In vivo imaging of the inflammatory receptor CD40 after cerebral ischemia using a fluorescent antibody.
OBJECTIVE
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.
Klohs
Jan
J
Department Experimental Neurology, Charité-University Medicine Berlin, Berlin, Germany. jan.klohs@charite.de
Gräfe
Michael
M
Graf
Kristof
K
Steinbrink
Jens
J
Dietrich
Thore
T
Stibenz
Dietger
D
Bahmani
Peyman
P
Kronenberg
Golo
G
Harms
Christoph
C
Endres
Matthias
M
Lindauer
Ute
U
Greger
Klaus
K
Stelzer
Ernst H K
EH
Dirnagl
Ulrich
U
Wunder
Andreas
A
D000818:Animals; D000911:Antibodies, Monoclonal; D002545:Brain Ischemia / Q000150:complications / Q000276:immunology* / Q000473:pathology; D019013:CD40 Antigens / Q000096:biosynthesis*; D002232:Carbocyanines; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes; D007150:Immunohistochemistry; D007249:Inflammation / Q000209:etiology / Q000276:immunology* / Q000473:pathology; D051379:Mice; D008817:Mice, Mutant Strains; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence / Q000379:methods
D000911:Antibodies, Monoclonal; D019013:CD40 Antigens; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D005456:Fluorescent Dyes