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International audience; Westernization of dietary habits has led to a progressive reduction in dietary intake of n-3 polyunsaturated fatty acids (n-3 PUFAs). Low maternal intake of n-3 PUFAs has been linked to neurodevelopmental disorders, conditions in which myelination processes are abnormal, leading to defects in brain functional connectivity. Only little is known about the role of n-3 PUFAs in oligodendrocyte physiology and white matter development. Here, we show that lifelong n-3 PUFA deficiency disrupts oligodendrocytes maturation and myelination processes during the postnatal period in mice. This has long-term deleterious consequences on white matter organization and hippocampus-prefrontal functional connectivity in adults, associated with cognitive and emotional disorders. Promoting developmental myelination with clemastine, a first-generation histamine antagonist and enhancer of oligodendrocyte precursor cell differentiation, rescues memory deficits in n-3 PUFA deficient animals. Our findings identify a novel mechanism through which n-3 PUFA deficiency alters brain functions by disrupting oligodendrocyte maturation and brain myelination during the neurodevelopmental period.

SUMMARYOmega-3 fatty acids (n-3 polyunsaturated fatty acids; n-3 PUFAs) are essential for the functional maturation of the brain. Westernization of dietary habits in both developed and developing countries is accompanied by a progressive reduction in dietary intake of n-3 PUFAs. Low maternal intake of n-3 PUFAs has been linked to neurodevelopmental diseases in epidemiological studies, but the mechanisms by which a n-3 PUFA dietary imbalance affects CNS development are poorly understood. Active microglial engulfment of synaptic elements is an important process for normal brain development and altered synapse refinement is a hallmark of several neurodevelopmental disorders. Here, we identify a molecular mechanism for detrimental effects of low maternal n-3 PUFA intake on hippocampal development. Our results show that maternal dietary n-3 PUFA deficiency increases microglial phagocytosis of synaptic elements in the developing hippocampus, through the activation of 12/15- lipoxygenase (LOX)/12-HETE signaling, which alters neuronal morphology and affects cognition in the postnatal offspring. While women of child bearing age are at higher risk of dietary n-3 PUFA deficiency, these findings provide new insights into the mechanisms linking maternal nutrition to neurodevelopmental disorders.One Sentence SummaryLow maternal omega-3 fatty acids intake impairs microglia-mediated synaptic refinementvia12-HETE pathway in the developing brain.

International audience; Maternal immune activation (MIA) and poor maternal nutritional habits are risk factors for the occurrence of neurodevelopmental disorders (NDD). Human studies show the deleterious impact of prenatal inflammation and low n-3 polyunsaturated fatty acid (PUFA) intake on neurodevelopment with long-lasting consequences on behavior. However, the mechanisms linking maternal nutritional status to MIA are still unclear, despite their relevance to the etiology of NDD. We demonstrate here that low maternal n-3 PUFA intake worsens MIA-induced early gut dysfunction, including modification of gut microbiota composition and higher local inflammatory reactivity. These deficits correlate with alterations of microglia-neuron crosstalk pathways and have long-lasting effects, both at transcriptional and behavioral levels. This work highlights the perinatal period as a critical time window, especially regarding the role of the gut-brain axis in neurodevelopment, elucidating the link between MIA, poor nutritional habits, and NDD. Fig. 1 EFFECT OF N-3 PUFA DEFICIENCY ON MIA-INDUCED BEHAVIORAL DEFICITS IN NEONATES AND IN ADULT OFFSPRING.: All graphs show Means ± SEM. a Experimental setup. b Average time spent by pups to achieve the Fox battery tests (negative geotaxis and righting reflex; 3 trials per day from PND4 to PND6). N = 14-19. Two-way ANOVA: MIA effect, F(1,62) = 11.67, p = 0.0011. c Average vocalization time (15-min sessions at PND7-8). N = 14-19. Kruskal-Wallis test followed by Mann-Whitney comparison; n-3 sufficient-Saline vs n-3 sufficient-LPS, **p

International audience; First evidence started to demonstrate the anxiolytic effects of low molecular weight peptides extracted from natural products, such as fish hydrolysate, but their underlying mechanisms remain to be elucidated. The objective of this study was to evaluate the effect of a chronic administration of fish hydrolysate on stress reactivity and to understand the mechanisms involved. Stress response (corticosterone secretion, expression of stress-responsive genes) was measured in Balb/c mice supplemented with fish hydrolysate (300 mg/kg body weight) or vehicle daily for 7 days before being submitted to an acute mild stress protocol. Our results demonstrated that 30 min after stress induction, fish hydrolysate decreased corticosterone level compared to control mice. Moreover, fish hydrolysate supplementation modulated expression of stress responsive genes involved in hypothalamic pituitary adrenal axis regulation, circadian rhythm and aging process. These findings suggest that fish hydrolysate represents an innovative strategy to prevent stress-induced aversive effects and participate in stress management.

Long-chain (LC) n-3 polyunsaturated fatty acids (PUFAs) have drawn attention in the field of neuropsychiatric disorders, in particular depression. However, whether dietary supplementation with LC n-3 PUFA protects from the development of mood disorders is still a matter of debate. In the present study, we studied the effect of a two-month exposure to isocaloric diets containing n-3 PUFAs in the form of relatively short-chain (SC) (6% of rapeseed oil, enriched in α-linolenic acid (ALA)) or LC (6% of tuna oil, enriched in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) PUFAs on behavior and synaptic plasticity of mice submitted or not to a chronic social defeat stress (CSDS), previously reported to alter emotional and social behavior, as well as synaptic plasticity in the nucleus accumbens (NAc). First, fatty acid content and lipid metabolism gene expression were measured in the NAc of mice fed a SC (control) or LC n-3 (supplemented) PUFA diet. Our results indicate that LC n-3 supplementation significantly increased some n-3 PUFAs, while decreasing some n-6 PUFAs. Then, in another cohort, control and n-3 PUFA-supplemented mice were subjected to CSDS, and social and emotional behaviors were assessed, together with long-term depression plasticity in accumbal medium spiny neurons. Overall, mice fed with n-3 PUFA supplementation displayed an emotional behavior profile and electrophysiological properties of medium spiny neurons which was distinct from the ones displayed by mice fed with the control diet, and this, independently of CSDS. Using the social interaction index to discriminate resilient and susceptible mice in the CSDS groups, n-3 supplementation promoted resiliency. Altogether, our results pinpoint that exposure to a diet rich in LC n-3 PUFA, as compared to a diet rich in SC n-3 PUFA, influences the NAc fatty acid profile. In addition, electrophysiological properties and emotional behavior were altered in LC n-3 PUFA mice, independently of CSDS. Our results bring new insights about the effect of LC n-3 PUFA on emotional behavior and synaptic plasticity.

