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A fluoro-Nissl dye identifies pericytes as distinct vascular mural cells during in vivo brain imaging

Nature Neuroscience volume 20, pages 1023–1032 (2017)Cite this article

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Abstract

Pericytes and smooth muscle cells are integral components of the brain microvasculature. However, no techniques exist to unambiguously identify these cell types, greatly limiting their investigation in vivo. Here we show that the fluorescent Nissl dye NeuroTrace 500/525 labels brain pericytes with specificity, allowing high-resolution optical imaging in the live mouse. We demonstrate that capillary pericytes are a population of mural cells with distinct morphological, molecular and functional features that do not overlap with precapillary or arteriolar smooth muscle actin-expressing cells. The remarkable specificity for dye uptake suggests that pericytes have molecular transport mechanisms not present in other brain cells. We demonstrate feasibility of longitudinal pericyte imaging during microvascular development and aging and in models of brain ischemia and Alzheimer's disease. The ability to easily label pericytes in any mouse model opens the possibility of a broad range of investigations of mural cells in vascular development, neurovascular coupling and neuropathology.

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Figure 1: NeuroTrace labels vascular cells lining capillaries in the live mouse brain.
Figure 2: NeuroTrace exclusively labels Pdgfrb-expressing capillary pericytes.
Figure 3: NeuroTrace labels capillary pericytes and not arteriolar smooth muscle cells.
Figure 4: NeuroTrace-labeled pericytes are embedded in the basal lamina.
Figure 5: NeuroTrace does not label α-SMA-expressing mural cells.
Figure 6: NeuroTrace-labeled pericytes do not exhibit spontaneous vasomotion.
Figure 7: In vivo imaging of pericytes during development and into advanced aging.
Figure 8: Pericyte imaging in Alzheimer's mouse models in vivo.

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Acknowledgements

We thank K. Hirschi (Yale University) for sharing SMA-mCherry mice, V. Lindner (Maine Medical Center Research Institute) for sharing Pdgfrb-cre mice, A. Nishiyama (University of Connecticut) for sharing NG2-cre mice and R. Vassar (Northwestern University) for sharing 5xFAD mice. We thank X. Liu and M. Graham at the biological electron microscopy facility core (Yale School of Medicine) for assistance with sample preparation and imaging. This work was supported by the following grants from the National Institutes of Health: R21NS087511, R21NS088411, R01NS0889734 and R21AG048181 to J.G. and F32NS090820 to R.A.H.

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Author notes

  1. Eyiyemisi C Damisah and Robert A Hill: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neurology, Yale School of Medicine, New Haven, Connecticut, USA

    Eyiyemisi C Damisah, Robert A Hill, Lei Tong, Katie N Murray & Jaime Grutzendler

  2. Department of Neurosurgery, Yale School of Medicine, New Haven, Connecticut, USA

    Eyiyemisi C Damisah

  3. Department of Neuroscience, Yale School of Medicine, New Haven, Connecticut, USA

    Robert A Hill, Lei Tong, Katie N Murray & Jaime Grutzendler

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Contributions

E.C.D., R.A.H. and J.G. made the initial observation and designed all experiments. E.C.D. and R.A.H. performed the morphological, developmental and functional characterization of dye-labeled cells. L.T. conducted Alzheimer's mouse model characterization and prepared tissue for electron microscopy. K.N.M. performed fixed tissue quantification and surgeries for carotid occlusion experiments. R.A.H. and J.G. wrote the manuscript with input from all authors. J.G. supervised the study.

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Correspondence to Jaime Grutzendler.

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Integrated supplementary information

Supplementary Figure 1 Pdgfrb-cre:Tomato transgenic mice exhibit complete labeling of vascular mural cells that are colabeled with Pdgfrb and NG2 antibodies.

a) Confocal fluorescence images captured from the cortex of Pdgfrb-cre:Tomato transgenic mice stained with an antibody against Pdgfrb (Pdgfrb Ab) showing complete overlap between Tomato expressing mural cells (arrowheads) and antibody staining.

(b) Confocal fluorescence images captured from the cortex of Pdgfrb-cre:Tomato transgenic mice stained with an antibody against NG2 (NG2 Ab) showing expected NG2 expression by mural cells (arrowheads) and also by NG2-glia and processes in the parenchyma (arrows).

Supplementary Figure 2 NeuroTrace-labeled pericytes are embedded in the basal lamina.

(a) Confocal fluorescence images captured from the cortex of Pdgfrb-cre:Tomato transgenic mice stained with collagen IV antibody showing detection of the double layer of the basal lamina completely surrounding capillary pericyte previously labeled with NeuroTrace dye. Single pericytes labeled with NeuroTrace and Tomato were imaged immediately after tissue sectioning but before immunostaining and then reimaged after immunostaining for collagen IV confirming that NeuroTrace labels capillary pericytes (n=11 cells). Top panel is the same cell shown in Figure 4c top only a single z plane. All images are single z-plane.

Supplementary Figure 3 Intracerebral injection results in rapid, exclusive labeling of capillary pericytes

(a-b) Schematic, in vivo images and time-lapse sequence showing intracerebral injection of NeuroTrace resulting in exclusive pericyte labeling minutes after dye injection (arrows). Location of injection site is indicated by drawing of the glass micropipette within the image.

(c) NeuroTrace fluorescence intensity measurements during intracerebral injection of NeuroTrace dye at 4 regions of interest (ROIs) showing specific targeting of capillary pericytes indicated by ROIs 2 and 4.

Supplementary Figure 4 Microvascular pathology during cerebral ischemia

(a) Schematic showing timeline and approach for in vivo imaging during transient cerebral ischemia.

(b-d) In vivo time-lapse sequences captured from the cerebral cortex showing collapse and reperfusion of SMC-covered (arrows) (SMA-mCherry+) but no change in diameter in pericyte-covered (NeuroTrace+) vessels.

(e) Percent changes in diameter from baseline in SMC and pericyte covered vessels showing significant vessel collapse/constriction during ischemia only on SMC-covered vessels (pericyte covered: 28 vessel locations, SMC covered: 23 vessel locations from n=3 mice, unpaired two-tailed student’s t-test p values as indicated during: t=6.244279 df=4, after: t=1.104133 df=4).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 1007 kb)

Supplementary Methods Checklist (PDF 397 kb)

NeuroTrace labeling in the live mouse cortex.

Video shows a z-stack of NeuroTrace labeling in the cortex of a live mouse with intravascular dye used to visualize the cortical blood vessels. Depth into the cortex is indicated in upper right corner, with NeuroTrace labeling reaching up to 400 μm into the tissue. (MP4 6873 kb)

Exclusive pericyte labeling after intracerebral NeuroTrace injection.

Video shows an in vivo time-lapse sequence captured during consecutive injections of NeuroTrace into the cerebral cortex, showing exclusive labeling of pericytes within minutes after parenchymal injection. Injections are indicated as green rectangles in upper right corner and time is indicated in seconds. (MP4 26634 kb)

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Damisah, E., Hill, R., Tong, L. et al. A fluoro-Nissl dye identifies pericytes as distinct vascular mural cells during in vivo brain imaging. Nat Neurosci 20, 1023–1032 (2017). https://doi.org/10.1038/nn.4564

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