Low-Dose Aspirin Augments the Anti-Inflammatory Effects of Low-Dose Lithium in Lipopolysaccharide-Treated Rats

Abstract

Mounting evidence suggests that immune-system dysfunction and inflammation play a role in the pathophysiology and treatment of mood-disorders in general and of bipolar disorder in particular. The current study examined the effects of chronic low-dose aspirin and low-dose lithium (Li) treatment on plasma and brain interleukin-6 and tumor necrosis factor-α production in lipopolysaccharide (LPS)-treated rats. Rats were fed regular or Li-containing food (0.1%) for six weeks. Low-dose aspirin (1 mg/kg) was administered alone or together with Li. On days 21 and 42 rats were injected with 1 mg/kg LPS or saline. Two h later body temperature was measured and rats were sacrificed. Blood samples, the frontal-cortex, hippocampus, and the hypothalamus were extracted. To assess the therapeutic potential of the combined treatment, rats were administered the same Li + aspirin protocol without LPS. We found that the chronic combined treatment attenuated LPS-induced hypothermia and significantly reduced plasma and brain cytokine level elevation, implicating the potential neuroinflammatory diminution purportedly present among the mentally ill. The combined treatment also significantly decreased immobility time and increased struggling time in the forced swim test, suggestive of an antidepressant-like effect. This preclinical evidence provides a potential approach for treating inflammation-related mental illness.

Keywords: aspirin; bipolar disorder; brain; inflammation; lipopolysaccharide; lithium.

Figure 3

Effects of Li and aspirin co-administration on LPS-induced hypothermia. Rats were fed regular food (control) or Li-containing food (0.1%) for 42 days. Low-dose aspirin (1 mg/kg, ip) was given alone or together with Li. On days 21 (a) and 42 (b) of the treatment protocol, at 2 h before sacrifice, LPS (1 mg/kg) was administered as described under Materials and Methods. BT was measured before and 2 h post LPS injection. The bars show the difference (delta) in BT between the two measurements. Results are means ± SEM of a representative experiment out of two demonstrating a similar pattern, with eight rats/group. (a) Day 21: One-way ANOVA, F = 13.24, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. LPS, LPS + Li and LPS + ASA, p < 0.006, Control vs. LPS + Li + ASA, NS; LPS vs. LPS + Li, p = 0.08; LPS vs. LPS + ASA and LPS + Li + ASA, p < 0.016; LPS + Li vs. LPS + ASA, NS; LPS + Li vs. LPS + Li + ASA, p = 0.0016; LPS + ASA vs. LPS + Li + ASA, p = 0.013. (b) Day 42: One-way ANOVA, F = 36.43, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. LPS, LPS + Li, LPS + ASA and LPS + Li + ASA, p < 0.0007; LPS vs. LPS + Li and LPS + ASA, NS; LPS vs. LPS + Li + ASA, p < 0.0001; LPS + Li vs. LPS + ASA, NS; LPS + Li vs. LPS + Li + ASA, p < 0.0001; LPS + ASA vs. LPS + Li + ASA, p < 0.0001. Asterisks and symbols denote the following: * p < 0.05 vs. Control; # p < 0.05 vs. LPS; ^ p < 0.05 vs. LPS + Li, § p < 0.05 vs. LPS + ASA. Abbreviations: ASA—acetylsalicylic acid, Li—lithium, LPS—lipopolysaccharide, NS—nonsignificant.

