{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:01:58Z","timestamp":1785402118557,"version":"3.56.0"},"posted":{"date-parts":[[2025,1,20]]},"group-title":"Neuroscience","reference-count":53,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,1,20]],"date-time":"2025-01-20T00:00:00Z","timestamp":1737331200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["K01HD083759"],"award-info":[{"award-number":["K01HD083759"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01 HD099397"],"award-info":[{"award-number":["R01 HD099397"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01 HD090064"],"award-info":[{"award-number":["R01 HD090064"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["T32NS007473"],"award-info":[{"award-number":["T32NS007473"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["F32NS134588"],"award-info":[{"award-number":["F32NS134588"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["James J. Gilliam Fellowships for Advanced Study"],"award-info":[{"award-number":["James J. Gilliam Fellowships for Advanced Study"]}],"id":[{"id":"https:\/\/ror.org\/006w34k90","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Pappendick Family Translational Research Program"},{"DOI":"10.13039\/100016376","name":"Hydrocephalus Association","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100016376","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,11,3]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  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 \u201cinfant\u201d piglets and more extensive tissue damage in PND30 \u201ctoddler\u201d 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\n                  <jats:italic>Slc12a2<\/jats:italic>\n                  and\n                  <jats:italic>Stk39,<\/jats:italic>\n                  and a decrease in\n                  <jats:italic>Slc12a5<\/jats:italic>\n                  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.\n                <\/jats:p>","DOI":"10.1101\/2025.01.20.633889","type":"posted-content","created":{"date-parts":[[2025,1,21]],"date-time":"2025-01-21T00:36:14Z","timestamp":1737419774000},"source":"Crossref","is-referenced-by-count":1,"title":["Temporal and cell-type specific SPAK-NKCC1 disruption following severe TBI in the developing gyrencephalic brain"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7550-9416","authenticated-orcid":false,"given":"Alexandra","family":"Hochstetler","sequence":"first","affiliation":[{"name":"Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115"},{"name":"Department of Pathology, Boston Children\u2019s Hospital and Harvard Medical School, Boston, MA 02115"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4562-500X","authenticated-orcid":false,"given":"Ya\u2019el","family":"Courtney","sequence":"additional","affiliation":[{"name":"Department of Pathology, Boston Children\u2019s Hospital and Harvard Medical School, Boston, MA 02115"},{"name":"Graduate Program in Neuroscience, Harvard Medical School, Boston, MA 02115"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-3475-7226","authenticated-orcid":false,"given":"Peace","family":"Oloko","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin","family":"Baskin","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew","family":"Ding-Su","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-8764-8733","authenticated-orcid":false,"given":"Tawny","family":"Stinson","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-4035-8061","authenticated-orcid":false,"given":"Declan","family":"McGuone","sequence":"additional","affiliation":[{"name":"Department of Pathology, Yale University School of Medicine, New Haven, CT, 06510"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4909-9585","authenticated-orcid":false,"given":"Robin","family":"Haynes","sequence":"additional","affiliation":[{"name":"Department of Pathology, Boston Children\u2019s Hospital and Harvard Medical School, Boston, MA 02115"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7243-2967","authenticated-orcid":false,"given":"Maria K.","family":"Lehtinen","sequence":"additional","affiliation":[{"name":"Department of Pathology, Boston Children\u2019s Hospital and Harvard Medical School, Boston, MA 02115"},{"name":"Graduate Program in Neuroscience, Harvard Medical School, Boston, MA 