A novel therapeutic strategy for experimental stroke using docosahexaenoic acid complexed to human albumin

Despite tremendous efforts in ischemic stroke research and significant improvements in patient care within the last decade, therapy is still insufficient. There is a compelling, urgent need for safe and effective neuroprotective strategies to limit brain injury, facilitate brain repair, and improve functional outcome. Recently, we reported that docosahexaenoic acid (DHA; 22:6, n-3) complexed to human albumin (DHA-Alb) is highly neuroprotective after temporary middle cerebral artery occlusion (MCAo) in young rats. This review highlights the potency of DHA-Alb therapy in permanent MCAo and aged rats and whether protection persists with chronic survival. We discovered that a novel therapy with DHA-Alb improved behavioral outcomes accompanied by attenuation of lesion volumes even when animals were allowed to survive three weeks after experimental stroke. This treatment might provide the basis for future therapeutics for patients suffering from ischemic stroke.


Neuroprotective strategies for ischemic stroke
Stroke is the fifth leading cause of death and disability in the United States.Every year, more than 795 000 people in the United States have a stroke.On average, one American dies from stroke every 4 min (Mozaffarian et al., 2015).Therapeutic options in the acute phase of a stroke are limited and the only approved treatment for ischemic stroke is intravenous recombinant tissue plasminogen activator (tPA), which open channels for blood flow through the occluded thrombi and improves neurological outcome (Heuschmann et al., 2004).
Correspondence: lbelay@lsuhsc.eduHowever only 3-5% of stroke patients are eligible for tPA therapy, which must be initiated within 4.5 h of stroke onset -otherwise effectiveness is limited and there is potential for hemorrhagic side effects (Fisher and Bastan, 2008).
While many neuroprotective drugs have been developed, none of these drugs have been translated to the clinical setting (O'Collins et al., 2006;Tymianski, 2013).This failure could be explained by the lack of consideration for the risk factors for stroke, including aging, permanent occlusion and long-term follow up in pre-clinical studies (Turner et al., 2013).Thus, animal studies which investigate the mechanisms and efficacy of novel treatments considering different risk factors for stroke is remain ideal.

Docosahexaenoic acid and stroke
Cerebral ischemia is characterized by a rapid accumulation (within minutes) of free fatty acids, including arachidonic acid (AA; 20:4, n-6) and docosahexaenoic acid (DHA; 22:6, n-3), due to increases in intracellular calcium and activation of phospholipases (Muralikrishna Adibhatla and Hatcher, 2006).This free pool of AA and DHA is then converted via enzymatic processes as well as free radical-mediated lipid peroxidation into a cascade of activated pro-and anti-inflammatory mediators, the makeup of which ultimately drives the cell toward survival or programmed cell death.
DHA is a natural element of our diet and is found in fish (salmon, tuna, mackerel, herring, sardines, halibut), other seafood (algae, krill).As a consequence, we depend on the dietary supply of DHA or of its precursor, linonenic acid (18:3, n-3).It is well established that DHA and other omega-3 fatty acids have an important role in heart health.Recent studies have established that omega-3 fatty acids reduce inflammation and may have beneficial effects in patients with coronary heart disease, asthma, rheumatoid arthritis, osteoporosis, sepsis, cancer, dry eye disease and age-related macular degeneration (Simopoulos, 2008).
DHA is a member of the essential omega-3 fatty acid family and is enriched in the membranes of the central nervous system (Bazan, 2006).It is also necessary for the development of the nervous system, including vision (Bazan, 2007).It has been shown to be involved in memory formation, synaptic membrane function, aging and neuroprotection (Bazan et al., 2011).We have discovered that DHA is a critical component of endogenous mechanisms that protect the brain after injury (Bazan, 2005(Bazan, , 2006).An early increase in unesterified (free) DHA during brain ischemia initiates a pathway for docosanoid biosynthesis, specifically neuroprotectin D1 (NPD1) (Bazan, 2003;Marcheselli et al., 2003).We have shown that NPD1 inhibits brain ischemia-reperfusion-mediated leukocyte infiltration and proinflammatory gene expression, and promotes cell survival both in vitro and in vivo (Bazan, 2007;Belayev et al., 2011;Marcheselli et al., 2003).Our recent studies demonstrated that DHA therapy in medium (5 mg/kg) dose improved neurological and histological outcome when treatment is initiated as late as 5 h after onset of stroke (Belayev et al., 2009(Belayev et al., , 2001)).

