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subcoeruleus following unilateral hindpaw inflammation in the rat
Brain Research Bulletin 78 (2009) 170–174
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Descending pathways from activated locus coeruleus/subcoeruleus following unilateral hindpaw inflammation in the rat Masako Maeda a , Masayoshi Tsuruoka a,∗ , Bunsho Hayashi a , Ikuko Nagasawa b , Tomio Inoue a a b
Department of Oral Physiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan Department of Anesthesiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
a r t i c l e
i n f o
Article history: Received 21 May 2008 Received in revised form 12 September 2008 Accepted 12 September 2008 Available online 14 October 2008 Keywords: Locus coeruleus Subcoeruleus Hyperalgesia Inflammation Descending modulation
a b s t r a c t We have previously shown that the descending pathways from the locus coeruleus (LC)/subcoeruleus (SC) to the spinal cord are activated during peripheral inflammation, and that activation of this coeruleospinal system decreases development of hyperalgesia. Anatomical evidence suggests that the descending modulation system from the LC/SC should be active bilaterally during inflammation when the LC/SC either ipsilateral or contralateral to the site of inflammation is activated. In the present study, the development of hyperalgesia following the induction of unilateral hindpaw inflammation was compared between rats with either bilateral or unilateral lesions of the LC/SC and rats with a sham operation. Four hours after carrageenan injection, in the inflamed paw, paw withdrawal latencies (PWLs) to thermal stimuli of the bilateral LC/SC-lesioned rats were significantly shorter than those of the unilateral LC/SC-lesioned and the sham-operated rats, whereas the decreased PWLs of the unilateral LC/SC-lesioned rats were equivalent to those of the sham-operated rats. A difference in PWL between the bilateral and the unilateral LC/SC-lesioned rats was not observed in the contralateral non-inflamed paw. The result suggests that in the LC/SC both ipsilateral and contralateral to the inflamed paw, only neurons which project to the dorsal horn ipsilateral to the inflamed paw were activated following peripheral inflammation. © 2008 Elsevier Inc. All rights reserved.
1. Introduction The nucleus locus coeruleus (LC), the A6 cell group, is located in the dorsolateral pons and has been shown to send noradrenergic projections to the spinal cord via descending pathways [1,13,16]. The nucleus subcoeruleus (SC), located ventrolateral to the A6 cell group in the rostral pons, also provides noradrenergic innervation of the spinal cord [1,17,18,23,2,6] These descending projections from the LC/SC have been demonstrated to be involved in the centrifugal modulation of pain [4,25,21,16,20,26,7]. Activation of the LC/SC either electrically or chemically can produce profound antinociception [23,21,16,26,7] and can inhibit nociceptive activity in dorsal horn neurons [4,2,29,25,20]. We have previously shown that the descending pathway from the LC/SC to the spinal cord is activated during peripheral inflammation, and that activation of this coeruleospinal system decreases development of hyperalgesia [15,24]. The present
study adds further knowledge concerning descending modulation from the LC/SC on nociceptive processing in the spinal cord during peripheral inflammation. Several lines of anatomical evidence have shown that the LC/SC sends a bilateral projection to the spinal cord dorsal horn [1,12,22,19,3]. Axons from the contralaterally projecting neurons of the LC/SC cross the midline within the brain and travel through the dorsolateral funiculus (DLF) to terminate in the dorsal horn on the side of the descending projection [12,22]. This finding suggests that the descending pathway from the LC/SC should be active bilaterally during inflammation when the LC/SC either ipsilateral or contralateral to the site of inflammation is activated. In the present study, the action of the descending modulation system from the LC/SC following unilateral hindpaw inflammation was examined in rats with either bilateral or unilateral lesions of the LC/SC.
2. Materials and methods
∗ Corresponding author. Tel.: +81 3 3784 8160; fax: +81 3 3784 8161. E-mail address: [email protected] (M. Tsuruoka). 0361-9230/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2008.09.005
The experiments in this study were approved by the Institutional Animal Care and Use Committee of Showa University and were in accordance with the International Association for the Study of Pain [9].
M. Maeda et al. / Brain Research Bulletin 78 (2009) 170–174 2.1. Animals
3. Results
Experiments were performed on male Sprague–Dawley rats weighing 250–330 g. Animals were housed in groups of 3–4 in a cage containing sawdust bedding, with free access to rat chow and water in a laboratory equipped with a 12 h/12 h (8 a.m./8 p.m.) light–dark cycle. Room temperature and humidity were maintained at 23 ± 0.5 ◦ C and 60%, respectively.
