3.1. The effect of different anti-inflammatory drugs on LPS-induced sickness behaviour
We previously showed that pre-treatment of mice with indomethacin is sufficient to inhibit LPS-induced changes in burrowing activity (Teeling et al., 2007
). In the present study, we aimed to further investigate these observations. We tested various well known anti-inflammatory drugs, including: indomethacin, ibuprofen, acetaminophen (paracetamol) and dexamethasone (), and measured their effect on LPS-induced changes in body temperature, burrowing and open-field activity, and production of inflammatory mediators. Mice were habituated to burrowing prior to the experiment. On the day of the experiments, mice received an intra-peritoneal injection of NSAID or saline, followed 30–60 min later by an intra-peritoneal injection of LPS or saline. Burrowing was assessed 1 and 3 h after injection of LPS, followed by measurement of open-field activity and body temperature. After the analysis of behavioural changes, mice were sacrificed and tissue collected for analysis of inflammatory mediators in serum and brain. All mice showed a similar baseline of burrowing and, as expected, systemic injection of LPS resulted in a marked suppression of burrowing (A, F(4,39)
= 40.99, p
< 0.001). This behavioural change was significantly inhibited by pre-treatment with indomethacin (15 mg/kg, p
< 0.001) and ibuprofen (30 mg/kg, p
< 0.001), while pre-treatment with acetaminophen (20 or 100 mg/kg (data not shown)) or dexamethasone (2 mg/kg) had no effect.
Anti-inflammatory drugs used in this study.
Fig. 1 Effect of anti-inflammatory drugs on LPS-induced behavioural changes. Burrowing: (A) Mice were pre-treated with indomethacin (15 mg/kg), ibuprofen (15 mg/kg), dexamethasone (2 mg/kg) or paracetamol (20 mg/kg) given by intra-peritoneal (more ...)
The open-field activity showed a similar effect; all mice showed a similar baseline and injection of LPS resulted in a marked suppression of the number of rears (data not shown) and the total distance travelled in an open field (B, F(4,39) = 23.57, p < 0.001). Pre-treatment with indomethacin (p < 0.001) and, albeit to a lesser degree, ibuprofen (p < 0.001) reversed the effect of LPS on open-field activity, while pre-treatment with acetaminophen or dexamethasone did not. To confirm the biological activity of the anti-inflammatory drugs used in our model, we measured PGE2 levels in the hypothalamus, body temperature and the circulating cytokine production. shows that LPS-induced PGE2 levels in the hypothalamus were completely blocked by indomethacin and significantly reduced by dexamethasone (A, F(3,24) = 10.92, p = 0.02). Although not statistically significant, ibuprofen also markedly reduced the LPS-induced PGE2 production in the brain. B shows that the LPS-induced hypothermia was completely blocked by dexamethasone and reduced by all other anti-inflammatory drugs tested. C shows the effect of two of the anti-inflammatory agents, indomethacin and dexamethasone, on systemic IL-6, IL-1β and TNF-α production. Indomethacin had no significant effect on LPS-induced cytokine production and even increased levels of circulating TNF-α were observed. Dexamethasone, on the other hand, completely abolished LPS-induced IL-1β, IL-6 and TNF-α production. These data suggest that, while all drugs tested were biologically active in our model, acute LPS-induced behavioural changes can only be inhibited by a subset of anti-inflammatory drugs, indomethacin and ibuprofen, and the changes in behaviour appear to be independent of blood-borne IL-6, IL-1β and TNF-α.
Fig. 2 Effect of NSAIDs on PGE2 production and fever response to LPS. (A) Effect of indomethacin, ibuprofen and dexamethasone pre-treatment on brain PGE2 protein expression levels measured in punches taken through the hypothalamus taken from brains 6 h (more ...)
3.2. Kinetics of inflammatory mediators during systemic inflammation
We next compared the kinetics of inflammatory mediator production in both the periphery and brain (). For circulating cytokines, we restricted our measurement to IL-6 since we previously showed that, in our model, this cytokine is reliably increased after LPS. Serum levels of IL-6 significantly increased at 2 h, (A, F(1,27) = 47.29, p < 0.0001), and declined sharply to return to baseline levels by 6 h. Comparable kinetics were found for brain IL-6 production in the brain. Brain IL-6 mRNA levels increased after systemic LPS challenge (C, F(5,24) = 6.381, p = 0.0007) showing a significant increase at 2 h and then returned to baseline by 4 h. Brain TNF-α mRNA levels increased significantly after systemic LPS challenge (B, F(5,24) = 5.144, p = 0.0026), peaking at 2 h, after which the cytokine mRNA levels declined sharply and returned to baseline levels by 6 h. No significant changes in brain IL-1β levels were observed (D, F(5,19) = 0.2683), although a trend toward increased levels was seen at 30 min.
