Intermittent fasting attenuates lipopolysaccharide-induced neuroinflammation and memory impairment

Andrea R Vasconcelos 0 Lidia M Yshii 0 Tania A Viel Hudson S Buck Mark P Mattson Cristoforo Scavone 0 Elisa M Kawamoto 0 0 Department of Pharmacology, Institute of Biomedical Science, University of Sao Paulo , Sao Paulo 05508-900 , Brazil 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-B 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-1, IL-1 and TNF- levels, and prevented the LPS-induced reduction of BDNF levels in the hippocampus. IF also significantly attenuated LPS-induced elevations of serum IL-1, IFN-, RANTES, TNF- 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. - Introduction Systemic inflammation/sepsis is a risk factor for cognitive impairment and dementia [1]. The elderly are vulnerable to the adverse effects of infections on cognitive function, and the aging process itself is associated with increased neuroinflammatory processes involving microglial activation and production of pro-inflammatory cytokines [2,3]. Inflammation also occurs in association with the pathological changes in the brains of patients with Alzheimers disease (AD) and ischemic stroke [4]. However, clinical trials of anti-inflammatory therapies for AD, including NSAIDs, TNF inhibitors and intravenous immunoglobulin [5,6], have not been encouraging [7-10]. NF-B, the activity of which is attributed to the Rel/ NF-B family proteins forming homo- and heterodimers through the combination of the subunits p65 (or RELA), p50, p52, cREL and RELB, can be activated by lipopolysaccharide (LPS), cytokines such as TNF- and IL-1, and reactive oxygen species [11,12]. NF-B, which is constitutively expressed in the cytoplasm, is inhibited by a family of molecules termed inhibitor B (IBs). IB binds NF-B and masks its nuclear localization signal, thus retaining it in the cytoplasm [13]. Inducers of NFB act by intracellular signaling pathways that activate the IB kinases (IKKs), which phosphorylate two specific N-terminal serines of IB, resulting in IB polyubiquitination and degradation in the 26S protease [14]. When IB is degraded, NF-B migrates to the nucleus, modulating the transcription of several genes associated with neurodegenerative or neuroprotective actions [15,16]. Bacterial infections activate innate immune signaling pathways involving toll-like receptor 4 (TLR4) and the transcription factor NF-B in microglia and macrophages, which induces the expression of pro-inflammatory cytokines and the production of nitric oxide [17]. These pathways play critical roles in the killing and degradation of the bacteria by immune cells, but can also adversely affect neurons. Studies of TLR4-deficient mice suggest that TLR4 signaling has a negative impact on hippocampusdependent cognitive function [18]. Data further suggest that TLR4 activation contributes to the degeneration of neurons in experimental models of AD [19,20] and stroke [21]. Lipopolysaccharide (LPS) is a major bacterial TLR4 ligand that activates the innate immune response to infection, and administration of LPS can cause cognitive impairment in animal models by mechanisms involving expression of pro-inflammatory cytokines and inhibition of neurotrophic factor production [22-26]. It is known that pro-inflammatory mediators disrupt hippocampal neuronal functions, including long-term potentiation and working memory consolidation [27,28]. Cytokines such as TNF-, IL-1 and IL-6 are involved in hippocampal longterm potentiation and dendritic branching, which are processes involved in memory formation and maintenance [29]. In addition, it has already been shown that systemic administration of TNF- reduces cell proliferation in the hippocampus, whereas no effect is observed with single doses of IL-1 and IL-6 [30]. IF can improve cognitive function in mouse models relevant to AD [31,32] and in old rats [33]. Also, IF reduces brain damage, increases levels of neurotrophic factors, and reduces markers of inflammation in a mouse model of focal ischemic stroke [34]. Here, we report that IF ameliorates cognitive deficits in a rat model of systemic bacterial infection, by a mechanism involving suppression of neuroinflammation and maintenance of neurotrophic support. Our findings suggest a potential for application of IF for the prevention and treatment of the cognitive deficits resulting from systemic inflammation. Material and methods Animals Adult 12-week-old male Wistar rats were kept under a 12-hour light/12-hour dark cycle and allowed free access to water. All treatments were administered between 9:00 and 10:00 am. Rats were randomly assigned to a normal feeding group and an alternate-day fasting diet (IF). Rats on the IF diet were deprived of food for 24 hours every other day for 30 days. On the 31st day, after being given ad libitum access to food for 24 hours to avoid the effects of acute fasting, each animal received 1 mg/kg of LPS (O111:B4) (Sigma-Aldrich, St Louis, MO, USA) or saline intravenously [35]. Thus, four groups were used: saline (Control), LPS treatment (LPS), IF with saline (IF) and IF with LPS treatment (IF + LPS). The LPS challenge was performed approximately three hours after lights on (ranging from 09:00 am to 10:00 am). Half of the rats were subjected to behavioral tests, and the other half were euthanized two hours after LPS administration [35] and the hippocampus was dissected and frozen for analyses, according to the timelines shown in Figure 1. This research was approved by the Biomedical College of Animal Experimentation (COBEA). All pr (...truncated)