Background: Two different species of sage, Salvia officinalis and Salvia lavandulaefolia, have demonstrated activities in cognitive function during preclinical and clinical studies related to impaired health situations or single administration. Different memory processes have been described to be significantly and positively impacted. Objective: Our objective is to explore the potential of these Salvia, and their additional activities, in healthy situations, and during prolonged administration, on memory and subsequent mechanisms of action related to putative effects. Design: This mouse study has implicated four investigational arms dedicated to control, Salvia officinalis aqueous extract, Salvia lavandulaefolia-encapsulated essential oil and a mix thereof (Cognivia&trade, ) for 2 weeks of administration. Cognitive functions have been assessed throughout Y-maze and Morris water maze models. The impact of supplementation on lipid peroxidation, oxidative stress, neurogenesis, neuronal activity, neurotrophins, neurotrophin receptors, CaM kinase II and glucocorticoid receptors has been assessed via post-interventional tissue collection. Results: All Salvia groups had a significant effect on Y-maze markers on day 1 of administration. Only the mix of two Salvia species demonstrated significant improvements in Morris water maze markers at the end of administration. Considering all biological and histological markers, we did not observe any significant effect of S. officinalis, S. lavandulaefolia and a mix of Salvia supplementation on lipid peroxidation, oxidative stress and neuronal plasticity (neurogenesis, neuronal activity, neurotrophins). Interestingly, CaM kinase II protein expression is significantly increased in animals supplemented with Salvia. Conclusion: The activities of Salvia alone after one intake have been confirmed, however, a particular combination of different types of Salvia have been shown to improve memory and present specific synergistic effects after chronic administration in healthy mice.

International audience; Aging is associated to cognitive decline, which can lead to loss of life quality, personal suffering, and ultimately neurodegenerative diseases. Neuroinflammation is one of the mechanisms explaining the loss of cognitive functions. Indeed, aging is associated to the activation of inflammatory signaling pathways, which can be targeted by specific nutrients with anti-inflammatory effects. Dietary n-3 polyunsaturated fatty acids (PUFAs) are particularly attractive as they are present in the brain, possess immunomodulatory properties, and are precursors of lipid derivates named specialized pro-resolving mediators (SPM). SPMs are crucially involved in the resolution of inflammation that is modified during aging, resulting in chronic inflammation. In this review, we first examine the effect of aging on neuroinflammation and then evaluate the potential beneficial effect of n-3 PUFA as precursors of bioactive derivates, particularly during aging, on the resolution of inflammation. Lastly, we highlight evidence supporting a role of n-3 PUFA during aging.

International audience; Rhodiola rosea L. (R. rosea) is an adaptogenic plant increasing body resistance to stress. Its efficacy has been evidenced mainly in chronic stress models, data concerning its effect in acute stress and underlying mechanisms being scarce. The objective was to investigate the effect of repeated doses of a R. rosea hydroethanolic root extract (HRE) on hypothalamic pituitary adrenal response in a murine model of acute mild stress and also the mechanisms involved. Stress response was measured in Balb/c mice having received by gavage HRE (5 g/kg) or vehicle daily for 2 weeks before being submitted to an acute mild stress protocol (open‐field test then elevated plus maze). Corticosterone was measured in plasma from mandibular vein blood drawn before and 30, 60, and 90 min after initiation of the stress protocol. Mice were sacrificed at 90 min, and the hippocampus, prefrontal cortex, and amygdala were excised for high‐frequency RT‐PCR gene expression analysis. At 30 min after acute mild stress induction, corticosterone level in mice having received the HRE was lower than in control mice and comparable to that in nonstressed mice in the HRE group. HRE administration induced brain structure‐dependent changes in expression of several stress‐responsive genes implicated in neuronal structure, HPA axis activation, and circadian rhythm. In the acute mild stress model used, R. rosea HRE decreased corticosterone level and increased expression of stress‐responsive genes, especially in the hippocampus and prefrontal cortex. These findings suggest that R. rosea HRE could be of value for modulating reactivity to acute mild stress.

International audience; The results of several meta-analyses suggest that eicosapentaenoic acid (EPA) supplementation is therapeutic in managing the symptoms of major depression. It was previously assumed that because EPA is extremely low in the brain it did not cross the blood-brain barrier and any therapeutic effects it exerted would be via the periphery. However, more recent studies have established that EPA does enter the brain, but is rapidly metabolised following entry. While EPA does not accumulate within the brain, it is present in microglia and homeostatic mechanisms may regulate its esterification to phospholipids that serve important roles in cell signaling. Furthermore, a variety of signaling molecules from EPA have been described in the periphery and they have the potential to exert effects within the brain. If EPA is confirmed to be therapeutic in major depression as a result of adequately powered randomized clinical trials, future research on brain EPA metabolism could lead to the discovery of novel targets for treating or preventing major depression.

International audience; Metabolic syndrome represents a major risk factor for severe comorbidities such as cardiovascular diseases or diabetes. It is also associated with an increased prevalence of emotional and cognitive alterations that in turn aggravate the disease and related outcomes. Identifying therapeutic strategies able to improve those alterations is therefore a major socioeconomical and public health challenge. We previously reported that both hippocampal inflammatory processes and neuronal plasticity contribute to the development of emotional and cognitive alterations in db/db mice, an experimental model of metabolic syndrome that displays most of the classical features of the syndrome. In that context, nutritional interventions with known impact on those neurobiological processes appear as a promising alternative to limit the development of neurobiological comorbidities of metabolic syndrome. We therefore tested here whether n-3 polyunsaturated fatty acids (n-3 PUFAs) associated with a cocktail of antioxidants can protect against the development of behavioral alterations that accompany the metabolic syndrome. Thus, this study aimed: 1) to evaluate if a diet supplemented with the plant-derived n-3 PUFA α-linolenic acid (ALA) and antioxidants (provided by n-3 PUFAs-rich rapeseed oil fortified with a mix of naturally constituting antioxidant micronutrients, including coenzyme Q10, tocopherol, and the phenolic compound canolol) improved behavioral alterations in db/db mice, and 2) to decipher the biological mechanisms underlying this behavioral effect. Although the supplemented diet did not improve anxiety-like behavior and inflammatory abnormalities, it reversed hippocampus-dependent spatial memory deficits displayed by db/db mice in a water maze task. It concomitantly changed subunit composition of glutamatergic AMPA and NMDA receptors in the hippocampus that has been shown to modulate synaptic function related to spatial memory. These data suggest that changes in local neuronal plasticity may underlie cognitive improvements in db/db mice fed the supplemented diet. The current findings might therefore provide valuable data for introducing new nutritional strategies for the treatment of behavioral complications associated with MetS.

