Probes for CCR2

the increased expression of 18kDa Translocator protein (TSPO) is one of the few available biomarkers of neuroinflammation that can be assessed in humans in vivo by positron emission tomography (PET). TSPO PET imaging of the central nervous system (CNS) has been widely undertaken, but to date no clear consensus has been reached about its utility in brain disorders. One reason for this could be because the interpretation of TSPO PET signal remains challenging, given the cellular heterogeneity and ubiquity of TSPO in the brain. the aim of the current study was to ascertain if TSPO PET imaging can be used to detect neuroinflammation induced by a peripheral treatment with endotoxin lipopolysaccharide (LPS) in a rat model (ip LPS), and investigate the origin of TSPO signal changes in terms of their cellular sources and regional distribution. An initial pilot study utilising both [18F]DPA-714 and [11C]PK11195 demonstrated [18F]DPA-714 to exhibit a significantly higher lesion-related signal in the intracerebral LPS rat model (ic LPS) than [11C]PK11195. Subsequently, [18F]DPA-714 was selected for use in the ip LPS study. twenty-four hours after ip LPS, there was an increased uptake of [18F]DPA-714 across the whole brain. Further analyses of regions of interest, using immunohistochemistry and RNAscope Multiplex fluorescence V2 in situ hybridization technology, showed TSPO expression in microglia, monocyte derived-macrophages, astrocytes, neurons and endothelial cells. The expression of TSPO was significantly increased after ip LPS in a region-dependent manner; with microglia, monocyte-derived macrophages and astrocytes in the substantia nigra, in contrast to the hippocampus where TSPO was mostly confined to microglia and astrocytes. in summary, our data demonstrate the robust detection of peripherally-induced neuroinflammation in the CNS utilizing the TSPO radioligand [18F]DPA-714, and importantly, confirm that the TSPO signal increase arises mostly from a combination of microglia, astrocytes and monocyte-derived macrophages.

The blood-brain barrier (BBB) consists of endothelial cells, astrocytic end feet, and pericytes to form a barrier that minimizes the entry of circulating proteins and cells into the brain. However, stroke is known to cause significant damage to the BBB, causing the barrier to become permeable, which allows immune cells and other substances to be extravasated into the brain parenchyma. Preservation of the BBB is associated with better ischemic stroke outcomes; therefore, this synopsis summarizes 3 new studies that aim to characterize specific mechanisms of BBB damage and identify potential therapeutic pathways to preserve barrier integrity. CD36 (cluster of differentiation 36) is a glycoprotein expressed by monocytes and macrophages, as well as by endothelial cells. Previous studies have shown that global knockout of CD36 prevents stroke-induced damage, and Kim et al in 2023 published a study in the Journal of Cerebral Blood Flow and Metabolism titled “Endothelial Cell CD36 Mediates Stroke-Induced Brain Injury via BBB Dysfunction and Monocyte Infiltration in Normal and Obese Conditions,” in which they explore the role of CD36 specifically in endothelial cells. Conditional deletion of CD36 in endothelial cells improved stroke outcomes, as indicated by reduced infarct size and hemispheric swelling. Moreover, this deletion improved survival and motor function. Additionally, CD36 deletion in endothelial cells reduced IgG expression in the brain, indicating improved vascular integrity. There was reduced monocyte infiltration into the brain and reduced MCP-1 (monocyte chemoattractant protein-1) and CCR2 (chemokine receptor type 2) expression in the mice with endothelial cell deletion of CD36. This reduced monocyte trafficking persisted even when normalized for infarct size, suggesting that vascular integrity is maintained independent of cell loss. Intriguingly, endothelial cell-specific deletion of CD36 also made mice resistant to developing an obesity phenotype, providing a potential molecular cause for obesity as a stroke risk factor. In the Proceedings of the National Academy for Science in a publication titled “Myeloid-Derived MIF Drives RIPK1-Mediated Cerebromicrovascular Endothelial Cell Death to Exacerbate Ischemic Brain Injury,” Li et al in 2023 describe how macrophage MIF (migration inhibitory