Effects of carrageenan and morphine on acute inflammation and pain in Lewis and Fischer rats

BRAIN, BEHAVIOR, and IMMUNITY Brain, Behavior, and Immunity 21 (2007) 68–78 www.elsevier.com/locate/ybrbi

Effects of carrageenan and morphine on acute inflammation and pain in Lewis and Fischer rats Karamarie Fecho

a,b,c,*

, Elizabeth L. Manning b, William Maixner

b,c

, Charles P. Schmitt

d

a

c

Department of Anesthesiology, Division of Pain Medicine, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA b Curriculum in Neurobiology, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA Center for Neurosensory Disorders, School of Dentistry, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA d Copperline Solutions, LLC, Chapel Hill, NC 27516, USA Received 19 December 2005; received in revised form 15 February 2006; accepted 16 February 2006 Available online 17 April 2006

Abstract The present study used inbred, histocompatible Fischer 344 (FIS) and Lewis (LEW) rats to begin to explore the role of the hypothalamic–pituitary–adrenal (HPA) axis in the immune processes and pain behavior associated with the carrageenan model of acute hindpaw inflammation. Because the HPA axis contributes in part to morphine’s analgesic and immunomodulatory properties, the present study also assessed the effects of morphine in carrageenan-inflamed LEW and FIS rats. The results showed that carrageenan-induced hindpaw swelling and pain behavior were greater in FIS than in LEW rats. The enhanced hindpaw swelling in FIS rats correlated with an increase in myeloperoxidase (MPO; a measure of neutrophils) in the inflamed hindpaw. FIS rats showed lower circulating levels of TNFa, higher IL-6 levels, and similar IL-1b and nitric oxide levels, when compared to LEW rats. Morphine produced a significant decrease in carrageenan-induced hindpaw swelling and MPO in both strains, but morphine did not significantly alter circulating cytokine/mediator levels. Morphine’s analgesic effects were greater in the inflamed than the noninflamed hindpaw, and they did not correlate with morphine’s antiinflammatory effects. In fact, low doses of morphine produced a mechanical allodynia and hyperalgesia in the noninflamed hindpaw of FIS, but not LEW, rats. These results suggest a positive relationship between HPA axis activity and acute inflammation and inflammatory pain. In contrast, little evidence is provided for HPA axis involvement in morphine’s anti-inflammatory or analgesic effects.  2006 Elsevier Inc. All rights reserved. Keywords: Lewis; Fischer 344; HPA axis; Pain; Allodynia; Hyperalgesia; Carrageenan; Inflammation; Morphine; Analgesia

1. Introduction Studies on inbred, histocompatible Lewis (LEW) and Fischer 344 (FIS) rats have identified a negative relationship between the responsiveness of the hypothalamic–pituitary– adrenal (HPA) axis and susceptibility to autoimmune and chronic inflammatory disorders. LEW rats are susceptible to the development of a variety of autoimmune and chronic inflammatory disorders, whereas FIS rats are resistant to the same disorders. This disparity is believed to reflect a differ-

*

Corresponding author. Fax: +1 919 966 4873. E-mail address: [email protected] (K. Fecho).

0889-1591/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2006.02.003

ence in HPA axis function. The HPA axis response to behavioral stressors or proinflammatory mediators is blunted in LEW rats, leading to reduced synthesis and secretion of corticosterone; the opposite is true for FIS rats (Dhabhar et al., 1993; Ferrick et al., 1991; Happ et al., 1988; Peers et al., 1993; Perretti et al., 1993; Rivest and Rivier, 1994; Sternberg et al., 1989a,b, 1992; Wei and Sternberg, 2004; Wei et al., 2002, 2003; Wilder et al., 1982). In general, corticosteroids produce immunosuppressive effects and suppress many aspects of inflammation (see, Riad et al., 2002 for review). Thus, FIS rats are believed to be inflammation-resistant due to heightened HPA axis activity and elevated corticosteroid levels. LEW rats, in contrast, are believed to be inflammation-susceptible due to blunted HPA axis

K. Fecho et al. / Brain, Behavior, and Immunity 21 (2007) 68–78

activity and reduced corticosteroid levels. In agreement with this hypothesis are studies showing that interruption of the HPA axis by pharmacological blockade of glucocorticoid receptors (Sternberg et al., 1989a) or adrenalectomy (Mason et al., 1990) can cause an inflammation-resistant host to become susceptible to chronic inflammation. Conversely, reconstitution of the HPA axis via transplantation of fetal hypothalamic tissue from resistant FIS rats into susceptible LEW rats restores the blunted HPA axis response in LEW rats and decreases susceptibility to chronic inflammatory disorders (Misiewicz et al., 1997). The relationship between HPA axis activity and acute inflammation is less understood. While corticosteroids are known to produce many immunosuppressive effects, recent evidence suggests that corticosteroids can enhance the acute inflammatory response. For example, corticosteroids can enhance neutrophil activity and prolong the neutrophil lifespan (Cox, 1995). Other studies show that acute stressors can act through the HPA axis to cause a redistribution of neutrophils and other leukocytes from the blood to peripheral sites of inflammation, thereby enhancing peripheral inflammatory responses (see Dhabhar and McEwen, 1997, 1999; Dhabhar et al., 1995, 1996). The aim of the present study was to use LEW and FIS rats to begin to decipher the role of the HPA axis in the immunological processes and pain behavior associated with a model of acute inflammation; namely, carrageenan-induced hindpaw inflammation. Carrageenan-induced hindpaw inflammation is a neutrophil-mediated acute inflammatory response that produces hindpaw swelling, erythema, and localized hyperthermia, with clinical symptoms peaking at 1 12 to 3 h after the intraplantar injection of carrageenan (Cunha et al., 1991, 1992, 1999, 2005; Di Rosa et al., 1971; Ferreira et al., 1988, 1993; Handy and Moore, 1998; Leung et al., 2001; Poole et al., 1999; Ribeiro et al., 2000; Tsuji et al., 2003; Vinegar et al., 1969; Wei et al., 1995; Winter et al., 1962). The carrageenan-inflamed hindpaw also is painful, and carrageenan-induced hindpaw pain can be reliably measured using established behavioral pain assays, such as the Hargreaves radiant heat test and the von Frey monofilament test (e.g., Fecho et al., 2005; Ferreira et al., 1988). Because morphine is known to modulate both pain and immunological processes (see Vallejo et al., 2004 for review), in part through effects on the HPA axis (see Mellon and Bayer, 1998; Kosten and Ambrosio, 2002; Kiefer and Wiedemann, 2004 for reviews), the present study also included an assessment of morphine’s effects in carrageenan-inflamed LEW and FIS rats. 2. Methods 2.1. Animals All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of North Carolina. Adult male LEW and FIS rats, weighing 175–225 g at 55–60 days old, were purchased from Charles-River Laboratories (Raleigh, NC). The rats were housed two per

