Ludmila Belayev

Background: In the United States, traumatic brain injury (TBI) contributes significantly to mortality and morbidity. Elovanoids (ELVs), a novel class of homeostatic lipid mediators we recently discovered and characterized, have demonstrated neuroprotection in experimental stroke models but have never been tested after TBI. Methods: A moderate fluid-percussion injury (FPI) model was used on male rats that were treated with ELVs by intravenous (IV) or intranasal (IN) delivery. In addition, using liquid chromatography-mass spectrometry (LC-MS/MS), we examined whether ELVs could be detected in brain tissue after IN delivery. Results: ELVs administered intravenously 1 h after FPI improved behavior on days 2, 3, 7, and 14 by 20, 23, 31, and 34%, respectively, and preserved hippocampal CA3 and dentate gyrus (DG) volume loss compared to the vehicle. Whole-brain tractography revealed that ELV-IV treatment increased corpus callosum white matter fibers at the injury site. In comparison to treatment with saline on days 2, 3, 7, and 14, ELVs administered intranasally at 1 h and 24 h after FPI showed improved neurological scores by 37, 45, 41, and 41%. T2-weighted imaging (T2WI) abnormalities, such as enlarged ventricles and cortical thinning, were reduced in rats treated by ELV-IN delivery compared to the vehicle. On day 3, ELVs were detected in the striatum and ipsilateral cortex of ELV-IN-treated rats. Conclusion: We have demonstrated that both ELV-IN and ELV-IV administration offer high-grade neuroprotection that can be selectively supplied to the brain. This discovery may lead to innovative therapeutic targets for secondary injury cascade prevention following TBI.

Neuroprotection to attenuate or block the ischemic cascade and salvage neuronal damage has been extensively explored for treating ischemic stroke. However, despite increasing knowledge of the physiologic, mechanistic, and imaging characterizations of the ischemic penumbra, no effective neuroprotective therapy has been found. This study focuses on the neuroprotective bioactivity of docosanoid mediators: Neuroprotectin D1 (NPD1), Resolvin D1 (RvD1), and their combination in experimental stroke. Molecular targets of NPD1 and RvD1 are defined by following dose–response and therapeutic window. We demonstrated that treatment with NPD1, RvD1, and combination therapy provides high-grade neurobehavioral recovery and decreases ischemic core and penumbra volumes even when administered up to 6 h after stroke. The expression of the following genes was salient: (a) Cd163, an anti-inflammatory stroke-associated gene, was the most differentially expressed gene by NPD1+RvD1, displaying more than a 123-fold upregulation in the ipsilesional penumbra (Lisi et al., Neurosci Lett 645:106–112, 2017); (b) 100-fold upregulation takes place in astrocyte gene PTX3, a key regulator of neurogenesis and angiogenesis after cerebral ischemia (. Rodriguez-Grande et al., J Neuroinflammation 12:15, 2015); and (c) Tmem119 and P2y12, two markers of homeostatic microglia, were found to be enhanced by ten- and fivefold, respectively (Walker et al. Int J Mol Sci 21:678, 2020). Overall, we uncovered that protection after middle cerebral artery occlusion (MCAo) by the lipid mediators elicits expression of microglia and astrocyte-specific genes (Tmem119, Fcrls, Osmr, Msr1, Cd68, Cd163, Amigo2, Thbs1, and Tm4sf1) likely participating in enhancing homeostatic microglia, modulating neuroinflammation, promoting DAMP clearance, activating NPC differentiation and maturation, synapse integrity and contributing to cell survival. Graphical Abstract

Despite displaying efficacy in experimental stroke studies, neuroprotection has failed in clinical trials. The translational difficulties include a limited methodological agreement between preclinical and clinical studies and the heterogeneity of stroke in humans compared to standardized strokes in animal models. Promising neuroprotective approaches based on a deeper understanding of the complex pathophysiology of ischemic stroke, such as blocking pro-inflammatory pathways plus pro-survival mediators, are now evaluated in preclinical studies. Combinatorial therapy has become increasingly attractive in recent years as recognizing the complexity of stroke progression becomes evident. The paper aimed to test the hypothesis that blocking pro-inflammatory platelet-activating factor receptor (PAF-R) with LAU-0901 plus administering a selected docosanoid, aspirin-triggered neuroprotectin D1 (AT-NPD1), which activates cell-survival pathways after middle cerebral artery occlusion (MCAo), would lead to neurological recovery. We have demonstrated that LAU-0901 plus AT-NPD1 treatment affords high-grade neuroprotection in MCAo, equaling or exceeding that afforded by LAU-0901 or AT-NPD1 alone at considerably moderate doses, and it has a broad therapeutic window extending to 6 hours after stroke onset.