02115"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7559-9378","authenticated-orcid":false,"given":"Beth","family":"Costine-Bartell","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041022063027000_2025.01.20.633889v2.1","first-page":"290","article-title":"A population-based study of inflicted traumatic brain injury in young children","volume":"6","year":"2003","journal-title":"JAMA. Aug"},{"issue":"5","key":"2026041022063027000_2025.01.20.633889v2.2","doi-asserted-by":"crossref","first-page":"931","DOI":"10.1016\/j.neuro.2007.01.014","article-title":"Extrapolating brain development from experimental species to humans","volume":"28","year":"2007","journal-title":"Neurotoxicology. Sep"},{"key":"2026041022063027000_2025.01.20.633889v2.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.neubiorev.2007.02.003"},{"key":"2026041022063027000_2025.01.20.633889v2.4","doi-asserted-by":"publisher","DOI":"10.3109\/08941938809141077"},{"key":"2026041022063027000_2025.01.20.633889v2.5","doi-asserted-by":"publisher","DOI":"10.1016\/0378-3782(79)90022-7"},{"key":"2026041022063027000_2025.01.20.633889v2.6","first-page":"36","article-title":"Development of a Model of Hemispheric Hypodensity (\u201dBig Black Brain\u201d)","volume":"1","year":"2019","journal-title":"J Neurotrauma. Mar"},{"key":"2026041022063027000_2025.01.20.633889v2.7","doi-asserted-by":"publisher","DOI":"10.1016\/S0079-6123(06)61020-0"},{"key":"2026041022063027000_2025.01.20.633889v2.8","doi-asserted-by":"crossref","unstructured":"Foster KA , Recker M , Lee P , Bell MJ , Tyler-Kabara E . (2014) Factors Associated with Hemispheric Hypodensity after Subdural Hematoma following Abusive Head Trauma in Children. J Neurotrauma. 2014 Oct 1;31(19):1625-31.","DOI":"10.1089\/neu.2014.3372"},{"issue":"279","key":"2026041022063027000_2025.01.20.633889v2.9","first-page":"187","article-title":"The GABA excitatory\/inhibitory developmental sequence: a personal journey","volume":"24","year":"2014","journal-title":"Neuroscience. Oct"},{"issue":"6","key":"2026041022063027000_2025.01.20.633889v2.10","doi-asserted-by":"crossref","first-page":"1287","DOI":"10.1016\/0896-6273(95)90008-X","article-title":"GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis","volume":"15","year":"1995","journal-title":"Neuron. Dec"},{"key":"2026041022063027000_2025.01.20.633889v2.11","doi-asserted-by":"publisher","DOI":"10.1097\/CCM.0B013E31816590C4"},{"key":"2026041022063027000_2025.01.20.633889v2.12","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.5139-11.2012"},{"issue":"5","key":"2026041022063027000_2025.01.20.633889v2.13","first-page":"276","article-title":"Chloride Dysregulation","volume":"40","year":"2017","journal-title":"Seizures, and Cerebral Edema: A Relationship with Therapeutic Potential. Trends Neurosci. May"},{"key":"2026041022063027000_2025.01.20.633889v2.14","doi-asserted-by":"crossref","unstructured":"Carney N , Totten AM , O\u2019Reilly C , Ullman J , Hawryluck GW , Bell MJ , Bratton SL , Chestnut R , Harriss OA , Kissoon NK , Rubiano AM , Shutter L , Tasker RC , Vavilala MS , Wiberger J , Wright DW , Ghajar J . Guidelines for the Management of Severe Traumatic Brain Injury, 4th Edition. (2016) Brain Trauma Foundation.","DOI":"10.1227\/NEU.0000000000001432"},{"issue":"6","key":"2026041022063027000_2025.01.20.633889v2.15","first-page":"1169","article-title":"Guidelines for the Management of Pediatric Severe Traumatic Brain Injury, Third Edition: Update of the Brain Trauma Foundation Guidelines","volume":"84","year":"2019","journal-title":"Executive Summary. Neurosrugery"},{"key":"2026041022063027000_2025.01.20.633889v2.16","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.21-24-09770.2001"},{"issue":"12","key":"2026041022063027000_2025.01.20.633889v2.17","doi-asserted-by":"crossref","first-page":"4574","DOI":"10.1093\/cercor\/bhv218","article-title":"Developmental Expression Patterns of KCC2 and Functionally Associated Molecules in the Human Brain","volume":"26","year":"2016","journal-title":"Cereb Cortex. Dec"},{"key":"2026041022063027000_2025.01.20.633889v2.18","doi-asserted-by":"publisher","DOI":"10.3390\/cells9010240"},{"key":"2026041022063027000_2025.01.20.633889v2.19","doi-asserted-by":"crossref","first-page":"179","DOI":"10.1016\/j.neubiorev.2021.01.024","article-title":"The postnatal GABA shift: A developmental perspective","volume":"124","year":"2021","journal-title":"Neurosci