + Pro, same as Phe + Pro plus a bolus of 1 mg/kg atropine (n = 8). Columns denote means with bars indicating SEM; *P , 0.05, **P , 0.01 versus before drug (paired ttest); n.t., not tested.
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U. Holzer-Petsche, B. Brodacz / Pain 80 (1999) 319–328 Table 1
Baseline values of mean arterial pressure (MAP) and intragastric pressure (IGP) immediately before and 10 min after i.v. administration of drugs. Values are means ± SEM; n in parentheses. The values before drug injection were not significantly different between the six groups. MAP (mmHg) Before saline (1 ml/kg i.v.) After saline (1 ml/kg i.v.) Before atropine (1 mg/kg i.v.) After atropine (1 mg/kg i.v.) Before phentolamine (1 mg/kg bolus followed by 1 mg/kg × h i.v.) After phentolamine (1 mg/kg bolus followed by 1 mg/kg × h i.v.) Before propranolol (0.3 mg/kg bolus followed by 0.3 mg/kg × h i.v.) After propranolol (0.3 mg/kg bolus followed by 0.3 mg/kg × h i.v.) Before phentolamine + propranolol After phentolamine + propranolol Before phentolamine + propranolol + atropine After phentolamine + propranolol + atropine
85 90 101 94 109 66 105 87 111 57 106 62
± ± ± ± ± ± ± ± ± ± ± ±
5 4 (6) 7 5 (6) 7 5** (6) 6 10* (7) 5 5** (10) 7 6** (8)
IGP (Pa) 885 871 840 917 833 790 711 800 718 772 772 753
± ± ± ± ± ± ± ± ± ± ± ±
64 73 (6) 50 73 (6) 97 74 (6) 42 59** (7) 43 72 (10) 82 85 (8)
*P , 0.05; **P , 0.01 versus before drug (paired t-test).
values falling ,50 mmHg were excluded from the following part of the study. 3.3. Afferent mechanisms Subcutaneous injection of 50 mg/kg capsaicin had no effect on the baseline values of MAP and IGP, whereas after vehicle basal MAP decreased within 10 min from 100 ± 5 to 92 ± 6 mmHg (n = 7, P , 0.05). These changes, however, were transient and basal IGP was not altered. Apart from an initial increase in the MAP response to traction and a significant increase of the IGP response to acid, nociception was not altered by s.c. treatment with vehicle. Capsaicin administration almost abolished the responses to the chemical stimulus, the MAP response to traction decreased significantly, but there was no effect on the IGP response to traction (Fig. 5). In order to test whether the observed reflexes were indeed a correlate of nociception, rats were treated with i.v. morphine. After morphine injection basal MAP gradually declined until the end of the experiments (from 101 ± 5 to 82 ± 7 and from 95 ± 7 to 61 ± 5 mmHg after 1 and 5 mg/ kg respectively, each n = 6, P , 0.05). Naloxone had no such effect. Basal IGP values were never significantly altered. Whereas, 1 mg/kg morphine reduced only the responses to the weaker stimuli, 5 mg/kg significantly reduced the reflex responses to all stimuli except proximal clamping (Fig. 6). Naloxone (5 mg/kg) itself was without effect but prevented the action of 5 mg/kg morphine except for the MAP responses to traction with 10 and 20 g. A possible involvement of prostaglandins in the observed reflexes was investigated by the use of indomethacin. Intravenous injection of 10 mg/kg indomethacin led to gradual decreases in MAP and increases in IGP (from 89 ± 5 to 65 ± 4 mmHg and from 294 ± 47 to 378 ± 70 Pa, n = 6, P , 0.05). Injection of 0.66% Na2CO3 as vehicle had no such effect, nor did it influence the reactions to noxious stimuli. Indomethacin only reduced the MAP responses to
traction with 10 g and to distal clamping (Fig. 7). At the end of the experiments, approximately 90 min after indomethacin injection, all rats presented with haemorrhagic streaks on their gastric mucosae at macroscopic inspection. Such lesions were never seen in any other group. In order to check whether the production of bradykinin occurred at some stage of the processing of noxious stimuli to the mesentery, the bradykinin B2-antagonist icatibant was used. In seven rats, i.v. injection of icatibant (100 nmol/kg) had no effect on basal parameters and did not influence the
Fig. 5. Effect of capsaicin (50 mg/kg s.c.) or its vehicle on the reflex responses to noxious stimulation of the mesentery. W response to traction (20 g) in vehicle-treated rats X response to traction (20 g) in capsaicintreated rats A response to acid in vehicle-treated rats B response to acid in capsaicin-treated rats. Symbols denote means with bars indicating SEM, n = 6 for capsaicin, n = 7 for vehicle. *P , 0.05 versus before s.c. injection, one-way RM ANOVA; †P , 0.05 versus vehicle, Student’s t-test.
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Fig. 6. Effect of morphine and naloxone on the reflex responses to noxious stimulation of the mesentery (abbreviations as in Fig. 4). White columns, 1st series of stimuli before drug; hatched columns, 2nd series of stimuli started 10 min after i.v. injection of drugs as indicated. NAL, 5 mg/kg naloxone; Mo1, 1 mg/kg morphine; Mo5, 5 mg/kg morphine; Mo5 + NAL, 5 mg/kg each naloxone plus morphine. Columns denote means with bars indicating SEM, each n = 6; *P , 0.05, **P , 0.01 versus before drug (paired t-test).
stimulus in humans. In the form applied in the present setup, it is reproducible, its intensity can be controlled and the response correlates with the size of the stimulus. It can be applied selectively to the mesentery without inadvertently influencing the gut beyond lifting up a piece of small intestine. This, however, appears not to arouse nocifensive reflexes on its own since there are definitely subthreshold intensities of traction during whose application the intestine is equally raised. Chemical stimuli can also be repeatedly employed, and under the present conditions no sensitization or desensitization phenomena have been observed. The use of phenobarbitone as a long acting anaesthetic produces a stable level of anaesthesia throughout experiments of up to 150 min, which is demonstrated by the reflex responses being reproducible after i.v. administration of NaCl. Since Sherrington (1906) pseudaffective reflexes have been used as a measure of nociception, also in anaesthetized rats (e.g. Cervero and McRitchie, 1982; Holzer et al., 1992; Holzer-Petsche, 1992). Alterations of blood pressure occur regularly, but their shape can vary depending on the type of anaesthesia (Ness and Gebhart, 1988). So far, no explanation has been found for the qualitative variations of the changes in MAP in the present study. Overall however, the size of both autonomic reflexes as evaluated in the present study correlates with the strength of the stimulus, similar to the rate of discharge of mesenteric fibres in the experiments by Morrison (1973). A good correlation of the size of the response with the stimulus intensity has also been observed by Ness and Gebhart (1988) after colorectal distension in awake rats. Thus, such a relationship seems to be valid for several types of mechanical stimuli. This fact leads to the inference that proximal clamping is a stronger stimulus than distal clamping. One reason could be that a clamp positioned close to the branching of the jejunal
reflex responses to traction, clamping or acid, although it abolished the changes in MAP and IGP in response to the application of 10 pmol bradykinin onto the mesenteric bundle (data not shown). A comprehensive list of drug effects on the reflex responses is shown in Table 2.
