Neonatal immune challenge alters nociception in the adult rat

Pain 119 (2005) 133–141 www.elsevier.com/locate/pain

Neonatal immune challenge alters nociception in the adult rat Lysa Boisse´ a, Sarah J. Spencer a,*, Abdeslam Mouihate a, Nathalie Vergnolle b, Quentin J. Pittman a a Department of Physiology and Biophysics, Hotchkiss Brain Institute, University of Calgary, Calgary, Alta., Canada T2N 4N1 Department of Pharmacology and Therapeutics, Faculty of Medicine, Hotchkiss Brain Institute, University of Calgary, Calgary, Alta., Canada

b

Received 1 June 2005; received in revised form 8 September 2005; accepted 19 September 2005

Abstract Intense pain or intense peripheral inflammation experienced during development can have pronounced effects upon adult pain sensation. However, little is known about the more commonly encountered mild systemic inflammation, such as that experienced with mild illness. Neonatal exposure to lipopolysaccharide (LPS), an established model of immune system activation, has been shown to affect febrile and cyclooxygenase-2 (COX-2) responses to a similar exposure in adulthood. Adult LPS also elicits a range of sickness behaviours, including enhanced responses to painful stimuli. We, therefore, hypothesized that adult sensation and pain responses could be affected by a neonatal LPS challenge. Male and female Sprague–Dawley rats were administered LPS at postnatal day 14 and were tested in adulthood for nociceptive responses to thermal and mechanical stimuli using, respectively, a plantar test apparatus and von Frey filaments, before and after adult LPS. Expression of dorsal root ganglion and lumbar spinal cord COX-2 was also examined. Animals treated as neonates with saline showed the expected hypersensitivity to painful stimuli after adult LPS as well as enhanced spinal cord COX-2. Neonatally LPS-treated rats, however, showed a significantly different profile. They displayed enhanced baseline nociception and elevated basal spinal cord COX-2 compared with neonatally saline-treated rats. Also, rather than the expected hyperalgesia after adult LPS, no changes in nociceptive responses and a reduction in spinal cord COX-2 expression were observed. These findings have important implications for the understanding of pain and its management and highlight the importance of the neonatal period in the development of pain pathways. q 2005 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Cyclooxygenase-2; Neonate; Nociception; Pain; Spinal cord

1. Introduction Disorders related to chronic pain, allodynia, and hyperalgesia are associated with prior exposure to a range of stressors, from childhood neglect to surgery in early life, or prior hepatitis C infection (Amir et al., 1997; Barkhuizen et al., 1999; Beckham et al., 1997; Buskila et al., 1997; Imbierowicz and Egle, 2003; Sherman et al., 2000; Taddio et al., 1997). However, not all people exposed to stress or infections develop chronic pain disorders and the ultimate causes of many such disorders are still unknown. It is now appreciated that a localized painful neonatal event can cause long-lasting hypersensitivity to nociceptive stimuli * Corresponding author. Tel.: C1 403 220 4497; fax: C1 403 283 2700. E-mail address: [email protected] (S.J. Spencer).

(Al Chaer et al., 2000; Ren et al., 2004; Ruda et al., 2000; Saab et al., 2004; Walker et al., 2003; Wang et al., 2004). Evidence is limited, however, as to the effects of a more mild and global neonatal inflammation, such as with a bacterial infection, on these pain responses. This relative omission is surprising given the demonstrated role of cytokines and prostaglandins in pain processing. Activation of cyclooxygenase-2 (COX-2) in the spinal cord, by pro-inflammatory cytokines such as interleukin-1b, is considered responsible for prostaglandin elevation after nociceptive stimuli, leading to increased spinal cord neuron excitability (Samad et al., 2001; Seybold et al., 2003; Vanegas and Schaible, 2001) and alteration of such cytokine function has been linked to chronic pain disorders such as fibromyalgia (Thompson and Barkhuizen, 2003). Our previous work has revealed that basal hypothalamic COX-2