The role of n-3 PUFAs has gained more importance these last decades, especially in inflammatory processes because they can display anti-inflammatory properties. Excessive inflammation can cause local tissue damage. This is especially the case for the brain for which the functional consequences of neuroinflammation include alterations in cognition, affect and behavior leading to a negative impact on the quality of life and well-being of patients. Hence, limiting the inflammation in the brain is a real strategy for neuroinflammatory disease therapy and treatment. Recent data show that n-3 PUFAs exert anti-inflammatory properties in part through the synthesis of specialized pro-resolving mediators such as resolvins that actively turned off the inflammatory response. This review first outlines basic concepts of the role of n-3 PUFAs in neuroinflammation, then summarizes the biosynthesis, signaling pathways and role of resolvins. We finally presented the possible regulation of the resolution of inflammation by dietary means and inflammation.

International audience; Intestinal microbiota plays a role in the communication between the gut and the brain and is now identified as a key player of brain pathologies, including neurodegenerative diseases. The causal link between dysbiosis and brain pathologies could be neuroinflammation, which is characterized by the production of inflammatory factors in the brain by the resident macrophages, microglia. Therefore, the microbiota and brain innate immune system are important factors in the etiopathology of neurodegenerative diseases. In this review, we discuss how probiotics could prevent and treat neurodegenerative diseases through their effect on neuroinflammatory pathways.; Le microbiote intestinal est un acteur clé de la communication entre les intestins et le cerveau et pourrait participer à la physiopathologie de plusieurs maladies du système nerveux, dont les maladies neurodégénératives. Les études des liens causaux entre le microbiote et les pathologies du cerveau font apparaître l’importance de la neuro-inflammation, qui se définit par les processus inflammatoires qui se produisent au sein du cerveau. Ainsi, le microbiote et le système immunitaire cérébral jouent un rôle clé dans l’étiopathogénie des maladies neurodégénératives. Dans cette revue, nous discutons du rôle des probiotiques, de leurs effets protecteurs vis-à-vis de la neuroinflammation et de leur impact sur la prévention et le traitement des pathologies neurodégénératives.

International audience; The brain is highly enriched in long chain polyunsaturated fatty acids (LC-PUFAs) that display immunomodulatory properties in the brain. At the periphery, the modulation of inflammation by LC-PUFAs occurs through lipid mediators called oxylipins which have anti-inflammatory and pro-resolving activities when derived from n-3 LC-PUFAs and pro-inflammatory activities when derived from n-6 LC-PUFAs. However, whether a diet rich in LC-PUFAs modulates oxylipins and neuroinflammation in the brain has been poorly investigated. In this study, the effect of a dietary n-3 LC-PUFA supplementation on oxylipin profile and neuroinflammation in the brain was analyzed. Mice were given diets deficient or supplemented in n-3 LC-PUFAs for a 2-month period starting at post-natal day 21, followed by a peripheral administration of lipopolysaccharide (LPS) at adulthood. We first showed that dietary n-3 LC-PUFA supplementation induced n-3 LC-PUFA enrichment in the hippocampus and subsequently an increase in n-3 PUFA-derived oxylipins and a decrease in n-6 PUFA-derived oxylipins. In response to LPS, n-3 LC-PUFA deficient mice presented a pro-inflammatory oxylipin profile whereas n-3 LC-PUFA supplemented mice displayed an anti-inflammatory oxylipin profile in the hippocampus. Accordingly, the expression of cyclooxygenase-2 and 5-lipoxygenase, the enzymes implicated in pro- and anti-inflammatory oxylipin synthesis, was induced by LPS in both diets. In addition, LPS-induced pro-inflammatory cytokine increase was reduced by dietary n-3 LC-PUFA supplementation. These results indicate that brain n-3 LC-PUFAs increase by dietary means and promote the synthesis of anti-inflammatory derived bioactive oxylipins. As neuroinflammation plays a key role in all brain injuries and many neurodegenerative disorders, the present data suggest that dietary habits may be an important regulator of brain cytokine production in these contexts.

International audience; Maternal immune activation (MIA) is a common environmental insult on the developing brain and represents a risk factor for neurodevelopmental disorders. Animal models of in utero inflammation further revealed a causal link between maternal inflammatory activation during pregnancy and behavioural impairment relevant to neurodevelopmental disorders in the offspring. Accumulating evidence point out that proinflammatory cytokines produced both in the maternal and fetal compartments are responsible for social, cognitive and emotional behavioral deficits in the offspring. Polyunsaturated fatty acids (PUFAs) are essential fatty acids with potent immunomodulatory activities. PUFAs and their bioactive derivatives can promote or inhibit many aspects of the immune and inflammatory response. PUFAs of the n-3 series ('n-3 PUFAs', also known as omega-3) exhibit anti-inflammatory/pro-resolution properties and promote immune functions, while PUFAs of the n-6 series ('n-6 PUFAs' or omega-6) favor pro-inflammatory responses. The present study aimed at providing insight into the effects of n-3 PUFAs on the consequences of MIA on brain development. We hypothesized that a reduction in n-3 PUFAs exacerbates both maternal and fetal inflammatory responses to MIA and later-life defects in memory in the offspring. Based on a lipopolysaccharide (LPS) model of MIA (LPS injection at embryonic day 17), we showed that n-3 PUFA deficiency 1) alters fatty acid composition of the fetal and adult offspring brain; 2) exacerbates maternal and fetal inflammatory processes with no significant alteration of microglia phenotype, and 3) induces spatial memory deficits in the adult offspring. We also showed a strong negative correlation between brain content in n-3 PUFA and cytokine production in MIA-exposed fetuses. Overall, our study is the first to address the deleterious effects of n-3 PUFA deficiency on brain lipid composition, inflammation and memory performances in MIA-exposed animals and indicates that it should be considered as a potent environmental risk factor for the apparition of neurodevelopmental disorders.

