Epolia Ramadan

Docosahexaenoic Acid (DHA) Incorporation into the Brain from Plasma, as an In Vivo Biomarker of Brain DHA Metabolism and Neurotransmission

Docosahexaenoic acid (DHA) is critical for maintaining normal brain structure and function, and is considered neuroprotective. Its brain concentration depends on dietary DHA content and hepatic conversion from its dietary derived n-3 precursor, α-linolenic acid (α-LNA). We have developed an in vivo method in rats using quantitative autoradiography and intravenously injected radiolabeled DHA to image net incorporation into the brain of unesterified plasma DHA, and showed with this method that the incorporation rate of DHA equals the rate of brain metabolic DHA consumption. The method has been extended for use in humans with positron emission tomography (PET). Furthermore, imaging in unanesthetized rats using DHA incorporation as a biomarker in response to acute N-methyl-D-aspartate administration confirms that regional DHA signaling is independent of extracellular calcium, and likely mediated by a calcium-independent phospholipase A(2) (iPLA(2)). Studies in mice in which iPLA(2)-VIA (β) was knocked out confirmed that this enzyme is critical for baseline and muscarinic cholinergic signaling involving DHA. Thus, quantitative imaging of DHA incorporation from plasma into brain can be used as an in vivo biomarker of brain DHA metabolism and neurotransmission.

Imaging Upregulated Brain Arachidonic Acid Metabolism in HIV-1 Transgenic Rats

Human immunodeficiency virus (HIV)-associated infection involves the entry of virus-bearing monocytes into the brain, followed by microglial activation, neuroinflammation, and upregulated arachidonic acid (AA) metabolism. The HIV-1 transgenic (Tg) rat, a noninfectious HIV-1 model, shows neurologic and behavioral abnormalities after 5 months of age. We hypothesized that brain AA metabolism would be elevated in older HIV-1 Tg rats in vivo. Arachidonic acid incorporation from the plasma into the brain of unanesthetized 7-to-9-month-old rats was imaged using quantitative autoradiography, after [1-14C]AA infusion. Brain phospholipase (PLA2) activities and eicosanoid concentrations were measured, and enzymes were localized by immunostaining. AA incorporation coefficients k* and rates Jin, measures of AA metabolism, were significantly higher in 69 of 81 brain regions in HIV-1 Tg than in control rats, as were activities of cytosolic (c)PLA2-IV, secretory (s)PLA2, and calcium independent (i)PLA2-VI, as well as prostaglandin E2 and leukotriene B4 concentrations. Immunostaining of somatosensory cortex showed elevated cPLA2-IV, sPLA2-IIA, and cyclooxygenase-2 in neurons. Brain AA incorporation and other markers of AA metabolism are upregulated in HIV-1 Tg rats, in which neurologic changes and neuroinflammation have been reported. Positron emission tomography with [1-11C]AA could be used to test whether brain AA metabolism is upregulated in HIV-1-infected patients, in relation to cognitive and behavioral disturbances.

Imaging decreased brain docosahexaenoic acid metabolism and signaling in iPLA2β (VIA)-deficient mice

Ca2+-independent phospholipase A2β (iPLA2β) selectively hydrolyzes docosahexaenoic acid (DHA, 22:6n-3) in vitro from phospholipid. Mutations in the PLA2G6 gene encoding this enzyme occur in patients with idiopathic neurodegeneration plus brain iron accumulation and dystonia-parkinsonism without iron accumulation, whereas mice lacking PLA2G6 show neurological dysfunction and neuropathology after 13 months. We hypothesized that brain DHA metabolism and signaling would be reduced in 4-month-old iPLA2β-deficient mice without overt neuropathology. Saline or the cholinergic muscarinic M1,3,5 receptor agonist arecoline (30 mg/kg) was administered to unanesthetized iPLA2β−/−, iPLA2β+/−, and iPLA2β+/+ mice, and [1-14C]DHA was infused intravenously. DHA incorporation coefficients k* and rates Jin, representing DHA metabolism, were determined using quantitative autoradiography in 81 brain regions. iPLA2β−/− or iPLA2β+/− compared with iPLA2β+/+ mice showed widespread and significant baseline reductions in k* and Jin for DHA. Arecoline increased both parameters in brain regions of iPLA2β+/+ mice but quantitatively less so in iPLA2β−/− and iPLA2β+/− mice. Consistent with iPLA2β’s reported ability to selectively hydrolyze DHA from phospholipid in vitro, iPLA2β deficiency reduces brain DHA metabolism and signaling in vivo at baseline and following M1,3,5 receptor activation. Positron emission tomography might be used to image disturbed brain DHA metabolism in patients with PLA2G6 mutations.

