Post-stroke administration of omega-3 polyunsaturated fatty acids promotes neurovascular restoration after ischemic stroke in mice: Efficacy declines with aging.

Post-stroke treatment with omega-3 polyunsaturated fatty acids (n-3 PUFAs) may be a promising therapy in young animals but this has not been tested in aged subjects, a population at most risk of ischemic stroke. Herein we examined the therapeutic efficacy of n-3 PUFAs after distal middle cerebral artery occlusion (dMCAO) in young (10-12 weeks old) and aged (18 months old) mice. Post-ischemic mice were randomly assigned to 4 groups that received: 1) regular food with low content of n-3 PUFAs, 2) intraperitoneal docosahexaenoic acid (DHA, a major component of n-3 PUFAs) injections, 3) Fish oil (FO, containing high concentration of n-3 PUFAs) dietary supplement, or 4) combined treatment with DHA and FO dietary supplement. Long-term neurorestoration induced by n-3 PUFA post-stroke administration and its underlying mechanism(s) were analyzed up to 35 days after dMCAO. Aged mice showed more severe neurological deficits than young mice after dMCAO with histological lesions extended to the striatum. Notably, post-stroke treatment with combined DHA injections and FO dietary supplementation was more effective in reducing brain injury and improving sensorimotor function in aged mice than either treatment alone, albeit to a lesser extent than in the young mice. Unlike the improvement in spatial cognitive function observed in young mice, the combined treatment regimen failed to improve cognitive function in aged mice. The reduction in stroke-induced neurological deficits with n-3 PUFA post-treatment was associated with enhanced angiogenesis, oligodendrogenesis, neuron survival and white matter restoration. Together, these results indicate that the neurological benefits of n-3 PUFA administration after stroke extend to older animals and are associated with improved neuronal survival and brain remodeling, therefore suggesting that post-stroke administration of n-3 PUFAs is a viable clinically relevant treatment option against stroke.

Promoting Neurovascular Recovery in Aged Mice after Ischemic Stroke - Prophylactic Effect of Omega-3 Polyunsaturated Fatty Acids.

The aged population is among the highest at risk for ischemic stroke, yet most stroke patients of advanced ages (>80 years) are excluded from access to thrombolytic treatment by tissue plasminogen activator, the only FDA approved pharmacological therapy for stroke victims. Omega-3 polyunsaturated fatty acids (n-3 PUFAs) robustly alleviate ischemic brain injury in young adult rodents, but have not yet been studied in aged animals. This study investigated whether chronic dietary supplementation of n-3 PUFAs protects aging brain against cerebral ischemia and improves long-term neurological outcomes. Aged (18-month-old) mice were administered n-3 PUFA-enriched fish oil in daily chow for 3 months before and up to 8 weeks after 45 minutes of transient middle cerebral artery occlusion (tMCAO). Sensorimotor outcomes were assessed by cylinder test and corner test up to 35 days and brain repair dynamics evaluated immunohistologically up to 56 days after tMCAO. Mice receiving dietary supplementation of n-3 PUFAs for 3 months showed significant increases in brain ratio of n-3/n-6 PUFA contents, and markedly reduced long-term sensorimotor deficits and chronic ischemic brain tissue loss after tMCAO. Mechanistically, n-3 PUFAs robustly promoted post-ischemic angiogenesis and neurogenesis, and enhanced white matter integrity after tMCAO. The Pearson linear regression analysis revealed that the enhancement of neurogenesis and white matter integrity both correlated positively with improved sensorimotor activities after tMCAO. This study demonstrates that prophylactic dietary supplementation of n-3 PUFAs effectively improves long-term stroke outcomes in aged mice, perhaps by promoting post-stroke brain repair processes such as angiogenesis, neurogenesis, and white matter restoration.

Repetitive and Prolonged Omega-3 Fatty Acid Treatment After Traumatic Brain Injury Enhances Long-Term Tissue Restoration and Cognitive Recovery.

