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On the role of galanin in mediating spinal flexor reflex excitability in inflammation
Pergamon
PII:
Neuroscience Vol. 85, No. 3, pp. 827–835, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00676-3
ON THE ROLE OF GALANIN IN MEDIATING SPINAL FLEXOR REFLEX EXCITABILITY IN INFLAMMATION I. S. XU, S. GRASS, X.-J. XU and Z. WIESENFELD-HALLIN* Department of Medical Laboratory Sciences and Technology, Division of Clinical Neurophysiology, Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden Abstract––The effects of exogenous and endogenous galanin on spinal flexor reflex excitability was evaluated in rats one to eight days after the induction of inflammation by subcutaneous injection of carrageenan into the sural nerve innervation area. In normal rats, electrical stimulation of C-fibres in the sural nerve elicited a brisk reflex discharge. Conditioning stimulation of C-fibres (1/s) generated a gradual increase in reflex magnitude (wind-up), which was followed by a period of reflex hyperexcitability. Intrathecal galanin dose-dependently blocked reflex hyperexcitability induced by C-fibre conditioning stimulation whereas i.t. M-35, a high-affinity galanin receptor antagonist, moderately potentiated this effect. At one to three days after the injection of carrageenen, when inflammation was at its peak, the magnitude of the reflex was significantly increased and discharge duration became prolonged. However, wind-up and reflex hyperexcitability were significantly reduced. Furthermore, reduced reflex excitability during conditioning stimulation (‘‘wind-down’’) and depression of the reflex were sometimes present, which are rarely observed in normal rats. Intrathecal galanin reduced hyperexcitability during inflammation, although its potency was weaker than in normals. However, the galanin receptor antagonist M-35 strongly enhanced wind-up and reflex hyperexcitability, similarly as in normal rats. The baseline flexor reflex, wind-up and C-fibre conditioning stimulation-induced facilitation were normalized four to eight days after carrageenan injection when signs of inflammation were diminishing. Interestingly, intrathecal galanin and M-35 failed to influence spinal excitability. The results suggest a complex functional plasticity in the role of endogenous galanin in mediating spinal excitability during inflammation. There appears to be an enhanced endogenous inhibitory control by galanin on C-afferent input during the peak of inflammation, which may explain the relative ineffectiveness of exogenous galanin. During the recovery phase there may be a reduction in galanin receptors, which may impair the action of endogenous and exogenous galanin. These results further support the notion that galanin is an endogenous inhibitory peptide in nociception. 1998 IBRO. Published by Elsevier Science Ltd. Key words: carrageenan, dorsal horn, galanin receptor antagonist, intrathecal, nociception, pain.
The neuropeptide galanin, originally isolated by Tatemoto et al.,33 has been recognized as an important neurotransmitter and/or neuromodulator in the nervous system.3 Galanin-like immunoreactivity (LI) and galanin mRNA has been found in dorsal root ganglion cells across many species.6,18,31 A network of galanin-LI fibres6,18,56 and galanin receptors16,55 have been localized in the superficial laminae of the dorsal horn. There are also dorsal horn interneurons which contain galanin, some of which also express the inhibitory transmitter GABA.6,18,30,57 These morphological findings imply that galanin may have a role in spinal nociceptive transmission and modulation. Functional studies examining the effect of galanin on spinal nociception have yielded conflicting results. Thus, while some early studies suggested that i.t. *To whom correspondence should be addressed. Abbreviations: CS, conditioning stimulation; EMG, electromyogram; LI, -like immunoreactivity; M-35, galanin (1– 12)-Pro-Bradykinin (2–9); NK, neurokinin. 827
galanin elicited hyperalgesia,17 others reported behavioural antinociception or depression of nociceptive reflexes in electrophysiological studies.7,24,26,27,45,54 Using the flexor reflex in decerebrate, spinalized rats as a tool, we have systematically assessed the effects of galanin on spinal reflex excitability. We observed that i.t. galanin had a dose-dependent biphasic facilitatory and inhibitory effect on the baseline flexor reflex.41,50 In addition, galanin dose-dependently suppressed the development of spinal cord hyperexcitability induced by intense activation of C-fibres, as well as by exogenously applied excitatory neuropeptides substance P and calcitonin gene-related peptide.41,50,51 Moreover, galanin also potentiated43 and is possibly involved28 in the spinal antinociceptive effect of morphine. Thus, we have suggested that galanin may be a predominantly inhibitory modulator in the spinal cord, particularly under states of hyperexcitability.42 A series of high-affinity antagonists of galanin receptors have been tested to address the role of endogenous galanin in the modulation of spinal
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nociception.1,2,44 Intrathecal administration of M-35 [galanin (1–12)-pro-bradykinin (2–9)], a high-affinity galanin receptor antagonist, blocked the spinal effects of galanin and moderately potentiated the C-fibre conditioning stimulus (CS)-induced facilitation of the flexor reflex in normal rats, indicating a tonic role for galanin in spinal inhibitory modulation.44 Interestingly, i.t. M-35-induced potentiation became significantly stronger after peripheral axotomy44 when galanin was up-regulated in sensory neurons,38,58 indicating that galanin-mediated inhibition is enhanced. Moreover, behavioural studies have also shown an endogenous inhibitory effect of galanin on neuropathic pain-related behaviours in rats after peripheral nerve injury.13,37 Peripheral inflammation induces complex changes in messengers in sensory neurons and in the spinal cord and many of these changes are different from those occurring after peripheral nerve injury.9,10 In contrast to the profound up-regulation of galanin in sensory neurons in axotomized rats,38,58 a decrease in the number of sensory neurons expressing galanin was observed three days after carrageenan-induced inflammation.14 In contrast, the number of dorsal horn neurons which express galanin mRNA are increased in the superficial laminae of the dorsal horn three days after inflammation.14 An earlier study has also shown that injecting formalin into the lips of rats induced an up-regulation of preprogalanin mRNA 6 h later in trigeminal nucleus caudalis in the dorsal horn.36 No changes in galanin binding sites were observed in the dorsal horn and in the dorsal root ganglia after inflammation.14 However, in a recent study mRNA coding for the galanin R1 receptor found in normal dorsal root ganglion cells in rat was shown to be transiently down-regulated during carrageenan-induced inflammation.53 Inflammation also alters the release of galanin. Thus, Hope et al.11 reported that joint inflammation increased the basal release of galanin in the dorsal horn and innocuous and noxious stimulation of the inflamed joints suppressed such release. The present study was undertaken to examine the effect of galanin on spinal flexor reflex excitability during inflammation, one to three days after application of subcutaneous carrageenan. The role of endogenous galanin was assessed with M-35. In view of the fact that peripheral inflammation induced by carrageenan is reversible, we have also studied the effects of galanin and M-35 in rats that recovered from inflammation (four to eight days after injection of carrageenan). The effects of inflammation, galanin and galanin receptor antagonist on spinal hyperexcitability was studied by observing both the gradual increase in reflex magnitude during the C-fibre CS (wind-up)19 and the facilitation of the flexor reflex following C-fibre CS (central sensitization).39 The relative significance of these measurements in studying spinal hyperexcitability has been discussed previously.48
EXPERIMENTAL PROCEDURES
Female Sprague–Dawley rats weighing 200–250 g (B & K Universal, Stockholm, Sweden) were used in the present experiments, which were conducted under the approval of the local research ethics committee. Induction and time-course of inflammation Inflammation was induced by subcutaneous injection of 0.1 ml 2% ë-carrageenan into the lateral side of the plantar skin under methohexital anaesthesia (Brietal, Lilly, Indianapolis, IN, U.S.A., 70 mg/kg; i.p.). Inflammation was verified by the presence of oedema and erythema and reached peak level 24–48 h after the injection and gradually recovered thereafter. Little or no signs of inflammation were observed five to six days after the injection of carrageenan. Flexor reflex experiments On the day of the acute experiment, normal rats or rats which has been injected with carrageenan one to eight days previously were briefly anaesthetized with methohexital, ventilated and decerebrated by aspiration of the forebrain and midbrain. The spinal cord was exposed by a laminectomy at mid-thoracic level and sectioned at Th8–9. An i.t. catheter (PE 10) was implanted caudal to the transaction with its tip on the lumbar spinal cord at L4–L5. All experiments started at least 1 h after spinal transection. The flexor reflex was elicited 1/min by electric shocks applied to the sural nerve (rectangular pulses of 0.5 ms duration, 10 mA) and was recorded as electromyogram (EMG) activity with stainless steel needle electrodes inserted into the ipsilateral posterior biceps femoris/semitendinosus muscles. Previous studies under similar experimental conditions have shown that such stimuli activate both A- and C-afferents.39 In some experiments, a conditioning stimulus (CS) train administered to the sural nerve (20 shocks at 1 Hz) was used to elicit wind-up (gradual increase in reflex magnitude during the CS train) and subsequent facilitation of the flexor reflex. For quantification of baseline reflex mangitude, the number of potentials exceeding the level of spontaneous EMG activity was counted using a window discriminator, integrated over 2 s and recorded on a chart recorder.41,49 The integration time was 1 s during the CS. A stable reflex baseline (defined as less than 15% variation) was established for at least 20–30 min before administration of drugs or the CS. Heart rate and rectal temperature of the rat were monitored. Peptides Porcine galanin was obtained from Bachem and M-35 was generously supplied by Prof. T. Bartfai (Department of Neurochemistry and Neurotoxicology, University of Stockholm). Galanin and M-35 were dissolved in 0.9% normal saline. The aliquoted peptide solutions were stored under 70C and used directly after thawing. The i.t. injections were made in 10 µl followed by 10 µl saline to flush the catheter. Data collection and statistics The effect of i.t. peptides and the nerve CS on the flexor reflex were expressed as percent change in reflex magnitude compared to baseline, which was defined as 100%. Data were analysed by one-way ANOVA followed by Fisher PLSD (protected least significant difference) test. Some data were also analysed by Wilcoxon signed ranks test or Mann– Whitney U-test or the ø2-test. Variability is expressed as S.E.M.
