A peripheral cannabinoid mechanism suppresses spinal fos protein expression and pain behavior in a rat model of inflammation

Neuroscience 117 (2003) 659 – 670

A PERIPHERAL CANNABINOID MECHANISM SUPPRESSES SPINAL FOS PROTEIN EXPRESSION AND PAIN BEHAVIOR IN A RAT MODEL OF INFLAMMATION A. G. NACKLEY, R. L. SUPLITA II AND A. G. HOHMANN*

geenan model of inflammation. Moreover, cannabinoid antagonists have been reported to prolong and enhance pain behavior resulting from acute tissue injury and inflammation (Calignano et al., 1998; Strangman et al., 1998; see also Beaulieu et al., 2000). Cannabinoids also act locally to suppress capsaicin-evoked immunoreactive calcitonin gene-related peptide release in rat hind paw skin (Richardson et al., 1998) and capsaicin-evoked hyperalgesia (Johanek et al., 2001). In addition, endocannabinoids are detected in peripheral tissue (Calignano et al., 1998). Taken together, these data suggest that peripheral cannabinoid mechanisms suppress inflammatory nociception. Activation of cannabinoid CB1 and CB2 receptors mediate peripheral cannabinoid actions. CB1 exists primarily in the CNS (Matsuda et al., 1990; Munro et al., 1993; Zimmer et al., 1999), whereas CB2 exists mainly in cells of the immune system (Munro et al., 1993; Lynn and Herkenham, 1994). Anatomical studies indicate that cannabinoid CB1 receptors are synthesized in dorsal root ganglion cells (Hohmann and Herkenham, 1999a,b; Ahluwalia et al., 2000) and are transported to peripheral nerve terminals (Hohmann and Herkenham, 1999a). CB2 receptors are likely to reside on nonneuronal cells in inflamed tissue where they may prevent the release of inflammatory mediators that excite nociceptors (Mazzari et al., 1996). Thus, cannabinoid receptors are anatomically situated to modulate peripheral cannabinoid actions. The role of CB1 and CB2 receptor subtypes in the modulation of inflammatory pain has been assessed through the development and use of high-affinity agonists and selective competitive antagonists. The selective CB1 antagonist SR141617A binds to cannabinoid receptors in rat brain with high affinity (Ki⫽1.98 nM), and displays minimal affinity for rat spleen or cloned human CB2 receptors (Ki⬎1000 nM; Rinaldi-Carmona et al., 1994). The selective CB2 antagonist SR144528 exhibits high affinity for rat spleen and cloned human CB2 receptors (Ki⫽0.6 nM), but does not bind readily to rat brain or cloned human CB1 receptors (Ki⫽400 nM; Rinaldi-Carmona et al., 1998). The potent cannabinoid agonist WIN55,212-2 shows high affinity for cannabinoid receptors in rat brain (Ki⫽9.94 nM) and spleen (Ki⫽16.2 nM; Rinaldi-Carmona et al., 1994). Behavioral studies indicate that peripheral antihyperalgesic actions of cannabinoids are mediated by CB1 (Richardson et al., 1998; Ko and Woods, 1999; Johanek et al., 2001). CB2 is also implicated in the modulation of inflammation (Facci et al., 1995; Mazzari et al., 1996) and in the antinociceptive effects of cannabinoids (Hanus et al., 1999; Malan et al., 2001).

Neuroscience and Behavior Program, Department of Psychology, The University of Georgia, Athens, GA 30602-3013, USA

Abstract—The present studies were conducted to test the hypothesis that systemically inactive doses of cannabinoids suppress inflammation-evoked neuronal activity in vivo via a peripheral mechanism. We examined peripheral cannabinoid modulation of spinal Fos protein expression, a marker of neuronal activity, in a rat model of inflammation. Rats received unilateral intraplantar injections of carrageenan (3%). In behavioral studies, carrageenan induced allodynia and mechanical hyperalgesia in response to stimulation with von Frey monofilaments. The cannabinoid agonist WIN55,212-2 (30 ␮g intraplantarly), administered concurrently with carrageenan, attenuated carrageenan-evoked allodynia and hyperalgesia relative to control conditions. In immunocytochemical studies, WIN55,212-2 suppressed the development of carrageenan-evoked Fos protein expression in the lumbar dorsal horn of the spinal cord relative to vehicle treatment. The same dose administered systemically or to the noninflamed contralateral paw failed to alter either carrageenanevoked allodynia and hyperalgesia or carrageenan-evoked Fos protein expression, consistent with a peripheral site of action. The suppressive effects of WIN55,212-2 (30 ␮g intraplantarly) on carrageenan-evoked Fos protein expression and pain behavior were blocked by local administration of either the CB2 antagonist SR144528 (30 ␮g intraplantarly) or the CB1 antagonist SR141716A (100 ␮g intraplantarly). WIN55,212-3, the enantiomer of the active compound, also failed to suppress carrageenan-evoked Fos protein expression. These data provide direct evidence that a peripheral cannabinoid mechanism suppresses the development of inflammation-evoked neuronal activity at the level of the spinal dorsal horn and implicate a role for CB2 and CB1 in peripheral cannabinoid modulation of inflammatory nociception. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: inflammation, dorsal horn, von Frey, anandamide, endocannabinoid.

Cannabinoids suppress nociception resulting from tissue injury and inflammation through a peripheral mechanism (Calignano et al., 1998; Richardson et al., 1998; Ko and Woods, 1999). Richardson et al. (1998) showed that local peripheral but not systemic administration of anandamide, a putative endogenous ligand for cannabinoid receptors, suppresses thermal hyperalgesia and edema in the carra*Corresponding author. Tel: ⫹1-706-542-2252; fax: ⫹1-706-5423275. E-mail address: [email protected] (A. G. Hohmann). Abbreviations: Fos-LI, Fos-like immunoreactivity; FLI, Fos-like immunoreactive; intraplantarly, i.p.l.

