Fig. 7: Visualization of the glia limitans in Aqp4-mRuby3 mice allows to distinguish CX3CR1-GFP+ BAMs and microglia in vivo.
From: In vivo imaging of the barrier properties of the glia limitans during health and neuroinflammation
a–e Representative images of cranial window preparation in CX3CR1-GFP; Aqp4-mRuby3 mice (n = 7). a XY MIP of the 2P-IVM of the cranial window showing the view from the top. The glia limitans (GL) is visible in red, the microglia and border-associated macrophages (BAMs) in green. b YZ MIP of the 2P-IVM showing lateral view from the field of view (FOV) in (a). The superficial GL separates the top compartment, including collapsed subarachnoid space (SAS) and dura mater, from the bottom compartment corresponding to the brain parenchyma. c–e 2P-IVM images depicting BAMs and microglia in optical sections (2 µm) at the levels shown in (b). c Distinct morphology of BAMs (green) resting on dura mater collagen fibers observed by faint signal from the second harmonic generation (SHG) in green (white arrowheads). d Spatial localization of BAMs (pink arrowheads) in the perivascular region, microglia (white arrowheads) in the parenchyma, and circulating monocytes (light blue arrowheads) within vessel lumens. e Left, distribution of brain microglia in relation to penetrating vessels. Right, the yellow inset shows thin microglial processes contacting the perivascular GL (white arrowheads). f–j Representative images of spinal cord window preparation in CX3CR1-GFP; Aqp4-mRuby3 mice (n = 6). f XY MIP showing an overview of the spinal cord CX3CR1-GFP+ resident myeloid cells (green) in area adjacent to the dorsal vein (DV). g YZ MIP from the FOV in (f) shows the SAS and parenchyma defined by the GL. h–j 2P-IVM images showing the position of BAMs and microglia in the spinal cord in the respective optical sections (2µm) indicated in (g). h Left, distribution of superficial BAMs resting on the spinal cord GL. Right, the yellow inset shows the ameboid shape of BAMs. i Spatial localization of BAMs (pink arrowhead) and microglia (white arrowhead) in the spinal cord. j Few microglia (white arrowhead) are visible in the spinal cord below the GL. Images in this figure were acquired with λex = 1000 nm. SAS subarachnoid space, GL glia limitans, DV dorsal vein, MIP maximum intensity projection.
