Comparison of intravital thinned skull and cranial window approaches to study CNS immunobiology in the mouse cortex

Abstract

Fluorescent imaging coupled with high-resolution femto-second pulsed infrared lasers allows for interrogation of cellular interactions deeper in living tissues than ever imagined. Intra-vital imaging of the central nervous system (CNS) has provided insights into neuronal development, synaptic transmission, and even immune interactions. In this review we will discuss the two most common intravital approaches for studying the cerebral cortex in the live mouse brain for pre-clinical studies, the thinned skull and cranial window techniques, and focus on the advantages and drawbacks of each approach. In addition, we will discuss the use of neuronal physiologic parameters as determinants of successful surgical and imaging preparation.

Keywords: CNS imaging; Thinned skull; cranial window; intravital microscopy; microglia; two-photon.

Figure 1. Comparison of microglia morphology using thinned skull and cranial window approaches. 2P-LSM was used to capture images within the CNS of CX3CR1^+/GFP mice. The cranial bone was thinned (TS) to either 5 µm (A-C) or 20 µm (D-F) and imaged immediately using a Leica SP5 fitted with a DM6000 stage and a 16W Ti/Sapphire IR laser (Chameleon, Coherent) through a 20x water immersion objective (NA: 1.0). Alternatively, cranial windows (CW) were implanted on adult (G-I; M-O) and day 10 neonatal mice (J-L). Adult animals were given 7 d and neonates were given 4 d to rest after CW implantation prior to imaging. (M-O) Mice underwent EAE induction as previously described. All images were collected at a resolution of 1024x1024 between 2–5 µm z-intervals. Vessels were highlighted with 150 kD TRITC dextran. The fluorescent imaging data were analyzed using Imaris (Bitplane, Inc.). (A,D,G,J) The xy axis demonstrates the normal, uniform distribution of microglia throughout the parenchyma. Scale bar = 40 μm. (M) Accumulation of microglia on day 5 after EAE induction. Note the difference of detectable microglia projections between preparations. Scale bar = 40 μm. (B,E,H,K,N) Same images as above in the xz dimension, demonstrating the imaging depth for each preparation. Scale bar = 40 μm. (B,E) Blue second harmonic signals show the intact bone. (C,F,I,L,O) Zoomed-in view of the cell body and projections of individual microglia. Scale bar = 15 μm. (C,F,I,L) In the homeostatic CNS, microglia projections emanate in all directions. The size of microglia cell body range from a maximum diameter of 7.5–15 µm. (O) In EAE, microglia shorten their projections and assume an amoeboid shape with an increased cell body size.