Biobehav Rev. May"},{"key":"2026041022063027000_2025.01.20.633889v2.20","first-page":"63","article-title":"Evaluating The Precocial-altricial Axis of Motor Skill at Birth in A Preterm Pig Model","volume":"15","year":"2023","journal-title":"Integr Comp Biol. Sep"},{"issue":"3","key":"2026041022063027000_2025.01.20.633889v2.21","first-page":"532","article-title":"Mother-Young Bonding: Neurobiological Aspects and Maternal Biochemical Signaling in Altricial Domesticated Mammals","volume":"13","year":"2023","journal-title":"Animals (Basel)"},{"key":"2026041022063027000_2025.01.20.633889v2.22","doi-asserted-by":"publisher","DOI":"10.1212\/WNL.0000000000208119"},{"issue":"5","key":"2026041022063027000_2025.01.20.633889v2.23","doi-asserted-by":"crossref","first-page":"585","DOI":"10.1007\/s00401-022-02414-7","article-title":"Proteomic differences in hippocampus and cortex of sudden unexplained death in childhood","volume":"143","year":"2022","journal-title":"Acta Neuropathol. May"},{"issue":"3","key":"2026041022063027000_2025.01.20.633889v2.24","doi-asserted-by":"crossref","first-page":"570","DOI":"10.1111\/epi.16833","article-title":"Seizure-related deaths in children: The expanding spectrum","volume":"62","year":"2021","journal-title":"Epilepsia. Mar"},{"issue":"9","key":"2026041022063027000_2025.01.20.633889v2.25","first-page":"762017","article-title":"Medullary Serotonergic Binding Deficits and Hippocampal Abnormalities in Sudden Infant Death Syndrome: One or Two Entities?","volume":"21","year":"2021","journal-title":"Front Pediatr. Dec"},{"key":"2026041022063027000_2025.01.20.633889v2.26","doi-asserted-by":"crossref","first-page":"102862","DOI":"10.1016\/j.autneu.2021.102862","article-title":"Review: Neuropathology findings in autonomic brain regions in SUDEP and future research directions","volume":"235","year":"2021","journal-title":"Auton Neurosci. Nov"},{"key":"2026041022063027000_2025.01.20.633889v2.27","doi-asserted-by":"crossref","first-page":"105334","DOI":"10.1016\/j.nbd.2021.105334","article-title":"A perfect storm: The distribution of tissue damage depends on seizure duration, hemorrhage, and developmental stage in a gyrencephalic, multi-factorial, severe traumatic brain injury model","volume":"154","year":"2021","journal-title":"Neurobiol Dis. Jul"},{"key":"2026041022063027000_2025.01.20.633889v2.28","doi-asserted-by":"publisher","DOI":"10.1159\/000536054"},{"key":"2026041022063027000_2025.01.20.633889v2.29","doi-asserted-by":"publisher","DOI":"10.1093\/cercor\/bhac470"},{"issue":"7","key":"2026041022063027000_2025.01.20.633889v2.30","doi-asserted-by":"crossref","first-page":"1092","DOI":"10.3390\/children9071092","article-title":"Magnetic Resonance Imaging Findings in Infants with Severe Traumatic Brain Injury and Associations with Abusive Head Trauma","volume":"9","author":"behalf of the ADAPT MRI Investigators","year":"2022","journal-title":"Children"},{"key":"2026041022063027000_2025.01.20.633889v2.31","doi-asserted-by":"publisher","DOI":"10.3390\/brainsci12040502"},{"key":"2026041022063027000_2025.01.20.633889v2.32","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-13851-6"},{"issue":"5","key":"2026041022063027000_2025.01.20.633889v2.33","doi-asserted-by":"crossref","first-page":"1720","DOI":"10.1161\/STROKEAHA.121.038351","article-title":"NF-\u03baB Signaling-Mediated Activation of WNK-SPAK-NKCC1 Cascade in Worsened Stroke Outcomes of Ang II-Hypertensive Mice","volume":"53","year":"2022","journal-title":"Stroke. May"},{"key":"2026041022063027000_2025.01.20.633889v2.34","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.4626-11.2012"},{"key":"2026041022063027000_2025.01.20.633889v2.35","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.1234-11.2011"},{"issue":"4","key":"2026041022063027000_2025.01.20.633889v2.36","doi-asserted-by":"crossref","first-page":"368","DOI":"10.1111\/j.1469-7580.2010.01274.x","article-title":"Subplate in the developing cortex of mouse and human","volume":"217","year":"2010","journal-title":"J Anat. Oct"},{"issue":"4","key":"2026041022063027000_2025.01.20.633889v2.37","doi-asserted-by":"crossref","first-page":"663","DOI":"10.1111\/j.1528-1167.2007.00986.x","article-title":"Cation-chloride cotransporters and GABA-ergic innervation in the human epileptic