Effect of intrathecal galanin and its putative antagonist M35 on pain behavior in a neuropathic pain model1

Brain Research 886 (2000) 67–72 www.elsevier.com / locate / bres

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Effect of intrathecal galanin and its putative antagonist M35 on pain behavior in a neuropathic pain model 1 * ¨ Hong-Xiang Liu, Tomas Hokfelt Department of Neuroscience, Karolinska Institutet, S-171 77 Stockholm, Sweden Accepted 8 August 2000

Abstract There is currently some debate over a possible role of galanin in pain processing. It was recently reported that the levels of galanin in dorsal root ganglia (DRGs) seem related to development of allodynia after unilateral sciatic nerve constriction injury. In our present study, we aimed at characterizing the effect of exogenous and endogenous galanin on pain behavior in allodynic and non-allodynic rats in which the levels of galanin in DRG neurons are low and high, respectively [28]. The results show that in allodynic rats, the mechanical threshold increases dose-dependently after intrathecal (i.t.) injection of galanin, while no significant changes were observed in groups treated with the putative galanin antagonist M35 or saline. In non-allodynic rats i.t. injection of M35 induced a significant mechanical allodynic state, which did not occur after injection of galanin, bradykinin, the bradykinin fragment(2–9) or saline. The results suggest that in the present experimental paradigm exogenous galanin has an anti-allodynic effect in the allodynic rats, and that endogenous galanin has a tonic inhibitory effect in the non-allodynic group.  2000 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation: pharmacology Keywords: Allodynia; Dorsal root ganglion; Nerve injury; Neuropeptide; Spinal cord

1. Introduction Neuropeptides are widely distributed in the nervous system, virtually always coexisting with one or more classic transmitters (see [11]). Their exact functional role has often not been defined, but they may both act as trophic molecules (see [26,32]) and / or as slow, mostly extrasynaptically released (see [34]) messengers (see [17,19]), especially in situations when neurons are exposed to, in the broadest sense, ‘stressful’ stimuli (see [11]). In the present study we focus on one such situation, neuropathic pain, and the possible involvement of the 29aminoacid peptide galanin [33] as a putative endogenous pain antagonist (see [41,44]). Immunohistochemical studies have shown that galanin1

Published on the World Wide Web on 13 September 2000. Abbreviations: ANOVA, analysis of variance; CCI, chronic constriction injury; DRG, dorsal root ganglion; i.t., intrathecal; i.p., intraperitoneal *Corresponding author. Tel.: 146-8-728-7070; fax: 146-8-331-692. ¨ E-mail address: [email protected] (T. Hokfelt).

like immunoreactivity (LI) [3,29], galanin mRNA [36] and galanin receptor mRNAs are expressed in dorsal root ganglion (DRG) neurons, and also in dorsal horn interneurons express galanin [3,7,21,23,25,27,30,37,45,49]. Galanin expression is strongly upregulated in DRG neurons [10,36], and its basal release is increased in the dorsal horn after peripheral nerve injury [4]. Many studies have focussed on a possible functional role of galanin in sensory processing in the dorsal horn of the spinal cord showing that the effects of galanin on nociception are complex, and both facilitory, inhibitory and bi-phasic responses have been reported (see [41,44]). Several models of partial nerve injury have been developed, which can be used for the assessment of the effects of analgesics on abnormal pain-like behaviors (allodynia and hyperalgesia). One example is loose chronic constriction nerve injury (CCI) [2]. In this so-called Bennett model allodynia is induced only in some rats, which allows comparison between the two groups treated in an apparently identical way, that is allodynic versus non-allodynic rats. Recently Shi et al. [28] found that

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following sciatic nerve CCI galanin expression in DRG neurons is more strongly upregulated in non-allodynic rats when compared to the allodynic group. In the present study we used the same model to examine the effect of i.t. galanin and a putative galanin antagonist, M35 [1], in both allodynic and non-allodynic rats.

manner from below at the center of the plantar surface of hindpaw ipsi-lateral to the nerve injury. Each filament was delivered three times with approximately 2-s intervals. The lowest force at which each of the three applications of the filament elicited a paw withdrawal was taken as the mechanical response threshold.

