Docosahexaenoic acid improves behavior and attenuates blood–brain barrier injury induced by focal cerebral ischemia in rats

In the present study, we have established a protective effect of DHA treatment against BBB disruption after experimental focal cerebral ischemia. Post-ischemic administration of DHA robustly protected against ischemia-induced BBB disruption, as evidenced by reduced EB extravasation and FITC-dextran leakage, improved the neurological deficits and attenuated infarct volume. Moreover, there were no adverse behavioral side effects observed with DHA administration. Our results suggest that this approach might be adaptable as a therapeutic avenue.

Cerebral ischemia results in rapid accumulation of free fatty acids, including arachidonic acid (ArAc; 20:4) and DHA [10,11]. These fatty acids are released from membrane phospholipids. Both fatty acids are derived from dietary essential fatty acids; however, only DHA, from the omega-3 polyunsaturated fatty acyl chain, is concentrated in phospholipids of various cells of the brain and retina [22]. Synaptic membranes and photoreceptors share the highest content of DHA of all cell membranes. DHA, one of the main structural lipids in the mammalian brain, plays crucial roles in the development and function of brain neurons [10,12]. It is involved in memory formation, promotes neurogenesis both in vitro and in vivo; and has been implicated in neuroprotection [8,9,23-25]. In vivo, the active DHA supply to the brain (provided by the liver through the blood stream) is necessary for cell development and function. It also may play a critical role in conditions where, due to enhanced oxidative stress, the polyunsaturated fatty acyl chains of membrane phospholipids are decreased as a consequence of lipid peroxidation, as occurs in aging, retinal degenerations, and neurodegenerations such as Alzheimer disease [26,27]. In ischemia, there is also loss of brain DHA due to breakdown of enhanced phospholipase A₂-activated DHA-containing phospholipids [23].

Beneficial effects of DHA in preventing and ameliorating stroke damage have been attributed to generation of the stereospecific derivative, neuroprotectin D1 (NPD1), which is a key survival signaling event leading to neuroprotection [22,28,29]. Recently, we have identified NPD1 following cerebral ischemia-reperfusion in the mouse [28]. NPD1 was found to serve an endogenous neuroprotective role by inhibiting apoptotic DNA damage, upregulating anti-apoptotic and downregulating pro-apoptotic proteins, and also binding toxic peroxides [26,28,29]. We demonstrated that NPD1 synthesis takes place in the ipsilateral side of ischemic brain and peaks at 8 h of reperfusion, and then decreases and is still detectable 25 h after reperfusion [24,28]. Although DHA in the brain can produce NPD1, additional DHA from acute administration is beneficial because lipidomic analysis shows that it potentiates NPD1 synthesis in the penumbra [9,30].

A variety of biologic effects of DHA have been demonstrated with fish or fish oil supplements in humans [31]. They have been found to reduce cholesterol, lower blood pressure, block clot-promoting platelet activation, prevent heart arrhythmias, prevent vascular inflammation and improve vascular function, and protect the heart muscle following a heart attack [32,33]. Epidemiologic studies provide evidence for a beneficial effect of omega-3 fatty acids on manifestations of coronary heart disease and ischemic stroke, particularly with respect to sudden cardiac death in patients with established disease [34]. Clinically important anti-inflammatory effects in humans are further suggested by trials demonstrating benefits of omega-3 fatty acids in rheumatoid arthritis, psoriasis, asthma, inflammatory bowel disorders, osteoporosis, sepsis and cancer [31].

Vascular damage during cerebral ischemia occurs early and it progresses in a biphasic manner [5], contributing to the development of brain edema, hemorrhagic transformation, and worsened clinical outcome in stroke patients [35]. The BBB has a central role in stroke pathogenesis and it is a therapeutic target. Many drugs have been screened to protect BBB permeability integrity and reduce ischemic brain damage by targeting different mechanisms [36]. Unfortunately, so far there are no effective therapeutic interventions for BBB disruption [3]. DHA presence in vascular endothelial cells in the brain suggests that this fatty acid is incorporated into these cells from the systemic circulation [37-39]. DHA in the cell membrane endows fluidity and proper functioning [40]. Furthermore, DHA enriched phospholipids in cellular membranes influences the function of BBB and signaling properties of neurons through fostering a dynamic environment to membrane-associated receptors or enzymes of the BBB compartment [41]. Little is known about the effect of DHA on BBB dysfunction after cerebral ischemia-reperfusion injury. The beneficial effects of pre-treatment with DHA have been reported on BBB disruption, brain edema and inflammatory cell infiltration [42,43]. In contrast, one study showed that post-treatment with DHA (500 nmol/kg, i.p.at 60 min after 90 min of MCAo) exacerbated cerebral ischemic injury and increased BBB permeability in the ischemic cortex at 24 h after reperfusion [44]; note that the DHA dosage applied was 100 times less than in our study. Our study revealed that DHA administration decreased EB extravasation in the ischemic hemisphere at 6 h (by 30%), 24 h (by 48%) and 72 h (by 38%) compared to vehicle-treated rats. In addition, EB extravasation was decreased by DHA treatment in the cortex at 72 h (by 36%) and subcortex at 6 h (by 32%), at 24 h (by 45%) and at 72 h (by 40%) as well as the reduction into the entire brain at 6 h (by 30%), at 24 h (by 49%) and at 72 h (by 31%) compared to the corresponding vehicle groups. We also found that treatment with DHA also suppressed FITC-dextran leakage into the cortex (by 67%) and entire hemisphere (by 71%) compared to the saline-treated animals at 72 h after onset of stroke. In addition, DHA attenuated cortical (by 50%) and total infarct volumes (by 38%) compared to the saline-treated rats at 72 h. These results strongly demonstrate that DHA protects the severely-damaged BBB, accompanied by a relief of abnormal BBB permeability in the delayed ischemic reperfusion damage.

The exact mechanism by which DHA protects the BBB requires further investigation. The beneficial effect of DHA has been shown in a well-controlled animal model of MCAo. In the present study, and in recently published observations [9,13], we have employed a poly-l-lysine coated filament and have found that this technique leads to a 100% incidence of infarction following 2 h of MCAo, predictable size and location of the infarct, and a high degree of inter-animal reproducibility of infarct size (coefficient of variation 8%) [18]. We demonstrated that DHA did not have direct effects on body and cranial temperatures or arterial blood gases because these variables were carefully controlled and did not differ among groups.