hippocampus","volume":"48","year":"2007","journal-title":"Epilepsia. Apr"},{"issue":"3","key":"2026041022063027000_2025.01.20.633889v2.38","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1016\/j.neurol.2015.01.563","article-title":"Hippocampus and epilepsy: Findings from human tissues","volume":"171","year":"2015","journal-title":"Rev Neurol (Paris). Mar"},{"key":"2026041022063027000_2025.01.20.633889v2.39","doi-asserted-by":"publisher","DOI":"10.1126\/scisignal.aaw9315"},{"issue":"12","key":"2026041022063027000_2025.01.20.633889v2.40","doi-asserted-by":"crossref","first-page":"726","DOI":"10.1016\/j.tins.2013.08.006","article-title":"Modulation of neuronal activity by phosphorylation of the K-Cl cotransporter KCC2","volume":"36","year":"2013","journal-title":"Trends Neurosci. Dec"},{"key":"2026041022063027000_2025.01.20.633889v2.41","doi-asserted-by":"publisher","DOI":"10.1126\/science.1245423"},{"key":"2026041022063027000_2025.01.20.633889v2.42","doi-asserted-by":"crossref","first-page":"747745","DOI":"10.3389\/fneur.2021.747745","article-title":"Efficacy and Safety of Levetiracetam vs. Phenobarbital for Neonatal Seizures: A Systematic Review and Meta-Analysis","volume":"12","year":"2021","journal-title":"Front Neurol"},{"key":"2026041022063027000_2025.01.20.633889v2.43","doi-asserted-by":"crossref","unstructured":"Normoyle KP , Lillis KP , Egawa K , McNally MA , Paulchakrabarti M , Coudhury BP , Lau L , Shiu FH , Staley KJ . (2024) Displacement of extracellular chloride by immobile anionic constituents of the brain\u2019s extracellular matrix. J Physiol. Dec 2.","DOI":"10.1113\/JP285463"},{"key":"2026041022063027000_2025.01.20.633889v2.44","doi-asserted-by":"publisher","DOI":"10.3390\/cells9122607"},{"key":"2026041022063027000_2025.01.20.633889v2.45","first-page":"10738584241282632","article-title":"Neuronal Death: Now You See It","volume":"24","year":"2024","journal-title":"Now You Don\u2019t. Neuroscientist. Sep"},{"issue":"11","key":"2026041022063027000_2025.01.20.633889v2.46","doi-asserted-by":"crossref","first-page":"e2446767","DOI":"10.1001\/jamanetworkopen.2024.46767","article-title":"Pediatric Traumatic Brain Injury and Microvascular Blood-Brain Barrier Pathology","volume":"7","year":"2024","journal-title":"JAMA Netw Open"},{"issue":"4","key":"2026041022063027000_2025.01.20.633889v2.47","doi-asserted-by":"crossref","first-page":"717","DOI":"10.3892\/mmr.2021.12356","article-title":"Inhibition of the p-SPAK\/p-NKCC1 signaling pathway protects the blood-brain barrier and reduces neuronal apoptosis in a rat model of surgical brain injury","volume":"24","year":"2021","journal-title":"Mol Med Rep. Oct"},{"key":"2026041022063027000_2025.01.20.633889v2.48","doi-asserted-by":"crossref","first-page":"113386","DOI":"10.1016\/j.expneurol.2020.113386","article-title":"Activated WNK3 induced by intracerebral hemorrhage deteriorates brain injury may be via WNK3\/SPAK\/NKCC1 pathway","volume":"332","year":"2020","journal-title":"Exp Neurol. Oct"},{"issue":"11","key":"2026041022063027000_2025.01.20.633889v2.49","doi-asserted-by":"crossref","first-page":"1943","DOI":"10.1089\/neu.2009.0956","article-title":"Scaled cortical impact in immature swine: effect of age and gender on lesion volume","volume":"26","year":"2009","journal-title":"J Neurotrauma. Nov"},{"issue":"9","key":"2026041022063027000_2025.01.20.633889v2.50","first-page":"371","article-title":"Development and regulation of chloride homeostasis in the central nervous system","volume":"24","year":"2015","journal-title":"Front Cell Neurosci. Sep"},{"key":"2026041022063027000_2025.01.20.633889v2.51","doi-asserted-by":"publisher","DOI":"10.1002\/ana.25959"},{"key":"2026041022063027000_2025.01.20.633889v2.52","doi-asserted-by":"publisher","DOI":"10.1111\/epi.17278"},{"key":"2026041022063027000_2025.01.20.633889v2.53","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-023-40638-z"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.01.20.633889","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T05:07:59Z","timestamp":1782277679000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.01.20.633889"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1,20]]},"references-count":53,"URL":"https:\/\/doi.org\/10.1101\/2025.01.20.633889","relation":{"is-preprint-of":[{"id-type":"doi","id":"10.1186\/s40478-026-02257-3","asserted-by":"subject"}]},"subject":[],"published":{"date-parts":[[2025,1,20]]},"subtype":"preprint"}}