International audience; The brain is highly enriched in long chain polyunsaturated fatty acids (LC-PUFAs) that are esterified into phospholipids, the major components of cell membranes. They accumulate during the perinatal period when the brain is rapidly developing. Hence, the levels of LC-PUFAs in the brains of the offspring greatly depend on maternal dietary intake. Perinatal n-3 PUFA consumption has been suggested to modulate the activity of microglial cells, the brain's innate immune cells which contribute to the shaping of neuronal network during development. However, the impact of maternal n-3 PUFA intake on microglial lipid composition in the offspring has never been studied. To investigate the impact of maternal dietary n-3 PUFA supply on microglia lipid composition, pregnant mice were fed with n-3 PUFA deficient, n-3 PUFA balanced or n-3 PUFA supplemented diets during gestation and lactation. At weaning, microglia were isolated from the pup's brains to analyze their fatty acid composition and phospholipid class levels. We here report that post-natal microglial cells displayed a distinctive lipid profile as they contained high levels of eicosapentaenoic acid (EPA), more EPA than docosahexaenoic acid (DHA) and large amount of phosphatidylinositol (PI) / phosphatidylserine (PS). Maternal n-3 PUFA supply increased DHA levels and decreased n-6 docosapentaenoic acid (DPA) levels whereas the PI/PS membrane content was inversely correlated to the quantity of PUFAs in the diet. These results raise the possibility of modulating microglial lipid profile and their subsequent activity in the developing brain.

International audience; Classically, polyunsaturated fatty acids (PUFA) were largely thought to be relatively inert structural components of brain, largely important for the formation of cellular membranes. Over the past 10 years, a host of bioactive lipid mediators that are enzymatically derived from arachidonic acid, the main n-6 PUFA, and docosahexaenoic acid, the main n-3 PUFA in the brain, known to regulate peripheral immune function, have been detected in the brain and shown to regulate microglia activation. Recent advances have focused on how PUFA regulate the molecular signaling of microglia, especially in the context of neuroinflammation and behavior. Several active drugs regulate brain lipid signaling and provide proof of concept for targeting the brain. Because brain lipid metabolism relies on a complex integration of diet, peripheral metabolism, including the liver and blood, which supply the brain with PUFAs that can be altered by genetics, sex, and aging, there are many pathways that can be disrupted, leading to altered brain lipid homeostasis. Brain lipid signaling pathways are altered in neurologic disorders and may be viable targets for the development of novel therapeutics. In this study, we discuss in particular how n-3 PUFAs and their metabolites regulate microglia phenotype and function to exert their anti-inflammatory and proresolving activities in the brain.

International audience; Aging is associated to cognitive decline that can lead to neurodegenerative diseases and constitutes one of the main social and economic issues of the 21st century. The loss of memory, orientation and processing abilities associated with aging are involved in the loss of autonomy and in the decline in the quality of life in the elderly. Brain structures involved in memory such as hippocampus, cortex and striatum, are particularly affected by molecular and cellular damage during this period. Lipid metabolism and neurofunctional alterations, including disturbances in synaptic plasticity and neurogenesis, chronic low-grade inflammation and increased oxidative stress, are partly to be involved in age-related cognitive decline. Actually, nutrition represents a strategy of choice to prevent or delay these impairments since many studies have provided valuable data concerning the effect of dietary patterns and specific nutrients on cognitive function. From all nutrients, some of them are particularly attractive. Indeed, n-3 polyunsaturated acids (PUFAs), especially docosahexaenoic acid (DHA), have been identified for their beneficial effects on cognition, notably by acting on brain plasticity (synaptic plasticity, neurogenesis), neuroinflammation and oxidative stress. Other nutrients such as vitamin A, vitamin E, vitamin D, polyphenols as well as pre- and probiotics have aroused a growing interest in decreasing cognitive disorders. As nutrition has to be taken as a whole, we first described the effects of the Mediterranean diet which constitutes the most complete association of nutrients and (DHA from fish, vitamins and polyphenols from fruits and vegetables) represents a global vision of nutrition, then we focused on the interest of combining DHA and micronutrients contained in this diet as well as pre- and probiotics, to prevent age-related cognitive decline and reported the synergistic effects of these associations. Finally, we completed with benefits from dairy products that increase DHA incorporation.

Over the last 20 years, it has become abundantly clear that the brain mounts robust inflammatory responses, mediated locally by glial populations including microglia and astrocytes. As more and more data emerge from genome wide association studies of neurodegenerative and neuropsychiatric conditions, it is evident that neuroinflammation is a key player in brain function and dysfunction. With the aging-related increase in neurodegenerative diseases occurrence, novel antiinflammatory strategies are required to prevent and/or dampen cognitive decline. Nowadays, nutritional approaches are pointed out and especially supplementation with the potent antiinflammatory omega-3 or n-3 long chain polyunsaturated fatty acids (n-3 PUFAs). This book chapter exposes the literature related to the mechanisms underlying the neuroinflammatory processes in cognitive disorders. We also summarizes results indicating that n-3 PUFAs are promising molecules in reducing neuroinflammation and age-related memory decline, and we describe the known mechanisms of action underlying their neuroprotective effects. As such, these nutrients are good candidates in the search of a new generation of drugs capable of counteracting neuroinflammation and protect from cognitive decline.

Background Neuroinflammatory processes are considered a double-edged sword, having both protective and detrimental effects in the brain. Microglia, the brain’s resident innate immune cells, are a key component of neuroinflammatory response. There is a growing interest in developing drugs to target microglia and control neuroinflammatory processes. In this regard, docosahexaenoic acid (DHA), the brain’s n-3 polyunsaturated fatty acid, is a promising molecule to regulate pro-inflammatory microglia and cytokine production. Several works reported that the bioavailability of DHA to the brain is higher when DHA is acylated to phospholipid. In this work, we analyzed the anti-inflammatory activity of DHA-phospholipid, either acetylated at the sn-1 position (AceDoPC, a stable form thought to have superior access to the brain) or acylated with palmitic acid at the sn-1 position (PC-DHA) using a lipopolysaccharide (LPS)-induced neuroinflammation model both in vitro and in vivo.[br/] Methods In vivo, adult C57Bl6/J mice were injected intravenously (i.v.) with either AceDoPC or PC-DHA 24 h prior to LPS (i.p.). For in vitro studies, immortalized murine microglia cells BV-2 were co-incubated with DHA forms and LPS. AceDoPC and PC-DHA effect on brain or BV-2 PUFA content was assessed by gas chromatography. LPS-induced pro-inflammatory cytokines interleukin IL-1β, IL-6, and tumor necrosis factor (TNF) α production were measured by quantitative PCR (qPCR) or multiplex. IL-6 receptors and associated signaling pathway STAT3 were assessed by FACS analysis and western-blot in vitro.[br/] Results In vivo, a single injection of AceDoPC or PC-DHA decreased LPS-induced IL-6 production in the hippocampus of mice. This effect could be linked to their direct effect on microglia, as revealed in vitro. In addition, AceDoPC or PC-DHA reduced IL-6 receptor while only AceDoPC decreased IL-6-induced STAT3 phosphorylation.[br/] Conclusions These results highlight the potency of administered DHA—acetylated to phospholipids—to rapidly regulate LPS-induced neuroinflammatory processes through their effect on microglia. In particular, both IL-6 production and signaling are targeted by AceDoPC in microglia.