Role of galanin in inflammation Table 1. The magnitude and duration of the flexor reflex in normal, inflamed and recovered rats
Normal Inflamed Recovered
n
Magnitude
Duration (s)
19 16 13
14.11.6** 36.48.7 16.12.3*
5.41.9** 15.12.2 6.02.2**
The relative magnitude and duration of the integrated EMG discharge recorded from ipsilateral hamstring muscles in response to electrical stimulation (0.5 ms, 10 mA) of the sural nerve at 1/min in normal, inflamed and recovered rats. Data are expressed as meanS.E.M. ANOVA indicated significant differences in both the magnitude (F2,45=5.6, P<0.01) and duration (F2,45=7.2, P<0.01) of the flexor reflex recorded from normal, inflamed and recovered rats. Individual comparisons were made with the Fisher PLSD test, *P<0.05, **P<0.01 compared to inflamed rats.
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Table 2. Number of rats showing the different forms of changes in reflex excitability during the C-fibre conditioning stimulus in normal, inflamed and recovered rats
Normal Inflamed Recovered
Wind-up >100%
Wind-up <100%
Wind-down
16 5 12
3 7 0
0 4 1
The ÷2-test indicated significant differences among the frequencies of occurrence of various types of excitability changes (÷2=14.2, d.f.=4, P<0.01).
RESULTS
The flexor reflex in normal, inflamed and recovered rats Electrical stimulation of the sural nerve elicited a brisk contraction of the ipsilateral hamstring muscles, which was quantified by recording EMG activity. Afterdischarges (defined as activity longer than 1 s after the stimulus artifact) were observed in more than 50% of the experiments in normal rats. However, in 80% of experiments, the duration of EMG activity generated by a single electric shock was less than 5 s (Table 1). At one to three days after injection of carrageenan the magnitude of the integrated flexor reflex was significantly increased compared to normals (Table 1). Moreover, the duration of EMG activity was also significantly prolonged with reflex discharges longer than 5 s in more than 90% of experiments (Table 1). The rate of ongoing EMG activity recorded during inflammation was higher than normal, although no particular attention was paid to this and the contribution of ongoing activity to the recorded reflex activity was eliminated by adjusting the window height on the discriminator to exclude ongoing EMG. At four to eight days after the injection of carrageenan, when the signs of inflammation were diminishing, the magnitude and duration of the reflex became normalized (Table 1), although prolonged discharges were observed more often than in normals. C-fibre conditioning stimulation-induced reflex windup and facilitation in normal, inflamed and recovered rats A CS train of 1 Hz for 20 s generated a gradual increase in reflex magnitude (wind-up) in all normal animals (Table 2, Fig. 1). At one to three days after induction of inflammation, the frequency of wind-up decreased and in some cases ‘‘wind-down’’ (decrease
Fig. 1. The magnitude of wind-up during the C-fibre CS (1 Hz, 20 shocks). The data are presented as mean increase in reflex magnitude over baseline reflex (defined as 100%) in blocks of five responses. In this and other figures, the number of rats examined is shown in the brackets. ANOVA indicated significant differences among the three experimental conditions for all four blocks (F2,37=3.3, 3.3, 5.2 and 5,6, respectively; P<0.05, 0.05, 0.01 and 0.01, respectively). Individul comparisons were with the Fisher PLSD test, *P<0.05 and **P<0.01 compared to inflamed rats.
in reflex magnitude during the CS) occurred (Table 2). During recovery (four to six days after injection of carrageenan), the frequency of wind-up became normalized (Table 2). The distribution of different response types to the CS train was very different among the three experimental groups (Table 2) and there were significant differences in the magnitude of wind-up during stimuli 11–15 and 16–20 between inflamed vs normal or recovered rats (Fig. 1). Single shocks applied 30 s and 1 min after the termination of the CS train generated a markedly enhanced reflex (post-CS facilitation) in normal rats (Fig. 2A). The duration of reflex facilitation was between 1–5 min in normals after which the reflex returned to baseline level (Fig. 2B). Similar findings were observed in recovered rats (Fig. 2). In contrast, in the inflamed rats the magnitude of post-CS facilitation was significantly reduced (Fig. 2). Moreover, in more than 60% of inflamed rats the CS generated depression or facilitation followed by depression of the flexor reflex was observed. Inhibition following C-fibre CS is rarely observed in normals (one of 19 or
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Fig. 2. The magnitude and duration of reflex hyperexcitability following CS. ANOVA indicated an overall significant difference in the magnitude, but not duration, of reflex facilitation induced by the C-fibre CS among the three experimental conditions (F2,37=6.1 and 2.6, respectively; P<0.01 and <0.05, respectively). Individual comparisons are with the Fisher PLSD test, *P<0.05 and **P<0.01 compared to inflamed rats.
5% in the present series of experiments). Inhibition was observed in two of 13 (6.5%) experiments in recovered rats. Effects of intrathecal galanin on the baseline flexor reflex in normal and inflamed rats In normal rats, 100 ng, 1 µg and 10 µg i.t. galanin caused a brief facilitation of the baseline flexor reflex. At the two highest doses (1 and 10 µg) depression was also frequently observed. Similar facilitatory effect of i.t. galanin on the baseline flexor reflex was found in inflamed and recovered rats (Fig. 3). In four normal rats the depressive effect of increasing doses of galanin on the baseline flexor reflex was examined in detail. At the lowest dose (100 ng), galanin did not depress the flexor reflex whereas at higher doses depression of the baseline flexor reflex was observed in some experiments (two of four at 1 µg, and three of three at 10 µg). Thus, the pattern of the effect of i.t. galanin on the baseline flexor reflex observed in the present experiments was similar to previous results.41 In three rats examined one to three days after inflammation, i.t. galanin similarly depressed the baseline flexor reflex (one of three at 1 µg and three of three at 10 µg). Effects of intrathecal galanin on the facilitation of the flexor reflex induced by C-fibre conditioning stimulation in normal, inflamed and recovered rats In normal rats, the reflex facilitation induced by the CS was dose-dependently blocked by i.t. galanin (Fig. 4), similar to previously published results.41,50 However, no significant effect on wind-up was observed for any doses of galanin (data not shown). In inflamed rats, as there was a decrease in the extent of CS-induced facilitation, only rats with marked reflex facilitation were used to examine the effect of
Fig. 3. The magnitude (A) and duration (B) of reflex facilitation induced by various doses of i.t. galanin. Data are from five to nine experiments for each dose of galanin under each experimental condition. No significant differences were found among the three experimental groups at any dose.
