Abstracts

Rationale: Millions of individuals endure a traumatic brain injury (TBI) every year in the United States alone. Beyond the initial injury and acute complications, TBIs cause secondary injuries that can cause a host of long-lasting neurological complications such as neurodegeneration, cognitive impairment, and post-traumatic epilepsy (PTE). Although PTE accounts for almost 20% of all symptomatic epilepsy cases, there are currently no FDA approved drugs to prevent the development of PTE or any other long-term neurological complications following TBI. Published data from our lab has demonstrated that in vivo glycolytic inhibition via a hexokinase inhibitor, 2-deoxy-d-glucose (2-DG), both attenuates inhibitory interneuron cell loss and cortical epileptiform activity – two hallmark complications associated with the rodent model of TBI called controlled cortical impact (CCI). We also found that acute application of 2-DG in vitro significantly reduced the intrinsic excitability of excitatory pyramidal neurons while having no effect on GABAergic interneurons of the cortex. This suggests that neuronal subtypes in the cortex may differentially rely on certain energy sources to support their function. With these findings in mind, we sought to determine the cell type-specific regulation of energy and metabolism after injury and following in vivo 2-DG treatment. Methods: We grouped 9 eleven-week-old male C57BL/6 mice into 3 cohorts. One cohort (CCI+2-DG) underwent CCI and subsequent daily intraperitoneal injections of 2-DG at 250 mg/kg starting an hour after injury. A second cohort (CCI+Saline) underwent CCI and subsequent daily injections of saline. The third cohort (Sham+Saline) underwent sham surgery without head impact and received daily saline injections. Following 3 days of treatment, mice were sacrificed, and brains were harvested quickly. Cortical tissue ipsilateral to the site of injury was dissected and used to prepare whole-tissue nuclei suspensions. Barcoded, genome-wide cDNA libraries were prepared from the nuclei suspensions using the 10x Genomics Chromium Single Cell 3’ system, and libraries were sequenced. Using the CellRanger pipeline, raw reads were aligned to the genome and gene-barcode matrices were generated to reveal single cell resolution transcriptome datasets for each cohort. R-Seurat was used for spatial projection and differential gene expression (DGE) analysis of the data. Ingenuity Pathway Analysis software was used for the probing of upstream regulators and gene ontology enrichment analysis. Results: A whole-tissue cortical extraction for single nucleus RNA-sequencing captures all major cell types within the cortex. DGE analysis of neurons between Sham+Saline and CCI+Saline reveals upregulation of several genes involved in glycolysis after injury that is attenuated to near Sham+Saline levels preferentially in excitatory pyramidal neurons and not GABAergic interneurons in the CCI+2-DG cohort. Glutamate transporter genes, ionotropic and metabotropic glutamate receptor genes, and enzymes involved in the glutamate-glutamine cycle are all found to be differentially regulated between all three cohorts in a cell type-specific manner. Conclusions: Single nucleus RNA-sequencing of injured and non-injured cortex provides an unbiased, unsupervised method to discover injury-induced changes and unveil potential pathways to manipulate for therapeutic benefit. This work also supports the idea that neuronal subtypes differentially rely on energy sources both physiologically and in disease states. Funding: NIH NINDS NS076885, DOD, American Epilepsy Society Research Grants