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

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).

References

  1. 1

    Kety, S.S. The general metabolism of the brain in vivo. in Metabolism of the Nervous System 221–237 (ed. D. Richter) (Elsevier, 1957).

  2. 2

    Sokoloff, L. The metabolism of the central nervous system in vivo. in Handbook of Physiology, Section I, Neurophysiology (eds. Field, J., Magoun, H. W. & Hall, V. E.) 3, 1843–1864 (American Physiological Society, 1960).

  3. 3

    Zlokovic, B.V. Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders. Nat. Rev. Neurosci. 12, 723–738 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Sweeney, M.D., Sagare, A.P. & Zlokovic, B.V. Blood–brain barrier breakdown in Alzheimer disease and other neurodegenerative disorders. Nat. Rev. Neurol. 14, 133–150 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Iadecola, C. The pathobiology of vascular dementia. Neuron 80, 844–866 (2013).

    CAS  PubMed  Google Scholar 

  6. 6

    Kisler, K., Nelson, A.R., Montagne, A. & Zlokovic, B.V. Cerebral blood flow regulation and neurovascular dysfunction in Alzheimer disease. Nat. Rev. Neurosci. 18, 419–434 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Arvanitakis, Z., Capuano, A.W., Leurgans, S.E., Bennett, D.A. & Schneider, J.A. Relation of cerebral vessel disease to Alzheimer's disease dementia and cognitive function in elderly people: a cross-sectional study. Lancet Neurol. 15, 934–943 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Iturria-Medina, Y. et al. Early role of vascular dysregulation on late-onset Alzheimer's disease based on multifactorial data-driven analysis. Nat. Commun. 7, 11934 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Montagne, A. et al. Brain imaging of neurovascular dysfunction in Alzheimer's disease. Acta Neuropathol. (Berl.) 131, 687–707 (2016).

    CAS  Google Scholar 

  10. 10

    Montagne, A., Zhao, Z. & Zlokovic, B.V. Alzheimer's disease: a matter of blood–brain barrier dysfunction? J. Exp. Med. 214, 3151–3169 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Murphy, M.J. et al. Widespread cerebral haemodynamics disturbances occur early in amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. 13, 202–209 (2012).

    PubMed  PubMed Central  Google Scholar 

  12. 12

    Ishikawa, T., Morita, M. & Nakano, I. Constant blood flow reduction in premotor frontal lobe regions in ALS with dementia - a SPECT study with 3D-SSP. Acta Neurol. Scand. 116, 340–344 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Yamashita, T. et al. Flow-metabolism uncoupling in the cervical spinal cord of ALS patients. Neurol. Sci. 38, 659–665 (2017).

    PubMed  PubMed Central  Google Scholar 

  14. 14

    Verfaillie, S.C.J. et al. Cerebral perfusion and glucose metabolism in Alzheimer's disease and frontotemporal dementia: two sides of the same coin? Eur. Radiol. 25, 3050–3059 (2015).

    PubMed  PubMed Central  Google Scholar 

  15. 15

    Malek, N. et al. Vascular disease and vascular risk factors in relation to motor features and cognition in early Parkinson's disease. Mov. Disord. 31, 1518–1526 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Al-Bachari, S., Vidyasagar, R., Emsley, H.C. & Parkes, L.M. Structural and physiological neurovascular changes in idiopathic Parkinson's disease and its clinical phenotypes. J. Cereb. Blood Flow Metab. 37, 3409–3421 (2017).

    PubMed  PubMed Central  Google Scholar 

  17. 17

    Drouin-Ouellet, J. et al. Cerebrovascular and blood-brain barrier impairments in Huntington's disease: potential implications for its pathophysiology. Ann. Neurol. 78, 160–177 (2015).

    PubMed  PubMed Central  Google Scholar 

  18. 18

    Chen, J.J., Salat, D.H. & Rosas, H.D. Complex relationships between cerebral blood flow and brain atrophy in early Huntington's disease. NeuroImage 59, 1043–1051 (2012).

    PubMed  PubMed Central  Google Scholar 

  19. 19

    Wardlaw, J.M., Smith, C. & Dichgans, M. Mechanisms of sporadic cerebral small vessel disease: insights from neuroimaging. Lancet Neurol. 12, 483–497 (2013).

