The mouse cortical meninges are the site of immune responses to many different pathogens, and are accessible to intravital imaging

Abstract

A wide range of viral and microbial infections are known to cause meningitis, and there is evidence that the meninges are the gateway to pathogenic invasion of the brain parenchyma. Hence observation of these regions has wide application to understanding host-pathogen interactions. Interactions between pathogens and cells of the immune response can be modified by changes in their environment, such as suppression of the flow of blood and lymph, and, particularly in the case of the meninges, with their unsupported membranes, invasive dissection can alter the tissue architecture. For these reasons, intravital imaging through the unperforated skull is the method of choice. We give a protocol for a simple method of two-photon microscopy through the thinned cortical skull of the anesthetized mouse to enable real-time imaging with sub-micron resolution through the meninges and into the superficial brain parenchyma. In reporter mice in which selected cell types express fluorescent proteins, imaging after infection with fluorescent pathogens (lymphocytic choriomeningitis virus, Trypanosoma brucei or Plasmodium berghei) has shown strong recruitment to the cortical meninges of immune cells, including neutrophils, T cells, and putative dendritic cells and macrophages. Without special labeling, the boundaries between the dura mater, the leptomeninx, and the parenchyma are not directly visualized in intravital two-photon microscopy, but other landmarks and characteristics, which we illustrate, allow the researcher to identify the compartment being imaged. While most infectious meningitides are localized mainly in the dura mater, others involve recruitment of immune cells to the leptomeninx.

Keywords: Dura mater; Immune response; Intravital; Leptomeninx; Multiphoton; Pathogen.

Graphical abstract

Fig. 1

A,B. Imaging several fluorophores. A. An example of an arrangement of dichroic mirrors and filters that was used for imaging second harmonic generation (SHG), EYFP, DsRed and 705 nm quantum dots. The dichroics and filters shown were chosen from those available and are not necessarily optimal. PM4 and PM5 are GaAsP photomultiplier tubes, which have higher sensitivity than the multi-alkali photomultipliers PM1-3 at long wavelengths; for this reason the long wavelength signal was directed to PM5 rather than PM1. B. 3D reconstruction of a four-color Z-stack obtained with the optics in (A). The mouse had been infected with Trypanosoma brucei 27 days earlier. T cells (red) and CD11c+ cells (yellow) are seen close under the skull (blue). Emission from the quantum dots in the blood plasma is shown as green. C. Planes from a Z-stack in a ‘DsRed’ mouse expressing DsRed under control of a β actin promoter and cytomegalovirus enhancer cassette . DsRed is shown as bronze and SHG from the skull as grey. An XY plane, slightly oblique to the plane of the meninges, is shown, including skull (lower right) and leptomeninx (upper left) and also one YZ plane and two XZ planes that cut the XY plane at the white dashed lines. There is labeling of vascular walls and, apparently, of the dural tissue. Arrowheads point to what we suggest is the arachnoid membrane. D. A vertical section showing (in blue) skull by SHG and nuclei labeled by i.v. injection of furamidine, and (in pink) blood plasma labeled with quantum dots. Three faintly visible intraparenchymal vessels are indicated by arrowheads. We suggest that the dashed white line follows the boundary between dura and leptomeninx. Excitation at 824 nm. E. A vertical section 59 min after infusion in the cisterna magna of Texas Red, which labels extracellular channels in the leptomeninx. Blue indicates the skull (by SHG) and nuclei labeled by i.v. injection of furamidine. We suggest that the dashed white line follows the boundary between dura and leptomeninx. Excitation at 810 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

In vivo images of T cells in hCD2⁺ DsRed mouse cranial meninges seen through the thinned skull. A-C show mainly the dura; D-F mainly the leptomeninx. A. Dural blood vessels (magenta), skull bone and collagen (blue, by SHG), dural cell nuclei (blue, labeled by i.v. injection of furamidine), two or three CD2⁺ T cells expressing DsRed. Small red spots are of unknown origin. B. The dura of a mouse infected 11 days earlier by i.p. injection of Trypanosoma brucei. T cells express DsRed, trypanosomes GFP. (A,B from .) C. Tracks of T-cells from a video lasting 21 min. 27 days post infection with T. brucei. The excitation was at 1116 nm (using an OPO beam - see Section 2.1.1). D. Large horizontal pial vessels lie in depressions in the cortical surface then plunge abruptly into the parenchyma where characteristically sinuous horizontal capillaries are seen. Examination of vertical reconstructions shows that the two DsRed T cells are in the leptomeninx. Blood plasma labeled with 705 nm Qdots. E. In a mouse infected 5 days earlier with Plasmodium berghei, T cells are recruited to spaces adjacent to leptomeningeal blood vessels. (From .) F. In a mouse with permanent occlusion of the middle cerebral artery, those T cells (faint GFP expression) that move are seen to remain close to blood leptomeningeal vessels. (From .) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

A system for holding the mouse skull by a ‘skull plate’ (bottom right) which is held by spring-loaded clamps on dovetails. The poly(methyl methacrylate) anesthesia mask is held over the mouse’s snout by the elasticity of the silicon tubes that deliver and extract the isofluorane anesthetic. For superfusion of the thinned skull with buffered saline, delivery and aspiration tubes are held on swivelling poly(methyl methacrylate) holders. The end of the aspiration tube should be cut at a bevel so that it sucks both air and liquid. The heating mat (not shown) is held by tape on the baseplate.

Fig. 4

The arrangement for temperature maintenance, anesthesia and superfusion during thinning of the skull under a dissecting micrscope.

Fig. 5

The mouse under the microscope. The superfusate is heated in an in-line heater. The anesthetic mask and its tubing are shown in the photograph, but not the diagram.