Volume 14, Issue 2, Pages 113-119 (February 2010)

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PGE2-induced lasting nociception to heat: Evidences for a selective involvement of A-delta fibres in the hyperpathic component of hyperalgesia

Received 15 December 2008; received in revised form 23 March 2009; accepted 4 April 2009. published online 08 May 2009.

Abstract

Animal models for mechanical pressure or heat nociception usually only measure the threshold response latency. In this study, the effect of typical sensitising treatments on the lasting nocifensive behaviour elicited after a supra-threshold heating stimulus – the hyperpathic component of hypernociception – was assessed. Male Wistar rats received either intra-plantar (i.pl.) injection of 350ng PGE2 (50μL) or topical application (t.a.) of 100% dimethylsulfoxide (DMSO), and 10mM capsaicin. One hour after the paw treatments the number of nocifensive events (NNE) was scored hourly (6h), for 5min, immediately after a hind paw immersion in hot water (50°C/7s). PGE2, DMSO and capsaicin increased the NNE -induced by the supra-threshold stimuli. Indomethacin (2.5mg/kg i.p.), given 30min before paw treatments, completely inhibited NNE in all groups (P<0.01). However, indomethacin given 60min after PGE2 did not reverse this sensitisation. PGE2 and DMSO did not lower the heat threshold in the paw withdrawal test, although carrageenan and capsaicin were effective (P<0.05). Capsaicin neonatal treatment (CNT) (50mg/kg) reduced the sensitisation induced by DMSO and capsaicin (P<0.01), but not that induced by PGE2. These data suggest that the heat-induced lasting nociception is probably conveyed by Að nociceptors, and PGE2 seems to be more selective to induce this phenomenon than the thermal threshold lowering. In addition, this hyperpathic effect induced by DMSO and capsaicin seems to be indirectly mediated by PGE2 and C-fibres.

Keywords: Hyperpathia, Prostaglandin E2, Nociception, Capsaicin, DMSO

Article Outline

• Abstract

• 1. Introduction

• 2. Methods

• 2.1. Animals

• 2.2. Drugs and dilutions

• 2.3. Lasting nociception evaluation

• 2.4. Thermal threshold evaluation

• 2.5. Degeneration of nociceptive C-fibres

• 2.6. Statistical analysis

• 3. Results

• 3.1. Definition of the temperature and time stimulation parameters

• 3.2. Indomethacin inhibited PGE, DMSO and capsaicin effects on heat-induced lasting nociception

• 3.3. PGE, DMSO and capsaicin effect on the heat nociceptive threshold

• 3.4. Effect of C-fibre degeneration on heat-induced lasting nociception

• 4. Discussion

• Acknowledgment

• References

• Copyright

1. Introduction

The application of thermal, mechanical or chemical stimuli to human subjects with inflammatory or chronic pain results in one or a combination of symptoms generally described in terms of hyperalgesia (increased pain sensitivity) or allodynia (pain due to non-nociceptive stimulus). Hyperalgesia may still include both a decrease in threshold and an increase in supra-threshold response (Loeser and Treede, 2008). In humans, these symptoms may be distinguished by verbal reports, as well as documented and quantified by the completion of pain questionnaires and visual analogue scales (Galer and Jensen, 1997, Krause and Backonja, 2003). In animal models, the nociception intensity is directly correlated to the threshold to elicit behavioural defensive responses after mechanical (von Frey fibres, paw pressure test) or thermal stimuli (heat or cold tests) (Le Bars et al., 2001). However, one important characteristic of the human hyperalgesic state, which is neglected by these models, is the lasting nociception after the end of the stimuli. This phenomenon is called hyperpathia when exacerbated by neuropathic conditions (Merskey and Bogduk, 1994, Galluzzi, 2007), although it also seems to be present in a moderated form in chronic inflammatory conditions – the so-called tenderness (Rigby and Wood, 1990, Chenitz, 1992, Scott and Houssien, 1996, Brosseau et al., 2003). One may judge the persistence of pain sensations more serious than the lowest threshold to elicit it (hyperalgesia), since the latter can easily be prevented by the withdrawal behaviour, but when a hyperpathic state is present the subject cannot avoid its complete manifestation even interrupting the eliciting stimulus. Hyperpathia has been evaluated in some kinds of neuropathic pain in humans (Defrin et al., 2001, Mitchell and Fallon, 2002, Ofek and Defrin, 2007), but in spite of its relevance, the vast majority of neuropathic and inflammatory pain studies only address the lowering of the nociceptive threshold for mechanical or thermal stimulation, as a correlate of hyperalgesia. In animal models, this goal is even more difficult due to the lack of a reliable relationship between stimulus and behavioural response. Indeed, we can find either a misuse of this term when employed as a correlate of the ongoing nociceptive effect of chemical agents (Wallace et al., 2002), or the nocifensive behaviour related to hyperpathia is taken as a secondary product of a model designed to evaluated threshold responses (Seltzer and Shir, 1988, Shir and Seltzer, 1990, Seltzer et al., 1990). In the former case, we cannot determine when the direct nociceptive action ends and the after-response begins (which is critical for hyperpathia definition), and in the latter case the duration of the trigger stimulus varies with the withdrawal response delay. In the present study, we aimed to develop a reliable method to approach the hyperpathic component of PGE2-induced nociceptive sensitisation to heat, and also elucidate some of its underlying mechanisms.

