Targeted ischemic stroke induction and mesoscopic imaging assessment of blood flow and ischemic depolarization in awake mice

Abstract

Despite advances in experimental stroke models, confounding factors such as anesthetics used during stroke induction remain. Furthermore, imaging of blood flow during stroke is not routinely done. We take advantage of bihemispheric transcranial windows for longitudinal mesoscopic imaging of cortical function to establish a protocol for focal ischemic stroke induction in target brain regions using photothrombosis in awake head-fixed mice. Our protocol does not require any surgical steps at the time of stroke induction or anesthetics during either head fixation or photoactivation. In addition, we performed laser speckle contrast imaging and wide-field calcium imaging to reveal the effect of cortical spreading ischemic depolarization after stroke in both anesthetized and awake animals over a spatial scale encompassing both hemispheres. With our combined approach, we observed ischemic depolarizing waves (3 to [Formula: see text]) propagating across the cortex 1 to 5 min after stroke induction in genetically encoded calcium indicator mice. Measures of blood flow by laser speckle were correlated with neurological impairment and lesion volume, suggesting a metric for reducing experimental variability. The ability to follow brain dynamics immediately after stroke as well as during recovery may provide a valuable guide to develop activity-dependent therapeutic interventions to be performed shortly after stroke induction.

Genetically Encoded Tools for Research of Cell Signaling and Metabolism under Brain Hypoxia

“…However, to date, there are only several proof-of-principle studies utilizing this approach, and they only employ Ca 2+ sensors of the GCaMP family. Murphy and colleagues have developed a protocol of focal stroke induction in awake head-fixed mice combined with laser speckle contrast imaging blood flow assessment and mesoscopic wide-field Ca 2+ imaging through bihemispheric transcranial windows [391]. Using transgenic GCaMP3 mice, they were able to directly observe the ischemic depolarizing wave within 5 min of stroke induction.…”

Cholinergic upregulation by optogenetic stimulation of nucleus basalis after photothrombotic stroke in forelimb somatosensory cortex improves endpoint and motor but not sensory control of skilled reaching in mice

“…Photothrombotic stroke was induced by injecting 10 mg/ml of Rose Bengal intraperitoneally 5 minutes prior to 20 minutes green laser (532 nm wavelength at power 10 mW) illumination at the thinned region as previously described [38][39][40] . The thinned area was covered with dental cement.…”

Awake chronic mouse model of targeted pial vessel occlusion via photothrombosis

“…Previous studies, albeit under anesthesia, have shown the ability to occlude surface arterioles or individual penetrating arterioles using photothrombosis by controlling the parameters of laser illumination in order to minimize photodamage to adjacent and deeper vessels. [26][27][28] Although photothrombosis has been performed in awake animals 29 and has also been used to target individual vessels, 26 these methods have not been previously combined. Here we combine the benefits of formerly used techniques to create a chronically prepared mouse photothrombosis stroke model that induces targeted ischemic stroke in awake mice while simultaneously monitoring macroscopic changes to CBF.…”