0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(02)00870-9

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In the present study, immunocytochemical studies were used to supplement behavioral studies by evaluating the consequences of peripheral cannabinoid actions on information processing at rostral levels of the nervous system. Fos, the protein product of the immediate early gene c-fos, is induced in the spinal cord by noxious stimulation (Hunt et al., 1987). Intraplantar (i.p.l.) carrageenan induces spinal expression of Fos protein-like immunoreactivity (Noguchi et al., 1991; Noguchi et al., 1992; Honore et al., 1995) and c-fos mRNA (Iadarola et al., 1988). Analgesic and antihyperalgesic agents suppress this indirect postsynaptic marker of neuronal activity in models of inflammatory nociception (Presley et al., 1990; Honore et al., 1996, 1997; Buritova et al., 1997). Fos protein-like immunoreactivity has also been used to examine cannabinoid modulation of noxious stimulus-evoked neuronal activity. Although Fos is not a marker for pain, noxious stimuli evoke the expression of c-fos in the superficial and deep nuclei of the lumbar dorsal horn, which are areas involved in the processing of nociceptive information (Hunt et al., 1987). Cannabinoid agonists reduce the number of neurons that express Fos in the presence of inflammation when administered systemically (Tsou et al., 1995) or spinally (Hohmann et al., 1999b; Martin et al., 1999). These data are consistent with neuroanatomical studies which have mapped the distribution of cannabinoid receptors in areas known to control nociceptive tracts, such as the spinal dorsal horn and the periaqueductal gray (Herkenham et al., 1991; Tsou et al., 1998). Electrophysiological studies also demonstrate that cannabinoids suppress noxious stimulus-evoked activity in nociceptive neurons in the spinal dorsal horn in intact (Hohmann et al., 1995, 1998, 1999a; Strangman and Walker, 1999; Drew et al., 2000; Kelly and Chapman, 2001) and inflamed (Drew et al., 2000; Harris et al., 2000) rats. Altogether, these data suggest that cannabinoids reduce behavioral responses to stimuli, in part, by decreasing spinal processing of nociceptive inputs. The present study was conducted to test the hypothesis that systemically inactive doses of cannabinoids modulate inflammation-evoked neuronal activity in vivo via a peripheral mechanism. Carrageenan-evoked Fos protein expression was used to identify the laminar distribution of spinal neurons modulated by a peripheral cannabinoid mechanism. We predicted that the selective cannabinoid agonist WIN55,212-2 would suppress the development of carrageenan-evoked Fos protein-like immunoreactivity, while the same dose administered systemically or to the contralateral paw would not alter c-fos expression. Pharmacological specificity was established in the present work using WIN55,212-3, the receptor inactive enantiomer of WIN55,212-2, as well as competitive antagonists for CB1 and CB2 (SR141716A and SR144528, respectively). Von Frey filament testing was performed in separate animals to provide behavioral correlates for the immunocytochemical studies.

EXPERIMENTAL PROCEDURES Subjects One hundred and forty six adult male Sprague–Dawley rats (Harlan, Indianapolis, IN, USA), weighing 275–350 g, were used in these experiments. All procedures were approved by the University of Georgia Animal Care and Use Committee and followed the guidelines for the treatment of animals of the International Association for the Study of Pain (Zimmermann, 1983). All efforts were made to minimize the number of animals and their suffering.

Drugs and chemicals Lambda carrageenan and WIN55,212-2 were obtained from Sigma Aldrich (St. Louis, MO, USA). SR141716A and SR144528 were generously provided by NIDA. Carrageenan was dissolved in saline and administered in a volume of 100 ␮L. Drugs were dispersed in a vehicle solution of emulphor:ethanol:saline (1:1:8) and administered in a volume of 50 ␮L. The c-fos polyclonal antibody (Ab-5) was purchased from Oncogene Research Products (San Diego, CA, USA). The antibody was raised in rabbit against residues 4 –17 of human Fos protein. A Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) was used to visualize Fos-like immunoreactive cells.

General experimental procedures Rats received i.p.l. injections of carrageenan (3%; 100 ␮L) in the right hind paw. The dose of carrageenan was selected based upon the ability of anandamide to suppress thermal hyperalgesia under similar conditions (Richardson et al., 1998). Injections (i.p.l.) of drug or vehicle were performed bilaterally in the behavioral studies and unilaterally in the immunocytochemical studies. A caliper square was used to measure peripheral paw edema at the site of the i.p.l. injection (Honore et al., 1995, 1996, 1997; Buritova et al., 1997) in experiments 2–5. Paw diameter was measured in duplicate to the nearest 0.1 mm in each group of rats just prior to carrageenan administration. Following induction of inflammation, paw diameter was reassessed in duplicate at 1.5 and 2.5 h in immunocytochemical and behavioral experiments, respectively. Edema was defined as an increase in paw diameter produced by i.p.l. carrageenan.

Behavioral studies Experiment 1 and 2: Assessment of allodynia and mechanical hyperalgesia. The 50% threshold for paw withdrawal to punctate mechanical stimulation was assessed using the up-down paradigm (Chaplan et al., 1994), a validated quantitative allodynia assessment technique. A series of nine filaments (with bending forces of 0.41, 0.69, 1.2, 2.04, 3.6, 5.5, 8.5, 15.1, and 28.8 g; Stoelting), with approximately equal spacing (in log units) between stimuli (mean⫾S.E.M.: 0.231⫾0.012 units) were presented to the hind paw in consecutive order, whether ascending or descending. Stimuli were presented perpendicular to the plantar surface of the hind paw until the filament just bent. Filaments were applied to the midplantar region for a duration of 5 s or until a withdrawal response occurred. Assessment was initiated with the middle hair of the series (3.6 g). In the absence of a paw withdrawal response, an incrementally stronger stimulus was presented. In the event of a paw withdrawal, an incrementally weaker stimulus was presented. After the initial response threshold was crossed, this procedure was repeated four times based upon continued presentation of stimuli that were varied sequentially up and down based upon the rat’s response. This technique permitted the assessment of six responses in the immediate vicinity of the threshold. The pattern of withdrawals (X) and absence of withdrawal (O) was noted together with the terminal filament used in the series of six