2.4. Experimental design 2. Materials and methods Adult male Sprague–Dawley rats (240–260 g; ALAB, Stockholm, Sweden) were used. The rats were housed in cages at room temperature (20–258C) under a 12 / 12 h light / dark cycle with free access to food and water. The experiments were conducted according to the Ethical Guidelines for Investigation of Experimental Pain in Conscious Animals [50] and were approved by the local ethics committee for animal research.

2.1. Unilateral sciatic nerve injury Unilateral sciatic nerve injury was produced under deep anesthesia with intraperitoneal (i.p.) injection of sodium pentobarbital (Mebumal  ; 60 mg / kg), as described by Bennett and Xie [2]. The common sciatic nerve was exposed and freed for about 10 mm at mid thigh level. Four ligatures (Ethicon  ; 4.0 plain gut) were tied loosely around the nerve with about 1-mm spacing. Great care was taken to tie the ligatures such that the nerve was barely constricted. The incision was closed in layers with 3-0 silk sutures.

2.2. Intrathecal catheterization and injection Seven days after sciatic nerve loose ligation, a chronic i.t. catheter was implanted according to a modification described by Storkson et al. [31]. After anesthesia with i.p. sodium pentobarbital, a catheter (PE 10, o.d. 0.61 mm) was inserted into the subarachnoid space through a guidecannula (Sterican  ; 20 G, 0.90340 mm) at the level of the gap between the L5 and L6 vertebrae. It was then carefully implanted rostrally with its tip at the lumbar enlargement. I.t. injection was given 7 days later. The proper location of the catheter was tested 24 h before the pharmacological experiments by assessing sensory and motor blockade after ¨ i.t. injection of 7 ml lidocaine (50 mg / ml; Astra, Sodertalje, Sweden).

The measurement of the mechanical threshold started 14 days after the nerve injury. The basal threshold was usually between 2.85 and 13.5 g (or higher), and then the rats were divided into two groups according to the threshold, nonallodynic ($8.8 g) and allodynic (#5.2 g) rats. In the first experiment, galanin was administered i.t. in four allodynic rats in a cumulative dose regime of 1, 9, and 20 mg, totally 30 mg. In non-allodynic rats, M35 was administered in a similar manner in doses of 0.1, 0.2, and 0.4 mg, totally 0.7 mg. The same volume of saline was injected in two allodynic and two non-allodynic rats. To further confirm the effects of galanin in allodynic, and of M35 in non-allodynic rats, a second series of experiments was performed blindly (six allodynic rats received galanin, and six saline; six non-allodynic rats received M35 and five saline). All testing procedures were the same as in the first series, except that the dose of M35 of the last injection was increased to 0.7 mg, to give a total cumulative dose of 1 mg. Additional control tests were carried out to check the effect of galanin (1, 9, 20 mg), bradykinin (0.047, 0.095, 0.33 mg), bradykinin fragment(2–9) (0.040, 0.080, 0.28 mg) in non-allodynic rats, and a high dose M35 (equimolar to the galanin, that is 0.7, 6.34, 14.1 mg) in allodynic rats in an alternate non-blind design. The experiments with bradykinin and the bradykinin fragment were applied, since bradykinin(2–9) represents the C-terminal part of M35 [1].

2.5. Drugs Rat galanin was purchased from Peninsula (Belmont, CA, USA) and M35 [galanin(1–13)-bradykinin(2–9) amide] from Bachem (Bubendorf, Switzerland). Bradykinin and bradykinin fragment (2–9) were purchased from ICN Biomedicals, Aurora, OH, USA. All the peptides were dissolved in distilled water and diluted with sterile saline and stored in aliquots at 2208C until use.

