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Pericyte degeneration leads to neurovascular uncoupling and limits oxygen supply to brain

Nature Neuroscience volume 20, pages 406–416 (2017)Cite this article

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Abstract

Pericytes are perivascular mural cells of brain capillaries. They are positioned centrally in the neurovascular unit between endothelial cells, astrocytes and neurons. This position allows them to regulate key neurovascular functions of the brain. The role of pericytes in the regulation of cerebral blood flow (CBF) and neurovascular coupling remains, however, under debate. Using loss-of-function pericyte-deficient mice, here we show that pericyte degeneration diminishes global and individual capillary CBF responses to neuronal stimuli, resulting in neurovascular uncoupling, reduced oxygen supply to the brain and metabolic stress. Neurovascular deficits lead over time to impaired neuronal excitability and neurodegenerative changes. Thus, pericyte degeneration as seen in neurological disorders such as Alzheimer's disease may contribute to neurovascular dysfunction and neurodegeneration associated with human disease.

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Figure 1: Delayed capillary dilation and reduced capillary red blood cell flow in response to a hindlimb stimulus in 1- to 2-month-old pericyte-deficient Pdgfrb+/− mice.
Figure 2: Impaired hemodynamic response in 1- to 2-month-old pericyte-deficient Pdgfrb+/− mice determined by IOS imaging (530 nm) at a time point when neuronal activity is unaffected as determined by LFP recordings.
Figure 3: Arteriolar vasoactivity and endothelium-dependent vasodilation in 1- to 2-month-old pericyte-deficient Pdgfrb+/− mice and age-matched littermate controls.
Figure 4: Diminished brain tissue oxygen levels and oxygen delivery in young pericyte-deficient Pdgfrb+/− mice.
Figure 5: Metabolic changes in 1- to 2-month-old pericyte-deficient Pdgfrb+/− mice.
Figure 6: Cortical neuronal activity, neuronal number, neuritic density and behavior in pericyte-deficient Pdgfrb+/− mice 1–2 and 6–8 months old, along with age-matched littermate controls.

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Acknowledgements

We would like to thank R. Angus for statistical discussions. This research was supported by National Institutes of Health grants R01 AG023084, R01 AG039452, R01 NS034467 and R01 NS100459 to B.V.Z.; R01 NS091230 to S.S.; and R01 EB000790, R24 NS092986 and P01 NS055104 to D.A.B.; National Natural Science Foundation of China grant 31371116 to Y.Z.; and American Heart Association grant SDG7600037 to S.S.

Author information

Author notes

  1. Kassandra Kisler, Amy R Nelson and Sanket V Rege: These authors contributed equally to this work.

Authors and Affiliations

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

    Kassandra Kisler, Amy R Nelson, Sanket V Rege, Anita Ramanathan, Yaoming Wang, Ashim Ahuja, Divna Lazic, Zhen Zhao & Berislav V Zlokovic

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

    Divna Lazic

  3. Department of Physics, University of California, San Diego, La Jolla, California, USA

    Philbert S Tsai

  4. Department of Neurobiology, Chongqing Key Laboratory of Neurobiology, Third Military Medical University, Shapingba District, Chongqing, China

    Yi Zhou

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

    David A Boas & Sava Sakadžić

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Contributions

K.K., A.R.N. and A.R. contributed to manuscript preparation and to experimental design and analysis, and conducted experiments. S.V.R. and S.S. contributed to experimental design and to data analysis and interpretation, and conducted experiments. A.A., D.L. and Y.W. conducted and analyzed experiments. P.S.T. contributed to three-dimensional angiography analysis and software. Z.Z. contributed to experimental design and analysis. Y.Z. contributed to data analysis. D.A.B. contributed to project design. B.V.Z. supervised and designed all experiments and analysis, and wrote the manuscript.

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Correspondence to Berislav V Zlokovic.

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Integrated supplementary information

Supplementary Figure 1 The average sigmoid parametric curves of capillary dilation time courses and average time to 50% peak capillary diameter dilation computed from individual capillary responses averaged per animal in control Pdgfrb+/+ mice (top, red traces, n = 12 mice, 37 individual capillaries) and pericyte-deficient Pdgfrb+/− mice (bottom, blue traces, n = 9 mice, 33 individual capillaries).