Traumatic brain injury (TBI) is one of the most disabling clinical conditions that could lead to neurocognitive disorders in survivors. Our group and others previously reported that prophylactic enrichment of dietary omega-3 polyunsaturated fatty acids (n-3 PUFAs) markedly ameliorate cognitive deficits after TBI. However, it remains unclear whether a clinically relevant therapeutic regimen with n-3 PUFAs administered after TBI would still offer significant improvement of long-term cognitive recovery. In the present study, we employed the decline of spatial cognitive function as a main outcome after TBI to investigate the therapeutic efficacy of post-TBI n-3 PUFA treatment and the underlying mechanisms. Mice were subjected to sham operation or controlled cortical impact, followed by random assignment to receive the following four treatments: (1) vehicle control; (2) daily intraperitoneal injections of n-3 PUFAs for 2 weeks, beginning 2 h after TBI; (3) fish oil dietary supplementation throughout the study, beginning 1 day after TBI; or (4) combination of treatments (2) and (3). Spatial cognitive deficits and chronic brain tissue loss, as well as endogenous brain repair processes such as neurogenesis, angiogenesis, and oligodendrogenesis, were evaluated up to 35 days after TBI. The results revealed prominent spatial cognitive deficits and massive tissue loss caused by TBI. Among all mice receiving post-TBI n-3 PUFA treatments, the combined treatment of fish oil dietary supplement and n-3 PUFA injections demonstrated a reproducible beneficial effect in attenuating cognitive deficits although without reducing gross tissue loss. Mechanistically, the combined treatment promoted post-TBI restorative processes in the brain, including generation of immature neurons, microvessels, and oligodendrocytes, each of which was significantly correlated with the improved cognitive recovery. These results indicated that repetitive and prolonged n-3 PUFA treatments after TBI are capable of enhancing brain remodeling and could be developed as a potential therapy to treat TBI victims in the clinic.

A Post-stroke Therapeutic Regimen with Omega-3 Polyunsaturated Fatty Acids that Promotes White Matter Integrity and Beneficial Microglial Responses after Cerebral Ischemia.

White matter injury induced by ischemic stroke elicits sensorimotor impairments, which can be further deteriorated by persistent proinflammatory responses. We previously reported that delayed and repeated treatments with omega-3 polyunsaturated fatty acids (n-3 PUFAs) improve spatial cognitive functions and hippocampal integrity after ischemic stroke. In the present study, we report a post-stroke n-3 PUFA therapeutic regimen that not only confers protection against neuronal loss in the gray matter but also promotes white matter integrity. Beginning 2 h after 60 min of middle cerebral artery occlusion (MCAO), mice were randomly assigned to receive intraperitoneal docosahexaenoic acid (DHA) injections (10 mg/kg, daily for 14 days), alone or in combination with dietary fish oil (FO) supplements starting 5 days after MCAO. Sensorimotor functions, gray and white matter injury, and microglial responses were examined up to 28 days after MCAO. Our results showed that DHA and FO combined treatment-facilitated long-term sensorimotor recovery and demonstrated greater beneficial effect than DHA injections alone. Mechanistically, n-3 PUFAs not only offered direct protection on white matter components, such as oligodendrocytes, but also potentiated microglial M2 polarization, which may be important for white matter repair. Notably, the improved white matter integrity and increased M2 microglia were strongly linked to the mitigation of sensorimotor deficits after stroke upon n-3 PUFA treatments. Together, our results suggest that post-stroke DHA injections in combination with FO dietary supplement benefit white matter restoration and microglial responses, thereby dictating long-term functional improvements.

Delayed Docosahexaenoic Acid Treatment Combined with Dietary Supplementation of Omega-3 Fatty Acids Promotes Long-Term Neurovascular Restoration After Ischemic Stroke.

