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bioRxiv · 10.1101/2025.01.20.633889

Acute temporal, regional, and cell-type specific NKCC1 disruption following severe TBI in the developing gyrencephalic brain.

Hochstetler, A. E.·Courtney, Y.·Oloko, P.·Baskin, B.·Ding-Su, A.·Stinson, T.·McGuone, D.·Haynes, R.·Lehtinen, M. K.·Costine-Bartell, B. A.
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Abstract

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality in infants and toddlers, with limited treatment options and persistent neurological sequelae. We developed a multi-pathoanatomic lesion multi-insult (MuLMI) severe TBI model in piglets that replicates age-dependent damage patterns to the cortical ribbon observed in human patients with less injury in postnatal day (PND) 7 "infant" piglets and more extensive tissue damage in PND30 "toddler" piglets. Given that neuronal chloride homeostasis influences excitability, seizure susceptibility, and edema, we examined the developmental and injury-induced regulation of key cation-chloride cotransporters and modulators: NKCC1 (sodium-potassium-2-chloride cotransporter), KCC2 (potassium-chloride cotransporter), and the regulatory kinase SPAK, which are biomarkers of neuronal chloride concentrations. This study is the first to define the spatiotemporal expression and phosphorylation profiles of these proteins in the developing piglet brain. We found a perinatal shift in the ratio of KCC2:NKCC1 across the brain, driven primarily by protein abundance, rather than transcriptional levels. We hypothesized that toddler piglets would exhibit an increase in cortical NKCC1 and SPAK causing hyperexcitability and perhaps explaining their more severe, unilateral cortical damage. Severe TBI induced a transcriptional increase in Slc12a2 and Stk39, and a decrease in Slc12a5 in toddler piglets, but not infant piglets. We further found that infant piglets, not toddler piglets, upregulated SPAK and Tyrosine Receptor Kinase B (TRKB) protein in cortex after TBI, with minimal changes in NKCC1 and KCC2. However, phosphorylated NKCC1 (pNKCC1) was significantly upregulated in surviving cortical neurons after TBI in infant piglets and was unchanged in toddlers, despite more severe injury. These findings suggest that cortical neuronal NKCC1 activation may play a role in post-traumatic excitability or resilience in the immature brain and identify NKCC1 and/or SPAK as a potential therapeutic target. In human tissue, the KCC2:NKCC1 ratio also increased postnatally, and TBI caused region and cell-type specific dysregulation of pNKCC1. Our results establish piglets as a valuable model for investigating age-specific mechanisms of pediatric TBI and for testing targeted interventions, particularly for infant populations where seizure control remains a major clinical challenge.

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Hochstetler, A. E., Courtney, Y., Oloko, P., Baskin, B., Ding-Su, A., Stinson, T., McGuone, D., Haynes, R., Lehtinen, M. K., Costine-Bartell, B. A.. 2025-01-20. Acute temporal, regional, and cell-type specific NKCC1 disruption following severe TBI in the developing gyrencephalic brain.. https://doi.org/10.1101/2025.01.20.633889

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