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is enhanced and the COX-2 response to lipopolysaccharide (LPS) attenuated in adult animals exposed to LPS as postnatal day (P)14 neonates (Boisse et al., 2004). In addition to changes in COX-2, LPS elicits a broad spectrum of behaviours known as sickness behaviours (Dantzer, 2001), which includes an exacerbated response, hyperalgesia, to painful stimuli (Watkins et al., 1994a,b). We therefore hypothesized that adult pain responses may also be affected by neonatal challenges to inflammatory pathways. Due to reported gender differences in response to inflammation (Bradshaw et al., 2000) and to neonatal intervention (Rosztoczy et al., 2003), we investigated both male and female rats here. Sprague–Dawley rats were injected at P14 with either LPS or pyrogen-free saline and examined in adulthood for nociceptive responses to thermal and mechanical stimuli. This neonatal age was chosen to enable us to compare responses to other changes we have previously observed in adult rats after injections at this time point (Boisse et al., 2004; Ellis et al., 2005; Heida et al., 2004). Given that COX-2 plays a key role in behaviours to nociceptive stimuli, we also tested whether COX-2 expression in the dorsal root ganglion (DRG) and lumbar spinal cord were affected by a neonatal immune challenge. Rats were tested as adults for responses at baseline and after a further injection of LPS to investigate changes in sickness behaviour. The results from this study have significant implications for the understanding and management of chronic pain and inflammatory disorders.

environment (Boisse et al., 2004; Ellis et al., 2005; Heida et al., 2004; Mouihate and Pittman, 2003), a temperature approximately that of the nest when litters are kept at normal room temperature. Ears were clipped for identification and the mother was returned to her pups. All were left undisturbed except for weaning and the usual cleaning and feeding procedures until experiments commenced. Approximately equal numbers of animals from each litter received LPS or saline and during the interval between injection and experimentation care was taken to ensure that each cage contained both LPS and saline pre-treated pups. 2.3. Nociceptive sensitivity testing Eight to 12 weeks after the neonatal treatment, the rats were brought in their home cages to the laboratory testing room. The same environmental parameters as the general animal care facility were maintained. We have previously shown that animals at this time show circadian rhythms, body temperature and activity levels that are identical whether injected with saline or LPS as pups (Boisse et al., 2004; Heida et al., 2004; Spencer et al., 2005). The rats were allowed to acclimatize overnight and between 11:30 am and 1:00 pm the following day were administered either LPS (50 mg/kg, i.p.) or an equivalent volume of pyrogen-free saline. After 2.5 h, the time point at which febrile temperatures are at their highest (Mouihate and Pittman, 2003) and LPS-induced hyperalgesia is at a stable plateau (Abe et al., 2001), all animals were tested for nociceptive sensitivity using either a plantar test apparatus or von Frey filaments. The following week the groups were switched so that those that had previously, as adults, received saline were now administered LPS and vice versa in a crossover design and the rats were tested again in one of the two tests of nociception sensitivity. All nociceptive sensitivity testing was done by an experimenter who was blind to the type of treatment the animals had received.

2. Materials and methods 2.1. Animals Pregnant Sprague–Dawley rats (Charles River) were maintained at 22 8C on a 12 h light/dark cycle (7:00 am–7:00 pm) with pelleted rat chow and water available ad libitum. On the day of birth, i.e. P0, litters were culled to 12 pups and pups were randomly distributed among the dams. All litters were weaned at P21 and male and female rats were subsequently housed separately, three to four animals per cage. All procedures were conducted in accordance with the Canadian Council on Animal Care regulations and were approved by the local University of Calgary Animal Care Committee. 2.2. Early life manipulations On P14 the dam was removed from her pups for approximately 5 min. The 46 randomly selected pups (taken from seven litters) were subjected to intraperitoneal (i.p.) injections of either LPS (nLPS; Escherichia coli; serotype 026:B6; L-3755; Sigma, St Louis, MO; 100 mg/kg) in pyrogen-free saline, or to an equivalent volume of pyrogen-free saline. We have previously shown that this dose of LPS gives a short lasting, reproducible fever that appears after a latency of approximately 2 h and has a peak of approximately 1.5 8C when pups are maintained in a 30 8C

