Robust docosahexaenoic acid-mediated neuroprotection in a rat model of transient, focal cerebral ischemia

Abstract

Background and purpose: Docosahexaenoic acid (DHA; 22:6n-3), an omega-3 essential fatty acid family member, is the precursor of neuroprotectin D1, which downregulates apoptosis and, in turn, promotes cell survival. This study was conducted to assess whether DHA would show neuroprotective efficacy when systemically administered in different doses after middle cerebral artery occlusion (MCAo) in rats.

Methods: Sprague-Dawley rats were anesthetized with isoflurane and subjected to 2 hour of MCAo. Animals were treated with either DHA (low doses=3.5 or 7 mg/kg; medium doses=16 or 35 mg/kg; and high dose=70 mg/kg) or an equivalent volume of saline intravenously 3 hours after MCAo onset. Neurologic status was evaluated during occlusion (60 minutes) and on days 1, 2, 3, and 7 after MCAo. Seven days after MCAo, brains were perfusion-fixed, and infarct areas and volumes were determined.

Results: Only the low and medium doses of DHA significantly improved the neurologic score compared with vehicle-treated rats at 24 hours, 48 hours, 72 hours, and 7 days. DHA markedly reduced total corrected infarct volume in all treated groups compared with vehicle-treated rats (3.5 mg/kg, 26+/-9 mm(3); 7 mg/kg, 46+/-12 mm(3); 16 mg/kg, 37+/-5 mm(3); and 35 mg/kg, 34+/-15 mm(3) vs vehicle, 94+/-12 mm(3)). Cortical and striatal infarct volumes were also significantly reduced by treatment with DHA. No neuroprotective effects were observed with 70 mg/kg DHA.

Conclusions: We conclude that DHA experimental therapy at low and medium doses improves neurologic and histologic outcomes after focal cerebral ischemia and might provide benefits in patients after ischemic stroke.

Figure 1

Schematic brain diagram showing locations of regions for GFAP and Nissl positive cell counts in the cortex (A, B and C) and striatum (S).

Figure 2

Total neurological score (normal score=0, maximal score=12) during MCAo and at various times after treatment. DHA or vehicle treatment was administered at 3 h after onset of ischemia. Values shown are means ± SEM. *, significantly different from corresponding vehicle group (p<0.05; repeated-measures ANOVA followed by Bonferroni tests).

Figure 3

Computer-generated MosaiX processed images (Zeiss Axio Imager.M1; AxioVision Release 4.6.3) of Nissl stained paraffin-embedded brain sections from rats treated with saline, DHA-3.5 mg/kg, DHA-7 mg/kg, DHA-14 mg/kg, DHA-35 mg/kg and DHA-70 mg/kg. Saline-treated rat shows large cortical and subcortical infarction. In contrast, rats treated with low and medium doses of DHA show less extensive damage, mostly in the subcortical area. Rat treated with high dose of DHA (70 mg/kg) shows a large infarct involving cortical and subcortical regions.

Figure 4

Cortical, subcortical and total infarct areas measured at 9 coronal levels and integrated infarct volumes in rats with 2-h MCAo and on day 7 of survival. Rostrocaudal distribution of cortical infarct area and cortical infarct volume (Panels A and B), subcortical infarct area and subcortical infarct volume (Panels C and D) and total infarct area and total corrected infarct volume (Panels E and F) in all groups. Data are presented as mean ± SEM. *, significantly different from corresponding vehicle group (p <0.05; repeated-measures ANOVA followed by Bonferroni tests).

Figure 5

Panel A: Representative Nissl and GFAP stained brain sections of rats treated with vehicle and DHA (14 mg/kg). Saline-treated rats show large cortical and subcortical infarction. In contrast, rats treated with DHA show less extensive damage and increased GFAP staining. Panel B: GFAP and Nissl positive cell counts in cortex (A, B and C) and striatum (S) (see Figure 1 for details). Treatment with DHA (14 mg/kg) significantly increased GFAP and Nissl positive cells compared to the saline-treated rats. Values shown are means ± SEM. *, significantly different from corresponding vehicle group (p<0.05; repeated-measures ANOVA followed by Bonferroni tests).