2.3. Measurement of mechanical allodynia

2.6. Statistics

Behavioral testing was performed during daytime (9.00– 18.00) 14 days after sciatic nerve constriction. Rats were placed in transparent plastic domes (838318 cm) on a metal mesh floor with a hole size of 333 mm. After 15 min of adaptation, a series of von Frey filaments (0.5, 0.88, 1.28, 2.85, 5.2, 7.5, 8.8, 13.5 g) were applied in ascending

The results were presented as median6median-derived absolute deviation (MAD). Friedman ANOVA (one-way repeated measures ANOVA on ranks) was used to analyze the data in each group with the time course. Kruskal– Wallis ANOVA test (one-way ANOVA on ranks) was used for the comparison of data among groups at the same time

¨ / Brain Research 886 (2000) 67 – 72 H.-X. Liu, T. Hokfelt

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point. A P value less than 0.05 was chosen as the significant level.

3. Results The data for galanin and saline in allodynic rats, and saline in non-allodynic rats were combined from the blind and non-blind series, since no differences were noted. The data for M35 in non-allodynic rats were blind. The rest of the data was non-blind.

3.1. Anti-allodynic effect of galanin in allodynic rats The effect of i.t. galanin or M35 on the behavioral thresholds for withdrawal of the hindpaw ipsilateral to the nerve injury was determined in allodynic rats. The results are shown as Fig. 1A and Table 1. Neither vehicle, nor M35, nor high dose M35 treatment had any significant effect on the mechanical threshold compared with baseline (Friedman ANOVA, P.0.05), whereas the threshold increased dose-dependently after i.t. injection of galanin (P,0.001). Galanin-treated rats had a significant increase in mechanical thresholds at 30 min after the last injection compared with saline and M35 groups (Kruskal–Wallis ANOVA, P,0.01).

3.2. M35 -induced allodynia in non-allodynic model rats Fig. 1. Effects of galanin and M35 on mechanical threshold in allodynic (A) and non-allodynic (B) rats. Galanin or M35 was given in a cumulative regime of 1, 9, 20 mg, totally 30 mg and 0.1, 0.2, 0.7 mg, totally 1 mg, respectively. In allodynic rats galanin dose-dependently induced a significant increase of mechanical threshold (repeated measures ANOVA on ranks, P,0.001), neither M35 nor saline had any effect (repeated measures ANOVA on ranks, P50.80, P50.18 respectively). In non-allodynic rats, M35 induced a dramatic drop of the mechanical threshold (P,0.0001) which did not change after injection of galanin or saline (P50.22, P50.57, respectively). *,** indicate P,0.05, P,0.01 compared among groups at the same time point (one-way ANOVA on ranks). The arrows indicate that i.t. injection was given immediately after the measurement of the value at the time point.

In non-allodynic rats with the same nerve injury operation, M35, galanin, bradykinin or its fragment bradykinin(2–9) were applied. M35 induced a dramatic dose-dependent drop of the mechanical threshold (Friedman ANOVA, P,0.0001), while no significant changes were observed for galanin, bradykinin, bradykinin(2–9) or saline (P.0.05) (Fig. 1B; Table 1). The mechanical threshold was lower than in the other groups at 30 min after 0.2 mg M35 and at 15 min, 30 min, 60 min after 0.7 mg M35 injection (Kruskal–Wallis ANOVA, P,0.001, P,0.001, P,0.01, P,0.05 respectively). In view of a bradykinin fragment being part of the M35

Table 1 Effects of high dose M35 on mechanical threshold in allodynic and of bradykinin, and bradykinin fragment(2–9) in non-allodynic rats a Group

Drug

0↓

15

30↓

45

60↓

75

90

120

150

Allodynic rats

High dose M35

2.8560.78

2.8560.78

2.8560.39

2.8561.04

2.8561.43

2.8561.43

2.8560.39

2.8560.26

2.8560.78

2.8560.0

P50.55

Bradykinin Bradykinin(2–9)

13.560.0 13.560.78

13.562.38 13.561.38

13.561.0 13.561.38

13.560.0 13.561.78

13.560.78 13.561.0

13.561.0 13.562.78

13.561.38 13.561.78

13.561.78 13.560.78

13.561.78 13.562.78

11.1562.78 13.561.0

P50.46 P50.52

Non-allodynic rats a

Time (minutes after i.t. injection)

Friedman ANOVA 180

The numerals in the table are median6median-derived absolute deviation (MAD). No changes were observed in each group (repeated measures ANOVA on ranks. P.0.05). The arrows indicate that i.t. injection was given immediately after the measurement of the value at the time point.

