The central nervous system (CNS) barriers ensure homeostasis of the brain and spinal cord and tightly regulate the exchange of immune cells and mediators between the CNS and the periphery. The leptomeninges and the glia limitans cover the surface of the brain and spinal cord and are important barriers regulating immune surveillance at the outer CNS borders. How these cellular layers establish CNS compartments with different accessibility to immune cells and immune mediators is, however, not well understood. We recently found that VE-cadherin in the junctions of the leptomeninges serves as a suitable landmark for in vivo imaging of the subarachnoid space (SAS). Here, we introduce a novel aquaporin-4 (AQP4)-mRuby3 knock-in reporter mouse that due to the polarized expression of AQP4 to astrocyte end-feet allows for visualization of the glia limitans.

We used confocal microscopy to characterize the novel reporter ex vivo and two-photon intravital microscopy in the brain and spinal cord to visualize the glia limitans in vivo and study its barrier properties to both immune cells and soluble molecules, in the context of health and in neuroinflammation models.

We demonstrate AQP4-mRuby3 co-localization with astrocytes and parenchymal basement membranes covering the surface of the parenchyma and the perivascular spaces. Moreover, crossing VE-cadherin-GFP knock-in mice with the AQP4-mRuby3 reporter mice allowed simultaneous in vivo imaging of blood vessels, the leptomeninges and the glia limitans along with their resulting compartments. Crossing AQP4-mRuby3 reporter mice into the CX3CR1-GFP mouse line, we could distinguish GFP+ microglia from GFP+border associated macrophages based on their location respective to the mRuby3+ glia limitans. Following T-cell migration in the brain and spinal cord of AQP4-mRuby3 reporter mice during immune surveillance and neuroinflammation allowed to observe that the glia limitans forms a barrier for T cells. Furthermore, imaging the distribution of fluorescent tracers of varying sizes allowed to analyze the barrier properties of the glia limitans for soluble tracers.

Collectively, our results show that the AQP4-mRuby3 reporter mouse is a valuable tool for in vivo imaging of the barrier properties of the glia limitans and will contribute to improve the understanding of the mechanisms maintaining CNS immune privilege.