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n-3 Polyunsaturated fatty acids in animal models with neuroinflammation
Prostaglandins, Leukotrienes and Essential Fatty Acids 88 (2013) 97–103
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n-3 Polyunsaturated fatty acids in animal models with neuroinflammation Sarah K. Orr, Marc-Olivier Tre´panier, Richard P. Bazinet n Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2
a r t i c l e i n f o
Keywords: Neuroinflammation Animal models Brain Docosahexaenoic acid Eicosapentaenoic acid
abstract Neuroinflammation is present in the majority of acute and chronic neurological disorders. Excess or prolonged inflammation in the brain is thought to exacerbate neuronal damage and loss. Identifying modulators of neuroinflammation is an active area of study since it may lead to novel therapies. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are anti-inflammatory in many non-neural tissues; their role in neuroinflammation is less studied. This review summarizes the relationship between n-3 PUFA and brain inflammation in animal models of brain injury and aging. Evidence by and large shows protective effects of n-3 PUFA in models of sickness behavior, stroke, aging, depression, Parkinson’s disease, diabetes, and cytokine- and irradiation-induced cognitive impairments. However, rigorous studies that test the direct effects of n-3 PUFA in neuroinflammation in vivo are lacking. Future research in this area is necessary to determine if, and if so which, n-3 PUFA directly target brain inflammatory pathways. n-3 PUFA bioactive metabolites may provide novel therapeutic targets for neurological disorders with a neuroinflammatory component. & 2012 Elsevier Ltd. All rights reserved.
1. Introduction Inflammation is a beneficial innate response to insults and injuries; however, an overly robust or chronic inflammatory response can contribute significantly to tissue damage. Excessive inflammation in the brain is thought to exacerbate acute injuries including stroke [1], and chronic diseases including multiple sclerosis and Alzheimer’s disease [2,3]. Elucidating neuroinflammatory pathways and identifying modulators is a growing area in brain research, since it may provide novel targets in disease prevention and treatment. The resolution of inflammation was originally considered a passive process that occurs through the dissipation of pro-inflammatory signals. More recently it has become clear that inflammation resolution is an active process, driven by its own mediators [4]. The most studied of these signaling molecules are Specialized Pro-resolving Mediators (SPM), which are enzymatically derived from omega-3 polyunsaturated fatty acids (n-3 PUFA). SPM include eicosapentaenoic acid (EPA; 20:5n-3)-derived resolvins, and docosahexaenoic acid (DHA; 22:6n-3)-derived resolvins, protectins, and Abbreviations: Ab, amyloid-b; ALA, a-linolenic acid; COX-2, cyclooxygenase-2; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; IFN-g, interferon-g; IL1b, interleukin-1b; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; n-3 PUFA, omega-3 polyunsaturated fatty acid; NF-kB, nuclear factor-kB; PPAR, peroxisome proliferator-activated receptor; TNF-a, tumor necrosis factor-a. n Correspondence to: Department of Nutritional Sciences, University of Toronto, FitzGerald Building, 150 College St., Room 306, Toronto, ON, Canada M5S 3E2. Tel.: þ416 946 8276; fax: þ416 978 5882. E-mail address: [email protected] (R.P. Bazinet). 0952-3278/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plefa.2012.05.008
maresins [5]. Through targeting inflammation resolution, SPM provide a potential mechanism to explain the benefits of n-3 PUFA seen in the prevention and treatment of neurodegenerative diseases. DHA is essential for neural and retinal development [6], and is highly enriched in brain compared to most other tissues, making up approximately 0.33% brain wet weight in adult human cortex (10– 12% of total fatty acids) [7,8]. DHA is similarly enriched in the brains of other animals, including non-human primates [9], rats [10,11], and mice [12,13]. EPA and its shorter chain n-3 PUFA precursor, alinolenic acid (ALA; 18:3n-3), are present at very low levels in the brain [11,12,14]. Rapid metabolism via b-oxidation appears to be a mechanism by which EPA and ALA are maintained at concentrations 200–500 fold lower than DHA [11,15], but what is unclear is the functional reason for their low levels in the brain. To date, mechanistic evidence from animal models shows that DHA is anti-apoptotic [16,17], neurotrophic [18], and important for synaptic plasticity [19]. There are, however, no studies testing the direct effects of DHA on the innate brain inflammatory response. The purpose of this short paper is to review the effect of n-3 PUFA modulation in animal models of brain injury and disease where inflammation is also measured (Table 1). In vitro research in this area is also briefly reviewed (for an in-depth review see [20]). Together, these models provide persuasive but indirect evidence of anti-inflammatory actions of n-3 PUFA in the brain.
2. n-3 PUFA and in vitro neuroinflammation Microglial cells are the main immune cell mediators of the brain [21]. BV-2 cell cultures are immortalized microglial cells.
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Table 1 Injury model Species
Comparison treatment
Kavenagh et al. (2004) [35]
Systemic LPS Wistar rats (i.p.)
Chow
Lonergan et al. (2004) [36]
Systemic LPS (i.p.) Systemic LPS (i.p.) Ischemia/ reperfusion
C57BL/6 mice
Yang et al. (2007) [40]
Ischemia/ reperfusion
SpragueDawley rats
Saline i.p.
Pan et al. (2009) [28]
Ischemia/ reperfusion
SpragueDawley rats
Saline i.p.
Belayev et al. (2009) [38]
Ischemia/ reperfusion Ischemia/ reperfusion
SpragueDawley rats SpragueDawley rats
Saline i.v.
Ischemia/ reperfusion
SpragueDawley rats
Saline i.v.
TLR2-flucGFP transgenic mice (C57BL/ 6) Wistar rats
Low n-3 PUFA (corn þ safflower oil) diet
Mingam et al. (2008) [37] Marcheselli et al. (2003) [30]
Belayev et al. (2011) [39]
Bazan et al. (2012) [45]
Lalancette-Hebert (2011) [13] Ischemia/ reperfusion
Wistar rats CD1 mice
PUFA treatment(s)
Chow supplemented with 50 mg/d eEPAn, 50 mg/d eGLA#, or 50 mg/d eEPA þeGLA Chow Chow supplemented with 500 mg/d eEPA 6% peanut oil (n-6) 6% peanutþ rapeseed oil (ndiet 6 þn-3) diet Vehicle i.c.v. 0.4–120 mg 10,17Sdihydroxy-DHAn; 2–200 mg DHA# i.c.v Per kg body weight: 250 nmol (71 mg) SA, 500 nmol (142 mg) SA, 250 nmol (76 mg) ARA, 500 nmol (152 mg) ARAn, 250 nmol (82 mg) DHA, or 500 nmol (164 mg) DHAn i.p. Per kg body weight: 100 nmol (33 mg), or 500 nmol (164 mg) nDHA i.p.
Treatment duration/time point
Brain n-3 PUFA
Primary outcome
Inflammatory outcome
4 wks
Not reported
n#mLTP
n# m HIP IL-10, IL-4 protein; n m HIP PPARg protein; 2 HIP IL-1b protein
4 wks
Not reported m CX DHA
m LTP; k apoptotic markers k Social interaction; 2 food intake nk Infarct size
k HIP IL-1b protein
Not reported
nm Infarct size
n m CX leukocyte/neutrophil accumulation; n m CX COX-2 mRNA
Not reported
nk Infarct size
nk CX leukocyte/neutrophil accumulation; n k CX IL-6 protein
Not reported nm PN NPD1 and 17-HDHA Not reported
k Infarct size
m CX GFAP
n#D k Infarct size; n#D
m neurologicalscore
n m CX and ST GFAP n k CX and ST CD68 (microglia)
k Infarct size; m neurologicalscore
m CX GFAP; k CX CD68; 2 SCX GFAP and CD68;
Gestation þ 8 wks post-natal 3 or 48 h continuous infusion postinjury 60 min postreperfusion
not reported
2 HIP IL-6 mRNA n#k CX and HIP leukocyte/neutrophil accumulation; n# k HIP NF-kB protein; n# k HIP COX-2 mRNA
1 d (single), 3 d (single), or 6 wks (daily) prior to ischemia Per kg body weight: 43 mmol 60 min post(14 mg) DHA i.v. reperfusion Per kg body weight: 15 mmol 3n h, 4 h#, 5 hD, (5 mg) DHA i.v. or 6 hp postischemia 60 min postPer kg body weight: reperfusion (924 nmol) 333 mg AT-NPD1 i.v. DHA supplemented (corn 12 wks oil þsafflower oil þDHA) diet
m Striatum k Infarct size DHA
Chow
Chow supplemented with 10 mg/d then 20 mg/d eEPA
3 wks (10 mg/ d)þ5 wks (20 mg/d) 3 wks (10 mg/ d)þ5 wks (20 mg/d) 4 wks
Not reported
m LTP; k apoptotic markers
k CX and HIP IL-1b
Not reported
k Apoptotic markers; k neurotrophic factors
k CX IL-1b protein; 2 CX IL-1RI protein; m CX IL-4 protein
4 wks
Not reported
m LTP
k k m k
Saline i.v.