2.3.1. Thermal nociception: plantar test The rats (nZ6 neonatally saline-treated (nSal) males, nZ6 neonatally LPS-treated (nLPS) males, nZ6 nSal females, nZ6 nLPS females) were assessed for nociceptive responses to a thermal stimulus using a plantar test apparatus (Stoelting, Chicago, Illinois, USA; (Hargreaves et al., 1988; Vergnolle et al., 2001)). Each rat was placed individually in a clear plastic testing box with a clear glass floor and was allowed to acclimatize to the new environment for 5 min (males) or 10 min (females). A source of radiant heat was then placed unobtrusively beneath the floor of the testing box, directly below one of the rat’s hind paws. The withdrawal reflex latency of each hind paw was measured in seconds, in random order, by means of a timer connected to the heat source and the average withdrawal latency of the two paws was taken. The timer switched off the heat when the paw was withdrawn, but was also programmed to automatically terminate the test after 25 s to avoid potential tissue damage due to long term heat exposure. The testing box was thoroughly cleaned between each animal. 2.3.2. Mechanical nociception: von Frey filament test Cutaneous sensitivity to mechanical stimuli was measured using the von Frey filament test (nZ5 nSal males, nZ6 nLPS males, nZ5 nSal females, nZ6 nLPS females), which uses a series of filaments of different size, each delivering a touch of different

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2.4. COX-2 Western blots On the last testing day, immediately after the final test, rats were deeply anaesthetised with sodium pentobarbital (80– 100 mg/kg i.p.) and perfused with 4 8C phosphate-buffered saline via the left cardiac ventricle. The DRGs and lumbar sections of the spinal cord (L4-6) were quickly removed over ice, snap-frozen in liquid nitrogen and stored at K80 8C until ready for use. Tissue was homogenized, proteins extracted, and homogenates (30 mg protein per well) were separated by 10% SDS polyacrylamide gel electrophoresis as previously described (Boisse et al., 2004; Mouihate and Pittman, 2003). Proteins were then transferred to a nitrocellulose membrane and incubated overnight at 4 8C in 5% fat-free milk in Tris-buffered saline, containing Tween 20 (TBS-T). The following day, the membrane was incubated for 2 h at room temperature in a 1:4000 affinity purified rabbit antiCOX-2 antibody (Cayman Chemical, Ann Arbor, MI) in 5% fatfree milk/TBS-T solution with a 1:40,000 affinity purified rabbit anti-actin antibody (Sigma). The membrane was then washed for 30 min in TBS-T and incubated for 1 h in goat anti-rabbit IgG horseradish peroxidase conjugate (1:4000; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature. Bound antibodies were revealed using an enhanced chemiluminescence assay (Amersham Biosciences, UK). The DRGs and lumbar spinal cord samples were run separately and all samples were run twice.

3. Results 3.1. Thermal nociception In response to a thermal stimulus (plantar test; nZ6 for each group), both male and female rats that received saline as neonates and were tested after a saline injection as adults displayed paw withdrawal latencies of approximately 13– 14 s (Fig. 1). This range was identical to that observed in naı¨ve animals that were not injected as neonates (data not shown). As expected, LPS in both male and female adults resulted in a significant reduction in withdrawal latency, to approximately 8–9 s, compared with the baseline (adult injection with saline) response for this group (P!0.05 for both; Fig. 1). This decreased withdrawal latency was characteristic of thermal hyperalgesia. This finding was also comparable to that seen with the naı¨ve animals not treated as neonates (data not shown). Adult rats that had received LPS as neonates, however, displayed a substantially different profile. In these animals, baseline paw withdrawal latency was significantly reduced, to approximately 8–9 s, compared to neonatally A - Males