3.1. Lesion verification
2.2. Neurotoxin-induced lesions of the LC/SC One week before the experiment, the rats received either bilateral or unilateral injections of 6 g of neurotoxin 6-hydroxydopamine (6-OHDA, Sigma) to specific lesion noradrenaline-containing neurons of the LC/SC. Animals were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and placed in a stereotaxic apparatus. One or two (bilateral) holes were made in the skull with a drill to insert a microinjection needle into the LC/SC (coordinates as defined by Paxinos and Watson [5]: 9.6 mm caudal to bregma; 1.15 mm lateral to the midline; 2.5 mm above the interaural axis). The 6-OHDA was dissolved in a 0.1% on higher solution of ascorbic acid immediately before use. A microinjection needle was attached by polyethylene tubing to a 10 l Hamilton syringe. 0.6 l of the drug solution (10 g/l) was infused at a rate of 0.1 l/min. Half of the solution (0.3 l) was delivered to the LC and half to the SC, after which the needle was left in place for 5 min. After completion of the injection, holes were covered with bone wax, and the incision sutured. For sham operations, the needle was lowered into the target, and the vehicle (artificial cerebrospinal fluid) was injected. After recovery from anesthesia, no obvious changes in gross behavior were obtained in lesioned and vehicle-injected rats.
2.3. Unilateral hindpaw inflammation Under halothane anesthesia (2.0% in air), lambda carrageenan (2 mg in 0.15 ml saline, Sigma) was injected subcutaneously into the plantar surface of the left hindpaw. The carrageenan-induced inflammation model is characterized by the rapid onset of inflammation causing a restricted distribution of hyperalgesia. In the injected hindpaw, maximum hyperalgesia occurs at 3–4 h after carrageenan administration, and the hyperalgesic inflammation is resolved substantially by 24–72 h [14,27,10]. Nociceptive measures in the present work were recorded 4 h after the administration of carrageenan.
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The location and extent of the lesions in the LC/SC are represented schematically for 20 experiments in Fig. 1. The lesions were clearly delineated in Nissl sections as areas containing reactive gliosis and degenerated neurons. In both cases of unilateral and bilateral lesions of the LC/SC, the rostrocaudal extent was between 0.8 and 1.5 mm, and the LC/SC was always completely destroyed ventrodorsally over all of its rostrocaudal extent. Rats with misplaced lesions were excluded from the data analysis. 3.2. Inflammation-induced activation of coeruleospinal system A total of 20 rats (6 with bilateral lesions of the LC/SC and 14 with unilateral lesions of the LC/SC) were selected on the basis of histological results which showed that the LC/SC was almost completely destroyed. In these rats, the weight slightly increased as compared with the baseline value when measured prior to the injection of carrageenan. Following the carrageenan injection, the development of edema in the injected left hindpaw was observed in all the rats tested. Within 4 h after carrageenan injection, paw thickness increased from 5.2 ± 0.1 to 11.3 ± 0.2 mm in the vehicle-injected rats (n = 20), from 5.0 ± 0.1 to 10.6 ± 0.2 mm in the bilateral LC/SC-lesioned rats (n = 6) and from 5.1 ± 0.1 to 10.9 ± 0.2 mm in the unilateral LC/SC-lesioned rats (n = 14), indicating that the development of inflammation did not differ between the three groups of rats. An
2.4. Nociceptive testing The rats were tested for behavioral nociception with radiant heat stimuli. Paw withdrawal latency (PWL) was determined by the method described by Hargreaves et al. [14], which measures cutaneous hyperalgesia in response to thermal stimuli. The rats were placed on an elevated glass surface under an inverted clear plastic chamber (13 cm × 13 cm × 14 cm), and a radiant heat source (projection lamp: 120 V, 300 W) was positioned under the glass floor directly beneath one hindpaw. The PWL, to the nearest 0.01 s, was recorded using an electronic timer circuit. Heating was terminated at 15 s to avoid tissue damage if an animal failed to withdraw its paw. The PWL, however, was within 15 s in all animals throughout the experiments. The advantage of this assay, using unilateral testing, is to enable side-to-side comparison of hyperalgesic or drug effects on inflamed and non-inflamed tissues within individual subjects under conditions of unilateral inflammation. The experiments were carried out in the light phase on independent groups of animals and all testing was conducted in a quiet room by the same person.
2.5. Histological reconstruction At the end of the experiments, the animals were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.). The brain stem and the spinal cord were perfused transcardially with 200 ml of saline followed by 500 ml 10% formaldehyde, and sectioned at 50 m in the coronal plane with a cryostat. Alternate tissue sections from the same experiment were used for dopamine--hydroxylase (DBH) immunocytochemistry and cresyl violet staining. Tissues from lesioned and vehicle-injected animals were processed for DBH immunocytochemistry by the avidin–biotin method of Hsu et al. [28] and simultaneously reacted with the same avidin–biotin reagents. A set of brain stem sections was mounted and stained with cresyl violet to verify cell histology. The location and extent of the lesions in the LC/SC were assessed by visualization of Nissl-stained tissues under high microscopic magnification.
