Near-infrared fluorescence imaging of inflammation in stroke

Brain inflammation is a hallmark of cerebral ischemia, where it has been implicated in tissue damage as well as in repair. Imaging technologies that specifically visualize these processes are highly desirable. In this PhD thesis, the feasibility of planar near-infrared fluorescence (NIRF) imaging to visualize inflammation in a mouse model of focal cerebral ischemia (middle cerebral artery occlusion, MCAO) was explored. First, the feasibility of NIRF to visualize fluorochromes in the brain of mice was evaluated using an in-vivo phantom. Secondly, the use of NIRF-labeled bovine serum albumin (NIRF-BSA) for the detection of blood-brain barrier (BBB) permeability after MCAO was tested. Thirdly, it was investigated whether the inflammatory receptor CD40 can be specifically visualized in mice after MCAO using a fluorescent monoclonal antibody. For preparing the in-vivo phantom, capsules containing different amounts of Cy5.5-dextran were implanted into the brain of mice and fluorescence reflectance imaging (FRI) and transillumination fluorescence imaging (TFI) were conducted. The detection limit of FRI and TFI was found to be 10 mol. TFI yielded significantly higher target-to-background ratios (TBR) than FRI at 10 mol (p<0.05). When background fluorescence is increased by intravenous injection of Cy5.5-dextran, the capsule is no longer detectable when the blood concentration reaches 0.4 x 10 mol/l. For NIRF imaging of BBB permeability, mice were subjected to MCAO and NIRF-BSA was injected together with Evans blue (EB) at 4, 8 or 12 h after reperfusion. FRI was performed 4 h after compound injection. Higher fluorescence intensities over the ischemic hemisphere compared with the contralateral side were detected in mice at 8 and 16 h after reperfusion. No differences between hemispheres were seen in mice at 12 h after reperfusion. A good correspondence between the detection of high fluorescence intensities over the ischemic hemisphere and EB extravasation detected in brain slices was observed. For NIRF imaging of the CD40 receptor, wild type and CD40-deficient mice were subjected to MCAO and were either intravenously injected with Cy5.5-CD40MAb or control Cy5.5IgGMAb 80 h after reperfusion. FRI was performed 16 h after injection of the compounds. Wild type MCAO mice that received Cy5.5-CD40MAb showed significantly higher TBR compared to CD40-deficient MCAO mice injected with Cy5.5-CD40MAb and wild type mice injected with Cy5.5-IgGMAb (p<0.001). Confocal microscopy demonstrated vascular and parenchymal cellular distribution of the injected Cy5.5-CD40MAb in the ischemic region, with partial co-localization with activated microglia and blood-derived cells. It was demonstrated that planar NIRF imaging can non-invasively detect fluorochromes in the brain of mice with high sensitivity. Using the technique, it was demonstrated that BBB permeability and the inflammatory receptor CD40 receptor can be specifically detected after cerebral ischemia.