A. G. Nackley et al. / Neuroscience 117 (2003) 659 – 670 responses. The contralateral paw was evaluated with this method prior to evaluation of the ipsilateral (carrageenan-injected) paw. The 50% g threshold was interpolated using the formula: 50% g threshold⫽(10[Xf⫹k␦])/10,000 where Xf⫽value (in log units) of the final von Frey hair used; k⫽tabular value of pattern of positive (X) and negative (O) responses, as annotated by Chaplan et al. (1994) and ␦⫽mean difference (in log units) between stimuli. Following determination of the response threshold, the frequency of paw withdrawal to punctate mechanical stimulation (calibrated bending force of approximately 48.1 g in experiment 1 and 65.3 in experiment 2) was assessed in the carrageenaninjected paw over 180 min. Stimuli were presented to the hind paw ten times for a duration of 1 s with an interstimulus interval of approximately 10 s. Only immediate, robust withdrawal responses from the stimulus were counted as withdrawal responses. Mechanical hyperalgesia was defined as an increase in the percentage frequency of paw withdrawal (i.e. [# of paw withdrawals observed/10]⫻100) evoked by stimulation with the von Frey monofilaments. Rats were placed in plexiglass cages positioned over an elevated wire mesh platform. Rats were habituated to the testing environment for at least 15 min prior to testing. After establishing baseline responsiveness to the series of von Frey filaments, rats received bilateral i.p.l. injections. In experiment 1, WIN55,212-2 (30 ␮g i.p.l.; n⫽6) or vehicle (50 ␮l; n⫽6) was administered to the paw concurrently with carrageenan (3 mg in 100 ␮l) in the same syringe. The opposite (noninflamed) paw received an i.p.l. injection of the vehicle (50 ␮l) concurrently with saline (100 ␮l). A separate group of rats received vehicle concurrently with carrageenan and WIN55,212-2 (30 ␮g i.p.l.) in the contralateral salineinjected paw (n⫽6). A separate group of rats receiving i.p.l. carageenan also received bilateral i.p.l. injections of the vehicle and the same dose of WIN55,212-2 (n⫽6) administered systemically (i.p.). In experiment 2, separate groups of rats received intaplantar injections of WIN55,212-2 (30 ␮g; n⫽7), SR141716A (100 ␮g; n⫽6), SR144528 (30 ␮g; n⫽7), SR141716A (100 ␮g) coadministered with WIN55,212-2 (n⫽7), SR144528 coadministered with WIN55,212-2 (n⫽7) or vehicle (50 ␮l; n⫽7) concurrently with carrageenan. Vehicle (50 ␮l) was administered to the contralateral paw concurrently with saline (100 ␮l). Responsiveness to von Frey filaments was reassessed over 180 min.

Immunocytochemical studies Experimental design. In experiment 3, the cannabinoid agonist WIN55,212-2 (30 ␮g; n⫽5), the receptor-inactive enantiomer WIN55,212-3 (30 ␮g; n⫽5) or vehicle (n⫽5) was administered locally in the paw concurrently with carrageenan. Separate groups of rats received the same dose of WIN55,212-2 locally in the noninflamed (contralateral) paw (n⫽5) or intraperitoneally (n⫽5). In experiment 4, rats received i.p.l. injections of WIN55,212-2 (30 ␮g; n⫽5), the CB1 antagonist SR141716A (30 ␮g; n⫽5), the CB2 antagonist SR144528 (30 ␮g; n⫽5), or either SR141716A (30 ␮g) or SR144528 (30 ␮g) coadministered with WIN55,212-2 (n⫽five and n⫽5, respectively) concurrently with carrageenan. In experiment 5, rats received i.p.l. injections of WIN55,212-2 (30 ␮g; n⫽4), SR141716A (100 ␮g; n⫽4), SR141716A (100 ␮g) coadministered with WIN55,212-2 (n⫽6) or vehicle (n⫽4) concurrently with carrageenan. Controls in non-inflamed rats. Control experiments were performed to verify that Fos-like immunoreactivity was not induced by drug manipulations in the absence of carrageenan. Separate groups of rats (n⫽2–3 per group) received i.p.l. injections of vehicle, WIN55,212-2 (30 ␮g), WIN55,212-3 (30 ␮g), SR141716A (30 or 100 ␮g), or SR144528 (30 ␮g) in the right hind paw in the absence of carrageenan.

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Immunocytochemistry Rats were deeply anesthetized with sodium pentobarbital (65 mg/kg i.p.) 1.5 h following i.p.l. administration of drug or vehicle. Animals were perfused intracardially with 300 –500 mL of ice-cold heparinized 0.1 M phosphate-buffered saline followed by 500 ml of fixative (4% paraformaldehyde in 0.1 M sodium phosphate buffer) at a rate of 20 ml/min. The lumbar-sacral segment of the spinal cord was removed, cryoprotected in 30% sucrose overnight, and embedded in matrix (⫺23 °C). Transverse sections (40 ␮m) of the spinal cord were cut at the lumbar L4 –L5 region using a cryostat. Alternate floating sections were collected in 0.1 M phosphate-buffered saline. The sections were washed twice with the same buffer and immersed in 0.3% H2O2 (30 min). Sections were pretreated with 3% normal goat serum (2 h) to block non-specific binding and then incubated with rabbit polyclonal Fos protein antibody (1:10,000 for 48 h at 0 °C). Sections were incubated in biotinylated goat-anti-rabbit secondary antibody (1:600; 2 h). Fos-like immunoreactivity was visualized by the avidin-biotin-peroxidase method (Hsu et al., 1981), using diaminobenzidine (0.05%) as the chromogen. The sections were mounted on gelatin-subbed slides, air-dried and coverslipped. Spinal cord sections from the different experimental conditions were processed simultaneously to control for differences in immunostaining across experiments. The specificity of the immunostaining was verified by preabsorbtion of the antibody with the peptide antigen and by omission of the primary antibody from the immunostaining protocols. Fos immunostaining was eliminated by either manipulation.

