Chapter 8 - Intravital Imaging of Autoreactive T Cells in Living Animals

Abstract

Developing technologies now allow observing cellular motility and functions in the living animal. Here, we introduce the method of intravital imaging by using two-photon microscopy. To acquire images with good quality, a highly stablilized tissue is required. Additionally, physiological parameters of the animal need to be controlled during the entire period of intravital imaging. We image rat autoreactive T cells within the spinal cord leptomeninges. Those autoantigen specific T cells were labeled in vitro by using fluorescent protein coding retroviral vectors. Adoptively transferred T cells migrate into the spinal cord with highly reproducible time kinetic. Intravital imaging was performed within the deeply anesthetized animals. Although two-photon microscopy is a powerful tool, the penetration depth in certain tissues, like the spinal cord parenchyma, is still limited. To overcome this issue, imaging of explanted acute spinal cord slices was performed under nearly physiological conditions. Results obtained from intravital imaging will strengthen the "snap shots" analysis such as FACS and quantitative PCR, and can provide new insight into cellular mechanisms in vivo.