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Nat Protoc. 2018 Jun;13(6):1377-1402. doi: 10.1038/nprot.2018.034. Epub 2018 May 24.

In vivo imaging and analysis of cerebrovascular hemodynamic responses and tissue oxygenation in the mouse brain.

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Department of Physiology and Neuroscience and the Zilkha Neurogenetic Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA.
Department of Neurobiology, Institute for Biological Research, University of Belgrade, Belgrade, Republic of Serbia.
Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Optics Division, MGH/HMS/MIT Athinoula A. Martinos Center for Biomedical Imaging, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, USA.
Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA.


Cerebrovascular dysfunction has an important role in the pathogenesis of multiple brain disorders. Measurement of hemodynamic responses in vivo can be challenging, particularly as techniques are often not described in sufficient detail and vary between laboratories. We present a set of standardized in vivo protocols that describe high-resolution two-photon microscopy and intrinsic optical signal (IOS) imaging to evaluate capillary and arteriolar responses to a stimulus, regional hemodynamic responses, and oxygen delivery to the brain. The protocol also describes how to measure intrinsic NADH fluorescence to understand how blood O2 supply meets the metabolic demands of activated brain tissue, and to perform resting-state absolute oxygen partial pressure (pO2) measurements of brain tissue. These methods can detect cerebrovascular changes at far higher resolution than MRI techniques, although the optical nature of these techniques limits their achievable imaging depths. Each individual procedure requires 1-2 h to complete, with two to three procedures typically performed per animal at a time. These protocols are broadly applicable in studies of cerebrovascular function in healthy and diseased brain in any of the existing mouse models of neurological and vascular disorders. All these procedures can be accomplished by a competent graduate student or experienced technician, except the two-photon measurement of absolute pO2 level, which is better suited to a more experienced, postdoctoral-level researcher.

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