IMAGING BRAIN SIGNAL TRANSDUCTION AND METABOLISM VIA ARACHIDONIC AND DOCOSAHEXAENOIC ACID IN ANIMALS AND HUMANS

The polyunsaturated fatty acids (PUFAs), arachidonic acid (AA, 20:4n-6) and docosahexaenoic acid (DHA, 22:6n-3), important second messengers in brain, are released from membrane phospholipid following receptor-mediated activation of specific phospholipase A(2) (PLA(2)) enzymes. We developed an in vivo method in rodents using quantitative autoradiography to image PUFA incorporation into brain from plasma, and showed that their incorporation rates equal their rates of metabolic consumption by brain. Thus, quantitative imaging of unesterified plasma AA or DHA incorporation into brain can be used as a biomarker of brain PUFA metabolism and neurotransmission. We have employed our method to image and quantify effects of mood stabilizers on brain AA/DHA incorporation during neurotransmission by muscarinic M(1,3,5), serotonergic 5-HT(2A/2C), dopaminergic D(2)-like (D(2), D(3), D(4)) or glutamatergic N-methyl-d-aspartic acid (NMDA) receptors, and effects of inhibition of acetylcholinesterase, of selective serotonin and dopamine reuptake transporter inhibitors, of neuroinflammation (HIV-1 and lipopolysaccharide) and excitotoxicity, and in genetically modified rodents. The method has been extended for the use with positron emission tomography (PET), and can be employed to determine how human brain AA/DHA signaling and consumption are influenced by diet, aging, disease and genetics.

Extracellular-derived calcium does not initiate in vivo neurotransmission involving docosahexaenoic acid

In vitro studies show that docosahexaenoic acid (DHA) can be released from membrane phospholipid by Ca2+-independent phospholipase A2 (iPLA2), Ca2+-independent plasmalogen PLA2 or secretory PLA2 (sPLA2), but not by Ca2+-dependent cytosolic PLA2 (cPLA2), which selectively releases arachidonic acid (AA). Since glutamatergic NMDA (N-methyl-D-aspartate) receptor activation allows extracellular Ca2+ into cells, we hypothesized that brain DHA signaling would not be altered in rats given NMDA, to the extent that in vivo signaling was mediated by Ca2+-independent mechanisms. Isotonic saline, a subconvulsive dose of NMDA (25 mg/kg), MK-801, or MK-801 followed by NMDA was administered i.p. to unanesthetized rats. Radiolabeled DHA or AA was infused intravenously and their brain incorporation coefficients k*, measures of signaling, were imaged with quantitative autoradiography. NMDA or MK-801 compared with saline did not alter k* for DHA in any of 81 brain regions examined, whereas NMDA produced widespread and significant increments in k* for AA. In conclusion, in vivo brain DHA but not AA signaling via NMDA receptors is independent of extracellular Ca2+ and of cPLA2. DHA signaling may be mediated by iPLA2, plasmalogen PLA2, or other enzymes insensitive to low concentrations of Ca2+. Greater AA than DHA release during glutamate-induced excitotoxicity could cause brain cell damage.

