Docosahexaenoic Acid therapy of experimental ischemic stroke

Abstract

We examined the neuroprotective efficacy of docosahexaenoic acid (DHA), an omega-3 essential fatty acid family member, in acute ischemic stroke; studied the therapeutic window; and investigated whether DHA administration after an ischemic stroke is able to salvage the penumbra. In each series described below, SD rats underwent 2 h of middle cerebral artery occlusion (MCAo). In series 1, DHA or saline was administered i.v. at 3, 4, 5, or 6 h after stroke. In series 2, MRI was conducted on days 1, 3 and 7. In series 3, DHA or saline was administered at 3 h, and lipidomic analysis was conducted on day 3. Treatment with DHA significantly improved behavior and reduced total infarct volume by a mean of 40% when administered at 3 h, by 66% at 4 h, and by 59% at 5 h. Total lesion volumes computed from T2-weighted images were reduced in the DHA group at all time points. Lipidomic analysis showed that DHA treatment potentiates neuroprotectin D1 (NPD1) synthesis in the penumbra 3 days after MCAo. DHA administration provides neurobehavioral recovery, reduces brain infarction and edema, and activates NPD1 synthesis in the penumbra when administered up to 5 h after focal cerebral ischemia in rats.

Fig. 1

Therapeutic window study: a Neurological score (normal score = 0; maximum score = 12) was improved after DHA administration when administered 3, 4, and 5 h after onset of stroke. Cortical (b), subcortical (c), and total corrected infarct volumes (d) on day 7. DHA reduced cortical and total infarct volumes when administered 3, 4, and 5 h after stroke. Data are means ± SEM. Asterisk, significantly different from saline (p < 0.05; repeated-measures ANOVA followed by Bonferroni tests). e Computer-generated MosaiX-processed images of Nissl stained paraffin-embedded brain sections from rats treated with saline or DHA at 3, 4, 5, and 6 h after the onset of ischemia. Saline-treated rat shows large cortical and subcortical infarction. In contrast, rats treated with DHA at 3, 4, and 5 h show less extensive damage, mostly in the subcortical area. DHA-treated rat at 6 h shows infarct involving cortical and subcortical regions. f Computer-generated MosaiX-processed images of GFAP (green), ED-1 (red), and GFAP/ED-1 double staining (overlay) on day 7 after 2 h of MCAo at a magnification ×10. Treatment with DHA or saline was given at 3 h after onset of stroke

Fig. 2

a Coronal brain diagram showing locations of regions for cell counts in cortex (1, 2, and 3), and striatum (S). b–d Number of GFAP-positive astrocytes, ED-1 positive microglia cells, and NeuN-positive neurons on day 7 after 2 h of MCAo. DHA or saline was given at 3 h after onset of stroke. DHA treatment decreased ED-1, increased NeuN- and GFAP-positive cell counts. Data are mean ± SEM. Asterisk, significantly different from saline (p < 0.05; repeated-measures ANOVA followed by Bonferroni tests)

Fig. 3

MRI study: a DHA improved neurological scores on days 3 and 7. b T2 hyperintensites were observed in the cortex and striatum of saline-treated rats, consistent with edema formation. In contrast, DHA reduced edema with little T2 hyperintensities at day 3 and not at all at day 7, resulting in an intact corpus callosum at day 7. c DHA reduced T2 values within the lesion computed from T2WI on days 1, 3, and 7, consistent with decreased edema. Data are means ± SEM. Asterisk, significantly different from saline-treated group (p < 0.05; repeated-measures ANOVA followed by Bonferroni tests). d 3D infarct volumes were computed from T2WI on days 1, 3, and 7 after MCAo. Saline-treated rats showed large cortical and subcortical infarct volumes that slowly decreased over the course of 7 days. By contrast, infarct volume was dramatically reduced in rats treated with DHA and was mostly localized in the subcortical areas. 3D reconstructions are from the same animal in each group over the 7-day time course

Fig. 4

The characterization and quantification of 17-HDHA and NPD1 in the ipsilateral penumbra 3 days after MCAo. a–b The fragmentation pattern is depicted and c the quantification is presented. The increased content of 17-HDHA and of NPD1 in the ipsilateral penumbra in animals injected with DHA is consistent with the activation of the biosynthesis of NPD1. d Enzyme-mediated oxygenation of DHA for the biosynthesis of NPD1. Phospholipase A2 releases DHA from the second C position of phospholipids during brain ischemia-reperfusion. 15-Lipoxygenase-1 catalyzes the synthesis of 17S-H(p)DHA, which in turn is converted to a 16(17)-epoxide and then is enzymatically hydrolyzed to NPD1