title: Endogenous Two-Photon Excited Fluorescence Provides Label-Free Visualization of the Inflammatory Response in the Rodent Spinal Cord.
abstract: Activation of CNS resident microglia and invasion of external macrophages plays a central role in spinal cord injuries and diseases. Multiphoton microscopy based on intrinsic tissue properties offers the possibility of label-free imaging and has the potential to be applied in vivo. In this work, we analyzed cellular structures displaying endogenous two-photon excited fluorescence (TPEF) in the pathologic spinal cord. It was compared qualitatively and quantitatively to Iba1 and CD68 immunohistochemical staining in two models: rat spinal cord injury and mouse encephalomyelitis. The extent of tissue damage was retrieved by coherent anti-Stokes Raman scattering (CARS) and second harmonic generation imaging. The pattern of CD68-positive cells representing postinjury activated microglia/macrophages was colocalized to the TPEF signal. Iba1-positive microglia were found in areas lacking any TPEF signal. In peripheral areas of inflammation, we found similar numbers of CD68-positive microglia/macrophages and TPEF-positive structures while the number of Iba1-positive cells was significantly higher. Therefore, we conclude that multiphoton imaging of unstained spinal cord tissue enables retrieving the extent of microglia activation by acquisition of endogenous TPEF. Future application of this technique in vivo will enable monitoring inflammatory responses of the nervous system allowing new insights into degenerative and regenerative processes.
authors.affiliation: Neurosurgery, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Ortrud
authors.initials: O
authors.lastname: Uckermann
authors.affiliation: Clinical Sensoring and Monitoring, Anesthesiology and Intensive Care Medicine, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Roberta
authors.initials: R
authors.lastname: Galli
authors.affiliation: Centre for Translational Bone, Joint and Soft Tissue Research, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Beiermeister
authors.affiliation: Neurosurgery, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Kerim-Hakan
authors.initials: KH
authors.lastname: Sitoci-Ficici
authors.affiliation: Neurosurgery, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Later
authors.affiliation: Neurosurgery, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Elke
authors.initials: E
authors.lastname: Leipnitz
authors.affiliation: Department of Clinical Pathobiochemistry, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Ales
authors.initials: A
authors.lastname: Neuwirth
authors.affiliation: Department of Clinical Pathobiochemistry, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany ; Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Fetscherstrasse 105, 01307 Dresden, Germany.
authors.forename: Triantafyllos
authors.initials: T
authors.lastname: Chavakis
authors.affiliation: Clinical Sensoring and Monitoring, Anesthesiology and Intensive Care Medicine, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany ; Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Fetscherstrasse 105, 01307 Dresden, Germany.
authors.forename: Edmund
authors.initials: E
authors.lastname: Koch
authors.affiliation: Neurosurgery, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Schackert
authors.affiliation: Clinical Sensoring and Monitoring, Anesthesiology and Intensive Care Medicine, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany.
authors.forename: Gerald
authors.initials: G
authors.lastname: Steiner
authors.affiliation: Neurosurgery, Carl Gustav Carus University Hospital, TU Dresden, Fetscherstraße 74, 01307 Dresden, Germany ; Center for Regenerative Therapies Dresden (CRTD), TU Dresden, Fetscherstrasse 105, 01307 Dresden, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Kirsch
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015214:Antigens, Differentiation, Myelomonocytic / Q000378:metabolism; D005260:Female; D005453:Fluorescence; D007249:Inflammation / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods; D064847:Multimodal Imaging / Q000379:methods; D017785:Photons; D051381:Rats; D017208:Rats, Wistar; D013059:Spectrum Analysis, Raman / Q000379:methods; D013116:Spinal Cord / Q000378:metabolism / Q000473:pathology*; D013119:Spinal Cord Injuries / Q000378:metabolism / Q000473:pathology*; D000097382:CD68 Molecule
chemical_list: D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; C067980:CD68 antigen, human; D000097382:CD68 Molecule