Preventing neuronal edema increases network excitability after traumatic brain injury
Punam A. Sawant-Pokam, … , Nick O. McKean, K.C. Brennan
Punam A. Sawant-Pokam, … , Nick O. McKean, K.C. Brennan
Published October 12, 2020
Citation Information: J Clin Invest. 2020;130(11):6005-6020. https://doi.org/10.1172/JCI134793.
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Preventing neuronal edema increases network excitability after traumatic brain injury

Abstract

Edema is an important target for clinical intervention after traumatic brain injury (TBI). We used in vivo cellular resolution imaging and electrophysiological recording to examine the ionic mechanisms underlying neuronal edema and their effects on neuronal and network excitability after controlled cortical impact (CCI) in mice. Unexpectedly, we found that neuronal edema 48 hours after CCI was associated with reduced cellular and network excitability, concurrent with an increase in the expression ratio of the cation-chloride cotransporters (CCCs) NKCC1 and KCC2. Treatment with the CCC blocker bumetanide prevented neuronal swelling via a reversal in the NKCC1/KCC2 expression ratio, identifying altered chloride flux as the mechanism of neuronal edema. Importantly, bumetanide treatment was associated with increased neuronal and network excitability after injury, including increased susceptibility to spreading depolarizations (SDs) and seizures, known agents of clinical worsening after TBI. Treatment with mannitol, a first-line edema treatment in clinical practice, was also associated with increased susceptibility to SDs and seizures after CCI, showing that neuronal volume reduction, regardless of mechanism, was associated with an excitability increase. Finally, we observed an increase in excitability when neuronal edema normalized by 1 week after CCI. We conclude that neuronal swelling may exert protective effects against damaging excitability in the aftermath of TBI and that treatment of edema has the potential to reverse these effects.

Authors

Punam A. Sawant-Pokam, Tyler J. Vail, Cameron S. Metcalf, Jamie L. Maguire, Thomas O. McKean, Nick O. McKean, K.C. Brennan

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Figure 1

Neuronal volume measurements and CCC expression in the perilesional area 2 hours, 48 hours, and 1 week after injury.

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Neuronal volume measurements and CCC expression in the perilesional area...
(A) Schematic showing a cranial window preparation with the CCI location (red) as well as the location for whole-cell and 2-photon recordings (red asterisk). Images show a cortical cavity at the injury site (red circles), but no overt injury to underlying structures was observed. Scale bars: 500 μm and 100 μm (enlarged inset shown in the third image). Tissue was stained with FJB, which labeled degenerating neurons after injury (red squares; n = 5 mice per group). (B) Plot quantifying recording sites in relation to the injury sites and FJB-positive staining. Neuronal degeneration on FJB staining extended approximately 1.5 mm from the lesion center; whole-cell and 2-photon recordings were located just outside this region in a “penumbral” area with an intact cortical structure and no overt neuronal injury (n = 4–5 mice per group). (C) Schematic of the experimental design for 2-photon excitation imaging (2P) after CCI. (D) 2-Photon microscopic images of representative neurons in sham- and CCI-treated Thy1-GCaMP6s mice showing morphological features of pyramidal neurons in layer 2/3 cortex. Scale bar: 20 μm. Plot shows that CCI-treated mice had significant increases in neuronal cross-sectional area 48 hours after CCI (**P = 0.006, 2-sided, unpaired t test; n = 68–75 neurons, n = 4–5 mice per group). (E) Plot shows no changes in neuronal cross-sectional area between sham-treated and CCI-treated Thy1-GCaMP6s mice at 1 week (P > 0.05, 2-sided, unpaired t test; n = 37–67 neurons, n = 4 mice per group). (F–H) Western blotting for NKCC1 and KCC2 two hours, 48 hours, and 1 week after CCI or sham treatment, respectively. Optical density (OD) analysis showed no change in NKCC1 but significant decreases in KCC2 expression at those time points. Plots showing OD (F) 2 hours after CCI or sham treatment (NKCC1, P > 0.05, KCC2, **P = 0.004; 2-sided Mann-Whitney U test; n = 5–6 mice per group); (G) 48 hours after CCI or sham treatment (NKCC1, P > 0.05, KCC2, **P = 0.004; 2-sided Mann-Whitney U test; n = 6 mice per group); and (H) 1 week after CCI or sham treatment (NKCC1, P > 0.05, KCC2, *P = 0.04; 2-sided Mann-Whitney U test; n = 6 mice per group). All lanes within the blots were run on the same gels.