Quantitative 3D Imaging of the Cranial Microvascular Environment at Single-Cell Resolution

Abstract

Vascularization is critical for skull development, maintenance, and healing. Yet, there remains a significant knowledge gap in the relationship of blood vessels to cranial skeletal progenitors during these processes. Here, we introduce a quantitative 3D imaging platform to enable visualization and analysis of high-resolution datasets (>100 GB) throughout the entire murine calvarium. Using this technique, we provide single-cell resolution 3D maps of vessel phenotypes and skeletal progenitors in the frontoparietal cranial bones. Through these high-resolution datasets, we demonstrate that CD31hiEmcnhi vessels are spatially correlated with both Osterix+ and Gli1+ skeletal progenitors during postnatal growth, healing, and stimulated remodeling, and are concentrated at transcortical canals and osteogenic fronts. Interestingly, this relationship was weakened in mice with a conditional knockout of PDGF-BB in TRAP+ osteoclasts, suggesting a potential role for osteoclasts in maintaining the native cranial microvascular environment. Our findings provide a foundational framework for understanding how blood vessels and skeletal progenitors spatially interact in cranial bone, and will enable more targeted studies into the mechanisms of skull disease pathologies and treatments. Additionally, our technique can be readily adapted to study numerous cell types and investigate other elusive phenomena in cranial bone biology.

Immune dynamics in the CNS and its barriers during homeostasis and disease*

“…These bone marrow niches maintain populations of immune cells in the meninges during homeostasis and allow for rapid recruitment in response to injury or autoimmunity. [29][30][31][32] Microscopy enables visualization of immune traffic through specialized ossified vascular channels. 29 These specialized bone marrow niches can imprint developing cells with distinct properties and play a role in tolerance to CNS antigens.…”

“…However, recent studies have shown that the skull and vertebral bodies are not simply structural barriers that protect against mechanical injuries; they contain specialized pockets of bone marrow that are connected to dural sinuses and meningeal veins (Figure 1). These bone marrow niches maintain populations of immune cells in the meninges during homeostasis and allow for rapid recruitment in response to injury or autoimmunity 29‐32 . Microscopy enables visualization of immune traffic through specialized ossified vascular channels 29 .…”

3D morphometric analysis of mouse skulls using microcomputed tomography and computer vision

“…When using intact skull methods in older animals, the gradual thickening of the skull can also result in lower quality imaging 20,21 . As a final note, there has been increased attention paid to skull microvasculature environment 22 and the interaction with the meninges. Our results demonstrating increased skull thickness with age indicate possible age-related effects on these interactions.…”

Label-free cleared tissue microscopy and machine learning for 3D histopathology of biomaterial implants

“…Tissues fixed with Bouin's solution have remnant yellow coloration that enhances the autofluorescence signal. However, Rindone et al have demonstrated adequate bone tissue clearing transparency without decalcification (8).…”