Craniula: A cranial window technique for prolonged imaging of brain surface vasculature with simultaneous adjacent intra
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FLUIDS AND BARRIERS OF THE CNS
Open Access
STUDY PROTOCOL
Craniula: A cranial window technique for prolonged imaging of brain surface vasculature with simultaneous adjacent intracerebral injection Viviana Zuluaga‑Ramirez1†, Slava Rom2† and Yuri Persidsky3*
Abstract Background: Imaging of the brain surface vasculature following inflammatory insults is critical to study structural and functional changes in the living brain under normal and pathological conditions. Although there have been pub‑ lished reports relating to the changes that occur in the blood brain barrier (BBB) during the inflammatory process, the ability to visualize and track such changes in vivo and over time has proven to be problematic. Different techniques have been used to achieve visualization of pial vessels, but the approach has limits, which can jeopardize the wellbeing of the animals. Development of the cranial window technique provided a major advance in the acquisition of live images of the brain vasculature and its response to different insults and treatments. Methods: We describe in detail a protocol for delivery of a localized inflammatory insult to the mouse brain via a craniula (cranial window and adjacent cannula) and subsequent imaging of the mouse brain vasculature by intravital microscopy and two-photon laser scanning microscopy. The surgical implantation of the craniula can be completed in 30-45 min and images can be acquired immediately and for several months thereafter. The technique is minimally invasive and permits serial injections directly to the brain, thereby allowing longitudinal imaging studies. The craniula technique permits the study of structural and functional changes of the BBB following inflammatory insult and as such has wide application to neuroscience research. Keywords: Blood Brain Barrier, Leukocyte–endothelial interaction, Intravital microscopy, 2-photon microscopy Background In vivo imaging of the brain vasculature to assess interactions of different leukocyte types within the blood brain barrier (BBB) has been challenging. Although there have been published reports related to the changes that occur in the BBB during the inflammatory process [1–4], the ability to visualize and track such changes over time and in vivo has proven to be problematic. Techniques such as skull-thinning [5] have been used to achieve visualization *Correspondence: [email protected] † Viviana Zuluaga-Ramirez and Slava Rom contributed equally to this work 3 Department of Pathology and Laboratory Medicine, Temple University School of Medicine, 3500 N Broad St, MERB 841, Philadelphia, PA 19140, USA Full list of author information is available at the end of the article
of pial vessels, but the approach has limits due to skin manipulation and wound management, which can jeopardize the well-being of the animals. On the other hand, this complication has not been reported on cranial windows, since the skin is actually removed from the skull and no further wound manipulation is required [6]. Development of the cranial win
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