Martin et al. (2002) [47]
Aging (4 vs. 22 mo)
Maher et al. (2004) [48]
Aging (4 vs. 22 mo)
Wistar rats
Chow
Chow supplemented with 10 mg/d then 20 mg/d eEPA
Lynch et al. (2007) [26]
Aging (4 vs. 22 mo)
Wistar rats
Chow supplemented with 125 mg/d eEPA
Ab (i.c.v.) in aged rats (22 mo)
Wistar rats
Chow supplemented with MUFA (isocaloric) Chow supplemented with MUFA (isocaloric)
Chow supplemented with 125 mg/d eEPA
Not reported
k TLR2 promoter induction (microglia activation); k brain IL-1b, IL-6, COX-2 protein; 2 brain TNF-a protein
HIP HIP HIP HIP
MHCII and CD40 (microglia activation); IL-1b, IFN-g protein; k HIP IL-1b mRNA; IL-4 protein and mRNA; IL-1b protein
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Authors (year)
Ab (i.c.v.)
Wistar rats
Aging (3 vs. 22 mo)
Wistar rats
Moranis et al. (2001) [49]
Aging (3–5n vs. 19– 23 mo)
CD1 mice
Song et al. (2009) [50]
Olfactory bulbectomy
SpragueDawley rats
Bousquet et al. (2011) [41]
MPTP (i.p.)
Minogue et al. (2007) [27]
4 wks
Not reported
m LTP
k HIP IFN-g, IL-1b protein; m HIP PPARg protein
Chow supplemented with 125 mg/d eEPA
4 wks
Not reported
p
m HIP PPARg protein
n-3 adequate diet (fatty acids 10.7% LA, 1.6% ALA)
3–5 mo or 19– 23 mo
m CX DHA
1% eEPA supplemented diet
7 wks
Not reported
k Depressive-behavior 2 CX IL-6 or IL-10 protein (FST)y; n m spatial memory; 2 age-induced spatial memory loss k Depressive-like k Hypothalamus cPLA2 mRNA and activity symptoms (MWM and OF)
Fat-1 transgenic mice on Wildtype littermates on high high n-6/low n-3 PUFA diet n-6/low n-3 PUFA diet
Life
m CX DHA
2 Striatal or nigral dopaminergic injuryC
Chow supplemented with Chow supplemented with 0.8% eEPA 0.8% palm oil
6 wks
k Hypokinesia and anxiety k Striatal TNF-a, IFN-g protein; k (RT, PT); m learning and midbrain IL-10 protein; 2 memory (MWM) striatal cPLA2, COX-2 mRNA1
2 wks
m CX EPA, DPAn-3; 2 CX DHA Not reported
1% palm oil supplemented diet
Ji et al. (2012) [55]
SN LPS (i.c.v.)
SpragueDawley rats
15% (wt) corn oil diet
15% (wt) fish oil diet (30% fish oil as EPA and DHA)
Song and Horrobin (2004) [56]
IL-1b (i.c.v.)
Wistar rats
5% coconut oil diet
Song et al. (2008) [57]
IL-1b (i.c.v.)
Wistar rats
5% palm oil diet
Lynch et al. (2003) [59]
Whole body irradiation
Wistar rats
Chow
5% soybean, 4.8% coconut oil 7 wks þ 0.2% eEPA, or 4% coconut oil þ1% eEPAn diet 7 wks 4.5% palm oil þ 0.5% eEPA, 4.5% palm oil þ 0.5% eGLA#, or 4% palm oilþ 1% AA-rich oil diet Chow supplemented with 4 wks 250 mg/d or 500 mg/d# eEPA
¨ Alvarez-Nolting et al. (2012) [62]
STZ (i.p.)
Wistar rats
Chow~
k CX GFAP
k Dopaminergic injury; k nigral dopaminergic neuron degeneration kMemory loss (MWM)
k SN OX42 protein (microglial activation); k SN TNF-a and IL-1b protein; k SN p65 (NF-kB subunit) protein kBrain PGE2
Not reported
kMemory loss (MWM); n k anxiety (EPM)
n# k HIP PGE2; n k amygdala PGF2; n m amygdala, hypothalamus IL-10 protein
Not reported
#
n# k HIP IL-1b, IL-1RI, IL- 1RAcP protein; n#
Not reported
kApoptotic markersp
2 HIP IRAK protein phosphorylation ratio; n m HIP IL-10 protein
Chow plus 13.3 mg/kg/d DHA by gavage
12 wks
Not reported
2 Blood glucose and glycated hemoglobin; m HIP neurogenesis; k HIP neuronal degeneration; k HIP oxidative stress; m learning and memory (MWM)
k HIP NF-kB protein
17-HDHA, 17-hydroxy-DHA (DHA derivative); Ab, amyloid-b; ALA, a-linolenic acid (18:3n-3); ARA, arachidonic acid (20:4n-6); AT-NPD1, aspirin triggered NPD1; COX-2, cyclooxygenase-2; cPLA2, cytosolic phospholipase A2; CX, cortex; DHA, docosahexaenoic acid (22:6n-3); DPAn-3, docosapentaenoic acid (22:5n-3); EPA, eicosapentaenoic acid (20:5n-3); eEPA, ethyl-EPA; eGLA, ethyl-gamma-linolenic acid (18:3n-6); EPM, elevated plus maze; FST, forced swim test; GFAP, glial fibrillary acidic protein; HIP, hippocampus; i.c.v., intracerebroventricular; IFN-g, interferon-g; IL, interleukin; IL-1RI, IL-1 receptor protein type I;, IL-1RAcP, IL-1 receptor accessory protein; IRAK, IL-1 receptor-associated kinase; LA, linoleic acid (18:2n-6); LPS, lipopolysaccharide; i.p., intraperitoneal; LTP, long term potentiation; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPTP-P, MPTP and probenecid; MWM, Morris water maze; MUFA, monounsaturated fatty acid; NF-kB, nuclear factor-kB; NPD1, neuroprotectin D1 (DHA derivative); OF, open field; PGE2, prostaglandin E2; PN, penumbra; PPARg, peroxisome proliferator activated protein g; PT, pole test; RT, rotorod test; SA, stearic acid (18:0); s.c., subcutaneous; SCX, subcortex; SN, substantia nigra; ST, striatum; TLR2, toll-like receptor 2; TNF-a, tumor necrosis factor-a; wt, weigth*#Dp indicates treatment group represented in outcome columns (brain n-3 PUFA, behavioral outcome, inflammatory outcome).1 EPA lowered COX-2 mRNA compared to palm oil in saline (control) injected animals.p inflammation was primary outcome.y main effect of n-3 adequate vs. n-3 deprived diet (no effect of age).C protection from nigral dopaminergic injury was correlated to brain DHA levels (secondary analysis).~ insulin-treated rats were also included in the study and were similar to DHA-only treated rats in all measures excluding weight, glycemic control, and oxidative stress.