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cage in plastic cages in a temperature- and humidity-controlled colony room under a 12 h day–night cycle. Food and water were provided ad libitum. The rats received a 1 week habituation period before the experimental procedures were initiated, during which time they were handled daily by the investigators, exposed to the behavioral testing room, and given one training session in each behavioral pain assay.

2.2. Morphine administration Morphine sulfate (National Institute on Drug Abuse, Bethesda, MD) was dissolved in sterile 0.9% saline and prepared to concentrations of 0 (saline vehicle alone), 2.5, and 5.0 mg/ml. Morphine was administered 1 h after intraplantar injection of carrageenan in a 1 ml/ kg volume by subcutaneous (sc) injection into the left lower abdomen, to achieve a final dose of 0 (control), 2.5 or 5.0 mg/kg. The behavioral pain assays were intiated 12 h after the morphine injection, in the order described below.

2.3. Carrageenan-elicited hindpaw inflammation On the test day, the width of the left and right hindpaw of each rat, defined as the distance from the plantar to the dorsal surface of the center of the hindpaw, was measured using manual calipers. Rats then received an intraplantar (ipl) injection of 100 ll of 3.5% (w/v) carrageenan in sterile 0.9% saline into the right hindpaw, using a 1-cc syringe and a 27-gauge needle. To control for the influence of the injection itself, 100 ll of sterile 0.9% saline was injected into the intraplantar region of the left hindpaw of each rat. Hindpaw width was measured again 4 h after the carrageenan injection, after which time the rats were euthanized. The data were presented as the change in hindpaw width (in mm) from pre-injection to 4 h post-injection.

2.4. Behavioral pain assays The behavioral pain assays were conducted in the order described below on the baseline and test days, with approximately 30 min of home cage rest between each assay. Baseline assessments of nociceptive sensitivity were taken for all rats 24 h before the test day. On the test day, the tailflick test was conducted at 1 12 h after the carrageenan injection (or 12 h after morphine), the Hargreaves test was initiated at 2 h after the carrageenan injection (or 1 h after morphine), and the von Frey monofilament test was initiated at 2 12 h after the carrageenan injection (or 1 12 h after morphine). All behavioral pain assays were conducted during the peak of the acute phase of the inflammatory response (Vinegar et al., 1969; Winter et al., 1962). Pilot studies using untreated rats confirmed that the order in which we ran the behavioral assays and the rest intervals that we included between assays resulted in stable responding that did not differ from baseline values (data not shown). 2.4.1. Hargreaves test for thermal hyperalgesia The Hargreaves radiant heat method (Hargreaves et al., 1988) was used to measure hindpaw sensitivity to a noxious thermal stimulus, applied via an IITC Model 336 Paw/Tail Stimulator Analgesia Meter (Woodland Hills, CA). Rats were placed in individual plexiglass cages on a clear glass platform and given 15 min to acclimate to the testing environment. The stimulator source was used to present a focused beam of radiant light onto the midplantar region of the hindpaw. The idle intensity of the light was set at 2% of the maximum intensity to permit accurate placement of the beam of light to the appropriate region of the hindpaw; the active intensity of the light was set at 50% maximum. The light stimulus was turned off manually when the rat withdrew the hindpaw from the noxious beam of light, or automatically if the 20 s cut-off time was reached. Each rat received 3 trials/hindpaw, with a period of 5–10 min separating each trial, and the results from the 3 trials were averaged for analysis. Data were expressed in terms of the latency (s) to hindpaw withdrawal and the percent maximum possible effect (MPE; defined as [(Latencytest Latencybaseline)/(20 Latencybaseline)] · 100).

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K. Fecho et al. / Brain, Behavior, and Immunity 21 (2007) 68–78

