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Carrageenan-induced thermal hyperalgesia in the mouse: role of nerve growth factor and the mitogen-activated protein kinase pathway
Brain Research 876 (2000) 48–54 www.elsevier.com / locate / bres
Research report
Carrageenan-induced thermal hyperalgesia in the mouse: role of nerve growth factor and the mitogen-activated protein kinase pathway Melanie J. Sammons, Pravin Raval, Philip T. Davey, Derek Rogers, Andrew A Parsons, Sharon Bingham* Neuroscience Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow CM19 5 AW, UK Accepted 13 June 2000
Abstract NGF is an important link between inflammation and hyperalgesia and interacts with many different mediators of inflammation, including the MAPK signaling pathway. In these studies, carrageenan-induced thermal hyperalgesia was evaluated in the mouse and the role of NGF and the MAPK pathway investigated. Carrageenan induced a time-dependent inflammation and thermal hyperalgesia, which was maximal 4 h post administration. Both indomethacin (0.3, 1.0 and 10 mg / kg s.c., 30 min pre-carrageenan) and morphine (0.4, 1.2, 4.0 mg / kg; s.c., 30 min pre-hyperalgesia measurement) significantly inhibited carrageenan-induced thermal hyperalgesia and indomethicin inhibited paw inflammation, demonstrating the model as suitable for the assessment of anti-hyperalgesic and antiinflammatory agents. Anti-NGF (0.67 mg / kg sc, 60 min pre-carrageenan) produced a significant inhibition of thermal hyperalgesia, but not inflammation. NGF itself produced a time-dependent hyperalgesia, but not inflammation, following intraplantar injection. The specific MAPK pathway inhibitor, PD98059 (0.1, 0.3 and 1 mg / kg sc, 30 min pre-carrageenan) significantly inhibited carrageenan-induced hyperalgesia, but not inflammation. These data demonstrate a role for both NGF and the MAPK signaling pathway in the production of thermal hyperalgesia, but not inflammation, in the mouse. 2000 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation: anatomy and physiology Keywords: Hyperalgesia; MAPK; NGF; Carrageenan; Mouse; Inflammation
1. Introduction Carrageenan is an inflammatory agent, used as a tool to investigate inflammatory hyperalgesia in rats and mice [19]. In rats, carrageenan, when injected subcutaneously into the plantar surface of a paw, produces a characteristic inflammation and associated hyperalgesia, which can be used to quantify the anti-inflammatory or anti-hyperalgesic actions of drugs. Studies in rats and mice show a similar time-course for inflammation in both species [12,31], but the thermal hyperalgesic response to carrageenan in mice is less well characterised Tissue damage following carrageenan injection is a complex phenomonen, incorporating many different medi*Corresponding author. Tel.: 144-1279-622-724; fax: 144-1279-622211. E-mail address: sharon [email protected] (S. Bingham). ]
ators and pathways to produce inflammatory hyperalgesia. Substances known to be involved in this process are products of arachadonic acid metabolism [5,28], mast cell contents (histamine, 5-HT, bradykinin) [6,9,17], neurokinins (Substance P) [7], cytokines (IL-1b) [15], nitric oxide (NO) [31], nerve growth factor (NGF) [26] and many others. However, the relative roles and significance of each of these substances in carrageenan-induced inflammation, is at present undetermined. NGF is described as an important link between inflammation and hyperalgesia, following injection of inflammatory agents [20]. NGF levels are elevated following induction of inflammation [32,35], and sequestration of NGF using anti-NGF [37] or an IgG-TrkA fusion molecule [27], significantly reduces the hyperalgesic response following an inflammatory insult. Similarly, intraplantar injection of NGF into the hindpaw of rats produces a time-dependant effect on thermal hyperalgesia [38,22] and
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inflammation [1,4], that is apparent within 30 min postinjection, and appears to have a later component at 7 h post injection [22]. Early effects of NGF, appear to be mediated peripherally, through inflammatory cells such as mast cells [22,38] and neutrophils [4], and / or peripheral nerve sensitisation [26] whereas the later phase appears to be mediated centrally and can be blocked by the NMDA channel blocker, MK801 [22]. NGF also activates the mitogen-activated protein kinase (MAPK) cascade, through interaction of the Grb / SOS complex with membrane bound ras and activation, of various members of the extracellular signal-regulated kinase (ERK) family [10]. MAPK activation is known to induce COX2 expression and subsequent release of the inflammatory mediator, PGE2 [23]. The aims of these studies were to: (i) characterise carrageenan-induced thermal hyperalgesia in the mouse and confirm the involvement of NGF in the induction of hyperalgesia; (ii) to determine whether these actions are mediated through the activation of the MAPK signaling cascade using the specific ERK1 / 2 inhibitor, PD 98059 [10].
2. Methods
2.1. Animals Male ICR mice (25–35 g; Harlan Olac, UK) were used in all experiments. All animals were housed in the animal unit at SmithKline Beecham Pharmaceuticals, Harlow at 218C on a 12 h light / dark cycle (06.00–18.00 h) and fed a pellet diet and tap water ad libitum. All experiments were performed in accordance with the Animal Scientific Procedures act of 1986 and subject to local ethical committee approval.
