Intravenous Treatment with a Long-Chain Omega-3 Lipid Emulsion Provides Neuroprotection in a Murine Model of Ischemic Stroke - A Pilot Study.

Single long-chain omega-3 fatty acids (e.g. docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA)) are known for their neuroprotective properties associated with ischemic stroke. This pilot study aimed to test the effectiveness of an acute treatment with a long-chain omega-3 lipid emulsion (Omegaven 10%®, OGV) that contains fish oil (DHA 18 mg/ml; EPA 21 mg/ml) and α-tocopherol (0.2 mg/ml) in a transient middle cerebral artery occlusion (MCAO) model of ischemic stroke in mice. For this purpose, female CD-1 mice were anesthetized and subjected to 90 minutes of MCAO. To reflect a clinically relevant situation for an acute treatment, either after induction of stroke or after reperfusion, a single dose of OGV was injected intravenously into the tail vein (5 ml/kg b.w.). A neurological severity score was used to assess motor function and neurological outcome. Stroke-related parameters were determined 24 hours after MCAO. Microdialysis was used to collect samples from extracellular space of the striatum. Mitochondrial function was determined in isolated mitochondria or dissociated brain cells. Inflammation markers were measured in brain homogenate. According to control experiments, neuroprotective effects could be attributed to the long-chain omega-3 content of the emulsion. Intravenous injection of OGV reduced size and severity of stroke, restored mitochondrial function, and prevented excitotoxic glutamate release. Increases of pro-inflammatory markers (COX-2 and IL-6) were attenuated. Neurological severity scoring and neurochemical data demonstrated that acute OGV treatment shortly after induction of stroke was most efficient and able to improve short-term neurological outcome, reflecting the importance of an acute treatment to improve the outcome. Summarising, acute treatment of stroke with a single intravenous dose of OGV provided strong neuroprotective effects and was most effective when given immediately after onset of ischemia. As OGV is an approved fishoil emulsion for parenteral nutrition in humans, our results may provide first translational data for a possible early management of ischemic stroke with administration of OGV to prevent further brain damage

Hepatic ketogenesis induced by middle cerebral artery occlusion in mice

BACKGROUND: Ketone bodies are known to substitute for glucose as brain fuel when glucose availability is low. Ketogenic diets have been described as neuroprotective. Similar data have been reported for triheptanoin, a fatty oil and anaplerotic compound. In this study, we monitored the changes of energy metabolites in liver, blood, and brain after transient brain ischemia to test for ketone body formation induced by experimental stroke. METHODS AND RESULTS: Mice were fed a standard carbohydrate-rich diet or 2 fat-rich diets, 1 enriched in triheptanoin and 1 in soybean oil. Stroke was induced in mice by middle cerebral artery occlusion for 90 minutes, followed by reperfusion. Mice were sacrificed, and blood plasma and liver and brain homogenates were obtained. In 1 experiment, microdialysis was performed. Metabolites (eg glucose, β-hydroxybutyrate, citrate, succinate) were determined by gas chromatography-mass spectrometry. After 90 minutes of brain ischemia, β-hydroxybutyrate levels were dramatically increased in liver, blood, and brain microdialysate and brain homogenate, but only in mice fed fat-rich diets. Glucose levels were changed in the opposite manner in blood and brain. Reperfusion decreased β-hydroxybutyrate and increased glucose within 60 minutes. Stroke-induced ketogenesis was blocked by propranolol, a β-receptor antagonist. Citrate and succinate were moderately increased by fat-rich diets and unchanged after stroke. CONCLUSIONS: We conclude that brain ischemia induces the formation of β-hydroxybutyrate (ketogenesis) in the liver and the consumption of β-hydroxybutyrate in the brain. This effect seems to be mediated by β-adrenergic receptors

Beneficial Effects of Ethanolic and Hexanic Rice Bran Extract on Mitochondrial Function in PC12 Cells and the Search for Bioactive Components