Data quantification Three sections from L4 –L5 qualitatively exhibiting the greatest number of labeled cells were selected from each rat. The number of Fos-like immunoreactive (FLI) cells was counted by an investigator blind to the experimental condition as described previously (Presley et al., 1990; Tsou et al., 1996). Cells were counted using an Olympus BH2 microscope at 100⫻ magnification. All cells exhibiting Fos-like immunoreactivity were counted, regardless of staining intensity. For each rat, the total number of cells was recorded as well as the subtotal in four cytoarchitectonically defined subdivisions of the spinal gray matter. The subdivisions used were the superficial laminae (laminae I and II), the nucleus proprius (laminae III and IV), the neck of the dorsal horn (laminae V and VI) and the ventral horn (laminae, VII, VIII, IX, and X) (Presley et al., 1990). The number of FLI cells in each subdivision of the spinal gray matter was counted twice to ensure accuracy; the separate determinations varied by less than 1% (mean⫾S.E.M.: 0.56⫾0.18%). The total number of FLI cells as well as the subtotals within each region (averaged across sections) were determined for each rat and subjected to analysis of variance (ANOVA). Example photomicrographs were obtained digitally for illustration purposes. Brightness contrast and levels were adjusted in Adobe Photoshop. All images were treated identically so that backgrounds appeared similar.

Statistical analysis Behavioral data were analyzed by ANOVA for repeated measures. ANOVA was used to assess the statistical significance of experimental differences in paw diameters as well as the total number of Fos-LI neurons and the subtotals in each spinal cord region. The Greenhouse-Geisser correction (Greenhouse and Geisser, 1959) was applied to all repeated factors to avoid spurious significance due to lack of homogeneity of variance and covariance in repeated factors. P⬍0.05 was considered to be statistically significant.

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Fig. 1. Intraplantar administration of the cannabinoid agonist WIN55,212-2 (WIN-2; 30 ␮g i.p.l.) suppresses the development of carrageenan-evoked allodynia and mechanical hyperalgesia. The same dose administered either systemically (i.p.) or to the noninflamed contralateral paw (contra i.p.l.) was inactive. (A) WIN-2 (ipsilateral i.p.l.) increased the 50% (g) withdrawal threshold relative to control conditions. (B) Administration of WIN-2 (i.pl.) suppressed the frequency of paw withdrawal to a single von Frey filament relative to control conditions. Data (mean⫾S.E.M.) are shown for the carrageenan-injected paw only. *P⬍0.05, **P⬍0.01 for all comparisons by ANOVA and Fisher’s PLSD post hoc test. XP⬍0.05 different from WIN-2 (ip). N⫽six rats per group.

RESULTS Behavioral studies In all studies, the threshold and frequency of paw withdrawal elicited in response to the von Frey monofilaments did not differ between groups prior to i.p.l. administration of carrageenan. Carrageenan lowered the threshold (P⬍0.0002 for each experiment) and increased the frequency (P⬍0.002 for each experiment) of paw withdrawal in response to stimulation with von Frey monofilaments. Experiment 1: Effects of local and systemic administration of WIN55,212-2 on the development of carrageenan-evoked allodynia and mechanical hyperalgesia In experiment 1, the threshold for paw withdrawal was higher in groups receiving the cannabinoid agonist WIN55,212-2 (30 ␮g i.p.l.) locally in the carrageenaninjected hind paw relative to all control conditions (F3,20⫽26.0, P⬍0.0002; P⬍0.0002 for all comparisons; Fig. 1A and 2A). Groups receiving local injections of WIN55,212-2 concurrently with carrageenan showed a lower frequency of paw withdrawal relative to control conditions (F3,20⫽8.84, P⬍0.0007; P⬍0.002 for all comparisons; Fig. 1B and 2B). The same dose of the cannabinoid administered either systemically (ip) or to the contralateral (noninflamed) paw was inactive (Fig. 1A, B). Withdrawal threshold and frequency did not differ in these latter groups from that observed in carrageenan-injected rats receiving local injections of vehicle.

Experiment 2: Pharmacological specificity of the antiallodynic and antihyeralgesic effects of WIN55,212-2 The suppression of carrageenan-evoked allodynia and hyperalgesia induced by WIN55,212-2 was blocked by both the CB1 antagonist SR141716A (100 ␮g i.p.l.; F5,35⫽15.69, P⬍0.0002 and F5,35⫽20.72, P⬍0.0002, respectively) and the CB2 antagonist SR144528 (30 ␮g i.p.l.; F5,35⫽15.69, P⬍0.0002 and F5,35⫽20.72, P⬍0.0002, respectively) (Fig. 2A, B). Withdrawal threshold and frequency in groups receiving either antagonist administered alone or together with WIN55,212-2 did not differ from vehicle. The threshold and frequency of paw withdrawal was also greater in groups receiving SR144528 alone compared with groups receiving SR141716A concurrently with WIN55,212-2 (P⬍0.05; Fig. 2A, B). Immunocytochemical studies General features of carrageenen-evoked c-Fos expression. The pattern of carrageenan-evoked Fos protein expression observed in the present study was consistent with other published reports (Honore et al., 1996, 1997; Buritova et al., 1997). Neurons expressing Fos-like immunoreactivity (Fos-LI) were located primarily in the ipsilateral dorsal horn, with the greatest levels of expression observed at the L4 –L5 region. Approximately 44% of labeled neurons were detected in the superficial laminae. Twenty-five percent of cells exhibiting Fos-LI were localized to the nucleus proprius, and 22% were observed in the neck region of the dorsal horn. The lowest level of Fos-LI occurred in the ventral horn, with neurons comprising less than 10% of labeled cells. Fos protein expression

