Docosahexaenoic acid complexed to human albumin in experimental stroke: neuroprotective efficacy with a wide therapeutic window

Stroke is a major cause of death and disability in industrialized countries. Pharmacological intervention of ischemic damage is critically important for controlling brain tissue deterioration. Tissue-type plasminogen activator (tPA) administered within 3 to 4.5 h of symptom onset is still the only thrombolytic agent approved for patients with ischemic stroke. However, the narrow therapeutic time window and the risk of intracerebral hemorrhage after tPA treatment pose major hurdles to its clinical use. Therefore, development of new therapeutic agents for stroke is essential.

Recent studies have revealed that omega-3 essential fatty acids (found in fish oils) may be beneficial in ameliorating cerebral ischemic injury [1, 2]. Docosahexaenoic acid (DHA; 22:6, n-3) is an essential omega-3-fatty acid and is vital for proper brain function. It is also necessary for the development of the nervous system, including vision, and has potent anti-inflammatory effects [3]. Several epidemiological studies indicate that consumption of fish is associated with reduced risk of ischemic stroke and coronary heart disease [4]. Recently, we have showed that DHA treatment can protect brain tissue and promote recovery in an experimental model of acute ischemic stroke in rats [5]. In addition, DHA administration provided neurobehavioral recovery, and reduced brain infarction and edema when administered up to 5 h after focal cerebral ischemia in rats [6].

Lately, special attention has been paid to human serum albumin (Alb) therapy, which has been reported to ameliorate neuronal damage during the acute phase of stroke [7, 8]. Alb has been shown to be highly neuroprotective in animal models of focal cerebral ischemia [8], global ischemia [9], hemorrhagic stroke [10, 11] and traumatic brain injury [12]. With its promising neuroprotective bioactivity, Alb can markedly improve neurological function, and reduce blood brain barrier (BBB) permeability and infarct volumes when administered up to 4 h after onset of stroke. The National Institutes of Health (NIH) funded clinical trials, the ALIAS (Albumin in Acute Stroke), based on these promising studies to evaluate efficacy of Alb treatment in patients with acute ischemic stroke [13, 14]. However, administration of high-dose Alb by expanding intravascular volume may lead to pulmonary edema and congestive heart failure in patients [15]. We suspected that if DHA were complexed with Alb, it might be possible to achieve neuroprotection at lower, more clinically achievable doses and extend the therapeutic window beyond 5 h after stroke onset.

In the first part of this study we have shown that DHA-Alb therapy substantially improves behavioral function, markedly reduces the volume of cerebral infarction, and promotes cell survival in animals with acute ischemic stroke when administered promptly at moderate doses, which are potentially amenable to clinical applications. In the second part of the study we have demonstrated the existence of a broad therapeutic window of neuroprotective efficacy with moderate-dose DHA-Alb therapy, such that treatment initiated even 7 hours after stroke onset of ischemia is highly effective.

Cerebral ischemia-reperfusion results in the rapid accumulation of free fatty acids (FFA), including arachidonic acid (AA; 20:4 n-6) and DHA, which are released from membrane phospholipids. Both AA and DHA are polyunsaturated fatty acids highly enriched in phospholipids from synaptic membranes, where they are reservoirs of potent signaling molecules and essential for overall membrane function [1]. DHA is necessary for ion channels, receptors, and transporters to maintain their proper physical conformation and is involved in memory, synaptic membrane biogenesis and function, and neuroprotection [5, 19, 20]. DHA is the precursor of neuroprotectin D1 (NPD1), which has been associated with reduction of neuroinflammation and activation of antiapoptotic pathways, two main mechanisms of action implicated in ischemic stroke [5, 21, 22]. Our studies reveal an early increase in unesterified (free) DHA in brain ischemia [19], which initiates a pathway for docosanoid biosynthesis [21]. Since inflammation is at the root of many chronic diseases, DHA treatment has been demonstrated to have beneficial effects in patients with coronary heart disease, rheumatoid arthritis, osteoporosis, sepsis, asthma, cancer and age-related macular degeneration, but its potential benefit in stroke was not known [23]. Recently, we have shown that DHA therapy in low (3.5 or 7 mg/kg) and medium (16 or 35 mg/kg) doses improves neurological and histological outcomes following focal cerebral ischemia [5]. Thus, we selected a dose in the middle of the effective dose range (5 mg/kg) for our studies. In addition, we demonstrated that DHA or saline administered intravenously at 3, 4 and 5 h after the onset of stroke reduced cerebral infarction and brain swelling, and facilitated neurobehavioral recovery even when treatment was delayed by up to 5 h [6].

Human serum albumin has been used for many years in the management of a diverse range of medical and surgical issues [24]. Alb administration has multiple effects including volume expansion, an increase in colloid osmotic pressure, and hemodilution. It is used in multiple clinical settings such as hypovolemia, shock, burns, surgery, trauma, cardiopulmonary bypass, acute respiratory distress syndrome, hemodialysis, acute liver failure, and ascites [25].

Preclinical studies in rodent models of ischemic stroke have established that that administration of Alb at high doses (25%, 2.5 g/kg, i.v.) decreases infarct volume, reduces brain swelling [7, 26] and improves local cerebral perfusion in affected tissue [27] Moderate doses of Alb therapy (0.63 or 1.25 g/kg) also improve neurological function and reduce infarct volume and brain swelling, even when treatment is delayed up to 4 hours after onset of ischemia in rats [8]. Albumin produces its neuroprotective effect through several mechanisms including brain swelling amelioration [26], BBB permeability reduction [10], local vascular dynamic and tissue perfusion improvement [28, 29] antioxidant activity [30], antithrombotic effects and anti-inflammatory activity [31], inhibiting endothelial apoptosis and maintenance of normal astrocytic function [32] and providing essential fatty acids to the injured brain [20].