galanin. As shown in Fig. 4, galanin still dosedependently suppressed reflex facilitation induced by C-fibre CS during inflammation, but its potency was reduced. Following recovery from inflammation, galanin did not produce significant blockade on the C-fibre CS-induced reflex facilitation at any dose (Fig. 4). As in normals, no effect of galanin on wind-up was observed in inflamed or recovered rats (data not shown). Effects of intrathecal galanin (1–12)-pro-bradykinin (2–9) on the flexor reflex, wind-up and reflex facilitation Intrathecal M-35 at 660 ng, a dose which effectively antagonizes the spinal effect of i.t. galanin,44 had a similar moderate facilitatory effect on the flexor reflex in normal, inflamed and recovered rats (Fig. 5). However, the duration of facilitation induced by M-35 was significantly shorter in inflamed and recovered rats than in normals (Fig. 5). Moreover, the magnitude of reflex facilitation induced by M-35 was significantly reduced in recovered rats (Fig. 5). Wind-up was not significantly influenced by M-35 (Fig. 6A), but the reflex facilitatory effect of the C-fibre CS was slightly enhanced in normals (Fig. 6B). After induction of inflammation, M-35 markedly enhanced wind-up in comparison to normals (Fig. 6A). The reduced wind-up observed during inflammation (Fig. 1) was increased to normal levels by M-35 (Fig. 6A). M-35 also potentiated the reflex
Role of galanin in inflammation
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Fig. 4. The effect of various doses of i.t. galanin on C-fibre CS-induced reflex facilitation in normal (n=5–7), inflamed (n=5) and recovered (n=6) rats. The data are expressed as % of control facilitation by CS (defined as 100%). The Mann–Whitney U-test indicated that the blocking effect of galanin on C-fibre CS-induced reflex facilitation in normal rats is significantly stronger than in inflamed rats at 10 µg or in recovered rats at 1 and 10 µg (*P<0.05 and **P<0.01 compared to normals).
Fig. 5. The magnitude (A) and duration (B) of reflex facilitation induced by 660 ng of i.t. M-35 in normal, inflamed and recovered rats. *P<0.05 and **P<0.01 compared to normal rats with Mann–Whitney U-test.
faciltiation induced by the C-fibre CS (Fig. 6B). Interestingly, M-35 did not prevent the occurrence of reflex depression sometimes generated by C-fibre CS in inflamed rats. In contrast, M-35 did not produce significant potentiation on wind-up and reflex facilitation in recovered rats (Fig. 6). DISCUSSION
Effects of inflammation on the baseline flexor reflex At one to three days after s.c. carrageenan, when inflammation was at its peak, the magnitude and
duration of the flexor reflex was significantly increased and prolonged in comparison to normals and to rats that had recovered from inflammation. We believe for several reasons that the observed enhanced reflex activity reflects spinal hyperexcitability during inflammation. (1) We have previously reported that s.c. carrageenan induced an increase in the magnitude and duration of the flexor reflex, which persisted for 5–6 h during acute experiments.52 Since inflammation persists during the experimental period (one to three days after injection), it is likely that the present results reflect a continuation of the spinal hyperexcitability observed within hours after induction of inflammation. (2) It is unlikely that the difference was due to different setting of the window discriminator because in inflamed rats there was higher rate of ongoing EMG activity, which was compensated by higher window height and lead in fact to underestimation of the absolute flexor reflex magnitude. (3) Similar findings have been previously reported after Freund’s adjuvantinduced inflammation.25 Moreover, studies using other approaches have also established that there is an increase in spinal cord excitability during persistent inflammation.12,22,23,35 Such increase in spinal excitability during persistent inflammation probably involves the increased release of tachykinins and activation of both neurokinin (NK)-1 and NK-2 receptors.15,22,23,25,35 The effect of inflammation on conditioning stimulation induced changes in the flexor reflex Despite an increase in baseline flexor reflex during inflammation, we observed a significant decrease in wind-up of the flexor reflex during the C-fibre CS. The post-CS reflex facilitation was also significantly reduced. Furthermore, the CS generated depression of the flexor reflex in some experiments in inflamed rats. There was, however, no correlation between the reduction in CS-induced wind-up and facilitation and
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Fig. 6. The effect of i.t. M-35 on wind-up (A) and post-CS reflex facilitation (B) in normal, inflamed and recovered rats. *P<0.05 compared to control wind-up and reflex facilitation with Wilcoxon signed-ranks test.
in CS-generated inhibition, indicating that these two phenomena may have different mechanisms. This is supported by the result that M-35 increased wind-up and reflex facilitation, but did not reverse CS-induced reflex suppression in most experiments. In a previous study, Stanfa et al. also reported that carrageenaninduced inflammation was associated with reduction of responses of some dorsal horn neurons.32 It can be argued that the reduced wind-up and reflex facilitation in inflamed rats reflects saturation due to higher baseline reflex level. However M-35 enhanced wind-up and reflex facilitation in inflamed rats to normal levels. Thus, even during the elevated baseline reflex, the CS was capable of generating normal levels of wind-up and facilitation in inflamed rats when endogenous galanin was antagonized. C-fibre input is capable of generating inhibition of spinal nociceptive activity. Although some forms of such inhibition may arise via a supraspinal loop,21,46 there is substantial evidence that some of these inhibitions are spinally mediated.20,34,47 The depression of the baseline flexor reflex induced by the C-fibre CS was rarely observed in normal rats. However, we have previously found that the CS in combination with galanin frequently induced reflex inhibition.41 Depression of the flexor reflex by the C-fibre CS can also be observed after pretreatment of tachykinin receptor antagonists which by themselves are not inhibitory (unpublished observations). Thus, it is likely that the CS generates inhibition which is normally masked by the strong excitatory influence of the CS. This inhibitory effect of the C-fibre CS was enhanced during inflammation, possibly as a result of up-regulation of endogenous inhibitory systems, including opioids.8,29,40 On the effect of exogenous and endogenous galanin on the flexor reflex, wind-up and C-fibre conditioning stimulation-induced reflex facilitation in inflamed rats In agreement with previous studies,41,50 i.t. galanin had dose-dependent facilitatory and inhibitory effects
on the baseline flexor reflex in normal rats. Moreover, galanin profoundly antagonized the reflex facilitation induced by the C-fibre CS in normal rats. During and after inflammation, i.t. galanin facilitated the flexor reflex similarly as in normals. Similarly, the depressive effect of high doses of i.t. galanin on the baseline flexor reflex was unchanged in inflamed rats. These results indicate that the mechanisms responsible for the effect of galanin on the baseline flexor reflex may be unchanged during inflammation. Galanin became less effective in blocking the facilitation of the flexor reflex induced by the CS during inflammation. It is possible that this effect is partially due to a reduction in galanin receptors in dorsal root ganglia,53 but not in the dorsal horn,14 during inflammation. However, M-35 still exerted a strong potentiating effect on wind-up and CS-induced facilitation, indicating that endogenous galanin was still effective. This may indicate that active receptors were still present in the terminals of afferents in the dorsal horn at a time when receptor protein was reduced in the cell bodies in the dorsal root ganglia. Furthermore, it is also possible that there was already a strong inhibitory control by endogenous galanin which reduced the effect of exogenous galanin. Intrathecal M-35 moderately potentiated reflex facilitation by the C-fibre CS in normal rats, in agreement with previous data.44 Wind-up during the CS was not significantly influenced by M-35, probably indicating that wind-up in normal rats is maximal and cannot be further increased. In inflamed rats, M-35 strongly enhanced wind-up and reflex facilitation, indicating a tonic galaninergic control on spinal hyperexcitability. Our data indicate that endogenous galanin reduced both the extent of wind-up and post-CS reflex facilitation. Increased basal release of galanin in the dorsal horn has been reported after joint inflammation.11 The source of the endogenous galanin is probably spinal interneurons. Thus, galanin has been found to co-exist with the inhibitory transmitter GABA in dorsal horn neurons in normal rats30 and peripheral
