title: In vivo imaging of the inflammatory receptor CD40 after cerebral ischemia using a fluorescent antibody.
abstract: 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.
authors.affiliation: Department Experimental Neurology, Charité-University Medicine Berlin, Berlin, Germany. jan.klohs@charite.de
authors.forename: Jan
authors.initials: J
authors.lastname: Klohs
authors.forename: Michael
authors.initials: M
authors.lastname: Gräfe
authors.forename: Kristof
authors.initials: K
authors.lastname: Graf
authors.forename: Jens
authors.initials: J
authors.lastname: Steinbrink
authors.forename: Thore
authors.initials: T
authors.lastname: Dietrich
authors.forename: Dietger
authors.initials: D
authors.lastname: Stibenz
authors.forename: Peyman
authors.initials: P
authors.lastname: Bahmani
authors.forename: Golo
authors.initials: G
authors.lastname: Kronenberg
authors.forename: Christoph
authors.initials: C
authors.lastname: Harms
authors.forename: Matthias
authors.initials: M
authors.lastname: Endres
authors.forename: Ute
authors.initials: U
authors.lastname: Lindauer
authors.forename: Klaus
authors.initials: K
authors.lastname: Greger
authors.forename: Ernst H K
authors.initials: EH
authors.lastname: Stelzer
authors.forename: Ulrich
authors.initials: U
authors.lastname: Dirnagl
authors.forename: Andreas
authors.initials: A
authors.lastname: Wunder
mesh_terms: 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
chemical_list: D000911:Antibodies, Monoclonal; D019013:CD40 Antigens; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D005456:Fluorescent Dyes