The time to 50% peak dilation corresponds to the time at which the sigmoid curve reaches 50% of its maximal value, as illustrated by the intercepts of the horizontal dashed line at 0.5 on the ordinate with the curves (diameter increase) and the vertical dashed line intercepts with the abscissa (time to 50% peak dilation). Arrows below the abscissa illustrate time intercepts showing 50% peak capillary diameter dilation per mouse in each group. As in Fig. 1c, diameter changes are expressed relative to the respective basal capillary diameter prior to stimulation (value set as 0) and maximal diameter after stimulation (value set as 1). The average sigmoid curves were derived from individual sigmoid curves fitted to individual capillary responses per animal. The sigmoid fits for individual capillary responses with their intercepts with the horizontal dashed line at 0.5 on the ordinate (diameter increase) and the vertical dashed line intercepts (time to 50% peak dilation) with the abscissa are not shown because of the high degree of overlap, which makes difficult to distinguish responses between individual capillaries. However, data (circles) in Fig. 1d were derived from averaging the individual capillary responses per mouse (time to 50% peak dilation) from sigmoid fits of individual vessel dilation time courses per animal, and then the individual times for 50% peak dilation values were averaged per animal.

Supplementary Figure 2 Pericyte capillary coverage in 1- to 2-month-old Pdgfrb+/− mice and age-matched littermate controls.

(a) Representative confocal microscopy images of pericyte coverage (CD13, magenta) of lectin-positive brain endothelial capillary profiles (< 6 μm in diameter; lectin, blue) in the stimulated cortical S1 area in Pdgfrb+/+ and Pdgfrb+/− mice. (b) Quantification of CD13-positive pericyte coverage of cortical capillaries in Pdgfrb+/+ and Pdgfrb+/− mice from a. Mean ± 95% CI; n = 5 Pdgfrb+/+ and 6 Pdgfrb+/− mice per group (t-test, single tail, equal variance: t = 11.36, p = 6.1e-7). In each animal 5 randomly selected fields from the cortex were analyzed in 6 non-adjacent sections (~100 μm apart) and averaged per mouse to obtain individual values (circles) as illustrated. (c) Vascular smooth muscle cell actin (SMa; yellow) and pericyte marker CD13 in Pdgfrb+/+ control mice illustrating that pericytes lining lectin-positive endothelial capillary profiles are largely negative for SMa. An SMα-positive arteriole (arrow) is shown for comparison.

Source data

Supplementary Figure 3 Baseline vessel diameters, RBC flow velocity and thickness of the arteriole smooth muscle cell layer in 1- to 2-month-old pericyte-deficient Pdgfrb+/− mice and age-matched littermate controls.

(a) Average baseline in vivo vessel diameters (mean ± 95% CI) prior to stimulation determined for 21 total arterioles from 9 Pdgfrb+/+ mice, 27 total arterioles from 11 Pdgfrb+/− mice, 57 total capillaries from 10 Pdgfrb+/+ mice and 40 total capillaries from 10 Pdgfrb+/− mice (arterioles: t-test, equal variance: t = 0.27, p = 0.79; capillaries: t-test, equal variance: t = 1.04, p = 0.31). (b) Average (mean ± 95% CI) baseline in vivo RBC velocity acquired prior to stimulation in 14 total arterioles from 6 Pdgfrb+/+ mice and 23 total arterioles from 10 Pdgfrb+/− mice, and 32 total capillaries from 9 Pdgfrb+/+ mice and 36 total capillaries from 12 Pdgfrb+/− mice (t-test, equal variance: arterioles: t = 0.23 p = 0.82, capillaries: t = 0.33, p = 0.74). (c) Representative images of the smooth muscle cell layer (SMα, red) thickness and lack of pericyte coverage (CD13, magenta) on arterioles. (d) Thickness of the arteriolar smooth muscle cell layer determined by confocal microscopy analysis. Mean ± 95% CI from 42 arterioles from 7 Pdgfrb+/+ mice and 45 arterioles from 6 Pdgfrb+/− mice (t-test, equal variance: t = 1.14, p = 0.28).Two-tail t-tests used.