    PubMed  PubMed Central  Google Scholar 

  20. 20

    Wardlaw, J.M. et al. Neuroimaging standards for research into small vessel disease and its contribution to ageing and neurodegeneration. Lancet Neurol. 12, 822–838 (2013).

    PubMed  PubMed Central  Google Scholar 

  21. 21

    Montine, T.J. et al. Recommendations of the Alzheimer's disease-related dementias conference. Neurology 83, 851–860 (2014).

    PubMed  PubMed Central  Google Scholar 

  22. 22

    Snyder, H.M. et al. Vascular contributions to cognitive impairment and dementia including Alzheimer's disease. Alzheimers Dement. 11, 710–717 (2015).

    PubMed  PubMed Central  Google Scholar 

  23. 23

    Hachinski, V. & World Stroke Organization Stroke and potentially preventable dementias proclamation: updated World Stroke Day Proclamation. Stroke 46, 3039–3040 (2015).

    PubMed  PubMed Central  Google Scholar 

  24. 24

    Faraco, G. & Iadecola, C. Hypertension: a harbinger of stroke and dementia. Hypertension 62, 810–817 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Last, D. et al. Global and regional effects of type 2 diabetes on brain tissue volumes and cerebral vasoreactivity. Diabetes Care 30, 1193–1199 (2007).

    PubMed  PubMed Central  Google Scholar 

  26. 26

    Ingrisch, M. et al. Quantification of perfusion and permeability in multiple sclerosis: dynamic contrast-enhanced MRI in 3D at 3T. Invest. Radiol. 47, 252–258 (2012).

    PubMed  PubMed Central  Google Scholar 

  27. 27

    De Roos, A., van der Grond, J., Mitchell, G. & Westenberg, J. Magnetic resonance imaging of cardiovascular function and the brain: is dementia a cardiovascular-driven disease? Circulation 135, 2178–2195 (2017).

    PubMed  PubMed Central  Google Scholar 

  28. 28

    Qiu, C. & Fratiglioni, L. A major role for cardiovascular burden in age-related cognitive decline. Nat. Rev. Cardiol. 12, 267–277 (2015).

    PubMed  PubMed Central  Google Scholar 

  29. 29

    Lok, J. et al. Targeting the neurovascular unit in brain trauma. CNS Neurosci. Ther. 21, 304–308 (2015).

    PubMed  PubMed Central  Google Scholar 

  30. 30

    Toth, P. et al. Traumatic brain injury-induced autoregulatory dysfunction and spreading depression-related neurovascular uncoupling: pathomechanisms, perspectives, and therapeutic implications. Am. J. Physiol. Heart Circ. Physiol. 311, H1118–H1131 (2016).

    PubMed  PubMed Central  Google Scholar 

  31. 31

    Najjar, S. et al. Neurovascular unit dysfunction and blood-brain barrier hyperpermeability contribute to Schizophrenia neurobiology: a theoretical integration of clinical and experimental evidence. Front. Psychiatry 8, 83 (2017).

    PubMed  PubMed Central  Google Scholar 

  32. 32

    Ances, B., Vaida, F., Ellis, R. & Buxton, R. Test-retest stability of calibrated BOLD-fMRI in HIV- and HIV+ subjects. NeuroImage 54, 2156–2162 (2011).

    PubMed  PubMed Central  Google Scholar 

  33. 33

    Iadecola, C. et al. SOD1 rescues cerebral endothelial dysfunction in mice overexpressing amyloid precursor protein. Nat. Neurosci. 2, 157–161 (1999).

    CAS  PubMed  Google Scholar 

  34. 34

    Niwa, K. et al. Abeta 1-40-related reduction in functional hyperemia in mouse neocortex during somatosensory activation. Proc. Natl. Acad. Sci. USA 97, 9735–9740 (2000).

    CAS  Google Scholar 

  35. 35

    Chow, N. et al. Serum response factor and myocardin mediate arterial hypercontractility and cerebral blood flow dysregulation in Alzheimer's phenotype. Proc. Natl. Acad. Sci. USA 104, 823–828 (2007).