Role of galanin in inflammation
inflammation induced an up-regulation of galanin mRNA in dorsal horn neurons.14 This may underlie the enhanced control by galanin on C-fibre CSinduced spinal hyperexcitability. However, it is unlikely that galanin in sensory neurons contributed to this process as galanin is present in only a few dorsal root ganglion cells in normal rats, which is further reduced after inflammation.14 Thus, inflammation markedly differs from peripheral nerve injury where marked up-regulation of galanin in sensory neurons occurs.38,42,44,58 On the effect of exogenous and endogenous galanin on the flexor reflex, wind-up and C-fibre conditioning stimulation-induced reflex facilitation in recovered rats At four to eight days after inflammation, when inflammation was reduced, the baseline flexor reflex was similar to normal rats, indicating that the spinal hyperexcitability generated during persistent inflammation is a reversible process and may be maintained by continuous discharges from inflamed peripheral tissues. The wind-up and reflex facilitation also returned to normal levels. However, surprisingly i.t. galanin did not block the reflex facilitatory effect of C-fibre CS as in normals. This is unlikely to be due to excess endogenous control by galanin as M-35 also did not potentiate wind-up and reflex facilitatory effect of the C-fibre CS. Thus, it appears that the C-fibre CS was no longer influenced by either exogenous or endogenous galanin. In a recent study galanin
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receptors in dorsal root ganglia were found to be reduced at three days and partially recovered at five days following carrageenan inflammation.53 However, it is possible that the newly synthesized receptors had not been transported to the afferent terminals from the cell body at five days after induction of inflammation. CONCLUSIONS
We have previous suggested that galanin is an endogenous inhibitory peptide in nociception, particularly after peripheral nerve injury where galanin is markedly up-regulated in primary afferents. The present study showed that during inflammation galanin has a similar inhibitory role, although the source of galanin is the dorsal horn. Inflammation induces up-regulation of opioid peptides in the spinal cord8,29,40 and there is also evidence that the function of the endogenous GABA system is also enhanced during inflammation.4,5 Thus, these inhibitory systems may act in concert to combat the input of excessive nociceptive information generated during persistent inflammation. Acknowledgements—The present study is supported by Swedish Medical Research Council (projects 07913 and 11038), the Bank of Sweden Tercentenary Foundation, Astra Pain Control AB, the Biomed II programme of the European Commission (project BMH4-CT950172) and research funds of the Karolinska Institute.
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Kuraishi Y., Kawamura M., Yamaguchi H., Houtani T., Kawabata S., Futaki S., Fujii N. and Satoh M. (1991) Intrathecal injections of galanin and its antiserum, affect nociceptive response of rat to mechanical, but not thermal, stimuli. Pain 44, 321–324. Melander T., Ho¨kfelt T. and Ro¨kaeus A r . (1986) Distribution of galanin-like immunoreactivity in the rat central nervous system. J. comp. Neurol. 248, 475–517. Mendell L. M. (1966) Physiological properties of nonmyelinated fibre projection to the spinal cord. Expl Neurol. 16, 316–332. Morris R. (1987) Inhibition of nociceptive responses of laminae V–VII dorsal horn neurones by stimulation of mixed and muscle nerves in the cat. Brain Res. 401, 365–370. Morton C. R., Maisch B. and Zimmermann M. (1987) Diffuse noxious inhibitory controls of lumbar spinal neurons involves a supraspinal loop in the cat. Brain Res. 410, 347–352. Neugebauer V., Rumenapp P. and Schaible H.-G. (1996) The role of spinal neurokinin-2 receptors in the processing of nociceptive information from the joint and in the generation and maintenance of inflammation-evoked hyperexcitability of dorsal horn neurons in the rat. Eur. J. Neurosci. 8, 249–260. Neugebauer V., Weiretter F. and Schaible H.-G. (1995) Involvement of substance P and neurokinin-1 receptors in the hyperexcitability of dorsal horn neurons during the development of acute arthritis in rat’s knee joint. J. Neurophysiol. 73, 1574–1583. Nussbaumer J.-C., Yanagisawa M. and Otsuka M. (1989) Pharmacological properties of a C-fibre response evoked by saphenous nerve stimulation in an isolated spinal cord-nerve preparation of the newborn rat. Br. J. Pharmac. 98, 373–382. Parsons A. M., Honda C., Jia Y.-P., Budai D., Xu X.-J., Wiesenfeld-Hallin Z. and Seybold V. S. (1996) Spinal NK1 receptors contribute to the increased excitability of the nociceptive flexor reflex during persistent peripheral inflammation. Brain Res. 739, 263–275. Post C., Alari L. and Ho¨kfelt T. (1988) Intrathecal galanin increases the latency in the tail flick and hot plate tests in mouse. Acta physiol. scand. 132, 583–584. Randic M., Gerber G., Ryu P. D. and Kangrga I. (1987) Inhibitory actions of galanin and somatostatin 28 on rat spinal dorsal horn neurons. Soc. Neurosci. Abstr. 17, 1308. Reimann W., Englberger W., Friderichs E., Selve N. and Wilffert B. (1994) Spinal antinociception by morphine is antagonised by galanin receptor antagonists. Naunyn-Schmiedeberg’s Arch. Pharmac. 350, 380–386. Ruda M. A., Iadarola M. J., Cohen L. V. and Young W. S. (1988) In situ hydbridization histochemistry and immunohistochemistry reveal increase in spinal dynorphin biosynthesis in a rat model of peripheral inflammation and hyperalgesia. Proc. natn. Acad. Sci. U.S.A. 85, 622–626. Simmons D. R., Spike R. C. and Todd A. J. (1995) Galanin is contained in GABAergic neurons in the rat spinal dorsal horn. Neurosci. Lett. 187, 119–122. Skofitsch G. and Jacobowitz D. (1985) Galanin-like immunoreactivity in capsaicin sensitive sensory neurons and ganglia. Brain Res. Bull. 15, 191–195. Stanfa L. C., Sullivan A. F. and Dickenson A. H. (1992) Alterations in neuronal excitability and the potency of spinal mu, delta and kappa opioids after carrageenan-induced inflammation. Pain 50, 345–354. Tatemoto K., Ro¨kaeus A r ., Jo¨rnvall H., McDonald W. and Mutt V. (1983) Galanin—a novel biologically active peptide from porcine intestine. Fedn Eur. biochem. Socs Lett. 164, 124–128. Taylor J. S., Neal R. I., Harris J., Ford T. W. and Clarke R. W. (1991) Prolonged inhibition of a spinal reflex after intense stimulation of distant peripheral nerves in the decerebrated rabbit. J. Physiol. 437, 71–83. Thompson S. N., Dray A. and Urban L. (1994) Injury-induced plasticity of spinal reflex activity: NK1 neurokinin receptor activation and enhanced A- and C-fiber mediated responses in the rat spinal cord. J. Neurosci. 14, 3672–3687. Togunaga A., Senba E., Manabe Y., Shida T., Ueda Y. and Tohyama M. (1992) Orofacial pain increas mRNA level for galanin in the trigeminal nucleus caudalis of the rat. Peptides 13, 1067–1072. Verge V. M., Xu X. J., Langel U } ., Ho¨kfelt T., Wiesenfeld-Hallin Z. and Bartfai T. (1993) Evidence for endogenous inhibition of autotomy by galanin in the rat after sciatic nerve section: demonstrated by chronic intrathecal infusion of a high affinity galanin receptor antagonist. Neurosci. Lett. 149, 193–197. Villar M. J., Corte´s R., Theodorsson E., Wiesenfeld-Hallin Z., Schalling M., Fahrenkrug J., Emson P. C. and Ho¨kfelt T. (1989) Neuropeptide expression in rat dorsal root ganglion cells and spinal cord after peripheral nerve injury with special reference to galanin. Neuroscience 33, 587–604. Wall P. D. and Woolf C. J. (1984) Muscle but not cutaneous C-afferent input produces prolonged increases in the excitability of the flexion reflex in the rat. J. Physiol. 356, 443–458. Weihe E., Millan M. J., Ho¨llt V., Nohr D. and Herz A. (1989) induction of the gene encoding pro-dynorphin by experimentally induced arthritis enhances staining for dynorphin in the spinal cord of rats. Neuroscience 31, 77–95. Wiesenfeld-Hallin Z., Villar M. J. and Ho¨kfelt T. (1989) The effect of intrathecal galanin and C-fiber stimulation on the flexor reflex in the rat. Brain Res. 486, 205–213. Wiesenfeld-Hallin Z., Bartfai T. and Ho¨kfelt T. (1992) Galanin in sensory neurons in the spinal cord. Frontiers Neuroendocr. 13, 319–343. Wiesenfeld-Hallin Z., Xu X.-J., Villar M. J. and Ho¨kfelt T. (1990) Intrathecal galanin potentiates the spinal analgesic effect of morphine: electrophysiological and behavioural studies. Neurosci. Lett. 109, 217–221.
