The Cellular Mechanisms of Neuronal Swelling Underlying Cytotoxic Edema

Authors: R.L. Rungta, H.B. Choi, J.R. Tyson, A. Malik, L. Dissing-Olesen, P.J.C. Lin, S.M. Cain, P.R. Cullis, T.P. Snutch and B.A. MacVicar

Journal: Cell

DOI: 10.14288/1.0167572

Publication - Summary

April 23, 2015


In this paper, the MacVicar Lab used Neuro9 siRNA cell transfection nanoparticles (Neuro9-siRNA) prepared using the NanoAssemblrTMBenchtop instrument to uncover the role of a specific chloride channel in cytotoxic edema, which is a major cause of death in traumatic and ischemic brain injury.  The study’s findings suggest a possible target for treatment of brain injury.  Previously published work had implicated Na+ and Cl- influx in neurons as a cause for neuronal swelling. However, with numerous ion channels at work in neurons, the precise molecular pathway was unknown. Understanding this pathway would allow targeted treatments for injuries involving cytotoxic edema to be developed. The MacVicar Lab tested pharmaceutical ion channel inhibitors and observed their effects on live cells using brain slice imaging to confirm a chloride channel was necessary for swelling. These data were combined with gene expression (qRT-PCR) data to create a short list of 4 chloride channels that might be involved. To identify the role of each of these channels, functional studies were required to observe the effect of inhibiting each channel independently. RNA interference is the only mechanism of inhibition with the degree of specificity necessary to test each ion channel independently, which requires transfecting neurons with short interfering RNA (siRNA) in vitro and in vivo. Neurons, however, are difficult to transfect because most transfection methods are toxic to neurons. Compounding the difficulty, a different transfection method would be required to perform the experiment in vivo.

Neuro9 transfection is unique among gene transfection methods, exhibiting no observable toxicity in primary neurons and proven to be effective in vitro and in vivo applications. The MacVicar lab used Neuro9-siRNA to validate siRNA sequences against the four Cl- channels in vitro. Neuro9 nanoparticles containing the validated sequences were then administered to rats by direct cortical injection. Brain slices were harvested 5-6 days after Neuro9 treatment and swelling measured by microscopy. SLC26A11 was the only candidate where significant reduction in swelling was observed following knockdown.  They verified protein knockdown by western blot. Additionally, Neuro9-siRNA was used in conjunction with whole cell voltage clamp electrophysiology to verify the phenotypic response to knockdown. These findings suggest that inhibition of SLC26A11 dependent Cl- influx can be exploited for treating traumatic and ischemic brain injury.


Cytotoxic brain edema is the principal cause of mortality following brain trauma and cerebral infarct yet the mechanisms underlying neuronal swelling are poorly understood. This thesis aims at identifying cellular mechanisms of neuronal swelling that cause cytotoxic edema (chapter 3) and describes a novel method for highly efficient neuronal transfection using lipid nanoparticle delivery of siRNA in vitro and in vivo (chapter 2). In chapter 2, we demonstrate that neurons accumulate lipid nanoparticles in an apolipoprotein E dependent fashion, resulting in very efficient uptake in cell culture (100%) with little apparent toxicity. In vivo, lipid nanoparticle delivery of siRNA resulted in knockdown of target genes in either discrete regions around the injection site following intracortical injections or in more widespread areas following intracerebroventricular injections with no apparent toxicity or immune reactions from the lipid nanoparticles. Effective targeted knockdown was demonstrated by showing that lipid nanoparticle delivery of siRNA against GRIN1 (encoding GluN1 subunit of the NMDA receptor) selectively reduced synaptic NMDA receptor currents in vivo as compared to synaptic AMPA receptor currents. Therefore, lipid nanoparticle delivery of siRNA rapidly manipulates expression of proteins involved in neuronal processes in vivo, possibly enabling development of gene therapies for neurological disorders. In chapter 3, we show that increasing intracellular sodium concentration ([Na⁺]i) by either activating voltage-gated sodium channels or NMDA receptors triggers a secondary Cl- influx that leads to neuronal swelling and death. Cl- but not Ca²⁺ entry was required for neuronal swelling and cell death. Pharmacological analyses indicated that a DIDS-sensitive HCO₃-/C1- exchanger was responsible for the majority of the Cl- influx. We used lipid nanoparticle-siRNA mediated knockdown (described in chapter 2) to determine the molecular identity of the Cl- influx pathway. Neuronal swelling was attenuated in brain slices by siRNA-mediated knockdown of the Cl-, SO₄²-, HCO₃- exchanger, SLC26A11, but not by knockdown of other HCO₃-/Cl- exchangers examined. We conclude that cytotoxic brain edema can occur when sufficient Na⁺ entry into neurons results in Cl- entry via SLC26A11 to trigger subsequent neuronal swelling.

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