2. Methods

2.1. Animals

Experiments were performed on male Wistar rats (200–250g), which were housed in temperature-controlled rooms (19–21°C) under a 12–12h light/dark cycle with free access to water and food. The animals were allowed a 20-min period of habituation to the experimental set, 24h before the experimental session, and were again transferred to the test room at least 1h prior to the experimental session, in order to minimise the stress. This study was conducted according to the ethical guidelines of the International Association for the Study of Pain (IASP, 1983), and also to the NIH guide for the care and use of laboratory animals (NIH publications no. 8023, revised 1978), and approved by the local ethical committee for the use of animals.

2.2. Drugs and dilutions

Prostaglandin E2 and phosphate buffered saline (PBS) were obtained from Sigma–Aldrich (USA), dimethylsulfoxide (DMSO) from MERCK (Germany), capsaicin from TOCRIS (USA), indomethacin from Prodome Química and Farmacêutica, (Brazil) and carrageenan multi-type κ/λ from BDH chemicals (UK). PGE2 was stored in an absolute ethanol solution (500μg/ml) and was freshly diluted in PBS immediately before its application. Capsaicin was diluted in a 50% ethanol solution. Indomethacin was diluted in a 1.29% sodium bicarbonate solution (pH=8). Carrageenan was diluted in PBS. The intra-plantar (i.pl.) injection was given in a volume of 50μL. Topical application (t.a.) was carried out with the aid of a cotton swab soaked in the drug. It was applied all around the right hind paw (dorsal and plantar surface). One hour after i.pl. injection, or topical application, the animals were assessed for heat sensitisation.

2.3. Lasting nociception evaluation

A compact electronic water heating apparatus keeps the water in constant circulation between a 2.5L heating chamber and an external open cuvette designed for the rat’s hind paw to sink into it (Fig. 1). Heat stimulus was applied by immersing the hind paw into the hot water (50°C±0.1°C) for 7s, after which the animals were placed into a Plexiglas chamber (29×29×29cm) for a 5-min period of paw shaking, lifting or licking movements counting (number of nocifensive events). This procedure was repeated hourly until the 6thh. The temperature and time of immersion were chosen based on previous pilot studies to induce a consistent nocifensive response only in the PGE2 injected paw, without sensitising the vehicle-treated paw. Other stimulation protocols were 45°C for 10s, 47°C for 10s, 50°C for 10s, and 50°C for 5s (Fig. 2).

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Fig. 1. Paw thermal bath apparatus.

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Fig. 2. Definition studies for hyperpathic sensitisation parameters. The animals received a 50μL i.pl. injection of 250ng PGE2/paw or vehicle (ethanol 1%). The number of nocifensive events induced after heat stimulation was scored in a 5-min observation period, 2h after PGE2 injection, and hourly thereafter until the 6thh. Water temperature and paw immersion time were as follow: (A) 45°C/10s; (B) 47°C/10s; (C) 50°C/5s; (D) 50°C/7s; (E) 50°C/10s. The points represent the mean±SEM of six animals. ∗P<0.05 and ∗∗P<0.01 between PGE2 and vehicle (whole curves compared by using paired Student’s t test).