Figure 4

Effects of Li and aspirin co-administration on plasma levels of IL-6 and TNF-α. Rats were fed regular food (control) or Li-containing food (0.1%) for 42 days. Low-dose aspirin (1 mg/kg, ip) was given alone or together with Li. On days 21 (a,c) and 42 (b,d) of the treatment protocol, 2 h before sacrifice, LPS (1 mg/kg) was administered as described under Materials and Methods. Determination of IL-6 and TNF-α was done using specific ELISA kits. Results are means ± SEM of a representative experiment out of two demonstrating a similar pattern, with eight rats/group. (a) Plasma IL-6 on day 21: One-way ANOVA, F = 15.53, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. LPS, LPS + Li and LPS + ASA, p < 0.003; Control vs. LPS + Li + ASA, NS; LPS vs. LPS + Li, LPS + ASA and LPS + Li + ASA, p <0.0002; LPS + Li vs. LPS + ASA, NS; LPS + Li vs. LPS + Li + ASA, p = 0.052; LPS + ASA vs. LPS + Li + ASA, NS. (b) Plasma IL-6 on day 42: One-way ANOVA, F = 33.90, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. LPS, LPS + Li, LPS + ASA and LPS + Li + ASA, p < 0.0001; LPS vs. LPS + Li and LPS + ASA, NS; LPS vs. LPS + Li + ASA, p < 0.0001; LPS + Li vs. LPS + ASA, NS; LPS + Li vs. LPS + Li + ASA, p = 0.005, LPS + ASA vs. LPS + Li + ASA, p = 0.0018. (c) Plasma TNF-α levels on day 21: One-way ANOVA, F = 14.23, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. LPS, LPS + Li, LPS + ASA and LPS + Li + ASA, p < 0.03; LPS vs. LPS + Li and LPS + ASA, NS; LPS vs. LPS + Li + ASA, p = 0.0006; LPS + Li vs. LPS + ASA, NS; LPS + Li vs. LPS + Li + ASA, p = 0.001; LPS + ASA vs. LPS + Li + ASA, p = 0.021. (d) Plasma TNF-α levels on day 42: One-way ANOVA, F = 15.55, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. LPS, LPS + Li, LPS + ASA and LPS + Li + ASA, p < 0.03; LPS vs. LPS + Li, LPS + ASA, NS; LPS vs. LPS + Li + ASA, p < 0.0001; LPS + Li vs. LPS + ASA, NS; LPS + Li vs. LPS + Li + ASA, p = 0.007; LPS + ASA vs. LPS + Li + ASA, p = 0.007. Asterisks and symbols denote the following: * p < 0.05 vs. Control, # p < 0.05 vs. LPS; ^ p < 0.05 vs. LPS + Li; § p < 0.05 vs. LPS + ASA. Abbreviations: ASA—acetyl salicylic acid; Li—lithium, LPS—lipopolysaccharide, NS—nonsignificant, UD—undetectable.

Figure 5

Effects of Li and aspirin co-administration on frontal cortex levels of IL-6 and TNF-α. Rats were fed regular food (control) or Li-containing food (0.1%) for 42 days. Low-dose aspirin (1 mg/kg, ip) was given alone or together with Li. On days 21 (a,c) and 42 (b,d) of the treatment protocol, 2 h before sacrifice, LPS (1 mg/kg) was administered as described under Materials and Methods. Determination of IL-6 and TNF-α was done using specific ELISA kits. Results are means ± SEM of a representative experiment out of two demonstrating a similar pattern, with eight rats/group. (a) FC IL-6 levels on day 21: One-way ANOVA, F = 7.199, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. LPS, LPS + Li and LPS + ASA, p < 0.002; Control vs. LPS + Li + ASA, NS; LPS vs. LPS + Li and LPS + ASA, NS; LPS vs. LPS + Li + ASA, p < 0.0001; LPS + Li vs. LPS + ASA, NS; LPS + Li vs. LPS + Li + ASA, p = 0.005; LPS + ASA vs. LPS + Li + ASA, p = 0.004. (b) FC IL-6 levels on day 42: One-way ANOVA, F = 5.131, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. LPS, p = 0.005; Control vs. LPS + Li, LPS + ASA and LPS + Li + ASA, NS; LPS vs. LPS + Li and LPS + ASA, NS; LPS vs. LPS + Li + ASA, p = 0.0037; LPS + Li vs. LPS + ASA and LPS + Li + ASA NS; LPS + ASA vs. LPS + Li + ASA, NS. (c) FC TNF-α levels on day 21: One-way ANOVA, F = 3.323, p = 0.005. Post-hoc Fisher’s LSD test: Control vs. Li, LPS, LPS + Li and LPS + ASA, p < 0.013; Control vs. LPS + Li + ASA, NS; LPS vs. LPS + Li, LPS + ASA, NS; LPS vs. LPS + Li + ASA, p = 0.0047; LPS + Li vs. LPS + ASA and LPS + Li + ASA, NS; LPS + ASA vs. LPS + Li + ASA, p = 0.023. (d) FC TNF-α on day 42: One-way ANOVA, F = 2.559, p = 0.023. Post-hoc Fisher’s LSD test: Control vs. LPS, LPS + Li, NS; Control vs. LPS + ASA, p = 0.032; Control vs. LPS + Li + ASA, NS; LPS vs. LPS + Li, p = 0.262; LPS vs. LPS + ASA, LPS + Li + ASA, NS; LPS + Li vs. LPS + ASA and LPS + Li + ASA, NS; LPS + ASA vs. LPS + Li + ASA, p = 0.043. Asterisks and symbols denote the following: * p < 0.05 vs. Control, # p < 0.05 vs. LPS; ^ p < 0.05 vs. LPS + Li; § p < 0.05 vs. LPS + ASA Abbreviations: ASA—acetylsalicylic acid, Li—lithium, LPS—lipopolysaccharide, NS—nonsignificant.