International audience; The medial prefrontal cortex (mPFC) is a key area for the regulation of numerous brain functions including stress response and cognitive processes. This brain area is also particularly affected by adversity during early life. Using an animal model in rats, we recently demonstrated that maternal exposure to a high-fat diet (HFD) prevents maternal separation (MS)-induced gene expression alterations in the developing PFC and attenuates several long-term deleterious behavioral effects of MS. In the present study, we ask whether maternal HFD could protect mPFC neurons of pups exposed to early life stress by examining dendritic morphology and spine density in juvenile [postnatal day (PND) 21] and adult rats submitted to MS. Dams were fed either a control or an HFD throughout gestation and lactation, and pups were submitted to MS from PND2 to PND14. We report that maternal HFD prevents MS-induced spine loss at PND21 and dendritic atrophy at adulthood. Furthermore, we show in adult MS rats that PFC-dependent memory extinction deficits are prevented by maternal HFD. Finally, perinatal HFD exposure reverses gut leakiness following stress in pups and seems to exert an anti-stress effect in dams. Overall, our work demonstrates that maternal HFD affects the developing brain and suggests that nutrition, possibly through gut–brain interactions, could modulate mPFC sensitivity to early stress.

Energy-dense, yet nutritionally poor food is a high-risk factor for mental health disorders. This is of particular concern during adolescence, a period often associated with increased consumption of low nutritional content food and higher prevalence of mental health disorders. Indeed, there is an urgent need to understand the mechanisms linking unhealthy diet and mental disorders. Deficiency in n-3 polyunsaturated fatty acids (PUFAs) is a hallmark of poor nutrition and mood disorders. Here, we developed a mouse model of n-3 PUFA deficiency lasting from adolescence into adulthood. Starting nutritional deficits in dietary n-3 PUFAs during adolescence decreased n-3 PUFAs in both medial prefrontal cortex (mPFC) and nucleus accumbens, increased anxiety-like behavior, and decreased cognitive function in adulthood. Importantly, we discovered that endocannabinoid/mGlu5-mediated LTD in the mPFC and accumbens was abolished in adult n-3-deficient mice. Additionally, mPFC NMDAR-dependent LTP was also lacking in the n-3-deficient group. Pharmacological enhancement of the mGlu5/eCB signaling complex, by positive allosteric modulation of mGlu5 or inhibition of endocannabinoid 2-arachidonylglycerol degradation, fully restored synaptic plasticity and normalized emotional and cognitive behaviors in malnourished adult mice. Our data support a model where nutrition is a key environmental factor influencing the working synaptic range into adulthood, long after the end of the perinatal period. These findings have important implications for the identification of nutritional risk factors for disease and design of new treatments for the behavioral deficits associated with nutritional n-3 PUFA deficiency.SIGNIFICANCE STATEMENT In a mouse model mimicking n-3 PUFA dietary deficiency during adolescence and adulthood, we found strong increases in anxiety and anhedonia which lead to decreases in specific cognitive functions in adulthood. We found that endocannabinoid/mGlu5-mediated LTD and NMDAR-dependent LTP were lacking in adult n-3-deficient mice. Acute positive allosteric modulation of mGlu5 or inhibition of endocannabinoid degradation normalized behaviors and synaptic functions in n-3 PUFA-deficient adult mice. These findings have important implications for the identification of nutritional risk for disease and the design of new treatments for the behavioral deficits associated with nutritional n-3 PUFAs' imbalance.

International audience; Within the central nervous system (CNS) the traditional role of microglia has been in brain infection and disease, phagocytosing debris and secreting factors to modify disease progression. This led to the concept of “resting” versus “activated” microglia. However, this is misleading because multiple phenotypic and morphological stages of microglia can influence neuronal structure and function in any condition and recent evidence extends their role to healthy brain homeostasis. The present work was thus aimed at reappraising the concept of morphofunctional activity of microglia in a context of peripheral acute immune challenge, where microglial activity is known to be modified, using the new state-of-the-art techniques available. To do so, mice were injected peripherally with lipopolysaccharide, a potent inducer of cerebral inflammation, and we assessed early cytokines production, phenotype, motility and morphology of microglial cells. Our results showed that LPS induced a widespread inflammatory response both peripherally and centrally, as revealed by the quantification of cytokines levels. We also found an alteration of microglial motility that was confirmed by in vivo studies showing an overall reduction of microglial processes length in the hippocampus of LPS-treated animals. Finally, analysis of various surface receptors expression revealed that LPS did not significantly impact microglial phenotype 2 h after the injection but rather induced an increase of CD11b+/CD45high cells. These latter may be at the vasculature, at the CNS vicinity, or may have invaded the CNS.

Energy-dense, yet nutritionally poor food is a high-risk factor for mental health disorders. This is of particular concern during adolescence, a period often associated with increased consumption of low nutritional content food and higher prevalence of mental health disorders. Indeed, there is an urgent need to understand the mechanisms linking unhealthy diet and mental disorders. Deficiency in n-3 polyunsaturated fatty acids (PUFAs) is a hallmark of poor nutrition and mood disorders. Here, we developed a mouse model of n-3 PUFA deficiency lasting from adolescence into adulthood. Starting nutritional deficits in dietary n-3 PUFAs during adolescence decreased n-3 PUFAs in both medial prefrontal cortex (mPFC) and nucleus accumbens, increased anxiety-like behavior, and decreased cognitive function in adulthood. Importantly, we discovered that endocannabinoid/mGlu(5)-mediated LTD in the mPFC and accumbens was abolished in adult n-3-deficient mice. Additionally, mPFC NMDAR-dependent LTP was also lacking in the n-3-deficient group. Pharmacological enhancement of the mGlu(5)/eCB signaling complex, by positive allosteric modulation of mGlu(5) or inhibition of endocannabinoid 2-arachidonylglycerol degradation, fully restored synaptic plasticity and normalized emotional and cognitive behaviors in malnourished adult mice. Our data support a model where nutrition is a key environmental factor influencing the working synaptic range into adulthood, long after the end of the perinatal period. These findings have important implications for the identification of nutritional risk factors for disease and design of new treatments for the behavioral deficits associated with nutritional n-3 PUFA deficiency. SIGNIFICANCE STATEMENT In a mouse model mimicking n-3 PUFA dietary deficiency during adolescence and adulthood, we found strong increases in anxiety and anhedonia which lead to decreases in specific cognitive functions in adulthood. We found that endocannabinoid/mGlu(5)-mediated LTD and NMDAR-dependent LTP were lacking in adult n-3-deficient mice. Acute positive allosteric modulation of mGlu(5) or inhibition of endocannabinoid degradation normalized behaviors and synaptic functions in n-3 PUFA-deficient adult mice. These findings have important implications for the identification of nutritional risk for disease and the design of new treatments for the behavioral deficits associated with nutritional n-3 PUFAs' imbalance.