2.4. Thermal threshold evaluation

For thermal threshold evaluation a Plantar Test apparatus (Ugo Basile, Model 7370, Milan, Italy) based on Hargreaves et al. (1988) was used. Briefly, each rat was placed into a Plexiglas chamber (28×40×35cm) with a thick transparent floor. A radiant heat beam (55W) was focused onto the middle of the plantar surface of the hind paw, through the chamber floor. The time elapsed between onset of the stimulus and manifestation of the paw withdrawal response was measured automatically and was taken as an index of the thermal nociceptive threshold. Significant decreases in paw withdrawal latency were interpreted as indicative of heat hypernociception. Nocifensive responsiveness to heat at each time point was calculated as the mean of three consecutive evaluations, carried out at 2-min intervals. A 20s cut-off time was used to avoid tissue damage. The first measure was taken 1h after intra-plantar injection or topical treatment, and then hourly over 6h. One day before the experiments, the animals were allowed a 20-min habituation period to minimise stress.

2.5. Degeneration of nociceptive C-fibres

Degeneration of unmyelinated sensory neurons was achieved by the capsaicin neonatal treatment as described by Jancsó (Jancsó et al., 1977). Briefly, 2-day old rats received subcutaneously 10μL/g of a solution (10% ethanol, 10% Tween 80 in physiological saline) containing 5mg of capsaicin per mL (0.5% w/v). These animals were subsequently subjected to testing at 60-days old. The degeneration effectiveness was confirmed using the wiping test described by Hammond and Ruda (1991). Briefly, 20μL of 0.01% (w/v) capsaicin was instilled into one eye, and the number of wiping reflexes that occurred in the subsequent 1-min period was counted. The animals that wiped their eyes only five times at most were used as the neonatal capsaicin-treated group in the experiments designed to assess the role of sensory afferent C-fibres.

2.6. Statistical analysis

All statistical analysis was carried out using the GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA). The Paired Student’s t test and one-way ANOVA for repeated measures was used for the whole time-course curve comparisons. When a significance level of at least P<0.05 was detected, the analysis was followed by the Dunnet’s post-hoc test. Results are expressed as the mean±SEM of six animals.

3. Results

3.1. Definition of the temperature and time stimulation parameters

In pilot studies, we evaluated different combinations of water temperature (°C) and periods of paw immersion (s) to find one that was able to evidence the effect of PGE2 on heat nociception, without producing nociceptor sensitisation by itself. (Fig. 2) shows some of these stimulation protocols. By using a stimulus of 45°C during 10s (45°C/10s), a temperature in the range of TRPV1 activation, there was no nocifensive response after each stimulation, even in the PGE2-treated group (Fig. 2A). On increasing the stimulus to 47°C/10s, a slightly significant difference between PGE2 and vehicle-treated animals could be observed (Fig. 2B). The difference between treated and untreated group was greater when the temperature was raised to 50°C. However, this higher stimulation temperature also elicited increasing lasting nociception in the vehicle-treated animals, indicating that the thermal stimulus could be damaging the tissue (Fig. 2E). By reducing the stimulation period to 5s, the 50°C stimulus did not elicit lasting nocifensive responses even in the PGE2-treated animals (Fig. 2C). The best temperature/period of stimulation was 50°C/7s, which caused a clear lasting response in the PGE2, but not in the vehicle-treated animals (Fig. 2D). Thus, over the 6-h period of the study it is unlikely that this kind of stimulation could be damaging the paw tissue. It was not possible to observe a clear PGE2-induced dose-dependent effect here. Instead, by increasing the PGE2 concentration we observed a more stable nocifensive response, but not an increasing in the response magnitude. In the subsequent experiments the PGE2 concentration was increased to 350ng.