Retraction Note to: Anti-Inflammatory Effects of Chronic Aspirin on Brain Arachidonic Acid Metabolites

Pro-inflammatory and anti-inflammatory mediators derived from arachidonic acid (AA) modulate peripheral inflammation and its resolution. Aspirin (ASA) is a unique non-steroidal anti-inflammatory drug, which switches AA metabolism from prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) to lipoxin A4 (LXA4) and 15-epi-LXA4. However, it is unknown whether chronic therapeutic doses of ASA are anti-inflammatory in the brain. We hypothesized that ASA would dampen increases in brain concentrations of AA metabolites in a rat model of neuroinflammation, produced by a 6-day intracerebroventricular infusion of bacterial lipopolysaccharide (LPS). In rats infused with LPS (0.5 ng/h) and given ASA-free water to drink, concentrations in high-energy microwaved brain of PGE2, TXB2 and leukotriene B4 (LTB4) were elevated. In rats infused with artificial cerebrospinal fluid, 6 weeks of treatment with a low (10 mg/kg/day) or high (100 mg/kg/day) ASA dose in drinking water decreased brain PGE2, but increased LTB4, LXA4 and 15-epi-LXA4 concentrations. Both doses attenuated the LPS effects on PGE2, and TXB2. The increments in LXA4 and 15-epi-LXA4 caused by high-dose ASA were significantly greater in LPS-infused rats. The ability of ASA to increase anti-inflammatory LXA4 and 15-epi-LXA4 and reduce pro-inflammatory PGE2 and TXB2 suggests considering aspirin further for treating clinical neuroinflammation.

Chronic valproate treatment blocks D2-like receptor-mediated brain signaling via arachidonic acid in rats.

BACKGROUND AND OBJECTIVE Hyperdopaminergic signaling and an upregulated brain arachidonic acid (AA) cascade may contribute to bipolar disorder (BD). Lithium and carbamazepine, FDA-approved for the treatment of BD, attenuate brain dopaminergic D(2)-like (D(2), D(3), and D(4)) receptor signaling involving AA when given chronically to awake rats. We hypothesized that valproate (VPA), with mood-stabilizing properties, would also reduce D(2)-like-mediated signaling via AA. METHODS An acute dose of quinpirole (1 mg/kg) or saline was administered to unanesthetized rats that had been treated for 30 days with a therapeutically relevant dose of VPA (200 mg/kg/day) or vehicle. Regional brain AA incorporation coefficients, k*, and incorporation rates, J(in), markers of AA signaling and metabolism, were measured by quantitative autoradiography after intravenous [1-(14)C]AA infusion. Whole brain concentrations of prostaglandin (PG)E(2) and thromboxane (TX)B(2) also were measured. RESULTS Quinpirole compared to saline significantly increased k* in 40 of 83 brain regions, and increased brain concentrations of PGE(2) in chronic vehicle-treated rats. VPA treatment by itself reduced concentrations of plasma unesterified AA and whole brain PGE(2) and TXB(2), and blocked the quinpirole-induced increments in k* and PGE(2). CONCLUSION These results further provide evidence that mood stabilizers downregulate brain dopaminergic D(2)-like receptor signaling involving AA.

Lamotrigine blocks NMDA receptor-initiated arachidonic acid signalling in rat brain: Implications for its efficacy in bipolar disorder

An up-regulated brain arachidonic acid (AA) cascade and a hyperglutamatergic state characterize bipolar disorder (BD). Lamotrigine (LTG), a mood stabilizer approved for treating BD, is reported to interfere with glutamatergic neurotransmission involving N-methyl-d-aspartate receptors (NMDARs). NMDARs allow extracellular calcium into the cell, thereby stimulating calcium-dependent cytosolic phospholipase A2 (cPLA2) to release AA from membrane phospholipid. We hypothesized that LTG, like other approved mood stabilizers, would reduce NMDAR-mediated AA signalling in rat brain. An acute subconvulsant dose of NMDA (25 mg/kg) or saline was administered intraperitoneally to unanaesthetized rats that had been treated p.o. daily for 42 d with vehicle or a therapeutically relevant dose of LTG (10 mg/kg.d). Regional brain AA incorporation coefficients k* and rates J in, and AA signals, were measured using quantitative autoradiography after intravenous [1-14C]AA infusion, as were other AA cascade markers. In chronic vehicle-treated rats, acute NMDA compared to saline increased k* and J in in widespread regions of the brain, as well as prostaglandin (PG)E2 and thromboxane B2 concentrations. Chronic LTG treatment compared to vehicle reduced brain cyclooxygenase (COX) activity, PGE2 concentration, and DNA-binding activity of the COX-2 transcription factor, NF-κB. Pretreatment with chronic LTG blocked the acute NMDA effects on AA cascade markers. In summary, chronic LTG like other mood stabilizers blocks NMDA-mediated signalling involving the AA metabolic cascade. Since markers of the AA cascade and of NMDAR signalling are up-regulated in the post-mortem BD brain, mood stabilizers generally may be effective in BD by dampening NMDAR signalling and the AA cascade.