2.4.2. Tail-flick test for thermal hyperalgesia An IITC Model 336 Paw/Tail Stimulator Analgesia Meter (Woodland Hills, CA) was used to measure the sensitivity of the tail to a noxious thermal stimulus. The animal was wrapped gently in a surgical cloth, the tail was placed in the tail groove directly under the stimulator source, and a focused beam of radiant light was directed to the tail, 2 inches from the tip. The light turned off automatically when the animal’s tail ‘‘flicked’’ and a pinhole sensor in the tail groove was exposed to the stimulation light, or when the cut-off time of 20 s was reached. Each animal was tested one time on the baseline and test days, and the data were expressed as the latency (s) to tail-flick and the percent maximum possible effect (MPE; defined as [(Latencytest Latencybaseline)/(20 Latencybaseline)] · 100). 2.4.3. Von frey monofilament test for mechanical allodynia and hyperalgesia Rats were placed into individual plexiglass cages on a wire-mesh table positioned approximately 1 foot above a standard laboratory bench. Animals were given 15 min to acclimate to the testing apparatus. A series of 11 calibrated von Frey monofilaments (0.40, 0.68, 1.1, 1.37, 2.1, 3.4, 5.7, 8.35, 10.1, 13.2, and 25.0 g; Stoelting, Wood Dale, IL) with approximately equal logarithmic spacing between stimuli (0.232 ± 0.04 U) were used to assess the threshold to mechanical stimulation. The monofilaments were placed perpendicularly onto the midplantar region of the hindpaw through the holes in the wire-mesh table, and pressure was applied just until the point of deflection of the monofilaments, after which the monofilament was immediately withdrawn. The ‘‘up-down’’ method of Chaplan et al. (1994) and the statistical equations of Dixon (1980) were used to calculate the 50% threshold (in grams), defined as the gram force at which presentation of the monofilament produced withdrawal of the hindpaw 50% of the time. Immediately after the von Frey threshold was determined, the frequency of hindpaw withdrawal to repetitive, punctate mechanical stimulation was measured. A von Frey monofilament with a calibrated bending force of 3.4 g (Lo) or 70.0 g (Hi) was presented to the hindpaw 10 times, with a 1 s duration and a 1 s interstimulus interval. The percent responses to the Lo or Hi monofilament was calculated as the # of hindpaw withdrawals/ 10 · 100. Mechanical allodynia was defined as an increase in the percent of hindpaw withdrawals using the Lo g force monofilament, and mechanical hyperalgesia was defined as an increase in the percent of hindpaw withdrawals using the Hi g force monofilament.

2.5. Western blots Hindpaw tissue from both the saline (control)-injected hindpaw and the carrageenan-injected hindpaw of each rat was isolated post mortem and immediately frozen in liquid nitrogen for storage at 80 C until processing at a later date. Samples were thawed on ice and homogenized manually, and protein was extracted using T-Per protein extraction reagent (Pierce, Rockford, IL) containing Halt protease inhibitor cocktail (Pierce). Protein levels were normalized using a BCA protein assay kit (Pierce). Normalized protein samples were reduced in Laemmli buffer containing 2 mM dithiothreitol, separated on 12% SDS–PAGE gel (Pierce), and transferred onto PVDF membranes (Hybond-P, Amersham Biosciences, Piscataway, NJ). Membranes were blocked with 5% (w/v) nonfat milk powder in Tris-buffered saline + 0.5% Tween for 1 h at room temperature and incubated overnight at 4 C with rabbit polyclonal anti-myeloperoxidase (MPO) antibody (#ab2088; 1:1000 dilution; Abcam Inc., Cambridge, MA; El-Chemaly et al., 2003) or rabbit polyclonal anti-actin (1–19) antibody (#sc-1616-R; 1:750 dilution; Santa Cruz Biotechnology, Inc., Paso Robles, CA; Doolittle, 1995). The blots then were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-rabbit IgG (Heavy and Light chain) secondary antibody (#AP307P; 1:15,000 dilution; Chemicon, Temecula, CA). The blots were developed with electrochemoluminescence solution (PerkinElmer, Boston, MA) and exposed to Kodak light film for visualization. For quantification, Adobe Photoshop cs2 (version 9.0; Adobe Systems, Inc., San Jose, CA) was used to measure the O.D. of labeled protein bands on inverted images from scanned blots, and the O.D. for each MPO band was expressed relative to the O.D. for the corresponding actin band for each sample. Results from four MPO and actin blots were quantified.

2.6. Plasma cytokine ELISAs Immediately after euthanasia, approximately 5 ml of blood was drawn from the inferior vena cava into heparinized syringes. Plasma was separated by centrifugation at 2500g for 10 min, removed into sterile 1.5 ml tubes, and frozen at 80 C until testing at a later date. An ELISA kit for rat IL6 (R&D Systems, Minneapolis, MN) was used to measure plasma IL-6 levels in duplicate samples, according to the manufacturer’s directions. Levels of rat TNFa and IL-1b were measured in duplicate plasma samples by the ImmunoTechnologies Core in the Center for Gastrointestinal Biology and Disease at UNC using ELISA kits from Biosource (Camarillo, CA) and the National Institute for Biological Standards and Controls (UK), respectively. Data were expressed as pg/ml and the duplicates were averaged for analysis. Preliminary results showed that plasma IL-6 and TNFa levels were undetectable or near background in untreated rats (data not shown). Plasma IL-1b levels in untreated rats were not measured.

2.7. Plasma nitric oxide (nitrite/nitrate) determinations Plasma was prepared as described above and combined levels of nitrite and nitrate, the major degradation products of nitric oxide, were measured using a colorimetric reaction. Briefly, nitrate was first converted to nitrite using nitrate reductase and concentrations of nitrite were measured using Griess reagent (Green et al., 1982; Weinberg et al., 1994). Absorbances were read at 560 nm on a Victor3 Multilabel Counter (Model #1420; PerkinElmer) plate reader and sample concentrations were determined using a standard curve prepared from known concentrations of sodium nitrate (Sigma). Data were expressed as lM nitrate/nitrite and the duplicates were averaged for analysis. Preliminary results indicated that plasma nitrate/nitrite levels were near background levels in untreated rats (data not shown).

2.8. Statistical treatment of the data The experiment was conducted three times, using a total of 7–14 rats per dose per strain. The data were analyzed two ways. The first analysis was a general Analysis of Variance (ANOVA), which assessed the overall influence of strain, treatment (morphine dose) and hindpaw (left, salineinjected or right, carrageenan-injected) as main factors. Examined interactions included the effects of strain · treatment and/or strain · treatment · hindpaw. In select cases, a post hoc general contrast comparison using Scheffe’s F statistic was used to compare specific groups. The second analysis involved computing linear regressions with linear and quadratic terms, and then using indicator variables to test the regressions for collinearity and cocurvature. The regression analysis was performed to compare the shape of the dose–response curve for each hindpaw and each strain (Kleinbaum et al., 1998). Values were excluded from the analysis if a technical problem was noted when that measure was taken or if the value was 3+ standard deviations away from the group mean. For all analyses, the level of significance was set at p < .05.