3.2. Indomethacin inhibited PGE2, DMSO and capsaicin effects on heat-induced lasting nociception

In this group of experiments we compared the sensitisation produced by two other chemical nociceptive substances, DMSO and capsaicin, which possess putative selective action on Að and C-fibres, respectively, to that produced by PGE2. Topical application of DMSO (100%) and capsaicin solution (10mM) induced hyperpathic sensitisation to the supra-threshold heating stimulus (50°C/7s) (P<0.01) as compared to vehicle-treated animals (Fig. 3A–C). Both treatments produced similar effects to that caused by PGE2. The involvement of cyclooxygenase products in the sensitisation mechanism of these chemicals was assessed by pre-treating the animals with indomethacin (2.5mg/kg, i.p.) 30min before the paw treatments. The sensitisation was reduced in all treatments between 2 and 6h (P<0.01) (Fig. 3A–C). Since these results indicated that endogenously produced prostanoids was a common pathway to all three sensitisers, we further verified the effect of indomethacin given 30 and 60min after PGE2 paw injection. Indomethacin partially reduced sensitisation when given 30min after PGE2 (P<0.01) (Fig. 4A), but not when given 60min after PGE2 (Fig. 4B).

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Fig. 3. Effect of indomethacin on PGE2, DMSO, and capsaicin-induced hyperpathic sensitisation. PGE2, DMSO, and capsaicin were applied to the hind paw and the number of nocifensive events induced by heat stimulation (50°C/7s) was scored in a 5-min observation period, 1h after PGE2 injection, and hourly thereafter until the 6thh. Indomethacin (2.5mg/kg, i.p.) was given 30min before the chemical stimuli. (A) PGE2 350ng/paw (i.pl.). (B) Capsaicin (10mM, t.a.). (C) DMSO 100% (t.a.). The points represent the mean±SEM of six animals. ∗∗P<0.01 between chemical stimulus and indomethacin; ##P<0.01 between chemical stimulus and vehicle (whole curves compared by using ANOVA for repeated measures followed by Dunnet’s test).

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Fig. 4. Effect of indomethacin post-treatment on PGE2-induced hyperpathic sensitisation. The animals were treated with indomethacin (2.5mg/kg i.p.) either 30min (panel A) or 60min (panel B) after PGE2 (350ng/paw) i.pl. injection. The number of nocifensive events induced by heat stimulation (50°C/7s) was scored in a 5-min observation period, 1h after PGE2 injection, and hourly thereafter until the 6thh. The points represent the mean±SEM of six animals. ∗∗P<0.01 between PGE2 and indomethacin post-treatment; ##P<0.01 between PGE2 and vehicle (whole curves compared by using ANOVA for repeated measures followed by Dunnet’s test).

3.3. PGE2, DMSO and capsaicin effect on the heat nociceptive threshold

In relation to our hypothesis that the lasting nociception induced by PGE2 could be due to a distinct mechanism not related to the threshold lowering, we further evaluated the effect of all three treatments on the heat-induced paw withdrawal test. In this assay, carrageenan (500μg/paw) was the positive control, and significantly reduced the withdrawal threshold when compared to the saline treated group (P<0.05) (Hargreaves et al., 1988). ANOVA for repeated measures was applied on the curves segment between the 2nd and the 6thh, since the carrageenan treated animals have shown decreased heat thresholds only from the 2ndh. Topical application of capsaicin 10mM also lowered the threshold when compared to the vehicle (P<0.05) (Fig. 5B), but PGE2 (350ng/paw) (Fig. 5A), and DMSO (100%) (Fig. 5B), did not lower the threshold for heat.

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Fig. 5. PGE2, DMSO and capsaicin effect on heat threshold for nociception. Heat-induced paw withdrawal was evaluated hourly after (A) 350ng PGE2/paw (i.pl.), (B) 10mM Capsaicin (t.a.), and 100% DMSO (t.a.). Carrageenan (500μg/paw, i.pl.) was used as a positive control, and the vehicle was topically applied 50% ethanol. The points represent the mean±SEM of six animals. ∗P<0.05 compared to vehicle, between the 2nd and 6thh (ANOVA for repeated measures followed by Dunnet’s test).