Alb is currently under clinical trials for treatment of focal cerebral ischemia [13, 14] and subarachnoid hemorrhage [33]. Phase I and II clinical trials have reported that higher doses of Alb correlated with better outcomes in subjects with acute ischemic stroke [15, 34], and a Phase III trial has begun [13, 14]. The only Alb-related adverse event encountered was mild-to-moderate pulmonary edema, which occurred in approximately 13% of patients [15]. We suspected that if DHA were complexed with Alb, it might be possible to achieve neuroprotection at lower and therefore more clinically achievable, doses and extend the therapeutic window beyond 5 h after stroke onset. The results of our experiments show that animals treated with moderate doses of DHA-Alb (0.63 and 1.25g/kg) significantly improved the neurological score compared to native Alb by 21 and 26% on day 1; by 28 and 29% on day 2; by 36 and 40% on day 3; and by 42 and 35% on day 7, respectively. All DHA-Alb doses markedly reduced infarct volumes compared to native Alb group. In the therapeutic window study, even when treatment was initiated as late as 7 h after onset of MCAo, DHA-Alb therapy led to significantly improved neurological scores and significant reductions of infarct volumes. Due to the challenges of treating patients in the timely manner, an extension of the therapeutic window to 7 h will offer great opportunities to clinicians treating ischemic stroke victims.

Acute stroke causes an irreversibly damaged ischemic core, and salvageable surrounding tissue called the ischemic penumbra [35]. The ischemic core is characterized by severe reduction of local cerebral blood flow (below 20%), disruption of ion homeostasis, massive release of glutamate, and activation of proteases and endonucleases, which may contribute to the acute neuronal death observed in the ischemic hemisphere [36]. The penumbra of ischemic stroke refers to the regions of brain tissue, surrounding the ischemic core, where blood flow is sufficiently reduced (below 40%) to result in hypoxia severe enough to arrest physiological function, but not so complete as to cause irreversible failure of energy metabolism and cellular necrosis [37]. The penumbra, which accounts for almost one-half of the initial ischemic lesion [38], deteriorates over a few hours following the initial ischemic core damage and is therefore the target of therapeutic intervention [36, 39]. Recent studies showed that DHA treatment decreased ED-1 –positive microglia/macrophages, increased GFAP –positive reactive astrocytes and NeuN positive neurons in the ischemic penumbra and core [11]. Our study demonstrates that DHA-Alb complex was able to salvage the ischemic penumbra. The effect of DHA-Alb was evident in the cortical (penumbral region) of the infarct through a significant 79% reduction of cortical infarct volume when administered at 5 h, 98% reduction at 6 h, and 76% at 7 h, as compared to Alb treated rats. This effect was associated with attenuated cellular death of both astrocytes and neurons and fewer activated microglia, as compared to vehicle-treated rats. GFAP expression was found in the boundary zone of infarct and was localized to the same areas where neurons are destined to survive the ischemic insult (as detected by NeuN positive cells count). These results indicate that DHA-Alb protects not only neurons but also astrocytes, which are critical for the maintenance and protections of neurons via secretion of growth factors and other neurotrophic mediators.

We have shown here that all DHA-Alb doses used in this study were highly neuroprotective, the therapeutic window was extended to 7 h by complexing DHA to human Alb (1.25 g/kg), and delayed DHA-Alb treatment (at 5-7h) was more effective than early treatment (at 2-4h) compared to native Alb. The exact mechanisms of DHA-Alb neuroprotection remain obscure. We can speculate however, that complexing DHA to human Alb provides additive neuroprotection in experimental stroke. Lipidomic analysis of DHA-Alb treated postischemic brains revealed a large accumulation of NPD1 in the ipsilateral hemisphere at 20 h of reperfusion after 2 h of MCAo in rats [17]. NPD1 is a potent inhibitor of polymorphonuclear leukocyte infiltration and pro-inflammatory gene expression, and an overall mediator of neuroprotection [6, 21]. In contrast NPD1 levels did not change in either ipsilateral or contralateral hemispheres in Alb treated rats [17]. Studies shows that DHA-Alb can also prevent injury to cultured human retinal pigment epithelial cells triggered by oxidative stress, and that this effect is mediated by NPD1 [40]. Albumin is actively involved in plasma transport of FFA [41]. Because transient MCAo triggers a massive loss of phospholipid-acyl groups, the systemic supply of FFA to the brain, mainly AA and DHA, may be essential to support the repair of neuronal membranes [42]. The direct delivery of FFA to brain cells by Alb may be favored by ischemia-induced disruption of the BBB [43]. In fact, previous studies have reported that the integrity of the BBB is preserved at 3–4 h after MCAo [44] but that progressive acute disruption of the BBB occurs between 5–6 h following MCAo [43]. In addition, immunohistochemical analysis reveals that following MCAo, viable-appearing cortical neurons and endothelial cells within the ischemic zone take up Alb [45]. Thus, this may be evidence in favor of a direct protective role of Alb, not only as a free-radical and toxic-product scavenger [31], but also as a supplier of DHA.

The current study has shown that moderate doses of DHA-Alb complex affords high-grade neuroprotection in focal cerebral ischemia, equaling or exceeding that afforded by native Alb or DHA. Importantly DHA-Alb therapy initiated as late as 7 h after onset of stroke improved behavioral scores and reduced infarct volume. This 7-hour time frame is clinically relevant in that it is logistically difficult to institute therapy in many patients with acute stroke at earlier times. We therefore suggest that this agent offers great promise in the therapy of cerebral ischemia and we propose that it may now be appropriate to consider early-phase clinical trials in patients with acute ischemic stroke.