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Luchman et al. (2012) [54]
Fat-1 transgenic mice (C57BL6/ C3H) MPTP-P (s.c.) C57BL/6 mice
Chow supplemented with 125 mg/d eEPA
Chow supplemented with MUFA (isocaloric) Chow supplemented with MUFA (isocaloric) n-3 deficient diet (fatty acids 60.5% LA, 0.1% ALA)
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In BV-2 cells, 3–30 mM DHA or EPA attenuates interferon- g (IFN- g)-induced up-regulation of pro-inflammatory marker gene expression, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and tumor necrosis factor- a (TNF- a) [22]. BV-2 microglial cells stimulated with lipopolysaccharide (LPS) have increased mRNA and protein levels of iNOS, COX-2, interleukin-1b (IL1b), and TNF- a, all of which are dose-dependently attenuated by the addition of 10, 20, or 30 mM of EPA [23]. Insight into DHA’s mechanism of action was provided by De SmedtPeyrusse et al. who demonstrated that 30 mM of DHA significantly downregulated the cell-surface expression of CD14 and toll-like receptor 4 (TLR4) in LPS-stimulated BV-2 microglial cells concomitant to reducing TNF- a protein expression, mature IL-1b protein expression, and activation of the proinflammatory gene transcription factor, nuclear factor-kB (NFkB) [24]. CD14 and TLR4 are co-receptors that bind LPS to initiate an innate immune response. Interestingly, EPA did not reduce CD14 or TLR4 cell-surface expression [24]. In C6 glioma cells, an astrocyte cancer cell line, EPA concentrations of 25–100 mM dose-dependently attenuate IL-1b-induced IL-6 gene expression, an effect also observed with 50 mM arachidonic acid (ARA; 20:4n-6) or DHA [25]. The effect of EPA on IL-6 was blocked by peroxisome proliferator-activated receptor-g (PPARg) and PPARa antagonists, suggesting PPAR signaling is necessary for the anti-inflammatory effects of EPA [25]. Similar effects of EPA were reported in primary hippocampal glial cell cultures from 1-day old rats, where 50 mM EPA increased IL-4 protein and mRNA, and attenuated subsequent LPS-induced increases in IL-1b protein [26]. In the same cell model, 75 mM EPA reduced LPS- and amyloid-b (Ab)-induced increase in IL-1b protein [27]. This effect was partially blocked by a PPARg antagonist [27]. Also using glial cell cultures from 1-day old rats, Pan et al. observed that 75 but not 10 mM DHA co-treatment with LPS/IFN-g attenuated increases in IL-6 protein [28]. Pretreating with 75 mM DHA was even more effective at inhibiting LPS/IFN-g IL-6 protein expression [28], suggesting incorporation of DHA into phospholipid membranes may optimize immune modulation. We identified three studies that measured the neuroinflammatory effects of SPM in vitro. In human neuronal-glial primary cell cultures, 10,17S-dihydroxy-DHA, a 15-lipoxygenase metabolite of DHA, downregulated COX-2, TNF-a, and IL-1b expression while increasing PPARg activity in a Ab42-induced cell culture model of Ab-deposition, which is a major pathology of Alzheimer’s disease [16,29]. In the same model, DHA downregulated COX-2, TNF-a and IL-1b expression [16]. In IL-1b-stimulated human neural progenitor cells, 10,17S-dihydroxy-DHA downregulated NF-kB activation and COX-2 gene expression; however, there was no effect of 0.03–3000 nM DHA on NF-kB activation in response to IL-1b [30]. In vitro studies support the hypothesis that n-3 PUFA and their derivatives are anti-inflammatory in the brain, and suggest that they target TLR4 signaling, and NF-kB, and PPAR pathways. Importantly, however, unesterified DHA occurs at concentrations of around 1–6 mM in brain and 1–40 mM in plasma; and phospholipid DHA occurs at concentrations of 10–150 mM in plasma and 7000–14,000 mM in the brain of rats and mice [10,31–34]. EPA occurs at even lower concentrations, particularly in the brain where unesterified EPA occurs below detection limits, and phospholipid EPA concentrations range from being undetectable to 115 mM [11,31,34]. Plasma unesterified and phospholipid EPA concentrations both range from undetected to 9 mM [11,31,34]. Whether these pathways are affected by DHA or EPA at fatty acid concentrations seen in brain tissue is not clear, and warrants in vivo study.
3. n-3 PUFA and neuroinflammation induced by systemic lipopolysaccharide We identified three studies that used intraperitoneally (i.p.) injected LPS to test the effects of n-3 PUFA on sickness behavior and/or brain inflammation. In the first, rats consumed one of four diets: standard chow, standard chow plus 50 mg/day of ethylEPA, standard chow plus 50 mg/day of ethyl-gamma linolenic acid (GLA), or standard chow plus 50 mg/day total of ethyl-EPA and ethyl-GLA for 4 weeks prior to 100 mg/kg i.p. LPS-induced hippocampal inflammation [35]. No dietary treatment protected against i.p. LPS-induced hippocampal IL-1b protein expression; however, all treatment diets prevented the reduction in hippocampal protein levels of anti-inflammatory cytokines IL-4 and IL10 compared to rats fed standard chow. Compared to standard chow, ethyl-EPA and ethyl-EPA þethyl-GLA supplemented rats significantly increased i.p. LPS-induced hippocampal protein expression of the anti-inflammatory transcription factor PPARg [35]. In a similar study, the consumption of 500 mg/day ethyl-EPA for 4 weeks did attenuate hippocampal increases in IL-1b protein induced by 100 mg/kg i.p. LPS [36]. While examining i.p. LPS-induced sickness behavior, Mingam et al. measured IL-6 in mice consuming isocaloric diets containing or deprived of n-3 PUFA. One diet included African peanut oil, rich in linoleic acid (18:2n-6; n-6 PUFA diet), and the second diet included African peanut oil plus rapeseed oil, rich in ALA (n-3 PUFAþn-6 PUFA diet). Mice consumed treatment diets from gestation to 8 weeks of age and sickness behavior was induced by 30 mg/kg i.p. LPS administration [37]. Mice consuming n-3þn6 PUFA had 2 to 3 fold higher cortical phospholipid DHA compared to mice fed n-6 PUFA alone. There was no consistent effect of diet on LPS-induced hippocampal IL-6 mRNA expression [37]. Overall, there is little evidence that increased DHA attenuates neuroinflammation following systemic LPS, bearing in mind there is a lack of studies. Ethyl-EPA and ethyl-GLA treatments appear to confer protection against neuroinflammation during systemic inflammation.
4. n-3 PUFA and neurionflammation induced by brain ischemia-reperfusion Inflammation is a major and detrimental pathophysiology of stroke [1]. Not surprisingly, then, inflammation is a common outcome measure in brain ischemia-reperfusion animal models. Seven studies have measured the effect of increased n-3 PUFA or SPM on brain inflammation resulting from brain ischemia-reperfusion. Only one study tested chronic dietary modulation of n-3 PUFA; in it, Laloncette-He´bert et al. reported that, following a 1-hour middle cerebral arterial occlusion, mice with higher brain DHA levels had attenuated infarct areas, decreased in vivo innate immune responses measured by TLR2-linked luciferase, and downregulated protein expression of IL-1b, IL-6, and COX-2 [13]. Similarly, Pan et al. reported 6 weeks of daily i.p. injection of DHA (500 nmol/kg) significantly attenuated infarct volume, leukocyte infiltration, and protein levels of IL-6 in rats [28]. Two other treatment groups received single i.p. injection treatments (500 nmol/kg), either 1 h or 3 days prior to ischemia. Both singleinjections mimicked the effect of the 6-weeks chronic treatment group [28]. There was no effect of 100 nmol DHA/kg in any of the three treatments [28]. No DHA measures were reported in this study, making it difficult to determine if brain or plasma accretion of DHA were similar between treatments, which might explain the similar responses. Four studies have infused DHA post-injury, by i.c.v. [30], i.v. [38,39], and i.p. [40] routes. Yang et al. reported DHA exacerbates infarct volume, loss in motor activity, leukocyte
S.K. Orr et al. / Prostaglandins, Leukotrienes and Essential Fatty Acids 88 (2013) 97–103
infiltration, and COX-2 expression [40]. A similar exacerbation occurred in rats treated with ARA, but not stearic acid (18:0). Oxidative damage increased in the ARA and DHA groups, but not the saturated fat-treated group, suggesting that PUFA contribute to oxidative stress [40]. In contrast, the other studies administering DHA post-ischemia found protective effects of DHA on infarct volume, behavioral deficits, and neurological scores [38,39]. Interestingly, the therapeutic window of post-ischemic DHA administration was identified as 3–5 h; by 6 h there was no longer an effect of treatment [39]. Regarding neuroinflammation, one reported a decrease in leukocyte/neutrophil infiltration, NFkB activation, and COX-2 expression in DHA-treated animals [30], while the others found an increase in glial fibrillary acidic protein (GFAP) in DHA-treated animals [38,39] concomitant to fewer activated microglia [39]. GFAP is expressed by astrocytes, and is more often reported as a marker of neuroinflammation [41–44], but also has been reported as neuro-protective [38,39]. While DHA can be protective in animal models of ischemia, the mechanism is unclear. One hypothesis is that DHA-derived SPM beneficially modulate immune responses to brain injury. Evidence for this comes from three studies. Marcheselli et al. reported that i.c.v. infusion of neuroprotectin D1 (NPD1, 10,17S-dihydroxyDHA) following brain ischemia reduced infarct volume, leukocyte infiltration, NF-kB activation, and COX-2 expression [30]. Similar, but less robust, effects were seen when infusing DHA at much higher doses, demonstrating the potency of 10,17S-dihydroxyDHA compared to DHA itself [30]. Belayev et al. report increased levels of NPD1 and a stable marker of its 17S-hydroperoxy-DHA precursor, 17-HDHA, in the penumbra of rats treated with DHA post-ischemia [39]. NPD1 and 17-HDHA production is inversely associated with stroke damage, neurological impairments, and cellular markers of neuroinflammation [39]. In a third study, i.v. infusing the aspirin triggered (AT) epimer of NPD1, AT-NPD1 (10,17R-dihydroxy-DHA), in both sodium salt and methyl ester forms, is similarly protective against stroke damage as NPD1, and similarly reduces cellular markers of neuroinflammation [45]. Taking all these studies into consideration, DHA is protective in animal models of stroke where it also attenuates neuroinflammation. However, there is some controversy around the benefit of post-ischemic treatment. In these models DHA acts, at least in part, through its 10,17-dihydroxy-DHA derivatives.