2.2. Nociceptive assay The Hargreaves plantar method [11], Ugo Basile (Italy), supplied by Linton (Norfolk, UK) for assessing thermal hyperalgesia was modified for use with mice. Red perspex boxes (Instrument Design Technologies, SmithKline Beecham, Harlow), with dimensions of 75 mm by 90 mm were used to house the animals during assessment. All measurements were made in red light. Mice were habituated to the testing boxes and paw withdrawal latencies (PWLs) measured in response to a noxious thermal stimulus measured. In initial experiments, different infra-red (IR) intensities (10, 20 and 30) were used to determine the intensity that elicited the most consistent and appropriate PWLs (|10 s). This intensity was used in subsequent experiments and an absolute cut-off time of 22.5 s was used to prevent tissue damage. PWLs were also determined at various time points following carrageenan administration. Prior to induction of hyperalgesia with
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either carrageenan or NGF, mice were habituated in the testing boxes three times, at least 24 h apart, for 15 min. Two plantar measurements were then made 24 h apart, with the second immediately prior to induction of hyperalgesia, according to [33]. Following the second plantar measurement, mice with PWLs that were within 3 s of the mean of the group were selected for inclusion in the experiment. The first PWL measurements were discarded and the second measurements used as baseline.
2.3. Paw depth measurements The degree of inflammation was assessed by changes in paw depth, using JOCAL callipers. Paw depth was measured immediately prior to carrageenan injection and following application of the noxious thermal stimulus at 4 h.
2.4. Characterisation of carrageenan-induced hyperalgesia in the mouse Hyperalgesia was induced by intraplantar injection of carrageenan (25 ml, 2%) into a randomly selected hind paw. Pilot studies were conducted to determine optimum parameters under which the effects of drugs of interest could be assessed in the model. Concentration (1 and 2%) and volume (15 and 25 ml) effects of carrageenan, on hyperalgesia and inflammation were determined and the effects of intraplantar saline injection on contralateral PWLs and paw depth, were assessed and compared to non-injected paws (data not shown). The time-course of the development of inflammation and hyperalgesia, following intraplantar carrageenan, was determined. Paw depth and PWLs were assessed at hourly intervals for up to 6 h followed by a single measurement at 24 h.
2.5. Pharmacological manipulation of carrageenaninduced thermal hyperalgesia Hyperalgesia was induced by 25 ml, 2% carrageenan and PWLs were obtained at 4 and 24 h post carrageenan, with paw depth assessed at 4.5 and 24.5 h. Indomethacin (0.3, 1, 10 mg / kg; s.c.; 30 min pre-carrageenan), morphine (0.4, 1.2, 4.0 mg / kg; s.c.; 30 min pre-test), the MAPK pathway inhibitor, PD 98059 (0.1, 0.3, 1.0 mg / kg; s.c.; 30 min pre-carrageenan) and anti-NGF (0.67 mg / kg; s.c.; 60 min pre-carrageenan) were administered in a volume of 5 ml / kg.
2.6. Effects of nerve growth factor on nociceptive threshold and paw depth Hyperalgesia was induced by intraplantar injection of NGF (1.0 mg / paw; 25 ml vol.) into a randomly selected hind paw. Thermal hyperalgesia was assessed at 15, 45, 75, 105 min and 24 h, according to the time-course
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observed in the rat [1] and mouse [8]. Paw depth was measured prior to and at 30 and 90 min following NGF administration.
2.7. Statistical analysis All results are expressed as mean6standard error of the mean (S.E.M.) for PWLs (s) and paw depth. Data was assessed for normality, as raw or transformed (log 10 ) data, as indicated. Normally distributed data was analysed for statistical significance using analysis of variance (ANOVA) followed by post hoc Duncans multiple range test (P, 0.05). Non normal data was assessed by Mann–Whitney U-test, and when significant, by Kruskall–Wallis.
2.8. Chemicals Lambda carrageenan, Nerve Growth Factor (2.5 s, from mouse submaxillary gland), indomethacin and morphine sulphate were all obtained from Sigma Chemical (Poole, Dorset, UK). PD98059 was obtained from Calbiochem (Nottingham, UK), and anti-NGF (2.5 s) from Promega (Southampton, UK). Lambda carrageenan was dissolved in 154 mM NaCl, to obtain a 2% stock solution and stored at 48C. NGF-2.5 s and anti-NGF-2.5 s were dissolved in 0.9% saline. Indomethacin and morphine were dosed as the free base. Indomethacin was dissolved in 10% sodium bicarbonate (pH 10). Morphine sulphate was dissolved in distilled water. PD 98059 was dissolved in PEG 400 and stored in amber bottles to protect from light.
3. Results
3.1. Time-course of carrageenan-induced hyperalgesia and inflammation Intraplantar carrageenan (25 ml, 2%) produced a timedependant thermal hyperalgesia and inflammation that persisted up to 24 h post injection, (Fig. 1). Saline injection had no effect on paw withdrawal latencies. A significant reduction in PWLs was observed at 3 h post carrageenan, reaching a maximum at 4 h, reducing PWLs from 9.460.7 s (baseline) to 2.760.4 s (4 h), (Fig. 1a). Similarly, a significant increase in paw depth was observed 1 h post-carrageenan, which also reached a maximum at 4 h post carrageenan, increasing paw depth from 1.7560.03 mm (baseline) to 3.7960.21 mm (4 h) (Fig. 1b). Contralateral PWLs and paw depths were unaffected at any time point.