Docosahexaenoic acid complexed to albumin (DHA-Alb) as a possible superior neuroprotectant
High-dose human albumin (Alb) therapy is strongly neuroprotective in animal models of focal cerebral ischemia (Belayev et al., 2001), as well as in global cerebral ischemia (Belayev et al., 1999).The neuroprotective efficacy of albumin is attributed to its multifunctional properties, which include antioxidant action, hemodilution and oncotic effects, binding of copper ions, fatty-acid transport, preservation of endothelial integrity, platelet anti-aggregatory effects and decreased red blood cell sedimentation under low-flow conditions (Belayev et al., 1997(Belayev et al., , 2002)).In addition, Alb administration following MCAo induces a systemic mobilization of free fatty acids (FFA) through the blood stream.Under these conditions, Alb may help to supply the n-3 polyunsaturated acids (PUFA) to the postischemic brain (Belayev et al., 2005;Rodriguez de Turco et al., 2002) and thereby contribute to replenishing PUFA lost from cellular membranes during ischemia (Bazan et al., 1992) and/or allow these PUFA to serve as an alternative source of energy (Rapoport et al., 2006).Preclinical studies in a rat focal cerebral ischemia stroke model (these were young rats) demonstrated that administration of Alb at high doses (2.5 g/kg) decreased infarct volume, reduced brain swelling (Belayev et al., 1997(Belayev et al., , 1998) ) and improved local cerebral perfusion in affected tissue (Belayev et al., 2002;Huh et al., 1998).These extremely-promising results led to the development of a human clinical trial testing Alb as a neuroprotectant after acute ischemic stroke.Unfortunately, a phase III clinical trial in humans was stopped because the high dose of Alb (2 g/kg) used in this trial led to pulmonary edema in 13% of patients (Ginsberg et al., 2011).This likely occurred because high-dose Alb expands intravascular volume and may exacerbate congestive heart failure in some elderly patients.
Recently, we tested a novel therapy -Alb (0.63 g/kg) complexed to DHA -in a young rat stroke model (Eady et al., 2012).The idea was to reduce the Alb dosage without reducing its protective effects.Excitingly, we found that this treatment with low/moderate doses of DHA-Alb led to improved neurological outcomes and significant reduction of infarct volume (especially in the salvageable penumbral region) even when treatment was initiated as late as 7 h after onset of MCAo (Eady et al., 2012), compared to 4 h for Alb (1.25 g/kg) (Belayev et al., 2001) and 5 h for DHA alone (Belayev et al., 2001).Thus, when albumin is complexed with DHA, it is possible to achieve neuroprotection at lower albumin doses; this takes on great clinical importance as the administration of high-dose albumin, by expanding intravascular volume, may on occasion precipitate acute pulmonary edema and congestive heart failure, particularly in patients with compromised cardiovascular function.

DHA-Alb therapy reduces injury after permanent MCAo
While human thromboembolic stroke commonly involves some degree of early or late reperfusion, in many cases the arterial occlusion remains permanent.The rate of spontaneous recanalization of cerebral artery occlusion in humans is at 17% by 6-8 h from onset (Kassem-Moussa and Graffagnino, 2002).Models resulting in permanent ischemia mimic clinical stroke without reperfusion.Occlusion is usually induced by cauterization of the MCA via a craniotomy or advancing a suture into the ICA to occlude the MCA at its origin from the circle of Willis.However, because of greater mortality with permanent occlusion models, most preclinical studies use transient MCAo.During preclinical investigations, it is vital that the potential for new therapies to ameliorate the consequences of clinically significant permanent occlusions are assessed in an animal model.As recommended by Stroke Therapy Academic Industry Roundtable, permanent MCAo models should be studied first, followed by transient (reperfusion) models (Stroke Therapy Academic Industry Roundtable, 1999).An exception to this recommendation exists for drugs with mechanisms of action requiring successful reperfusion.
Our recent studies demonstrated that DHA-Alb is highly neuroprotective after temporary MCAo, but whether a similar effect occurs in permanent MCAo is unknown.The argument for applying DHA-Alb in human ischemic stroke would be strengthened by the demonstration of its efficacy in permanent arterial occlusion.
Male Sprague-Dawley rats underwent permanent MCAo and behavioral function was evaluated on days 1, 2 and 3 after MCAo.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 were administered i.v. at 3 hours after onset of stroke.Histopathology was conducted on day 3.Our study demonstrated that rats treated with low and moderate doses of DHA-Alb showed improved neurological score compared to corresponding Alb groups on days 2 and 3 (Fig. 1a).In addition, the total corrected infarct volumes were reduced by both doses of DHA-Alb compared to the corresponding Alb groups (Fig. 1b).Histological evaluation of the brains exhibited a consistent pannecrotic lesion involving both cortical and subcortical (mainly striatal) regions of the right hemisphere in saline-treated rats.In contrast, infarct size was attenuated in rats treated with DHA-Alb (Fig. 1c).In conclusion, DHA-Alb therapy is neuroprotective in permanent MCAo in rats.This treatment can provide the basis for future therapeutics for patients suffering from ischemic stroke without reperfusion (Eady et al., 2013).