5. n-3 PUFA and neuroinflammation in aging Cognitive decline with aging is associated with neuroinflammation and an increase in pro-inflammatory cytokines, including IL-1b [46,47]. In rats, consumption of a 10 then 20 mg/day ethylEPA-enriched diet over 8 weeks prevents age-induced increases in cortical and hippocampal IL-1b protein [47,48], and the ageinduced decrease in cortical IL-4 protein [48]. Consumption of 125 mg/day ethyl-EPA for 4 weeks attenuated age-induced increases in hippocampal IL-1b mRNA and protein, IFN-g protein, and age-induced decreases in IL-4 mRNA and protein [26]. The same EPA treatment protected aged rats (22 months old) from further increases in hippocampal IL-1b protein induced by Ab [26]. Giving mechanistic insight, 4 weeks of 125 mg/day EPA consumption attenuated age-induced decreases in PPARg protein and Abinduced decreases in PPARg in adult (3 months old) rats [27]. Lifelong consumption of ALA increased cortical DHA levels in adult (3–5 month old) and aged (19–23 month old) mice compared to mice that consumed an n-3 PUFA deprived diet [49]. Increased cortical DHA protected against depressive-like symptoms, but did not prevent age-related memory deficits, and did not dampen age-related increases in cortical IL-6 or decreases in IL-10 [49].
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6. n-3 PUFA and neuroinflammation in other models The effect of n-3 PUFA supplementation on inflammation has also been studied in the olfactory bulbectomized rat model of depression [50]. The majority of inflammatory markers were made in serum; however, hypothalamic calcium-dependent cytosolic phospholipase A2 (cPLA2) mRNA and protein activity were measured. cPLA2 is an enzyme of the ARA cascade that increases during neuroinflammation and thus can be used as a neuroinflammatory marker [51]. A 1% EPA diet for seven weeks protected olfactory bulbectomized rats from the increased cPLA2 mRNA and activity that was observed in palm oil-fed olfactory bulbectomized rats [50]. There was also an attenuation of systemic inflammatory measures and depressive-like behavioral measures in EPA-fed rats. Neuroinflammation is present in Parkinson’s disease [52], and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of Parkinson’s disease [53]. Compared to a palm oilsupplemented chow, consumption of a 0.8% ethyl-EPA-supplemented chow for 6 weeks protected mice against MPTP-induced hypokinesia and other behavioral deficits [54]. Mice that consumed ethyl-EPA also had no increase in striatal proteins TNF-a, IFN-g, or midbrain IL-10 protein, while mice consuming palm oil had increases in all three [54]. Conversely, fat-1 transgenic mice, which endogenously convert n-6 to n-3 PUFA, are not protected against MPTP-induced dopaminergic injury compared to their wildtype littermates [41]. However, secondary analysis that grouped fat-1 and wildtype mice found a significant inverse correlation between brain DHA levels and nigral dopaminergic damage [41]. In the substantia nigra (SN) LPS injection model of Parkinson’s disease, rats fed a 15% fish oil diet for 2 weeks prior to SN-LPS were protected against SN dopaminergic injury, microglial activation, and TNF-a and IL-1b protein expression [55]. Overall, elevated dietary or brain DHA levels appear to be protective against dopaminergic damage and neuroinflammation in models of Parkinson’s disease. Cytokines are endogenously produced molecules that act as autocoids and paracoids to coordinate immune responses. IL-1b is a pro-inflammatory cytokine produced by astrocytes, microglia, and neurons in response to injury or infection. Two studies have examined the modulatory effects of n-3 PUFA on memory impairment induced by an i.c.v. injection of IL-1b, and also measured brain inflammation. In the first, rats consumed diets containing 5% coconut oil (n-3 PUFA deprived, n-6 PUFA low), 5% soybean oil (n-3 PUFA low, n-6 PUFA rich), 4.8% coconut oilþ0.2% ethyl-EPA, or 4% coconut oilþ1% ethyl-EPA. After 7 weeks of dietary treatment, 15 ng IL-1b was injected i.c.v. on 3 different days throughout 7 days of behavioral testing, which was the main outcome of the study [56]. One hour following the final IL-1b treatment, brains were removed and hippocampal PGE2 levels were measured. Rats consuming the 1% ethyl-EPA diet, but not the 0.2% diet, had decreased IL-1b-induced PGE2 compared to rats on the coconut oil diet. In a subsequent study by the same group, 0.5% ethyl-EPA diet consumption was found to protect rats against i.c.v. IL-1b-induced increases in PGE2 concentrations and decreases in IL-10 protein in several brain regions including the hippocampus [57]. In a different locally-induced rat model of neuroinflammation, four-week consumption of 125 mg/day ethyl-EPA attenuated i.c.v. Ab-induced increases in IFN-g and IL1b protein [27]. Irradiation as a therapy for cancers, particularly brain tumors, can lead to cognitive deficits that animal studies suggest are the result of neuronal apoptosis [58]. Inflammation has been investigated as a mechanism contributing to brain damage following irradiation, and one study has looked specifically at the effects of n-3 PUFA supplementation on brain inflammation pathways
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following irradiation [59]. Four weeks on a 1% (250 mg/d) or 2% (500 mg/d) EPA diet significantly attenuated hippocampal inflammation in rats following gamma irradiation [59]. EPA-fed rats had attenuated irradiation-induced increases in IL-1b and IL-1 receptor proteins, along with attenuated irradiation-induced decreases in IL-10 protein compared to n-3 PUFA deprived controls [59]. Cognitive deficits are associated with diabetes [60,61]. In a streptozotocin-induced model of diabetes, daily gavaging 13.3 mg/kg/d of DHA for 12 weeks protected rats from spatial learning and memory deficits, though DHA did not protect rats from diabetes-associated weight loss or hyperglycemia [62]. DHA treatment increased neurogenesis, and decreased neuronal loss and oxidative stress in the hippocampus. NF-kB activation is also attenuated in the hippocampi of DHA-treated rats, suggesting that they have less neuroinflammation [62].
7. Conclusion This review has summarized data on the effects of n-3 PUFA on brain inflammation outcomes in animal brain injury models of sickness behavior, stroke, cognitive impairment, Parkinson’s disease, depression, aging, and irradiation. n-3 PUFA are antineuroinflammatory in all of these brain disease models. However, we cannot conclude that n-3 PUFA are acting directly in neuroinflammatory pathways, since there was also a consistent attenuation of the primary injury (i.e. systemic inflammation, ischemic area, dopaminergic system injury, behavioral and cognitive deficits). While these results are promising because disease models are likely more relevant to human pathologies, studies on the effects of n-3 PUFA in models of direct and focused neuroinflammation are necessary. Further, there is a paucity of studies on the mechanisms of DHA or EPA on neuroinflammation pathways in vivo. It is hypothesized that n-3 PUFA are anti-inflammatory via their enzymatically-derived metabolites, however comprehensive lipidomics profiling during neuroinflammation has yet to be reported in the literature. SPM, SPM receptors, and the pathways that they mediate should be investigated as they may provide novel targets for the modulation of neuroinflammation.