3.2. Validation of the mouse carrageenan model with the standard agents, indomethacin and morphine The COX inhibitor, indomethacin (0.3, 1 and 10 mg / kg; 30 min pre-carrageenan; n510), administered 30 min prior
Fig. 1. Time-course of carrageenan-induced hyperalgesia (a) and inflammation (b) in contralateral and ipsilateral hind paws in the mouse. Data represents mean6S.E.M. (n59).
to carrageenan injection, dose-dependently inhibited carrageenan-induced hyperalgesia, measured at 4 h post-carrageenan. PWLs were significantly increased to 6.161.0 s at 1 mg / kg and to 5.560.6 s at 10 mg / kg, compared to vehicle (2.260.4 s) (Table 1). Similarly, indomethacin dose-dependently inhibited inflammation compared to vehicle, with 10 mg / kg decreasing paw depth from 3.6860.17 mm to 2.9760.15 mm at 4 h post-carrageenan (Table 1). Indomethacin had no effect on contralateral PWLs or paw depth, measured at 4 h. The opiate agent, morphine (0.4, 1.2, 4.0 mg / kg s.c.; n59) administered 30 min prior to the measurement of the noxious thermal stimulus response dose-dependently inhibited carrageenan-induced hyperalgesia, without affecting inflammation (Table 1) in the mouse hind paw. Significant inhibition (P,0.05) was obtained with both the
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Table 1 The effect of the standard drugs, indomethacin and morphine on carrageenan-induced hyperalgesia and inflammation, at 4 h post carrageenan in the mouse hind paw a Dose (mg / kg)
PWL (s)
Paw depth (mm)
Baseline
4h
Baseline
4h
Indomethacin Vehicle 0.3 1.0 10.0
8.461.0 8.561.0 10.061.0 10.660.7
2.260.4 4.360.6 6.161.0* 5.560.6*
1.6060.04 1.6160.05 1.5960.03 1.8360.05
3.6860.17 3.5260.22 2.8260.11* 2.9560.12*
Morphine Vehicle 0.4 1.2 4.0
11.060.6 9.460.7 9.260.6 9.160.7
2.860.3 3.460.4 5.460.3* 5.960.3*
1.6960.03 1.7260.03 1.7560.03 1.6660.03
3.2360.10 3.4060.10 3.3160.13 3.0260.17
a
Data represents mean6S.E.M., P,0.05 compared to baseline, ANOVA followed by post hoc Dunnett’s test.
1.2 mg / kg (PWL55.460.3 s) and the 4.0 mg / kg dose (5.960.3 s) compared to vehicle (2.860.3).
3.3. Anti-NGF inhibits carrageenan-induced thermal hyperalgesia but not inflammation Anti-NGF, at the given dose (0.67 mg / kg; s.c.; 60 min pre-carrageenan; n510), significantly inhibited hyperalgesia (Fig. 2a), without affecting inflammation (Fig. 2b). At 4 h post-carrageenan, PWLs were significantly increased from a vehicle value of 3.560.3 s, to 6.060.4 s, similar to the effect seen with morphine (4 mg / kg sc). Anti-NGF had no significant effect on contralateral PWLs at 4 h.
3.4. NGF-induced hyperalgesia in the mouse hind paw Intraplantar administration of NGF (1 mg / paw) produced a time-dependent reduction of PWLs in response to the noxious thermal stimulus (Fig. 3a), without an inflammatory effect (Fig. 3b). Following administration of 1 mg / paw NGF, significant and maximum reductions in PWLs occurred 45 min post injection (baseline 10.060.3 s; 45 min 4.760.4 s; n510) and returned to baseline by 105 min. Contralateral PWLs were unaffected by intraplantar NGF administration.
Fig. 2. Effect of anti-NGF (0.67 mg / kg; i.p.; 60 min pre-carrageenan) on carrageenan-induced hyperalgesia (a) and inflammation (b) in the mouse hind paw at 4 h post-carrageenan. Data represents mean6S.E.M. (n510).
5.060.8, 5.760.8 s and 5.160.5 s at 0.1, 0.3 and 1 mg / kg respectively (Fig. 4a). However, PD98059 administration had no effect on carrageenan-induced inflammation, with no change observed in paw depth measurements when compared to control values (Fig. 4b). PD98059 per se had no effect on contralateral PWLs or paw depth.
3.5. The specific MAPK pathway inhibitor PD98059 inhibits carrageenan-induced thermal hyperalgesia but not inflammation
3.6. The specific MAPK pathway inhibitor PD98059 inhibits NGF-induced thermal hyperalgesia
The specific MEK inhibitor, PD98059 (0.1, 0.3 and 1.0 mg / kg s.c., 30 min pre-carrageenan) produced a significant inhibition of hyperalgesia 4 h post carrageenan. PD98059 increased PWLs from control values of 2.660.3 s to
PD98059 (0.1, 0.3 and 1.0 mg / kg s.c., 30 min pre-NGF) also significantly inhibited thermal hyperalgesia at 1 mg / kg sc when measured 45 min post-NGF (Fig. 5). PWL was increased from 3.960.4 s to 4.760.4 s.