Role of galanin in inflammation 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58.
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Wiesenfeld-Hallin Z., Xu X.-J., Langel U } ., Bedecs K, Ho¨kfelt T. and Bartfai T. (1992) Galanin-mediated control of pain: enhanced role after nerve injury. Proc. natn. Acad. Sci. U.S.A. 89, 3334–3337. Wiesenfeld-Hallin Z., Xu X. J., Hao J. X. and Ho¨kfelt T. (1993) The behavioural effects of intrathecal galanin on tests of thermal and mechanical nociception in the rat. Acta physiol. scand. 147, 457–458. Willer J. C., Roby A. and Le Bars D. (1984) Psychophysiological and electorphysiological approaches to the pain-relieving effects of heterotopic nociceptive stimuli. Brain 107, 1095–1112. Woolf C. J. (1983) C-primary afferent fibre mediated inhibition in the dorsal horn of the decerebrate-spinal rat. Expl Brain Res. 51, 283–290. Woolf C. J. (1996) Wind-up and central sensitization are not equivalent. Pain 66, 105–108. Xu X.-J., Dalsgaard C.-J. and Wiesenfeld-Hallin Z. (1992) Intrathecal CP-96,345 blocks reflex facilitation induced in rats by substance P and C-fiber-conditioning stimulation. Eur. J. Pharmac. 216, 337–344. Xu X.-J., Wiesenfeld-Hallin Z., Villar M. J., Fahrenkrug J. and Ho¨kfelt T. (1990) On the role of galanin, substance P and other neuropeptides in primary sensory neurons of the rat: studies on spinal reflex excitability and peripheral axotomy. Eur. J. Neurosci. 2, 733–743. Xu X.-J., Wiesenfeld-Hallin Z. and Ho¨kfelt T. (1991) Intrathecal galanin blocks the prolonged increase in spinal cord flexor reflex induced by conditioning stimulation of umyelinated muscle afferents in the rat. Brain Res. 541, 350–353. Xu X.-J., Elfvin A. and Wiesenfeld-Hallin Z. (1995) Subcutaneous carrageenan, but not formalin, increase spinal flexor reflex excitability in the rat. Neurosci. Lett. 196, 116–118. Xu Z.-Q., Shi T.-J., Landry M. and Ho¨kfelt T. (1996) Evidence for galanin receptors in primary sensory neurones and effect of axotomy and inflammation. NeuroReport 8, 237–242. Yanagisawa M., Yagi N., Otsuka M., Yanaihara C. and Yanaihara N. (1986) Inhibitory effects of galanin on the isolated spinal cord of the newborn rat. Neurosci. Lett. 70, 278–282. Zhang X., Ji R. R., Nilsson S., Villar M., Ubink R., Ju G., Wiesenfeld-Hallin Z. and Ho¨kfelt T. (1995) Neuropeptide Y and galanin binding sites in rat and monkey lumbar dorsal root ganglia and spinal cord and effect of peripheral axotomy. Eur. J. Neurosci. 7, 367–380. Zhang X., Nicholas A. P. and Ho¨kfelt T. (1995) Ultrastructural studies on peptides in the dorsal horn of the spinal cord. 1. Co-existence of galanin with other peptides in primary afferents in normal rats. Neuroscience 57, 365–384. Zhang X., Nicholas A. P. and Ho¨kfelt T. (1995) Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord. 2. Coexistence of galanin with other peptides in local neurons. Neuroscience 64, 875–891. Zhang X., Bean A. J., Wiesenfeld-Hallin Z., Xu W. and Ho¨kfelt T. (1995) Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord. 3. Effects of peripheral axotomy with special reference to galanin. Neuroscience 64, 893–915. (Accepted 5 December 1997)
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Neuroscience Vol. 85, No. 3, pp. 827–835, 1998 Copyright 1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00676-3
ON THE ROLE OF GALANIN IN MEDIATING SPINAL FLEXOR REFLEX EXCITABILITY IN INFLAMMATION I. S. XU, S. GRASS, X.-J. XU and Z. WIESENFELD-HALLIN* Department of Medical Laboratory Sciences and Technology, Division of Clinical Neurophysiology, Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden Abstract––The effects of exogenous and endogenous galanin on spinal flexor reflex excitability was evaluated in rats one to eight days after the induction of inflammation by subcutaneous injection of carrageenan into the sural nerve innervation area. In normal rats, electrical stimulation of C-fibres in the sural nerve elicited a brisk reflex discharge. Conditioning stimulation of C-fibres (1/s) generated a gradual increase in reflex magnitude (wind-up), which was followed by a period of reflex hyperexcitability. Intrathecal galanin dose-dependently blocked reflex hyperexcitability induced by C-fibre conditioning stimulation whereas i.t. M-35, a high-affinity galanin receptor antagonist, moderately potentiated this effect. At one to three days after the injection of carrageenen, when inflammation was at its peak, the magnitude of the reflex was significantly increased and discharge duration became prolonged. However, wind-up and reflex hyperexcitability were significantly reduced. Furthermore, reduced reflex excitability during conditioning stimulation (‘‘wind-down’’) and depression of the reflex were sometimes present, which are rarely observed in normal rats. Intrathecal galanin reduced hyperexcitability during inflammation, although its potency was weaker than in normals. However, the galanin receptor antagonist M-35 strongly enhanced wind-up and reflex hyperexcitability, similarly as in normal rats. The baseline flexor reflex, wind-up and C-fibre conditioning stimulation-induced facilitation were normalized four to eight days after carrageenan injection when signs of inflammation were diminishing. Interestingly, intrathecal galanin and M-35 failed to influence spinal excitability. The results suggest a complex functional plasticity in the role of endogenous galanin in mediating spinal excitability during inflammation. There appears to be an enhanced endogenous inhibitory control by galanin on C-afferent input during the peak of inflammation, which may explain the relative ineffectiveness of exogenous galanin. During the recovery phase there may be a reduction in galanin receptors, which may impair the action of endogenous and exogenous galanin. These results further support the notion that galanin is an endogenous inhibitory peptide in nociception. 1998 IBRO. Published by Elsevier Science Ltd. Key words: carrageenan, dorsal horn, galanin receptor antagonist, intrathecal, nociception, pain.