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Fig. 2. WIN55,212-2 (WIN; 30 ␮g i.p.l.) attenuates carrageenan-evoked allodynia and mechanical hyperalgesia through actions at CB1 and CB2. The CB1 antagonist SR141716A (100 ␮g i.p.l.) and the CB2 antagonist SR144528 (30 ␮g i.p.l.) block the (A) anti-allodynic and (B) antihyperalgesic effects of WIN when coadministered with the agonist. Data (mean⫾S.E.M.) are shown for the carrageenan-injected paw only. **P⬍0.01 for all comparisons by ANOVA and Fisher’s PLSD post hoc test. XP⬍0.05 different from vehicle, SR141716A and SR144528. #P⬍0.05 different from vehicle, SR141716A, SR144528 and WIN55,212-2⫹SR144528. N⫽6 –7 rats per group.

was absent or minimal (mean⫾S.E.M.: 8.87⫾3.85 FLI cells) in the spinal cord contralateral to the carrageenaninjected paw. Moreover, i.p.l. administration of drug or vehicle failed to induce Fos in the spinal cord in the absence of carrageenan (mean⫾S.E.M.: 4.33⫾0.33 FLI cells). In all studies the number of FLI cells differed between groups (P⬍0.003) and the laminar distribution of cells exhibiting carrageenan-evoked Fos protein expression was differentially affected by the experimental treatment (P⬍0.02).

Experiment 3: Effects of local and systemic administration of WIN55,212-2 on carrageenanevoked Fos protein expression WIN55,212-2 (30 ␮g i.p.l.) suppressed the development of carrageenan-evoked Fos protein expression in the lumbar dorsal horn relative to vehicle treatment (Fig. 3). The total number of FLI cells was lower in rats receiving i.p.l. WIN55,212-2 concurrently with carrageenan compared with all other experimental groups (F4,20⫽4.95, P⬍0.007; P⬍0.02 for all comparisons; Fig. 3A). By contrast, the effective dose

Fig. 3. WIN55,212-2 (WIN-2; 30 ␮g i.p.l.) administered locally in the paw concurrently with carrageenan suppressed the development of inflammationevoked Fos protein expression. The same dose administered either systemically (i.p.) or locally in the noninflamed (contralateral) paw was inactive. (A) The total number of FLI cells was lower in rats receiving i.p.l. WIN-2 compared with control conditions. (B) WIN55,212-2 (WIN-2; ipsilateral i.pl.) suppressed Fos protein-expression in the superficial dorsal horn and nucleus proprius but not in the ventral horn. Data are expressed as mean⫾S.E.M. **P⬍0.01 for all comparisons by ANOVA and Fisher’s LSD post hoc test. XP⬍0.05, WIN-3 different from WIN-2 or vehicle. N⫽five rats per group.

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of WIN55,212-2 administered either systemically or to the noninflamed contralateral paw failed to reduce Fos proteinlike immunoreactivity relative to vehicle treatment. Local administration of WIN55,212-3, the receptor-inactive enantiomer, similarly failed to suppress carrageenan-evoked Fos protein expression in rat lumbar spinal cord (Fig. 3A). No significant differences in the total number of labeled cells were observed between groups of rats that received WIN55,212-3 or vehicle concurrently with carrageenan. Administration (i.p.l.) of WIN55,212-2 suppressed carrageenan-evoked Fos-LI in the superficial dorsal horn (F4,20⫽6.07, P⬍0.003; Fig. 3B) and in the nucleus proprius (F4,20⫽4.95, P⬍0.007) by 71 and 50%, respectively. In lamina V and VI, greater numbers of FLI cells were observed (F4,20⫽3.54, P⬍0.03) following administration of the enantiomer WIN55,212-3 compared with other groups receiving intraplantar injections of WIN55,212-2 or vehicle (P⬍0.05 for each comparison). Intraplantar administration of WIN55,212-2 did not alter Fos protein expression in the ventral horn. Administration of the same dose of WIN55,212-2 to the noninflamed contralateral paw or intraperitoneally failed to suppress Fos protein expression in any spinal cord region. Example photomicrographs depict the suppression of carrageenan-evoked Fos protein expression in rat lumbar spinal cord by local but not systemic injections of the cannabinoid agonist (Fig. 4). Experiment 4: Pharmacological specificity of the suppression of carrageenan-evoked Fos protein expression by WIN55,212-2 The total number of cells displaying Fos-LI was reduced approximately 70% in rats that received WIN55,212-2 concurrently with carrageenan compared with groups receiving i.p.l. injections of either SR141716A or SR144528 alone, or SR144528 coadministered with WIN55,212-2 (F4,20⫽8.02, P⬍0.0006; P⬍0.002 for all comparisons; Fig. 5A). By contrast, groups receiving i.p.l. WIN55,212-2 coadministered with SR141716A (30 ␮g i.p.l.) showed lower levels of FLI cells compared with other groups receiving local injections of either the CB1 or CB2 antagonist alone or SR144528 coadministered with WIN55,212-2 (P⬍0.02 for all comparisons; Fig. 5A). Intaplantar administration of WIN55,212-2 suppressed carrageenan-evoked Fos protein expression in the superficial (F4,20⫽5.55, P⬍0.004) and neck region of the dorsal horn (F4,20⫽6.95, P⬍0.002) and in the nucleus proprius (F4,20⫽4.88, P⬍0.007) (Fig. 5B). Administration (i.p.l.) of WIN55,212-2 also suppressed FLI in the ventral horn (F4,20⫽14.85, P⬍0.0002), where only small numbers of FLI cells were observed. Levels of Fos protein expression following administration of either the CB1 antagonist SR141716A (30 ␮g i.p.l.) or the CB2 antagonist SR144528 (30 ␮g i.p.l.) were comparable to that observed previously following i.p.l. injections of the vehicle. The suppression of carrageenan-evoked Fos protein expression induced by i.p.l. WIN55,212-2 was blocked by SR144528 (Fig. 5B). In all spinal laminae, the number of FLI cells was higher in groups receiving SR144528 (30 ␮g i.p.l.)