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Fig. 3. Time-course of NGF-induced (1.0 mg / paw; 25 ml vol.) hyperalgesia (a) and inflammation (b) in contralateral and ipsilateral hind paws in the mouse. Data represents mean6S.E.M. (n58 per group).
4. Discussion These data show that in the mouse, 25 ml of a 2% solution of carrageenan elicits a time-dependent inflammation and thermal hyperalgesia that is maximal at 4 h. This is consistent with previous studies on inflammation in mice [12], and also similar to the time-course of carrageenaninduced hyperalgesia observed in the rat [11,18]. The inflammatory and / or hyperalgesic effects of carrageenan in mice were blocked by the prostaglandin inhibitor, indomethacin and the opiate agent morphine, similar to the situation in rat and confirms the mouse is suitable for the assessment of anti-inflammatory and anti-hyperalgesic drugs. To evaluate the involvement of NGF in carrageenaninduced hyperalgesia, in the mouse, anti-NGF antibodies
Fig. 4. The effect of PD98059 (0.1, 0.3, 1.0 mg / kg; s.c.; 30 min pre-carrageenan) on carrageenan-induced hyperalgesia (a) and inflammation (b) in the mouse hind paw measured 4 h post-carrageenan. Data represents mean6S.E.M. (n58).
were administered prior to carrageenan injection. These results produced an inhibition of thermal hyperalgesia comparable to that of morphine but had no effect on inflammation produced by carrageenan. This is similar to inflammation induced by complete Freund’s adjuvant in the rat, which was also not modified by Anti-NGF whereas hyperalgesia was prevented [37]. As in the rat, NGF clearly has a pivotal role in producing thermal hyperalgesia in mice. In the rat, the NGF-mediated early events involve activation of mast cells and release of inflammatory and sensitising agents [25], sensitisation of peripheral nerve terminals [30]. There may also be a sympathetic component to its action [2,38]. Later effects of NGF are thought to be mediated centrally as they are blocked by NMDA receptor antagonists and are present 7 h post NGF administration [22]. In addition, NGF transport and transcriptional events, which may include NGF-induced upregulation of SP in sensory
M. J. Sammons et al. / Brain Research 876 (2000) 48 – 54
Fig. 5. The effect of PD98059 (0.1, 0.3, 1.0 mg / kg; s.c.; 30 min pre-NGF) on NGF-induced hyperalgesia (1.0 mg / paw; 25 ml vol.) in the mouse hind paw measured 45 min post-NGF. Data represents mean6S.E.M. (n58 per group).
neurones [36] and SP and CGRP release in the spinal cord [24], are also postulated to occur over a time period longer than 4 h [18]. This suggests a peripheral action of NGF in the present experiments, although relative differences in the timing of these events between the rat and the mouse cannot be ruled out. Intraplantar NGF itself did not produce inflammation in the mouse hind paw. This is in contrast to previous studies in rats [1,3], where inflammation is observed following an intraplantar injection of NGF. This suggests that in mice the inflammatory component of the acute response to NGF is small or absent. NGF is known to degranulate rat mast cells [14,34] and in rats this leads to the production of inflammation and hyperalgesia via release of inflammatory mediators such as bradykinin, 5-HT, histamine, prostaglandin E(2) and leukotrienes [3,14,34]. The reduced number of mast cells in mice, in comparison to rats, and the relative insensitivity of mice to histamine [29], may indicate a minor contribution of mast cell degranulation products, during the early phase of NGF-induced hyperalgesia in mice. NGF activates the MAPK signaling pathway, which results in gene expression [13]. For example, in dorsal root ganglia, NGF induces changes in expression of a number of neuropeptides and factors implicated in the generation of persistent pain [36]. MAPK pathway activation induces COX2 expression and subsequent release of the inflammatory mediator, PGE2 [23]. The MAPK pathway inhibitor, PD98059 [10] decreased the hyperalgesic response to carrageenan and NGF, to a similar degree to that obtained with anti-NGF and morphine. This strongly implicates the MAPK pathway in the production of thermal hyperalgesia in the mouse with little involvement in the inflammatory response to carrageenan,
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since PD98059 had no effect on paw depth. A recent publication [16] in rat, shows transient ERK1 / 2 activation in the spinal cord following C-fibre stimulation and application of the noxious chemicals, capsaicin and formalin, an effect that was also blocked by PD98059. Taken together with the present study, these data suggest that phosphorylation of ERK1 / 2 may contribute to inflammation-induced hyperalgesia. However, PD98059 only inhibited NGF-induced hyperalgesia at 1 mg / kg whereas it was effective against carrageenan-induced thermal hyperalgesia over a wider dose range (0.3–1 mg / kg). This might suggest that during carrageenan-induced thermal hyperalgesia, the MAPK pathway is also activated through interactions other than NGF. Alternatively, PD98059 may have been less capable of inhibiting the effects of exogenously applied NGF than that liberated endogenously during inflammation. These studies show, that in the mouse, NGF plays a pivotal role in the production of hyperalgesia but not inflammation by carrageenan. Inhibition of the MAPK pathway produces a marked reduction of thermal hyperalgesia and indicates a role for this signaling pathway in the development of hyperalgesia but not inflammation.