    CAS  PubMed  Google Scholar 

  36. 36

    Miyazaki, K. et al. Early and progressive impairment of spinal blood flow—glucose metabolism coupling in motor neuron degeneration of ALS model mice. J. Cereb. Blood Flow Metab. 32, 456–467 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Kleinberger, G. et al. The FTD-like syndrome causing TREM2 T66M mutation impairs microglia function, brain perfusion, and glucose metabolism. EMBO J. 36, 1837–1853 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Toth, P. et al. Age-related autoregulatory dysfunction and cerebromicrovascular injury in mice with angiotensin II-induced hypertension. J. Cereb. Blood Flow Metab. 33, 1732–1742 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Faraco, G. et al. Hypertension enhances Aβ-induced neurovascular dysfunction, promotes β-secretase activity, and leads to amyloidogenic processing of APP. J. Cereb. Blood Flow Metab. 36, 241–252 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Kisler, K. et al. Pericyte degeneration leads to neurovascular uncoupling and limits oxygen supply to brain. Nat. Neurosci. 20, 406–416 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Kim, T.N. et al. Line-scanning particle image velocimetry: an optical approach for quantifying a wide range of blood flow speeds in live animals. PLoS One 7, e38590 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Harrison, T.C., Sigler, A. & Murphy, T.H. Simple and cost-effective hardware and software for functional brain mapping using intrinsic optical signal imaging. J. Neurosci. Methods 182, 211–218 (2009).

    PubMed  Google Scholar 

  43. 43

    Hillman, E.M.C. Optical brain imaging in vivo: techniques and applications from animal to man. J. Biomed. Opt. 12, 051402 (2007).

    PubMed  PubMed Central  Google Scholar 

  44. 44

    Sirotin, Y.B., Hillman, E.M.C., Bordier, C. & Das, A. Spatiotemporal precision and hemodynamic mechanism of optical point spreads in alert primates. Proc. Natl. Acad. Sci. USA 106, 18390–18395 (2009).

    CAS  PubMed  Google Scholar 

  45. 45

    Frostig, R.D., Lieke, E.E., Ts'o, D.Y. & Grinvald, A. Cortical functional architecture and local coupling between neuronal activity and the microcirculation revealed by in vivo high-resolution optical imaging of intrinsic signals. Proc. Natl. Acad. Sci. USA 87, 6082–6086 (1990).

    CAS  PubMed  Google Scholar 

  46. 46

    Kasischke, K.A., Vishwasrao, H.D., Fisher, P.J., Zipfel, W.R. & Webb, W.W. Neural activity triggers neuronal oxidative metabolism followed by astrocytic glycolysis. Science 305, 99–103 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Mayevsky, A. & Rogatsky, G.G. Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies. Am. J. Physiol. Cell Physiol. 292, C615–C640 (2006).

    PubMed  PubMed Central  Google Scholar 

  48. 48

    Finikova, O.S. et al. Oxygen microscopy by two-photon-excited phosphorescence. Chemphyschem 9, 1673–1679 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Gama Sosa, M.A. et al. Age-related vascular pathology in transgenic mice expressing Presenilin 1-associated familial Alzheimer's disease mutations. Am. J. Pathol. 176, 353–368 (2010).

    PubMed  PubMed Central  Google Scholar 

  50. 50

    Blair, L.J. et al. Tau depletion prevents progressive blood-brain barrier damage in a mouse model of tauopathy. Acta Neuropathol. Commun. 3 (2015).

  51. 51

    Lewis, J. et al. Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein. Nat. Genet. 25, 402–405 (2000).

    CAS  PubMed  Google Scholar 

  52. 52

    Bell, R.D. et al. Apolipoprotein E controls cerebrovascular integrity via cyclophilin A. Nature 485, 512–516 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Alata, W., Ye, Y., St-Amour, I., Vandal, M. & Calon, F. Human apolipoprotein Ež4 expression impairs cerebral vascularization and blood-brain barrier function in mice. J. Cereb. Blood Flow Metab. 35, 86–94 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Nelson, A.R., Sweeney, M.D., Sagare, A.P. & Zlokovic, B.V. Neurovascular dysfunction and neurodegeneration in dementia and Alzheimer's disease. Biochim. Biophys. Acta 1862, 887–900 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Park, L. et al. Brain and circulating levels of A 1-40 differentially contribute to vasomotor dysfunction in the mouse brain. Stroke 44, 198–204 (2013).

