DHA but Not EPA Emulsions Preserve Neurological and Mitochondrial Function after Brain Hypoxia-Ischemia in Neonatal Mice

Background and Purpose Treatment with triglyceride emulsions of docosahexaenoic acid (tri-DHA) protected neonatal mice against hypoxia-ischemia (HI) brain injury. The mechanism of this neuroprotection remains unclear. We hypothesized that administration of tri-DHA enriches HI-brains with DHA/DHA metabolites. This reduces Ca2+-induced mitochondrial membrane permeabilization and attenuates brain injury. Methods: 10-day-old C57BL/6J mice following HI-brain injury received tri-DHA, tri-EPA or vehicle. At 4–5 hours of reperfusion, mitochondrial fatty acid composition and Ca2+ buffering capacity were analyzed. At 24 hours and at 8–9 weeks of recovery, oxidative injury, neurofunctional and neuropathological outcomes were evaluated. In vitro, hyperoxia-induced mitochondrial generation of reactive oxygen species (ROS) and Ca2+ buffering capacity were measured in the presence or absence of DHA or EPA. Results: Only post-treatment with tri-DHA reduced oxidative damage and improved short- and long-term neurological outcomes. This was associated with increased content of DHA in brain mitochondria and DHA-derived bioactive metabolites in cerebral tissue. After tri-DHA administration HI mitochondria were resistant to Ca2+-induced membrane permeabilization. In vitro, hyperoxia increased mitochondrial ROS production and reduced Ca2+ buffering capacity; DHA, but not EPA, significantly attenuated these effects of hyperoxia. Conclusions: Post-treatment with tri-DHA resulted in significant accumulation of DHA and DHA derived bioactive metabolites in the HI-brain. This was associated with improved mitochondrial tolerance to Ca2+-induced permeabilization, reduced oxidative brain injury and permanent neuroprotection. Interaction of DHA with mitochondria alters ROS release and improves Ca2+ buffering capacity. This may account for neuroprotective action of post-HI administration of tri-DHA

DHA but Not EPA Emulsions Preserve Neurological and Mitochondrial Function after Brain Hypoxia-Ischemia in Neonatal Mice.

Treatment with triglyceride emulsions of docosahexaenoic acid (tri-DHA) protected neonatal mice against hypoxia-ischemia (HI) brain injury. The mechanism of this neuroprotection remains unclear. We hypothesized that administration of tri-DHA enriches HI-brains with DHA/DHA metabolites. This reduces Ca2+-induced mitochondrial membrane permeabilization and attenuates brain injury.10-day-old C57BL/6J mice following HI-brain injury received tri-DHA, tri-EPA or vehicle. At 4-5 hours of reperfusion, mitochondrial fatty acid composition and Ca2+ buffering capacity were analyzed. At 24 hours and at 8-9 weeks of recovery, oxidative injury, neurofunctional and neuropathological outcomes were evaluated. In vitro, hyperoxia-induced mitochondrial generation of reactive oxygen species (ROS) and Ca2+ buffering capacity were measured in the presence or absence of DHA or EPA.Only post-treatment with tri-DHA reduced oxidative damage and improved short- and long-term neurological outcomes. This was associated with increased content of DHA in brain mitochondria and DHA-derived bioactive metabolites in cerebral tissue. After tri-DHA administration HI mitochondria were resistant to Ca2+-induced membrane permeabilization. In vitro, hyperoxia increased mitochondrial ROS production and reduced Ca2+ buffering capacity; DHA, but not EPA, significantly attenuated these effects of hyperoxia.Post-treatment with tri-DHA resulted in significant accumulation of DHA and DHA derived bioactive metabolites in the HI-brain. This was associated with improved mitochondrial tolerance to Ca2+-induced permeabilization, reduced oxidative brain injury and permanent neuroprotection. Interaction of DHA with mitochondria alters ROS release and improves Ca2+ buffering capacity. This may account for neuroprotective action of post-HI administration of tri-DHA