4. Discussion 4.1. General aspects The experimental model presented here allows to study the effects of different types of mechanical and chemical noxious stimuli in the peritoneal cavity, at a location that is considered to be supplied by visceral afferent nerves. The present setup is aimed primarily at investigating the sites and transmitter mechanisms involved in acute mesenteric nociception. Mesenteric traction is known to be a noxious
Fig. 7. The effect of indomethacin on the reflex responses to noxious stimulation of the mesentery (abbreviations as in Fig. 4). White columns, 1st series of stimuli before drug; hatched columns, 2nd series of stimuli started 30 min after i.v. injection of drugs as indicated. Na2CO3, 1 ml/kg, 0.66% sodium carbonate (solvent for indomethacin) (n = 5); INDO, 10 mg/kg indomethacin (n = 6). Columns denote means with bars indicating SEM; *P , 0.05 versus before drug (paired t-test).
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U. Holzer-Petsche, B. Brodacz / Pain 80 (1999) 319–328 Table 2
Summary of the effects of manipulations on changes in mean arterial pressure (MAP) and intragastric pressure (IGP) induced by noxious stimuli. For detailed explanations of the treatments see Section 2 Treatment
Effect on changes in MAP (mmHg)
Traction (g)
10
20
30
10
20
30
NaCl Atropine Phentolamine Propranolol Phe + Pro Phe + Pro + Atr Capsaicin (60 min) Naloxone Morphine (5 mg/kg) MO + NAL Indomethacin Icatibant
n.c. n.c. n.c. n.c. –– n.t. n.t. n.c. –– –– –– n.c.
n.c. n.c. n.c. n.c. –– –– –– n.c. –– –– n.c. n.c.
n.c. n.c. n.c. n.c. – n.t. n.t. n.c. –– n.c. n.c. n.c.
n.c. n.c. n.c. n.c. n.c. n.t. n.t. n.c. –– n.c. n.c. n.c.
n.c. n.c. n.c. n.c. n.c. – n.c. n.c. –– n.c. n.c. n.c.
+ n.c. n.c. n.c. n.c. n.t. n.t. n.c. –– n.c. n.c. n.c.
D
P
Acid
D
P
Acid
n.c. n.c. n.c. n.c. n.c. n.c. n.t. n.c. –– n.c. –– n.c.
n.c. n.c. n.c. n.c. n.c. – n.t. n.c. n.c. n.c. n.c. n.c.
n.c. n.c. – n.c. –– –– –– n.c. –– n.c. n.c. n.c.
n.c. n.c. + + n.c. n.c. n.c. n.t. n.c. –– n.c. n.c. n.c.
n.c. n.c. n.c. n.c. n.c. – n.t. n.c. n.c. n.c. n.c. n.c.
n.c. n.c. n.c. n.c. – –– –– n.c. – n.c. n.c. n.c.
NaCl Atropine Phentolamine Propranolol Phe + Pro Phe + Pro + Atr Capsaicin (65 min) Naloxone Morphine (5 mg/kg) MO + NAL Indomethacin Icatibant
Effect on fall of IGP (Pa)
D, distal clamp; P, proximal clamp; n.c., no change; +/−, statistically significant (P , 0.05) increase or decrease of the response by ,50%; ++/−−, statistically significant increase or decrease of the response by .50%; n.t., not tested.
arteries from the superior mesenteric artery compresses a greater amount of tissue and thus stimulates a greater number of nociceptors than a clamp at the distal end of the jejunal vessels. On the other hand, a contribution of intestinal hypoxia cannot be excluded since the piece of gut supplied by the blood vessels under investigation became hypoxic during the 3-min stimulation as judged from the darkening of the tissue. In line with this, Longhurst and Dittman (1987) found that visceral afferents sensitive to hypoxia already started to fire after a mean latency of 63 s. In the present setup the threshold for the mechanoreceptors seems to be rather high, since there was practically no response to traction with 2 g although the mesenteric vessels appeared well stretched. The different shapes of the MAP responses to application of tension or clamping could indicate a qualitatively different processing of the two types of stimuli. It might be speculated that tension is mediated by fibres adapting more rapidly than the fibres excited by clamping the tissue. As to chemical stimuli, no attempt has been made to look for a dose-response relationship in the present setup. Instead, the concentration of acetic acid matched that used in writhing tests (Berkenkopf and Weichman, 1988).
4.2. Alteration of autonomic functions As the sympathetic nervous system is always activated when the organism has to cope with stressful situations, the importance of patent adrenergic mechanisms for the responses to visceral nociceptive stimuli has been investigated. Phentolamine or propranolol alone did not change the reflex responses measured. This is unlikely to be caused by an insufficient dose of the compounds since either of them significantly altered basal MAP. Only the combination of the a- with the b-receptor antagonist was able to reduce the cardiovascular responses to nociceptive stimulation. This is in line with a previous study, where changes in MAP in response to i.p. chemical stimuli were abolished by the combination of pretreatment with guanethidine and adrenalectomy but not by either procedure alone (Holzer-Petsche, 1992). In contrast, either adrenoceptor antagonist alone significantly decreased cardiovascular reflex responses to colorectal distension in awake rats (Ness and Gebhart, 1988) and the gastric relaxation in response to noxious pressure applied to a testicle in rats anaesthetized with chloralose (Bojo¨ et al., 1992). Possibly the type of anaesthesia may modify the contribution of the sympathetic nervous system in the reflex response and thereby the apparent sensitivity of
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the animals to the adrenoceptor blockers in the present experiments. Muscarinic receptors seem not to play a major role in these reflex responses as judged by the lack of effect of atropine alone. This is in line with earlier observations by Radhakrishnan et al. (1985), where the reflex change in blood pressure to serosal application of noxious chemicals was not altered by atropine. The latter study had been performed in rats anaesthetized with urethane. It could, therefore, be argued that under general anaesthesia there is little influence of the parasympathetic nervous system on cardiovascular reflexes to noxious stimulation. The two adrenoceptor blockers did not impair the fall in IGP except when evoked by acid, nor did atropine pretreatment have a major effect on the gastric response to noxious stimuli. Similar observations have been made by Glise and Abrahamsson (1980) in cats where neither the gastric inhibition by noxious mechanical stimulation of the gut nor that induced by i.p. hydrochloric acid was blocked by antiadrenergic or anticholinergic drugs. It needed the administration of atropine on top of both adrenoceptor blockers to significantly reduce the fall in IGP after all stimuli but distal clamping. Thus, both a reduction in parasympathetic and an increase in sympathetic tone are responsible for the gastric relaxation occurring with nociception. Overall, although the observed pseudaffective reflexes obviously depend on the integrity of the autonomic nervous system, they seem to be rather robust towards elimination of only one of its components (a- or b-adrenergic or muscarinergic transmission). Furthermore, the observations indicate that measurements of the above reflexes are reliable over a great range of basal MAP or IGP and make the experimental model suitable for testing drugs influencing nociceptive mechanisms. 4.3. Antinociceptive measures The sensory neurotoxin capsaicin has been shown to attenuate or abolish a number of nociceptive reflexes, both somatic and visceral ones (see Holzer, 1991). In a model similar to the one used here, cardiovascular and gastric reflex responses to i.p. acid and bradykinin were markedly decreased in rats treated systemically with capsaicin 2 weeks earlier (Holzer-Petsche, 1992). Furthermore, these capsaicin-sensitive afferents have been shown to mediate the gastric inhibition occurring after laparotomy (Holzer et al., 1992). Also in the present experiments the reflexes to the application of acetic acid were almost abolished after s.c. injection of capsaicin with a time course corresponding to the kinetics of capsaicin as measured by Saria et al. (1982) and to the disappearance of the capsaicin-sensitive micturition reflex (Holzer-Petsche and Lembeck, 1984). However, there was no unequivocal effect on the reflexes induced by mesenteric traction: while the gastric response was unchanged both after capsaicin and vehicle, there was a reduction of the MAP response after capsaicin injection.