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Withdrawal latencies for the plantar test were compared using a two-way analysis of variance (ANOVA), with neonatal treatment and adult treatment as the between factors. Where a significant main effect was found, this was followed by a one-way ANOVA and Student Neuman-Keuls post hoc comparisons. Scores for the von Frey filament test were also compared using a two-way ANOVA followed by a one-way ANOVA and Student NeumanKeuls post hoc tests comparing the area under the curve with neonatal treatment and adult treatment as the between factors. Levels of COX-2 expression in each region were quantified by densitometric analysis of specific bands detected at the molecular weight of COX-2 and actin. COX-2 to actin ratios were compared using a two-way ANOVA with neonatal treatment and adult treatment as the between factors followed by a one-way ANOVA and Student Neuman-Keuls post hoc tests. Statistical significance was assumed when P!0.05. Data are presented as meanG standard error of the mean.

*

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pressure, with the larger filaments exerting greater pressure (Asfaha et al., 2002; Vergnolle et al., 2001). Each rat was placed individually in a clear plastic testing box with a metal grid floor and was allowed to acclimatize to the new environment for 5 min (males) or 10 min (females). Nine von Frey filaments ranging from 0.31 to 0.81 mm in diameter with marking forces of 2.04–125.89 g were applied onto one of the rat’s hind paws in sequential order of size. The filaments were applied three times in random paw order for 1–2 s. A score was assigned based on the animal’s response: 0Zno movement; 1Zremoval of the paw; 2Zremoval of the paw and vocalisation, licking or holding of the paw. The mean score was then taken for the three applications. The testing box was thoroughly cleaned between each animal.

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Fig. 1. Withdrawal latencies in response to thermal stimulation using a plantar test apparatus for (A) males and (B) females after treatment as neonates with either saline (nSal) or lipopolysaccharide (nLPS) followed by treatment in adulthood with either saline (aSal—white bars) or LPS (aLPS—black bars). nZ6 for each group. * P!0.05.

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rats, and hyperalgesia in their exaggerated responses to the larger diameter filaments. Also, in agreement with the results seen in response to thermal stimulation, neonatally LPS-treated males did not seem to respond to LPS treatment in terms of nociceptive changes. No significant difference in nociceptive score was observed after LPS-treatment in adult males that received LPS as neonates, compared to neonatally LPS-treated males given saline as adults. The same trends were seen with von Frey filament stimulation with the female rats (nZ5, nSal; nZ6, nLPS; Fig. 2(C) and (D)), but despite a significant interaction with the two-way ANOVA (P!0.05), subsequent post hoc comparisons did not reveal where the differences lay.

saline-treated rats (P!0.05 for males and females; Fig. 1). Neonatally LPS-treated animals also displayed a very different response to adult administration of LPS. That is, rather than the hyperalgesia normally associated with LPS, no change in withdrawal latency was observed after an adult LPS injection in these rats compared with baseline (Fig. 1). 3.2. Mechanical nociception Neonatally saline-treated male rats (nZ5, nSal; nZ6, nLPS) given saline as adults were unresponsive to the mild von Frey filament stimulation of diameters 0.31–0.41 mm (2.04–8.51 g marking force) and, as expected, nociceptive scores increased with increasing filament size thereafter. Adult treatment with LPS in these neonatally saline-treated animals led to a significant increase in nociceptive score in response to the von Frey filament stimulation, characteristic of mechanical hyperalgesia (P!0.01; Fig. 2). As was seen with thermal stimulation, males treated with LPS as neonates showed an increase in baseline mechanical nociceptive sensitivity relative to neonatally saline-treated rats, characteristic of hyperalgesia (P!0.05; Fig. 2(A) and (B)). These rats displayed both allodynia, manifested in nociceptive responses to small (0.31–0.41 mm) sized filaments, which did not cause a response in saline-treated