Présentation «flash» : projet de recherche (Oxylino) en cours inter-collaboratif du Dr Frédéric Capel; National audience

International audience; Mimicking the breast milk lipid composition appears to be necessary for infant formula to cover the brain's needs in n-3 PUFA. In this study, we evaluated the impact of partial replacement of vegetable oil (VL) in infant formula by dairy fat (DL) on docosahexaenoic acid (DHA) brain level, neuroplasticity and corticosterone in mice. Mice were fed with balanced VL or balanced DL diets enriched or not in DHA and arachidonic acid (ARA) from the first day of gestation. Brain DHA level, microglia number, neurogenesis, corticosterone and glucocorticoid receptor expression were measured in the offsprings. DL diet increased DHA and neuroplasticity in the brain of mice at postnatal day (PND) 14 and at adulthood compared to VL. At PND14, ARA and DHA supplementation increased DHA in VL but not in DL mice brain. Importantly, DHA and ARA supplementation further improved neurogenesis and decreased corticosterone level in DL mice at adulthood. In conclusion, dairy lipids improve brain DHA level and neuroplasticity.

The aim of the study was to determine the effect of maternal diets administered since day 1 of gestation and containing dairy lipids or vegetable oils differing in the supply of n-3 polyunsaturated fatty acids (n-3 PUFAs) (equilibrated or deficient) and of Lactobacillus fermentum (L. fermentum) on the docosahexaenoic acid (DHA) accretion in the pups at postnatal day 14 in the prefrontal cortex (PFC) and hippocampus (HC) for brain structures and in the liver and adipose tissue for peripheral tissues. Maternal milk fatty acid composition was also assessed by analyzing the fatty acid composition of the gastric content of the pups. DHA was higher in mice supplemented with L. fermentum than in mice in the deficient group in HC and PFC and also in liver and adipose tissue. This increase could be linked to the slight but significant increase in C18:3n-3 in the maternal milk. This proportion was comparable in the dairy lipid group for which the brain DHA level was the highest. L. fermentum may have a key role in the protection of the brain during the perinatal period via the neuronal accretion of n-3 PUFAs, especially during n-3 PUFA deficiency.

The role of n-3 PUFAs has gained more importance these last decades, especially in inflammatory processes because they can display anti-inflammatory properties. Inflammation is a protective response of the body in controlling infection and promoting tissue repair. However, excessive inflammation can cause local tissue damage. This is especially the case for the brain for which the functional consequences of neuroinflammation include alterations in cognition, affect and behavior leading to a negative impact on the quality of life and well-being of patients (Dantzer, 2001, 2008). Hence, limiting the inflammation in the brain is a real strategy for neuroinflammatory disease therapy and treatment. Recent data show that n-3 PUFAs exert anti-inflammatory properties in part through the synthesis of specialized pro-resolving mediators such as resolvins that actively turned off the inflammatory response. This review first outlines basic concepts of neuroinflammation and the role of n-3 PUFAs in this process and then summarizes the biosynthesis, signaling pathways and role of resolvins., Le rôle des AGPI n-3 a considérablement augmenté ces dernières années, en particulier dans les processus inflammatoires en raison de leurs propriétés anti-inflammatoires. L’inflammation est une réponse protectrice de l’organisme visant à contrôler l’infection et à favoriser la réparation des tissus. Cependant, une inflammation excessive peut avoir de graves conséquences au niveau des tissus. C’est notamment le cas pour le cerveau pour lequel les conséquences fonctionnelles de la neuro-inflammation comprennent des altérations de la cognition, de l’affect et du comportement, conduisant à un impact négatif sur la qualité de vie et le bien-être des patients (Dantzer, 2001, 2008). Par conséquent, limiter l’inflammation dans le cerveau représente une véritable stratégie dans le cadre de la prévention et du traitement des maladies neuro-inflammatoires. Des données récentes montrent que les AGPI n-3 exercent leurs propriétés anti-inflammatoires en partie via la synthèse de médiateurs lipidiques spécialisés tels que les résolvines, qui participent activement à réduire la réponse inflammatoire. Cette revue rappelle d’abord les concepts de base de la réponse inflammatoire et le rôle des AGPI n-3 dans ce processus et présente ensuite la biosynthèse, les voies de signalisation et le rôle des résolvines.

International audience; Polyunsaturated fatty acids (PUFAs) are essential fatty acids, which are critical for brain development and later life cognitive functions. The main brain PUFAs are docosahexaenoic acid (DHA) for the n-3 family and arachidonic acid (ARA) for the n-6 family, which are provided to the post-natal brain by breast milk or infant formula. Recently, the use of dairy lipids (DL) in replacement of vegetable lipids (VL) was revealed to potently promote the accretion of DHA in the developing brain. Brain DHA, in addition to be a key component of brain development, display potent anti-inflammatory activities, which protect the brain from adverse inflammatory events. In this work, we evaluated the protective effect of partial replacement of VL by DL, supplemented or not with DHA and ARA, on post-natal inflammation and its consequence on memory. Mice were fed with diets poor in vegetal n-3 PUFA (Def VL), balanced in vegetal n-3/n-6 PUFA (Bal VL), balanced in dairy lipids (Bal DL) or enriched in DHA and ARA (Supp VL; Supp DL) from the first day of gestation until adulthood. At post-natal day 14 (PND14), pups received a single administration of the endotoxin lipopolysaccharide (LPS) and brain cytokine expression, microglia phenotype and neurogenesis were measured. In a second set of experiments, memory and neurogenesis were measured at adulthood. Overall, our data showed that lipid quality of the diet modulates early life LPS effect on microglia phenotype, brain cytokine expression and neurogenesis at PND14 and memory at adulthood. In particular, Bal DL diet protects from the adverse effect of early life LPS exposure on PND14 neurogenesis and adult spatial memory.