    PubMed  PubMed Central  Google Scholar 

  56. 56

    Poliakova, T., Levin, O., Arablinskiy, A., Vasenina, E. & Zerr, I. Cerebral microbleeds in early Alzheimer's disease. J. Neurol. 263, 1961–1968 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Manaenko, A., Chen, H., Zhang, J.H. & Tang, J. Comparison of different preclinical models of intracerebral hemorrhage. in Intracerebral Hemorrhage Research (eds. Zhang, J. & Colohan, A.) 111, 9–14 (Springer, 2011).

    Google Scholar 

  58. 58

    Petraglia, A.L., Marky, A.H., Walker, C., Thiyagarajan, M. & Zlokovic, B.V. Activated protein C is neuroprotective and mediates new blood vessel formation and neurogenesis after controlled cortical impact. Neurosurgery 66, 165–171 (2010).

    PubMed  PubMed Central  Google Scholar 

  59. 59

    Toda, N. & Okamura, T. Hyperhomocysteinemia impairs regional blood flow: involvements of endothelial and neuronal nitric oxide. Pflugers Arch. 468, 1517–1525 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Sudduth, T.L., Powell, D.K., Smith, C.D., Greenstein, A. & Wilcock, D.M. Induction of hyperhomocysteinemia models vascular dementia by induction of cerebral microhemorrhages and neuroinflammation. J. Cereb. Blood Flow Metab. 33, 708–715 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61

    Hardigan, T., Hernandez, C., Ward, R., Hoda, M.N. & Ergul, A. TLR2 knockout protects against diabetes-mediated changes in cerebral perfusion and cognitive deficits. Am. J. Physiol. Regul. Integr. Comp. Physiol. 312, R927–R937 (2017).

    PubMed  PubMed Central  Google Scholar 

  62. 62

    Iordanova, B., Li, L., Clark, R.S.B. & Manole, M.D. Alterations in cerebral blood flow after resuscitation from cardiac arrest. Front. Pediatr. 5, 174 (2017).

    PubMed  PubMed Central  Google Scholar 

  63. 63

    Khan, M.B. et al. Chronic remote ischemic conditioning is cerebroprotective and induces vascular remodeling in a VCID model. Transl. Stroke Res. 9, 51–63 (2017).

    PubMed  PubMed Central  Google Scholar 

  64. 64

    Zhao, Z., Nelson, A.R., Betsholtz, C. & Zlokovic, B.V. Establishment and dysfunction of the blood-brain barrier. Cell 163, 1064–1078 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. 65

    Winkler, E.A. et al. GLUT1 reductions exacerbate Alzheimer's disease vasculo-neuronal dysfunction and degeneration. Nat. Neurosci. 18, 521–530 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. 66

    Guemez-Gamboa, A. et al. Inactivating mutations in MFSD2A, required for omega-3 fatty acid transport in brain, cause a lethal microcephaly syndrome. Nat. Genet. 47, 809–813 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. 67

    Ben-Zvi, A. et al. Mfsd2a is critical for the formation and function of the blood-brain barrier. Nature 509, 507–511 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. 68

    Lacombe, P., Oligo, C., Domenga, V., Tournier-Lasserve, E. & Joutel, A. Impaired cerebral vasoreactivity in a transgenic mouse model of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy arteriopathy. Stroke 36, 1053–1058 (2005).

    PubMed  PubMed Central  Google Scholar 

  69. 69

    Joutel, A. et al. Cerebrovascular dysfunction and microcirculation rarefaction precede white matter lesions in a mouse genetic model of cerebral ischemic small vessel disease. J. Clin. Invest. 120, 433–445 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. 70

    Sharma, A. & Shiras, A. Cancer stem cell-vascular endothelial cell interactions in glioblastoma. Biochem. Biophys. Res. Commun. 473, 688–692 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. 71

    Dai, M., Yang, Y. & Shi, X. Lactate dilates cochlear capillaries via type V fibrocyte-vessel coupling signaled by nNOS. Am. J. Physiol. Heart Circ. Physiol. 301, H1248–H1254 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. 72