3.4. Effect of C-fibre degeneration on heat-induced lasting nociception

Another attempt to establish the role of different kinds of nociceptors in this lasting response was carried out using the capsaicin neonatal treatment (CNT). This treatment is considered to eliminate a significant amount of C-fibres and some Að-fibres. CNT did not prevent PGE2 sensitisation of the heat-induced lasting nociception (Fig. 6A), but inhibited the sensitisation produced by DMSO (Fig. 6B). Surprisingly, CNT prevented only partially the sensitisation produced by topically applied capsaicin on the heat-induced lasting nociception (P<0.01, between 3 and 6h) (Fig. 6C). As explained in method section, all these CNT animals presented less than five wipings after capsaicin instillation in the eye, thus confirming C-fibre degeneration. In addition, the topical application of capsaicin in CNT animals did not induce significant nocifensive behaviours immediately after paw treatment, as occurred in the control group. The mean number of nocifensive events immediately after topical application of capsaicin in naïve animals was 7.67±1.7 (vehicle=1.5±0.42), while in rats neonatally-treated with capsaicin it was 2.83±0.79 (vehicle=0.6±0.24).

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Fig. 6. Influence of capsaicin neonatal treatment on the hyperpathic sensitisation. The number of nocifensive events induced by heat stimulation (50°C/7s) was scored in a 5-min observation period, 1h after (A) PGE2 (350ng/paw, i.pl.), (B) 100% DMSO (t.a.), and (C) 10mM capsaicin (t.a.), and hourly thereafter until the 6thh, in 60-day old rats, neonatally-treated with capsaicin (CNT; 50mg/kg, s.c.). The points represent the mean±SEM of six animals. ∗∗P<0.01 between CNT and non-CNT experimental groups; ##P<0.01 when compared to vehicle group. ANOVA for repeated measures followed by Dunnet’s test was applied on the whole curves (A and B), or the curve segments between 3 and 6h (C).

4. Discussion

In the previous sections it was shown that after sustained heat stimulation of one hind paw it is possible to observe a lasting nocifensive response that was potentiated by prostaglandin E2. We assume that a sustained nociceptive input after the end of the heat stimulation maintains the nocifensive behaviour, and it is likely that an after-discharge phenomenon in the nociceptors provides this sustained input (Devor and Seltzer, 1999, Devor et al., 2002). The term lasting nociception was given to this behaviour since it seems to be associated with the duration of the nociception-related behaviour rather than to the threshold for the nociception.

The first evidence that the lasting nociception observed here could be mediated by a mechanism other than threshold lowering was given by the effect of PGE2 alone. PGE2 did not lower the nociceptive threshold for heat in the paw withdrawal test at the same dose at which it produced the nocifensive responses after the sustained heat stimulation. This suggests that this lasting nociceptive phenomenon may be more sensitive to the PGE2 effects than the lowering of the nociceptive threshold. This reasoning led us to consider that PGE2 could be acting in a different population of afferent fibres, which did not lower its nociceptive threshold, but enhanced its after-discharge. Electrophysiological studies have demonstrated that a long-lasting heat stimulus causes an early discharge in sensory C-fibres, which adapts within a few seconds to a lower level. On the other hand, Að afferents, which are initially unresponsive, vigorously discharge after a couple of seconds delay until the peak, sustaining this activity without adaptation (Meyer and Campbell, 1981, Treede et al., 1995). In addition, a sustained supra-threshold heat stimulation was even seen to increase the nociceptive threshold of C-fibres, although enhancing the Að-fibres firing response to heat (Meyer and Campbell, 1981). Recent developments in heat thermal stimulation have clearly allowed the discrimination between C and Að-fibre mediated nociception. Slow heating rate procedures have been shown to selectively activate C-fibres, while a fast heating rate seems to selectively activate Að-fibres (Yeomans et al., 1996, Yeomans and Proudfit, 1996, McMullan et al., 2004). The present model should be considered as a very fast heating rate procedure, since the paw was immersed in a water bath with the constant 50°C temperature. Given these characteristics, our hypothesis is that Að-fibres are the best suited to convey the nociceptive information responsible for the lasting nociception observed here. Supporting this hypothesis, DMSO, which is thought to be a selective sensitising stimulus for Að-fibres (Tzabazis et al., 2005, Leith et al., 2007) also produced lasting nociception, but did not lower the threshold for heat-induced paw withdrawal in an apparatus regulated to a slow heating rate. However, capsaicin, which is known to sensitise C-fibres (Holzer, 1988), also lowered the paw withdrawal threshold.