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Research report
Carrageenan-induced thermal hyperalgesia in the mouse: role of nerve growth factor and the mitogen-activated protein kinase pathway Melanie J. Sammons, Pravin Raval, Philip T. Davey, Derek Rogers, Andrew A Parsons, Sharon Bingham* Neuroscience Research, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow CM19 5 AW, UK Accepted 13 June 2000
Abstract NGF is an important link between inflammation and hyperalgesia and interacts with many different mediators of inflammation, including the MAPK signaling pathway. In these studies, carrageenan-induced thermal hyperalgesia was evaluated in the mouse and the role of NGF and the MAPK pathway investigated. Carrageenan induced a time-dependent inflammation and thermal hyperalgesia, which was maximal 4 h post administration. Both indomethacin (0.3, 1.0 and 10 mg / kg s.c., 30 min pre-carrageenan) and morphine (0.4, 1.2, 4.0 mg / kg; s.c., 30 min pre-hyperalgesia measurement) significantly inhibited carrageenan-induced thermal hyperalgesia and indomethicin inhibited paw inflammation, demonstrating the model as suitable for the assessment of anti-hyperalgesic and antiinflammatory agents. Anti-NGF (0.67 mg / kg sc, 60 min pre-carrageenan) produced a significant inhibition of thermal hyperalgesia, but not inflammation. NGF itself produced a time-dependent hyperalgesia, but not inflammation, following intraplantar injection. The specific MAPK pathway inhibitor, PD98059 (0.1, 0.3 and 1 mg / kg sc, 30 min pre-carrageenan) significantly inhibited carrageenan-induced hyperalgesia, but not inflammation. These data demonstrate a role for both NGF and the MAPK signaling pathway in the production of thermal hyperalgesia, but not inflammation, in the mouse. 2000 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation: anatomy and physiology Keywords: Hyperalgesia; MAPK; NGF; Carrageenan; Mouse; Inflammation
1. Introduction Carrageenan is an inflammatory agent, used as a tool to investigate inflammatory hyperalgesia in rats and mice [19]. In rats, carrageenan, when injected subcutaneously into the plantar surface of a paw, produces a characteristic inflammation and associated hyperalgesia, which can be used to quantify the anti-inflammatory or anti-hyperalgesic actions of drugs. Studies in rats and mice show a similar time-course for inflammation in both species [12,31], but the thermal hyperalgesic response to carrageenan in mice is less well characterised Tissue damage following carrageenan injection is a complex phenomonen, incorporating many different medi*Corresponding author. Tel.: 144-1279-622-724; fax: 144-1279-622211. E-mail address: sharon [email protected] (S. Bingham). ]
ators and pathways to produce inflammatory hyperalgesia. Substances known to be involved in this process are products of arachadonic acid metabolism [5,28], mast cell contents (histamine, 5-HT, bradykinin) [6,9,17], neurokinins (Substance P) [7], cytokines (IL-1b) [15], nitric oxide (NO) [31], nerve growth factor (NGF) [26] and many others. However, the relative roles and significance of each of these substances in carrageenan-induced inflammation, is at present undetermined. NGF is described as an important link between inflammation and hyperalgesia, following injection of inflammatory agents [20]. NGF levels are elevated following induction of inflammation [32,35], and sequestration of NGF using anti-NGF [37] or an IgG-TrkA fusion molecule [27], significantly reduces the hyperalgesic response following an inflammatory insult. Similarly, intraplantar injection of NGF into the hindpaw of rats produces a time-dependant effect on thermal hyperalgesia [38,22] and
0006-8993 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02596-8
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inflammation [1,4], that is apparent within 30 min postinjection, and appears to have a later component at 7 h post injection [22]. Early effects of NGF, appear to be mediated peripherally, through inflammatory cells such as mast cells [22,38] and neutrophils [4], and / or peripheral nerve sensitisation [26] whereas the later phase appears to be mediated centrally and can be blocked by the NMDA channel blocker, MK801 [22]. NGF also activates the mitogen-activated protein kinase (MAPK) cascade, through interaction of the Grb / SOS complex with membrane bound ras and activation, of various members of the extracellular signal-regulated kinase (ERK) family [10]. MAPK activation is known to induce COX2 expression and subsequent release of the inflammatory mediator, PGE2 [23]. The aims of these studies were to: (i) characterise carrageenan-induced thermal hyperalgesia in the mouse and confirm the involvement of NGF in the induction of hyperalgesia; (ii) to determine whether these actions are mediated through the activation of the MAPK signaling cascade using the specific ERK1 / 2 inhibitor, PD 98059 [10].
2. Methods
2.1. Animals Male ICR mice (25–35 g; Harlan Olac, UK) were used in all experiments. All animals were housed in the animal unit at SmithKline Beecham Pharmaceuticals, Harlow at 218C on a 12 h light / dark cycle (06.00–18.00 h) and fed a pellet diet and tap water ad libitum. All experiments were performed in accordance with the Animal Scientific Procedures act of 1986 and subject to local ethical committee approval.