3. Results 3.1. Carrageenan-induced inflammation and morphine’s antiinflammatory effects in FIS and LEW rats Fig. 1A shows the degree of hindpaw swelling, expressed as change in hindpaw width (mm), measured 4 h after the ipl injection of carrageenan or saline in FIS and LEW rats at each dose of morphine. The ipl injection of carrageenan caused a significant amount of hindpaw swelling in both FIS and LEW rats (F (1, 100) = 299.12, p < .0001). Carrageenan-induced hindpaw swelling was significantly greater in FIS rats than in LEW rats (F (1, 100) = 12.85, p < .001). Morphine produced a significant decrease in

K. Fecho et al. / Brain, Behavior, and Immunity 21 (2007) 68–78

A 8

B 3.0 FIS Left HP (SAL) FIS Right HP (CARR) LEW Left HP (SAL) LEW Right HP (CARR)

2.5

MPO/Actin

6

ΔPaw Width (mm)

71

4

2

0

2.0 1.5 1.0 0.5 0.0

-2 0.0

2.5

0.0

5.0

Morphine (mg/kg)

2.5

5.0

Morphine (mg/kg)

Fig. 1. Carrageenan produced a significant increase in hindpaw (HP) swelling and MPO levels, and the effect was greatest in FIS rats. Morphine decreased carrageenan-induced hindpaw swelling and MPO levels in both LEW and FIS rats. (A) Means ± SEM change in hindpaw width (in mm), on the y-axis, from the pre-injection width to 4 h after ipl injection of carrageenan (CARR) into the right hindpaw (filled symbols) and saline (SAL) into the left hindpaw (open symbols) of FIS (squares) and LEW (triangles) rats (n = 7–14) treated with 0 (control), 2.5 or 5.0 mg/kg morphine, on the x-axis. (B) The quantification results from four Western blots for MPO and actin (n = 5). Means ± SEM O.D. of MPO relative to actin is shown on the y-axis for salineinjected (open symbols) and carrageenan-injected (filled symbols) hindpaws of FIS (squares) and LEW (triangles) rats at each dose of morphine, on the x-axis.

carrageenan-induced hindpaw swelling in both strains (F (2, 100) = 9.57, p < .001). Regression analyses showed that the dose–response function for morphine was similar in LEW and FIS rats. In contrast, morphine’s dose–response function for the left and right hindpaws of each strain differed in collinearity and cocurvature (F (5, 50) > 2.49, p < .05). Indeed, morphine produced a decrease in hindpaw swelling in the carrageenan-inflamed hindpaw of both strains, but morphine did not significantly alter the width of the saline-injected hindpaw in either strain. Additional experiments were conducted to determine whether alterations in the immune response to carrageenan account for the difference in carrageenan-induced hindpaw swelling in FIS and LEW rats, and in the anti-inflammatory effects of morphine on hindpaw swelling. Western blot analysis was used to measure hindpaw levels of MPO, an indirect measure of neutrophils (Leung et al., 2001; Segal, 2005). Representative Western blot results are shown in Fig. 2, and the quantification of the Western blot results is shown in Fig. 1B. Hindpaw MPO levels were significantly higher in the carrageenan-injected hindpaw than in the saline-injected hindpaw in both strains (F (1, 80) = 57.55, p < .0001). MPO levels also were higher in FIS rats than in LEW rats (F (1, 80) = 5.84, p < .05). Morphine produced a significant decrease in hindpaw MPO levels in both strains (F (2, 80) = 3.26, p < .05), and although the effect of morphine appeared to be greater in the carrageenan-injected hindpaws, this difference did not reach significance. In accordance, the regression analysis showed that the morphine dose–response function was similar in both LEW and FIS rats and in both saline-injected and carrageenan-injected hindpaws. Table 1 shows circulating levels of the proinflammatory cytokines TNFa, IL-1b, and IL-6, and nitrate/nitrite (an

Left HP (SAL)

A

LEW

FIS

Right HP (CARR) LEW

FIS

MPO actin

B

Molecular Weight Markers (kDa) MPO (glycosylated precursor, 92 kDa) actin (43 kDa)

Fig. 2. MPO levels in the left, saline-injected and right, carrageenaninjected hindpaws of LEW and FIS rats. (A) Representative Western blot results for hindpaw MPO and actin levels in the left, saline-injected (SAL) and right, carrageenan-injected (CARR) hindpaws of LEW and FIS rats. (B) The mobilities of MPO and actin, with respect to the molecular weight markers used in the Western blots.

indirect measure of nitric oxide) measured 4 h after intraplantar injection of carrageenan in saline- or morphine-treated FIS and LEW rats. These proinflammatory cytokines/ mediators were selected on the basis of their known involvement in carrageenan-induced hindpaw inflammation (Cunha et al., 1991, 1992, 2005; Ferreira et al., 1988, 1993; Handy and Moore, 1998; Leung et al., 2001; Tsuji et al., 2003; Wei et al., 1995). Plasma levels of IL-1b and nitrate/nitrite were similar in FIS and LEW rats. In contrast, FIS rats showed lower plasma levels of TNFa (F (1, 16) = 33.78, p < .0001) and higher plasma levels of IL-6 (F (1, 11) = 6.97, p < .05), when compared to LEW

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K. Fecho et al. / Brain, Behavior, and Immunity 21 (2007) 68–78