factor) exacerbates endothelial cell death and increases BBB permeability after middle cerebral artery occlusion (MCAo). By treating endothelial cells with MIF and subjecting them to oxygen-glucose deprivation followed by reoxygenation, the authors demonstrated that MIF promotes endothelial cell death specifically by activating RIPK1 (receptor-interacting protein kinase 1). Surgical trauma in both mice and humans increases circulating MIF, and the authors use a perioperative ischemic stroke model to see how this surgically induced increase in MIF affects outcomes after distal MCAo. Two-photon imaging showed that perioperative ischemic stroke mice showed increased adhesion of myeloid cells to ischemic microvascular endothelial cells, and RNAscope analysis showed that MIF expression was increased in microglia surrounding endothelial cells in perioperative ischemic stroke mice. Perioperative ischemic stroke mice also exhibited larger infarct volumes and exacerbated BBB damage. The authors then used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) to delete MIF in myeloid cells, which resulted in reduced expression of necrosis markers phosphorylated RIPK1, phosphorylated RIPK3, and CC3 (cleaved caspase 3) in endothelial cells. Mice with myeloid-deleted MIF showed reduced zonulin-1 loss, a marker of endothelial tight junctions, and sensorimotor deficits after distal MCAo. Peripheral blood mononuclear cells from mice with myeloid-deleted MIF were given after MCAo and resulted in smaller infarct volume and reduced IgG extravasation as compared with mice who were given peripheral blood mononuclear cells from wild types. Endothelial cells were cocultured with peripheral blood mononuclear cells from mice with myeloid-deleted MIF and wild types, and zonulin-1 expression was preserved in endothelial cocultured with peripheral blood mononuclear cells from myeloid-deleted MIF mice. Administration of MIF inhibitor before stroke reduced infarct volume, prevented IgG extravasation, preserved tight junction integrity, and prevented endothelial cell death. Collectively, these data show that myeloid-derived MIF is detrimental to BBB integrity after stroke and deleting this source of MIF can improve outcomes through preserving BBB health. Finally, Li et al in 2023 show in ACS Nano in a publication titled “Inducible Pluripotent Stem Cell-Derived Small Extracellular Vesicles Rejuvenate Senescent Blood-Brain Barrier to Protect Against Ischemic Stroke in Aged Mice” that small extracellular vesicles (sEVs) from induced pluripotent stem cells (iPSCs) are able to restore BBB function in old mice by reversing cellular senescence. Mice treated with iPSC-sEVs showed reduced senescence-associated β-galactosidase, p16, p53, p21, and γ-H2AX (histone family member X), all of which are markers associated with cellular senescence. Pretreatment with iPSC-sEVs before MCAo reduced infarct volume in the aged mice, improved neurological score, and reduced sensorimotor deficits, indicating improved stroke outcomes. Moreover, these mice showed decreased leakage of Evans blue dye into the brain parenchyma and preservation of tight junction proteins, indicating that these sEVs preserve BBB integrity after stroke. Mice treated with sEVs showed decreased immune cell infiltration after stroke and attenuated expression of tumor necrosis factor-α, IL (interleukin)-17, IL-6, and IL-1β, Moreover, sEV treatment reduced ischemia-induced apoptosis of oligodendrocytes and neurons. Reversal of the senescent phenotype of the BBB was tested in vitro, by chemically inducing senescence using D-galactose in endothelial cell cultures and subsequent treatment with iPSC-sEVs. sEV treatment reduced senescence markers and prevented loss of tight junction proteins. Oxygen-glucose deprivation was then used to mimic stroke conditions in these cultures, and sEV treatment preserved angiogenic properties of endothelial cells and reduced dextran leakage in a transwell assay. These experiments affirm the in vivo findings that sEV treatment reverses BBB senescence. The 3 studies summarized in this synopsis show 3 different potential pathways that may serve as a target for preserving BBB function after stroke. Deleting endothelial cell CD36, deleting myeloid-derived MIF, or reversing BBB senescence using iPSC-sEVs resulted in improved vascular integrity and overall better stroke outcomes.