together with WIN55,212-2 (30 ␮g i.p.l.) compared with groups receiving the agonist alone (P⬍0.02 for all comparisons). In general, the number of FLI cells did not differ in groups receiving either antagonist alone or SR144528 coadministered with WIN55,212. However, in the ventral horn, SR144528 produced a modest but significant increase in the Fos-LI relative to groups receiving concurrent treatment with WIN55,212-2 or the CB1 antagonist (P⬍0.03 for all comparisons). Groups receiving local injections of either the CB1 or CB2 antagonist showed greater carrageenan-evoked Fos protein expression compared with local administration of the agonist alone (P⬍0.02 for all comparisons). Coadministration of SR141716A (30 ␮g i.p.l.) together with WIN55,212-2 (30 ␮g i.p.l.) did not block the cannabinoid-induced suppression of Fos protein expression in rat lumbar spinal cord (Fig. 5A, B). Significant reductions in Fos-LI were observed in this group in the nucleus proprius, neck region and ventral horn (P⬍0.02 for relevant comparisons) relative to treatment with either antagonist alone or WIN55,212-2 coadministered with SR144528. The number of FLI cells observed following coadministration of SR141716A (30 ␮g i.p.l.) with WIN55,212-2 was similar to that observed following administration of WIN55,212-2 alone. In the superficial dorsal horn, the number of Fos immunoreactive cells was lower in groups receiving SR141716A coadministered with WIN55,212-2 relative to groups receiving either the CB1 or CB2 antagonist alone (P⬍0.004 for all comparisons). Experiment 5: Role of CB1 in the suppression of carrageenan-evoked Fos protein expression by local administration of WIN55,212-2 The total number of cells displaying Fos-LI was reduced nearly 70% in rats that received WIN55,212-2 concurrently with carrageenan compared with groups receiving vehicle, SR141716A (100 ␮g i.p.l.) alone or SR141716A (100 ␮g i.p.l.) coadministered with WIN55,212-2 (F3,14⫽7.81, P⬍0.002, P⬍0.002 for all comparisons; Fig. 6A). The total number of labeled cells was similar in groups that received i.p.l. injections of either the vehicle, SR141716A (100 ␮g i.p.l.) alone or SR141716A together with WIN55,212-2 in the carrageenan-injected paw. The suppression of inflammation-evoked Fos protein expression induced by i.p.l. WIN55,212-2 was blocked by the high dose of SR141716A. WIN55,212-2 reduced Fos protein expression in the superficial laminae (F3,14⫽17.14, P⬍0.0001), nucleus proprius (F3,14⫽5.83, P⬍0.009) and neck region (F3,14⫽3.48, P⬍0.05). In each region, the number of FLI cells was lower in groups receiving WIN55,212-2 alone compared with groups receiving vehicle, SR141716A (100 ␮g i.p.l.) alone or SR141716A coadministered with WIN55,212-2 (P⬍0.02 for all comparisons; Fig. 6B). The number of labeled cells was similar in spinal cord sections derived from carrageenan-injected rats that received vehicle, SR141716A alone or SR141716A coadministered with WIN55,212-2. Example photomicrographs show the ability of SR144528 and the high dose of SR141716A to block the suppressive effects of the cannabinoid on carrageenanevoked Fos protein expression (Fig. 7).

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Fig. 4. Photomicrographs depicting carrageenan-evoked Fos protein expression in the lumbar spinal cord of rats receiving i.p.l. injections of (A) vehicle, (B) the cannabinoid agonist WIN55,212-2 administered concurrently with carrageenan (30 ␮g ipsilateral i.p.l.), (C) WIN55,212-2 administered systemically (30 ␮g i.p.), (D) WIN55,212-2 administered to the noninflamed contralateral paw (30 ␮g contralateral i.p.l.) or (E) the enantiomer WIN55,212-3 administered concurrently with carrageenan (30 ␮g ipsilateral i.p.l.). (F) High power photomicrograph showing carrageenan-evoked Fos protein expression in superficial dorsal horn of rat shown in A. (G) Diagram of a hemisection of the lumbar spinal cord, adapted from Presley et al. (1990) and Paxinos and Watson (1998), showing the subdivisions used to quantify carrageenan-evoked Fos protein expression. The scale bar equals 100 ␮m.

Assessment of peripheral edema Prior to i.p.l. administration of carrageenan, diameters of the left and right paws did not differ between groups in either the immunocytochemical (mean⫾S.E.M.: 5.40⫾ 0.09 vs. 5.38⫾0.07 mm, left vs. right paw) or behavioral

(mean⫾S.E.M.: 5.18⫾0.06 vs. 5.14⫾0.07 mm left vs. right paw) experiments. Carrageenan increased paw diameter in the injected paw at 1.5 (average mean⫾S.E.M.: 8.48⫾0.32 mm) and 2.5 h (average mean⫾S.E.M.: 8.86⫾0.19 mm) following the i.p.l. injection (P⬍0.0002 for each comparison). I.p.l. WIN55,212-2 (30 ␮g i.p.l.) was

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Fig. 5. SR144528 blocks the suppression of carrageenan-evoked Fos-LI induced by i.p.l. administration of WIN55,212-2 (WIN-2). (A) The total number of FLI cells was lower in rats receiving WIN-2 (30 ␮g) compared with groups receiving SR141716A (SR1; 30 ␮g i.p.l.) or SR144528 (SR2; 30 ␮g i.p.l.) alone or SR2 coadministered with WIN-2. The total number of FLI cells was lower in groups receiving SR1 (30 ␮g i.p.l.) together with WIN-2 compared with either antagonist alone. (B) i.p.l. administration of WIN-2 suppressed carrageenan-evoked Fos protein-expression in the superficial dorsal horn, the nucleus proprius, neck region of the dorsal horn and the ventral horn. Data are expressed as mean⫾S.E.M. **P⬍0.01, *P⬍0.05 different from SR1, SR2 and WIN-2⫹SR2 by ANOVA and Fisher’s PLSD post hoc test. #P⬍0.05, different from SR1 and SR2 conditions. N⫽five rats per group.

associated with a reduction in peripheral edema at 2.5 h (F3,24⫽3.19, P⬍0.05; Table 1) but not at 1.5 h post-carrageenan (P⬍0.33) relative to vehicle treatment. Paw diameter was similar in carrageenan-inflamed rats receiving local injections of WIN55,212-2 together with either SR141716A (100 ␮g i.p.l.) or SR144528 (30 ␮g i.p.l.) compared with groups receiving vehicle or WIN55,212-2 administered alone (Table 1).