Table 1 FIS rats have lower plasma levels of TNF-a and higher plasma levels of IL-6, but equal plasma levels of IL-1b and nitrate/nitrite, when compared to LEW rats. Morphine does not significantly alter circulating cytokine/mediator levels in carrageenan-treated rats Plasma cytokine/mediator levelsa

Fischer Saline Morphineb Lewis Saline Morphinesb

TNFa (pg/ml)

IL-6 (pg/ml)

IL-1b (pg/ml)

Nitrate/Nitrite (lM)

6.23 ± 1.66 5.63 ± 0.91

55.21 ± 13.34 52.95 ± 12.02

34.71 ± 4.18 42.22 ± 10.03

34.98 ± 3.03 38.78 ± 2.32

18.68 ± 2.32 18.46 ± 3.02

27.79 ± 6.36 29.29 ± 5.13

30.26 ± 2.86 38.13 ± 4.74

28.65 ± 4.41 31.74 ± 2.73

a Plasma samples were collected 4 h after the intraplantar injection of carrageenan and they were analyzed for levels of TNFa, IL-6, IL-1b and nitrate/ nitrite. Preliminary results indicated that plasma levels of TNFa, IL-6 and nitrate/nitrite were undetectable or near background levels in untreated rats (data not shown). Plasma IL-1b levels in untreated rats were not determined. b The data for plasma IL-1b and TNFa levels are from FIS and LEW rats that received either 0 (saline) or 2.5 mg/kg morphine 1 h after carrageenan. The data for plasma IL-6 and nitrate/nitrite levels are from FIS and LEW rats that received 0 (saline) or 5.0 mg/kg morphine 1 h after carrageenan. (n = 4–8).

rats. Morphine did not significantly alter circulating levels of nitrate/nitrite or any of the tested proinflammatory cytokines. 3.2. Carrageenan-induced pain behavior and morphine’s analgesic effects in FIS and LEW rats In the Hargreaves radiant heat assay (Fig. 3), the withdrawal latency of the carrageenan-injected hindpaw was shorter than that of the saline-injected hindpaw in both strains, indicating that carrageenan produced a thermal hyperalgesia, but only FIS rats showed a significant degree of thermal hyperalgesia at the 0 mg/kg dose of morphine (F (1, 100) = 4.59, p < .05 for raw data; F (1,100) = 2.75, p < .07 for MPE). Morphine produced significant analgesic effects in the Hargreaves radiant heat assay in both strains (F (2, 100) = 68.09, p < .0001 for raw data; F (2, 100) = 54.07, p < .0001 for MPE). The regression analyses showed that morphine’s dose–response function was similar in LEW and FIS rats, but the dose–response curves for the

B 20

% Maximum Possible Effect

Paw Withdrawal Latency (sec)

A

left and right hindpaws of each strain differed in collinearity and cocurvature (F (5, 50) > 3.27, p < .05 for raw data; F (5, 50) > 2.08, p < .09 for MPE). These findings indicate that morphine’s analgesic effects were greater in the inflamed hindpaw than in the noninflamed hindpaw in FIS and LEW rats. The tail-flick test was conducted to determine whether carrageenan altered thermal pain sensitivity in the tail, as opposed to the site of inflammation, and whether thermal pain sensitivity in the tail differed between FIS and LEW rats (Fig. 4). Tail-flick latencies did not change from baseline after carrageenan treatment (7.85 + 0.36 baseline vs. 8.68 + 0.34 after carrageenan for FIS rats; 8.18 + 0.45 baseline vs. 8.30 + 0.59 after carrageenan for LEW rats) and were similar in FIS and LEW rats at the 0 mg/kg dose of morphine, indicating that carrageenan did not alter pain sensitivity in the tail. Morphine produced significant analgesic effects in the tail-flick test in both strains (F (2, 50) = 57.07, p < .0001 for raw data; F (2, 50) = 57.81, p < .0001 for MPE), but LEW rats showed a greater

15

10

5

80 60

FIS Left HP (SAL) FIS Right HP (CARR) LEW Left HP (SAL) LEW Right HP (CARR)

40 20 0 -20

0 0.0

2.5

Morphine (mg/kg)

5.0

0.0

2.5

5.0

Morphine (mg/kg)

Fig. 3. Carrageenan-induced thermal hyperalgesia was greater in FIS rats than LEW rats. Morphine increased hindpaw withdrawal latencies in both strains, and morphine’s analgesic effects were most pronounced in the carrageenan-inflamed hindpaw. In (A), the figure depicts the paw withdrawal latency (in s), on the y-axis, in the Hargreaves radiant heat test for the saline (SAL)-injected (open symbols) and carrageenan (CARR)-injected (filled symbols) hindpaws, measured 1 h after 0 (control), 2.5 or 5.0 mg/kg morphine, on the x-axis, in FIS (squares) and LEW (triangles) rats. The cut-off time for the assay was 20 s and is indicated by the dashed line. (B) The same results expressed as percent maximum possible effect (MPE). Results represent means ± SEM for each group (n = 7–14).

