Purinergic Receptor Stimulation Reduces Cytotoxic Edema and Brain Infarcts in Mouse Induced by Photothrombosis by Energizing Glial Mitochondria

Wei Zheng,Christi A Walter,Deborah M Holstein,James D Lechleiter,Lora Talley Watts,Suresh I Prajapati,Eileen H Grass,Charles Keller,David Finkelstein

doi:10.1371/journal.pone.0014401

Wei Zheng, Christi A Walter + Show 7 more

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https://doi.org/10.1371/journal.pone.0014401

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Abstract

Treatments to improve the neurological outcome of edema and cerebral ischemic stroke are severely limited. Here, we present the first in vivo single cell images of cortical mouse astrocytes documenting the impact of single vessel photothrombosis on cytotoxic edema and cerebral infarcts. The volume of astrocytes expressing green fluorescent protein (GFP) increased by over 600% within 3 hours of ischemia. The subsequent growth of cerebral infarcts was easily followed as the loss of GFP fluorescence as astrocytes lysed. Cytotoxic edema and the magnitude of ischemic lesions were significantly reduced by treatment with the purinergic ligand 2-methylthioladenosine 5′ diphosphate (2-MeSADP), an agonist with high specificity for the purinergic receptor type 1 isoform (P2Y1R). At 24 hours, cytotoxic edema in astrocytes was still apparent at the penumbra and preceded the cell lysis that defined the infarct. Delayed 2MeSADP treatment, 24 hours after the initial thrombosis, also significantly reduced cytotoxic edema and the continued growth of the brain infarction. Pharmacological and genetic evidence are presented indicating that 2MeSADP protection is mediated by enhanced astrocyte mitochondrial metabolism via increased inositol trisphosphate (IP3)-dependent Ca2+ release. We suggest that mitochondria play a critical role in astrocyte energy metabolism in the penumbra of ischemic lesions, where low ATP levels are widely accepted to be responsible for cytotoxic edema. Enhancement of this energy source could have similar protective benefits for a wide range of brain injuries.

Highlights

Brain edema and infarctions result from multiple insults, but are typically preceded by cytotoxic edema [1,2,3]
We demonstrated that occlusion of single blood vessels by photothrombosis leads to rapid cytotoxic edema in astrocytes followed by a progressive cell lysis and brain infarction
Our evidence suggested that the mechanism by which 2MeSADP-reduced both cytotoxic edema and brain infarctions was dependent on astrocyte mitochondrial metabolism

Summary

Introduction

Brain edema and infarctions result from multiple insults, but are typically preceded by cytotoxic edema [1,2,3]. Astrocytes are known to play a crucial role in supporting and protecting neuronal function as well as modulating brain energy metabolism [8,9,10,11]. Inhibition of glial mitochondria is known to increase astrocyte swelling and lead to oncotic cell death [15,16]. Taken together, these reports are consistent with the hypothesis that glial mitochondrial metabolism is a key determinant of cytotoxic edema, necrosis and neuronal survival during cerebral ischemic stroke. The metabotropic IP3-mediated intracellular Ca2+ signaling pathway provides a very efficient mechanism to rapidly increase mitochondrial metabolism by activation of Ca2+ sensitive matrix dehydrogenases [17,18]. We found that the resistance of astrocytes and co-cultured neurons to oxidative stress was enhanced in a mitochondrial energy dependent manner [22]

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