2.2. Nociceptive assay The Hargreaves plantar method [11], Ugo Basile (Italy), supplied by Linton (Norfolk, UK) for assessing thermal hyperalgesia was modified for use with mice. Red perspex boxes (Instrument Design Technologies, SmithKline Beecham, Harlow), with dimensions of 75 mm by 90 mm were used to house the animals during assessment. All measurements were made in red light. Mice were habituated to the testing boxes and paw withdrawal latencies (PWLs) measured in response to a noxious thermal stimulus measured. In initial experiments, different infra-red (IR) intensities (10, 20 and 30) were used to determine the intensity that elicited the most consistent and appropriate PWLs (|10 s). This intensity was used in subsequent experiments and an absolute cut-off time of 22.5 s was used to prevent tissue damage. PWLs were also determined at various time points following carrageenan administration. Prior to induction of hyperalgesia with
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either carrageenan or NGF, mice were habituated in the testing boxes three times, at least 24 h apart, for 15 min. Two plantar measurements were then made 24 h apart, with the second immediately prior to induction of hyperalgesia, according to [33]. Following the second plantar measurement, mice with PWLs that were within 3 s of the mean of the group were selected for inclusion in the experiment. The first PWL measurements were discarded and the second measurements used as baseline.
2.3. Paw depth measurements The degree of inflammation was assessed by changes in paw depth, using JOCAL callipers. Paw depth was measured immediately prior to carrageenan injection and following application of the noxious thermal stimulus at 4 h.
2.4. Characterisation of carrageenan-induced hyperalgesia in the mouse Hyperalgesia was induced by intraplantar injection of carrageenan (25 ml, 2%) into a randomly selected hind paw. Pilot studies were conducted to determine optimum parameters under which the effects of drugs of interest could be assessed in the model. Concentration (1 and 2%) and volume (15 and 25 ml) effects of carrageenan, on hyperalgesia and inflammation were determined and the effects of intraplantar saline injection on contralateral PWLs and paw depth, were assessed and compared to non-injected paws (data not shown). The time-course of the development of inflammation and hyperalgesia, following intraplantar carrageenan, was determined. Paw depth and PWLs were assessed at hourly intervals for up to 6 h followed by a single measurement at 24 h.
2.5. Pharmacological manipulation of carrageenaninduced thermal hyperalgesia Hyperalgesia was induced by 25 ml, 2% carrageenan and PWLs were obtained at 4 and 24 h post carrageenan, with paw depth assessed at 4.5 and 24.5 h. Indomethacin (0.3, 1, 10 mg / kg; s.c.; 30 min pre-carrageenan), morphine (0.4, 1.2, 4.0 mg / kg; s.c.; 30 min pre-test), the MAPK pathway inhibitor, PD 98059 (0.1, 0.3, 1.0 mg / kg; s.c.; 30 min pre-carrageenan) and anti-NGF (0.67 mg / kg; s.c.; 60 min pre-carrageenan) were administered in a volume of 5 ml / kg.
2.6. Effects of nerve growth factor on nociceptive threshold and paw depth Hyperalgesia was induced by intraplantar injection of NGF (1.0 mg / paw; 25 ml vol.) into a randomly selected hind paw. Thermal hyperalgesia was assessed at 15, 45, 75, 105 min and 24 h, according to the time-course
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observed in the rat [1] and mouse [8]. Paw depth was measured prior to and at 30 and 90 min following NGF administration.
2.7. Statistical analysis All results are expressed as mean6standard error of the mean (S.E.M.) for PWLs (s) and paw depth. Data was assessed for normality, as raw or transformed (log 10 ) data, as indicated. Normally distributed data was analysed for statistical significance using analysis of variance (ANOVA) followed by post hoc Duncans multiple range test (P, 0.05). Non normal data was assessed by Mann–Whitney U-test, and when significant, by Kruskall–Wallis.
2.8. Chemicals Lambda carrageenan, Nerve Growth Factor (2.5 s, from mouse submaxillary gland), indomethacin and morphine sulphate were all obtained from Sigma Chemical (Poole, Dorset, UK). PD98059 was obtained from Calbiochem (Nottingham, UK), and anti-NGF (2.5 s) from Promega (Southampton, UK). Lambda carrageenan was dissolved in 154 mM NaCl, to obtain a 2% stock solution and stored at 48C. NGF-2.5 s and anti-NGF-2.5 s were dissolved in 0.9% saline. Indomethacin and morphine were dosed as the free base. Indomethacin was dissolved in 10% sodium bicarbonate (pH 10). Morphine sulphate was dissolved in distilled water. PD 98059 was dissolved in PEG 400 and stored in amber bottles to protect from light.
3. Results
3.1. Time-course of carrageenan-induced hyperalgesia and inflammation Intraplantar carrageenan (25 ml, 2%) produced a timedependant thermal hyperalgesia and inflammation that persisted up to 24 h post injection, (Fig. 1). Saline injection had no effect on paw withdrawal latencies. A significant reduction in PWLs was observed at 3 h post carrageenan, reaching a maximum at 4 h, reducing PWLs from 9.460.7 s (baseline) to 2.760.4 s (4 h), (Fig. 1a). Similarly, a significant increase in paw depth was observed 1 h post-carrageenan, which also reached a maximum at 4 h post carrageenan, increasing paw depth from 1.7560.03 mm (baseline) to 3.7960.21 mm (4 h) (Fig. 1b). Contralateral PWLs and paw depths were unaffected at any time point.