Figure 5

Effects of Li and aspirin co-administration on frontal cortex levels of IL-6 and TNF-α. Rats were fed regular food (control) or Li-containing food (0.1%) for 42 days. Low-dose aspirin (1 mg/kg, ip) was given alone or together with Li. On days 21 (a,c) and 42 (b,d) of the treatment protocol, 2 h before sacrifice, LPS (1 mg/kg) was administered as described under Materials and Methods. Determination of IL-6 and TNF-α was done using specific ELISA kits. Results are means ± SEM of a representative experiment out of two demonstrating a similar pattern, with eight rats/group. (a) FC IL-6 levels on day 21: One-way ANOVA, F = 7.199, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. LPS, LPS + Li and LPS + ASA, p < 0.002; Control vs. LPS + Li + ASA, NS; LPS vs. LPS + Li and LPS + ASA, NS; LPS vs. LPS + Li + ASA, p < 0.0001; LPS + Li vs. LPS + ASA, NS; LPS + Li vs. LPS + Li + ASA, p = 0.005; LPS + ASA vs. LPS + Li + ASA, p = 0.004. (b) FC IL-6 levels on day 42: One-way ANOVA, F = 5.131, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. LPS, p = 0.005; Control vs. LPS + Li, LPS + ASA and LPS + Li + ASA, NS; LPS vs. LPS + Li and LPS + ASA, NS; LPS vs. LPS + Li + ASA, p = 0.0037; LPS + Li vs. LPS + ASA and LPS + Li + ASA NS; LPS + ASA vs. LPS + Li + ASA, NS. (c) FC TNF-α levels on day 21: One-way ANOVA, F = 3.323, p = 0.005. Post-hoc Fisher’s LSD test: Control vs. Li, LPS, LPS + Li and LPS + ASA, p < 0.013; Control vs. LPS + Li + ASA, NS; LPS vs. LPS + Li, LPS + ASA, NS; LPS vs. LPS + Li + ASA, p = 0.0047; LPS + Li vs. LPS + ASA and LPS + Li + ASA, NS; LPS + ASA vs. LPS + Li + ASA, p = 0.023. (d) FC TNF-α on day 42: One-way ANOVA, F = 2.559, p = 0.023. Post-hoc Fisher’s LSD test: Control vs. LPS, LPS + Li, NS; Control vs. LPS + ASA, p = 0.032; Control vs. LPS + Li + ASA, NS; LPS vs. LPS + Li, p = 0.262; LPS vs. LPS + ASA, LPS + Li + ASA, NS; LPS + Li vs. LPS + ASA and LPS + Li + ASA, NS; LPS + ASA vs. LPS + Li + ASA, p = 0.043. Asterisks and symbols denote the following: * p < 0.05 vs. Control, # p < 0.05 vs. LPS; ^ p < 0.05 vs. LPS + Li; § p < 0.05 vs. LPS + ASA Abbreviations: ASA—acetylsalicylic acid, Li—lithium, LPS—lipopolysaccharide, NS—nonsignificant.

