Docosahexaenoic acid complexed to albumin (DHA-Alb) is highly neuroprotective after temporary middle cerebral artery occlusion (MCAo), but whether a similar effect occurs in permanent MCAo is unknown. Male Sprague-Dawley rats (270–330 g) underwent permanent MCAo. Neurological function was evaluated on days 1, 2 and 3 after MCAo. We studied six groups: DHA (5 mg/kg), Alb (0.63 or 1.25 g/kg), DHA-Alb (5 mg/kg+0.63 g/kg or 5 mg/kg+1.25 g/kg) or saline. Treatment was administered i.v. at 3 h after onset of stroke (n = 7–10 per group). Ex vivo imaging of brains and histopathology were conducted on day 3. Saline- and Alb-treated rats developed severe neurological deficits but were not significantly different from one another. In contrast, rats treated with low and moderate doses of DHA-Alb showed improved neurological score compared to corresponding Alb groups on days 2 and 3. Total, cortical and subcortical lesion volumes computed from T2 weighted images were reduced following a moderate dose of DHA-Alb (1.25 g/kg) by 25%, 22%, 34%, respectively, compared to the Alb group. The total corrected, cortical and subcortical infarct volumes were reduced by low (by 36–40%) and moderate doses (by 34–42%) of DHA-Alb treatment compared to the Alb groups. In conclusion, DHA-Alb therapy is highly neuroprotective in permanent MCAo in rats. This treatment can provide the basis for future therapeutics for patients suffering from ischemic stroke.

Introduction: Ring finger protein 146, also called Iduna, has been identified, as a neuroprotective protein. Iduna facilitates DNA repair and protects against cell death induced by NMDA receptor-mediated glutamate excitotoxicity or cerebral ischemia. Recently, we have shown that docosahexaenoic acid (DHA; 22:6n-3) therapy improves functional and histological outcomes following experimental stroke. This study evaluated the time course expression of Iduna in the ischemic penumbra and the role of DHA in cerebral ischemia and its potential mechanism. METHODS: Thirty-six male SD rats were anesthetized with isoflurane and subjected to 2 h of middle cerebral artery occlusion (MCAo) by poly-L-lysine-coated intraluminal suture. DHA (5 mg/kg) or vehicle (saline) was administered IV at 3 h after the onset of MCAo and animals were sacrificed on days 1, 3 and 7 (n=6 rats per group). The neurological function was evaluated during occlusion (60 min), and on days 1, 3 and 7 after MCAo; a grading scale of 0-12 was employed (0=normal and 12=maximal deficit). Western blot and double immunostaining (Iduna/NeuN and Iduna/GFAP) were used to analyze Iduna expression in the ischemic penumbra on days 1, 3 and 7. RESULTS: All animals showed similar values for rectal and cranial temperatures, arterial blood gases, and plasma glucose during and after MCAo. Behavioral deficit was significantly improved by treatment with DHA compared to vehicle on days 1 (by 30%), 3 (by 31%) and 7 (by 32%). Western-blot analysis showed that DHA treatment increased Iduna expression in the ischemic penumbra compared to vehicle on day 1 (2.2±0.3 vs. 0.6±0.1) and day 3 (0.44±0.05 vs. 0.3±0.01, respectively). There were no differences in Iduna expression on day 7 between DHA and vehicle-treated groups (0.33±0.02 and 0.35±0.02). Immunostaining revealed that Iduna expression was increased in the penumbra of DHA-treated rats. Conclusions: DHA protected the brain from severe damage caused by MCAo. This effect may be through upregulation of Iduna expression in the ischemic penumbra. Thus, it is reasonable to hypothesize that DHA has potential for the effective treatment of ischemic stroke in patients.