Fig. 3 Kinetics of cytokine production in response to systemic immune challenge with LPS. (A) Effect of LPS (100 μg/kg) on expression levels of circulating IL-6 measured in serum samples taken at different time points following intra-peritoneal (more ...)
Circulating PGE2 metabolite levels increased significantly after systemic LPS challenge (E, F(1,27) = 14.25, p < 0.0001) starting at 30 min, and levels remained high for 2 h. At 6 h, PGE2 metabolite levels returned to baseline levels. We measured the hippocampal levels of COX-1 and COX-2 mRNA, the genes that encode the key enzymes responsible for the formation of prostanoids. All NSAIDs inhibited PGE2 levels in the hypothalamus () and since behavioural changes were inhibited by indomethacin and ibuprofen only, we assessed the hippocampus for COX and cytokine expression levels. COX-1, changed modestly after systemic LPS challenge (F, F(5,22) = 2.865, p = 0.0134), however, no statistically significant changes were found between t = 0 and any other time point after LPS. In contrast, the levels of COX-2 mRNA increased after systemic LPS challenge (G, F(5,22) = 2.865, p = 0.0386). A small, non-significant increase was found 1 h after LPS injection and a second significant increase was observed 6 h post LPS challenge. These data suggest that PGE2 levels in the serum precede IL-6 production and that cytokine levels in the brain peak at 2 h.
3.3. The effect of specific inhibitors on LPS-induced behaviour, cytokine and prostaglandin production
To further investigate the biological mechanisms underlying the inhibitory effects of indomethacin and ibuprofen on LPS-induced behavioural changes, we used a series of selective inhibitors, including inhibitors of thromboxane, COX-1, COX-2 and a PPAR-γ agonist. Brain and serum samples were collected 3 h after LPS injection, immediately after the burrowing task when expression of most inflammatory mediators is still increased. shows the results of pre-treatment with the thromboxane synthase inhibitors, ozagrel, picotamide, furegrelate, and the thromboxane receptor antagonist BM 567 on LPS-induced changes in burrowing. The selective inhibitors only modestly affected the LPS-induced changes in burrowing, and none of these changes were significantly different from mice treated with LPS alone (all p > 0.05). These data suggest that increased production of thromboxane cannot explain the effects of LPS on behavioural changes. Pre-treatment of mice with the potent and selective PPAR-γ ligand ciglitazone had no effect on LPS-induced behavioural changes (p > 0.05). These data suggest that direct activation of PPAR-γ does not play a role in the inhibitory effects of indomethacin on LPS-induced behavioural changes.
Fig. 4 Role of thromboxane and PPAR-γ in LPS-induced behavioural changes. Mice were pre-treatment with an intra-peritoneal injection of furegrelate, picotamide, BM 567, ozagrel, ciglitazone or saline as described in Section 2, followed by a intra-peritoneal (more ...)
3.4. Role of COX-1 and COX-2
Thus far, our data suggest a role for COX in LPS-induced changes in burrowing and open-field activity. To investigate the role of the different isoforms of COX we next compared the effect of selective COX-1 and COX-2 inhibitors on LPS-induced behaviour changes. shows the changes in burrowing tested 1–3 h after injection of LPS. Administration of LPS alone significantly decreased burrowing (, F(5,25) = 4.851, p = 0.0046) and mice pre-treated with the COX-1 selective inhibitors piroxicam and sulindac no longer differed from saline-treated mice. In contrast, pre-treatment with the selective COX-2 inhibitor nimesulide or niflumic acid had no effect and mice were still significantly impaired in the burrowing task.
Fig. 5 Role of COX-1 and COX-2 in LPS-induced behavioural changes. Effect of the selective COX-1 inhibitors piroxicam (10 mg/kg) and sulindac (10 mg/kg), or the selective COX-2 inhibitors nimesulide (10 mg/kg) and nuflimic acid (10 mg/kg) (more ...)