DHA-Alb therapy provides neuroprotection after stroke in aged rats
Age is the most important independent risk factor for stroke.Both the incidence of stroke and level of negative outcomes associated with stroke (in terms of disability and mor-tality rate) increase with age.With the growth of the global elderly population, the prevalence of stroke will rise also.Reports indicate that 75-89% of strokes occur in individuals aged > 65 years (Feigin et al., 2003).Of these strokes, 50% occur in people who are aged 70 years and nearly 25% occur in individuals who are aged > 85 years.Risk factor profiles and mechanisms of ischemic injury vary between young and old patients with stroke.It is well known that elderly patients tend to have a worse outcome than younger patients because they have comorbidities that heavily affect outcome.In addition, elderly patients often receive less-effective treatment than younger individuals.Although age is one of the most significant prognostic markers for poor outcome, very few studies have been performed in aged animals, especially in animals over 15 months of age.Studies on aged animals (e.g.rodents) can closely mimic the clinical features of older patients with stroke.Aged animals show greater infarction volumes and a higher mortality rate than young animals following MCAo (Chen et al., 2010).While animals of all ages have shown neurological deficits after MCAo, aged animals exhibited poorer performances than young animals in functional tests (Liu et al., 2009).Aged mice of both sexes showed considerably reduced stroke-induced edema compared with young animals.This finding is consistent with the clinical observation that young patients with stroke are more likely to develop fatal brain edema than older patients (Wagner and Lutsep, 2005).
Unfortunately, most experimental studies are conducted on healthy young animals under rigorously controlled laboratory conditions.However, the typical stroke patient is elderly with numerous risk factors and complicating diseases (e.g.diabetes, hypertension and heart diseases).Stroke researchers frequently avoid using aged animals for MCAo due to the more complex surgical procedure and high cost of purchasing and raising animals.Very few experimental studies exist in the literature that have examined middle aged and aging animals, and these have led to somewhat inconsistent results (Liu and McCullough, 2011).This might partly explain the translational failure of neuroprotective drugs in humans.
Recently we have shown that DHA-Alb is neuroprotective after experimental stroke in young rats.To determine whether treatment with DHA-Alb would be protective in aged rodents, SD aged rats (18-months old) received 2 h of MCAo.The behavior was conducted on days 1, 2, 3 and 7 after MCAo.DHA (5 mg/kg), Alb (0.63 g/kg), DHA-Alb (5 mg/kg + 0.63 g/kg) or saline was administered i.v. 3 h after onset of stroke.Ex vivo T2-weighted imaging (T2WI) and histopathology were conducted on day 7. DHA-Alb treatment improved behavioral scores on day 1, 2, 3 and day 7 (Fig. 2a).A reduction in lesion volume was observed in DHA-Alb-treated rats by ex vivo MRI (Fig. 2b) and histopathology (Figs. and has potential for the effective treatment of ischemic stroke in aged individuals.