Acknowledgements This work was funded by a Canadian Institutes of Health Research grant to RPB and a Natural Sciences and Engineering Research Council scholarship to SKO. References [1] C. Iadecola, J. Anrather, The immunology of stroke: from mechanisms to translation, Nat. Med. 17 (7) (2011) 796–808. [2] H. Akiyama, S. Barger, S. Barnum, B. Bradt, J. Bauer, G.M. Cole, N.R. Cooper, P. Eikelenboom, M. Emmerling, B.L. Fiebich, C.E. Finch, et al., Inflammation and Alzheimer’s disease, Neurobiol. Aging 21 (3) (2000) 383–421. [3] C. Stadelmann, C. Wegner, W. Bruck, Inflammation, demyelination, and degeneration—recent insights from MS pathology, Biochim. Biophys. Acta 1812 (2) (2011) 275–282. [4] C.N. Serhan, N. Chiang, T.E. Van Dyke, Resolving inflammation: dual antiinflammatory and pro-resolution lipid mediators, Nat. Rev. Immunol. 8 (5) (2008) 349–361. [5] G. Bannenberg, C.N. Serhan, Specialized pro-resolving lipid mediators in the inflammatory response: an update, Biochim. Biophys. Acta 1801 (12) (2010) 1260–1273. [6] D.R. Hoffman, J.A. Boettcher, D.A. Diersen-Schade, Toward optimizing vision and cognition in term infants by dietary docosahexaenoic and arachidonic acid supplementation: a review of randomized controlled trials, Prostaglandins Leukot. Essen. Fatty acids 81 (2–3) (2009) 151–158. [7] M. Igarashi, K. Ma, F. Gao, H.W. Kim, D. Greenstein, S.I. Rapoport, J.S. Rao, Brain lipid concentrations in bipolar disorder, J. Psychiatr. Res. 44 (3) (2010) 177–182.
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Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
n-3 Polyunsaturated fatty acids in animal models with neuroinflammation Sarah K. Orr, Marc-Olivier Tre´panier, Richard P. Bazinet n Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, ON, Canada M5S 3E2
a r t i c l e i n f o
Keywords: Neuroinflammation Animal models Brain Docosahexaenoic acid Eicosapentaenoic acid
abstract Neuroinflammation is present in the majority of acute and chronic neurological disorders. Excess or prolonged inflammation in the brain is thought to exacerbate neuronal damage and loss. Identifying modulators of neuroinflammation is an active area of study since it may lead to novel therapies. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are anti-inflammatory in many non-neural tissues; their role in neuroinflammation is less studied. This review summarizes the relationship between n-3 PUFA and brain inflammation in animal models of brain injury and aging. Evidence by and large shows protective effects of n-3 PUFA in models of sickness behavior, stroke, aging, depression, Parkinson’s disease, diabetes, and cytokine- and irradiation-induced cognitive impairments. However, rigorous studies that test the direct effects of n-3 PUFA in neuroinflammation in vivo are lacking. Future research in this area is necessary to determine if, and if so which, n-3 PUFA directly target brain inflammatory pathways. n-3 PUFA bioactive metabolites may provide novel therapeutic targets for neurological disorders with a neuroinflammatory component. & 2012 Elsevier Ltd. All rights reserved.
1. Introduction Inflammation is a beneficial innate response to insults and injuries; however, an overly robust or chronic inflammatory response can contribute significantly to tissue damage. Excessive inflammation in the brain is thought to exacerbate acute injuries including stroke [1], and chronic diseases including multiple sclerosis and Alzheimer’s disease [2,3]. Elucidating neuroinflammatory pathways and identifying modulators is a growing area in brain research, since it may provide novel targets in disease prevention and treatment. The resolution of inflammation was originally considered a passive process that occurs through the dissipation of pro-inflammatory signals. More recently it has become clear that inflammation resolution is an active process, driven by its own mediators [4]. The most studied of these signaling molecules are Specialized Pro-resolving Mediators (SPM), which are enzymatically derived from omega-3 polyunsaturated fatty acids (n-3 PUFA). SPM include eicosapentaenoic acid (EPA; 20:5n-3)-derived resolvins, and docosahexaenoic acid (DHA; 22:6n-3)-derived resolvins, protectins, and Abbreviations: Ab, amyloid-b; ALA, a-linolenic acid; COX-2, cyclooxygenase-2; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; IFN-g, interferon-g; IL1b, interleukin-1b; IL-6, interleukin-6; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; n-3 PUFA, omega-3 polyunsaturated fatty acid; NF-kB, nuclear factor-kB; PPAR, peroxisome proliferator-activated receptor; TNF-a, tumor necrosis factor-a. n Correspondence to: Department of Nutritional Sciences, University of Toronto, FitzGerald Building, 150 College St., Room 306, Toronto, ON, Canada M5S 3E2. Tel.: þ416 946 8276; fax: þ416 978 5882. E-mail address: [email protected] (R.P. Bazinet). 0952-3278/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.plefa.2012.05.008
maresins [5]. Through targeting inflammation resolution, SPM provide a potential mechanism to explain the benefits of n-3 PUFA seen in the prevention and treatment of neurodegenerative diseases. DHA is essential for neural and retinal development [6], and is highly enriched in brain compared to most other tissues, making up approximately 0.33% brain wet weight in adult human cortex (10– 12% of total fatty acids) [7,8]. DHA is similarly enriched in the brains of other animals, including non-human primates [9], rats [10,11], and mice [12,13]. EPA and its shorter chain n-3 PUFA precursor, alinolenic acid (ALA; 18:3n-3), are present at very low levels in the brain [11,12,14]. Rapid metabolism via b-oxidation appears to be a mechanism by which EPA and ALA are maintained at concentrations 200–500 fold lower than DHA [11,15], but what is unclear is the functional reason for their low levels in the brain. To date, mechanistic evidence from animal models shows that DHA is anti-apoptotic [16,17], neurotrophic [18], and important for synaptic plasticity [19]. There are, however, no studies testing the direct effects of DHA on the innate brain inflammatory response. The purpose of this short paper is to review the effect of n-3 PUFA modulation in animal models of brain injury and disease where inflammation is also measured (Table 1). In vitro research in this area is also briefly reviewed (for an in-depth review see [20]). Together, these models provide persuasive but indirect evidence of anti-inflammatory actions of n-3 PUFA in the brain.
2. n-3 PUFA and in vitro neuroinflammation Microglial cells are the main immune cell mediators of the brain [21]. BV-2 cell cultures are immortalized microglial cells.
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Table 1 Injury model Species
Comparison treatment
Kavenagh et al. (2004) [35]
Systemic LPS Wistar rats (i.p.)
Chow
Lonergan et al. (2004) [36]
Systemic LPS (i.p.) Systemic LPS (i.p.) Ischemia/ reperfusion
C57BL/6 mice
Yang et al. (2007) [40]
Ischemia/ reperfusion
SpragueDawley rats
Saline i.p.
Pan et al. (2009) [28]
Ischemia/ reperfusion
SpragueDawley rats
Saline i.p.
Belayev et al. (2009) [38]
Ischemia/ reperfusion Ischemia/ reperfusion
SpragueDawley rats SpragueDawley rats
Saline i.v.
Ischemia/ reperfusion
SpragueDawley rats
Saline i.v.
TLR2-flucGFP transgenic mice (C57BL/ 6) Wistar rats
Low n-3 PUFA (corn þ safflower oil) diet
Mingam et al. (2008) [37] Marcheselli et al. (2003) [30]
Belayev et al. (2011) [39]
Bazan et al. (2012) [45]
Lalancette-Hebert (2011) [13] Ischemia/ reperfusion
Wistar rats CD1 mice
PUFA treatment(s)
Chow supplemented with 50 mg/d eEPAn, 50 mg/d eGLA#, or 50 mg/d eEPA þeGLA Chow Chow supplemented with 500 mg/d eEPA 6% peanut oil (n-6) 6% peanutþ rapeseed oil (ndiet 6 þn-3) diet Vehicle i.c.v. 0.4–120 mg 10,17Sdihydroxy-DHAn; 2–200 mg DHA# i.c.v Per kg body weight: 250 nmol (71 mg) SA, 500 nmol (142 mg) SA, 250 nmol (76 mg) ARA, 500 nmol (152 mg) ARAn, 250 nmol (82 mg) DHA, or 500 nmol (164 mg) DHAn i.p. Per kg body weight: 100 nmol (33 mg), or 500 nmol (164 mg) nDHA i.p.