Altered lipid concentrations of liver, heart and plasma but not brain in HIV-1 transgenic rats.

Disturbed lipid metabolism has been reported in antiretroviral-naive HIV-1-infected patients suggesting a direct effect of the virus on lipid metabolism. To test that the HIV-1 virus alone could alter lipid concentrations, we measured these concentrations in an HIV-1 transgenic (Tg) rat model of human HIV-1 infection, which demonstrates peripheral and central pathology by 7-9 months of age. Concentrations were measured in high-energy microwaved heart, brain and liver from 7-9 month-old HIV-1 Tg and wildtype rats, and in plasma from non-microwaved rats. Plasma triglycerides and liver cholesteryl ester and total cholesterol concentrations were significantly higher in HIV-1 Tg rats than controls. Heart and plasma fatty acid concentrations reflected concentration differences in liver, which showed higher n-3 and n-6 polyunsaturated fatty acid (PUFA) concentrations in multiple lipid compartments. Fatty acid concentrations were increased or decreased in heart and liver phospholipid subfractions. Brain fatty acid concentrations differed significantly between the groups for minor fatty acids such as linoleic acid and n-3 docosapentaenoic acid. The profound changes in heart, plasma and liver lipid concentrations suggest a direct effect of chronic exposure to the HIV-1 virus on peripheral lipid (including PUFA) metabolism.

Upregulated expression of brain enzymatic markers of arachidonic and docosahexaenoic acid metabolism in a rat model of the metabolic syndrome

BackgroundIn animal models, the metabolic syndrome elicits a cerebral response characterized by altered phospholipid and unesterified fatty acid concentrations and increases in pro-apoptotic inflammatory mediators that may cause synaptic loss and cognitive impairment. We hypothesized that these changes are associated with phospholipase (PLA2) enzymes that regulate arachidonic (AA, 20:4n-6) and docosahexaenoic (DHA, 22:6n-6) acid metabolism, major polyunsaturated fatty acids in brain. Male Wistar rats were fed a control or high-sucrose diet for 8 weeks. Brains were assayed for markers of AA metabolism (calcium-dependent cytosolic cPLA2 IVA and cyclooxygenases), DHA metabolism (calcium-independent iPLA2 VIA and lipoxygenases), brain-derived neurotrophic factor (BDNF), and synaptic integrity (drebrin and synaptophysin). Lipid concentrations were measured in brains subjected to high-energy microwave fixation.ResultsThe high-sucrose compared with control diet induced insulin resistance, and increased phosphorylated-cPLA2 protein, cPLA2 and iPLA2 activity and 12-lipoxygenase mRNA, but decreased BDNF mRNA and protein, and drebrin mRNA. The concentration of several n-6 fatty acids in ethanolamine glycerophospholipids and lysophosphatidylcholine was increased, as was unesterified AA concentration. Eicosanoid concentrations (prostaglandin E2, thromboxane B2 and leukotriene B4) did not change.ConclusionThese findings show upregulated brain AA and DHA metabolism and reduced BDNF and drebrin, but no changes in eicosanoids, in an animal model of the metabolic syndrome. These changes might contribute to altered synaptic plasticity and cognitive impairment in rats and humans with the metabolic syndrome.