3.2. Validation of the mouse carrageenan model with the standard agents, indomethacin and morphine The COX inhibitor, indomethacin (0.3, 1 and 10 mg / kg; 30 min pre-carrageenan; n510), administered 30 min prior
Fig. 1. Time-course of carrageenan-induced hyperalgesia (a) and inflammation (b) in contralateral and ipsilateral hind paws in the mouse. Data represents mean6S.E.M. (n59).
to carrageenan injection, dose-dependently inhibited carrageenan-induced hyperalgesia, measured at 4 h post-carrageenan. PWLs were significantly increased to 6.161.0 s at 1 mg / kg and to 5.560.6 s at 10 mg / kg, compared to vehicle (2.260.4 s) (Table 1). Similarly, indomethacin dose-dependently inhibited inflammation compared to vehicle, with 10 mg / kg decreasing paw depth from 3.6860.17 mm to 2.9760.15 mm at 4 h post-carrageenan (Table 1). Indomethacin had no effect on contralateral PWLs or paw depth, measured at 4 h. The opiate agent, morphine (0.4, 1.2, 4.0 mg / kg s.c.; n59) administered 30 min prior to the measurement of the noxious thermal stimulus response dose-dependently inhibited carrageenan-induced hyperalgesia, without affecting inflammation (Table 1) in the mouse hind paw. Significant inhibition (P,0.05) was obtained with both the
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Table 1 The effect of the standard drugs, indomethacin and morphine on carrageenan-induced hyperalgesia and inflammation, at 4 h post carrageenan in the mouse hind paw a Dose (mg / kg)
PWL (s)
Paw depth (mm)
Baseline
4h
Baseline
4h
Indomethacin Vehicle 0.3 1.0 10.0
8.461.0 8.561.0 10.061.0 10.660.7
2.260.4 4.360.6 6.161.0* 5.560.6*
1.6060.04 1.6160.05 1.5960.03 1.8360.05
3.6860.17 3.5260.22 2.8260.11* 2.9560.12*
Morphine Vehicle 0.4 1.2 4.0
11.060.6 9.460.7 9.260.6 9.160.7
2.860.3 3.460.4 5.460.3* 5.960.3*
1.6960.03 1.7260.03 1.7560.03 1.6660.03
3.2360.10 3.4060.10 3.3160.13 3.0260.17
a
Data represents mean6S.E.M., P,0.05 compared to baseline, ANOVA followed by post hoc Dunnett’s test.
1.2 mg / kg (PWL55.460.3 s) and the 4.0 mg / kg dose (5.960.3 s) compared to vehicle (2.860.3).
3.3. Anti-NGF inhibits carrageenan-induced thermal hyperalgesia but not inflammation Anti-NGF, at the given dose (0.67 mg / kg; s.c.; 60 min pre-carrageenan; n510), significantly inhibited hyperalgesia (Fig. 2a), without affecting inflammation (Fig. 2b). At 4 h post-carrageenan, PWLs were significantly increased from a vehicle value of 3.560.3 s, to 6.060.4 s, similar to the effect seen with morphine (4 mg / kg sc). Anti-NGF had no significant effect on contralateral PWLs at 4 h.
3.4. NGF-induced hyperalgesia in the mouse hind paw Intraplantar administration of NGF (1 mg / paw) produced a time-dependent reduction of PWLs in response to the noxious thermal stimulus (Fig. 3a), without an inflammatory effect (Fig. 3b). Following administration of 1 mg / paw NGF, significant and maximum reductions in PWLs occurred 45 min post injection (baseline 10.060.3 s; 45 min 4.760.4 s; n510) and returned to baseline by 105 min. Contralateral PWLs were unaffected by intraplantar NGF administration.
Fig. 2. Effect of anti-NGF (0.67 mg / kg; i.p.; 60 min pre-carrageenan) on carrageenan-induced hyperalgesia (a) and inflammation (b) in the mouse hind paw at 4 h post-carrageenan. Data represents mean6S.E.M. (n510).
5.060.8, 5.760.8 s and 5.160.5 s at 0.1, 0.3 and 1 mg / kg respectively (Fig. 4a). However, PD98059 administration had no effect on carrageenan-induced inflammation, with no change observed in paw depth measurements when compared to control values (Fig. 4b). PD98059 per se had no effect on contralateral PWLs or paw depth.
3.5. The specific MAPK pathway inhibitor PD98059 inhibits carrageenan-induced thermal hyperalgesia but not inflammation
3.6. The specific MAPK pathway inhibitor PD98059 inhibits NGF-induced thermal hyperalgesia
The specific MEK inhibitor, PD98059 (0.1, 0.3 and 1.0 mg / kg s.c., 30 min pre-carrageenan) produced a significant inhibition of hyperalgesia 4 h post carrageenan. PD98059 increased PWLs from control values of 2.660.3 s to
PD98059 (0.1, 0.3 and 1.0 mg / kg s.c., 30 min pre-NGF) also significantly inhibited thermal hyperalgesia at 1 mg / kg sc when measured 45 min post-NGF (Fig. 5). PWL was increased from 3.960.4 s to 4.760.4 s.