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molecule, a high dose (10 mg) of bradykinin or its fragment was injected in non-allodynic rats. All five rats receiving this dose of bradykinin started to struggle in the domes within 1 min and this behavior lasted for about 1 min. No behavioral response was observed in the four rats that received 10 mg of bradykinin(2–9).

4. Discussion The function of galanin in pain processing at the spinal level has been subject of many studies since its discovery in 1983 by Tatemoto and collaborators [33], and complex functional patterns have emerged, whereby the endogenous galanin levels, alternatively the dose of exogenously applied galanin appear to be important factors. Under normal circumstances, galanin can only be detected in less than 5% of sensory neurons in adult dorsal root ganglia [3,29], but may be synthesized at a low rate in a considerably larger number of neurons [16]. These galanin neurons give predominantly rise to small diameter fibers, presumably nociceptors with unmyelinated, slowly conducting axons that also coexpress the neuropeptide calcitonin gene-related peptide [12,48]. Using the microprobe technique, originally described by Duggan et al. [6], basal release of galanin-LI has been demonstrated in the dorsal horn [9], although it is not clear if this galanin originates from primary afferents, or local dorsal horn neurons, or both. Functional studies have been carried out to characterize the role of galanin in normal animals, that is animals with low galanin DRG neuron levels. Wiesenfeld-Hallin and colleagues [42] found that i.t. injection of the putative galanin receptor antagonist M35 potentiated the facilitation of the flexor reflex by conditioning stimulation of cutaneous unmyelinated afferents in rats with intact nerves. In accordance with this, Kerr et al. [15] recently reported that the sensitivity to noxious stimuli is significantly higher in galanin null mutant mice than wildtype controls. These results indicate that endogenous galanin plays a tonic inhibitory role in nociceptive processing in the spinal cord under normal conditions. In all animal models based on peripheral nerve injury known to be associated with neuropathic pain behaviors there is upregulation of galanin in DRG neurons. This is true for complete axotomy [10,13,36], nerve crush [36], CCI [20,22,28], local treatment with mitosis inhibitor [13] and partial nerve ligation [20,28]. In agreement enhanced immunoreactive galanin release was found in the superficial dorsal horn ipsi-lateral to the sciatic nerve injury [4]. The association of galanin expression with the development of neuropathic pain behaviors following peripheral nerve damage suggests that galanin may modulate the sensory transmission in the spinal cord, especially after nerve injury [35,39,40,42]; (see [41,44]). Wiesenfeld-Hallin et al. [38,39] examined the effect of i.t. galanin on the nocifensive flexor reflex in decerebrate, spinalized, unanesthetized rats and found that low doses of galanin (10