2.6. Data analysis Results are presented as the mean ± S.E.M. Statistical analysis was carried out using analysis of variance (ANOVA) with repeated measures. If significance was obtained, the Scheffe’s t-test was used for a post-hoc analysis of differences between individual points. A value of P < 0.05 was considered significant.
Fig. 1. Schematic representation of neurotoxin-induced lesions of the LC/SC contralateral (A), ipsilateral (B) and bilateral (C) to the site of inflammation. Drawings are simplified from Paxinos and Watson [3]. The extent of lesions (dotted lines) is revealed by Nissl staining in the LC/SC. Keys for medullary structures are indicated by abbreviations in (A) LC, locus coeruleus; SC, subcoeruleus; Pr5, principal sensory trigeminal nucleus.
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Fig. 2. Schematic representation of the LC/SC lesions (blackened areas) and projections to the spinal cord. Treatment A, there is no lesion and carrageenan is injected into the left hindpaw (hatched areas); Treatment B, there is a bilateral lesion with carrageenan injected into the same paw; Treatment C, there is an LC/SC lesion on the ipsilateral side; Treatment D, there is an LC/SC lesion on the contralateral side. The lines from the LC/SC to the spinal cord represent schematic axons of LC/SC neurons.
increase in paw thickness was not observed in the contralateral uninjected (non-inflamed) paws. Schematic representations of the lesions and projections to the spinal cord are shown in Fig. 2. In Treatment A, there is no lesion and carrageenan is injected into the left hindpaw. Progressing along, in Treatment B, there is a bilateral lesion with carrageenan injected into the same paw. Treatment C then shows a LC/SC lesion on the ipsilateral side whereas the last treatment (Treatment D) shows a lesion on the contralateral side. In the inflamed paw (Fig. 3A), prior to injection of carrageenan, PWLs did not differ between the four treatments. Four hours after carrageenan injection, PWLs decreased significantly in all four treatments tested (F1,36 = 255.14, P = 0.001), demonstrating that hypersensitivity to thermal stimuli had developed. Statistical analysis revealed that decreased PWLs in rats of Treatment B were significantly shorter than those in rats of Treatments A, C and D, whereas PWLs did not differ significantly between Treatments A, C and D. In the contralateral non-inflamed paws (Fig. 3B), no decrease in PWL following injection of carrageenan was observed in all the rats tested. At 4 h after injection, there was no significant difference in PWLs between the four treatments. In some experiments, rats received a subcutaneous injection of 0.15 ml saline into the left hindpaw. No significant change in PWL was observed before, during and 4 h post-injection in the vehicle-injected, the bilateral LC/SC-lesioned and the unilateral LC/SC-lesioned rats (not shown).
4. Discussion 4.1. Ipsilateral effect In a previous study [15,24], we showed that carrageenaninduced inflammation activated the descending modulation system from the LC region. This finding was derived from the observation that PWLs in the inflamed paw of the bilateral LC/SC-lesioned rats were significantly shorter than those of the vehicle-injected rats and that a difference in PWL between the two groups of rats was not observed in paws before the induction of inflammation. A similar result can also be seen in the present study (Fig. 3A). An important finding of the present study is that 4 h after carrageenan injection, in the inflamed paws, PWLs of the unilateral LC/SC-lesioned rats were significantly longer than those of the bilateral LC/SC-lesioned rats and were equivalent to those of the vehicle-injected rats. This result indicates that the unilateral LC/SClesioned rats can be distinguished from the bilateral LC/SC-lesioned rats in the degree of thermal hyperalgesia. Four hours after carrageenan injection, in the inflamed paws, no significant difference in PWLs was seen between the unilateral LC/SC-lesioned and the vehicle-injected rats. This finding suggests that unilateral activation of the LC/SC is sufficient to modulate nociceptive processing in the dorsal horn. At this time, we can-
M. Maeda et al. / Brain Research Bulletin 78 (2009) 170–174
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with morphological data. Wei et al. [8] reported that after LC/SC lesions there were changes in spinal Fos expression contralateral to the inflamed paw, as well as ipsilateral alterations in Fos expression. This discrepancy in contralateral effects between behavioral hyperalgesia and Fos protein expression may be because the magnitude of LC/SC lesions in the Fos experiment is not sufficient compared to that in the behavioral experiment, as the authors have described in their report [8]. Anatomical studies have shown that the LC/SC sends a bilateral projection to the spinal cord dorsal horn [1,12,22,19,3]. For the contralaterally projecting neurons, axons from the LC/SC cross the midline within the brain and travel through the DLF with axonal terminals ipsilaterally in the dorsal horn [12,22]. On the basis of anatomical evidence, we suggest that unilateral hindpaw inflammation activates descending axons that originate from the LC/SC both ipsilateral and contralateral to the inflamed paw and travel through the DLF ipsilateral to the inflamed paw. In addition, a neurochemical study using a microdialysis technique [11] has demonstrated that the norepinephrine level does not change significantly in the dorsal horn contralateral to the site of inflammation for 4 h from induction of inflammation, whereas the norepinephrine level increases significantly in the dorsal horn ipsilateral to the site of inflammation even 1 h after the induction of inflammation. This indicates that descending neurons from the LC/SC which project to the dorsal horn contralateral to the site of inflammation are inactive during inflammation. This neurochemical finding appears to be consistent with the findings of the present study that in the LC/SC both ipsilateral and contralateral to the inflamed paw, only neurons which project to the dorsal horn ipsilateral to the inflamed paw were activated following peripheral inflammation. Fig. 3. Changes in PWLs following unilateral injection of carrageenan in rats of Treatments A–D. (A) PWLs in the inflamed paw. (B) PWLs in the contralateral noninflamed paw. # P < 0.01, significantly different from PWLs before injection. *P < 0.01, significantly different between four treatments.