Prophylactic dietary intake of omega-3 polyunsaturated fatty acids (n-3 PUFAs) has been shown to remarkably ameliorate ischemic brain injury. However, the therapeutic efficacy of n-3 PUFA administration post-stroke, especially its impact on neurovascular remodeling and long-term neurological recovery, has not been fully characterized thus far. In this study, we investigated the effect of n-3 PUFA supplementation, as well as in combination with docosahexaenoic acid (DHA) injections, on long-term stroke outcomes. Mice were subjected to transient middle cerebral artery occlusion (MCAO) before randomly assigned to four groups to receive the following: (1) low dose of n-3 PUFAs as the vehicle control, (2) intraperitoneal DHA injections, (3) n-3 PUFA dietary supplement, or (4) combined treatment of (2) and (3). Neurological deficits and brain atrophy, neurogenesis, angiogenesis, and glial scar formation were assessed up to 28 days after MCAO. Results revealed that groups 2 and 3 showed only marginal reduction in post-stroke tissue loss and attenuation of cognitive deficits. Interestingly, group 4 exhibited significantly reduced tissue atrophy and improved cognitive functions compared to groups 2 and 3 with just a single treatment. Mechanistically, the combined treatment promoted post-stroke neurogenesis and angiogenesis, as well as reduced glial scar formation, all of which significantly correlated with the improved spatial memory in the Morris water maze. These results demonstrate an effective therapeutic regimen to enhance neurovascular restoration and long-term cognitive recovery in the mouse model of MCAO. Combined post-stroke DHA treatment and n-3 PUFA dietary supplementation thus may be a potential clinically translatable therapy for stroke or related brain disorders.

Omega-3 polyunsaturated fatty acids mitigate blood-brain barrier disruption after hypoxic-ischemic brain injury.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to protect the neonatal brain against hypoxic/ischemic (H/I) injury. However, the mechanism of n-3 PUFA-afforded neuroprotection is not well understood. One major determinant of H/I vulnerability is the permeability of the blood-brain barrier (BBB). Therefore, we examined the effects of n-3 PUFAs on BBB integrity after neonatal H/I. Female rats were fed a diet with or without n-3 PUFA enrichment from day 2 of pregnancy to 14days after parturition. H/I was introduced in 7day-old offspring. We observed relatively rapid BBB penetration of the small molecule cadaverine (640Da) at 4h post-H/I and a delayed penetration of larger dextrans (3kD-40kD) 24-48h after injury. Surprisingly, the neonatal BBB was impermeable to Evans Blue or 70kD dextran leakage for up to 48h post-H/I, despite evidence of IgG extravasation at this time. As expected, n-3 PUFAs ameliorated H/I-induced BBB damage, as shown by reductions in tracer efflux and IgG extravasation, preservation of BBB ultrastructure, and enhanced tight junction protein expression. Furthermore, n-3 PUFAs prevented the elevation in matrix metalloproteinase (MMP) activity in the brain and blood after H/I. Thus, n-3 PUFAs may protect neonates against BBB damage by blunting MMPs activation after H/I.

Dietary supplementation with omega-3 polyunsaturated fatty acids robustly promotes neurovascular restorative dynamics and improves neurological functions after stroke.

Stroke is a devastating neurological disease with no satisfactory therapies to preserve long-term neurological function, perhaps due to the sole emphasis on neuronal survival in most preclinical studies. Recent studies have revealed the importance of protecting multiple cell types in the injured brain, such as oligodendrocytes and components of the neurovascular unit, before long-lasting recovery of function can be achieved. For example, revascularization in the ischemic penumbra is critical to provide various neurotrophic factors that enhance the survival and activity of neurons and other progenitor cells, such as oligodendrocyte precursor cells. In the present study, we hypothesized that chronic dietary supplementation with fish oil promotes post-stroke angiogenesis, neurogenesis, and oligodendrogenesis, thereby leading to long-term functional improvements. Mice received dietary supplementation with n-3 PUFA-enriched fish oil for three months before and up to one month after stroke. As expected, dietary n-3 PUFAs significantly increased levels of n-3 PUFAs in the brain and improved long-term behavioral outcomes after cerebral ischemia. n-3 PUFAs also robustly improved revascularization and angiogenesis and boosted the survival of NeuN/BrdU labeled newborn neurons up to 35days after stroke injury. Furthermore, these pro-neurogenic effects were accompanied by robust oligodendrogenesis. Thus, this is the first study to demonstrate that chronic dietary intake of n-3 PUFAs is an effective prophylactic measure not only to protect against ischemic injury for the long term but also to actively promote neurovascular restorative dynamics and brain repair.