Figure 6

Effects of Li and aspirin co-administration onhippocampal levels of IL-6 and TNF-α. Rats were fed regular food (control) or Li-containing food (0.1%) for 42 days. Low-dose aspirin (1 mg/kg, ip) was given alone or together with Li. On days 21 (a,c) and 42 (b,d) of the treatment protocol, 2 h before sacrifice, LPS (1 mg/kg) was administered as described under Materials and Methods. Determination of IL-6 and TNF-α was done using specific ELISA kits. Results are means ± SEM of a representative experiment out of two demonstrating a similar pattern, with eight rats/group. (a) HC IL-6 levels on day 21: One-way ANOVA, F = 5.421, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. Li, ASA and Li + ASA, p < 0.003; Control vs. LPS and LPS + Li, NS; Control vs. LPS + ASA and LPS + Li + ASA, p < 0.024; LPS vs. LPS + Li, NS; LPS vs. LPS + ASA and LPS + Li + ASA, p < 0.011; LPS + Li vs. LPS + ASA and LPS + Li + ASA, p < 0.045; LPS + ASA vs. LPS + Li + ASA, NS. (b) HC IL-6 levels on day 42: One-way ANOVA, F = 5.439, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. LPS and LPS + Li + ASA, p < 0.005; Control vs. LPS + Li and LPS + ASA, NS; LPS vs. LPS + Li, LPS + ASA and LPS + Li + ASA p < 0.0002; LPS + Li vs. LPS + ASA, NS; LPS + Li vs. LPS + Li + ASA, p = 0.025; LPS + ASA vs. LPS + Li + ASA, NS. (c) HC TNF-α levels on day 21: One-way ANOVA, F = 2.270, p = 0.042. Post-hoc Fisher’s LSD test: Control vs. Li, Li + ASA, LPS, LPS + Li, NS; Control vs. LPS + ASA and LPS + Li + ASA, p < 0.01; LPS vs. LPS + Li, NS; LPS vs. LPS + ASA and LPS + Li + ASA, p < 0.013; LPS + Li vs. LPS + ASA and LPS + Li + ASA, NS; LPS + ASA vs. LPS + Li + ASA, NS. (d) HC TNF-α on day 42: One-way ANOVA, F = 8.160, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. LPS, p < 0.0001; Control vs. LPS + Li, LPS + ASA and LPS + Li + ASA, NS; LPS vs. LPS + Li, LPS + ASA and LPS + Li + ASA, p < 0.002; LPS + Li vs. LPS + ASA, NS; LPS + Li vs. LPS + Li + ASA, p = 0.005; LPS + ASA vs. LPS + Li + ASA, NS. Asterisks and symbols denote the following: * p < 0.05 vs. Control, # p < 0.05 vs. LPS; ^ p < 0.05 vs. LPS + Li. Abbreviations: ASA—acetylsalicylic acid, Li—lithium, LPS—lipopolysaccharide, NS—nonsignificant.