not explain the result of the present study in which PWLs are equivalent between the vehicle-injected and the unilateral LC/SClesioned rats. Our previous study [11], however, may serve as a clue to the present result. In our present research, the concentration of norepinephrine in the dorsal horn ipsilateral to the site of inflammation during unilateral hindpaw inflammation was measured using the microdialysis technique. The norepinephrine level in the dorsal horn increased with the increase of the development of inflammation, indicating that the activity of descending neurons from the LC increases with the increase of the development of inflammation. This finding suggests that the activity of descending LC/SC neurons is regulated by nociceptive inputs through a positive feedback loop which consists of connections between collaterals of ascending nociceptive projection neurons and the LC/SC neurons [13]. In this context, the equivalence of PWLs between the vehicle-injected and the unilateral LC/SC-lesioned rats may be due to the increased activity of LC/SC neurons contralateral to the site of lesion, which is induced by the feedback mechanism. 4.2. Contralateral effect Four hours after carrageenan injection, no difference in PWL between the vehicle-injected and the unilateral LC/SC-lesioned rats was observed in the contralateral non-inflamed paw, as well as in the case between the vehicle-injected and the bilateral LC/SClesioned rats. This result indicates that the descending modulation system from the LC/SC is not active in the dorsal horn contralateral to the inflamed paw. However, this finding is not consistent
Acknowledgements The authors thank Dr. Yukiko Hiruma for her critical reading of the manuscript. This work supported by a grant from The Science Research Promotion Fund of Japan. References [1] F.M. Clark, H.K. Proudfit, The projection of locus coeruleus neurons to the spinal cord in the rat determined by anterograde tracing combined with immunocytochemistry, Brain Res. 538 (1991) 231–245. [2] F.M. Clark, H.K. Proudfit, Anatomical evidence for genetic differences in the innervation of the rat spinal cord by noradrenergic locus coeruleus neurons, Brain Res. 591 (1992) 44–53. [3] F.M. Clark, D.C. Yeomans, H.K. Proudfit, The noradrenergic innervation of the spinal cord: differences between two substrains of Sprague–Dawley rats determined using retrograde tracers combined with immunocytochemistry, Neurosci. Lett. 125 (1991) 155–158. [4] A.D. Craig, Distribution of brain stem projection from spinal lamina I neurons in the cat and the monkey, J. Comp. Neurol. 361 (1995) 225–248. [5] J.-M. Fritschy, R. Grzanna, Demonstration of two separate descending noradrenergic pathways to the rat spinal cord: evidence for an intragriseal trajectory of locus coeruleus axons in the superficial layers of the dorsal horn, J. Comp. Neurol. 291 (1990) 553–582. [6] P.G. Guyenet, The coeruleospinal noradrenergic neurons: anatomical and electrophysiological studies in the rat, Brain Res. 189 (1980) 121–133. [7] K. Hargreaves, R. Dubner, F. Brown, C. Flores, J. Joris, A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia, Pain 32 (1988) 77–88. [8] C.J. Hodge Jr., A.V. Apkarian, R. Stevens, G. Vogelsang, H.J. Wisnicki, Locus coeruleus modulation of dorsal horn unit responses to cutaneous stimulation, Brain Res. 204 (1981) 415–420. [9] S.M. Hsu, L. Raind, H. Fanger, Use of avidin–biotin–peroxidase complex (ABC) in immunoperoxidase technique. A comparison between ABC and unlabeled antibody (PAP) procedures, J. Histochem. Cytochem. 29 (1981) 577–580. [10] J.L.K. Hylden, D.A. Thomas, M.J. Iadarola, R.L. Nahin, R. Dubner, Spinal opioid analgesic effects are enhanced in a model of unilateral inflammation/hyperalgesia: possible involvement of noradrenergic mechanisms, Eur. J. Pharmacol. 194 (1991) 135–143. [11] S.L. Jones, Descending noradrenergic influences on pain, Prog. Brain Res. 88 (1991) 381–394.