While there is consensus about the desensitization of chemonociceptors by capsaicin (Cervero and McRitchie, 1982; Holzer et al., 1992), there is conflicting evidence about the effect of capsaicin on mechanosensitive nerve fibres. Small diameter, high-threshold mechanosensitive fibres which, in addition respond to heat, are also sensitive to capsaicin (Szolcsa´nyi et al., 1988; Gallar et al., 1993; Seno and Dray, 1993). However, there are no intestinal afferents known to react to heat stimuli (see Cervero, 1994), and somatic fibres sensitive to mechanical but not to heat stimuli can neither be excited by low doses, nor desensitized by high doses of capsaicin (Szolcsa´nyi et al., 1988; Seno and Dray, 1993). In some cases, such as the response of articular mechanoreceptors (He et al., 1990), the block of the response by systemic capsaicin was variable. Contradictory observations were also made on afferents of abdominal organs. Whereas the slow depolarization in neurones of the inferior mesenteric ganglion evoked by distention of the ureter and the change in blood pressure in response to distension of the biliopancreatic duct were abolished by capsaicin (Amann et al., 1988; Griesbacher, 1994), gastric mechanoreceptors appeared to be insensitive to capsaicin (Berthoud et al., 1997). In the present study there was a striking difference between the responses of the cardiovascular system and the stomach to the mechanical stimulus after capsaicin. Since the reflex can reasonably have only one afferent pathway, one could speculate that processes in the CNS, at the point of divergence of the reflex to the various effector systems, are differentially sensitive to capsaicin. A similar dissociation between the responses of different end organs has been mentioned by Gebhart and Ness (1991). In their (awake) rats, which had been treated with capsaicin as neonates, the distension threshold of the colon to elicit a visceromotor reflex was doubled, but no change was reported for the cardiovascular reflex. However, one should take care not to equate capsaicin treatment of neonates with that of adult animals. Loss of capsaicin-sensitive nerves in the first days of life leads to compensatory changes in the afferent innervation that are absent in adult animals (Holzer, 1991). The present experimental model has also been tested for its response to morphine, in order to verify that the reflexes under investigation are nocifensive. Morphine is effective in inhibiting the responses to traction, distal clamping and acetic acid, but does not attenuate that to proximal clamping. However, the latter certainly was the strongest stimulus applied in the present study and there is evidence that the effectiveness of a particular dose of morphine is inversely related to the strength of the stimulus (Saeki and Yaksh, 1993). Naloxone at the rather high dose of 5 mg/kg i.v. had no effect of its own. It did, however, counteract the effect of morphine, although not in the case of MAP after 10 and 20 g traction. Most likely a persisting effect of morphine against the two low stimuli is responsible for this remaining antinociception. It cannot be due to pharmacokinetic differences between morphine and naloxone, because
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firstly they were administered together i.v. and secondly because the effect of morphine on the gastric response measured simultaneously was abolished by naloxone. Indomethacin was ineffective in blocking the nocifensive reflexes after visceral stimulation, in contrast to its antinociceptive action when acetic acid is used as stimulus in the writhing test (Berkenkopf and Weichman, 1988), However, in the latter test, a relatively large volume of the irritant is present in the peritoneal cavity for several minutes before the rat’s behaviour is evaluated. Thus, there is plenty of time for the synthesis of prostaglandins to take place and to participate in the noxious stimulation. In the present setup, in contrast, the immediate autonomic reactions to a small volume of acid were measured, making it unlikely for any prostaglandins to contribute to the stimulation before the acid was diluted and buffered. Nevertheless, the dose of indomethacin was high enough to cause gastric mucosal lesions, whose development might have been facilitated by the ligation of the pylorus around the gastric cannula. In models of acute nociception the action of bradykinin has been shown to be mediated by B2 receptors (Hall, 1992). In the present setting the B2-antagonist icatibant blocks only the reflexes induced by mesenteric application of bradykinin, and not those induced by the other types of stimuli. Similarly, gastric motor inhibition induced by HCl was unaffected by icatibant (Hoe-140), whereas, bradykinininduced inhibition was blocked (Holzer, 1992). Thus, bradykinin via B2 receptors does not play a role in the transmission of mechanical and acute chemical stimuli.
5. Conclusions The present data confirm that both the cardiovascular system and the stomach react in response to visceral noxious stimuli. Mesenteric traction can be used as a model for visceral mechanonociception and the stimuli described can be compared with other types of noxious input. This setup can serve as a basis for further pharmacological investigations into the mechanisms of nocifensive reflexes.
References Amann, R., Dray, A. and Hankins, M.W., Stimulation of afferent fibres of the guinea-pig ureter evokes potentials in inferior mesenteric ganglion neurones, J. Physiol. (Lond.), 402 (1988) 543–553. Berkenkopf, J.W. and Weichman, B.M., Production of prostacyclin in mice following intraperitoneal injection of acetic acid, phenylbenzoquinone and zymosan: its role in the writhing response, Prostaglandins, 36 (1988) 693–709. Berthoud, H.-R., Patterson, L.M., Willing, A.E., Mueller, K. and Neuhuber, W.L., Capsaicin-resistant vagal afferent fibers in the rat gastrointestinal tract: anatomical identification and functional integrity, Brain Res., 746 (1997) 195–206.
