Molecular Imaging of Stroke

Currently, we are working on three major projects: the development of new optical imaging devices for small animal imaging, the improvement of small animal SPECT imaging, and the non-invasive visualisation of physiology and pathophysiology in small animals with, a focus on stroke using a combination of our available imaging modalities.

A depiction of our self-made planar near-infrared fluorescence (NIRF) imaging system for non-invasive small animal imaging including the anatomical location of capsules implanted into the brain of live mice containing different amounts of NIRF dyes, and corresponding non-invasive NIRF images of the heads of the mice. The capsules (ID = Inner Diameter, OD = Outer Diameter, L = Length, V = Volume) were implanted 3-4 mm into the left hemisphere 2 ± 0.5 mm caudal from bregma (A, B). The fluorescence reflectance imaging (FRI) system () consisted of two light sources that illuminate the head of the mouse from above and a CCD camera to collect the fluorescence light, both located on the same side of the animal. The transillumination fluorescence imaging (TFI) system () consisted of one light source below the head of the mouse and a CCD camera located on the opposite side of the animal (B). Sagital (C) and axial (D) MR images of each mouse were recorded to show the exact anatomical position of the capsules. Color-coded NIRF images of the heads of mice (E) implanted with capsules containing different amounts of NIRF dye (10-13, 10-12, and 10-11 mol Cy5.5) are shown below. From each mouse, a fluorescence reflectance image (FRI, E1-E3) and a transillumination fluorescence image (TFI, E4-E6) were recorded. Examples of the regions of interest (ROIs) drawn are delineated in E2. Capsules containing 10-13 mol could not be detected. In contrast, capsules containing 10-12 and 10-11 mol NIRF dye were clearly visible. Figure adapted from (1).

A coronal slice of the tomographically reconstructed fluorescence from a capsule containing a near-infrared fluorescent dye implanted into the brain of a live mouse overlaid on the corresponding MR slice. The study shows that tomographic reconstruction of the distribution of an injected fluorochrome within the brain of live mice, is feasible. Figure adapted from (2).

Non-invasive NIRF images of the head of a live mouse after experimentally induced cerebral ischemia in the left hemisphere 2 h after i.v.-injection of NIRF-labeled albumin, a marker for blood-brain barrier impairment. Images after illumination with light of 670 or 633 nm, the scalped image, and the extracted brain are shown. When illuminated with 670 nm and 633 nm, the NIRF images are dominated by the signal from the confluens sinuum. Only the scalped image reveals the accumulation of fluorescent albumin in the area of infarction, which can be clearly seen on the fluorescence image of the extracted brain as well. Figure adapted from (3).

SPECT/CT image of a mouse 1 h after i.v.-injection of a long-circulating marker labeled with 111In. The heart, bigger vessels and organs with a good blood supply show high amounts of radioactivity.

Non-invasive near-infrared fluorescence (NIRF) imaging of stroke-induced blood-brain barrier (BBB) impairment in a mouse model of cerebral ischemia using fluorescently labeled serum albumin as a marker. Mice were subjected to transient middle cerebral artery occlusion (MCAO) and intravenously injected with NIRF-labeled albumin and Evans blue (EB) at different time points (4, 8, 12 hours) after MCAO. NIRF imaging was performed 4 hours post injection. Target-to-background ratios (TBR) from non-invasive NIRF images were calculated by dividing the mean fluorescence intensities measured in regions of interest (ROIs) selected over the left and the right hemisphere. Non-invasive NIRF images of the head of mice (A and B) and corresponding ex-vivo NIRF imaging of the brains removed from the skull (C and D). Significantly higher fluorescence intensities over the ischemic hemisphere, compared to the contralateral side, were detected in MCAO mice at 4-8 hours (A,C) and 12-16 hours post MCAO, indicative of an impaired BBB. However, no differences between the hemispheres were seen at 8-12 hours (B,D). TBR calculated at different time points post injection are shown in the lower panel. The dotted line indicates the lowest TBR, at which differences between the ischemic and the non-ischemic hemisphere can be observed (E). A significant correlation between the TBR and the amount of EB detected in tissue homogenates of the ischemic hemispheres was found (F). The data demonstrates that non-invasive NIRF imaging with fluorescent albumin is a good analogue to EB, which is a standard marker for BBB impairment, but requires sacrificing the animal under study. Figure adapted from (4).

Non-invasive near-infrared (NIRF) fluorescence imaging of stroke-induced brain inflammation in a mouse model of cerebral ischemia targeting the pro-inflammatory CD40 receptor. Wild type or CD40 knock-out mice were subjected to transient middle cerebral artery occlusion (MCAO) or sham operation and were intravenously injected either with Cy5.5-labeled CD40-antibody or control IgG 80 hours after reperfusion. Target-to-background ratios (TBR) from non-invasive NIRF images were calculated by dividing the mean fluorescence intensities measured in rectangular regions of interest (ROIs) selected over the left and the right hemisphere. Non-invasive NIRF images of the head of mice (top row) and corresponding ex-vivo NIRF images of the brains removed from the skull (bottom row) 16 hours after injection of the compounds (A) showed significantly higher fluorescence intensities over the ischemic hemisphere compared to the contralateral side (A and B). Selective plane illumination microscopy demonstrated vascular and parenchymal cellular distribution of the injected CD40-antibody in the ischemic region (C). Confocal microscopy showed partial co-localization of the signal form the injected compound with activated microglia/macrophages (Iba1-positive cells) (D). Figure adapted from (5).

Non-invasive near-infrared fluorescence (NIRF) imaging of matrixmetalloproteinase (MMP) activity in a mouse model of cerebral ischemia. Representative in vivo (A, upper row) or ex vivo (A, bottom row) NIRF images. Imaging was performed 24 h after 1 h of MCAO. (A) No differences between the hemispheres were seen in vivo and ex vivo in sham-operated mice injected with the MMP-activatable probe. Slightly higher fluorescence intensities over the ischemic hemisphere compared with the contralateral side were detected in MCAO mice injected with the nonactivatable probe as control. Strong fluorescence was seen over the ipsilateral side of MCAO mice injected with the MMP-activatable probe. The asterisk indicates unspecific fluorescence of circulating probe in the superior sagittal sinus and transverse sinus. (B) Scatter plot showed a strong linear correlation (R2=0.833; n=10) between mean fluorescence intensities calculated from the ischemic hemisphere of ex vivo NIRF images and TBR obtained from noninvasive NIRF images of MCAO mice injected with the MMP-activatable probe. (C) Target-to-background ratio calculated from ROI analysis of noninvasive NIRF images. MCAO mice that received the MMP-activatable probe showed significantly higher TBR (mean ± s.d.; Kruskal–Wallis test, *P<0.05). (D) Distribution of the injected MMP-activatable probe 24 h after 1 h of MCAO. The left panel shows a representative ex vivo NIRF image of a coronal brain slice and an image of the same slice after TTC staining. The right panel shows images of higher magnification, taken with fluorescence microscopy at the area indicated by the box in the TTC image. FITC-BSA had been coadministered before sacrifice of the animal to investigate BBB permeability. Intense, diffuse Cy5.5 fluorescence was observed in addition to strong fluorescence of the extravasated FITC-BSA in the ischemic area of the cortex, whereas fluorescence in both channels is scarcely visible in the nonischemic area (D, right panel). The border between the ischemic and nonischemic area is clearly delineated after TTC and hematoxylin staining. Figure from (5).