Aims: Mesencephalic astrocyte‐derived neurotrophic factor (MANF) is a secretory neurotrophic factor protein that promotes repair after neuronal injury. The microglia cell surface receptor (triggering receptor expressed on myeloid cells‐2; TREM2) regulates the production of pro‐ and antiinflammatory mediators after stroke. Here, we study MANF and TREM2 expression after middle cerebral artery occlusion (MCAo) and explore if docosahexaenoic acid (DHA) treatment exerts a potentiating effect.Methods: We used 2 hours of the MCAo model in rats and intravenously administered DHA or vehicle at 3 hours after the onset of MCAo. Neurobehavioral assessment was performed on days 1, 3, 7, and 14; MANF and TREM2 expression was measured by immunohistochemistry and Western blotting.Results: MANF was upregulated in neurons and astrocytes on days 1, 7, and 14, and TREM2 was expressed on macrophages in the ischemic penumbra and dentate gyrus (DG) on days 7 and 14. DHA improved neurobehavioral recovery, attenuated infarct size on days 7 and 14, increased MANF and decreased TREM2 expression in ischemic core, penumbra, DG, and enhanced neurogenesis on Day 14.Conclusion: MANF and TREM2 protein abundance is robustly increased after MCAo, and DHA treatment potentiated MANF abundance, decreased TREM2 expression, improved neurobehavioral recovery, reduced infarction, and provided enhanced neuroprotection.

OBJECTIVE: Acute ischemic stroke triggers complex neurovascular, neuroinflammatory, and synaptic alterations. This study explores whether blocking pro-inflammatory platelet-activating factor receptor (PAF-R) plus selected docosanoids after middle cerebral artery occlusion (MCAo) would lead to neurological recovery. The following small molecules were investigated: (a) LAU-0901, a PAF-R antagonist that blocks pro-inflammatory signaling; and (b) derivatives of docosahexaenoic acid (DHA), neuroprotectin D1 (NPD1), and aspirin-triggered NPD1 (AT-NPD1), which activates cell survival pathways and are exert potent anti-inflammatory activity in the brain. MATERIALS AND METHODS: Sprague-Dawley rats received 2 h MCAo and LAU-0901 (30 or 60 mg/kg, 2 h after stroke), NPD1, and AT-NPD1 (333 μg/kg), DHA (5 mg/kg), and their combination were administered intravenous at 3 h after stroke. Behavior testing and ex vivo magnetic resonance imaging were conducted on day 3 or 14 to assess lesion characteristics and lipidomic analysis on day 1. Series 1 (LAU-0901 + NPD1, 14d), Series 2 (LAU-0901 + AT-NPD1, 3d), and Series 3 (LAU-0901 + DHA, 1d). RESULTS: All combinatory groups improved behavior compared to NPD1, AT-NPD1, or DHA treatments alone. Total lesion volumes were reduced with LAU-0901 + NPD1 by 62% and LAU-0901 + AT-NPD1 by 90% treatments versus vehicle groups. LAU-0901 and LAU-0901 + DHA increased the production of vasoactive lipid mediators (prostaglandins: PGE₂, PGF_2-α, 6-keto-PGF_1-α, and PGD₂) as well an inflammatory regulating mediator hydroxyoctadecadienoic acid. In contrast, LAU-0901 and LAU-0901 + DHA decreased the production of 12-hydroxyeicosatetraenoic acid, a pro-inflammatory mediator. CONCLUSION: Combination therapy with LAU-0901 and selected docosanoids is more effective than the single therapy, affording synergistic neuroprotection, with restored pro-homeostatic lipid mediators and improved neurological recovery. Altogether, our findings support the combinatory therapy as the basis for future therapeutics for ischemic stroke.

Docosahexaenoic acid (DHA) and neuroprotectin D1 (NPD1) are neuroprotective after experimental ischemic stroke. To explore underlying mechanisms, SD rats underwent 2 h of middle cerebral artery occlusion (MCAo) and treated with DHA (5 mg/kg, IV) or NPD1 (5 μg/per rat, ICV) and vehicles 1 h after. Neuro-behavioral assessments was conducted on days 1, 2, and 3, and on week 1, 2, 3, or 4. BrdU was injected on days 4, 5, and 6, immunohistochemistry was performed on week 2 or 4, MRI on day 7, and lipidomic analysis at 4 and 5 h after onset of stroke. DHA improved short- and long-term behavioral functions and reduced cortical, subcortical, and total infarct volumes (by 42, 47, and 31%, respectively) after 2 weeks and reduced tissue loss by 50% after 4 weeks. DHA increased the number of BrdU⁺/Ki-67⁺, BrdU⁺/DCX⁺, and BrdU⁺/NeuN⁺ cells in the cortex, subventricular zone, and dentate gyrus and potentiated NPD1 synthesis in the penumbra at 5 h after MCAo. NPD1 improved behavior, reduced lesion volumes, protected ischemic penumbra, increased NeuN, GFAP, SMI-71-positive cells and vessels, axonal regeneration in the penumbra, and attenuated blood-brain barrier (BBB) after MCAo. We conclude that docosanoid administration increases neurogenesis and angiogenesis, activates NPD1 synthesis in the penumbra, and diminishes BBB permeability, which correlates to long-term neurobehavioral recovery after experimental ischemic stroke.