Neuroprotective lipid mediators target penumbra in experimental ischemic stroke - Abstract Ischemic stroke is a leading cause of long-term disability in both genders and lacks effective therapeutic options. We propose to study elovanoids (ELVs), which are stereospecific dihydroxylated derivatives of the free fatty acids (FFAs) 32:6n-3 or 34:6n-3 that yield ELV-N32 and ELV-N34, respectively, to protect the stroke penumbra from irreversible damage. The precursors of ELVs are made by the enzyme ELOVL4 (elongation of very long- chain fatty acids protein-4). Our preliminary data showed improved behavior, decreased infarct size, restored blood-brain barrier (BBB) integrity, and upregulation of 76 genes in the ischemic core and penumbra by ELV precursors 32:6n-3 or 34:6n-3 when intranasally delivered (IND) after ischemic stroke in rats. Our central hypothesis is that the FFAs precursors 32:6n-3 or 34:6n-3 activate ELV synthesis, promoting cell survival in the penumbra. We postulate that ELV bioactivity is elicited by targeting gene clusters in neurons, astrocytes, and microglia/macrophage phenotypes. Our goal is to identify specific cell survival events using ELVs instead of blocking inflammation, as is often the case. In this project, we will use the rat middle cerebral artery occlusion (MCAo) model, MRI to assess brain edema, the ischemic core and penumbra volumes, single-nuclei Multiome Gene Expression plus ATAC sequencing (snRNA + snATAC), Spatially Resolved Transcriptomics (SRT) using Visium and Xenium, and Jess technology to identify the cells and the cell-specific mechanisms that limit core expansion into the penumbra and assessment of the BBB permeability after administration ELVs. Aim 1: To test the hypothesis that ELV precursors 34:6n-3 or 32:6n-3 protect the ischemic brain by activating ELV biosynthesis in the ischemic penumbra. We will explore the concept that the failure of penumbra and ischemic core cells to survive ischemia-reperfusion damage results from the inability to generate specific ELVs. We will investigate dose-response, therapeutic window, use of deuterated 32:6n-3 or 34:6n-3 to assess endogenous pool sizes of the fatty acids, identify pathways using LC-MS/MS, and characterize the ischemic penumbra in young and aged rats. Aim 2: Define the molecular mechanisms by which IND ELV precursors (32:6n-3 or 34:6n-3) and ELVs (ELV-N32 or ELV-N34) sustain penumbra integrity and limit core expansion into the penumbra. We will determine: 1) How ELVs modulate the inflammatory response leading to penumbra protection by specific proteins using Jess technology. 2) How ELVs mediate cell-specific transcriptional landscape in astrocytes, microglia/macrophages, and neurons using snRNA + snATAC, SRT using Visium and Xenium to identify gene expression networks and determine cell phenotypes. 3) Whether blocking BBB leakage with LMs restores the NVU and BBB integrity by suppressing inflammation, preventing hemorrhagic transformation, and improving behavioral functions. The results of this project have the potential to develop novel therapeutics to protect the ischemic stroke penumbra and improve the quality of life of young and older individuals of both genders after ischemic stroke, which remains among the most significant challenges to public health.