DISCUSSION The present studies demonstrate that systemically inactive doses of cannabinoids suppress the development of inflammation-evoked neuronal activity in rat lumbar spinal

cord in vivo through a peripheral mechanism. Administration (i.p.l.) of the selective cannabinoid agonist WIN55,212-2 suppressed carrageenan-evoked allodynia and hyperalgesia as well as carrageenan-evoked Fos protein expression in the rat lumbar dorsal horn. These effects were mediated by a peripheral mechanism because the effective dose of WIN55,212-2 administered to the noninflamed contralateral paw or intraperitoneally failed to suppress carrageenan-evoked allodynia and hyperalgesia or carrageenan-evoked Fos protein expression. These data demonstrate that the local actions of WIN55,212-2 in the carrageenan-injected paw cannot be attributed to leakage of drug into the systemic circulation.

Fig. 6. SR141716A (SR1; 100 ␮g i.p.l.) blocks the suppression of carrageenan-evoked Fos protein expression induced by WIN55,212-2 (WIN-2; 30 ␮g i.p.l.). (A) The total number of FLI cells was lower in rats receiving i.p.l. WIN-2 compared with groups receiving vehicle, the CB1 antagonist SR1 (100 ␮g i.p.l.) or SR1 coadministered with WIN-2. (B) WIN-2 suppressed Fos in the superficial dorsal horn, nucleus proprius and neck region of the dorsal horn but not in the ventral horn. Data are expressed as mean⫾S.E.M. **P⬍0.01, *P⬍0.05 for all comparisons by ANOVA and Fisher’s PLSD post hoc test. N⫽4 – 6 rats per group.

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Fig. 7. Photomicrographs depicting carrageenan-evoked Fos-LI in the lumbar spinal cord of rats receiving i.p.l. injections of (A) vehicle, (B) the cannabinoid agonist WIN55,212 (30 ␮g i.p.l.), WIN55,212-2 (30 ␮g i.p.l.) coadministered with (C) SR144528 (30 ␮g i.p.l.), or (D) SR141716A (100 ␮g i.p.l.), and (E) SR144528 (30 ␮g i.p.l.) or (F) SR141716A (100 ␮g i.p.l.) administered alone.

The WIN55,212-2-induced attenuation of carrageenan-evoked allodynia and hyperalgesia was blocked by competitive antagonists for CB1 and CB2. The competitive CB1 antagonist SR141716A blocks the antihyperalgesic and/or antiallodynic effects of cannabinoids in tissue injury models of persistent pain induced by capsaicin (Ko and Woods, 1999; Johanek et al., 2001), complete Freund’s ad-

juvant (Martin et al., 1999) and carrageenan (Richardson et al., 1998). Our data thereby extend previous observations of a cannabinoid suppression of thermal hyperalgesia in the carrageenan model of inflammation that is mediated by peripheral CB1 receptors (Richardson et al., 1998). Moreover, our data also add to a recent literature implicating CB2 in the modulation of behavioral responses to acute and chronic

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Table 1. Intraplantar WIN55,212-2 reduces carrageenan-induced edema at 2.5 h Condition

Pre-Carrageenan

Post-Carrageenan

Vehicle WIN55,212-2 (30 ␮g i.p.l.) WIN55,212-2⫹ SR141716A (100 ␮g i.p.l.) WIN55,212-2⫹ SR144528 (30 ␮g i.p.l.)

5.13⫾0.06 5.16⫾0.05

9.36⫾0.17 8.48⫾0.27*

5.08⫾0.05

8.82⫾0.23

5.20⫾0.09

8.85⫾0.11

Data show paw diameter (mean⫾S.E.M.) in mm. * Different from vehicle, P⬍0.05.

inflammation (Hanus et al., 1999; Farquhar-Smith and Rice, 2001; Farquhar Smith et al., 2002). In the immunocytochemical studies, pharmacological specificity was established by examining effects of a receptor-inactive enantiomer as well as coadministration of the active enantiomer with competitive antagonists for CB1 and CB2. Administration (i.p.l.) of WIN55,212-3 failed to suppress carrageenan-evoked Fos protein expression in the rat lumbar spinal cord relative to vehicle treatment. The lack of efficacy of the receptor inactive enantiomer is consistent with a receptor-mediated effect. This finding is in accordance with previous studies that demonstrate that WIN55,212-3 is inactive in in vivo models (Compton et al., 1992; Hohmann et al., 1995; Martin et al., 1996; Tsou et al., 1996; Herzberg et al., 1997; Strangman and Walker, 1999; Johanek et al., 2001). Consistent with the observed behavioral data, the suppressive effects of WIN55,212-2 on carrageenan-evoked Fos protein expression were blocked by either the CB2 antagonist or the CB1 antagonist in the absence of the other drug. However, a dose–response relationship of each antagonist is required to make definitive conclusions about the relative potencies of the antagonists in blocking the actions of WIN55,212-2 in the carrageenan model of inflammation. It should be noted that SR144528 and SR141716A bind to different cannabinoid receptors and have different binding affinities and therefore no conclusions can be drawn from the present data about the relative importance of CB1 and CB2 in the observed effects of WIN55,212-2. Our data suggest that a peripheral cannabinoid mechanism suppresses carrageenan-induced edema, as described previously for i.p.l. administration of anandamide (Richardson et al., 1998). In the present study apparent anti-inflammatory actions of i.p.l. WIN55,212-2 were observed subsequent to the attenuation of carrageenanevoked neuronal activity and pain behavior. A peripheral cannabinoid mechanism suppressed inflammation-evoked Fos protein expression, allodynia and hyperalgesia at 1.5 h following i.p.l. carrageenan although reductions in peripheral edema, as assessed by measurements of paw diameter, were only apparent at 2.5 post-carrageenan. This observation is consistent with other reports showing