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Fig. 3. Time-course of NGF-induced (1.0 mg / paw; 25 ml vol.) hyperalgesia (a) and inflammation (b) in contralateral and ipsilateral hind paws in the mouse. Data represents mean6S.E.M. (n58 per group).
4. Discussion These data show that in the mouse, 25 ml of a 2% solution of carrageenan elicits a time-dependent inflammation and thermal hyperalgesia that is maximal at 4 h. This is consistent with previous studies on inflammation in mice [12], and also similar to the time-course of carrageenaninduced hyperalgesia observed in the rat [11,18]. The inflammatory and / or hyperalgesic effects of carrageenan in mice were blocked by the prostaglandin inhibitor, indomethacin and the opiate agent morphine, similar to the situation in rat and confirms the mouse is suitable for the assessment of anti-inflammatory and anti-hyperalgesic drugs. To evaluate the involvement of NGF in carrageenaninduced hyperalgesia, in the mouse, anti-NGF antibodies
Fig. 4. The effect of PD98059 (0.1, 0.3, 1.0 mg / kg; s.c.; 30 min pre-carrageenan) on carrageenan-induced hyperalgesia (a) and inflammation (b) in the mouse hind paw measured 4 h post-carrageenan. Data represents mean6S.E.M. (n58).
were administered prior to carrageenan injection. These results produced an inhibition of thermal hyperalgesia comparable to that of morphine but had no effect on inflammation produced by carrageenan. This is similar to inflammation induced by complete Freund’s adjuvant in the rat, which was also not modified by Anti-NGF whereas hyperalgesia was prevented [37]. As in the rat, NGF clearly has a pivotal role in producing thermal hyperalgesia in mice. In the rat, the NGF-mediated early events involve activation of mast cells and release of inflammatory and sensitising agents [25], sensitisation of peripheral nerve terminals [30]. There may also be a sympathetic component to its action [2,38]. Later effects of NGF are thought to be mediated centrally as they are blocked by NMDA receptor antagonists and are present 7 h post NGF administration [22]. In addition, NGF transport and transcriptional events, which may include NGF-induced upregulation of SP in sensory
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Fig. 5. The effect of PD98059 (0.1, 0.3, 1.0 mg / kg; s.c.; 30 min pre-NGF) on NGF-induced hyperalgesia (1.0 mg / paw; 25 ml vol.) in the mouse hind paw measured 45 min post-NGF. Data represents mean6S.E.M. (n58 per group).
neurones [36] and SP and CGRP release in the spinal cord [24], are also postulated to occur over a time period longer than 4 h [18]. This suggests a peripheral action of NGF in the present experiments, although relative differences in the timing of these events between the rat and the mouse cannot be ruled out. Intraplantar NGF itself did not produce inflammation in the mouse hind paw. This is in contrast to previous studies in rats [1,3], where inflammation is observed following an intraplantar injection of NGF. This suggests that in mice the inflammatory component of the acute response to NGF is small or absent. NGF is known to degranulate rat mast cells [14,34] and in rats this leads to the production of inflammation and hyperalgesia via release of inflammatory mediators such as bradykinin, 5-HT, histamine, prostaglandin E(2) and leukotrienes [3,14,34]. The reduced number of mast cells in mice, in comparison to rats, and the relative insensitivity of mice to histamine [29], may indicate a minor contribution of mast cell degranulation products, during the early phase of NGF-induced hyperalgesia in mice. NGF activates the MAPK signaling pathway, which results in gene expression [13]. For example, in dorsal root ganglia, NGF induces changes in expression of a number of neuropeptides and factors implicated in the generation of persistent pain [36]. MAPK pathway activation induces COX2 expression and subsequent release of the inflammatory mediator, PGE2 [23]. The MAPK pathway inhibitor, PD98059 [10] decreased the hyperalgesic response to carrageenan and NGF, to a similar degree to that obtained with anti-NGF and morphine. This strongly implicates the MAPK pathway in the production of thermal hyperalgesia in the mouse with little involvement in the inflammatory response to carrageenan,
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since PD98059 had no effect on paw depth. A recent publication [16] in rat, shows transient ERK1 / 2 activation in the spinal cord following C-fibre stimulation and application of the noxious chemicals, capsaicin and formalin, an effect that was also blocked by PD98059. Taken together with the present study, these data suggest that phosphorylation of ERK1 / 2 may contribute to inflammation-induced hyperalgesia. However, PD98059 only inhibited NGF-induced hyperalgesia at 1 mg / kg whereas it was effective against carrageenan-induced thermal hyperalgesia over a wider dose range (0.3–1 mg / kg). This might suggest that during carrageenan-induced thermal hyperalgesia, the MAPK pathway is also activated through interactions other than NGF. Alternatively, PD98059 may have been less capable of inhibiting the effects of exogenously applied NGF than that liberated endogenously during inflammation. These studies show, that in the mouse, NGF plays a pivotal role in the production of hyperalgesia but not inflammation by carrageenan. Inhibition of the MAPK pathway produces a marked reduction of thermal hyperalgesia and indicates a role for this signaling pathway in the development of hyperalgesia but not inflammation.
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