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

Nature Protocols volume 13, pages 1377–1402 (2018)Cite this article

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Abstract

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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Figure 1: A workflow describing different in vivo methods for studying brain hemodynamic responses and tissue oxygenation in mice.
Figure 2: Two-photon microscopy measurements of individual brain capillary diameter changes in the somatosensory cortex in response to electrical stimulus in an anesthetized wild-type mouse (Step 18B).
Figure 3: Two-photon microscopy measurements of vascular RBC velocity changes in response to electrical stimulus in the somatosensory cortex of an anesthetized wild-type mouse (Step 18B).
Figure 4: IOS imaging of regional hemodynamic response to green 530-nm illumination in in the somatosensory cortex of an anesthetized mouse (Step 18A).
Figure 5: IOS imaging of regional hemodynamic response with red 630-nm illumination of the somatosensory cortex in an anesthetized mouse (Step 18A).
Figure 6: Dissecting microscope configured for IOS imaging.
Figure 7: Two-photon measurements of NADH fluorescence intensity changes in response to a stimulus in the somatosensory cortex of an anesthetized pericyte-deficient mouse (carrying a single platelet-derived growth factor receptor β allele; Pdgfrb+/−), which exhibits altered metabolic responses to a stimulus (Step 18C).
Figure 8: Two-photon acquisition of absolute tissue pO2 values in the mouse cortex using PtP-C343 phosphorescent dye (Box 1).
Figure 9: Cranial window preparation for in vivo imaging (Steps 1–17).

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Acknowledgements

This work was supported by US National Institutes of Health grants R01AG023084, R01NS090904, R01NS034467, R01AG039452, R01NS100459, and P01AG052350 to B.V.Z.; grants R24NS092986, R01EB018464, and R01NS091230 to S.S., S.A.V., and D.A.B.; by funding from the Alzheimer's Association and Cure Alzheimer's fund to B.V.Z.; and by funding from the Fondation Leducq Transatlantic Network of Excellence for the Study of Perivascular Spaces in Small Vessel Disease (ref. no. 16 CVD 05) to B.V.Z. We thank R. Jaswal for helping to create Figure 8. We gratefully acknowledge the feedback, forum posts, and questions from our peers regarding the techniques presented here, which provided the inspiration for the writing of the manuscript.

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Author notes

  1. Sava Sakadži´ and Berislav V Zlokovic: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physiology and Neuroscience and the Zilkha Neurogenetic Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA

    Kassandra Kisler, Divna Lazic, Melanie D Sweeney & Berislav V Zlokovic

  2. Department of Neurobiology, Institute for Biological Research, University of Belgrade, Belgrade, Republic of Serbia

    Divna Lazic

  3. Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Shane Plunkett, Mirna El Khatib & Sergei A Vinogradov

  4. Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Shane Plunkett, Mirna El Khatib & Sergei A Vinogradov

  5. Optics Division, Department of Radiology, MGH/HMS/MIT Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, USA

    David A Boas & Sava Sakadži´

  6. Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA

    David A Boas

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  1. Kassandra Kisler
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Contributions

K.K. and B.V.Z. conceived the concept presented here. K.K., S.S., and B.V.Z. contributed to the experimental design. K.K. and S.S. performed experiments and analyzed the data. D.L. and M.D.S. performed experiments and contributed to cranial window protocol development, and S.P. and M.E.K. contributed to PtP-C343 dye development. D.A.B. and S.A.V. contributed to the project design. B.V.Z. contributed to the project design and supervised the project. K.K., S.S., and B.V.Z. wrote the manuscript with input from all authors. S.S. and B.V.Z. shared senior authorship responsibilities.

Corresponding author

Correspondence to Berislav V Zlokovic.

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The authors declare no competing financial interests.

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Kisler, K., Lazic, D., Sweeney, M. et al. In vivo imaging and analysis of cerebrovascular hemodynamic responses and tissue oxygenation in the mouse brain. Nat Protoc 13, 1377–1402 (2018). https://doi.org/10.1038/nprot.2018.034

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