Role of NKCC1 and KCC2 during hypoxia-induced neuronal swelling in the neonatal neocortex
Abstract number :
1.439
Submission category :
1. Basic Mechanisms / 1D. Mechanisms of Therapeutic Interventions
Year :
2022
Submission ID :
2232925
Source :
www.aesnet.org
Presentation date :
12/3/2022 12:00:00 PM
Published date :
Nov 22, 2022, 05:28 AM
Authors :
Yusuke Takezawa, MD, PhD – The University of Iowa; Rachel Langton, BS – Research assistant, Department of Pediatrics, The University of Iowa; Samuel Baule, - – Department of Biomedical Engineering – The University of Iowa; Miriam Zimmerman, PhD – Department of Biostatistics – The University of Iowa; Stephen Baek, PhD – Associate professor, School of Data Science, University of Virginia; Joseph Glykys, MD, PhD – Associate Professor, Department of Pediatrics, The University of Iowa
This is a Late Breaking abstract
Rationale: Neonatal hypoxic-ischemic encephalopathy (HIE) can cause neonatal seizures acutely and epilepsy later in life. Hypoxia causes cytotoxic neuronal swelling through the entry of water and ions, including Cl-. Since neuronal cytotoxic edema correlates with cell injury and high intracellular Cl- concentration ([Cl-]i) increases seizure susceptibility, preventing these changes during hypoxia could reduce brain injury and seizure incidence in HIE. Importantly, neurons do not have water channels, and their membrane water permeability is low. The cation-chloride cotransporters (CCCs), including KCC2 and NKCC1, move water in different cells, but it is unclear if they do in neonatal neurons. We studied the effect of pharmacological modulation of CCCs on neuronal swelling and intracellular [Cl-]i in the neocortex (layer IV/V) of neonatal mice (post-natal day 9-13) during prolonged and brief hypoxia.
Methods: We used acute brain slices from Clomeleon mice which encode a ratiometric fluorophore sensitive to Cl- and exposed them to oxygen-glucose deprivation (OGD) while imaging neuronal size and [Cl-]i by multiphoton microscopy. We identified neurons using a convolutional neural network algorithm. The changes in neuronal area and [Cl-]i were evaluated with a linear mixed model for repeated measures. CCCs were modulated pharmacologically using CLP257 (KCC2 enhancer), VU0463271 (KCC2 blocker), bumetanide (NKCC1 blocker), or furosemide (broad-spectrum CCCs blocker).
Results: We observed neuronal swelling and Cl- accumulation starting 10 minutes after OGD, which worsened in prolonged OGD or returned to baseline during reperfusion. Neuronal swelling was dependent on [Cl-]o. Compared to when no drug was perfused: (1) enhancing KCC2 did not alter neuronal swelling but prevented Cl- accumulation, (2) blocking KCC2 increased Cl- accumulation during prolonged OGD and aggravated neuronal swelling during reoxygenation, (3) blocking NKCC1 reduced neuronal swelling during early but not after prolonged OGD, while it aggravated Cl- accumulation during prolonged OGD, and (4) furosemide reduced both swelling and Cl- accumulation in prolonged and brief OGD. However, blocking simultaneously NKCC1 and KCC2 with their specific antagonists aggravated neuronal swelling during prolonged OGD.
Conclusions: We conclude that CCCs are involved in water movement in neocortical neurons during OGD in the neonatal period. Blocking NKCC1 decreases neuronal swelling early during OGD. Yet, furosemide is more effective in preventing neuronal swelling than only blocking NKCC1 during prolonged hypoxia, suggesting additional non-CCC water pathways at this age. Thus, bumetanide and furosemide alleviate neuronal swelling and [Cl-]i accumulation during neonatal HIE.
Funding: Post-doctoral research fellowship by the American Epilepsy Society, NIH/NINDS R01NS115800, and The Iowa Neuroscience Institute
Basic Mechanisms