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Bessou, P. and Perl, E.R., A movement receptor of the small intestine, J. Physiol. (Lond.), 182 (1966) 404–426. Bojo¨, L., Cassuto, J. and Nellga˚rd, P., Pain-induced inhibition of gastric motility is mediated by adrenergic and vagal non-adrenergic reflexes in the rat, Acta Physiol. Scand., 146 (1992) 377–383. Cervero, F. and McRitchie, H.A., Neonatal capsaicin does not affect unmyelinated efferent fibers of the autonomic nervous system: functional evidence, Brain Res., 239 (1982) 283–288. Cervero, F., Sensory innervation of the viscera: peripheral basis of visceral pain, Physiol. Rev., 74 (1994) 95–138. Gallar, J., Pozo, M.A., Tuckett, R.P. and Belmonte, C., Response of sensory units with unmyelinated fibres to mechanical, thermal and chemical stimulation of the cat’s cornea, J. Physiol. (Lond.), 468 (1993) 609– 622. Gebhart, G.F. and Ness, T.J., Central mechanisms of visceral pain, Can. J. Physiol. Pharmacol., 69 (1991) 627–634. Glise, H. and Abrahamsson, H., Spino-vagal non-adrenergic inhibition of gastric motility elicited by abdominal nociceptive stimulation in the cat, Scand. J. Gastroenterol., 15 (1980) 665–672. Griesbacher, T., Blood pressure reflexes following activation of capsaicinsensitive afferent neurones in the biliopancreatic duct of rats, Br. J. Pharmacol., 111 (1994) 547–554. Hall, J.M., Bradykinin receptors: pharmacological properties and biological roles, Pharmac. Ther., 56 (1992) 131–190. He, X., Schepelmann, K., Schaible, H.G. and Schmidt, R.F., Capsaicin inhibits responses of fine afferents from the knee joint of the cat to mechanical and chemical stimuli, Brain. Res., 530 (1990) 147– 150. Holzer, P., Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons, Pharmacol. Rev., 43 (1991) 143– 201. Holzer, P., Reflex gastric motor inhibition caused by intraperitoneal bradykinin: antagonism by Hoe 140, a bradykinin antagonist, Peptides, 13 (1992) 1073–1077. Holzer, P., Lippe, I.T. and Amann, R., Participation of capsaicin-sensitive afferent neurons in gastric motor inhibition caused by laparotomy and intraperitoneal acid, Neuroscience, 48 (1992) 715–722. Holzer-Petsche, U. and Lembeck, F., Systemic capsaicin treatment impairs the micturition reflex in the rat, Br. J. Pharmacol., 83 (1984) 935– 941. Holzer-Petsche, U., Blood pressure and gastric motor responses to bradykinin and hydrochloric acid injected into somatic or visceral tissues, Naunyn-Schmiedeberg’s Arch. Pharmacol., 346 (1992) 219–225. Holzer-Petsche, U. and Brodacz, B., Mesenteric stretch as a model of visceral nociception, Br. J. Pharmacol., 116 (Suppl.)(1995) 92P. Holzer-Petsche, U. and Brodacz, B., A model for the pharmacological investigation of mesenteric mechano- and chemonociception, NaunynSchmiedeberg’s Arch. Pharmacol., 355 (Suppl.)(1997) R105. Lewis, T., Pain, London, McMillan, 1942. Longhurst, J.C. and Dittman, L.E., Hypoxia, bradykinin, and prostaglandins stimulate ischemically sensitive visceral afferents, Am. J. Physiol., 253 (1987) H556–H567. Morrison, J.F.B., Splanchnic slowly adapting mechanoreceptors with punctate receptive fields in the mesentery and gastrointestinal tract of the cat, J. Physiol. (Lond.), 233 (1973) 349–361. Ness, T.J. and Gebhart, G.F., Colorectal distension as a noxious visceral stimulus: physiologic and pharmacologic characterization of pseudaffective reflexes in the rat, Brain Res., 450 (1988) 153–169. Ness, T.J. and Gebhart, G.F., Visceral pain: a review of experimental studies, Pain, 41 (1990) 167–234. Radhakrishnan, V., Shankar, N., Gogia, M. and Sharma, K.N., Cardiorespiratory changes following chemical applications to gut serosa, J. Autonom. Nerv. Syst., 14 (1985) 363–377. Saeki, S. and Yaksh, T.L., Suppression of nociceptive responses by spinal m opioid agonists: effects of stimulus intensity and agonist efficacy, Anesth. Analg., 77 (1993) 265–274. Saria, A., Skofitsch, G. and Lembeck, F., Distribution of capsaicin in rat
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tissues after systemic administration, J. Pharm. Pharmacol., 34 (1982) 273–275. Seno, N. and Dray, A., Capsaicin-induced activation of fine afferent fibres from rat skin in vitro, Neuroscience, 55 (1993) 563–569. Sherrington, C.S., The Integrative Action of the Nervous System, Yale University Press, New Haven, CT, 1906.
Szolcsa´nyi, J., Anton, F., Reeh, P.W. and Handwerker, H.O., Selective excitation by capsaicin of mechano-heat sensitive nociceptors in rat skin, Brain Res., 446 (1988) 262–268.
Traction on the mesentery as a model of visceral nociception Ulrike Holzer-Petsche*, Birgit Brodacz Department of Experimental and Clinical Pharmacology, Karl-Franzens-University, Universita¨tsplatz 4, A-8010 Graz, Austria Received 2 March 1998; received in revised form 25 September 1998; accepted 27 October 1998
Abstract Traction of the mesentery is known to induce strong autonomic reactions in patients undergoing abdominal surgery. An experimental model using this stimulus in anaesthetized rats has been developed, which allows the comparison of noxious mechanical and chemical stimulation of the mesentery. Graded traction on a bundle of jejunal vessels with 2–30 g led to reflex changes in blood pressure and intragastric pressure, the size of which correlated with the strength of the stimulus. Comparable responses were elicited by clamping the same vessels either at their distal or proximal end or by applying 100 ml 0.6% acetic acid or 0.1 mM bradykinin. These reflexes are fairly insensitive towards impairment of the autonomic system. Only the combination of phentolamine and propranolol reduced the cardiovascular responses to all stimuli but at the same time, significantly lowered basal blood pressure. The adrenoceptor antagonists affected the gastric response to acid only. Atropine on its own was ineffective. Administered together with the combination of adrenoceptor blockers it had no further influence on the cardiovascular reflexes but significantly reduced the gastric responses to stretch, proximal clamping and acid. Acute desensitization of small diameter afferents with capsaicin almost abolished the reflex responses to acid. The cardiovascular, but not the gastric, response to traction was reduced by capsaicin. Morphine led to dose-dependent reductions of the reflex responses in a naloxone-reversible manner, whereas indomethacin was inactive. The bradykinin B2-antagonist icatibant abolished the reflex in response to the application of bradykinin but not to acid or traction. It is concluded that the measurement of the cardiovascular and gastric responses of anaesthetized rats to traction on the mesentery is a suitable method to investigate acute visceral nociception. Chemical stimuli to the mesentery are transmitted by capsaicin-sensitive afferents, but there is a dichotomy regarding capsaicin’s influence on visceral mechanonociception. Opioid mechanisms are always involved, whereas prostaglandins or bradykinin have no role in the reflexes evoked by acid or traction. Intact a- or b-adrenergic (as tested with unselective receptor antagonists) or muscarinic mechanisms are required for the reaction of the end organs in the reflex but they have no role in the afferent or central processing. 1999 International Association for the Study of Pain. Published by Elsevier Science B.V. Keywords: Visceral pain; Mechanonociception; Chemonociception
1. Introduction The method most frequently used to study visceral mechanonociception is distension of a viscus. In the case of the intestine, however, this procedure leads not only to the stimulation of stretch receptors in the intestinal wall but often also to a displacement of the gut with concomitant
* Corresponding author. Tel.: +43-316-380-4510; fax: +43-316-3809645; e-mail: [email protected]
excitation of mesenteric mechanoreceptors (Morrison, 1973). Thus, experiments using distension most likely measure the effects of a mixture of visceral stimuli. Here, another model to study visceral nociception in anaesthetized rats is characterized, which allows to study the effects of traction on the mesentery. Such a stimulus has already been described in 1942 by Lewis (Lewis, 1942) to induce pain in humans and is well known from the clinical setting to induce strong autonomic reactions in patients undergoing abdominal surgery. The mechanoreceptors that sense these stimuli are situated near the jejunal arteries, in particular close to their branching points, and the fibres travel with
0304-3959/99/$ - see front matter 1999 International Association for the Study of Pain. Published by Elsevier Science B.V. PII: S03 04-3959(98)002 33-4
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the splanchnic nerves (Bessou and Perl, 1966; Morrison, 1973). The experiments were done in rats under barbiturate anaesthesia. In these animals pseudaffective reflexes, i.e. changes in blood pressure and intragastric pressure, served as indicators of nociception. Since the animals’ ability to react in such ways strongly depends on the general status of the autonomic nervous system, the influence of drugs inhibiting a- or b-adrenergic receptors or muscarinic receptors was investigated. By these means the reliability of the responses could be judged under different baseline states of the cardiovascular system and the stomach. Only then was the experimental model tested for its sensitivity towards drugs known to inhibit nociceptive mechanisms. Preliminary results have been published in abstract form (Holzer-Petsche and Brodacz, 1995, 1997).