Source data

Supplementary Figure 4 Astrocyte coverage of capillary wall, astrocyte and microglia numbers and capillary length in 1- to 2-month-old pericyte-deficient Pdgfrb+/− mice and age-matched littermate controls.

(a) Confocal microscopy of immunolabeled aquaporin-4-positive astrocytic endfoot coverage of microvessels (lectin), GFAP-positive astrocytes, and Iba1-positive microglia in the S1 cortical region. (b-d) Quantification (mean ± 95% CI) of aquaporin-4-positive (AQP4+) astrocytic endfoot coverage (t-test, equal variance: t = 0.24, p = 0.82) (b), the number of GFAP-positive astrocytes (t-test, equal variance: t = 0.93, p = 0.38) (c), and Iba1-positive microglia (t-test, equal variance: t = 0.26, p = 0.80) (d) from 5 Pdgfrb+/+ and 5 Pdgfrb+/− mice at 1-2 months of age; in each animal 6 randomly selected fields from the cortex were analyzed in 5 nonadjacent sections (~100 μm apart) and data were averaged per mouse to obtain individual values (circles) as illustrated. (e) 3D reconstructions of 880 x 330 x 1000 μm sections of Pdgfrb+/+ and Pdgfrb+/− vasculature in the S1 somatosensory cortex region. Scale bar = 200 μm. Insets: Single 50 μm thick slices horizontally through cortex cortex layer IV. (f) Comparison of vascular density with depth in cortex of Pdgfrb+/− and Pdgfrb+/+ mice for all vessels <35 μm diameter (top; t-test, single tail, equal variance: t = 1.68, p = 0.07), and vascular density of capillaries (< 6 μm diameter; t-test, single tail, equal variance: t = 1.77, p = 0.06) and larger vessels between 6-35 μm (bottom; t-test, two tail, equal variance: t = 0.10, p = 0.92) measured in Pdgfrb+/− and Pdgfrb+/+ mice. Lines represent average values ± 95% CI from n = 4 mice per group. T-tests based on average vessel density through all cortical layers per mouse.

Source data

Supplementary Figure 5 Capillary length, pericyte coverage and vessel dilation in young Meox2+/− mice.

(a) Representative image of pericyte coverage (CD13, magenta; left) of lectin-positive brain endothelial capillary profiles (< 6 μm in diameter; lectin, blue; middle) and overlay (right) in Meox2+/− and Meox2+/+ control mice. (b,c) Quantification (mean ± 95% CI) of total capillary length calculated from the length of lectin-positive endothelial profiles < 6 μm in diameter (b, Mann-Whitney U test, single tail, p = 0.05), and pericyte coverage of lectin-positive brain endothelial capillary profiles (c, Mann-Whitney U test, p = 1.00) in 3 control Meox2+/+ and 3 Meox2+/− mice (b), and 5 control Meox2+/+ and 3 Meox2+/− mice (c). In each animal 6 randomly selected fields from the cortex were analyzed in 5 nonadjacent sections (~100 μm apart) and data were averaged per mouse to obtain individual values (circles) as illustrated. (d, e) Average time (mean ± CI) to 50% peak capillary diameter (d, Mann-Whitney U test, p = 0.79) (d) or 50% peak arteriole diameter (e, Mann-Whitney U test, p = 0.55) was determined after an electrical hind limb stimulation (10 s, 10 Hz, 2 ms pulse duration) in capillaries from 7 control Meox2+/+ (16 total capillaries) mice and 3 Meox2+/− mice (15 total capillaries), and arterioles from 6 control Meox2+/+ mice (13 total arterioles) and 3 Meox2+/− mice (12 total arterioles). Sigmoid parametric fit analysis was performed as for figure 1 c–e. Bootstrapped in panels b–e.

Source data

Supplementary Figure 6 Lactate and glucose plasma levels in 1- to 2-month-old pericyte-deficient Pdgfrb+/− mice and age-matched littermate controls.

(a) Serum lactate levels (mean + 95% CI) in 5 Pdgfrb+/+ and 5 Pdgfrb+/− mice (t-test, equal variance: t = 0.51, p = 0.63). (b) Serum glucose levels (mean ± 95% CI.) in 6 Pdgfrb+/+ and 4 Pdgfrb+/− mice (t-test, equal variance: t = 0.08, p = 0.94).