ng–1 mg) increased reflex excitability. It has also been reported that in normal rats a single i.t. injection of galanin (0.1 and 1 nmol) decreases the nociceptive threshold for mechanical stimulation [18]. Recently Kerr et al. [15] found that following full sciatic transection or partial nerve injury, spontaneous and evoked neuropathic pain behaviors are largely eliminated or severely compromised in galanin null mutant mice. Furthermore, chronic intrathecal delivery of low dose (25 ng / h, 14 d) exogenous galanin to nerveintact adult rats, which should mimic nerve injury-induced upregulation of galanin, induces persistent mechanical hypersensitivity. These data suggest that the upregulation of galanin is associated with the development of neuropathic pain after the peripheral nerve injury. However, the putative galanin receptor antagonist M35 given i.t. did not significantly alter pain behavior after photochemically induced ischaemic peripheral nerve injury [8]. In agreement, our present study shows no significant effect of i.t. M35 on the mechanical threshold in allodynic rats with unilateral CCI of sciatic nerve in a wide range of doses. It is possible that M35 blocked both inhibitory and facilitory effects of galanin. There is evidence that high levels / doses of galanin have antinociceptive effects. Thus, i.t. administration of high doses (.1 mg) of galanin inhibits the nocifensive reflex in spinalized rats [39,40] and normal rats [5,43], in contrast to the facilitory effect of low doses mentioned above. Moreover, very high doses of exogenous galanin alleviate the neuropathic pain behaviors following peripheral nerve injury [8,47]. The question therefore arises to what extent high levels of endogenous galanin really can act as an analgesic factor in the spinal cord. In our present study, we examined the effect of exogenous and endogenous galanin on pain behavior in non-allodynic and allodynic Bennett model rats, in which the levels of galanin in DRG have been reported to be different, that is 43% of neuron profiles expressed galanin in non-allodynic rats versus 23% in the allodynic group [28]. We found that i.t. galanin induced an anti-allodynic effect in allodynic rats which is identical with other reports [8,47]. Moreover, in non-allodynic rats the putative galanin receptor antagonist M35, when given i.t., dose-dependently induced a long-lasting allodynic state, indicating that high-level endogenous galanin exerts a tonic inhibition of pain processing in the spinal cord following nerve injury. It should, however, be remembered that the presently available galanin ‘antagonists’, including M35, have been shown to exert agonist activity in many models, at least at high doses (see e.g. [14,24,46] and below). Taken together, galanin’s role in pain processing in the spinal cord seems to include inhibition and excitation. The mechanisms underlying varying response properties to either endogenous or exogenous galanin are unknown. It has been reported that all the three galanin receptor subtype mRNAs are expressed within DRGs and spinal cord [7,23,25,27,30,37,45], where their distribution and expression level differ. Therefore, it is likely that endogen-

¨ / Brain Research 886 (2000) 67 – 72 H.-X. Liu, T. Hokfelt

ous and exogenous galanin may have pre- and post-synaptic actions in the spinal cord. Maybe the differential distribution and regulation of galanin receptors underlie the wide range of behavioral and electrophysiological responses. The chimeric peptide M35 [galanin(1–13)-bradykinin(2–9) amide] is a putative high-affinity galanin receptor antagonist acting in various central and peripheral tissues ¨ [1]. For example, the studies of Ogren’s group indicate that a low dose of M35 (1 nmol) acts as a galanin receptor antagonist, whereas higher doses behave as a mixed agonist–antagonist in vivo in the rat striatum (see [24]). M35 also has a dual effect on the galanin-mediated inhibition of forskolin-stimulated cAMP production in the rat pancreatic beta-cell line Rin m 5F: at low concentrations M35 antagonizes the effect of galanin, while at concentrations above 10 nM M35 acts as a galanin receptor agonist [14]. In the locus coeruleus it also exhibits agonist action [46]. In our present study, no similarity in the effects of M35 and galanin was found after i.t. administration, neither in allodynic nor in non-allodynic rats at a cumulative dose of 1 mg (even over 20 mg in non-allodynic rats). The results suggest that in our model and at the doses used M35 had no agonistic effect in the spinal cord. This is in agreement with earlier results by Hao et al. [8]. In this study we also paid attention to the possible activity of the bradykinin fragment included in the M35 molecule. Bradykinin is a well-known potent algesic factor. To exclude bradykinin-like algesic activity of its fragment (2–9) in non-allodynic rats, we used the same molecular doses of bradykinin and bradykinin(2–9) as control, but no significant changes of the mechanical threshold were observed. This indicates that the allodynic effect induced by M35 in non-allodynic rats represents a galanin receptor-related action. In summary, the results in our study suggest that exogenous galanin can play an anti-allodynic role in allodynic rats with peripheral nerve injury. Moreover, M35 dose-dependently changed non-allodynic rats into an allodynic state, indicating that high endogenous galanin levels in non-allodynic model rats play a tonic inhibitory role in the neuropathic pain processing in the spinal cord.

Acknowledgements This study was supported by an Unrestricted BristolMyers Squibb Neuroscience Grant, Marianne and Marcus Wallenberg’s Foundation and the Swedish MRC (04X2887).

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