April 2017 | Volume 8 | Article 406 1 METHODS published: 11 April 2017 doi: 10.3389/fmmu.2017.00406 Frontiers in Immunology | www.frontiersin.org Edited by: V. Wee Yong, University of Calgary, Canada Reviewed by: Seema TiwariWoodruff, University of California, Riverside, USA Luc Vallieres, Laval University, Canada *Correspondence: Britta Engelhardt bengel@tki.unibe.ch Specialty section: This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Immunology Received: 22 December 2016 Accepted: 22 March 2017 Published: 11 April 2017 Citation: Haghayegh Jahromi N, Tardent H, Enzmann G, Deutsch U, Kawakami N, Bittner S, Vestweber D, Zipp F, Stein JV and Engelhardt B (2017) A Novel Cervical Spinal Cord Window Preparation Allows for Two-Photon Imaging of T-Cell Interactions with the Cervical Spinal Cord Microvasculature during Experimental Autoimmune Encephalomyelitis. Front. Immunol. 8:406. doi: 10.3389/fmmu.2017.00406 A Novel Cervical Spinal Cord Window Preparation Allows for Two-Photon Imaging of T-Cell Interactions with the Cervical Spinal Cord Microvasculature during Experimental Autoimmune Encephalomyelitis Neda Haghayegh Jahromi 1 , Heidi Tardent 1 , Gaby Enzmann 1 , Urban Deutsch 1 , Naoto Kawakami 2,3 , Stefan Bittner 4 , Dietmar Vestweber 5 , Frauke Zipp 4 , Jens V. Stein 1 and Britta Engelhardt 1 * 1 Theodor Kocher Institute, University of Bern, Bern, Switzerland, 2 Max Planck Institute of Neurobiology, Martinsried, Germany, 3 Institute of Clinical Neuroimmunology, Biomedical Center and University Hospital, Ludwig-Maximilians University of Munich, Martinsried, Germany, 4 Focus Program Translational Neuroscience (FTN) and Immunotherapy (FZI), Rhine Main Neuroscience Network (rmn 2 ), University Medical Center of the Johannes Gutenberg University, Mainz, Germany, 5 Max-Planck-Institute of Molecular Biomedicine, Münster, Germany T-cell migration across the blood–brain barrier (BBB) is a crucial step in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Two-photon intravital microscopy (2P-IVM) has been established as a powerful tool to study cell–cell interactions in infammatory EAE lesions in living animals. In EAE, central nervous system infammation is strongly pronounced in the spinal cord, an organ in which 2P-IVM imaging is technically very challenging and has been limited to the lumbar spinal cord. Here, we describe a novel spinal cord window preparation allowing to use 2P-IVM to image immune cell interactions with the cervical spinal cord microvascular endothelium during EAE. We describe differences in the angioarchitecture of the cervical spinal cord versus the lumbar spinal cord, which will entail different hemo- dynamic parameters in these different vascular beds. Using T cells as an example, we demonstrate the suitability of this novel methodology in imaging the post-arrest multistep T-cell extravasation across the cervical spinal cord microvessels. The novel methodology includes an outlook to the analysis of the cellular pathway of T-cell diapedesis across the BBB by establishing visualization of endothelial junctions in this vascular bed. Keywords: cervical spinal cord window, two-photon intravital microscopy, experimental autoimmune encephalomyelitis, blood–brain barrier, T-cell migration