3.3. Spinal cord and DRG COX-2 expression Densitometric analysis of lumbar spinal cord Western blots for COX-2 in the males revealed a pattern that reflected the results from the nociception tests. A very low constitutive COX-2 expression was seen in neonatally saline-treated animals, that was significantly increased after LPS injection in adulthood (P!0.01 Fig. 3(A) and (B)). Neonatally LPS-treated males, however, had a significantly higher baseline expression of COX-2 than did the neonatally saline-treated males (P!0.001). In contrast to the increased

A - Males 1.25

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Filament Size Fig. 2. Absolute and area under the curve (AUC) nociceptive scores in response to mechanical stimulation with von Frey filaments for (A, B) males and (C, D) females after treatment as neonates with either saline (nSal) or lipopolysaccharide (nLPS) followed by treatment in adulthood with either saline (aSal) or LPS (aLPS). nSal, nZ5; nLPS, nZ6 for both males and females. *P!0.05, **P!0.01.

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Fig. 4. COX-2 expression in male dorsal root ganglia as identified by Western blots with semi-quantitative densitometric analysis. (A) Sample Western blot of male dorsal root ganglion (B) densitometric analysis for males after treatment as neonates with either saline (nSal) or lipopolysaccharide (nLPS) followed by treatment in adulthood with either saline (aSal) or LPS (aLPS). nZ4 for each group.

all groups that was not different between any of the groups (Fig. 4). Western blots for the females were therefore not conducted.

4. Discussion Fig. 3. Lumbar spinal cord COX-2 expression as identified by Western blots with semi-quantitative densitometric analysis. (A) Sample Western blot of male spinal cord (B) densitometric analysis for males and (C) densitometric analysis for females after treatment as neonates with either saline (nSal) or lipopolysaccharide (nLPS) followed by treatment in adulthood with either saline (aSal) or LPS (aLPS). nZ4 for each group. *P!0.05, **P!0.01, ***P!0.001.

expression observed after adult LPS treatment in neonatally saline-treated rats, COX-2 expression in the lumbar spinal cord was significantly reduced after the adult LPS treatment in neonatally LPS-treated rats (P!0.05). Levels of COX-2 expression in the female rats were more variable and thus no statistically significant differences were seen, but the same trends as for the males were maintained (Fig. 3(B) and (C)). Investigation of COX-2 expression in male rat DRGs by Western blot analysis revealed a high COX-2 expression in

Previous investigations have documented the impact of early-life pain, psychological trauma, and intense peripheral inflammation on pathways processing responses to painful stimuli. For instance, localized painful inflammation in neonates, such as injection of the paw with complete Freund’s adjuvant (Ruda et al., 2000; Walker et al., 2003) or carrageenan (Ren et al., 2004; Wang et al., 2004), or an intensely painful experience, such as repeated colonic irrigation (Al Chaer et al., 2000; Saab et al., 2004), all cause long-lasting alterations in pain processing and are associated with adult hyperalgesia to further painful stimuli. Nonetheless, the literature remains confusing. The present investigation is the first, to our knowledge, to show that a mild inflammation, such as with LPS, can also cause such nociceptive disorders. We demonstrate that a single LPS exposure during development impacts upon