DHA-Alb treatment confers enduring neuroprotection
Postischemic histologic and neurologic responses to ischemia persist for weeks after perfusion has been restored (Sheng et al., 2009).This is relevant to translation of preclinical studies to clinical trials, which typically assess outcome at intervals of several months after insult.Thus, observations made in the first few days after experimental stroke may not predict efficacy in long-term outcome clinical trials.Chronic or late-stage behavioral testing is especially critical when assessing neurorestorative agents (Herson and Traystman, 2014).Not only may benefits of some agents appear only after a period of time, but extended testing can determine whether the beneficial effects of a compound are sustained or simply delay the appearance of a deficit.There are examples of compounds shown to be initially effective after a short survival time but those initial beneficial effects were lost over time (Valtysson et al., 1994).It should be emphasized that some behaviors initially impaired by stroke recover naturally in rodent models.Thus, it is especially important to be able to distinguish between a test compound's ability to speed normal recovery versus its ability to produce recovery of functions otherwise lost.Several studies have now demonstrated that it is necessary to follow animals for much longer time periods.
Recently we demonstrated that DHA-Alb is highly neuroprotective when animals were allowed to survive during one week.To determine whether treatment with DHA-Alb would persist with chronic survival, SD rats (4-months old) received 2 h of MCAo.The behavior was conducted on days 1, 2, 3 and weeks 1, 2, 3 and 4 after treatment.DHA (5 mg/kg), Alb (0.63 g/kg), DHA-Alb (5 mg/kg + 0.63 g/kg) or saline was administered i.v. 3 h after onset of stroke.All treatments improved neurologic score beginning on day 1, which persisted throughout four weeks survival period compared to the salinetreated group (Fig. 3a).DHA-Alb complex enhanced recovery of behavioral function on day 1 and weeks 1, 2 and 4 and reduced tissue loss on week 4 compared to corresponding Alb-treated group (Fig. 3b).In conclusion, DHA-Alb therapy confers marked and enduring neuroprotection, as assessed by both neurobehavioral and histological methods, in animals followed for 30 days after stroke.

Conclusions
Stroke research in animal models has made useful contributions to our understanding of the disease, but animal research and clinical practice still seem to be distant from each other.The clinical failure is mostly caused by the choice of drug and its pre-clinical evaluation, or clinical trial design and analysis.Crucial issues remain unresolved regarding the translation of preclinical developments to the bedside.
We reported here that a novel DHA-Alb therapy is highly neuroprotective in permanent and temporary MCAo in middleaged rats and persist with chronic survival.This treatment might provide the basis for future therapeutics for patients suffering from ischemic stroke.

Fig. 1 .
Fig. 1.Effect of DHA-Alb on permanent MCAo.(a) Total neurological score (normal score = 0, maximal score = 12) during MCAo (60 min) and on days 1, 2 and 3 after treatment; (b) total infarct volume and (c) computer-generated MosaiX-processed images of Nissl stained paraffinembedded brain sections on day 3 after stroke.Treatment with DHA-Alb significantly improved neurological scores (48 and 72 h), showed less extensive damage, mostly in the subcortical area and reduced total lesion volumes compared to the corresponding Alb-treated group.Values shown are means ± SEM. # p < 0.05 versus saline group; * p < 0.05 versus Alb (1.25 g/kg) group (two-way repeated-measures ANOVA).

Fig. 2 .
Fig. 2. Effect of DHA-Alb after MCAo in aged rats.(a) Total behavioral score (normal score = 0, maximal score = 12) during MCAo (60 min) and on days 1, 2, 3 and 7 after treatment; (b) 3D reconstructions of MRI-derived lesion volumes from high resolution T2 weighted images; (c) computer-generated MosaiX processed images of Nissl stained paraffin-embedded brain sections; (d) total infarct volume.DHA-Alb treatment significantly improved neurological score, reduced MRI lesion volume and attenuated infarct size compared to the saline group during the 7-day survival period.Values shown are means ± SEM. # p < 0.05 versus saline group; * p < 0.05 DHA-Alb versus Alb group (repeated measures ANOVA followed by Bonferroni tests).

Fig. 3 .
Fig. 3. Effect of DHA-Alb on long term survival after MCAo.(a) Behavioral outcome after stroke (normal score = 0, maximal deficit = 12) during 60 min of MCAo and various time after MCAo.(b) Histopathology at three weeks after stroke.DHA-Alb treatment improved total neurological score and reduced tissue loss on week four after stroke.Values shown are means ± SEM. # p < 0.05 versus saline group; * p < 0.05 DHA-Alb versus Alb group (repeated measures ANOVA followed by Bonferroni tests).