Reinforced thinned-skull window for repeated imaging of the neonatal mouse brain

Abstract

. Significance: Two-photon microscopy is a powerful tool for in vivo imaging of the mammalian brain at cellular to subcellular resolution. However, resources that describe methods for imaging live newborn mice have remained sparse. Aim: We describe a non-invasive cranial window procedure for longitudinal imaging of neonatal mice. Approach: We demonstrate construction of the cranial window by iterative shaving of the calvarium of P0 to P12 mouse pups. We use the edge of a syringe needle and scalpel blades to thin the bone to thickness. The window is then reinforced with cyanoacrylate glue and a coverslip to promote stability and optical access for at least a week. The head cap also includes a light-weight aluminum flange for head-fixation during imaging. Results: The resulting chronic thinned-skull window enables in vivo imaging to a typical cortical depth of without disruption of the intracranial environment. We highlight techniques to measure vascular structure and blood flow during development, including use of intravenous tracers and transgenic mice to label the blood plasma and vascular cell types, respectively. Conclusions: This protocol enables direct visualization of the developing neurogliovascular unit in the live neonatal brain during both normal and pathological states.

Virally induced CRISPR/Cas9-based knock-in of fluorescent albumin allows long-term visualization of cerebral circulation in infant and adult mice

“…We showed how transcranial two-photon imaging following injection of CRISPR/Cas9 AAVs in PN3 mice enables assessment of vasculature structure, blood flow and vasomotion from PN10. Thinned skull or cranial windows adapted for chronic imaging [41,42] and/or injection of the same AAV dosage at earlier time points would offer the chance to image neonate mice even before PN10.…”

Meningeal origins and dynamics of perivascular fibroblast development on the mouse cerebral vasculature

“…For lineage tracing experiments, P1 mice were injected into the milk sac with 50 µL of 1 mg/mL ( Ai14-flox ) or 1.5 mg/mL ( Brainbow-flox ) of tamoxifen (Sigma, Cat#: T5648) in corn oil (Sigma, Cat#: C8267) to achieve Cre-activated recombination. For two-photon imaging studies on P8 Col1a1-GFP pups, we followed our previous methods that are comprehensively described in Coelho-Santos et al 2021 and Coelho-Santos et al 2022 (Coelho-Santos et al, 2021a, 2022). For EdU-based proliferation assays, we intraperitoneally injected 2.5 mg/mL EdU (Thermo Fisher, Cat#: NC1495577) into pregnant mice for embryonic collections (150 µL) or postnatal mice (50 µL) 2 hours prior to collection.…”

Does Perinatal Intermittent Hypoxia Affect Cerebrovascular Network Development?

“…However, the response of neurovascular cell types in white matter remains poorly characterized. By leveraging the growing array of mouse models with fluorescent labeling of cell types within the NVU [[103], [108]], it will be possible to examine these questions both in histology and with in vivo imaging.…”

“…In vivo physiological studies are also needed to understand the quality of blood perfusion through vascular networks, both in the state of heightened capillary density and in "normalized" networks following reoxygenation. This is where in vivo high-resolution imaging of vascular networks in mouse pups can shed light on blood flow [103][104][105]. The flow of blood cells can be measured at the single capillary level and neurovascular coupling responses to external sensory stimuli can be assessed.…”