Source data

Supplementary Figure 7 Cortical neuronal activity in 1- to 2-month-old pericyte-deficient Pdgfrb+/− mice and age-matched littermate controls.

(a) Representative pseudo-colored voltage-sensitive dye (VSD) image sequences of cortical neuronal activity in the S1 region in response to a 300 ms mechanical hind limb stimulus in Pdgfrb+/+ and Pdgfrb+/− mice. Hind limb region (HL) is indicated by dashed line. Scale bar = 0.5 mm. (b-d) Representative VSD intensity traces (b), time to peak (Mann-Whitney U test, p = 0.84; bootstrapped) (c) and peak fluorescence change (t-test, two tailed, equal variance: t = 0.11, p = 0.92) (d) in Pdgfrb+/+ and Pdgfrb+/− mice. Arrow in (b) indicates peak VSD signal for Pdgfrb+/− (blue) and littermate control Pdgfrb+/+ (red) mice. Mean ± 95% CI, from n = 5 mice per group.

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Supplementary Figure 8 Cerebral blood flow response and pericyte coverage in 6- to 8-month-old pericyte-deficient Pdgfrb+/− mice and age-matched littermate controls.

(a) Cerebral blood flow (CBF) response to an electrical hind limb stimulus (60 s, 7 Hz, 2 ms pulse duration) determined by laser doppler flowmetry (LDF) as a percentage of baseline change in 4 Pdgfrb+/− mice and 4 Pdgfrb+/+ littermate controls at 6-8 months of age. Circles denote individual values derived from 3 independent LDF measurements per mouse; mean + 95% CI (Mann-Whitney U test, p = 0.03; bootstrapped). (b) Representative confocal microscopy images of pericyte coverage (CD13, magenta) of lectin-positive brain endothelial capillary profiles (< 6 μm in diameter; lectin, blue) in the S1 cortical area in Pdgfrb+/+ and Pdgfrb+/− mice at 6-8 mo of age. (c) Quantification (mean ± 95% CI) of CD13-positive pericyte coverage of cortical capillaries in 4 Pdgfrb+/+ and 4 Pdgfrb+/− mice as in b (t-test, single tail, equal variance: t = 4.89, p = 0.001). In each animal 6 randomly selected fields from the cortex were analyzed in 5 nonadjacent sections (~100 μm apart) and data were averaged per mouse to obtain individual values (circles) as illustrated.

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Supplementary Figures 1–8 and Supplementary Table 1 (PDF 1134 kb)

Supplementary Methods Checklist (PDF 539 kb)

Illustration of diameter change upon stimulus in a pericyte-covered capillary in vivo in a pericyte-deficient Pdgfrb+/−;dsRed mouse using TPLSM.

Beginning of the movie shows a still image indicating pericyte location on a vessel before zooming to the magnification used for time-lapse imaging. The second part of the movie shows time-lapse imaging of vessel dilation in response to stimulus. Dashed lines indicate vessel's pre-stimulus diameter. Arrowheads indicate maximal extent of dilation during stimulus. Appearance of white dot in upper left corner indicates application of stimulus. Images were acquired at 1.57 s/frame and played at 2 fps. (AVI 1048 kb)

Illustration of a minimal diameter change upon stimulus in a pericyte-absent capillary in vivo in a pericyte-deficient Pdgfrb+/−;dsRed mouse using TPLSM.

Beginning of the movie shows a still image of a pericyte-free vessel before zooming to the magnification used for time-lapse imaging. The second part of the movie shows time-lapse imaging of vessel dilation in response to stimulus. Dashed lines indicate vessel's pre-stimulus diameter. Arrowheads indicate maximal extent of dilation during stimulus. Appearance of white dot in upper left corner indicates application of stimulus. Images were acquired at 1.57 s/frame and played at 2 fps. (AVI 969 kb)

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Kisler, K., Nelson, A., Rege, S. et al. Pericyte degeneration leads to neurovascular uncoupling and limits oxygen supply to brain. Nat Neurosci 20, 406–416 (2017). https://doi.org/10.1038/nn.4489

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