    Martinez Sosa, S. & Smith, K.J. Understanding a role for hypoxia in lesion formation and location in the deep and periventricular white matter in small vessel disease and multiple sclerosis. Clin. Sci. Lond. 131, 2503–2524 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. 73

    Tang, P. et al. In vivo two-photon imaging of axonal dieback, blood flow, and calcium influx with methylprednisolone therapy after spinal cord injury. Sci. Rep. 5, 9691 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  74. 74

    Montagne, A. et al. Pericyte degeneration causes white matter dysfunction in the mouse central nervous system. Nat. Med. 24, 326–337 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. 75

    Uhlirova, H. et al. Cell type specificity of neurovascular coupling in cerebral cortex. eLife 5 e14315 (2016).

    PubMed  PubMed Central  Google Scholar 

  76. 76

    Kasischke, K.A. et al. Two-photon NADH imaging exposes boundaries of oxygen diffusion in cortical vascular supply regions. J. Cereb. Blood Flow Metab. 31, 68–81 (2011).

    CAS  PubMed  Google Scholar 

  77. 77

    Sakadži´, S. et al. Large arteriolar component of oxygen delivery implies a safe margin of oxygen supply to cerebral tissue. Nat. Commun. 5, 5734 (2014).

    Google Scholar 

  78. 78

    Shahram, M. & Milanfar, P. Imaging below the diffraction limit: a statistical analysis. IEEE Trans. Image Process. 13, 677–689 (2004).

    PubMed  PubMed Central  Google Scholar 

  79. 79

    Ram, S., Ward, E.S. & Ober, R.J. Beyond Rayleigh's criterion: a resolution measure with application to single-molecule microscopy. Proc. Natl. Acad. Sci. USA 103, 4457–4462 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. 80

    Rayleigh, L. XXXI: Investigations in optics, with special reference to the spectroscope. Philos. Mag. Ser. 5 8, 261–274 (1879).

    Google Scholar 

  81. 81

    Nyquist, H. Certain topics in telegraph transmission theory. Trans. Am. Inst. Electr. Eng. 47, 617–644 (1928).

    Google Scholar 

  82. 82

    Shannon, C.E. A mathematical theory of communication. Bell Syst. Tech. J. 27, 379–423 (1948).

    Google Scholar 

  83. 83

    Pawley, J.B. Points, pixels, and gray levels: digitizing image data. in Handbook of Biological Confocal Microscopy (ed. Pawley, J.B.) 59–79 (Springer, 2006).

  84. 84

    Hall, C.N. et al. Capillary pericytes regulate cerebral blood flow in health and disease. Nature 508, 55–60 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. 85

    Mishra, A. et al. Astrocytes mediate neurovascular signaling to capillary pericytes but not to arterioles. Nat. Neurosci. 19, 1619–1627 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. 86

    Otsu, Y. et al. Calcium dynamics in astrocyte processes during neurovascular coupling. Nat. Neurosci. 18, 210–218 (2015).

    CAS  PubMed  Google Scholar 

  87. 87

    Kornfield, T.E. & Newman, E.A. Regulation of blood flow in the retinal trilaminar vascular network. J. Neurosci. 34, 11504–11513 (2014).

    PubMed  PubMed Central  Google Scholar 

  88. 88

    Biesecker, K.R. et al. Glial cell calcium signaling mediates capillary regulation of blood flow in the retina. J. Neurosci. 36, 9435–9445 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. 89

    Drew, P.J., Shih, A.Y. & Kleinfeld, D. Fluctuating and sensory-induced vasodynamics in rodent cortex extend arteriole capacity. Proc. Natl. Acad. Sci. USA 108, 8473–8478 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. 90

    Takano, T. et al. Astrocyte-mediated control of cerebral blood flow. Nat. Neurosci. 9, 260–267 (2006).

    CAS  PubMed  Google Scholar 

  91. 91

    Hill, R.A. et al. Regional blood flow in the normal and ischemic brain is controlled by arteriolar smooth muscle cell contractility and not by capillary pericytes. Neuron 87, 95–110 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. 92

    Damisah, E.C., Hill, R.A., Tong, L., Murray, K.N. & Grutzendler, J. A fluoro-Nissl dye identifies pericytes as distinct vascular mural cells during in vivo brain imaging. Nat. Neurosci. 20, 1023–1032 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. 93

    Wei, H.S. et al. Erythrocytes are oxygen-sensing regulators of the cerebral microcirculation. Neuron 91, 851–862 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. 94

    Chang, C.-I., Du, Y., Wang, J., Guo, S.-M. & Thouin, P.D. Survey and comparative analysis of entropy and relative entropy thresholding techniques. IEE Proc. - Vis. Image Signal Process. 153, 837 (2006).