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[22] K.A. Sluka, K.N. Westlund, Spinal projections of the locus coeruleus and the nucleus subcoeruleus in the Harlan and the Sasco Sprague–Dawley rat, Brain Res. 579 (1992) 67–73. [23] M. Tsuruoka, W.D. Willis, Descending modulation from the region of the locus coeruleus on nociceptive sensitivity in a rat model of inflammatory hyperalgesia, Brain Res. 743 (1996) 86–92. [24] M. Tsuruoka, W.D. Willis, Bilateral lesions in the area of the nucleus locus coeruleus affect the development of hyperalgesia during carrageenan-induced inflammation, Brain Res. 726 (1996) 233–236. [25] M. Tsuruoka, T. Hitoto, Y. Hiruma, Y. Matsui, Neurochemical evidence for inflammation-induced activation of the coeruleospinal modulation system in the rat, Brain Res. 821 (1999) 236–240. [26] F. Wei, R. Dubner, K. Ren, Nucleus reticularis gigantocellularis and nucleus raphe magnus in the brain stem exert opposite effects on behavioral hyperalgesia and spinal Fos protein expression after peripheral inflammation, Pain 80 (1999) 127–141. [27] W.L. West, D.C. Yeomans, H.K. Proudfit, The function of noradrenergic neurons in mediating antinociception induced by electrical stimulation of the locus coeruleus in two different sources of Sprague–Dawley rats, Brain Res. 626 (1993) 127–135. [28] K.N. Westlund, R.M. Bowker, M.G. Ziegler, J.D. Coulter, Origins and terminations of descending noradrenergic projections to the spinal cord of monkey, Brain Res. 292 (1984) 1–16. [29] M. Zimmermann, Ethical guidelines for investigation of experimental pain in conscious animals, Pain 16 (1983) 109–110.
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
Descending pathways from activated locus coeruleus/subcoeruleus following unilateral hindpaw inflammation in the rat Masako Maeda a , Masayoshi Tsuruoka a,∗ , Bunsho Hayashi a , Ikuko Nagasawa b , Tomio Inoue a a b
Department of Oral Physiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan Department of Anesthesiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
a r t i c l e
i n f o
Article history: Received 21 May 2008 Received in revised form 12 September 2008 Accepted 12 September 2008 Available online 14 October 2008 Keywords: Locus coeruleus Subcoeruleus Hyperalgesia Inflammation Descending modulation
a b s t r a c t We have previously shown that the descending pathways from the locus coeruleus (LC)/subcoeruleus (SC) to the spinal cord are activated during peripheral inflammation, and that activation of this coeruleospinal system decreases development of hyperalgesia. Anatomical evidence suggests that the descending modulation system from the LC/SC should be active bilaterally during inflammation when the LC/SC either ipsilateral or contralateral to the site of inflammation is activated. In the present study, the development of hyperalgesia following the induction of unilateral hindpaw inflammation was compared between rats with either bilateral or unilateral lesions of the LC/SC and rats with a sham operation. Four hours after carrageenan injection, in the inflamed paw, paw withdrawal latencies (PWLs) to thermal stimuli of the bilateral LC/SC-lesioned rats were significantly shorter than those of the unilateral LC/SC-lesioned and the sham-operated rats, whereas the decreased PWLs of the unilateral LC/SC-lesioned rats were equivalent to those of the sham-operated rats. A difference in PWL between the bilateral and the unilateral LC/SC-lesioned rats was not observed in the contralateral non-inflamed paw. The result suggests that in the LC/SC both ipsilateral and contralateral to the inflamed paw, only neurons which project to the dorsal horn ipsilateral to the inflamed paw were activated following peripheral inflammation. © 2008 Elsevier Inc. All rights reserved.