The neuropeptide galanin, originally isolated by Tatemoto et al.,33 has been recognized as an important neurotransmitter and/or neuromodulator in the nervous system.3 Galanin-like immunoreactivity (LI) and galanin mRNA has been found in dorsal root ganglion cells across many species.6,18,31 A network of galanin-LI fibres6,18,56 and galanin receptors16,55 have been localized in the superficial laminae of the dorsal horn. There are also dorsal horn interneurons which contain galanin, some of which also express the inhibitory transmitter GABA.6,18,30,57 These morphological findings imply that galanin may have a role in spinal nociceptive transmission and modulation. Functional studies examining the effect of galanin on spinal nociception have yielded conflicting results. Thus, while some early studies suggested that i.t. *To whom correspondence should be addressed. Abbreviations: CS, conditioning stimulation; EMG, electromyogram; LI, -like immunoreactivity; M-35, galanin (1– 12)-Pro-Bradykinin (2–9); NK, neurokinin. 827
galanin elicited hyperalgesia,17 others reported behavioural antinociception or depression of nociceptive reflexes in electrophysiological studies.7,24,26,27,45,54 Using the flexor reflex in decerebrate, spinalized rats as a tool, we have systematically assessed the effects of galanin on spinal reflex excitability. We observed that i.t. galanin had a dose-dependent biphasic facilitatory and inhibitory effect on the baseline flexor reflex.41,50 In addition, galanin dose-dependently suppressed the development of spinal cord hyperexcitability induced by intense activation of C-fibres, as well as by exogenously applied excitatory neuropeptides substance P and calcitonin gene-related peptide.41,50,51 Moreover, galanin also potentiated43 and is possibly involved28 in the spinal antinociceptive effect of morphine. Thus, we have suggested that galanin may be a predominantly inhibitory modulator in the spinal cord, particularly under states of hyperexcitability.42 A series of high-affinity antagonists of galanin receptors have been tested to address the role of endogenous galanin in the modulation of spinal
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nociception.1,2,44 Intrathecal administration of M-35 [galanin (1–12)-pro-bradykinin (2–9)], a high-affinity galanin receptor antagonist, blocked the spinal effects of galanin and moderately potentiated the C-fibre conditioning stimulus (CS)-induced facilitation of the flexor reflex in normal rats, indicating a tonic role for galanin in spinal inhibitory modulation.44 Interestingly, i.t. M-35-induced potentiation became significantly stronger after peripheral axotomy44 when galanin was up-regulated in sensory neurons,38,58 indicating that galanin-mediated inhibition is enhanced. Moreover, behavioural studies have also shown an endogenous inhibitory effect of galanin on neuropathic pain-related behaviours in rats after peripheral nerve injury.13,37 Peripheral inflammation induces complex changes in messengers in sensory neurons and in the spinal cord and many of these changes are different from those occurring after peripheral nerve injury.9,10 In contrast to the profound up-regulation of galanin in sensory neurons in axotomized rats,38,58 a decrease in the number of sensory neurons expressing galanin was observed three days after carrageenan-induced inflammation.14 In contrast, the number of dorsal horn neurons which express galanin mRNA are increased in the superficial laminae of the dorsal horn three days after inflammation.14 An earlier study has also shown that injecting formalin into the lips of rats induced an up-regulation of preprogalanin mRNA 6 h later in trigeminal nucleus caudalis in the dorsal horn.36 No changes in galanin binding sites were observed in the dorsal horn and in the dorsal root ganglia after inflammation.14 However, in a recent study mRNA coding for the galanin R1 receptor found in normal dorsal root ganglion cells in rat was shown to be transiently down-regulated during carrageenan-induced inflammation.53 Inflammation also alters the release of galanin. Thus, Hope et al.11 reported that joint inflammation increased the basal release of galanin in the dorsal horn and innocuous and noxious stimulation of the inflamed joints suppressed such release. The present study was undertaken to examine the effect of galanin on spinal flexor reflex excitability during inflammation, one to three days after application of subcutaneous carrageenan. The role of endogenous galanin was assessed with M-35. In view of the fact that peripheral inflammation induced by carrageenan is reversible, we have also studied the effects of galanin and M-35 in rats that recovered from inflammation (four to eight days after injection of carrageenan). The effects of inflammation, galanin and galanin receptor antagonist on spinal hyperexcitability was studied by observing both the gradual increase in reflex magnitude during the C-fibre CS (wind-up)19 and the facilitation of the flexor reflex following C-fibre CS (central sensitization).39 The relative significance of these measurements in studying spinal hyperexcitability has been discussed previously.48
EXPERIMENTAL PROCEDURES
Female Sprague–Dawley rats weighing 200–250 g (B & K Universal, Stockholm, Sweden) were used in the present experiments, which were conducted under the approval of the local research ethics committee. Induction and time-course of inflammation Inflammation was induced by subcutaneous injection of 0.1 ml 2% ë-carrageenan into the lateral side of the plantar skin under methohexital anaesthesia (Brietal, Lilly, Indianapolis, IN, U.S.A., 70 mg/kg; i.p.). Inflammation was verified by the presence of oedema and erythema and reached peak level 24–48 h after the injection and gradually recovered thereafter. Little or no signs of inflammation were observed five to six days after the injection of carrageenan. Flexor reflex experiments On the day of the acute experiment, normal rats or rats which has been injected with carrageenan one to eight days previously were briefly anaesthetized with methohexital, ventilated and decerebrated by aspiration of the forebrain and midbrain. The spinal cord was exposed by a laminectomy at mid-thoracic level and sectioned at Th8–9. An i.t. catheter (PE 10) was implanted caudal to the transaction with its tip on the lumbar spinal cord at L4–L5. All experiments started at least 1 h after spinal transection. The flexor reflex was elicited 1/min by electric shocks applied to the sural nerve (rectangular pulses of 0.5 ms duration, 10 mA) and was recorded as electromyogram (EMG) activity with stainless steel needle electrodes inserted into the ipsilateral posterior biceps femoris/semitendinosus muscles. Previous studies under similar experimental conditions have shown that such stimuli activate both A- and C-afferents.39 In some experiments, a conditioning stimulus (CS) train administered to the sural nerve (20 shocks at 1 Hz) was used to elicit wind-up (gradual increase in reflex magnitude during the CS train) and subsequent facilitation of the flexor reflex. For quantification of baseline reflex mangitude, the number of potentials exceeding the level of spontaneous EMG activity was counted using a window discriminator, integrated over 2 s and recorded on a chart recorder.41,49 The integration time was 1 s during the CS. A stable reflex baseline (defined as less than 15% variation) was established for at least 20–30 min before administration of drugs or the CS. Heart rate and rectal temperature of the rat were monitored. Peptides Porcine galanin was obtained from Bachem and M-35 was generously supplied by Prof. T. Bartfai (Department of Neurochemistry and Neurotoxicology, University of Stockholm). Galanin and M-35 were dissolved in 0.9% normal saline. The aliquoted peptide solutions were stored under 70C and used directly after thawing. The i.t. injections were made in 10 µl followed by 10 µl saline to flush the catheter. Data collection and statistics The effect of i.t. peptides and the nerve CS on the flexor reflex were expressed as percent change in reflex magnitude compared to baseline, which was defined as 100%. Data were analysed by one-way ANOVA followed by Fisher PLSD (protected least significant difference) test. Some data were also analysed by Wilcoxon signed ranks test or Mann– Whitney U-test or the ø2-test. Variability is expressed as S.E.M.
Role of galanin in inflammation Table 1. The magnitude and duration of the flexor reflex in normal, inflamed and recovered rats
Normal Inflamed Recovered
n
Magnitude
Duration (s)
19 16 13
14.11.6** 36.48.7 16.12.3*
5.41.9** 15.12.2 6.02.2**
The relative magnitude and duration of the integrated EMG discharge recorded from ipsilateral hamstring muscles in response to electrical stimulation (0.5 ms, 10 mA) of the sural nerve at 1/min in normal, inflamed and recovered rats. Data are expressed as meanS.E.M. ANOVA indicated significant differences in both the magnitude (F2,45=5.6, P<0.01) and duration (F2,45=7.2, P<0.01) of the flexor reflex recorded from normal, inflamed and recovered rats. Individual comparisons were made with the Fisher PLSD test, *P<0.05, **P<0.01 compared to inflamed rats.