    Google Scholar 

  95. 95

    Drew, P.J., Blinder, P., Cauwenberghs, G., Shih, A.Y. & Kleinfeld, D. Rapid determination of particle velocity from space-time images using the Radon transform. J. Comput. Neurosci. 29, 5–11 (2010).

    PubMed  PubMed Central  Google Scholar 

  96. 96

    Summers, P.M., Taylor, Z.J. & Shih, A.Y. Two-photon imaging of cerebral vasodynamics in awake mice during health and disease. in Advances in Intravital Microscopy (ed. Weigert, R.) 25–43 (Springer, 2014).

  97. 97

    Art, J. Photon detectors for confocal microscopy. in Handbook of Biological Confocal Microscopy (ed. Pawley, J.B.) 251–264 (Springer, 2006).

  98. 98

    Baran, U. & Wang, R.K. Review of optical coherence tomography based angiography in neuroscience. Neurophotonics 3, 010902 (2016).

    PubMed  PubMed Central  Google Scholar 

  99. 99

    Srinivasan, V.J. et al. OCT methods for capillary velocimetry. Biomed. Opt. Express 3, 612–629 (2012).

    PubMed  PubMed Central  Google Scholar 

  100. 100

    Ma, Y. et al. Wide-field optical mapping of neural activity and brain haemodynamics: considerations and novel approaches. Philos. Trans. R. Soc. Lond. B Biol. Sci. 371, 20150360 (2016).

    Google Scholar 

  101. 101

    Wang, Y. et al. 3K3A-activated protein C stimulates postischemic neuronal repair by human neural stem cells in mice. Nat. Med. 22, 1050–1055 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. 102

    Berndt, N., Kann, O. & Holzhütter, H.-G. Physiology-based kinetic modeling of neuronal energy metabolism unravels the molecular basis of NAD(P)H fluorescence transients. J. Cereb. Blood Flow Metab. 35, 1494–1506 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  103. 103

    Yaseen, M.A. et al. In vivo imaging of cerebral energy metabolism with two-photon fluorescence lifetime microscopy of NADH. Biomed. Opt. Express 4, 307–321 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  104. 104

    Yaseen, M.A. et al. Fluorescence lifetime microscopy of NADH distinguishes alterations in cerebral metabolism in vivo. Biomed. Opt. Express 8, 2368–2385 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  105. 105

    Baraghis, E. et al. Two-photon microscopy of cortical NADH fluorescence intensity changes: correcting contamination from the hemodynamic response. J. Biomed. Opt. 16, 106003 (2011).

    PubMed  PubMed Central  Google Scholar 

  106. 106

    Zhao, Y. et al. In vivo monitoring of cellular energy metabolism using SoNar, a highly responsive sensor for NAD+/NADH redox state. Nat. Protoc. 11, 1345–1359 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  107. 107

    Mongeon, R., Venkatachalam, V. & Yellen, G. Cytosolic NADH-NAD(+) redox visualized in brain slices by two-photon fluorescence lifetime biosensor imaging. Antioxid. Redox Signal. 25, 553–563 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  108. 108

    Vanderkooi, J.M., Maniara, G., Green, T.J. & Wilson, D.F. An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence. J. Biol. Chem. 262, 5476–5482 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  109. 109

    Becker, W. Fluorescence lifetime imaging - techniques and applications: fluorescence lifetime imaging. J. Microsc. 247, 119–136 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  110. 110

    Sakadzi´, S. et al. Simultaneous imaging of cerebral partial pressure of oxygen and blood flow during functional activation and cortical spreading depression. Appl. Opt. 48, D169–D177 (2009).

