A Long‐Term Clearing Cranial Window for Longitudinal Imaging of Cortical and Calvarial Ischemic Injury through the Intact Skull

Abstract

Skull is a reservoir for supplying immune cells that mediate brain immune surveillance. However, during intravital optical imaging of brain, conventional cranial windows requiring skull thinning or removal disrupt brain immunity integrity. Here, a novel long‐term clearing cranial window (LCCW) based on the intact skull, dedicated to chronic skull transparency maintenance, is proposed. It significantly improves optical imaging resolution and depth, by which the cortical and calvarial vascular injury and regeneration processes after ischemic injury are longitudinally monitored in awake mice. Results show that calvarial blood vessels recover earlier than the cortex. And the transcriptome analysis reveals that gene expression patterns and immune cells abundances exist substantial differences between brain and skull after ischemic injury, which may be one of the causes for the time lag between their vascular recovery. These findings bring great enlightenment to vascular regeneration and reconstruction. Moreover, LCCW provides a minimally invasive approach for imaging the brain and skull bone marrow.

Systematic analysis of brain and skull ischemic injury expression profiles reveals associations of the tumor immune microenvironment and cell death with ischemic stroke

“…In this work, the ischemic injury modeling was consistent with our recent study ( 39 ), and the time points we selected for brain and skull sampling were based on the vascular regeneration patterns reported. Since we needed to compare and analyze the changes in the brain and skull after ischemic injury, we controlled the degree of ischemic injury; thus, the PT-induced ischemic injury model was quite suitable.…”

“…Here, we established the ischemic injury model using PT for the brain and skull and performed whole-transcriptome sequencing, including the injured skull (second and fourth days), the skull with brain injury (second and sixth days), the injured brain (second and sixth days), the brain with skull injury (second and fourth days), and the skull and brain in the control groups ( Figure 1A ). The time points for the sample selection referred to our previous work ( 39 ). In order to verify the effectiveness of model establishment and to evaluate brain recovery after ischemic injury, we evaluated the changes in the blood–brain barrier after ischemic injury from the acute phase (second day) to the chronic phase (sixth day) based on the area of dye leakage ( Figure 1B ).…”

“…The results showed that the disruption of the blood–brain barrier was severe on the second day after PT, but was greatly restored on the sixth day after PT. Moreover, we combined the transcriptome data of the untreated brain and skull as control groups from our previous work ( 39 ) (GSE191183). We performed removal of batch effects for the transcriptome dataset before further analysis, as shown in Supplementary Figure S1 .…”

Dissecting calvarial bones and sutures at single‐cell resolution

“…In addition, both Gli1 + and Osx/Sp7 + progenitors were preferentially associated with CD31 hi Emcn hi blood vessels during the bone healing process. Most recently, a novel long‐term clearing cranial window (LCCW) has been developed based on the intact calvarial bones (Zhang, Liu & Feng, 2022). It enables the non‐invasive monitoring of cortical and calvarial dynamics through the intact skull without disrupting the CNS homeostatic microenvironment.…”

“…Therefore, the LCCW method dramatically improves imaging depth and resolution, enabling precise and longitudinal observations of the calvarial and cortical blood vessels and even of a variety of cells, such as neurons, microglia, and monocytes, in fully conscious mice with better animal welfare. The LCCW method has provided information on the structure of bone marrow cavities in parietal and interparietal bone, showing that the latter contains more abundant monocytes and vessels (Zhang et al ., 2022). This unique immune microenvironment might contribute to the rapid regeneration of blood vessels after cerebral photothrombotic ischemic injury.…”