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adult pain and sensory processing, leading to a decrease in nociceptive thresholds and increases in responses to painful and even innocuous stimuli in both male and female rats in adulthood. Biochemical evidence, in that baseline spinal cord COX-2 expression was increased in neonatally LPStreated animals relative to saline-treated, suggests a mechanism for these alterations involving changes in COX-2 expression or activity at the spinal cord, but not the DRG level. 4.1. Adult baseline pain responses after neonatal inflammation Prostaglandins play an important role in mechanisms of pain processing and hyperalgesia at the level of the spinal cord, as well as peripherally (Vanegas and Schaible, 2001). COX-2, a rate-limiting enzyme in prostaglandin synthesis, is constitutively expressed in spinal cord neurons and glia and is upregulated by painful stimuli or peripheral inflammation, leading to increased prostaglandin synthesis and spinal cord neuronal hyperexcitability (Seybold et al., 2003; Vanegas and Schaible, 2001). Conversely, COX-2 inhibitors can limit prostaglandin production and ameliorate hyperalgesia associated with many sources of pain (Choi et al., 2003; Fox et al., 2004; Seybold et al., 2003; Suyama et al., 2004). Thus, it seems likely that the baseline hyperalgesic responses in neonatally LPS-treated rats in this study are due to permanent alterations in COX-2 expression, and consequently prostaglandin production, which would result in hyperexcitability of the spinal cord neurons responsible for pain processing. The mechanism that would result in a permanently altered level of COX-2, i.e. producing what is essentially constitutive COX-2, is still obscure. The cox-2 gene contains a number of well known promoter regions that could be altered (Tanabe and Tohnai, 2002). In addition, there are downstream regulators of this enzyme that are a part of the prostaglandin pathway, for example, PGJ2 that could be subject to alteration (Inoue et al., 2000). Long-term alterations to the brain, functioning independently of or in addition to spinal cord COX-2 could also contribute to altered nociception after neonatal LPS. For instance we have previously demonstrated neurochemical changes in the hypothalamus with neonatal LPS exposure (Boisse et al., 2004) and this could affect the integration of pain responses via connections with medullary and brainstem sites (Buller, 2003; Buller et al., 2003; Jiang and Behbehani, 2001) providing descending modulation of spinal and nociceptive reflexes (Mason, 2001; Millan, 2002; Willis and Westlund, 1997). In addition, central serotonin pathways, important in pain modulation (Suzuki et al., 2004), appear to be a particular target of several types of early life adversity (Anseloni et al., 2005; Butkevich et al., 2005; Gartside et al., 2003). Other neonatal interventions can alter opioid receptor-like receptors in hypothalamic areas (Neal et al., 2003), and possible changes in

corticolimbic-brainstem pain circuitry have been demonstrated with neonatal stimulation of pain pathways (Ren et al., 2004). Other possible mechanisms for long-term changes in nociception include changes in spinal cord release of substance P or calcitonin gene-related peptide, or changes in glial activation potentially leading to induction of the pro-inflammatory cytokine cascade (Kreutzberg, 1996; Wieseler-Frank et al., 2005). In addition, peripheral inflammation can alter neuronal circuitry by causing changes in the density of nociceptive primary afferents in the DRG and spinal cord, as well as changes in their excitability (Peng et al., 2003; Ruda et al., 2000). This alteration is thought to occur through changes in release patterns of various crucial growth factors after peripheral inflammation (Ruda et al., 2000). However, these effects were not seen with CFA as late in development as P14 (Ruda et al., 2000), the time point used in this study. It is probable that even locally injected CFA, which contains mycobacterial organisms, has similar effects on the immune system to those seen with LPS, thus it is likely that alternative or additional mechanisms to changes in DRG and spinal cord nociceptive primary afferents are responsible for the appearance of increased spinal cord COX-2 after neonatal LPS exposure. In agreement with the CFA study (Ruda et al., 2000), we could not detect peripheral changes in COX-2 expression (in DRG) in adult rats neonatally pre-treated with LPS, either at baseline or after adult LPS. However, many investigations have indicated that, of the two isoforms, COX-2 plays an important role in pain processing in the spinal cord (Ghilardi et al., 2004; Ohtori et al., 2004; Yaksh et al., 2001) whereas in the DRG, COX-1 has the more prominent role (Chopra et al., 2000; Dou et al., 2004). It would be interesting therefore to examine the role of COX-1 in the changes to pain responses seen in this investigation. 4.2. Adult pain responses with LPS after neonatal inflammation During illness, animals display a well-characterised array of responses known as sickness behaviour (Dantzer, 2001), including typically displaying hyperalgesia (Watkins et al., 1994a,b). Indeed, a hyperalgesic response to nociceptive stimuli was observed here in neonatally saline-treated animals after adult LPS exposure. However, this LPSinduced hyperalgesia was not seen in neonatally LPStreated animals and spinal cord COX-2 instead of being enhanced was reduced or unaltered in these animals. Thus, neonatal LPS reduced at least one component of sickness behaviour in LPS-treated adults. It is important to note that this lack of hyperalgesia after adult LPS is different from the hyperalgesia seen with the localized paw carrageenan in adult rats treated with carregeenan as young neonates (Ren et al., 2004). Further experiments will be required to determine if responses to systemic inflammation (i.e. LPS) differ from those to localized

