A method for longitudinal, transcranial imaging of blood flow and remodeling of the cerebral vasculature in postnatal mice

Abstract

In the weeks following birth, both the brain and the vascular network that supplies it undergo dramatic alteration. While studies of the postnatal evolution of the pial vasculature and blood flow through its vessels have been previously done histologically or acutely, here we describe a neonatal reinforced thin‐skull preparation for longitudinally imaging the development of the pial vasculature in mice using two‐photon laser scanning microscopy. Starting with mice as young as postnatal day 2 (P2), we are able to chronically image cortical areas >1 mm2, repeatedly for several consecutive days, allowing us to observe the remodeling of the pial arterial and venous networks. We used this method to measure blood velocity in individual vessels over multiple days, and show that blood flow through individual pial venules was correlated with subsequent diameter changes. This preparation allows the longitudinal imaging of the developing mammalian cerebral vascular network and its physiology.

Postnatal development of cerebrovascular structure and the neurogliovascular unit

“…Intussusceptive pillars may then further widen to change the structure of the two daughter vessels. Imaging data from brain suggest the presence of similar events occur during pial venule development (Letourneur et al, ), evident as the formation of small black “holes” in a vascular tube (Figure a,b), similar to that noted by imaging studies of Djonov and colleagues in the chicken CAM (Djonov, Galli, & Burri, ), and more recently in the zebrafish caudal vein plexus (Karthik et al, ). However, rather than increase vascular network complexity, this intussusceptive process results in pruning of the initially amorphous venous plexus into a hierarchical tree‐like structure for effective blood drainage.…”

“…In contrast to arterioles, the pial venular network begins as a dense vascular plexus covering most of the cortical surface at birth (Figures and a; Fehér, Schulte, Weigle, Kampine, & Hudetz, ; Letourneur et al, ; Wang et al, ). This plexus is composed of small diameter, short distance loops that have been referred to as a superficial “capillary‐like” network, although they appear distinct from true parenchymal capillaries (Wang et al, ).…”

Time to wake up: Studying neurovascular coupling and brain-wide circuit function in the un-anesthetized animal

“…Two-photon microscopy is sensitive to animal movement, but movement during voluntary locomotion are typically small enough to be corrected using motion correction software (Dombeck et al, 2007; Gao and Drew, 2016). Head-fixation and optical imaging can be longitudinally done on very young animals, starting as early at postnatal day 3 (P3) (Letourneur et al, 2014). Using two-photon microscopy, we are able to image locomotion-induced vasodilation in the somatosensory cortex (Fig.…”

Title: Time to wake up: Studying neurovascular coupling and brain-wide circuit function in the un-anesthetized animal

“…Two-photon microscopy is sensitive to animal movement, but movement during voluntary locomotion are typically small enough to be corrected using motion correction software (Dombeck et al, 2007;Gao and Drew, 2016). Head-fixation and optical imaging can be longitudinally done on very young animals, starting as early at postnatal day 3 (P3) (Letourneur et al, 2014).…”