2. Methods 2.1. Experimental setup The experiments were approved by the Commission for Animal Experiments of the Austrian Ministry of Science. Female Sprague–Dawley rats (200–300 g body weight; Forschungsinstitut fu¨r Versuchstierkunde, Himberg, Austria) were fasted overnight with free access to tap water. The experimental setup is shown in Fig. 1. Under phenobarbitone anaesthesia (250 mg/kg i.p.) the trachea was cannulated to keep the airways patent and the oesophagus ligated in the neck. Mean arterial pressure (MAP) was monitored from a carotid artery. A jugular vein was cannulated for continuous i.v. infusion of isotonic saline (1.5 ml/h) and for drug injections. After laparotomy, a cannula was introduced into the stomach through an incision in the duodenum and fixed by a ligation around the pylorus for measurement of intragastric pressure (IGP) at a constant volume of 5 ml isotonic saline. The body temperature was kept at 37–38°C by means of a heating pad. A hook was positioned around the superior mesenteric
artery just before its division into the jejunal arteries to prevent displacement of the major vessels. A thread was tied loosely around a jejunal artery and vein just proximal to their branching to the gut. The other end of the thread was led over a pulley in order to attach weights of 2–30 g for stretching the vessels. The intestinal loop was placed onto the abdominal wall, on a piece of cotton soaked in saline, and covered with parafilm. As judged by inspection, this procedure did not compress the blood vessels, neither did it seem it to induce any mechanical irritation, since there were no alterations to the cardiovascular or gastric parameters. The effect of traction on the jejunal vessels for 1 min was compared with the effect of clamping the same vessels with a bulldog clamp right before their branching into the arcades (‘distal clamp’) for 1 min or at their leaving the mesenteric artery (‘proximal clamp’) for 3 min. The effect of the ‘distal clamp’ corresponds with a pure mechanical stimulus comparable with pinching the skin, whereas the ‘proximal clamp’ also leads to a transient hypoxia in a short gut segment as judged by the darkening and reddening of the supplied tissue. A further stimulus consisted in dropping 100 ml of 0.6% (v/v) acetic acid onto the same bundle of jejunal vessels (‘acid’). In one set of experiments, 10 pmol bradykinin was applied in a volume of 100 ml. Chemicals applied in such a way did not get into contact with the parietal peritoneum, thus the stimulus was purely visceral. After a first series of stimuli at 5 min-intervals (2, 5, 10, 20 and 30 g, distal and proximal clamp and acid) the experimental animals were injected i.v. with a single drug or a drug combination (1 ml/kg) and 10 min later (30 min in the case of indomethacin) the series of stimuli were repeated. Changes in MAP and IGP were taken as signs of pseudaffective reflexes (Cervero and McRitchie, 1982; HolzerPetsche, 1992). The reflex size after drug injection was then compared with the value obtained during the first series of stimuli, thereby compensating for interindividual variations. In the experiments with capsaicin the test stimuli were traction with 20 g and acetic acid, each applied once as a control stimulus. At time 0 capsaicin was injected s.c. (50 mg/kg). After capsaicin administration, traction was repeated every 10 min up to 60 min after capsaicin. The chemical stimulus was repeated 35 and 65 min after capsaicin. Both types of stimuli were therefore tested in the same rats. 2.2. Statistics
Fig. 1. Diagram of the experimental setup. A detailed description is given in Section 2.
The results are presented as mean ± SEM. If there was no response to a particular stimulus, the value was entered into the calculation as 0. The software SigmaStat was used to calculate Student’s t-test, paired t-test, one-way ANOVA, or one-way repeated measure ANOVA (followed by Dunnett’s test), as appropriate. Relationships between the strength of traction and the size of the reflex responses were determined
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chloride, Aldrich (Steinheim, Germany); atropine sulfate, Merck (Darmstadt, Germany); capsaicin, Serva (Heidelberg, Germany); morphine hydrochloride, Diosynth (Apeldoorn, The Netherlands); naloxone hydrochloride, DuPont (Geneva, Switzerland); indomethacin, Merck Sharp and Dohme (Rahway, NJ, USA); bradykinin, Bachem (Bubendorf, Switzerland); icatibant, Hoechst (Frankfurt, Germany). Capsaicin was dissolved in 10% ethanol, 10% Tween 80 and 80% isotonic NaCl at 25 mg/ml and injected s.c. at a volume of 2 ml/kg. Indomethacin was dissolved in 0.66% Na2CO3. All other drugs were dissolved in, and diluted with, isotonic saline and injected i.v. at 1 ml/kg.
3. Results 3.1. Characterization of the experimental model
Fig. 2. Tracings of mean arterial pressure (MAP) and intragastric pressure (IGP) during mesenteric stimulation. The duration of the mechanical stimuli is indicated by black bars, application of acetic acid by arrowhead. Asterisks indicate spontaneous contractions. The evaluation of the response is shown for the 30 g stimulus.
by a first order regression using the least squares method, and the Pearson product moment correlation coefficient was calculated as an indicator for the strength of the association. 2.3. Materials The chemicals were obtained from the following sources: phentolamine methane-sulfonate and propranolol hydro-
Graded traction on the jejunal vessels with weights of 5– 30 g led to prompt and transient changes in MAP and IGP (Fig. 2). Traction with 2 g was subthreshold in all cases but one. The changes in MAP usually consisted of an immediate fall followed by a rise, whereas the stomach reacted only with relaxations reflected in a transient lowering of the IGP, similar to those observed in a previous study (HolzerPetsche, 1992). Between animals it was unpredictable whether an increase or a decrease of MAP would prevail, in some animals there was even only a monophasic response. However, there was no correlation between the basal blood pressure at the time of the stimulus and the quality of the response. Only when MAP fell below 50 mmHg, did the responses to noxious stimulation cease consistently. Therefore, the total deviation of MAP from baseline (absolute value of rise plus absolute value of fall) rather than the isolated falls or rises was evaluated (see Fig. 2). By this method, a highly significant correlation was obtained between the magnitude of the responses and the weight
Fig. 3. Pseudaffective reflex responses to mesenteric traction with weights of 2–30 g. Individual values are plotted (n = 9 for MAP n = 6 for IGP). The line shows the first order regression calculated by the least squares method. The Pearson product–moment correlation coefficient (r) is inserted in each graph. All correlations are statistically significant (P , 0.001).
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applied (Fig. 3). However, after traction with 5 g or distal clamping responses often seemed random, although basal values were not significantly altered. Therefore, in the experiments testing drug effects, only stimuli .5 g were evaluated. When a clamp was put on the bundle of vessels, the reflex responses to the ‘distal clamp’ were generally smaller than those to the ‘proximal clamp’, probably as an indication of the different number of nociceptors stimulated at the two clamping sites. Furthermore, the time course of the responses differed from those to traction: during clamping MAP and IGP, after the peak, remained at a plateau value for the duration of the stimulus. After application of a chemical stimulus, a prompt change of MAP and IGP was seen, after which both parameters gradually returned to baseline, most likely reflecting the time course of elimination of the chemical agent. Although respiration was not recorded, the rats breathed faster and deeper during a stimulus (see Ness and Gebhart, 1990). At the end of the first experiments, after euthanasia, the maximum weight tolerated by a bundle of mesenteric vessels before rupture was evaluated. In 11 rats, mesenteric vessels tolerated traction with at least 75 g, in one rat, however, they ruptured at 40 g. Therefore no weight higher than 30 g was applied. After i.v. injection of isotonic saline (1 ml/kg) there was a tendency for greater responses to most stimuli (Fig. 4 top panels). These differences reached statistical significance for the IGP response to traction with 30 g. Since, however, the anticipated effect of the test drugs was to reduce rather than enhance nociception, the responses of the animals to mesenteric noxious stimuli were evaluated before and after systemic administration of drugs.