International audience; Several genetic causes of autism spectrum disorder (ASD) have been identified. However, more recent work has highlighted that certain environmental exposures early in life may also account for some cases of autism. Environmental insults during pregnancy, such as infection or malnutrition, seem to dramatically impact brain development. Maternal viral or bacterial infections have been characterized as disruptors of brain shaping, even if their underlying mechanisms are not yet fully understood. Poor nutritional diversity, as well as nutrient deficiency, is strongly associated with neurodevelopmental disorders in children. For instance, imbalanced levels of essential fatty acids, and especially polyunsaturated fatty acids (PUFAs), are observed in patients with ASD and other neurodevelopmental disorders (e.g., attention deficit hyperactivity disorder (ADHD) and schizophrenia). Interestingly, PUFAs, and specifically n-3 PUFAs, are powerful immunomodulators that exert anti-inflammatory properties. These prenatal dietary and immunologic factors not only impact the fetal brain, but also affect the microbiota. Recent work suggests that the microbiota could be the missing link between environmental insults in prenatal life and future neurodevelopmental disorders. As both nutrition and inflammation can massively affect the microbiota, we discuss here how understanding the crosstalk between these three actors could provide a promising framework to better elucidate ASD etiology.

International audience; The purpose of this study was to compare the susceptibility of the retina and the exorbital lacrimal gland to dietary supplies of long-chain omega-3 (omega 3) and omega-6 (omega 6) polyunsaturated fatty acids (LC-PUFAs). Male Wistar rats were fed a 5% lipid diet containing: (1) 10% eicosapentaenoic acid (EPA) and 7% docosahexaenoic acid (DHA), or (2) 10% gamma-linolenic acid (GLA), or (3) 10% EPA, 7% DHA and 10% GLA or (4) a balanced diet deprived of EPA, DHA and GLA for 3 months. Lipids were extracted from plasma phospholipids, retina and exorbital lacrimal gland, and fatty acid composition was determined by gas chromatography. Dietary supplementation with EPA and DHA increased omega 3 PUFA levels in plasma phospholipids as well as in the retina and the exorbital lacrimal gland. By contrast, GLA supplementation favored omega 6 PUFA incorporation, and particularly the incorporation of the end-chain omega 6 product, docosapentaenoic acid (DPA), into all tissues. Supplementation with EPA, DHA and GLA increased the levels of DHA, EPA and dihomo-GLA (dGLA), whereas arachidonic acid (AA) was unchanged and DPA decreased in the retina and the lacrimal gland. The ability of both tissues to incorporate PUFAs from blood was evaluated. The results showed that the retina was more selective than the lacrimal gland for EPA. In spite of the different susceptibility of the retina and the lacrimal gland to dietary PUFAs, these results suggest that the concomitant use of dietary omega 3 and omega 6 PUFAs may be useful in modulating inflammation in both tissues.

International audience; Sustained inflammation in the brain together with microglia activation can lead to neuronal damage. Hence limiting brain inflammation and activation of microglia is a real therapeutic strategy for inflammatory disease. Resolvin D1 (RvD1) and resolvin E1 (RvE1) derived from n-3 long chain polyunsaturated fatty acids are promising therapeutic compounds since they actively turn off the systemic inflammatory response. We thus evaluated the anti-inflammatory activities of RvD1 and RvE1 in microglia cells in vitro. BV2 cells were pre-incubated with RvD1 or RvE1 before lipopolysaccharide (LPS) treatment. RvD1 and RvE1 both decreased LPS-induced proinflammatory cytokines (TNF-α, IL-6 and IL-1β) gene expression, suggesting their proresolutive activity in microglia. However, the mechanisms involved are distinct as RvE1 regulates NFκB signaling pathway and RvD1 regulates miRNAs expression. Overall, our findings support that pro-resolving lipids are involved in the resolution of brain inflammation and can be considered as promising therapeutic agents for brain inflammation.

International audience; The central nervous system (CNS) has previously been regarded as an immune-privileged site with the absence of immune cell responses but this dogma was not entirely true. Microglia are the brain innate immune cells and recent findings indicate that they participate both in CNS disease and infection as well as facilitate normal CNS function. Microglia are highly plastic and play integral roles in sculpting the structure of the CNS, refining neuronal circuitry and connectivity, and contribute actively to neuronal plasticity in the healthy brain. Interestingly, psychological stress can perturb the function of microglia in association with an impaired neuronal plasticity and the development of emotional behavior alterations. As a result it seemed important to describe in this review some findings indicating that the stress-induced microglia dysfunction may underlie neuroplasticity deficits associated to many mood disorders.This article is part of a Special Issue entitled ‘Neuroimmunology and Synaptic Function’.

International audience; Polyunsaturated fatty acids (PUFAs) display immunomodulatory properties in the brain, n-3 PUFAs being able to reduce inflammation whereas n-6 PUFAs are more pro-inflammatory. It has been extensively demonstrated that exposure to a peripheral immune challenge leads to the production and release of inflammatory mediators in the brain in association with cognitive deficits. The question arises whether n-3 PUFA supplementation could downregulate the brain inflammatory response and subsequent cognitive alterations. In this study, we used a genetically modified mouse line carrying the fat-1 gene from the roundworm Caenorhabditis elegans, encoding an n-3 PUFA desaturase that catalyzes conversion of n-6 into n-3 PUFA. Consequently, these mice display endogenously elevated n-3 PUFA tissue contents. Fat-1 mice or wild-type (WT) littermates were injected peripherally with lipopolysaccharide (LPS), a bacterial endotoxin, to induce an inflammatory episode. Our results showed that LPS altered differently the phenotype of microglia and the expression of cytokines and chemokines in Fat-1 and WT mice. In Fat-1 mice, pro-inflammatory factors synthesis was lowered compared with WT mice, whereas anti-inflammatory mechanisms were favored 24 h after LPS treatment. Moreover, LPS injection impaired spatial memory in WT mice, whereas interestingly, the Fat-1 mice showed normal cognitive performances. All together, these data suggest that the central n-3 PUFA increase observed in Fat-1 mice modulated the brain innate immune system activity, leading to the protection of animals against LPS-induced pro-inflammatory cytokine production and subsequent spatial memory alteration.

