Advances in imaging of new targets for pharmacological intervention in stroke: real-time tracking of T-cells in the ischaemic brain

Abstract

Background and purpose: T-cells may play a role in the evolution of ischaemic damage and repair, but the ability to image these cells in the living brain after a stroke has been limited. We aim to extend the technique of real-time in situ brain imaging of T-cells, previously shown in models of immunological diseases, to models of experimental stroke.

Experimental approach: Male C57BL6 mice (6-8 weeks) (n= 3) received a total of 2-5 x 10(6) carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled lymphocytes from donor C57BL6 mice via i.v. injection by adoptive transfer. Twenty-four hours later, recipient mice underwent permanent left distal middle cerebral artery occlusion (MCAO) by electrocoagulation or by sham surgery under isoflurane anaesthesia. Female hCD2-green fluorescent protein (GFP) transgenic mice that exhibit GFP-labelled T-cells underwent MCAO. At 24 or 48 h post-MCAO, a sagittal brain slice (1500 microm thick) containing cortical branches of the occluded middle cerebral artery (MCA) was dissected and used for multiphoton laser scanning microscopy (MPLSM).

Key results: Our results provide direct observations for the first time of dynamic T-cell behaviour in living brain tissue in real time and herein proved the feasibility of MPLSM for ex vivo live imaging of immune response after experimental stroke.

Conclusions and implications: It is hoped that these advances in the imaging of immune cells will provide information that can be harnessed to a therapeutic advantage.

Figure 1

Representative images of the area of multiphoton laser scanning microscopy (MPLSM) imaging (white oval) containing the cortical branches of the occluded MCA (arrow) (A) and of a 1500 µm sagittal slice glued onto a coverslip for MPLSM imaging (B).

Figure 2

(A,B) Three-dimensional reconstruction of lymphocyte infiltration in the cortex post-middle cerebral artery occlusion (MCAO). At 24 h post-MCAO, fluorescently CFSE-labelled lymphocytes (green) were detectable in a cortical artery, and only few cells were infiltrating the parenchyma (A). On the contrary, lymphocyte infiltration in the parenchyma is clearly detectable at 48 h post-MCAO (B). Each imaged volume consisted of 13–21 planes, 2.5 µm apart. (C) Tracking of T-cells in the cortex post-MCAO. At 48 h post-MCAO, endogenous hCD2-GFP T-cells were clearly detectable in the brain with tracking (yellow lines, red circle) shown in six consecutive snapshots representative of 9 min of imaging of the same field of view. Bar = 25 µm. The scans were acquired with 500 lps and between 256 × 256 pixel boxes. Images were analysed using Volocity software (Improvision). The movie of T-cell movement in the ischaemic brain tissue is also available as online supplemental data (movie 1; view using Quick Time or Real Player).