Omega-3 polyunsaturated fatty acids enhance cerebral angiogenesis and provide long-term protection after stroke.

Stroke is a devastating neurological disorder and one of the leading causes of death and serious disability. After cerebral ischemia, revascularization in the ischemic boundary zone provides nutritive blood flow as well as various growth factors to promote the survival and activity of neurons and neural progenitor cells. Enhancement of angiogenesis and the resulting improvement of cerebral microcirculation are key restorative mechanisms and represent an important therapeutic strategy for ischemic stroke. In the present study, we tested the hypothesis that post-stroke angiogenesis would be enhanced by omega-3 polyunsaturated fatty acids (n-3 PUFAs), a major component of dietary fish oil. To this end, we found that transgenic fat-1 mice that overproduce n-3 PUFAs exhibited long-term behavioral and histological protection against transient focal cerebral ischemia (tFCI). Importantly, fat-1 transgenic mice also exhibited robust improvements in revascularization and angiogenesis compared to wild type littermates, suggesting a potential role for n-3 fatty acids in post-stroke cerebrovascular remodeling. Mechanistically, n-3 PUFAs induced upregulation of angiopoietin 2 (Ang 2) in astrocytes after tFCI and stimulated extracellular Ang 2 release from cultured astrocytes after oxygen and glucose deprivation. Ang 2 facilitated endothelial proliferation and barrier formation in vitro by potentiating the effects of VEGF on phospholipase Cγ1 and Src signaling. Consistent with these findings, blockade of Src activity in post-stroke fat-1 mice impaired n-3 PUFA-induced angiogenesis and exacerbated long-term neurological outcomes. Taken together, our findings strongly suggest that n-3 PUFA supplementation is a potential angiogenic treatment capable of augmenting brain repair and improving long-term functional recovery after cerebral ischemia.

Omega-3 fatty acids protect the brain against ischemic injury by activating Nrf2 and upregulating heme oxygenase 1.

Ischemic stroke is a debilitating clinical disorder that affects millions of people, yet lacks effective neuroprotective treatments. Fish oil is known to exert beneficial effects against cerebral ischemia. However, the underlying protective mechanisms are not fully understood. The present study tests the hypothesis that omega-3 polyunsaturated fatty acids (n-3 PUFAs) attenuate ischemic neuronal injury by activating nuclear factor E2-related factor 2 (Nrf2) and upregulating heme oxygenase-1 (HO-1) in both in vitro and in vivo models. We observed that pretreatment of rat primary neurons with docosahexaenoic acid (DHA) significantly reduced neuronal death following oxygen-glucose deprivation. This protection was associated with increased Nrf2 activation and HO-1 upregulation. Inhibition of HO-1 activity with tin protoporphyrin IX attenuated the protective effects of DHA. Further studies showed that 4-hydroxy-2E-hexenal (4-HHE), an end-product of peroxidation of n-3 PUFAs, was a more potent Nrf2 inducer than 4-hydroxy-2E-nonenal derived from n-6 PUFAs. In an in vivo setting, transgenic mice overexpressing fatty acid metabolism-1, an enzyme that converts n-6 PUFAs to n-3 PUFAs, were remarkably resistant to focal cerebral ischemia compared with their wild-type littermates. Regular mice fed with a fish oil-enhanced diet also demonstrated significant resistance to ischemia compared with mice fed with a regular diet. As expected, the protection was associated with HO-1 upregulation, Nrf2 activation, and 4-HHE generation. Together, our data demonstrate that n-3 PUFAs are highly effective in protecting the brain, and that the protective mechanisms involve Nrf2 activation and HO-1 upregulation by 4-HHE. Further investigation of n-3 PUFA neuroprotective mechanisms may accelerate the development of stroke therapies.