International audience; Dietary n-3 polyunsaturated fatty acids (PUFAs) are critical components of inflammatory response and memory impairment. However, the mechanisms underlying the sensitizing effects of low n-3 PUFAs in the brain for the development of memory impairment following inflammation are still poorly understood. In this study, we examined how a 2-month n-3 PUFAs deficiency from pre-puberty to adulthood could increase vulnerability to the effect of inflammatory event on spatial memory in mice. Mice were given diets balanced or deficient in n-3 PUFAs for a 2-month period starting at post-natal day 21, followed by a peripheral administration of lipopolysaccharide (LPS), a bacterial endotoxin, at adulthood. We first showed that spatial memory performance was altered after LPS challenge only in n-3 PUFA-deficient mice that displayed lower n-3/n-6 PUFA ratio in the hippocampus. Importantly, long-term depression (LTD), but not long-term potentiation (LTP) was impaired in the hippocampus of LPS-treated n-3 PUFA-deficient mice. Proinflammatory cytokine levels were increased in the plasma of both n-3 PUFA-deficient and n-3 PUFA-balanced mice. However, only n-3 PUFA-balanced mice showed an increase in cytokine expression in the hippocampus in response to LPS. In addition, n-3 PUFA-deficient mice displayed higher glucocorticoid levels in response to LPS as compared with n-3 PUFA-balanced mice. These results indicate a role for n-3 PUFA imbalance in the sensitization of the hippocampal synaptic plasticity to inflammatory stimuli, which is likely to contribute to spatial memory impairment.

Epidemiological observations report an increase in fat consumption associated with low intake of n-3 relative to n-6 polyunsaturated fatty acids (PUFAs) in women of childbearing age. However, the impact of these maternal feeding habits on cognitive function in the offspring is unknown. This study aims to investigate the impact of early exposure to a high-fat diet (HFD) with an unbalanced n-6/n-3 PUFAs ratio on hippocampal function in adult rats. Furthermore, we explored the effects of perinatal HFD combined with exposure to HFD after weaning. Dams were fed a control diet (C, 12% of energy from lipids, n-6/n-3 PUFAs ratio: 5) or HFD (HF, 39% of energy from lipids, n-6/n-3 PUFAs ratio: 39) throughout gestation and lactation. At weaning, offspring were placed either on control (C-C, HF-C) or high-fat (HF-HF) diets. In adulthood, hippocampus-dependent memory was assessed using the water-maze task and potential hippocampal alterations were determined by studying PUFA levels, gene expression, neurogenesis and astrocyte morphology. Perinatal HFD induced long-lasting metabolic alterations and some changes in gene expression in the hippocampus, but had no effect on memory. In contrast, spatial memory was impaired in animals exposed to HFD during the perinatal period and maintained on this diet. HF-HF rats also exhibited low n-3 and high n-6 PUFA levels, decreased neurogenesis and downregulated expression of several plasticity-related genes in the hippocampus. To determine the contribution of the perinatal diet to the memory deficits reported in HF-HF animals, an additional experiment was conducted in which rats were only exposed to HFD starting at weaning (C-HF). Interestingly, memory performance in this group was similar to controls. Overall, our results suggest that perinatal exposure to HFD with an unbalanced n-6/n-3 ratio sensitizes the offspring to the adverse effects of subsequent high-fat intake on hippocampal function.

Trans polyunsaturated fatty acids are formed during heat treatments of vegetable oils from polyunsaturated fatty acids containing cis double bonds. After dietary intake, they are dis- tributed in the body and are incorporated into nervous tissues including the retina. Since nervous tissues are known to be rich in n-3 fatty acids such as docosahexaenoic acid (DHA), we studied the ability of the retina and the brain to incorporate trans isomers of DHA formed in vivo from the dietary precursor trans α-linolenic acid. Wistar rats were fed with trans isomers of α-linolenic acid for 21 months. A linear incorporation of trans DHA and a decrease in cis DHA was observed in the retina, whereas no major changes were observed in the brain. In parallel to the modifications in reti- nal cis and trans DHA levels, the retinal functionality evaluated by the electroretinogram showed defects in animals that consumed trans α-linolenic acid. These results suggest that the mechanisms leading to the incorporation of cis and trans fatty acids are quite different in the retina when com- pared to the brain and the liver, the retina being more susceptible to changes in the dietary lipid contribution. dietary trans polyunsaturated fatty acids / rat / retina / cerebral cortex / electroretinography

Les mecanismes physiopathologiques de la degenerescence maculaire liee a l’âge (DMLA) sont encore peu connus. Comme d’autres pathologies du vieillissement telles que la maladie d’Alzheimer ou l’atherosclerose, la DMLA est associee a l’apparition de depots macromoleculaires anormaux appeles drusen. Ces derniers sont localises au niveau de la membrane de Bruch. De recentes investigations sur la nature de ces depots, leurs relations avec les reactions inflammatoires et immunitaires ainsi que sur le role important du stress oxydatif ont permis d’elargir les hypotheses sur les mecanismes physiopathologiques conduisant aux lesions de DMLA. De nombreuses analogies avec les mecanismes impliques dans la genese des lesions d’atherosclerose suggerent des similitudes physiopathologiques entre ces deux maladies. Le role pro-atherogene du cholesterol et de ses derives oxydes, les oxysterols, a ete largement demontre dans les processus atheromateux. Il semblerait que ces composes interviennent egalement dans la genese de la DMLA entrainant des activites cytotoxiques, pro-oxydantes et pro-inflammatoires.

Pure individual phytosterols were prepared using reversed-phase HPLC in order to obtain the oxidized compounds of sitosterol, campesterol, stigmasterol and brassicasterol. 7-Hydroxy-, 7-keto-, 5,6-epoxy-, 4beta-hydroxy-, 4-ene-6-hydroxy-, 6-keto- and 5alpha,6beta-dihydroxyphytosterols were obtained as well as analogous compounds of cholesterol. The gas chromatographic properties as well as the electronic impact mass spectra of these compounds (as trimethylsilyl ether derivatives) were studied. These data were used to identify oxyphytosterols in a spread enriched in phytosterols: the oxyphytosterols represented no more than 68 microg/g of spread (about 0.08% of phytosterols were oxidised).

The oxidised derivatives of phytosterols (oxyphytosterols) were identified in plasma samples from thirteen healthy human volunteers, using MS. All the samples contained noticeable quantities of (24R)-5b,6b-epoxy-24-ethylcholestan-3b-ol (b-epoxysitostanol) and (24R)-ethylcholestan-3b,5a,6b-triol (sitostanetriol) and also trace levels of (24R)-5a,6a-epoxy-24-ethylcholestan-3b-ol (a-epoxysitostanol), (24R)-methylcholestan-3b,5a,6b-triol (campestanetriol) and (24R)-ethylch olest-5-en-3b-ol-7-one(7-ketositosterol). The amounts of these oxyphytosterols in plasma varied from 4·8 to 57·2 ng/ml. There are two possibilities concerning the origin of these compounds. First, they could come from the small amounts of oxyphytosterols in food. Second, they could originate from the in vivo oxidation of phytosterols in plasma. Very few data actually exist concerning these compounds. Their identification in human samples suggests that further research is necessary in this field. Oxyphytosterols: Phytosterols: Human plasma: Mass spectrometry