Intermittent fasting attenuates lipopolysaccharide-induced neuroinflammation and memory impairment

Abstract

Background: Systemic bacterial infections often result in enduring cognitive impairment and are a risk factor for dementia. There are currently no effective treatments for infection-induced cognitive impairment. Previous studies have shown that intermittent fasting (IF) can increase the resistance of neurons to injury and disease by stimulating adaptive cellular stress responses. However, the impact of IF on the cognitive sequelae of systemic and brain inflammation is unknown.

Methods: Rats on IF for 30 days received 1 mg/kg of lipopolysaccharide (LPS) or saline intravenously. Half of the rats were subjected to behavioral tests and the other half were euthanized two hours after LPS administration and the hippocampus was dissected and frozen for analyses.

Results: Here, we report that IF ameliorates cognitive deficits in a rat model of sepsis by a mechanism involving NF-κB activation, suppression of the expression of pro-inflammatory cytokines, and enhancement of neurotrophic support. Treatment of rats with LPS resulted in deficits in cognitive performance in the Barnes maze and inhibitory avoidance tests, without changing locomotor activity, that were ameliorated in rats that had been maintained on the IF diet. IF also resulted in reduced levels of mRNAs encoding the LPS receptor TLR4 and inducible nitric oxide synthase (iNOS) in the hippocampus. Moreover, IF prevented LPS-induced elevation of IL-1α, IL-1β and TNF-α levels, and prevented the LPS-induced reduction of BDNF levels in the hippocampus. IF also significantly attenuated LPS-induced elevations of serum IL-1β, IFN-γ, RANTES, TNF-α and IL-6 levels.

Conclusions: Taken together, our results suggest that IF induces adaptive responses in the brain and periphery that can suppress inflammation and preserve cognitive function in an animal model of systemic bacterial infection.

Figures

Figure 1

Design of experiments to determine the effects of intermittent fasting (IF) on cognitive function and hippocampal neuroinflammation. (A) For the biochemical experiments, rats were subjected to IF or ad libitum Control diets for 30 days. To determine whether IF modulated the inflammatory response to a subsequent challenge, rats were injected intravenously with either 1 mg/kg lipopolysaccharide (LPS) or saline on day 31, and 2 hours later brain tissue and serum samples were collected for subsequent analysis. (B) For the behavioral experiments, rats were subjected to the habituation session of the Barnes maze and were immediately injected intravenously with either 1 mg/kg LPS or saline on day 31. Barnes maze testing sessions and inhibitory avoidance tests were performed on days 32 to 36 and 37 to 38, respectively. Locomotor activity test was performed 24 hours after LPS administration and rotarod tests were performed on days 32 to 35.

Figure 2

Intermittent fasting (IF) prevents lipopolysaccharide (LPS)-induced memory impairment in the Barnes maze and inhibitory avoidance task. (A) Barnes maze test results (***P < 0.001 versus IF and IF + LPS groups). Data are represented as the escape latency (time in seconds that the rat takes to find the correct hole containing the burrow). (B) Inhibitory avoidance test results (*P < 0.05 IF test session versus its training session; **P < 0.01 Control test session versus its training session; ***P < 0.001 IF + LPS test session versus its training session; #P < 0.05 IF + LPS versus LPS test sessions). Both tests showed that IF attenuated memory impairment elicited by LPS challenge. (C) Beginning 24 hours after LPS injection, mice were tested daily for motor performance on a rotarod. The latency to fall during the testing period (four days) are shown. (D, E) Results of the analysis of spontaneous locomotor activity in the electronic animal activity meter showing the total locomotion (D) and total rearing (E) 24 hours after LPS administration. N = 8 to 10 for each experimental group.

Figure 3

Lipopolysaccharide LPS) treatment and intermittent fasting (IF) increase the NF-κB nuclear translocation and binding activity. Electrophoretic mobility shift assay (EMSA) and Western blot were performed using nuclear extract (10 μg) from rat hippocampus. (A) Western blot results using antibodies to RELA and β-ACTIN showed that IF and LPS treatment with or without IF increased the nuclear translocation of RELA. (B) EMSA showed that LPS treatment increased the NF-κB binding activity in rats submitted or not to IF protocol. IF by itself also increased NF-κB binging activity. Competition and supershift assays of NF-κB activation by LPS treatment (C), IF (D) and IF with LPS treatment (E) were carried out. Competition studies were performed using the nuclear extract in the absence or presence of unlabeled specific (NF-κB consensus sequence, 20-fold molar excess) or nonspecific (TFIID consensus sequence, 20-fold molar excess) oligonucleotide, as indicated. Supershift assays were performed with the same nuclear extract incubated in the absence or presence of antibodies (1:20 dilution) against subunits p50, RELA, and cREL as indicated. Antibodies were added 20 minutes prior to addition of the radiolabeled NF-κB consensus oligonucleotide. The positions of specific NF-κB/DNA and non-specific (NS) binding complexes are indicated. *P < 0.05 versus Control; #P < 0.05 versus IF. N = 5 for each experimental group.

Figure 4

Intermittent fasting reduces toll-like receptor 4 (TLR4) and attenuates lipopolysaccharide (LPS)-induced synthase (iNOS) up-regulation in the hippocampus. (A) Levels of Tlr-4 mRNA (*P = 0.008 versus Control; **P = 0.004 versus Control; #P = 0.016 versus LPS). (B) Levels of iNos mRNA (**P = 0.015 versus Control; *P = 0.013 versus Control; #P = 0.045 versus LPS). The mRNA levels were measured by quantitative RT-PCR in samples from hippocampus. N = 5 for each experimental group.

Figure 5

Intermittent fasting (IF) inhibits lipopolysaccharide LPS)-induced increases in inflammatory mediators in the hippocampus and serum. Levels of hippocampal IL-1α (A), IL-1β (B), TNF-α (C), IL-10 (D) and RANTES (E), and levels of serum IL-1β (F), TNF-α (G), IL-6 (H), RANTES (I) and IFN-γ (J) were measured by multiplex or ELISA (*P < 0.05 versus Control; **P < 0.01 versus Control; ***P < 0.001 versus Control; #P < 0.05 versus LPS; ##P < 0.01 versus LPS and ###P < 0.001 versus LPS. N = 5 to 10 for each experimental group.

Figure 6

Intermittent fasting (IF) prevents lipopolysaccharide (LPS)-induced depletion of hippocampal brain-derived neurotrophic factor (BDNF). Levels of BDNF were measured in hippocampal extracts by ELISA (*P < 0.05 versus Control, IF, and IF + LPS groups. N = 10 for each experimental group.