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inflammation (i.e. paw carrageenan), or if neonatal exposure at different periods results in a different adult phenotype. The mechanism affected in this reduced response to LPS is unclear and may be distinct from that contributing to the baseline hyperalgesia described above. It is possible that the hyper-sensitive baseline response to painful stimuli is already maximal and not capable of further enhancement by LPS. However, the latency for withdrawal to the thermal stimulus was approximately 9 s and the maximal response to stimulation with the von Frey filaments well below the maximal possible score. Previous studies have reported similar baseline withdrawal latencies that were reduced to as little as 2–3 s (Vergnolle et al., 2001), indicating that the rats are capable of responding much faster. It is also possible that long term alterations in the hypothalamus, an area known to mediate LPS-induced hyperalgesia (Abe et al., 2001), could cause enhanced corticosterone release (Anisman et al., 1998), leading to a relative suppression of spinal cord nuclear factor kB-induced COX-2 expression in response to inflammation (Auphan et al., 1995; Umland et al., 2002). Alternatively, alterations in the profile of proor anti-inflammatory cytokines may result in reduced stimulation of downstream inflammatory events. Recent evidence from our laboratory indicates that levels of LPSinduced cytokines are substantially reduced in animals treated as neonates with LPS (Ellis et al., 2005). 4.3. Gender differences in responses to nociceptive stimuli Many previous investigations have reported differences between male and female responses to inflammation and painful stimuli (Aloisi, 2003; Bradshaw et al., 2000; Riley et al., 1999). There is also considerable evidence for different effects of a number of early interventions on the male and female adult (O’Regan et al., 2004; Rosztoczy et al., 2003; Smythe et al., 1994). We, therefore, thought it pertinent, in the present study, to examine the responses of both genders. Perhaps surprisingly, we found no real differences between genders, either in their baseline responses or after adult LPS. Thermal stimulus withdrawal responses in females were identical to those in males and, trends in the mechanical stimulation test and spinal cord COX-2 expression were also identical. Female rats did display more variable responses. However, oestrogens and other steroid hormones are known to have a very important role in nociception (Aloisi, 2003; Riley et al., 1999) and it is very probable that the rats in this study were in different stages of their oestrous cycle, a factor that may have contributed substantially to the variability of the groups.

5. Conclusion The results of the present investigation demonstrate that neonatal LPS exposure alters the perception of nociceptive stimuli in adulthood, both at baseline and after further LPS

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exposure. These effects on behavioural responses to pain are accompanied by alterations in spinal cord, but not DRG COX-2 expression, providing a cellular mechanism via which behavioural changes may occur. These findings have serious implications for the understanding of analgesia as well as chronic pain syndromes, and indicate the importance of early-life events on adulthood.

Acknowledgements This work was supported by the Canadian Institutes of Health Research (CIHR). L.B. was an Alberta Heritage Foundation for Medical Research (AHFMR) student, S.J.S. is an AHFMR postdoctoral fellow, N.V. is a CIHR New Investigator and an AHFMR Scholar and Q.J.P. is an AHFMR Medical Scientist. We would like to thank Drs C. Cahill and K. Jhamandas for their comments on the manuscript.

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