AAV Systems and Mouse Models for Investigating Ectopic Expression of Neurod1 in Transduced Cells at Subacute and Chronic Times Post-Ischemic Stroke.

Ectopic expression of neurogenic factors in vivo has emerged as a promising approach for replacing lost neurons in disease models. The use of neural basic helix-loop-helix (bHLH) transcription factors via non-propagating virus-like particle systems, including retrovirus, lentivirus, and adeno-associated virus (AAV), has been extensively reported. For in vivo experiments, AAVs are increasingly used due to their low pathogenicity and potential for translatability. This protocol describes two AAV systems for investigating the ectopic expression of transcription factors in transduced cells post-ischemic stroke. In both systems, Neurod1 expression is controlled by the short GFAP (gfaABC(1)D) promoter, which is upregulated in reactive astrocytes post-stroke as well as in endogenous neurons when combined with neurogenic factor expression. In the ischemic stroke model described, focal ischemia is induced by injecting endothelin-1 (ET-1) into the motor cortex of mice, creating a lesion surrounded by reactive GFAP-expressing astrocytes and surviving neurons. Intracerebral injections of AAV are performed to ectopically induce the expression of Neurod1 in the subacute (7 days) and chronic (21 days) phases post-stroke. Within weeks following AAV injection, a significantly higher number of neurons among transduced cells are identified in mice ectopically expressing Neurod1 compared to mice receiving AAV control viruses. The AAV-based strategies used replicated observed outcomes of increased numbers of neurons expressing the reporter gene in a model of mild-to-moderate cortical stroke. This protocol establishes a standard platform for exploring the effects of ectopic expression of transcription factors delivered with AAV-based systems, contributing to the understanding of neurogenic factor expression in the context of stroke.