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Single intranasal dose of L-MYC–immortalized human neural stem cells enhances neuroprotection and tissue repair after controlled cortical impact in male rats

ABSTRACT: Traumatic brain injury (TBI) initiates cellular and molecular cascades that impair neurogenesis and promote chronic neuroinflammation, ultimately contributing to cognitive and functional decline. We previously demonstrated that L-myc–expressing human neural stem (LMNSC01) cells migrate to sites of injury and modulate biomarkers associated with neuroprotection and repair after repeated intranasal (IN) administration. Here, we tested whether a single bulk IN dose of LMNSC01 is sufficient to induce neuroprotective changes in gene and protein expression following injury. Immunocompetent male rats underwent controlled cortical impact (CCI) or sham surgery, followed by a single bulk IN dose of LMNSC01 or vehicle on postoperative day 7. Transcriptomic profiling of brain tissue from injured and LMNSC01-treated rats revealed upregulation of immune modulation and tissue repair genes (Il22ra1, Il1rn, Gfi1, Ddx60) and downregulation of genes involved in microglial activation and cytokine signaling (Xcr1, Ccl21a, Ccl9) compared to controls. Proteomic analysis confirmed reduced levels of inflammatory cytokines and endothelial activation markers (ICAM-1, PDGF-AA, TCK-1). Multiplex immunohistochemistry showed LMNSC01-mediated changes in microglial activation, macrophage infiltration, and vascular remodeling. These findings highlight the potential of single IN LMNSC01 dose to reprogram neuroinflammatory and tissue remodeling networks and offer a clinically translatable strategy to improve long-term outcomes after TBI.

ORGANISM(S): Mus musculus Rattus norvegicus

PROVIDER: GSE322819 | GEO | 2026/04/01

REPOSITORIES: GEO

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Project description:Traumatic brain injury (TBI) is a leading cause of death and disability globally. Several studies have shown that 5-lipoxygenase (5-LOX) inhibition reduces leukotriene (LT) release and the inflammatory response, attenuating the development of respiratory diseases, myocardial infarction, and ischemic cerebral injury. However, its role in the pathophysiology of TBI remains unclear. In this study, controlled cortical impact injury was induced to construct a mouse model of TBI. Pericontusional brain tissue samples from sham and TBI mice at 7 days after injury were used for RNA-seq analysis. Altered gene enrichment following TBI, based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, was quantified through real-time polymerase chain reaction (RT-PCR). Immunocytochemistry, Western blotting, and single-cell sequencing experiments were also performed to analyze 5-LOX protein expression. Arachidonic acid (AA) was detected through liquid chromatography mass spectrometry/mass spectrometry. Enzyme-linked immunosorbent assay was used to detect LTB4 release after TBI with or without zileuton treatment. Brain damage, blood–brain barrier disruption, and neuronal apoptosis were detected through histological examination. Neurological outcomes were determined through rotarod and fear conditioning tests. TBI induced significant upregulation of genes related to the AA metabolic pathway, particularly the AA/5-LOX/LT axis, as verified by RT-PCR. AA and LTB4 production increased significantly after TBI. The expression levels of Pla2g4a, which hydrolyses phospholipids to release AA, and 5-LOX, which in turn act downstream to convert AA to LT, were dramatically upregulated up to 7 days after TBI. 5-LOX accumulated in the cytoplasm of activated ameboid microglial cells. In vivo, 5-LOX inhibition with zileuton blocked LT release and reduced microglial activation and the production of inflammatory cytokines, including Il-1β, Ccl7, Spp1, Ccr1, Ccl2, and Il-10. Zileuton also reduced TBI-induced lipid ROS and neuronal cell apoptosis, ameliorating brain damage compared to the vehicle group and improving neurological outcomes after TBI. Mechanically, TBI-induced LT upregulation may stimulate BV2 microglial activation through the ERK, NF-κB, and Akt pathways. Our findings demonstrated the role of 5-LOX in TBI and its potential as a therapeutic target in TBI treatment.

Project description:The problem of traumatic brain injury (TBI) heterogeneity has been a critical barrier to successful translation of therapies in the field. TBI heterogeneity exists in the patient substrate pre-injury (genetics, sex, comorbidities), external injury characteristics (severity, mechanism), and resultant post-injury host response that is responsible for deleterious secondary injury processes (seizures, neuroinflammation, neurodegeneration) and repair/regeneration. Identification of final common molecular pathways and signatures that integrate this vast heterogeneity could be valuable for guiding biomarkers, therapeutic targets, and predictive enrichment. In this study, we present the first large-scale searchable murine single-cell atlas of the transcriptomic response to TBI in 339,357 cells as a foundational step in molecularly deconstructing TBI heterogeneity. We identify 23 cell types with massive heterogeneity in the single-cell response across extrinsicand intrinsic factors, that has been underestimated. Majority of response to TBI was unique to individual cell populations with minimal overlap even within a single injury-model thus highlighting the importance of cell-level resolution. Through this effort, we report novel cell-specific targets and a previously unrecognized role for specific microglial and ependymal subtypes in post-TBI pathophysiology that is highly variable depending on the extrinsic and intrinsic factors studied. One ependymal subtype was a hub of neuroinflammatory signaling after contusional-TBI, particularly related to Il-1b. A single microglial-lineage along pseudotime (comprising 3 microglial subtypes) was a key mediator of host-response after TBI, and shared features with disease associated microglia noted in Alzheimer’s disease and other neurodegenerative disorders, potentially providing a link between TBI and accelerated neurodegeneration. One microglial subtype within this lineage emerged as a key target – it was the only cell type of all 23 that retained persistent and marked gene expression changes 6 months post contusional-TBI. We identify sexually dimorphic gene expression and pathway vulnerabilities with cell-specific differences in both immune and non-immune biological processes. These likely contribute to sex-based outcome and warrant further study to facilitate discovery of cell- and sex-specific druggable targets. Active changes in brain regions distal from the site of primary TBI impact included infiltration of specific microglial populations as well as cell-specific transcriptomic changes in several genes and inflammatory processes distinct from both the peri-contusional and naïve signatures. This atlas validates several known contributors in TBI pathophysiology, and also identifies previously unrecognized targets and avenues for further research. Beyond our presented exemplar analyses (including pathways of clinical interest like sulfonylurea-receptor-1), the companion searchable atlas serves as a foundation for countless future efforts to understand cell-specific heterogeneity after TBI (https://shiny.crc.pitt.edu/cerebri/) as well as numerous other neurological diseases with overlapping pathophysiology.

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Single intranasal dose of L-MYC–immortalized human neural stem cells enhances neuroprotection and tissue repair after controlled cortical impact in male rats

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