K. Fecho et al. / Brain, Behavior, and Immunity 21 (2007) 68–78

B

% Maximum Possible Effect

Tail-Flick Latency (sec)

A 25 20 15 10 5 0

100

73

FIS LEW

80 60 40 20 0 -20

-5 0.0

2.5

5.0

0.0

2.5

5.0

Morphine (mg/kg)

Morphine (mg/kg)

Fig. 4. Morphine increased tail-flick latencies after carrageenan in both strains, but morphine’s analgesic effects were greater in LEW rats. (A) The tailwithdrawal latency (in s), on the y-axis, measured 30 min after 0 (control), 2.5 or 5.0 mg/kg morphine, on the x-axis, in FIS (filled squares) and LEW (open triangles) rats. The dashed line marks the assay’s cut-off time of 20 s. (B) The same results expressed as percent maximum possible effect (MPE). Results represent means ± SEM for each group (n = 7–14).

response to morphine than FIS rats (F (2, 50) = 3.26, p < .05 for raw data; F (2, 50) = 3.59, p < .05 for MPE). The regression analyses showed that morphine’s dose–response function for LEW and FIS rats differed in both collinearity and cocurvature (F (5, 50) > 3.26, p < .05), reflecting the fact that FIS rats did not respond to the 2.5 mg/kg dose of morphine. Carrageenan produced an increase in mechanical sensitivity in the von Frey threshold (Fig. 5) and frequency (Fig. 6) tests. The von Frey threshold was lower in the carrageenan-injected hindpaw than in the saline-injected hindpaw in both strains, but only FIS rats showed a significant

von Frey Threshold (g)

30 25 20 15 10 FIS Left HP (SAL) FIS Right HP (CARR) LEW Left HP (SAL) LEW Right HP (CARR)

5 0 0.0

2.5

5.0

Morphine (mg/kg) Fig. 5. Carrageenan produced an increase in mechanical sensitivity in the von Frey threshold test and the effect was greatest in FIS rats. Morphine reversed the carrageenan-induced increase in mechanical sensitivity in the carrageenan-inflamed hindpaw and morphine’s dose–response function was similar in FIS and LEW rats. The 2.5 mg/kg dose of morphine increased mechanical sensitivity in the noninflamed hindpaw of FIS, but not LEW, rats. The figure shows the von Frey threshold (in grams), on the y-axis, for the saline (SAL)-injected (open symbols) and carrageenan (CARR)-injected (filled symbols) hindpaws, measured 1 12 h after 0 (control), 2.5 or 5.0 mg/kg morphine, on the x-axis, in FIS (squares) and LEW (triangles) rats. The dashed line marks the upper limit of the assay (25 g). Results represent means ± SEM for each group (n = 7–14).

effect of carrageenan at the 0 mg/kg dose of morphine (F (1, 98) = 13.37, p < .0001). The frequency of hindpaw withdrawal in the von Frey frequency tests was greater for the carrageenan-injected hindpaw than the saline-injected hindpaw in both strains, and again, the effect reached significance only in FIS rats (F (1, 100) > 16.07, p < .0001). Morphine produced significant analgesic effects in the von Frey threshold test (F (2, 98) = 5.97, p < .01) and in the von Frey frequency tests (F (2, 100) > 3.83, p < .05), in both strains. The regression analyses showed that the morphine dose–response curves for the carrageenan-inflamed hindpaws of FIS and LEW rats were similar in the threshold test and in the frequency test for mechanical allodynia (Lo; Fig. 6A). The dose–response functions for FIS and LEW rats in the frequency test for mechanical hyperalgesia (Hi) differed in collinearity and cocurvature (Hi; Fig. 6B; (F (5, 50) = 3.15, p < .05), which likely reflects the small response to morphine in LEW rats. Because LEW rats showed very little carrageenan-induced mechanical allodynia/hyperalgesia, and because of the limits of the von Frey threshold and frequency tests (with 25 g being the maximal response for the threshold test and 0% being the minimal response for the frequency tests), LEW rats would be expected to show a smaller analgesic effect of morphine. Morphine’s effects on the saline-injected, noninflamed hindpaw differed dramatically in LEW and FIS rats. Morphine’s dose–response function for the saline-injected hindpaws of FIS and LEW rats differed in collinearity and cocurvature for the von Frey threshold (F (5, 49) = 3.75, p < .01) and frequency tests (F (5, 50) > 2.82, p < .05). Indeed, while the morphine dose–response function for the inflamed hindpaw is mostly linear in both strains, the morphine dose–response function for the noninflamed hindpaw is quadratic for FIS rats, with the low dose of morphine producing an increase in mechanical sensitivity in the threshold test and a mechanical allodynia and hyperalgesia in the frequency tests. The noninflamed hindpaws

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K. Fecho et al. / Brain, Behavior, and Immunity 21 (2007) 68–78 90 FIS Left HP (SAL) FIS Right HP (CARR) LEW Left HP (SAL) LEW Right HP (CARR)

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50

Response Rate-Hi (%)

Response Rate-Lo (%)

60

40 30 20 10 0

70 60 50 40 30 20 10 0

0.0

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0.0

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Fig. 6. Carrageenan produced a mechanical allodynia and hyperalgesia in the von Frey frequency tests and the effect was greatest in FIS rats. Morphine decreased responding in the carrageenan-inflamed hindpaw, and morphine’s analgesic dose–response function was generally similar in LEW and FIS rats. In addition, the 2.5 mg/kg dose of morphine produced an increase in responding in the noninflamed hindpaw of FIS, but not LEW, rats. The figure shows the response rate (in percent), on the y-axis, in the von Frey frequency tests for allodynia (Lo; (A)) and hyperalgesia (Hi; (B)), for the saline (SAL)-injected (open symbols) and carrageenan (CARR)-injected (filled symbols) hindpaws, measured 1 12 h after 0 (control), 2.5 or 5.0 mg/kg morphine, on the x-axis, in FIS (squares) and LEW (triangles) rats. Results represent means ± SEM for each group (n = 7–14).

