Visualization of cell death in mice with focal cerebral ischemia using fluorescent annexin A5, propidium iodide, and TUNEL staining.

To monitor stroke-induced brain damage and assess neuroprotective therapies, specific imaging of cell death after cerebral ischemia in a noninvasive manner is highly desirable. Annexin A5 has been suggested as a marker for imaging cell death under various disease conditions including stroke. In this study, C57BL6/N mice received middle cerebral artery occlusion (MCAO) and were injected intravenously with either active or inactive Cy5.5-annexin A5 48 hours after reperfusion. Some mice also received propidium iodide (PI), a cell integrity marker. Only in mice receiving active Cy5.5-annexin A5 were fluorescence intensities significantly higher over the hemisphere ipsilateral to MCAO than on the contralateral side. This was detected noninvasively and ex vivo 4 and 8 hours after injection. The majority of cells positive for fluorescent annexin A5 were also positive for PI and fragmented DNA as detected by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining. This study demonstrates the high specificity of annexin A5 for visualization of cell death in a mouse model of stroke. To our knowledge, this is the first study to compare the distribution of injected active and inactive annexin A5, PI, and TUNEL staining. It provides important information on the experimental and potential clinical applications of annexin A5-based imaging agents in stroke.

Non-invasive surface-stripping for epifluorescence small animal imaging.

Non-invasive near-infrared fluorescence (NIRF) imaging is a powerful tool to study pathophysiology in a wide variety of animal disease models including brain diseases. However, especially in NIRF imaging of the brain or other deeper laying target sites, background fluorescence emitted from the scalp or superficial blood vessels can impede the detection of fluorescence in deeper tissue. Here, we introduce an effective method to reduce the impact of fluorescence from superficial layers. The approach uses excitation light at two different wavelengths generating two images with different depth sensitivities followed by an adapted subtraction algorithm. This technique leads to significant enhancement of the contrast and the detectability of fluorochromes located in deep tissue layers in tissue simulating phantoms and murine models with stroke.

In vivo imaging of the inflammatory receptor CD40 after cerebral ischemia using a fluorescent antibody.

BACKGROUND AND PURPOSE: Brain inflammation is a hallmark of stroke, 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 study, we explored whether the inflammatory receptor CD40 can be noninvasively and specifically visualized in mice after cerebral ischemia using a fluorescent monoclonal antibody, which we labeled with the near-infrared fluorescence dye Cy5.5 (Cy5.5-CD40MAb). METHODS: Wild-type and CD40-deficient mice were subjected to transient middle cerebral artery occlusion. Mice were either intravenously injected with Cy5.5-CD40MAb or control Cy5.5-IgGMAb. Noninvasive and ex vivo near-infrared fluorescence imaging was performed after injection of the compounds. Probe distribution and specificity was further assessed with single-plane illumination microscopy, immunohistochemistry, and confocal microscopy. RESULTS: Significantly higher fluorescence intensities over the stroke-affected hemisphere, compared to the contralateral side, were only detected noninvasively in wild-type mice that received Cy5.5-CD40MAb, but not in CD40-deficient mice injected with Cy5.5-CD40MAb or in wild-type mice that were injected with Cy5.5-IgGMAb. Ex vivo near-infrared fluorescence showed an intense fluorescence within the ischemic territory only in wild-type mice injected with Cy5.5-CD40MAb. In the brains of these mice, single-plane illumination microscopy demonstrated vascular and parenchymal distribution, and confocal microscopy revealed a partial colocalization of parenchymal fluorescence from the injected Cy5.5-CD40MAb with activated microglia and blood-derived cells in the ischemic region. CONCLUSIONS: The study demonstrates that a CD40-targeted fluorescent antibody enables specific noninvasive detection of the inflammatory receptor CD40 after cerebral ischemia using optical techniques.

Noninvasive near-infrared imaging of fluorochromes within the brain of live mice: an in vivo phantom study.

Near-infrared fluorescence (NIRF) imaging has great potential for studying physiological and pathophysiological processes noninvasively in several locations of the body. In this study, we evaluated the feasibility of NIRF imaging to visualize fluorescent compounds within the brains of live mice commonly used in brain research. To simulate the presence of a molecular NIRF reporter agent at the site of a lesion, we developed a new in vivo phantom model wherein capsules containing different amounts of an NIRF dye (Cy5.5) were stereotactically implanted deep into the left hemispheres of living mice. To precisely locate the implanted capsules, magnetic resonance imaging (MRI) was performed. Fluorescence reflectance imaging (FRI) and transillumination fluorescence imaging (TFI) were conducted to analyze and compare sensitivity and target-to-background ratios of the two methods. The sensitivities of FRI and TFI to background fluorescence from circulating dye was tested by imaging fluorescent capsules in mice intravenously injected with increasing amounts of long-circulating Cy5.5-dextran. The results show that capsules containing dye amounts as low as 10(-12) mol can be detected. TFI yielded significantly higher target-to-background ratios than FRI at 10(-11) mol (p < .05). Comparatively low amounts of fluorescence in the blood vessels can extinguish the signal. We conclude that keeping the signal from circulating NIRF dye low, NIRF imaging offers high sensitivity in detecting fluorochromes noninvasively within brains of mice, especially by using TFI. This encourages the application of NIRF for molecular imaging in the mouse brain using NIRF reporters.