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.