Injection of atropine (1 mg/kg i.v.) alone had no effect on basal measurements, in combination with the adrenoceptor blockers it lowered basal MAP but did not change basal IGP (Table 1). At the end of the experiment 2/8 rats in the latter group had a basal MAP ,50 mmHg. On its own, atropine did not influence the responses to the noxious stimuli. If given in combination with the a- and badrenoceptor blockers it led to a reduction of all reflex responses, which was statistically significant except for the response to distal clamping (Fig. 4 bottom panels). Quantitatively, the MAP responses did not differ from those observed with the adrenoceptor blockers alone, whereas the gastric responses became smaller than in the former group. These observations also show that reliable reflex responses could be obtained over a wide range of basal MAP and that lowering of basal MAP per se did not necessarily abolish the reflex response. In individual rats it was noted that the MAP responses ceased consistently only when basal MAP fell well below 50 mmHg, regardless of the type of drug injected. For this reason, rats with MAP
3.2. Efferent mechanisms Phentolamine (1 mg/kg i.v. as bolus followed by an infusion of 1 mg/kg × h) significantly reduced basal MAP while propranolol (0.3 mg/kg i.v. followed by 0.3 mg/kg × h) reduced basal MAP and increased IGP (Table 1). As expected, administration of phentolamine plus propranolol strongly modified basal MAP as detailed in Table 1. In this group, 4/10 rats had a basal MAP ,50 mmHg by the end of the experiment, while in none of the control rats or the rats injected with only one compound such a profound alteration of basal MAP was observed. Basal intragastric pressure was not significantly altered after phentolamine plus propranolol. Administration of either phentolamine or propranolol alone had no influence on the reflexes induced by the various noxious stimuli. The combination of the a- and badrenoceptor antagonists reduced the changes in MAP after all stimuli, but the reduction of the responses to clamping did not reach statistical significance (Fig. 4 middle panel). The gastric response was significantly reduced by the antagonists only when acetic acid was used as a stimulus (Fig. 4 middle panel).
Fig. 4. Reflex changes of mean arterial pressure (MAP, left panels) and intragastric pressure (IGP, right panels) in response to traction of the mesentery with weights of 10, 20 and 30 g, clamping of the vessels under investigation close to the gut for 1 min (‘D’ distal), clamping close to the branching from the superior mesenteric artery for 3 min (‘P’ proximal) and to application of 100 ml 0.6% acetic acid (‘Ac’). White columns, 1st series of stimuli before drug; hatched columns, 2nd series of stimuli started 10 min after i.v. injection of drugs as indicated. NaCl, 1 ml/kg isotonic saline (n = 6); Phe + Pro, 1 mg/kg phentolamine as bolus followed by 1 mg/kg × h together with 0.3 mg/kg propranolol as bolus followed by 0.3 mg/kg × h (n = 10); Atr + Phe + Pro, same as Phe + Pro plus a bolus of 1 mg/kg atropine (n = 8). Columns denote means with bars indicating SEM; *P , 0.05, **P , 0.01 versus before drug (paired ttest); n.t., not tested.
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Baseline values of mean arterial pressure (MAP) and intragastric pressure (IGP) immediately before and 10 min after i.v. administration of drugs. Values are means ± SEM; n in parentheses. The values before drug injection were not significantly different between the six groups. MAP (mmHg) Before saline (1 ml/kg i.v.) After saline (1 ml/kg i.v.) Before atropine (1 mg/kg i.v.) After atropine (1 mg/kg i.v.) Before phentolamine (1 mg/kg bolus followed by 1 mg/kg × h i.v.) After phentolamine (1 mg/kg bolus followed by 1 mg/kg × h i.v.) Before propranolol (0.3 mg/kg bolus followed by 0.3 mg/kg × h i.v.) After propranolol (0.3 mg/kg bolus followed by 0.3 mg/kg × h i.v.) Before phentolamine + propranolol After phentolamine + propranolol Before phentolamine + propranolol + atropine After phentolamine + propranolol + atropine
85 90 101 94 109 66 105 87 111 57 106 62
± ± ± ± ± ± ± ± ± ± ± ±
5 4 (6) 7 5 (6) 7 5** (6) 6 10* (7) 5 5** (10) 7 6** (8)
IGP (Pa) 885 871 840 917 833 790 711 800 718 772 772 753
± ± ± ± ± ± ± ± ± ± ± ±
64 73 (6) 50 73 (6) 97 74 (6) 42 59** (7) 43 72 (10) 82 85 (8)
*P , 0.05; **P , 0.01 versus before drug (paired t-test).
values falling ,50 mmHg were excluded from the following part of the study. 3.3. Afferent mechanisms Subcutaneous injection of 50 mg/kg capsaicin had no effect on the baseline values of MAP and IGP, whereas after vehicle basal MAP decreased within 10 min from 100 ± 5 to 92 ± 6 mmHg (n = 7, P , 0.05). These changes, however, were transient and basal IGP was not altered. Apart from an initial increase in the MAP response to traction and a significant increase of the IGP response to acid, nociception was not altered by s.c. treatment with vehicle. Capsaicin administration almost abolished the responses to the chemical stimulus, the MAP response to traction decreased significantly, but there was no effect on the IGP response to traction (Fig. 5). In order to test whether the observed reflexes were indeed a correlate of nociception, rats were treated with i.v. morphine. After morphine injection basal MAP gradually declined until the end of the experiments (from 101 ± 5 to 82 ± 7 and from 95 ± 7 to 61 ± 5 mmHg after 1 and 5 mg/ kg respectively, each n = 6, P , 0.05). Naloxone had no such effect. Basal IGP values were never significantly altered. Whereas, 1 mg/kg morphine reduced only the responses to the weaker stimuli, 5 mg/kg significantly reduced the reflex responses to all stimuli except proximal clamping (Fig. 6). Naloxone (5 mg/kg) itself was without effect but prevented the action of 5 mg/kg morphine except for the MAP responses to traction with 10 and 20 g. A possible involvement of prostaglandins in the observed reflexes was investigated by the use of indomethacin. Intravenous injection of 10 mg/kg indomethacin led to gradual decreases in MAP and increases in IGP (from 89 ± 5 to 65 ± 4 mmHg and from 294 ± 47 to 378 ± 70 Pa, n = 6, P , 0.05). Injection of 0.66% Na2CO3 as vehicle had no such effect, nor did it influence the reactions to noxious stimuli. Indomethacin only reduced the MAP responses to
traction with 10 g and to distal clamping (Fig. 7). At the end of the experiments, approximately 90 min after indomethacin injection, all rats presented with haemorrhagic streaks on their gastric mucosae at macroscopic inspection. Such lesions were never seen in any other group. In order to check whether the production of bradykinin occurred at some stage of the processing of noxious stimuli to the mesentery, the bradykinin B2-antagonist icatibant was used. In seven rats, i.v. injection of icatibant (100 nmol/kg) had no effect on basal parameters and did not influence the
Fig. 5. Effect of capsaicin (50 mg/kg s.c.) or its vehicle on the reflex responses to noxious stimulation of the mesentery. W response to traction (20 g) in vehicle-treated rats X response to traction (20 g) in capsaicintreated rats A response to acid in vehicle-treated rats B response to acid in capsaicin-treated rats. Symbols denote means with bars indicating SEM, n = 6 for capsaicin, n = 7 for vehicle. *P , 0.05 versus before s.c. injection, one-way RM ANOVA; †P , 0.05 versus vehicle, Student’s t-test.