Figure 7

Effects of Li and aspirin co-administration on hypothalamic levels of IL-6 and TNF-α. Rats were fed regular food (control) or Li-containing food (0.1%) for 42 days. Low-dose aspirin (1 mg/kg, ip) was given alone or together with Li. On days 21 (a,c) and 42 (b,d) of the treatment protocol, 2 h before sacrifice, LPS (1 mg/kg) was administered as described under Materials and Methods. Determination of IL-6 and TNF-α was done using specific ELISA kits. Results are means ± SEM of a single representative experiment out of two demonstrating a similar pattern, with eight rats/group. (a) HT IL-6 levels on day 21: One-way ANOVA, F = 5.847, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. Li, Li + ASA, LPS + Li, LPS + ASA and LPS + Li + ASA, NS; Control vs. ASA, p = 0.015; Control vs. LPS, p = 0.025; LPS vs. LPS + Li, LPS + ASA, NS; LPS vs. LPS + Li + ASA, p = 0.004; LPS + Li vs. LPS + ASA, NS; LPS + Li vs. LPS + Li + ASA, p = 0.015; LPS + ASA vs. LPS + Li + ASA, p = 0.02. (b) HT IL-6 levels on day 42: One-way ANOVA, F = 5.566, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. ASA and Li + ASA, p < 0.0025; Control vs. Li, LPS, LPS + Li, LPS + ASA and LPS + Li + ASA, NS; LPS vs. LPS + Li, LPS + ASA, LPS + Li + ASA, NS; LPS + Li vs. LPS + ASA and LPS + Li + ASA, NS; LPS + ASA vs. LPS + Li + ASA, NS. (c) HT TNF-α levels on day 21: One-way ANOVA, F = 2, 714, p = 0.017. Post-hoc Fisher’s LSD test: Control vs. Li + ASA, LPS + Li, LPS + ASA and LPS + Li + ASA, NS; Control vs. LPS, p = 0.047; LPS vs. LPS + Li and LPS + ASA, NS; LPS vs. LPS + Li + ASA, p = 0.022; LPS + Li vs. LPS + ASA and LPS + Li + ASA, NS; LPS + ASA vs. LPS + Li + ASA, NS. (d) HT TNF-α on day 42: One-way ANOVA, F = 8.325, p < 0.0001. Post-hoc Fisher’s LSD test: Control vs. Li + ASA, LPS + ASA and LPS + Li + ASA, NS; Control vs. LPS and LPS + Li, p < 0.0058; LPS vs. LPS + Li, LPS + ASA, NS; LPS vs. LPS + Li + ASA, p = 0.0002, LPS + Li vs. LPS + ASA and LPS + Li + ASA, p ≤ 0.001; LPS + ASA vs. LPS + Li + ASA, p = 0.037. Asterisks and symbols denote the following: * p < 0.05 vs. Control, # p < 0.05 vs. LPS; ^ p < 0.05 vs. LPS + Li, § p < 0.05 vs. LPS + ASA. Abbreviations: ASA—acetylsalicylic acid, Li—lithium, LPS—lipopolysaccharide, NS—nonsignificant.

Figure 8

Effects of Li and aspirin co-administration on amphetamine-induced hyperactivity. On day 40 of the treatment protocol, rats were injected with amphetamine (as described in Materials and Methods) after which they were placed in an open field arena for 20 min. Total distance traveled (a) and mean velocity (b) during the last 10 min of the sessions were assessed by a video-tracking system. Results are means ± SEM of a single representative experiment out of two demonstrating a similar pattern, with 12 rats per group. (a) Total distance traveled: one-way ANOVA, p = NS. (b) Mean velocity: one-way ANOVA, p = NS. Abbreviations: ASA—acetylsalicylic acid, Li—lithium, NS—nonsignificant.

Figure 9

Effects of Li and aspirin co-administration on rats’ performance in the FST. On day 42 of the treatment protocol, rats were subjected to a 5 min FST session. Rats’ behavior was videotaped and subsequently analyzed by a video-tracking system. Immobility time and struggling time were measured as described in Materials and Methods. Results are means ± SEM of a single representative experiment out of two demonstrating a similar pattern, with 12 rats per group. (a) Immobility time: one-way ANOVA, p = NS. Student’s-t test for the difference between the Control and the Li + ASA group, p = 0.0235. (b) Struggling time: one-way ANOVA, p = 0.029. Post-hoc Fisher’s LSD test: Control and the Li + ASA group, F = 3.298, p = 0.011. Asterisks and symbols denote the following: * p < 0.05 vs. Control. Abbreviations: ASA—acetylsalicylic acid, FST—forced swim test, Li—lithium, NS—nonsignificant.

Figure 1

Timeline of the LPS-induced inflammation experiment. BC = blood collection; D = day; LPS = lipopolysaccharide; TC = tissue collection; W = week.

Figure 2

Timeline of the behavior experiments. AHT = amphetamine-induced hyperactivity test; D = day; FST = forced swim test; OFT = open field test; W = week.