1. Introduction The nucleus locus coeruleus (LC), the A6 cell group, is located in the dorsolateral pons and has been shown to send noradrenergic projections to the spinal cord via descending pathways [1,13,16]. The nucleus subcoeruleus (SC), located ventrolateral to the A6 cell group in the rostral pons, also provides noradrenergic innervation of the spinal cord [1,17,18,23,2,6] These descending projections from the LC/SC have been demonstrated to be involved in the centrifugal modulation of pain [4,25,21,16,20,26,7]. Activation of the LC/SC either electrically or chemically can produce profound antinociception [23,21,16,26,7] and can inhibit nociceptive activity in dorsal horn neurons [4,2,29,25,20]. We have previously shown that the descending pathway from the LC/SC to the spinal cord is activated during peripheral inflammation, and that activation of this coeruleospinal system decreases development of hyperalgesia [15,24]. The present
study adds further knowledge concerning descending modulation from the LC/SC on nociceptive processing in the spinal cord during peripheral inflammation. Several lines of anatomical evidence have shown that the LC/SC sends a bilateral projection to the spinal cord dorsal horn [1,12,22,19,3]. Axons from the contralaterally projecting neurons of the LC/SC cross the midline within the brain and travel through the dorsolateral funiculus (DLF) to terminate in the dorsal horn on the side of the descending projection [12,22]. This finding suggests that the descending pathway from the LC/SC should be active bilaterally during inflammation when the LC/SC either ipsilateral or contralateral to the site of inflammation is activated. In the present study, the action of the descending modulation system from the LC/SC following unilateral hindpaw inflammation was examined in rats with either bilateral or unilateral lesions of the LC/SC.
2. Materials and methods
∗ Corresponding author. Tel.: +81 3 3784 8160; fax: +81 3 3784 8161. E-mail address: [email protected] (M. Tsuruoka). 0361-9230/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2008.09.005
The experiments in this study were approved by the Institutional Animal Care and Use Committee of Showa University and were in accordance with the International Association for the Study of Pain [9].
M. Maeda et al. / Brain Research Bulletin 78 (2009) 170–174 2.1. Animals
3. Results
Experiments were performed on male Sprague–Dawley rats weighing 250–330 g. Animals were housed in groups of 3–4 in a cage containing sawdust bedding, with free access to rat chow and water in a laboratory equipped with a 12 h/12 h (8 a.m./8 p.m.) light–dark cycle. Room temperature and humidity were maintained at 23 ± 0.5 ◦ C and 60%, respectively.
3.1. Lesion verification
2.2. Neurotoxin-induced lesions of the LC/SC One week before the experiment, the rats received either bilateral or unilateral injections of 6 g of neurotoxin 6-hydroxydopamine (6-OHDA, Sigma) to specific lesion noradrenaline-containing neurons of the LC/SC. Animals were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and placed in a stereotaxic apparatus. One or two (bilateral) holes were made in the skull with a drill to insert a microinjection needle into the LC/SC (coordinates as defined by Paxinos and Watson [5]: 9.6 mm caudal to bregma; 1.15 mm lateral to the midline; 2.5 mm above the interaural axis). The 6-OHDA was dissolved in a 0.1% on higher solution of ascorbic acid immediately before use. A microinjection needle was attached by polyethylene tubing to a 10 l Hamilton syringe. 0.6 l of the drug solution (10 g/l) was infused at a rate of 0.1 l/min. Half of the solution (0.3 l) was delivered to the LC and half to the SC, after which the needle was left in place for 5 min. After completion of the injection, holes were covered with bone wax, and the incision sutured. For sham operations, the needle was lowered into the target, and the vehicle (artificial cerebrospinal fluid) was injected. After recovery from anesthesia, no obvious changes in gross behavior were obtained in lesioned and vehicle-injected rats.
2.3. Unilateral hindpaw inflammation Under halothane anesthesia (2.0% in air), lambda carrageenan (2 mg in 0.15 ml saline, Sigma) was injected subcutaneously into the plantar surface of the left hindpaw. The carrageenan-induced inflammation model is characterized by the rapid onset of inflammation causing a restricted distribution of hyperalgesia. In the injected hindpaw, maximum hyperalgesia occurs at 3–4 h after carrageenan administration, and the hyperalgesic inflammation is resolved substantially by 24–72 h [14,27,10]. Nociceptive measures in the present work were recorded 4 h after the administration of carrageenan.