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Table 2. Number of rats showing the different forms of changes in reflex excitability during the C-fibre conditioning stimulus in normal, inflamed and recovered rats
Normal Inflamed Recovered
Wind-up >100%
Wind-up <100%
Wind-down
16 5 12
3 7 0
0 4 1
The ÷2-test indicated significant differences among the frequencies of occurrence of various types of excitability changes (÷2=14.2, d.f.=4, P<0.01).
RESULTS
The flexor reflex in normal, inflamed and recovered rats Electrical stimulation of the sural nerve elicited a brisk contraction of the ipsilateral hamstring muscles, which was quantified by recording EMG activity. Afterdischarges (defined as activity longer than 1 s after the stimulus artifact) were observed in more than 50% of the experiments in normal rats. However, in 80% of experiments, the duration of EMG activity generated by a single electric shock was less than 5 s (Table 1). At one to three days after injection of carrageenan the magnitude of the integrated flexor reflex was significantly increased compared to normals (Table 1). Moreover, the duration of EMG activity was also significantly prolonged with reflex discharges longer than 5 s in more than 90% of experiments (Table 1). The rate of ongoing EMG activity recorded during inflammation was higher than normal, although no particular attention was paid to this and the contribution of ongoing activity to the recorded reflex activity was eliminated by adjusting the window height on the discriminator to exclude ongoing EMG. At four to eight days after the injection of carrageenan, when the signs of inflammation were diminishing, the magnitude and duration of the reflex became normalized (Table 1), although prolonged discharges were observed more often than in normals. C-fibre conditioning stimulation-induced reflex windup and facilitation in normal, inflamed and recovered rats A CS train of 1 Hz for 20 s generated a gradual increase in reflex magnitude (wind-up) in all normal animals (Table 2, Fig. 1). At one to three days after induction of inflammation, the frequency of wind-up decreased and in some cases ‘‘wind-down’’ (decrease
Fig. 1. The magnitude of wind-up during the C-fibre CS (1 Hz, 20 shocks). The data are presented as mean increase in reflex magnitude over baseline reflex (defined as 100%) in blocks of five responses. In this and other figures, the number of rats examined is shown in the brackets. ANOVA indicated significant differences among the three experimental conditions for all four blocks (F2,37=3.3, 3.3, 5.2 and 5,6, respectively; P<0.05, 0.05, 0.01 and 0.01, respectively). Individul comparisons were with the Fisher PLSD test, *P<0.05 and **P<0.01 compared to inflamed rats.
in reflex magnitude during the CS) occurred (Table 2). During recovery (four to six days after injection of carrageenan), the frequency of wind-up became normalized (Table 2). The distribution of different response types to the CS train was very different among the three experimental groups (Table 2) and there were significant differences in the magnitude of wind-up during stimuli 11–15 and 16–20 between inflamed vs normal or recovered rats (Fig. 1). Single shocks applied 30 s and 1 min after the termination of the CS train generated a markedly enhanced reflex (post-CS facilitation) in normal rats (Fig. 2A). The duration of reflex facilitation was between 1–5 min in normals after which the reflex returned to baseline level (Fig. 2B). Similar findings were observed in recovered rats (Fig. 2). In contrast, in the inflamed rats the magnitude of post-CS facilitation was significantly reduced (Fig. 2). Moreover, in more than 60% of inflamed rats the CS generated depression or facilitation followed by depression of the flexor reflex was observed. Inhibition following C-fibre CS is rarely observed in normals (one of 19 or
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Fig. 2. The magnitude and duration of reflex hyperexcitability following CS. ANOVA indicated an overall significant difference in the magnitude, but not duration, of reflex facilitation induced by the C-fibre CS among the three experimental conditions (F2,37=6.1 and 2.6, respectively; P<0.01 and <0.05, respectively). Individual comparisons are with the Fisher PLSD test, *P<0.05 and **P<0.01 compared to inflamed rats.
5% in the present series of experiments). Inhibition was observed in two of 13 (6.5%) experiments in recovered rats. Effects of intrathecal galanin on the baseline flexor reflex in normal and inflamed rats In normal rats, 100 ng, 1 µg and 10 µg i.t. galanin caused a brief facilitation of the baseline flexor reflex. At the two highest doses (1 and 10 µg) depression was also frequently observed. Similar facilitatory effect of i.t. galanin on the baseline flexor reflex was found in inflamed and recovered rats (Fig. 3). In four normal rats the depressive effect of increasing doses of galanin on the baseline flexor reflex was examined in detail. At the lowest dose (100 ng), galanin did not depress the flexor reflex whereas at higher doses depression of the baseline flexor reflex was observed in some experiments (two of four at 1 µg, and three of three at 10 µg). Thus, the pattern of the effect of i.t. galanin on the baseline flexor reflex observed in the present experiments was similar to previous results.41 In three rats examined one to three days after inflammation, i.t. galanin similarly depressed the baseline flexor reflex (one of three at 1 µg and three of three at 10 µg). Effects of intrathecal galanin on the facilitation of the flexor reflex induced by C-fibre conditioning stimulation in normal, inflamed and recovered rats In normal rats, the reflex facilitation induced by the CS was dose-dependently blocked by i.t. galanin (Fig. 4), similar to previously published results.41,50 However, no significant effect on wind-up was observed for any doses of galanin (data not shown). In inflamed rats, as there was a decrease in the extent of CS-induced facilitation, only rats with marked reflex facilitation were used to examine the effect of
Fig. 3. The magnitude (A) and duration (B) of reflex facilitation induced by various doses of i.t. galanin. Data are from five to nine experiments for each dose of galanin under each experimental condition. No significant differences were found among the three experimental groups at any dose.
galanin. As shown in Fig. 4, galanin still dosedependently suppressed reflex facilitation induced by C-fibre CS during inflammation, but its potency was reduced. Following recovery from inflammation, galanin did not produce significant blockade on the C-fibre CS-induced reflex facilitation at any dose (Fig. 4). As in normals, no effect of galanin on wind-up was observed in inflamed or recovered rats (data not shown). Effects of intrathecal galanin (1–12)-pro-bradykinin (2–9) on the flexor reflex, wind-up and reflex facilitation Intrathecal M-35 at 660 ng, a dose which effectively antagonizes the spinal effect of i.t. galanin,44 had a similar moderate facilitatory effect on the flexor reflex in normal, inflamed and recovered rats (Fig. 5). However, the duration of facilitation induced by M-35 was significantly shorter in inflamed and recovered rats than in normals (Fig. 5). Moreover, the magnitude of reflex facilitation induced by M-35 was significantly reduced in recovered rats (Fig. 5). Wind-up was not significantly influenced by M-35 (Fig. 6A), but the reflex facilitatory effect of the C-fibre CS was slightly enhanced in normals (Fig. 6B). After induction of inflammation, M-35 markedly enhanced wind-up in comparison to normals (Fig. 6A). The reduced wind-up observed during inflammation (Fig. 1) was increased to normal levels by M-35 (Fig. 6A). M-35 also potentiated the reflex
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Fig. 4. The effect of various doses of i.t. galanin on C-fibre CS-induced reflex facilitation in normal (n=5–7), inflamed (n=5) and recovered (n=6) rats. The data are expressed as % of control facilitation by CS (defined as 100%). The Mann–Whitney U-test indicated that the blocking effect of galanin on C-fibre CS-induced reflex facilitation in normal rats is significantly stronger than in inflamed rats at 10 µg or in recovered rats at 1 and 10 µg (*P<0.05 and **P<0.01 compared to normals).