INTRODUCTION: Docosahexaenoic acid (DHA; 22:6n-3) is an omega-3 essential fatty acid family member and the precursor of neuroprotectin D1, a lipid mediator which downregulates apoptosis and promotes cell survival. Recently, we have shown that DHA therapy improves functional and histological outcomes following experimental stroke. We examined the effect of DHA on blood-brain barrier (BBB) integrity after middle cerebral artery occlusion (MCAo) in rats. Damage to the BBB was judged by extravasation of Evans Blue (EB) dye. METHODS: Male SD rats were anesthetized with isoflurane and subjected to 2 h of MCAo by retrograde insertion of an intraluminal suture. DHA (5 mg/kg) or vehicle (saline) was administered IV at 3 h after the onset of MCAo and animals were sacrificed at 6, 24 or 72 h. Behavioral tests were performed at 5, 24, 48 or 72 h. EB (2% in saline, 4 ml/kg) was administered IV either at 5, 23 or 71 h after onset of MCAo. Fluorometric quantitation of EB was performed 1 h later in six brain regions. RESULTS: Physiological variables were stable and showed no significant differences between groups. In the 6 h series (n=9 per group), DHA decreased EB extravasation in the posterior ischemic hemisphere compared to saline (9.2±1.1 vs. 14.3± 1.5, respectively). In the 24 h series (n=8-9 per group), DHA improved behavioral scores on day 1 and decreased EB extravasation in the posterior ischemic hemisphere (4.9±0.41 vs. 8.5±1.0) and cortical area (3.0±0.4 vs. 8.6± 2.0, respectively) compared to saline. In the 72 h series (n=12 per group), DHA improved behavioral scores on days 1, 2 and 3, and decreased EB extravasation in the anterior (6.1±0.8 vs. 9.6±0.9), posterior (5.6±0.7 vs. 9.1±1.2, respectively) ischemic hemispheres compared to saline. In addition, EB extravasation was decreased by DHA in the cortical area (6.2±1.0 vs. 10.1±1.1) and total hemisphere (11.7±1.3 vs. 18.7±1.9, respectively) compared to vehicle. CONCLUSION: These data have demonstrated that DHA is an effective neuroprotective drug, diminishing BBB damage in a model of severe focal ischemia. Thus, it is reasonable to hypothesize that DHA may have potential use in treating focal ischemic stroke in the clinical setting. This study was supported by NIH, NINDS grant R01 NS065786 (LB) and R01 NS046741 (NGB).

Introduction: Docosahexaenoic acid (DHA; 22:6n-3) is an omega-3 essential fatty acid family member and the precursor of neuroprotectin D1, a lipid mediator which downregulates apoptosis and promotes cell survival. Recently, we have shown that DHA improved behavior in animals allowed to survive for 7 d after stroke. This study was conducted to establish whether the behavioral improvement induced by DHA persists with chronic survival. In addition, we examined the effect of DHA on blood-brain barrier (BBB) integrity using Evans Blue (EB) and FITC-dextran markers. METHODS: Physiologically-controlled SD rats received 2 h middle cerebral artery occlusion (MCAo). DHA (5 mg/kg; n=10) or vehicle (saline; n=9) was administered i.v. at 3 h after onset of MCAo. In behavioral studies, the composite 12-point neuroscore, rota-rod, Y-maze and beam walking test were conducted 1, 2 and 3 weeks after MCAo. Ex vivo imaging of the brains and histopathology were carried out on day 21. In BBB studies, fluorometric quantitation of Evans Blue (EB) was performed in six brain regions at 6, 24 or 72 h after stroke. FITC dextran leakage was analyzed on day 3 after MCAo. RESULTS: Physiological variables were stable and showed no significant differences between groups. DHA treatment significantly improved the 12-point neuroscore compared to vehicle on day 1 (by 16%), day 2 (by 19%), day 3 (by 22%), week 1 (by 20%), week 2 (by 22%) and week 3 (by 33%) respectively. Treatment with DHA prolonged latency time in the rota-rod test on weeks 1-3 (by 44-68%), enhanced the score in the balance beam (by 29%) and improved Y-maze performance by 19% compared to the saline group. DHA decreased EB extravasation in the posterior ischemic hemisphere at 6 h (by 34%), 24 h (by 42%) and 72 h (by 38%). EB extravasation was decreased by DHA in the cortex and total hemisphere as well. FITC dextran leakage was reduced by DHA treatment on day 3 by 68% compared to the saline group. CONCLUSION: DHA therapy accelerates long-lasting behavioral recovery and diminishes BBB damage in a model of experimental stroke. Thus, it is reasonable to hypothesize that DHA may have potential use in treating focal ischemic stroke in the clinical setting.