J Vis Exp. 2025 Nov 14;(225). doi: 10.3791/69418.
ABSTRACT
Stroke is a leading cause of death and disability across the globe. During ischemic stroke, an obstructed cerebral blood vessel causes focal metabolic failure, neuronal membrane potential breakdown, and dramatic neuronal Ca2+ influx as part of the anoxic depolarization. Without timely reperfusion, prolonged depolarization in the anoxic core is terminal. The dysregulated metabolic environment, aberrant cellular activity, and subsequent tissue infarction associated with ischemic stroke cause both acute and lasting symptoms. This study presents an all-optical method for inducing ischemia and monitoring neuronal Ca2+ in the emerging stroke core of awake and freely behaving mice by delivering and collecting light through a single optical fiber. Here, stereotaxic fiberoptic cannula implantation and sufficient recovery time in advance of experimentation permit flexibility in generating a focal stroke in a brain region of interest, without the need for anesthesia. Mice are then tethered to a fiberoptic patch cord for scalable focal ischemia induction via Rose Bengal photothrombosis. Strokes are performed in transgenic Thy1-GCaMP6f mice, featuring sparse expression of the genetically encoded Ca2+ biosensor GCaMP6f in pyramidal neurons. To record neuronal Ca2+ dynamics in the emerging stroke core, the same optical fiber is utilized for simultaneous GCaMP photometry. This murine stroke model is flexible for any brain region of interest, allowing scalability with laser illumination power and duration, as well as simultaneous behavioral assessment, and is amenable to any green fluorescent biosensor. Together, combining photothrombosis and fiber photometry in behaving mice enables a precise and region-specific investigation of symptomology during acute stroke, in the absence of the neuroprotective effects of anesthesia.
PMID:41325293 | DOI:10.3791/69418