Mitochondria are involved in the aging processes that ultimately lead to neurodegeneration and the development of Alzheimer’s disease (AD). A healthy lifestyle, including a diet rich in antioxidants and polyphenols, represents one strategy to protect the brain and to prevent neurodegeneration. We recently reported that a stabilized hexanic rice bran extract (RBE) rich in vitamin E and polyphenols (but unsuitable for human consumption) has beneficial effects on mitochondrial function in vitro and in vivo (doi:10.1016/j.phrs.2013.06.008, 10.3233/JAD-132084). To enable the use of RBE as food additive, a stabilized ethanolic extract has been produced. Here, we compare the vitamin E profiles of both extracts and their effects on mitochondrial function (ATP concentrations, mitochondrial membrane potential, mitochondrial respiration and mitochondrial biogenesis) in PC12 cells. We found that vitamin E contents and the effects of both RBE on mitochondrial function were similar. Furthermore, we aimed to identify components responsible for the mitochondria-protective effects of RBE, but could not achieve a conclusive result. α-Tocotrienol and possibly also γ-tocotrienol, α-tocopherol and δ-tocopherol might be involved, but hitherto unknown components of RBE or a synergistic effect of various components might also play a role in mediating RBE’s beneficial effects on mitochondrial function

Effects of OGV and control emulsions when applied at reperfusion.

<p>Saline, Lipofundin® (LPF) and d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) in doses equal to OGV when injected at reperfusion (a.r.). (A) Infarct areas and (B) differences in grayscale for LPF, TPGS, and Omegaven 10% (OGV) vs. Saline; n = 6. Mean ± SEM, p*<0.05; p**<0.01; p***<0.001; One-Way ANOVA with Tukey post-test. (C) Representative striatal brain slices for determination of differences in grayscale for each group. Density of grayscale in a representative area from core of infarction (solid circle) was subtracted from corresponding grayscale of contralateral control area (dashed circle). </p

Western Blot analysis of brain homogenates.

<p>Sham-operated mice (Control) versus control group that received saline (Stroke) and treatment group that received OGV at stroke (a.s.). (A) COX-2-, (B) IL-6-, and (C) IL-10 protein levels, n = 8. Mean ± SEM, p*<0.05; p**<0.01; p***<0.001; One-Way ANOVA with Tukey post-test.</p

Comparison of average main-ingredients of Omegaven 10% and Lipofundin 10% MCT.

<p>Comparison of average main-ingredients of Omegaven 10% and Lipofundin 10% MCT.</p

Marker of mitochondrial function after treatment at stroke.

<p>Sham mice (Control) versus stroke control group that received saline (Stroke) and stroke treatment group that received OGV at stroke (a.s.). (A) Mitochondrial membrane potential (MMP)- and (B) Adenosine triphosphate (ATP)-levels as measured in dissociated brain cells 24 hours after reperfusion. Energy metabolite levels in striatal core region of stroke for (C) glucose and (D) glutamate as determined by microdialysis 30 minutes before (PRE), 90 minutes during (Stroke) and 30 minutes after (POST) stroke surgery, n = 8. Mean ± SEM, p*<0.05; p**<0.01;p***<0.001; One-Way ANOVA with Tukey post-test.</p

Marker of mitochondrial function after treatment at reperfusion.

<p>Sham-operated mice. (Control) versus stroke control group that received saline (Stroke) and stroke treatment group that received OGV at reperfusion (a.r.). (A) Mitochondrial membrane potential (MMP)- and (B) Adenosine triphosphate (ATP)-levels as measured 24 hours after reperfusion in dissociated brain cells, n = 8; (C) Respiration [pmol oxygen/(s* mg protein)] of different complexes of the respiratory chain were determined in isolated mitochondria, n = 6; (D) Respiratory control ratio (RCR) that indicates the coupling of mitochondrial respiration chain and citrate synthase activity that represents a quantitative marker for mitochondrial mass, n = 6. Mean ± SEM, p*<0.05; p**<0.01; p***<0.001; One-Way ANOVA with Tukey post-test.</p

Effects of OGV when given at stroke (a.s.).

<p>(A) Neurobehavioral assessment (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167329#pone.0167329.s006" target="_blank">S1 Table</a>) for sham-operated mice (Control) versus control group that received saline (Stroke) and treatment group that received OGV at stroke (a.s.), (B) Representative brain slices for determination of infarct volume and differences in grayscale. (C) Effect on infarct volume and grayscale levels, n = 8. Mean ± SEM, p*<0.05; p**<0.01; p***<0.001; (A) One-Way ANOVA with Tukey post-test, (C) t-test.</p