Treatment duration/time point
Brain n-3 PUFA
Primary outcome
Inflammatory outcome
4 wks
Not reported
n#mLTP
n# m HIP IL-10, IL-4 protein; n m HIP PPARg protein; 2 HIP IL-1b protein
4 wks
Not reported m CX DHA
m LTP; k apoptotic markers k Social interaction; 2 food intake nk Infarct size
k HIP IL-1b protein
Not reported
nm Infarct size
n m CX leukocyte/neutrophil accumulation; n m CX COX-2 mRNA
Not reported
nk Infarct size
nk CX leukocyte/neutrophil accumulation; n k CX IL-6 protein
Not reported nm PN NPD1 and 17-HDHA Not reported
k Infarct size
m CX GFAP
n#D k Infarct size; n#D
m neurologicalscore
n m CX and ST GFAP n k CX and ST CD68 (microglia)
k Infarct size; m neurologicalscore
m CX GFAP; k CX CD68; 2 SCX GFAP and CD68;
Gestation þ 8 wks post-natal 3 or 48 h continuous infusion postinjury 60 min postreperfusion
not reported
2 HIP IL-6 mRNA n#k CX and HIP leukocyte/neutrophil accumulation; n# k HIP NF-kB protein; n# k HIP COX-2 mRNA
1 d (single), 3 d (single), or 6 wks (daily) prior to ischemia Per kg body weight: 43 mmol 60 min post(14 mg) DHA i.v. reperfusion Per kg body weight: 15 mmol 3n h, 4 h#, 5 hD, (5 mg) DHA i.v. or 6 hp postischemia 60 min postPer kg body weight: reperfusion (924 nmol) 333 mg AT-NPD1 i.v. DHA supplemented (corn 12 wks oil þsafflower oil þDHA) diet
m Striatum k Infarct size DHA
Chow
Chow supplemented with 10 mg/d then 20 mg/d eEPA
3 wks (10 mg/ d)þ5 wks (20 mg/d) 3 wks (10 mg/ d)þ5 wks (20 mg/d) 4 wks
Not reported
m LTP; k apoptotic markers
k CX and HIP IL-1b
Not reported
k Apoptotic markers; k neurotrophic factors
k CX IL-1b protein; 2 CX IL-1RI protein; m CX IL-4 protein
4 wks
Not reported
m LTP
k k m k
Saline i.v.
Martin et al. (2002) [47]
Aging (4 vs. 22 mo)
Maher et al. (2004) [48]
Aging (4 vs. 22 mo)
Wistar rats
Chow
Chow supplemented with 10 mg/d then 20 mg/d eEPA
Lynch et al. (2007) [26]
Aging (4 vs. 22 mo)
Wistar rats
Chow supplemented with 125 mg/d eEPA
Ab (i.c.v.) in aged rats (22 mo)
Wistar rats
Chow supplemented with MUFA (isocaloric) Chow supplemented with MUFA (isocaloric)
Chow supplemented with 125 mg/d eEPA
Not reported
k TLR2 promoter induction (microglia activation); k brain IL-1b, IL-6, COX-2 protein; 2 brain TNF-a protein
HIP HIP HIP HIP
MHCII and CD40 (microglia activation); IL-1b, IFN-g protein; k HIP IL-1b mRNA; IL-4 protein and mRNA; IL-1b protein
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Authors (year)
Ab (i.c.v.)
Wistar rats
Aging (3 vs. 22 mo)
Wistar rats
Moranis et al. (2001) [49]
Aging (3–5n vs. 19– 23 mo)
CD1 mice
Song et al. (2009) [50]
Olfactory bulbectomy
SpragueDawley rats
Bousquet et al. (2011) [41]
MPTP (i.p.)
Minogue et al. (2007) [27]
4 wks
Not reported
m LTP
k HIP IFN-g, IL-1b protein; m HIP PPARg protein
Chow supplemented with 125 mg/d eEPA
4 wks
Not reported
p
m HIP PPARg protein
n-3 adequate diet (fatty acids 10.7% LA, 1.6% ALA)
3–5 mo or 19– 23 mo
m CX DHA
1% eEPA supplemented diet
7 wks
Not reported
k Depressive-behavior 2 CX IL-6 or IL-10 protein (FST)y; n m spatial memory; 2 age-induced spatial memory loss k Depressive-like k Hypothalamus cPLA2 mRNA and activity symptoms (MWM and OF)
Fat-1 transgenic mice on Wildtype littermates on high high n-6/low n-3 PUFA diet n-6/low n-3 PUFA diet
Life
m CX DHA
2 Striatal or nigral dopaminergic injuryC
Chow supplemented with Chow supplemented with 0.8% eEPA 0.8% palm oil
6 wks
k Hypokinesia and anxiety k Striatal TNF-a, IFN-g protein; k (RT, PT); m learning and midbrain IL-10 protein; 2 memory (MWM) striatal cPLA2, COX-2 mRNA1
2 wks
m CX EPA, DPAn-3; 2 CX DHA Not reported
1% palm oil supplemented diet
Ji et al. (2012) [55]
SN LPS (i.c.v.)
SpragueDawley rats
15% (wt) corn oil diet
15% (wt) fish oil diet (30% fish oil as EPA and DHA)
Song and Horrobin (2004) [56]
IL-1b (i.c.v.)
Wistar rats
5% coconut oil diet
Song et al. (2008) [57]
IL-1b (i.c.v.)
Wistar rats
5% palm oil diet
Lynch et al. (2003) [59]
Whole body irradiation
Wistar rats
Chow
5% soybean, 4.8% coconut oil 7 wks þ 0.2% eEPA, or 4% coconut oil þ1% eEPAn diet 7 wks 4.5% palm oil þ 0.5% eEPA, 4.5% palm oil þ 0.5% eGLA#, or 4% palm oilþ 1% AA-rich oil diet Chow supplemented with 4 wks 250 mg/d or 500 mg/d# eEPA
¨ Alvarez-Nolting et al. (2012) [62]
STZ (i.p.)
Wistar rats
Chow~
k CX GFAP
k Dopaminergic injury; k nigral dopaminergic neuron degeneration kMemory loss (MWM)
k SN OX42 protein (microglial activation); k SN TNF-a and IL-1b protein; k SN p65 (NF-kB subunit) protein kBrain PGE2
Not reported
kMemory loss (MWM); n k anxiety (EPM)
n# k HIP PGE2; n k amygdala PGF2; n m amygdala, hypothalamus IL-10 protein
Not reported
#
n# k HIP IL-1b, IL-1RI, IL- 1RAcP protein; n#
Not reported
kApoptotic markersp
2 HIP IRAK protein phosphorylation ratio; n m HIP IL-10 protein
Chow plus 13.3 mg/kg/d DHA by gavage
12 wks
Not reported
2 Blood glucose and glycated hemoglobin; m HIP neurogenesis; k HIP neuronal degeneration; k HIP oxidative stress; m learning and memory (MWM)
k HIP NF-kB protein
17-HDHA, 17-hydroxy-DHA (DHA derivative); Ab, amyloid-b; ALA, a-linolenic acid (18:3n-3); ARA, arachidonic acid (20:4n-6); AT-NPD1, aspirin triggered NPD1; COX-2, cyclooxygenase-2; cPLA2, cytosolic phospholipase A2; CX, cortex; DHA, docosahexaenoic acid (22:6n-3); DPAn-3, docosapentaenoic acid (22:5n-3); EPA, eicosapentaenoic acid (20:5n-3); eEPA, ethyl-EPA; eGLA, ethyl-gamma-linolenic acid (18:3n-6); EPM, elevated plus maze; FST, forced swim test; GFAP, glial fibrillary acidic protein; HIP, hippocampus; i.c.v., intracerebroventricular; IFN-g, interferon-g; IL, interleukin; IL-1RI, IL-1 receptor protein type I;, IL-1RAcP, IL-1 receptor accessory protein; IRAK, IL-1 receptor-associated kinase; LA, linoleic acid (18:2n-6); LPS, lipopolysaccharide; i.p., intraperitoneal; LTP, long term potentiation; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPTP-P, MPTP and probenecid; MWM, Morris water maze; MUFA, monounsaturated fatty acid; NF-kB, nuclear factor-kB; NPD1, neuroprotectin D1 (DHA derivative); OF, open field; PGE2, prostaglandin E2; PN, penumbra; PPARg, peroxisome proliferator activated protein g; PT, pole test; RT, rotorod test; SA, stearic acid (18:0); s.c., subcutaneous; SCX, subcortex; SN, substantia nigra; ST, striatum; TLR2, toll-like receptor 2; TNF-a, tumor necrosis factor-a; wt, weigth*#Dp indicates treatment group represented in outcome columns (brain n-3 PUFA, behavioral outcome, inflammatory outcome).1 EPA lowered COX-2 mRNA compared to palm oil in saline (control) injected animals.p inflammation was primary outcome.y main effect of n-3 adequate vs. n-3 deprived diet (no effect of age).C protection from nigral dopaminergic injury was correlated to brain DHA levels (secondary analysis).~ insulin-treated rats were also included in the study and were similar to DHA-only treated rats in all measures excluding weight, glycemic control, and oxidative stress.