We next tested the effect of the inhibitors at various time points after injection of LPS to investigate the possibility that LPS-induced burrowing and open-field activity are differentially regulated over time as was previously reported for other behaviours (Swiergiel and Dunn, 2002
). shows the effect of LPS on burrowing and open-field activity at 2–4, 5–7 and 24 h after injection of LPS in mice pre-treated with the COX-1 specific inhibitor piroxicam or the COX-2 specific inhibitor nimesulide. The anti-inflammatory drugs were suspended in the same vehicle and given 30 min prior to LPS. Administration of LPS significantly reduced burrowing at all time points tested. Piroxicam significantly reversed the effect of LPS on burrowing when tested between 2 and 4 h (, F(1,12)
= 36.91, p
< 0.0001). At later time points piroxicam was no longer protective, which may be explained by the short half life of drug in mice (T1/2
= 1.7 h) (Milne and Twomey, 1980
). Nimesulide (T1/2
= 2–3 h) (Hull et al., 2005
) did not significantly reverse the LPS-induced changes in burrowing at any time point tested (). Similar results were observed for open-field activity: a clear trend towards protection of piroxicam at 2–4 h which disappeared at later time points. Pre-treatment with the drugs alone did not have an effect on burrowing or open-field activity. Interestingly, mice pre-treated with the COX-2 inhibitor appeared to recover better 24 h after LPS injection, compared to LPS-treated only or piroxicam pre-treated mice. The changes did not, however, reach significance. These results suggest that LPS-induced changes in burrowing and open-field activity between 2 and 4 h are largely mediated by COX-1 activity and show a minimal role for COX-2.
Fig. 6 Kinetics of COX-1 and COX-2 in LPS-induced behavioural changes. Effect of the selective COX-1 inhibitor piroxicam (10 mg/kg), or the selective COX-2 inhibitor nimesulide (10 mg/kg) on burrowing and open-field activity measured 1–3, (more ...)
3.5. Cytokines and prostaglandin production
Having established a key role of COX-1 in LPS-induced changes in burrowing and open-field activity, we next investigated the effect of piroxicam and nimesulide on cytokine and PG production. LPS increased serum IL-6 levels measured 3 h post challenge (A, F(3,16) = 5.893, p = 0.0091). Pre-treatment with piroxicam or nimesulide did not affect the serum levels of IL-6. In contrast, circulating PGE2 levels, which were significantly increased 3 h after LPS (B, F(3,17) = 7.885, p = 0.0025), were completely inhibited by pre-treatment with piroxicam (p < 0.05). Selective COX-2 inhibition had no effect on circulating PGE2 levels. Next, we measured cytokine mRNA levels in the brain. TNF-α mRNA was significantly increased 3 h after LPS challenge (C, F(5,25) = 3.723, p = 0.0035). Pre-treatment with piroxicam did not change the mRNA levels of TNF-α in the brain, while, pre-treatment with nimesulide significantly inhibited TNF-α mRNA expression. IL-6 mRNA levels were also increased after LPS challenge (D, F(3,17) = 6.263, p = 0.0064), and like TNF-α, only inhibited by nimesulide pre-treatment. Finally, we measured COX-2 mRNA levels, which were significantly up-regulated 3 h post LPS challenge (E, F(3,18) = 4.674, p = 0.0017). Both piroxicam and nimesulide equally reduced COX-2 mRNA expression and were no longer different from saline-treated mice. The mechanism to explain these unexpected changes in COX-2 remain unknown, but it is possible that measurement at 3 h is too early to detect effects of the anti-inflammatory drugs tested. These data suggest that LPS-induced behavioural changes arise independent of cytokine production, and depend on COX-1 mediated peripheral and/or central PGE2 production. Furthermore, it suggests that cytokine synthesis in the brain, after intra-peritoneal challenge with LPS, largely depend on COX-2 signalling, and not on COX-1.
Fig. 7 Role of COX-1 and COX-2 in LPS-induced behavioural changes and inflammatory mediator production. Effect of the selective COX-1 inhibitor piroxicam (10 mg/kg), or the selective COX-2 inhibitor nimesulide (10 mg/kg) pre-treatment on expression (more ...)