of LEW rats do not show mechanical allodynia or hyperalgesia after morphine. 4. Discussion LEW and FIS rats have been used in numerous studies to establish a role for the HPA axis in autoimmune and chronic inflammatory disorders. The HPA axis response to emotional or immunological stressors is blunted in LEW rats, leading to low circulating levels of corticosterone and a consequential increase in susceptibility to autoimmune disorders and chronic inflammation. In contrast, the HPA axis response is enhanced in FIS rats, leading to high circulating levels of corticosterone and a decrease in susceptibility to autoimmunity and chronic inflammation (Dhabhar et al., 1993; Ferrick et al., 1991; Happ et al., 1988; Mason et al., 1990; Misiewicz et al., 1997; Peers et al., 1993; Perretti et al., 1993; Rivest and Rivier, 1994; Sternberg et al., 1989a,b, 1992; Wei and Sternberg, 2004; Wei et al., 2002, 2003; Wilder et al., 1982). Less clear is the relationship between HPA axis activity and acute inflammation. The aim of the present study was to use LEW and FIS rats to begin to decipher the role of the HPA axis in the immunological processes and pain behavior associated with carrageenan-induced acute hindpaw inflammation. The results indicated that compared to LEW rats, FIS rats show more hindpaw swelling and neutrophil accumulation (as measured by MPO levels), and correspondingly greater pain behavior, in the carrageenan-injected hindpaw. Our findings are suggestive of a positive relationship between HPA axis activity and acute inflammation, in contrast to prior findings establishing a negative relationship between HPA axis activity and chronic inflammation or autoimmunity. Differences between acute inflammatory responses and autoimmune or chronic inflammatory

responses might explain the proposed differential role of the HPA axis. In particular, the immune response to intraplantar carrageenan involves the activation of resident macrophages, mast cells and endothelial cells, which results in the release of a number of proinflammatory cytokines and mediators (including TNFa, IL-1b, IL-6, and nitric oxide). Neutrophils are the first immune cells recruited from the circulation to the inflamed hindpaw. Neutrophils reach peak levels 1 12 to 3 h after carrageenan and the period of neutrophil influx is followed by a period of macrophage influx. Macrophages initiate the resolution phase of the innate immune response, via the phagocytosis of apoptotic neutrophils (Cunha et al., 1991, 1992, 2005; Di Rosa et al., 1971; Ferreira et al., 1988, 1993; Handy and Moore, 1998; Leung et al., 2001; Sampson, 2000; Tsuji et al., 2003; Vinegar et al., 1969; Walker et al., 2005; Winter et al., 1962). By definition, models of autoimmunity or chronic inflammation do not resolve, but instead persist. Furthermore, the autoimmune or inflammatory disorders generally develop over an extensive time course after an initial exposure to antigen or another type of insult, they entail a dysregulation in the acquired (as opposed to the innate) immune response, and they involve cells of both the innate and acquired immune systems, particularly T and B lymphocytes (Blach-Olszewska, 2005; Goodnow et al., 2005; Kasama et al., 2005; Tonelli et al., 2001). While corticosterone produces well established immunosuppressive effects on T and B lymphocytes, corticosterone’s effects on innate immune cells, like neutrophils, are not necessarily immunosuppressive, but can be immunoenhancing. In fact, corticosteroids can cause a redistribution of neutrophils from the blood to sites of inflammation, enhance neutrophil function, and extend the extremely short lifespan of neutrophils (which is approximately 24 h after emigration from the blood), both in vivo and in vitro (Cox, 1995; Dhabhar and McEwen, 1997, 1999; Dhabhar et al., 1995, 1996).

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Therefore, FIS rats, with a hyperactive HPA axis, might be expected to simultaneously show an enhanced acute inflammatory response and a decreased susceptibility to autoimmune and chronic inflammatory disorders, whereas LEW rats, with a hypoactive HPA axis, might be expected to show the opposite relationships. The present results fully support this hypothesis. Further support comes from a study by Allen et al. (1983), who showed that the acute immune response to streptococcal cell wall injection was slightly greater in FIS rats, compared to LEW rats, despite the fact that FIS rats did not develop polyarthritis, while LEW rats did. The hypothesis that corticosteroids, in particular, and stressors, in general, can produce both proinflammatory and anti-inflammatory effects has been championed by several investigators over the past few years (Dhabhar, 2002; Straub et al., 2005) and it is slowly gaining experimental support. Nevertheless, not all studies provide supporting evidence for this hypothesis and alternative explanations of our current findings are possible. In particular, a study by Karalis et al. (1995) using the air pouch model of carrageenan showed the opposite relationship, with LEW rats showing a greater acute inflammatory response than FIS rats. However, the air pouch carrageenan model differs significantly from the hindpaw carrageenan model used in our study. While both models involve an accumulation of neutrophils at the site of inflammation, the subcutaneous air pouch environment is quite distinct from that of the hindpaw, including the cell type(s) that responds initially to the carrageenan injection and the vascularization (Cronstein et al., 1993). Corticosterone might exert different effects in the air pouch versus the hindpaw carrageenan models. Alternatively, the different results reported in the present study and that of Karalis et al. (1995) might reflect the involvement of different mediators in the two models. Differences in adrenergic function between LEW and FIS rats have recently been reported (Herrado´n et al., 2006, 2003) and the possibility exists that those differences, in addition to or in lieu of differences in HPA axis function, account for the observed differences in the acute inflammatory response to carrageenan in the air pouch versus the hindpaw models. The present study also demonstrated differences in circulating levels of proinflammatory cytokines and mediators in FIS and LEW rats, but the relationship to carrageenan-induced hindpaw inflammation was less clear. We expected to see a positive relationship between proinflammatory mediator levels and carrageenan-induced inflammation. Instead, FIS rats had higher levels of IL-6, lower levels of TNFa, and similar levels of IL-1b and nitrate/nitrite, when compared to LEW rats. Each of the tested proinflammatory mediators is believed to play a role in the acute inflammatory response to carrageenan, although the time course for each mediator varies (Cunha et al., 1991, 1992, 2005; Ferreira et al., 1988, 1993; Handy and Moore, 1998; Leung et al., 2001; Ribeiro et al., 2000; Tsuji et al., 2003; Wei et al., 1995). Therefore, the possibility