    Google Scholar 

  111. 111

    Wilson, D.F. et al. Effect of hyperventilation on oxygenation of the brain cortex of newborn piglets. J. Appl. Physiol. 70, 2691–2696 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  112. 112

    Wilson, D.F., Gomi, S., Pastuszko, A. & Greenberg, J.H. Microvascular damage in the cortex of cat brain from middle cerebral artery occlusion and reperfusion. J. Appl. Physiol. 74, 580–589 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. 113

    Yaseen, M.A. et al. Optical monitoring of oxygen tension in cortical microvessels with confocal microscopy. Opt. Express 17, 22341–22350 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  114. 114

    Sakadzi´, S. et al. Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue. Nat. Methods 7, 755–759 (2010).

    Google Scholar 

  115. 115

    Lecoq, J. et al. Simultaneous two-photon imaging of oxygen and blood flow in deep cerebral vessels. Nat. Med. 17, 893–898 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  116. 116

    Devor, A. et al. 'Overshoot' of O2 is required to maintain baseline tissue oxygenation at locations distal to blood vessels. J. Neurosci. 31, 13676–13681 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  117. 117

    Lyons, D.G., Parpaleix, A., Roche, M. & Charpak, S. Mapping oxygen concentration in the awake mouse brain. eLife 5, e12024 (2016).

    PubMed  PubMed Central  Google Scholar 

  118. 118

    Kazmi, S.M.S. et al. Three-dimensional mapping of oxygen tension in cortical arterioles before and after occlusion. Biomed. Opt. Express 4, 1061 (2013).

    PubMed  PubMed Central  Google Scholar 

  119. 119

    Spencer, J.A. et al. Direct measurement of local oxygen concentration in the bone marrow of live animals. Nature 508, 269–273 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  120. 120

    Yaseen, M.A. et al. Multimodal optical imaging system for in vivo investigation of cerebral oxygen delivery and energy metabolism. Biomed. Opt. Express 6, 4994–5007 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  121. 121

    Kalmbach, A.S. & Waters, J. Brain surface temperature under a craniotomy. J. Neurophysiol. 108, 3138–3146 (2012).

    PubMed  PubMed Central  Google Scholar 

  122. 122

    Shirey, M.J. et al. Brief anesthesia, but not voluntary locomotion, significantly alters cortical temperature. J. Neurophysiol. 114, 309–322 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. 123

    Goldey, G.J. et al. Removable cranial windows for long-term imaging in awake mice. Nat. Protoc. 9, 2515–2538 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. 124

    Rumsey, W.L., Vanderkooi, J.M. & Wilson, D.F. Imaging of phosphorescence: a novel method for measuring oxygen distribution in perfused tissue. Science 241, 1649–1651 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  125. 125

    Mik, E.G., van Leeuwen, T.G., Raat, N.J. & Ince, C. Quantitative determination of localized tissue oxygen concentration in vivo by two-photon excitation phosphorescence lifetime measurements. J. Appl. Physiol. 97, 1962–1969 (2004).

    PubMed  PubMed Central  Google Scholar 

  126. 126

    Holtmaat, A. et al. Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window. Nat. Protoc. 4, 1128–1144 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  127. 127

    Polesskaya, O. et al. Detection of microregional hypoxia in mouse cerebral cortex by two-photon imaging of endogenous NADH fluorescence. J. Vis. Exp. (60) (2012).

  128. 128

    Shih, A.Y., Mateo, C., Drew, P.J., Tsai, P.S. & Kleinfeld, D. A polished and reinforced thinned-skull window for long-term imaging of the mouse brain. J. Vis. Exp. 61, 3742 (2012).

    Google Scholar 

  129. 129

    Tallquist, M.D., French, W.J. & Soriano, P. Additive effects of PDGF receptor beta signaling pathways in vascular smooth muscle cell development. PLoS Biol. 1, E52 (2003).

    PubMed  PubMed Central  Google Scholar 

  130. 130

    Schneider, C.A., Rasband, W.S. & Eliceiri, K.W. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  131. 131

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  132. 132

    Sakadži´, S. et al. Cerebral blood oxygenation measurement based on oxygen-dependent quenching of phosphorescence. J. Vis. Exp. (2011), (51).

  133. 133

    Sinks, L.E. et al. Two-photon microscopy of oxygen: polymersomes as probe carrier vehicles. J. Phys. Chem. B 114, 14373–14382 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

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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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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.

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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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