Synthesis of the 16<i>S</i>,17<i>S</i>‑Epoxyprotectin Intermediate in the Biosynthesis of Protectins by Human Macrophages

The n-3 polyunsaturated fatty acids act as substrates during the resolution phase of acute inflammation for the biosynthesis of specialized pro-resolving lipid mediators. One premier example is the C22-dihydroxy-polyunsaturated fatty acid protectin D1 (<b>1</b>). The human 15-lipoxygenase type I, via stereoselective processes and with docosahexaenoic acid as the substrate, enables the formation of this specialized pro-resolving lipid mediator. Herein, based on results from LC/MS-MS metabololipidomics, support is presented for the apprehended biosynthesis of <b>1</b> in human macrophages occurring via the intermediate 16<i>S</i>,17<i>S</i>-epoxyprotectin (<b>5</b>). Stereochemically pure <b>5</b> was obtained using the Katsuki–Sharpless epoxidation protocol, establishing the chirality at the C16 and C17 atoms, one <i>Z</i>-selective reduction, and <i>E</i>- and <i>Z</i>-stereoselective Wittig reactions. In addition, information on the nonenzymatic aqueous hydrolysis products and the half-life of 16<i>S</i>,17<i>S</i>-epoxyprotectin (<b>5</b>) is presented

Mitochondrial production of ROS in vitro and markers for oxidative brain injury in vivo.

<p>(A) Mitochondrial H₂O₂ emission rates in Normox; O₂+Veh; O₂ +DHA:Alb 7:1; O₂ +DHA:Alb 1:1; and O₂ +EPA:Alb 7:1. N = 5 in all groups (B) Representative tracing of mitochondrial H₂O₂ production (groups are indicated). (C) 3-nitrotyrosine immunopositive cells / total cells ratio in HI+Veh, HI+EPA and HI+DHA. N = 4 in all groups. * p = 0.02. (D) Representative images of 3-nitrotyrosine staining in HI+Veh, HI+EPA and HI+DHA mice.</p

Long term neurological outcomes.

<p>(A) Cumulative latency time over 3 days of training and (B) Navigational memory performance in Naive (n = 16); HI+Veh (n = 20); HI+tri-EPA (n = 16) and HI+tri-DHA (n = 22) adult mice. (C) and (D) Nissl-stained cerebral coronal sections and residual ipsilateral hemisphere volume in the adult HI+Veh (n = 6), HI+EPA (n = 5) or HI+tri-DHA (n = 5) mice.</p

Short term neurological outcomes.

<p>(A) Infarct volumes in HI+Veh (n = 30), HI+tri-EPA (n = 20), or HI+tri-DHA (n = 20) mice. (B) Representative TTC-stained cerebral sections from the same groups of mice. (C) Righting, and (D) Negative geotaxis reflex performance in naïve (n = 12); Veh+HI (n = 18); HI+tri-EPA (n = 13); and HI+tri-DHA (n = 18) mice.</p

Mitochondrial DHA content and function following hyperoxic stress in vitro.

<p>(A) Mitochondrial DHA content following in vitro incubation with Veh, DHA:Alb 1:1, and DHA:Alb 7:1. * p < 0.05. (B) Mitochondrial Ca²⁺ buffering capacity: Normoxia, O₂+Veh; O₂ +DHA:Alb 1:1; and O₂ +DHA:Alb 7:1. * p ≤ 0.0005. (C) Mitochondrial Ca²⁺ buffering capacity in Normoxia; O₂+Veh; O₂ +EPA:Alb 7:1; and O₂ +DHA:Alb 7:1. * p < 0.05. (D) Representative tracings of mitochondrial Ca²⁺ buffering capacity. Groups are indicated. N = 4 in all groups.</p

DHA-metabolites content in the brain.

<p>(A) Cerebral content of DHA metabolites in naïve (n = 4) or HI-mice treated with either vehicle or tri-DHA (n = 3). (B) DHA metabolites spectra used for calculation of their concentration in the brains of mice (groups are indicated above).</p