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exists that the proinflammatory cytokines/mediators were not measured at times when they reached peak levels, in which case FIS rats might have shown higher peak levels. Another possibility is that systemic levels of proinflammatory cytokines/mediators do not accurately reflect local levels, and local levels of cytokines/mediators might correlate directly with hindpaw swelling and neutrophil influx in FIS and LEW rats. Indeed, other investigators have reported only small increases in circulating levels of proinflammatory cytokines/mediators after intraplantar carrageenan (Vajja et al., 2004) and the proinflammatory mediator levels we report here are only slightly above background levels or the levels seen in untreated rats (Fecho, unpublished observations). Morphine’s effects on carrageenan-induced inflammatory immune processes and pain also were assessed in FIS and LEW rats. Morphine’s modulatory effects on both pain and immune status are well established (see Vallejo et al., 2004 for review). Morphine also is known to activate the HPA axis (Pechnick, 1993), and the HPA axis is believed to play a partial role in morphine’s immunomodulatory, analgesic, and rewarding effects (see Kiefer and Wiedemann, 2004; Kosten and Ambrosio, 2002; Mellon and Bayer, 1998 for reviews). For example, our earlier findings support a role for the HPA axis in morphine’s suppressive effects on the proliferation of blood, but not splenic, lymphocytes (Fecho et al., 1996). Others have demonstrated a direct relationship between HPA axis activity and tolerance to morphine’s analgesic effects (Vaccarino and Couret, 1995) and acquisition of morphine self-administration (Martin et al., 1999). The present results do not provide evidence for HPA axis involvement in morphine’s effects on carrageenan-induced inflammation and pain, in that morphine’s effects were generally similar in FIS and LEW rats. Morphine decreased carrageenan-induced hindpaw swelling and neutrophil accumulation to an equal extent in FIS and LEW rats, and morphine did not alter circulating levels of IL-6, TNFa, IL-1b or nitrate/nitrite in either strain. Interestingly, the morphine-induced decrease in hindpaw swelling and neutrophil accumulation did not correlate with morphine’s analgesic effects on hindpaw pain sensitivity after carrageenan. In particular, morphine’s anti-inflammatory effects on the carrageenan-injected hindpaw were similar at the 2.5 and 5.0 mg/kg doses of morphine, but morphine’s analgesic effects on the inflamed hindpaw showed a dose-dependent increase in both strains. Furthermore, in FIS rats, morphine produced biphasic effects on pain in the noninflamed hindpaw, with low doses of morphine producing an increase in mechanical sensitivity and higher doses producing analgesia; this effect was not seen in LEW rats. These results demonstrate a dissociation between morphine’s anti-inflammatory effects and morphine’s analgesic effects, implying distinct mechanisms. The results also provide evidence for a hyperalgesic effect of low doses of morphine in FIS rats. Previous studies by our group and others have likewise demonstrated

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excitatory or hyperalgesic effects of low doses of morphine in vitro and in vivo (Cano et al., 1999; Crain and Shen, 2001; Herrado´n et al., 2003; Holtman and Wala, 2005; Suarez-Roca and Maixner, 1992, 1993, 1995). Allodynic and hyperalgesic effects of morphine are increasingly recognized as potential explanations for the variability of morphine and other opioids to reduce pain and as potential factors influencing tolerance (DeLeo et al., 2004; Mao, 2002; Ossipov et al., 2003; Simonnet and Rivat, 2003). The mechanism(s) by which this occurs is not known, but the present findings suggest that FIS rats are good subjects for experiments specifically designed to study opioid-induced allodynia/hyperalgesia. Morphine’s analgesic effects in the inflamed hindpaw differed from those in the noninflamed hindpaw. In the Hargreaves radiant heat test, the inflamed hindpaw was significantly more sensitive to morphine than the noninflamed hindpaw, and this effect was similar in both strains. Other investigators have demonstrated similar findings (e.g., Hylden et al., 1991; Kayser and Guilbaud, 1983; Stanfa and Dickenson, 1993; Stein et al., 1988). These findings are important, for they suggest that accurate, preclinical assessments of the predictive, clinical analgesic effects of opioids are best examined in animals models that produce a pathological pain response. Although several groups have demonstrated a negative relationship between sensitivity to nociceptive stimuli and the analgesic response to morphine (Elmer et al., 1998; Mogil et al., 1996; Wilson et al., 2003), the present study found that FIS rats behaved similarly to LEW rats in terms of sensitivity of the inflamed hindpaw to morphine analgesia, despite the fact that carrageenan-induced allodynia and hyperalgesia were more pronounced in FIS rats. Nonetheless, the noninflamed hindpaw of FIS (but not LEW) rats showed an anti-analgesic or hyperalgesic response to morphine, as discussed above. LEW rats also showed a greater analgesic response to morphine in the tail-flick test, primarily because FIS rats did not show an effect of the low dose of morphine in that test. Thus, our results neither entirely support nor negate the proposed inverse relationship between nociceptive sensitivity and morphine analgesia. However, our present findings do suggest that the relationship depends on the particular pain model and assay, as others have emphasized (Mogil, 2004). In summary, the present findings on FIS and LEW rats suggest that the HPA axis plays an important role in acute inflammation, although the observed differences between FIS and LEW rats might reflect the contribution of other systems (e.g., the sympathetic nervous system) instead of, or in addition to, the HPA axis. Nonetheless, the current results suggest that activation of the HPA axis promotes the development of the immune response during acute inflammation. Thus, HPA axis hyper-reactive FIS rats show an enhancement in the acute inflammatory response and HPA axis hypo-reactive LEW rats show the opposite relationship. The proposed facilitatory role of the HPA axis in the development of acute inflammation has func-

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