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Fig. 6. Effect of morphine and naloxone on the reflex responses to noxious stimulation of the mesentery (abbreviations as in Fig. 4). White columns, 1st series of stimuli before drug; hatched columns, 2nd series of stimuli started 10 min after i.v. injection of drugs as indicated. NAL, 5 mg/kg naloxone; Mo1, 1 mg/kg morphine; Mo5, 5 mg/kg morphine; Mo5 + NAL, 5 mg/kg each naloxone plus morphine. Columns denote means with bars indicating SEM, each n = 6; *P , 0.05, **P , 0.01 versus before drug (paired t-test).
stimulus in humans. In the form applied in the present setup, it is reproducible, its intensity can be controlled and the response correlates with the size of the stimulus. It can be applied selectively to the mesentery without inadvertently influencing the gut beyond lifting up a piece of small intestine. This, however, appears not to arouse nocifensive reflexes on its own since there are definitely subthreshold intensities of traction during whose application the intestine is equally raised. Chemical stimuli can also be repeatedly employed, and under the present conditions no sensitization or desensitization phenomena have been observed. The use of phenobarbitone as a long acting anaesthetic produces a stable level of anaesthesia throughout experiments of up to 150 min, which is demonstrated by the reflex responses being reproducible after i.v. administration of NaCl. Since Sherrington (1906) pseudaffective reflexes have been used as a measure of nociception, also in anaesthetized rats (e.g. Cervero and McRitchie, 1982; Holzer et al., 1992; Holzer-Petsche, 1992). Alterations of blood pressure occur regularly, but their shape can vary depending on the type of anaesthesia (Ness and Gebhart, 1988). So far, no explanation has been found for the qualitative variations of the changes in MAP in the present study. Overall however, the size of both autonomic reflexes as evaluated in the present study correlates with the strength of the stimulus, similar to the rate of discharge of mesenteric fibres in the experiments by Morrison (1973). A good correlation of the size of the response with the stimulus intensity has also been observed by Ness and Gebhart (1988) after colorectal distension in awake rats. Thus, such a relationship seems to be valid for several types of mechanical stimuli. This fact leads to the inference that proximal clamping is a stronger stimulus than distal clamping. One reason could be that a clamp positioned close to the branching of the jejunal
reflex responses to traction, clamping or acid, although it abolished the changes in MAP and IGP in response to the application of 10 pmol bradykinin onto the mesenteric bundle (data not shown). A comprehensive list of drug effects on the reflex responses is shown in Table 2.
4. Discussion 4.1. General aspects The experimental model presented here allows to study the effects of different types of mechanical and chemical noxious stimuli in the peritoneal cavity, at a location that is considered to be supplied by visceral afferent nerves. The present setup is aimed primarily at investigating the sites and transmitter mechanisms involved in acute mesenteric nociception. Mesenteric traction is known to be a noxious
Fig. 7. The effect of indomethacin on the reflex responses to noxious stimulation of the mesentery (abbreviations as in Fig. 4). White columns, 1st series of stimuli before drug; hatched columns, 2nd series of stimuli started 30 min after i.v. injection of drugs as indicated. Na2CO3, 1 ml/kg, 0.66% sodium carbonate (solvent for indomethacin) (n = 5); INDO, 10 mg/kg indomethacin (n = 6). Columns denote means with bars indicating SEM; *P , 0.05 versus before drug (paired t-test).
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U. Holzer-Petsche, B. Brodacz / Pain 80 (1999) 319–328 Table 2
Summary of the effects of manipulations on changes in mean arterial pressure (MAP) and intragastric pressure (IGP) induced by noxious stimuli. For detailed explanations of the treatments see Section 2 Treatment
Effect on changes in MAP (mmHg)
Traction (g)
10
20
30
10
20
30
NaCl Atropine Phentolamine Propranolol Phe + Pro Phe + Pro + Atr Capsaicin (60 min) Naloxone Morphine (5 mg/kg) MO + NAL Indomethacin Icatibant
n.c. n.c. n.c. n.c. –– n.t. n.t. n.c. –– –– –– n.c.
n.c. n.c. n.c. n.c. –– –– –– n.c. –– –– n.c. n.c.
n.c. n.c. n.c. n.c. – n.t. n.t. n.c. –– n.c. n.c. n.c.
n.c. n.c. n.c. n.c. n.c. n.t. n.t. n.c. –– n.c. n.c. n.c.
n.c. n.c. n.c. n.c. n.c. – n.c. n.c. –– n.c. n.c. n.c.
+ n.c. n.c. n.c. n.c. n.t. n.t. n.c. –– n.c. n.c. n.c.
D
P
Acid
D
P
Acid
n.c. n.c. n.c. n.c. n.c. n.c. n.t. n.c. –– n.c. –– n.c.
n.c. n.c. n.c. n.c. n.c. – n.t. n.c. n.c. n.c. n.c. n.c.
n.c. n.c. – n.c. –– –– –– n.c. –– n.c. n.c. n.c.
n.c. n.c. + + n.c. n.c. n.c. n.t. n.c. –– n.c. n.c. n.c.
n.c. n.c. n.c. n.c. n.c. – n.t. n.c. n.c. n.c. n.c. n.c.
n.c. n.c. n.c. n.c. – –– –– n.c. – n.c. n.c. n.c.
NaCl Atropine Phentolamine Propranolol Phe + Pro Phe + Pro + Atr Capsaicin (65 min) Naloxone Morphine (5 mg/kg) MO + NAL Indomethacin Icatibant
Effect on fall of IGP (Pa)
D, distal clamp; P, proximal clamp; n.c., no change; +/−, statistically significant (P , 0.05) increase or decrease of the response by ,50%; ++/−−, statistically significant increase or decrease of the response by .50%; n.t., not tested.
arteries from the superior mesenteric artery compresses a greater amount of tissue and thus stimulates a greater number of nociceptors than a clamp at the distal end of the jejunal vessels. On the other hand, a contribution of intestinal hypoxia cannot be excluded since the piece of gut supplied by the blood vessels under investigation became hypoxic during the 3-min stimulation as judged from the darkening of the tissue. In line with this, Longhurst and Dittman (1987) found that visceral afferents sensitive to hypoxia already started to fire after a mean latency of 63 s. In the present setup the threshold for the mechanoreceptors seems to be rather high, since there was practically no response to traction with 2 g although the mesenteric vessels appeared well stretched. The different shapes of the MAP responses to application of tension or clamping could indicate a qualitatively different processing of the two types of stimuli. It might be speculated that tension is mediated by fibres adapting more rapidly than the fibres excited by clamping the tissue. As to chemical stimuli, no attempt has been made to look for a dose-response relationship in the present setup. Instead, the concentration of acetic acid matched that used in writhing tests (Berkenkopf and Weichman, 1988).