Experiments with the cyclooxygenase inhibitor indomethacin have shown that ongoing production of prostanoids are needed for the development of the lasting nociception state induced by PGE2, DMSO and capsaicin, at least in an early stage, since indomethacin treatment given after this time was ineffective to reverse the PGE2 effects. In addition, the capsaicin neonatal treatment (CNT) did not prevent PGE2-induced lasting nociception. Considering that CNT destroys most of the C-fibres (Lawson, 1987), this finding is our strongest evidence that PGE2-induced lasting nociception is an Að-mediated response. In the case of capsaicin stimulation, the inhibitory effect of indomethacin supports the notion that the lasting nociception state induced by the vanilloid was indirect, and probably caused by prostaglandins sensitising Að-fibres. Capsaicin sensitive fibres may be directly or indirectly involved in the peripheral release of prostaglandin E2 (Chopra et al., 2000, Averbeck et al., 2001), and thus it is conceivable that C-fibres can play an important role here. In the case of PGE2 stimulation, since indomethacin pretreatment, but not CNT, produced a marked inhibitory effect, it is unlikely that the endogenous prostaglandin induced by PGE2 injection was mediated by C-fibres to a significant extent.

However, it was intriguing that the capsaicin effects in CNT rats was significantly, but not completely inhibited due to fibre degeneration. Considering that these animals failed to respond in the eye-wiping test, and in addition they showed no nocifensive reaction immediately after the capsaicin hind paw injection, we assume that the lasting nociception to heat induced by topical application of capsaicin might not be due to an action on neuronal TRPV1 receptors (Juan et al., 1980). Indeed, non-neuronal TRPV1 expression has been reported for many cutaneous structures, such as mast cells, epidermal keratinocytes, dermal blood vessels, the inner root sheet and the infundibulum of hair follicles, differentiated sebocytes, sweat gland ducts, and the secretory portion of eccrine sweat glands (Ständer et al., 2004, Gunthorpe and Szallasi, 2008). In the present case, mast cells, as well as other cutaneous structures, may be a significant source of prostaglandins, which could mediate the capsaicin effect. Finally, DMSO-induced lasting nociception was totally inhibited by indomethacin, and by CNT. The mechanism by which DMSO could act selectively on Að-fibres is not yet known, but this substance may have direct stimulatory effects on prostaglandin E series production (LaHann and Horita, 1975). In the present case, this potential selectivity for Að-fibres could be indirect, therefore, due to the PGE release by a C-fibre-mediated process.

The data reported herein suggest that Að-fibres, once sensitised by prostaglandins, may play an important role in inflammatory pain by supplying nociceptive input after the end of a supra-threshold stimulus – the hyperpathic state. In addition, C-fibres could act as an important prostaglandin source responsible for Að-fibre sensitisation. A better understanding of the molecular and neural basis would be of great value not only for improving the management of inflammatory pain, but also of neuropathic pain, since the exaggerated hyperpathia following nerve lesions may share some mechanisms involved in the present model. Indeed, even the role of prostaglandins in neuropathic conditions, formerly neglected, has now being considered more accurately (Ma and Quirion, 2008).

Acknowledgements

This study was conducted with the financial support of the Conselho Nacional de Pesquisa (CNPq), Coordenação de Aperfeiçoamento do Pessoal do Ensino Superior (CAPES), and FAPESC (Pronex), Brazil.

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Department of Pharmacology, Federal University of Santa Catarina, Florianópolis – Santa Catarina 88040-900, Brazil

Corresponding author. Tel.: +55 48 3721 9491x218; fax: +55 48 3337 5479.

PII: S1090-3801(09)00079-2

doi:10.1016/j.ejpain.2009.04.002

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