Preventing neuronal edema increases network excitability after traumatic brain injury
Punam A. Sawant-Pokam, Tyler J. Vail, Cameron S. Metcalf, Jamie L. Maguire, Thomas O. McKean, Nick O. McKean, K.C. Brennan
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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Research Article Neuroscience

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 4

The CCC inhibitor bumetanide eliminates neuronal swelling and increases neuronal intrinsic and excitatory activity.

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The CCC inhibitor bumetanide eliminates neuronal swelling and increases ...
(A) Images for each group. Scale bar: 20 μm. Quantification of neuronal cross-sectional area showed a reversal of edema with bumetanide (Bum) treatment (P = 0.001, 1-way ANOVA with Bonferroni’s multiple-comparisons test; CCI-bumetanide vs. vehicle [Veh]: ***P < 0.001; CCI-vehicle vs. sham-vehicle: **P < 0.01; n = 54–64 neurons, n = 5–6 mice per group). (B) Consistent with a reversal of edema, membrane capacitance was reduced in CCI-bumetanide neurons compared with CCI-vehicle neurons (P = 0.0009, Bonferroni’s multiple-comparisons test; CCI-bumetanide vs. vehicle: **P = 0.002; CCI vehicle vs. sham-vehicle: **P = 0.001; n = 8–10 neurons, n = 6–8 mice per group).The following membrane properties were measured: (C) input resistance (P = 0.01, Bonferroni’s multiple-comparisons test; CCI-bumetanide vs. vehicle: **P = 0.004; CCI-vehicle vs. sham-vehicle: *P = 0.03; n = 8–10 neurons, n = 6–8 mice per group); (D) plot of the mean firing frequency as a function of current intensity (n = 8–10 neurons, n = 6–8 mice per group); (E) I-V slope (P = 0.01, Bonferroni’s multiple-comparisons test; CCI-bumetanide vs. vehicle: **P = 0.005; CCI-vehicle vs. sham-vehicle: *P = 0.01; n = 8–10 neurons, n = 6–8 mice per group); and (F) rheobase (P = 0.005, Bonferroni’s multiple-comparisons test; CCI-bumetanide vs. vehicle: **P = 0.001; CCI-vehicle vs. sham-vehicle: *P = 0.02; n = 8–10 neurons, n = 6–8 mice per group). These measurements showed increases in intrinsic membrane excitability in CCI-bumetanide neurons compared with CCI-vehicle neurons. (G) Typical recordings of spontaneous excitatory currents. Scale bar: 200 pA, 10 seconds. Traces on the right show expanded currents (i–iv). Scale bar: 200 pA, 500 ms. (H) IEIs were shorter (frequencies were higher) in the CCI-bumetanide–treated group compared with the CCI-vehicle–treated group (P = 0.005, Kruskal-Wallis with Dunn’s multiple-comparisons test; CCI-bumetanide vs. vehicle: **P < 0.01; CCI-vehicle vs. sham-vehicle: P > 0.05; n = 7–9 cells, n = 5–8 mice per group). Box-and-whisker plot on the right shows a larger AUC of excitatory currents in CCI-bumetanide neurons (P = 0.01, Kruskal-Wallis with Dunn’s multiple-comparisons test; CCI-bumetanide vs. vehicle: *P < 0.05; CCI-vehicle vs. sham-vehicle: *P < 0.05; n = 7–9 cells, n = 5–8 mice per group).