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The ubiquitous volume-regulated anion channels (VRACs), which are composed of LRRC8 proteins, facilitate cell volume homeostasis, and contribute to many other physiological processes. Prior studies demonstrated that treatment with non-specific VRAC blockers, or brain-specific deletion of the essential VRAC subunit LRRC8A, are highly protective in rodent stroke. In this work, we tested the widely accepted idea that harmful effects of VRACs in the brain are mediated by pathological release of the excitatory transmitter glutamate. We used two molecular genetic strategies to ablate LRRC8A expression in either brain astrocytes only (inducible deletion ofLrrc8aflox/floxwithAldh1l1CreERT2) or the majority of brain cells (neurons, astrocytes, and oligodendrocytes withNestinCre). To produce stroke, genetically modified mice were subjected to a 40-minute occlusion of the middle cerebral artery. The inducible deletion of astrocytic LRRC8A yielded no histological or behavioral protection. In contrast, the brain-wide LRRC8A knockout reduced ischemic infarction by ~50% in both heterozygotes (Het) and the fullLrrc8aknockout (KO) as compared to the controlLrrc8aflox/+genotype. However, despite identical brain damage, Het and KO mice dramatically differed in their VRAC activities. Het mice had full swelling-activated glutamate release, while KO animals showed its virtual absence. These new findings refute the notion that VRAC-mediated glutamate release plays significant role in ischemic brain injury.

This is a pre-registration of research design for the subproject exploring if tamoxifen is neuroprotective in a mouse MCAO model of stroke

In this arm of our study, we will test the HYPOTHESIS that inducible elimination of the LRRC8A subunit of VRAC in astrocytes protects mouse brain against ischemic damage caused by transient occlusion of the middle cerebral artery.

This registration describes experimental design for one ARM of the study exploring the role of volume regulated anion channel (VRAC) in a mouse model of stroke. In this arm we will test the neuroprotective effects of the brain-wide conditional knockout of Lrrc8a (Nestin-Cre-driven deletion)

This is a revised version of the previously registered manuscript https://osf.io/wcd2n