Effect of poly I:C on IL-1ra mRNA expression in the various brain regions.

<p>Tissue samples (cerebral cortex, hippocampus, hypothalamus, and cerebellum) were prepared 5 h and 1, 3, 5, and 7 days after poly I:C injection. The bar plots show the levels of IL-1ra mRNA as the fold change relative to the corresponding vehicle-injected group after calibration with the GAPDH mRNA level. N = 4–5 per experimental group. *P<0.05 versus vehicle-injected group.</p

Transient fever and decreased spontaneous activity in response to rat recombinant (rr) IL-1β injection.

<p>Rats were injected with rrIL-1β (30 µg/kg, i.p.) or the vehicle (saline) at time (<i>t</i>)  = 0 min. (A) The line plots depict the rectal temperature just before and 1, 3, 5, 7, and 24 h after the injection. (B) The line plot represents the percent change in spontaneous activity from baseline. Spontaneous activity was measured from the onset of and throughout the dark period. Arrows depicts the time of injection. N = 5–8 per group. *P<0.05 versus vehicle-injected group.</p

Effect of i.c.v. infusion of rrIL-1ra on poly I:C-induced decrease in spontaneous activity.

<p>Poly I:C-induced spontaneous activity in the presence of i.c.v. infusion of rat recombinant IL-1 receptor antagonist (rrIL-1ra, 1 µg/day). Infusion (at a rate of 1 µl/h) was started 1 day before poly I:C injection and continued for 5 days. The line plots represent the percent change in spontaneous activity from baseline. Spontaneous activity was measured from the onset of and throughout the dark period. Arrows depict the time of poly I:C injection. N = 5–8 per experimental group. *P<0.05, versus poly I:C + vehicle infusion group.</p

Effect of i.c.v. infusion of anti-IL-1ra neutralizing antibody on poly I:C-induced decreased spontaneous activity.

<p>(A) Immunoblotting result. rrIL-1ra (300, 1000 pg) was detected by cross-reactivity of the anti-mouse neutralizing antibody. Infusion (100 ng/µl, at a rate of 1 µl/h) was started 7 days before poly I:C injection and continued for 5 days. (B) Before injection and (C) after injection of poly I:C or the vehicle. The line plots represent the percent change in spontaneous activity from baseline. Spontaneous activity was measured from the onset of and throughout the dark period. N = 4–8 per experimental group. Arrows depict the time of poly I:C or the vehicle injection. *P<0.05 versus poly I:C + i.c.v. infusion of non-immune IgG group. In this procedure, we injected poly I:C or the vehicle three times (10:00, 18:00, and 10:00 on the next day) to precisely evaluate the effect of neutralizing antibody on the recovery speed of spontaneous activity. We infused antibody from 7 days before poly I:C injection, due to slow diffusion speed of antibody with a big chemical structure.</p

Transient fever and decreased spontaneous activity in response to poly I:C injection, and the effect of NS-398.

<p>Rats were injected with the poly I:C (3 mg/kg, i.p.) or the vehicle (saline) at time (<i>t</i>)  =  0 min. NS-398 was injected i.p. 5 min before and 4 h after poly I:C injection. (A) The line plot depicts the rectal temperature just before and 1, 3, 5, 7, and 24 h after poly I:C injection. (B) The line plots represent the percent change in spontaneous activity from baseline. Spontaneous activity was measured from the onset of and throughout the dark period. Arrows depict the time of injections. N = 5–8 per group. *P<0.05 versus vehicle-injected group, ^#P<0.05 versus poly I:C-injected group.</p

Brain Interleukin-1β and the Intrinsic Receptor Antagonist Control Peripheral Toll-Like Receptor 3-Mediated Suppression of Spontaneous Activity in Rats

