Targeted Over-Expression of Glutamate Transporter 1 (GLT-1) Reduces Ischemic Brain Injury in a Rat Model of Stroke

Brandon K Harvey,Jason Hinzman,Mikko Airavaara,Emily M Wires,Matthew J Chiocco,Hui Shen,Yun Wang,Douglas B Howard,Greg Gerhardt,Barry J Hoffer

doi:10.1371/journal.pone.0022135

Abstract

Following the onset of an ischemic brain injury, the excitatory neurotransmitter glutamate is released. The excitotoxic effects of glutamate are a major contributor to the pathogenesis of a stroke. The aim of this study was to examine if overexpression of a glutamate transporter (GLT-1) reduces ischemic brain injury in a rat model of stroke. We generated an adeno-associated viral (AAV) vector expressing the rat GLT-1 cDNA (AAV-GLT1). Functional expression of AAV-GLT1 was confirmed by increased glutamate clearance rate in non-stroke rat brain as measured by in vivo amperometry. AAV-GLT1 was injected into future cortical region of infarction 3 weeks prior to 60 min middle cerebral artery occlusion (MCAo). Tissue damage was assessed at one and two days after MCAo using TUNEL and TTC staining, respectively. Behavioral testing was performed at 2, 8 and 14 days post-stroke. Animals receiving AAV-GLT1, compared to AAV-GFP, showed significant decreases in the duration and magnitude of extracellular glutamate, measured by microdialysis, during the 60 minute MCAo. A significant reduction in brain infarction and DNA fragmentation was observed in the region of AAV-GLT1 injection. Animals that received AAV-GLT1 showed significant improvement in behavioral recovery following stroke compared to the AAV-GFP group. We demonstrate that focal overexpression of the glutamate transporter, GLT-1, significantly reduces ischemia-induced glutamate overflow, decreases cell death and improves behavioral recovery. These data further support the role of glutamate in the pathogenesis of ischemic damage in brain and demonstrate that targeted gene delivery to decrease the ischemia-induced glutamate overflow reduces the cellular and behavioral deficits caused by stroke.

Highlights

An ischemic stroke begins with obstruction of an arterial vessel in the brain, progresses through a cascade of cellular and molecular events, and leads to cell death
The rat cDNA for the glutamate transporter 1 was successfully subcloned into an associated viral (AAV) vector to generate pssAAV-GLT1 as determined by DNA sequencing
Expression of the rat GLT1 protein was confirmed by western blot analysis of protein extracts prepared from HEK293 cells transfected with the pAAV-GLT1 packing plasmid into HEK293 cells (Figure 1A)

Summary

Introduction

An ischemic stroke begins with obstruction of an arterial vessel in the brain, progresses through a cascade of cellular and molecular events, and leads to cell death. The altered ion homeostasis activates voltage-dependent calcium channels and the depolarization of the neuronal membrane can cause massive release of neurotransmitters such as glutamate. Ischemia causes the release of glutamate into the synaptic cleft and extracellular space. Our lab has previously reported that blocking glutamate release or glutamate-mediated post-synaptic excitability reduces neural degeneration in stroke rats [6,7]. Taken together, these data support that regulation of glutamate overflow during the ischemic phase can alter outcomes in stroke animals, clinical trials based on iGluR antagonists have failed with adverse CNS effects and they possibly impede endogenous neurorepair mechanisms [8]

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