that i.p.l. morphine reduces paw and ankle diameter at 3 h but not at 1.5 h post-carrageenan (Honore et al., 1996). Our results suggest that the cannabinoid suppression of inflammation-evoked neuronal activity and pain behavior observed here does not result solely from an anti-inflammatory effect of WIN55,212-2. Of course, more sensitive techniques such as measurements of plasma extravasation and paw volume (Joris et al., 1990) are required to establish beyond doubt that i.p.l. WIN55,212-2 suppresses peripheral edema. Our data suggest that WIN55,212-2 acts at the site of inflammation to suppress carrageenan-evoked neuronal activity by direct and/or indirect actions at CB1 and CB2. It is possible that the site of action for CB1 is neural (e.g. nerve terminals) and the site of action for CB2 is local inflammatory cells. Although the cannabinoid antagonists were applied locally in the paw, the present studies did not evaluate whether antagonism of peripheral cannabinoid actions occurred through a peripheral action; our experiments do not preclude the possibility that the high dose of SR141716A reached the systemic circulation to block the WIN55,212-2 induced attenuation of carrageenan-evoked Fos protein expression and allodynia and hyperalgesia. Activation of CB1 attenuates neuronal excitability via inhibition of calcium channels (Mackie and Hille, 1992; Ross et al., 2001) and activation of inwardly rectifying potassium channels (Mackie et al., 1995). However, CB2 is likely to act through a different mechanism as it is not coupled to Q-type calcium or inwardly rectifying potassium channels (Felder et al., 1995). The positioning of CB2 on immune cells in inflamed tissue may allow CB2 mechanisms to inhibit the release of inflammatory mediators that excite nociceptors (Mazzari et al., 1996). A recent study also suggests that a CB2 mechanism specifically modulates the nerve growth factor driven component of inflammatory hyperalgesia (Farquhar-Smith et al., 2002). By using Fos as an indirect marker of inflammationevoked neuronal activity, we identified the laminar distribution of spinal neurons that are modulated downstream of peripheral cannabinoid actions. The suppression of Fos-LI induced by WIN55,212-2 was most pronounced in the superficial laminae and was also observed in the nucleus proprius and neck region of the dorsal horn. This pattern of suppression of carrageenan-evoked Fos-LI is similar to that observed at the same time point following i.p.l. administration of morphine (Honore et al., 1996). Whereas activation of small-diameter cutaneous sensory afferents by noxious heat or chemical stimuli results in the rapid appearance of c-fos-protein-like immunoreactivity in the superficial layers of the dorsal horn, activation of low-threshold cutaneous afferents results in fewer FLI cells with a different laminar distribution (Hunt et al., 1987). Because immunocytochemical and behavioral studies were performed in separate animals in the present work, any possible nonspecific induction of Fos by repetitive testing could not mask effects of pharmacological manipulations and contribute to the observed results. It is also unlikely that artifacts were introduced into the quantification of the number of FLI cells because means of the

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separate determinations varied by only 1%, indicating that our methods for quantifying Fos-LI were reliable. Moreover, because alternate 40-␮m sections were analyzed in the present study, the nuclei of the same cell cannot appear in more than one section in the present analysis and bias estimates of the number of labeled neurons (Tsou et al., 1996). However, physiological studies are required to establish beyond doubt that the suppression of carrageenan-evoked neuronal activity observed here represents a suppression of nociceptive transmission. Activation of dynorphin biosynthesis in spinal cord is a feature associated with hyperalgesia and peripheral inflammation that is induced by diverse inflammatory agents including carrageenan (Iadarola et al., 1988). The laminar distribution of these neurons is coincident with the distribution of cells exhibiting increased Fos protein expression in rat models of peripheral inflammation and hyperalgesia. Greater than 80% of neurons exhibiting increases in dynorphin or enkephalin following carrageenan inflammation also exhibit carrageenan-induced Fos protein expression (Noguchi et al., 1991, 1992). However, the number of neurons displaying increased Fos-like immunoreactivity is also significantly greater than the number of neurons expressing increased dynorphin or increased enkephalin (Noguchi et al., 1991, 1992). Activation of Fos-related proteins may be related to the induction of dynorphin and enkephalin gene expression in a subpopulation of spinal cord neurons following peripheral inflammation and hyperalgesia. Thus, interpretation of the Fos studies is limited by the nonspecific nature of Fos. The results from these experiments provide further support for the notion that the transmission of inflammation-evoked neuronal activity in the spinal dorsal horn is suppressed by peripheral cannabinoid actions. Taken together, these results raise the possibility that activation of these peripheral cannabinoid mechanisms represent novel therapeutic interventions for inflammatory pain states and alternatives to current pain therapies (see Simone and Seybold, 2000; Hohmann, 2002). For example, adjunct therapies combining systemically inactive doses of CB1 agonists with CB2 agonists may produce synergistic antihyperalgesic effects. Moreover, local administration of cannabinoid agonists may reduce clinical pain associated with inflammation without producing unwanted side-effects typical of centrally acting cannabinoids or opiates.

CONCLUSIONS In conclusion, the present studies provide direct evidence that systemically inactive doses of cannabinoids suppress the development of inflammation-evoked neuronal activity in rat lumbar spinal cord in vivo through a peripheral mechanism. These data are consistent with the ability of local but not systemic injections of a cannabinoid agonist to attenuate the development of behavioral sensitization evoked by punctate mechanical stimuli in the carrageenan model of inflammation. Fos protein expression, a postsynaptic marker of neuronal activity, is suppressed in the superficial dorsal horn, the neck region of the spinal cord and in the nucleus proprius in a rat model of inflammation by this peripheral cannabinoid

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mechanism. Locally administered cannabinoids suppress inflammation-evoked neuronal activity and pain behavior through actions at CB1 and CB2. Acknowledgements—Supported by UGARF and DA014265 (AGH). The authors are grateful to Mark H. Neely for assistance with data analysis.

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(Accepted 23 October 2002)