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Luchman et al. (2012) [54]
Fat-1 transgenic mice (C57BL6/ C3H) MPTP-P (s.c.) C57BL/6 mice
Chow supplemented with 125 mg/d eEPA
Chow supplemented with MUFA (isocaloric) Chow supplemented with MUFA (isocaloric) n-3 deficient diet (fatty acids 60.5% LA, 0.1% ALA)
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In BV-2 cells, 3–30 mM DHA or EPA attenuates interferon- g (IFN- g)-induced up-regulation of pro-inflammatory marker gene expression, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and tumor necrosis factor- a (TNF- a) [22]. BV-2 microglial cells stimulated with lipopolysaccharide (LPS) have increased mRNA and protein levels of iNOS, COX-2, interleukin-1b (IL1b), and TNF- a, all of which are dose-dependently attenuated by the addition of 10, 20, or 30 mM of EPA [23]. Insight into DHA’s mechanism of action was provided by De SmedtPeyrusse et al. who demonstrated that 30 mM of DHA significantly downregulated the cell-surface expression of CD14 and toll-like receptor 4 (TLR4) in LPS-stimulated BV-2 microglial cells concomitant to reducing TNF- a protein expression, mature IL-1b protein expression, and activation of the proinflammatory gene transcription factor, nuclear factor-kB (NFkB) [24]. CD14 and TLR4 are co-receptors that bind LPS to initiate an innate immune response. Interestingly, EPA did not reduce CD14 or TLR4 cell-surface expression [24]. In C6 glioma cells, an astrocyte cancer cell line, EPA concentrations of 25–100 mM dose-dependently attenuate IL-1b-induced IL-6 gene expression, an effect also observed with 50 mM arachidonic acid (ARA; 20:4n-6) or DHA [25]. The effect of EPA on IL-6 was blocked by peroxisome proliferator-activated receptor-g (PPARg) and PPARa antagonists, suggesting PPAR signaling is necessary for the anti-inflammatory effects of EPA [25]. Similar effects of EPA were reported in primary hippocampal glial cell cultures from 1-day old rats, where 50 mM EPA increased IL-4 protein and mRNA, and attenuated subsequent LPS-induced increases in IL-1b protein [26]. In the same cell model, 75 mM EPA reduced LPS- and amyloid-b (Ab)-induced increase in IL-1b protein [27]. This effect was partially blocked by a PPARg antagonist [27]. Also using glial cell cultures from 1-day old rats, Pan et al. observed that 75 but not 10 mM DHA co-treatment with LPS/IFN-g attenuated increases in IL-6 protein [28]. Pretreating with 75 mM DHA was even more effective at inhibiting LPS/IFN-g IL-6 protein expression [28], suggesting incorporation of DHA into phospholipid membranes may optimize immune modulation. We identified three studies that measured the neuroinflammatory effects of SPM in vitro. In human neuronal-glial primary cell cultures, 10,17S-dihydroxy-DHA, a 15-lipoxygenase metabolite of DHA, downregulated COX-2, TNF-a, and IL-1b expression while increasing PPARg activity in a Ab42-induced cell culture model of Ab-deposition, which is a major pathology of Alzheimer’s disease [16,29]. In the same model, DHA downregulated COX-2, TNF-a and IL-1b expression [16]. In IL-1b-stimulated human neural progenitor cells, 10,17S-dihydroxy-DHA downregulated NF-kB activation and COX-2 gene expression; however, there was no effect of 0.03–3000 nM DHA on NF-kB activation in response to IL-1b [30]. In vitro studies support the hypothesis that n-3 PUFA and their derivatives are anti-inflammatory in the brain, and suggest that they target TLR4 signaling, and NF-kB, and PPAR pathways. Importantly, however, unesterified DHA occurs at concentrations of around 1–6 mM in brain and 1–40 mM in plasma; and phospholipid DHA occurs at concentrations of 10–150 mM in plasma and 7000–14,000 mM in the brain of rats and mice [10,31–34]. EPA occurs at even lower concentrations, particularly in the brain where unesterified EPA occurs below detection limits, and phospholipid EPA concentrations range from being undetectable to 115 mM [11,31,34]. Plasma unesterified and phospholipid EPA concentrations both range from undetected to 9 mM [11,31,34]. Whether these pathways are affected by DHA or EPA at fatty acid concentrations seen in brain tissue is not clear, and warrants in vivo study.
3. n-3 PUFA and neuroinflammation induced by systemic lipopolysaccharide We identified three studies that used intraperitoneally (i.p.) injected LPS to test the effects of n-3 PUFA on sickness behavior and/or brain inflammation. In the first, rats consumed one of four diets: standard chow, standard chow plus 50 mg/day of ethylEPA, standard chow plus 50 mg/day of ethyl-gamma linolenic acid (GLA), or standard chow plus 50 mg/day total of ethyl-EPA and ethyl-GLA for 4 weeks prior to 100 mg/kg i.p. LPS-induced hippocampal inflammation [35]. No dietary treatment protected against i.p. LPS-induced hippocampal IL-1b protein expression; however, all treatment diets prevented the reduction in hippocampal protein levels of anti-inflammatory cytokines IL-4 and IL10 compared to rats fed standard chow. Compared to standard chow, ethyl-EPA and ethyl-EPA þethyl-GLA supplemented rats significantly increased i.p. LPS-induced hippocampal protein expression of the anti-inflammatory transcription factor PPARg [35]. In a similar study, the consumption of 500 mg/day ethyl-EPA for 4 weeks did attenuate hippocampal increases in IL-1b protein induced by 100 mg/kg i.p. LPS [36]. While examining i.p. LPS-induced sickness behavior, Mingam et al. measured IL-6 in mice consuming isocaloric diets containing or deprived of n-3 PUFA. One diet included African peanut oil, rich in linoleic acid (18:2n-6; n-6 PUFA diet), and the second diet included African peanut oil plus rapeseed oil, rich in ALA (n-3 PUFAþn-6 PUFA diet). Mice consumed treatment diets from gestation to 8 weeks of age and sickness behavior was induced by 30 mg/kg i.p. LPS administration [37]. Mice consuming n-3þn6 PUFA had 2 to 3 fold higher cortical phospholipid DHA compared to mice fed n-6 PUFA alone. There was no consistent effect of diet on LPS-induced hippocampal IL-6 mRNA expression [37]. Overall, there is little evidence that increased DHA attenuates neuroinflammation following systemic LPS, bearing in mind there is a lack of studies. Ethyl-EPA and ethyl-GLA treatments appear to confer protection against neuroinflammation during systemic inflammation.
4. n-3 PUFA and neurionflammation induced by brain ischemia-reperfusion Inflammation is a major and detrimental pathophysiology of stroke [1]. Not surprisingly, then, inflammation is a common outcome measure in brain ischemia-reperfusion animal models. Seven studies have measured the effect of increased n-3 PUFA or SPM on brain inflammation resulting from brain ischemia-reperfusion. Only one study tested chronic dietary modulation of n-3 PUFA; in it, Laloncette-He´bert et al. reported that, following a 1-hour middle cerebral arterial occlusion, mice with higher brain DHA levels had attenuated infarct areas, decreased in vivo innate immune responses measured by TLR2-linked luciferase, and downregulated protein expression of IL-1b, IL-6, and COX-2 [13]. Similarly, Pan et al. reported 6 weeks of daily i.p. injection of DHA (500 nmol/kg) significantly attenuated infarct volume, leukocyte infiltration, and protein levels of IL-6 in rats [28]. Two other treatment groups received single i.p. injection treatments (500 nmol/kg), either 1 h or 3 days prior to ischemia. Both singleinjections mimicked the effect of the 6-weeks chronic treatment group [28]. There was no effect of 100 nmol DHA/kg in any of the three treatments [28]. No DHA measures were reported in this study, making it difficult to determine if brain or plasma accretion of DHA were similar between treatments, which might explain the similar responses. Four studies have infused DHA post-injury, by i.c.v. [30], i.v. [38,39], and i.p. [40] routes. Yang et al. reported DHA exacerbates infarct volume, loss in motor activity, leukocyte
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infiltration, and COX-2 expression [40]. A similar exacerbation occurred in rats treated with ARA, but not stearic acid (18:0). Oxidative damage increased in the ARA and DHA groups, but not the saturated fat-treated group, suggesting that PUFA contribute to oxidative stress [40]. In contrast, the other studies administering DHA post-ischemia found protective effects of DHA on infarct volume, behavioral deficits, and neurological scores [38,39]. Interestingly, the therapeutic window of post-ischemic DHA administration was identified as 3–5 h; by 6 h there was no longer an effect of treatment [39]. Regarding neuroinflammation, one reported a decrease in leukocyte/neutrophil infiltration, NFkB activation, and COX-2 expression in DHA-treated animals [30], while the others found an increase in glial fibrillary acidic protein (GFAP) in DHA-treated animals [38,39] concomitant to fewer activated microglia [39]. GFAP is expressed by astrocytes, and is more often reported as a marker of neuroinflammation [41–44], but also has been reported as neuro-protective [38,39]. While DHA can be protective in animal models of ischemia, the mechanism is unclear. One hypothesis is that DHA-derived SPM beneficially modulate immune responses to brain injury. Evidence for this comes from three studies. Marcheselli et al. reported that i.c.v. infusion of neuroprotectin D1 (NPD1, 10,17S-dihydroxyDHA) following brain ischemia reduced infarct volume, leukocyte infiltration, NF-kB activation, and COX-2 expression [30]. Similar, but less robust, effects were seen when infusing DHA at much higher doses, demonstrating the potency of 10,17S-dihydroxyDHA compared to DHA itself [30]. Belayev et al. report increased levels of NPD1 and a stable marker of its 17S-hydroperoxy-DHA precursor, 17-HDHA, in the penumbra of rats treated with DHA post-ischemia [39]. NPD1 and 17-HDHA production is inversely associated with stroke damage, neurological impairments, and cellular markers of neuroinflammation [39]. In a third study, i.v. infusing the aspirin triggered (AT) epimer of NPD1, AT-NPD1 (10,17R-dihydroxy-DHA), in both sodium salt and methyl ester forms, is similarly protective against stroke damage as NPD1, and similarly reduces cellular markers of neuroinflammation [45]. Taking all these studies into consideration, DHA is protective in animal models of stroke where it also attenuates neuroinflammation. However, there is some controversy around the benefit of post-ischemic treatment. In these models DHA acts, at least in part, through its 10,17-dihydroxy-DHA derivatives.