Fig. 5. The magnitude (A) and duration (B) of reflex facilitation induced by 660 ng of i.t. M-35 in normal, inflamed and recovered rats. *P<0.05 and **P<0.01 compared to normal rats with Mann–Whitney U-test.
faciltiation induced by the C-fibre CS (Fig. 6B). Interestingly, M-35 did not prevent the occurrence of reflex depression sometimes generated by C-fibre CS in inflamed rats. In contrast, M-35 did not produce significant potentiation on wind-up and reflex facilitation in recovered rats (Fig. 6). DISCUSSION
Effects of inflammation on the baseline flexor reflex At one to three days after s.c. carrageenan, when inflammation was at its peak, the magnitude and
duration of the flexor reflex was significantly increased and prolonged in comparison to normals and to rats that had recovered from inflammation. We believe for several reasons that the observed enhanced reflex activity reflects spinal hyperexcitability during inflammation. (1) We have previously reported that s.c. carrageenan induced an increase in the magnitude and duration of the flexor reflex, which persisted for 5–6 h during acute experiments.52 Since inflammation persists during the experimental period (one to three days after injection), it is likely that the present results reflect a continuation of the spinal hyperexcitability observed within hours after induction of inflammation. (2) It is unlikely that the difference was due to different setting of the window discriminator because in inflamed rats there was higher rate of ongoing EMG activity, which was compensated by higher window height and lead in fact to underestimation of the absolute flexor reflex magnitude. (3) Similar findings have been previously reported after Freund’s adjuvantinduced inflammation.25 Moreover, studies using other approaches have also established that there is an increase in spinal cord excitability during persistent inflammation.12,22,23,35 Such increase in spinal excitability during persistent inflammation probably involves the increased release of tachykinins and activation of both neurokinin (NK)-1 and NK-2 receptors.15,22,23,25,35 The effect of inflammation on conditioning stimulation induced changes in the flexor reflex Despite an increase in baseline flexor reflex during inflammation, we observed a significant decrease in wind-up of the flexor reflex during the C-fibre CS. The post-CS reflex facilitation was also significantly reduced. Furthermore, the CS generated depression of the flexor reflex in some experiments in inflamed rats. There was, however, no correlation between the reduction in CS-induced wind-up and facilitation and
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Fig. 6. The effect of i.t. M-35 on wind-up (A) and post-CS reflex facilitation (B) in normal, inflamed and recovered rats. *P<0.05 compared to control wind-up and reflex facilitation with Wilcoxon signed-ranks test.
in CS-generated inhibition, indicating that these two phenomena may have different mechanisms. This is supported by the result that M-35 increased wind-up and reflex facilitation, but did not reverse CS-induced reflex suppression in most experiments. In a previous study, Stanfa et al. also reported that carrageenaninduced inflammation was associated with reduction of responses of some dorsal horn neurons.32 It can be argued that the reduced wind-up and reflex facilitation in inflamed rats reflects saturation due to higher baseline reflex level. However M-35 enhanced wind-up and reflex facilitation in inflamed rats to normal levels. Thus, even during the elevated baseline reflex, the CS was capable of generating normal levels of wind-up and facilitation in inflamed rats when endogenous galanin was antagonized. C-fibre input is capable of generating inhibition of spinal nociceptive activity. Although some forms of such inhibition may arise via a supraspinal loop,21,46 there is substantial evidence that some of these inhibitions are spinally mediated.20,34,47 The depression of the baseline flexor reflex induced by the C-fibre CS was rarely observed in normal rats. However, we have previously found that the CS in combination with galanin frequently induced reflex inhibition.41 Depression of the flexor reflex by the C-fibre CS can also be observed after pretreatment of tachykinin receptor antagonists which by themselves are not inhibitory (unpublished observations). Thus, it is likely that the CS generates inhibition which is normally masked by the strong excitatory influence of the CS. This inhibitory effect of the C-fibre CS was enhanced during inflammation, possibly as a result of up-regulation of endogenous inhibitory systems, including opioids.8,29,40 On the effect of exogenous and endogenous galanin on the flexor reflex, wind-up and C-fibre conditioning stimulation-induced reflex facilitation in inflamed rats In agreement with previous studies,41,50 i.t. galanin had dose-dependent facilitatory and inhibitory effects
on the baseline flexor reflex in normal rats. Moreover, galanin profoundly antagonized the reflex facilitation induced by the C-fibre CS in normal rats. During and after inflammation, i.t. galanin facilitated the flexor reflex similarly as in normals. Similarly, the depressive effect of high doses of i.t. galanin on the baseline flexor reflex was unchanged in inflamed rats. These results indicate that the mechanisms responsible for the effect of galanin on the baseline flexor reflex may be unchanged during inflammation. Galanin became less effective in blocking the facilitation of the flexor reflex induced by the CS during inflammation. It is possible that this effect is partially due to a reduction in galanin receptors in dorsal root ganglia,53 but not in the dorsal horn,14 during inflammation. However, M-35 still exerted a strong potentiating effect on wind-up and CS-induced facilitation, indicating that endogenous galanin was still effective. This may indicate that active receptors were still present in the terminals of afferents in the dorsal horn at a time when receptor protein was reduced in the cell bodies in the dorsal root ganglia. Furthermore, it is also possible that there was already a strong inhibitory control by endogenous galanin which reduced the effect of exogenous galanin. Intrathecal M-35 moderately potentiated reflex facilitation by the C-fibre CS in normal rats, in agreement with previous data.44 Wind-up during the CS was not significantly influenced by M-35, probably indicating that wind-up in normal rats is maximal and cannot be further increased. In inflamed rats, M-35 strongly enhanced wind-up and reflex facilitation, indicating a tonic galaninergic control on spinal hyperexcitability. Our data indicate that endogenous galanin reduced both the extent of wind-up and post-CS reflex facilitation. Increased basal release of galanin in the dorsal horn has been reported after joint inflammation.11 The source of the endogenous galanin is probably spinal interneurons. Thus, galanin has been found to co-exist with the inhibitory transmitter GABA in dorsal horn neurons in normal rats30 and peripheral
Role of galanin in inflammation
inflammation induced an up-regulation of galanin mRNA in dorsal horn neurons.14 This may underlie the enhanced control by galanin on C-fibre CSinduced spinal hyperexcitability. However, it is unlikely that galanin in sensory neurons contributed to this process as galanin is present in only a few dorsal root ganglion cells in normal rats, which is further reduced after inflammation.14 Thus, inflammation markedly differs from peripheral nerve injury where marked up-regulation of galanin in sensory neurons occurs.38,42,44,58 On the effect of exogenous and endogenous galanin on the flexor reflex, wind-up and C-fibre conditioning stimulation-induced reflex facilitation in recovered rats At four to eight days after inflammation, when inflammation was reduced, the baseline flexor reflex was similar to normal rats, indicating that the spinal hyperexcitability generated during persistent inflammation is a reversible process and may be maintained by continuous discharges from inflamed peripheral tissues. The wind-up and reflex facilitation also returned to normal levels. However, surprisingly i.t. galanin did not block the reflex facilitatory effect of C-fibre CS as in normals. This is unlikely to be due to excess endogenous control by galanin as M-35 also did not potentiate wind-up and reflex facilitatory effect of the C-fibre CS. Thus, it appears that the C-fibre CS was no longer influenced by either exogenous or endogenous galanin. In a recent study galanin
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receptors in dorsal root ganglia were found to be reduced at three days and partially recovered at five days following carrageenan inflammation.53 However, it is possible that the newly synthesized receptors had not been transported to the afferent terminals from the cell body at five days after induction of inflammation. CONCLUSIONS
We have previous suggested that galanin is an endogenous inhibitory peptide in nociception, particularly after peripheral nerve injury where galanin is markedly up-regulated in primary afferents. The present study showed that during inflammation galanin has a similar inhibitory role, although the source of galanin is the dorsal horn. Inflammation induces up-regulation of opioid peptides in the spinal cord8,29,40 and there is also evidence that the function of the endogenous GABA system is also enhanced during inflammation.4,5 Thus, these inhibitory systems may act in concert to combat the input of excessive nociceptive information generated during persistent inflammation. Acknowledgements—The present study is supported by Swedish Medical Research Council (projects 07913 and 11038), the Bank of Sweden Tercentenary Foundation, Astra Pain Control AB, the Biomed II programme of the European Commission (project BMH4-CT950172) and research funds of the Karolinska Institute.
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