Open Collections

Cellular mechanisms of neuronal swelling underlying cytotoxic edema

University of British Columbia

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.

Text

2015-06-30

Attribution-NonCommercial-NoDerivs 2.5 Canada

http://hdl.handle.net/2429/48501

Neuroscience

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/2.5/ca/