5. n-3 PUFA and neuroinflammation in aging Cognitive decline with aging is associated with neuroinflammation and an increase in pro-inflammatory cytokines, including IL-1b [46,47]. In rats, consumption of a 10 then 20 mg/day ethylEPA-enriched diet over 8 weeks prevents age-induced increases in cortical and hippocampal IL-1b protein [47,48], and the ageinduced decrease in cortical IL-4 protein [48]. Consumption of 125 mg/day ethyl-EPA for 4 weeks attenuated age-induced increases in hippocampal IL-1b mRNA and protein, IFN-g protein, and age-induced decreases in IL-4 mRNA and protein [26]. The same EPA treatment protected aged rats (22 months old) from further increases in hippocampal IL-1b protein induced by Ab [26]. Giving mechanistic insight, 4 weeks of 125 mg/day EPA consumption attenuated age-induced decreases in PPARg protein and Abinduced decreases in PPARg in adult (3 months old) rats [27]. Lifelong consumption of ALA increased cortical DHA levels in adult (3–5 month old) and aged (19–23 month old) mice compared to mice that consumed an n-3 PUFA deprived diet [49]. Increased cortical DHA protected against depressive-like symptoms, but did not prevent age-related memory deficits, and did not dampen age-related increases in cortical IL-6 or decreases in IL-10 [49].
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6. n-3 PUFA and neuroinflammation in other models The effect of n-3 PUFA supplementation on inflammation has also been studied in the olfactory bulbectomized rat model of depression [50]. The majority of inflammatory markers were made in serum; however, hypothalamic calcium-dependent cytosolic phospholipase A2 (cPLA2) mRNA and protein activity were measured. cPLA2 is an enzyme of the ARA cascade that increases during neuroinflammation and thus can be used as a neuroinflammatory marker [51]. A 1% EPA diet for seven weeks protected olfactory bulbectomized rats from the increased cPLA2 mRNA and activity that was observed in palm oil-fed olfactory bulbectomized rats [50]. There was also an attenuation of systemic inflammatory measures and depressive-like behavioral measures in EPA-fed rats. Neuroinflammation is present in Parkinson’s disease [52], and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of Parkinson’s disease [53]. Compared to a palm oilsupplemented chow, consumption of a 0.8% ethyl-EPA-supplemented chow for 6 weeks protected mice against MPTP-induced hypokinesia and other behavioral deficits [54]. Mice that consumed ethyl-EPA also had no increase in striatal proteins TNF-a, IFN-g, or midbrain IL-10 protein, while mice consuming palm oil had increases in all three [54]. Conversely, fat-1 transgenic mice, which endogenously convert n-6 to n-3 PUFA, are not protected against MPTP-induced dopaminergic injury compared to their wildtype littermates [41]. However, secondary analysis that grouped fat-1 and wildtype mice found a significant inverse correlation between brain DHA levels and nigral dopaminergic damage [41]. In the substantia nigra (SN) LPS injection model of Parkinson’s disease, rats fed a 15% fish oil diet for 2 weeks prior to SN-LPS were protected against SN dopaminergic injury, microglial activation, and TNF-a and IL-1b protein expression [55]. Overall, elevated dietary or brain DHA levels appear to be protective against dopaminergic damage and neuroinflammation in models of Parkinson’s disease. Cytokines are endogenously produced molecules that act as autocoids and paracoids to coordinate immune responses. IL-1b is a pro-inflammatory cytokine produced by astrocytes, microglia, and neurons in response to injury or infection. Two studies have examined the modulatory effects of n-3 PUFA on memory impairment induced by an i.c.v. injection of IL-1b, and also measured brain inflammation. In the first, rats consumed diets containing 5% coconut oil (n-3 PUFA deprived, n-6 PUFA low), 5% soybean oil (n-3 PUFA low, n-6 PUFA rich), 4.8% coconut oilþ0.2% ethyl-EPA, or 4% coconut oilþ1% ethyl-EPA. After 7 weeks of dietary treatment, 15 ng IL-1b was injected i.c.v. on 3 different days throughout 7 days of behavioral testing, which was the main outcome of the study [56]. One hour following the final IL-1b treatment, brains were removed and hippocampal PGE2 levels were measured. Rats consuming the 1% ethyl-EPA diet, but not the 0.2% diet, had decreased IL-1b-induced PGE2 compared to rats on the coconut oil diet. In a subsequent study by the same group, 0.5% ethyl-EPA diet consumption was found to protect rats against i.c.v. IL-1b-induced increases in PGE2 concentrations and decreases in IL-10 protein in several brain regions including the hippocampus [57]. In a different locally-induced rat model of neuroinflammation, four-week consumption of 125 mg/day ethyl-EPA attenuated i.c.v. Ab-induced increases in IFN-g and IL1b protein [27]. Irradiation as a therapy for cancers, particularly brain tumors, can lead to cognitive deficits that animal studies suggest are the result of neuronal apoptosis [58]. Inflammation has been investigated as a mechanism contributing to brain damage following irradiation, and one study has looked specifically at the effects of n-3 PUFA supplementation on brain inflammation pathways
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following irradiation [59]. Four weeks on a 1% (250 mg/d) or 2% (500 mg/d) EPA diet significantly attenuated hippocampal inflammation in rats following gamma irradiation [59]. EPA-fed rats had attenuated irradiation-induced increases in IL-1b and IL-1 receptor proteins, along with attenuated irradiation-induced decreases in IL-10 protein compared to n-3 PUFA deprived controls [59]. Cognitive deficits are associated with diabetes [60,61]. In a streptozotocin-induced model of diabetes, daily gavaging 13.3 mg/kg/d of DHA for 12 weeks protected rats from spatial learning and memory deficits, though DHA did not protect rats from diabetes-associated weight loss or hyperglycemia [62]. DHA treatment increased neurogenesis, and decreased neuronal loss and oxidative stress in the hippocampus. NF-kB activation is also attenuated in the hippocampi of DHA-treated rats, suggesting that they have less neuroinflammation [62].
7. Conclusion This review has summarized data on the effects of n-3 PUFA on brain inflammation outcomes in animal brain injury models of sickness behavior, stroke, cognitive impairment, Parkinson’s disease, depression, aging, and irradiation. n-3 PUFA are antineuroinflammatory in all of these brain disease models. However, we cannot conclude that n-3 PUFA are acting directly in neuroinflammatory pathways, since there was also a consistent attenuation of the primary injury (i.e. systemic inflammation, ischemic area, dopaminergic system injury, behavioral and cognitive deficits). While these results are promising because disease models are likely more relevant to human pathologies, studies on the effects of n-3 PUFA in models of direct and focused neuroinflammation are necessary. Further, there is a paucity of studies on the mechanisms of DHA or EPA on neuroinflammation pathways in vivo. It is hypothesized that n-3 PUFA are anti-inflammatory via their enzymatically-derived metabolites, however comprehensive lipidomics profiling during neuroinflammation has yet to be reported in the literature. SPM, SPM receptors, and the pathways that they mediate should be investigated as they may provide novel targets for the modulation of neuroinflammation.
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