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The location and extent of the lesions in the LC/SC are represented schematically for 20 experiments in Fig. 1. The lesions were clearly delineated in Nissl sections as areas containing reactive gliosis and degenerated neurons. In both cases of unilateral and bilateral lesions of the LC/SC, the rostrocaudal extent was between 0.8 and 1.5 mm, and the LC/SC was always completely destroyed ventrodorsally over all of its rostrocaudal extent. Rats with misplaced lesions were excluded from the data analysis. 3.2. Inflammation-induced activation of coeruleospinal system A total of 20 rats (6 with bilateral lesions of the LC/SC and 14 with unilateral lesions of the LC/SC) were selected on the basis of histological results which showed that the LC/SC was almost completely destroyed. In these rats, the weight slightly increased as compared with the baseline value when measured prior to the injection of carrageenan. Following the carrageenan injection, the development of edema in the injected left hindpaw was observed in all the rats tested. Within 4 h after carrageenan injection, paw thickness increased from 5.2 ± 0.1 to 11.3 ± 0.2 mm in the vehicle-injected rats (n = 20), from 5.0 ± 0.1 to 10.6 ± 0.2 mm in the bilateral LC/SC-lesioned rats (n = 6) and from 5.1 ± 0.1 to 10.9 ± 0.2 mm in the unilateral LC/SC-lesioned rats (n = 14), indicating that the development of inflammation did not differ between the three groups of rats. An
2.4. Nociceptive testing The rats were tested for behavioral nociception with radiant heat stimuli. Paw withdrawal latency (PWL) was determined by the method described by Hargreaves et al. [14], which measures cutaneous hyperalgesia in response to thermal stimuli. The rats were placed on an elevated glass surface under an inverted clear plastic chamber (13 cm × 13 cm × 14 cm), and a radiant heat source (projection lamp: 120 V, 300 W) was positioned under the glass floor directly beneath one hindpaw. The PWL, to the nearest 0.01 s, was recorded using an electronic timer circuit. Heating was terminated at 15 s to avoid tissue damage if an animal failed to withdraw its paw. The PWL, however, was within 15 s in all animals throughout the experiments. The advantage of this assay, using unilateral testing, is to enable side-to-side comparison of hyperalgesic or drug effects on inflamed and non-inflamed tissues within individual subjects under conditions of unilateral inflammation. The experiments were carried out in the light phase on independent groups of animals and all testing was conducted in a quiet room by the same person.
2.5. Histological reconstruction At the end of the experiments, the animals were deeply anesthetized with sodium pentobarbital (100 mg/kg, i.p.). The brain stem and the spinal cord were perfused transcardially with 200 ml of saline followed by 500 ml 10% formaldehyde, and sectioned at 50 m in the coronal plane with a cryostat. Alternate tissue sections from the same experiment were used for dopamine--hydroxylase (DBH) immunocytochemistry and cresyl violet staining. Tissues from lesioned and vehicle-injected animals were processed for DBH immunocytochemistry by the avidin–biotin method of Hsu et al. [28] and simultaneously reacted with the same avidin–biotin reagents. A set of brain stem sections was mounted and stained with cresyl violet to verify cell histology. The location and extent of the lesions in the LC/SC were assessed by visualization of Nissl-stained tissues under high microscopic magnification.
2.6. Data analysis Results are presented as the mean ± S.E.M. Statistical analysis was carried out using analysis of variance (ANOVA) with repeated measures. If significance was obtained, the Scheffe’s t-test was used for a post-hoc analysis of differences between individual points. A value of P < 0.05 was considered significant.
Fig. 1. Schematic representation of neurotoxin-induced lesions of the LC/SC contralateral (A), ipsilateral (B) and bilateral (C) to the site of inflammation. Drawings are simplified from Paxinos and Watson [3]. The extent of lesions (dotted lines) is revealed by Nissl staining in the LC/SC. Keys for medullary structures are indicated by abbreviations in (A) LC, locus coeruleus; SC, subcoeruleus; Pr5, principal sensory trigeminal nucleus.
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Fig. 2. Schematic representation of the LC/SC lesions (blackened areas) and projections to the spinal cord. Treatment A, there is no lesion and carrageenan is injected into the left hindpaw (hatched areas); Treatment B, there is a bilateral lesion with carrageenan injected into the same paw; Treatment C, there is an LC/SC lesion on the ipsilateral side; Treatment D, there is an LC/SC lesion on the contralateral side. The lines from the LC/SC to the spinal cord represent schematic axons of LC/SC neurons.
increase in paw thickness was not observed in the contralateral uninjected (non-inflamed) paws. Schematic representations of the lesions and projections to the spinal cord are shown in Fig. 2. In Treatment A, there is no lesion and carrageenan is injected into the left hindpaw. Progressing along, in Treatment B, there is a bilateral lesion with carrageenan injected into the same paw. Treatment C then shows a LC/SC lesion on the ipsilateral side whereas the last treatment (Treatment D) shows a lesion on the contralateral side. In the inflamed paw (Fig. 3A), prior to injection of carrageenan, PWLs did not differ between the four treatments. Four hours after carrageenan injection, PWLs decreased significantly in all four treatments tested (F1,36 = 255.14, P = 0.001), demonstrating that hypersensitivity to thermal stimuli had developed. Statistical analysis revealed that decreased PWLs in rats of Treatment B were significantly shorter than those in rats of Treatments A, C and D, whereas PWLs did not differ significantly between Treatments A, C and D. In the contralateral non-inflamed paws (Fig. 3B), no decrease in PWL following injection of carrageenan was observed in all the rats tested. At 4 h after injection, there was no significant difference in PWLs between the four treatments. In some experiments, rats received a subcutaneous injection of 0.15 ml saline into the left hindpaw. No significant change in PWL was observed before, during and 4 h post-injection in the vehicle-injected, the bilateral LC/SC-lesioned and the unilateral LC/SC-lesioned rats (not shown).