<div><p>During acute viral infections such as influenza, humans often experience not only transient fever, but also prolonged fatigue or depressive feelings with a decrease in social activity for days or weeks. These feelings are thought to be due to neuroinflammation in the brain. Recent studies have suggested that chronic neuroinflammation is a precipitating event of various neurological disorders, but the mechanism determining the duration of neuroinflammation has not been elucidated. In this study, neuroinflammation was induced by intraperitoneal injection of polyriboinosinic:polyribocytidylic acid (poly I:C), a Toll-like receptor-3 agonist that mimics viral infection in male Sprague-Dawley rats, and then investigated how the neuroinflammation shift from acute to the chronic state. The rats showed transient fever and prolonged suppression of spontaneous activity for several days following poly I:C injection. NS-398, a cyclooxygenase-2 inhibitor, completely prevented fever, but did not improve spontaneous activity, indicating that suppression of spontaneous activity was not induced by the arachidonate cascade that generated the fever. The animals overexpressed interleukin (IL)-1β and IL-1 receptor antagonist (IL-1ra) in the brain including the cerebral cortex. Blocking the IL-1 receptor in the brain by intracerebroventricular (i.c.v.) infusion of recombinant IL-1ra completely blocked the poly I:C-induced suppression of spontaneous activity and attenuated amplification of brain interferon (IFN)-α expression, which has been reported to produce fatigue-like behavior by suppressing the serotonergic system. Furthermore, i.c.v. infusion of neutralizing antibody for IL-1ra prolonged recovery from suppression of spontaneous activity. Our findings indicated that IL-1β is the key trigger of neuroinflammation and that IL-1ra prevents the neuroinflammation entering the chronic state.</p></div

Effect of i.c.v. infusion of rrIL-1ra on brain IFN-α mRNA expression in the brain.

<p>On day 1 and day 7 after poly I:C injection, tissue samples (cerebral cortex, hippocampus, and hypothalamus) were prepared. The bar plots show the levels of IFN-α mRNA as the fold change relative to the corresponding vehicle-injected group after calibration with the GAPDH mRNA level. N = 4–5 per experimental group. *P<0.05.</p

Alteration in IL-1β mRNA expression in the several brain regions following poly I:C injection.

<p>Tissue samples (cerebral cortex, hippocampus, hypothalamus, and cerebellum) were prepared 5 h and 1, 3, 5, and 7 days after poly I:C injection. The bar plots show the levels of IL-1β mRNA as the fold change relative to the corresponding vehicle-injected group after calibration with the GAPDH mRNA level. N = 4–5 per experimental group. *P<0.05 versus vehicle-injected group.</p

Potential Biomarkers of Fatigue Identified by Plasma Metabolome Analysis in Rats

<div><p>In the present study, prior to the establishment of a method for the clinical diagnosis of chronic fatigue in humans, we validated the utility of plasma metabolomic analysis in a rat model of fatigue using capillary electrophoresis-mass spectrometry (CE-MS). In order to obtain a fatigued animal group, rats were placed in a cage filled with water to a height of 2.2 cm for 5 days. A food-restricted group, in which rats were limited to 10 g/d of food (around 50% of the control group), was also assessed. The food-restricted group exhibited weight reduction similar to that of the fatigued group. CE-MS measurements were performed to evaluate the profile of food intake-dependent metabolic changes, as well as the profile in fatigue loading, resulting in the identification of 48 metabolites in plasma. Multivariate analyses using hierarchical clustering and principal component analysis revealed that the plasma metabolome in the fatigued group showed clear differences from those in the control and food-restricted groups. In the fatigued group, we found distinctive changes in metabolites related to branched-chain amino acid metabolism, urea cycle, and proline metabolism. Specifically, the fatigued group exhibited significant increases in valine, leucine, isoleucine, and 2-oxoisopentanoate, and significant decreases in citrulline and hydroxyproline compared with the control and food-restricted groups. Plasma levels of total nitric oxide were increased in the fatigued group, indicating systemic oxidative stress. Further, plasma metabolites involved in the citrate cycle, such as cis-aconitate and isocitrate, were reduced in the fatigued group. The levels of ATP were significantly decreased in the liver and skeletal muscle, indicative of a deterioration in energy metabolism in these organs. Thus, this comprehensive metabolic analysis furthered our understanding of the pathophysiology of fatigue, and identified potential diagnostic biomarkers based on fatigue pathophysiology.</p></div

Random forest analysis of the plasma metabolome.

<p>The mean decrease in accuracy from the random forest analysis was used to rank metabolites according to their prognostic importance for fatigue status. The 15 most important metabolites among three groups (control, food-restricted, and fatigued) or two groups (fatigued and a combined control and food-restricted group) are shown in A and B, respectively.</p