4.2. Alteration of autonomic functions As the sympathetic nervous system is always activated when the organism has to cope with stressful situations, the importance of patent adrenergic mechanisms for the responses to visceral nociceptive stimuli has been investigated. Phentolamine or propranolol alone did not change the reflex responses measured. This is unlikely to be caused by an insufficient dose of the compounds since either of them significantly altered basal MAP. Only the combination of the a- with the b-receptor antagonist was able to reduce the cardiovascular responses to nociceptive stimulation. This is in line with a previous study, where changes in MAP in response to i.p. chemical stimuli were abolished by the combination of pretreatment with guanethidine and adrenalectomy but not by either procedure alone (Holzer-Petsche, 1992). In contrast, either adrenoceptor antagonist alone significantly decreased cardiovascular reflex responses to colorectal distension in awake rats (Ness and Gebhart, 1988) and the gastric relaxation in response to noxious pressure applied to a testicle in rats anaesthetized with chloralose (Bojo¨ et al., 1992). Possibly the type of anaesthesia may modify the contribution of the sympathetic nervous system in the reflex response and thereby the apparent sensitivity of
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the animals to the adrenoceptor blockers in the present experiments. Muscarinic receptors seem not to play a major role in these reflex responses as judged by the lack of effect of atropine alone. This is in line with earlier observations by Radhakrishnan et al. (1985), where the reflex change in blood pressure to serosal application of noxious chemicals was not altered by atropine. The latter study had been performed in rats anaesthetized with urethane. It could, therefore, be argued that under general anaesthesia there is little influence of the parasympathetic nervous system on cardiovascular reflexes to noxious stimulation. The two adrenoceptor blockers did not impair the fall in IGP except when evoked by acid, nor did atropine pretreatment have a major effect on the gastric response to noxious stimuli. Similar observations have been made by Glise and Abrahamsson (1980) in cats where neither the gastric inhibition by noxious mechanical stimulation of the gut nor that induced by i.p. hydrochloric acid was blocked by antiadrenergic or anticholinergic drugs. It needed the administration of atropine on top of both adrenoceptor blockers to significantly reduce the fall in IGP after all stimuli but distal clamping. Thus, both a reduction in parasympathetic and an increase in sympathetic tone are responsible for the gastric relaxation occurring with nociception. Overall, although the observed pseudaffective reflexes obviously depend on the integrity of the autonomic nervous system, they seem to be rather robust towards elimination of only one of its components (a- or b-adrenergic or muscarinergic transmission). Furthermore, the observations indicate that measurements of the above reflexes are reliable over a great range of basal MAP or IGP and make the experimental model suitable for testing drugs influencing nociceptive mechanisms. 4.3. Antinociceptive measures The sensory neurotoxin capsaicin has been shown to attenuate or abolish a number of nociceptive reflexes, both somatic and visceral ones (see Holzer, 1991). In a model similar to the one used here, cardiovascular and gastric reflex responses to i.p. acid and bradykinin were markedly decreased in rats treated systemically with capsaicin 2 weeks earlier (Holzer-Petsche, 1992). Furthermore, these capsaicin-sensitive afferents have been shown to mediate the gastric inhibition occurring after laparotomy (Holzer et al., 1992). Also in the present experiments the reflexes to the application of acetic acid were almost abolished after s.c. injection of capsaicin with a time course corresponding to the kinetics of capsaicin as measured by Saria et al. (1982) and to the disappearance of the capsaicin-sensitive micturition reflex (Holzer-Petsche and Lembeck, 1984). However, there was no unequivocal effect on the reflexes induced by mesenteric traction: while the gastric response was unchanged both after capsaicin and vehicle, there was a reduction of the MAP response after capsaicin injection.
While there is consensus about the desensitization of chemonociceptors by capsaicin (Cervero and McRitchie, 1982; Holzer et al., 1992), there is conflicting evidence about the effect of capsaicin on mechanosensitive nerve fibres. Small diameter, high-threshold mechanosensitive fibres which, in addition respond to heat, are also sensitive to capsaicin (Szolcsa´nyi et al., 1988; Gallar et al., 1993; Seno and Dray, 1993). However, there are no intestinal afferents known to react to heat stimuli (see Cervero, 1994), and somatic fibres sensitive to mechanical but not to heat stimuli can neither be excited by low doses, nor desensitized by high doses of capsaicin (Szolcsa´nyi et al., 1988; Seno and Dray, 1993). In some cases, such as the response of articular mechanoreceptors (He et al., 1990), the block of the response by systemic capsaicin was variable. Contradictory observations were also made on afferents of abdominal organs. Whereas the slow depolarization in neurones of the inferior mesenteric ganglion evoked by distention of the ureter and the change in blood pressure in response to distension of the biliopancreatic duct were abolished by capsaicin (Amann et al., 1988; Griesbacher, 1994), gastric mechanoreceptors appeared to be insensitive to capsaicin (Berthoud et al., 1997). In the present study there was a striking difference between the responses of the cardiovascular system and the stomach to the mechanical stimulus after capsaicin. Since the reflex can reasonably have only one afferent pathway, one could speculate that processes in the CNS, at the point of divergence of the reflex to the various effector systems, are differentially sensitive to capsaicin. A similar dissociation between the responses of different end organs has been mentioned by Gebhart and Ness (1991). In their (awake) rats, which had been treated with capsaicin as neonates, the distension threshold of the colon to elicit a visceromotor reflex was doubled, but no change was reported for the cardiovascular reflex. However, one should take care not to equate capsaicin treatment of neonates with that of adult animals. Loss of capsaicin-sensitive nerves in the first days of life leads to compensatory changes in the afferent innervation that are absent in adult animals (Holzer, 1991). The present experimental model has also been tested for its response to morphine, in order to verify that the reflexes under investigation are nocifensive. Morphine is effective in inhibiting the responses to traction, distal clamping and acetic acid, but does not attenuate that to proximal clamping. However, the latter certainly was the strongest stimulus applied in the present study and there is evidence that the effectiveness of a particular dose of morphine is inversely related to the strength of the stimulus (Saeki and Yaksh, 1993). Naloxone at the rather high dose of 5 mg/kg i.v. had no effect of its own. It did, however, counteract the effect of morphine, although not in the case of MAP after 10 and 20 g traction. Most likely a persisting effect of morphine against the two low stimuli is responsible for this remaining antinociception. It cannot be due to pharmacokinetic differences between morphine and naloxone, because
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firstly they were administered together i.v. and secondly because the effect of morphine on the gastric response measured simultaneously was abolished by naloxone. Indomethacin was ineffective in blocking the nocifensive reflexes after visceral stimulation, in contrast to its antinociceptive action when acetic acid is used as stimulus in the writhing test (Berkenkopf and Weichman, 1988), However, in the latter test, a relatively large volume of the irritant is present in the peritoneal cavity for several minutes before the rat’s behaviour is evaluated. Thus, there is plenty of time for the synthesis of prostaglandins to take place and to participate in the noxious stimulation. In the present setup, in contrast, the immediate autonomic reactions to a small volume of acid were measured, making it unlikely for any prostaglandins to contribute to the stimulation before the acid was diluted and buffered. Nevertheless, the dose of indomethacin was high enough to cause gastric mucosal lesions, whose development might have been facilitated by the ligation of the pylorus around the gastric cannula. In models of acute nociception the action of bradykinin has been shown to be mediated by B2 receptors (Hall, 1992). In the present setting the B2-antagonist icatibant blocks only the reflexes induced by mesenteric application of bradykinin, and not those induced by the other types of stimuli. Similarly, gastric motor inhibition induced by HCl was unaffected by icatibant (Hoe-140), whereas, bradykinininduced inhibition was blocked (Holzer, 1992). Thus, bradykinin via B2 receptors does not play a role in the transmission of mechanical and acute chemical stimuli.
5. Conclusions The present data confirm that both the cardiovascular system and the stomach react in response to visceral noxious stimuli. Mesenteric traction can be used as a model for visceral mechanonociception and the stimuli described can be compared with other types of noxious input. This setup can serve as a basis for further pharmacological investigations into the mechanisms of nocifensive reflexes.
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