4. Discussion 4.1. Ipsilateral effect In a previous study [15,24], we showed that carrageenaninduced inflammation activated the descending modulation system from the LC region. This finding was derived from the observation that PWLs in the inflamed paw of the bilateral LC/SC-lesioned rats were significantly shorter than those of the vehicle-injected rats and that a difference in PWL between the two groups of rats was not observed in paws before the induction of inflammation. A similar result can also be seen in the present study (Fig. 3A). An important finding of the present study is that 4 h after carrageenan injection, in the inflamed paws, PWLs of the unilateral LC/SC-lesioned rats were significantly longer than those of the bilateral LC/SC-lesioned rats and were equivalent to those of the vehicle-injected rats. This result indicates that the unilateral LC/SClesioned rats can be distinguished from the bilateral LC/SC-lesioned rats in the degree of thermal hyperalgesia. Four hours after carrageenan injection, in the inflamed paws, no significant difference in PWLs was seen between the unilateral LC/SC-lesioned and the vehicle-injected rats. This finding suggests that unilateral activation of the LC/SC is sufficient to modulate nociceptive processing in the dorsal horn. At this time, we can-
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with morphological data. Wei et al. [8] reported that after LC/SC lesions there were changes in spinal Fos expression contralateral to the inflamed paw, as well as ipsilateral alterations in Fos expression. This discrepancy in contralateral effects between behavioral hyperalgesia and Fos protein expression may be because the magnitude of LC/SC lesions in the Fos experiment is not sufficient compared to that in the behavioral experiment, as the authors have described in their report [8]. Anatomical studies have shown that the LC/SC sends a bilateral projection to the spinal cord dorsal horn [1,12,22,19,3]. For the contralaterally projecting neurons, axons from the LC/SC cross the midline within the brain and travel through the DLF with axonal terminals ipsilaterally in the dorsal horn [12,22]. On the basis of anatomical evidence, we suggest that unilateral hindpaw inflammation activates descending axons that originate from the LC/SC both ipsilateral and contralateral to the inflamed paw and travel through the DLF ipsilateral to the inflamed paw. In addition, a neurochemical study using a microdialysis technique [11] has demonstrated that the norepinephrine level does not change significantly in the dorsal horn contralateral to the site of inflammation for 4 h from induction of inflammation, whereas the norepinephrine level increases significantly in the dorsal horn ipsilateral to the site of inflammation even 1 h after the induction of inflammation. This indicates that descending neurons from the LC/SC which project to the dorsal horn contralateral to the site of inflammation are inactive during inflammation. This neurochemical finding appears to be consistent with the findings of the present study that in the LC/SC both ipsilateral and contralateral to the inflamed paw, only neurons which project to the dorsal horn ipsilateral to the inflamed paw were activated following peripheral inflammation. Fig. 3. Changes in PWLs following unilateral injection of carrageenan in rats of Treatments A–D. (A) PWLs in the inflamed paw. (B) PWLs in the contralateral noninflamed paw. # P < 0.01, significantly different from PWLs before injection. *P < 0.01, significantly different between four treatments.
not explain the result of the present study in which PWLs are equivalent between the vehicle-injected and the unilateral LC/SClesioned rats. Our previous study [11], however, may serve as a clue to the present result. In our present research, the concentration of norepinephrine in the dorsal horn ipsilateral to the site of inflammation during unilateral hindpaw inflammation was measured using the microdialysis technique. The norepinephrine level in the dorsal horn increased with the increase of the development of inflammation, indicating that the activity of descending neurons from the LC increases with the increase of the development of inflammation. This finding suggests that the activity of descending LC/SC neurons is regulated by nociceptive inputs through a positive feedback loop which consists of connections between collaterals of ascending nociceptive projection neurons and the LC/SC neurons [13]. In this context, the equivalence of PWLs between the vehicle-injected and the unilateral LC/SC-lesioned rats may be due to the increased activity of LC/SC neurons contralateral to the site of lesion, which is induced by the feedback mechanism. 4.2. Contralateral effect Four hours after carrageenan injection, no difference in PWL between the vehicle-injected and the unilateral LC/SC-lesioned rats was observed in the contralateral non-inflamed paw, as well as in the case between the vehicle-injected and the bilateral LC/SClesioned rats. This result indicates that the descending modulation system from the LC/SC is not active in the dorsal horn contralateral to the inflamed paw. However, this finding is not consistent
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