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Imaging pericytes and capillary diameter in brain slices and isolated retinae

Nature Protocols volume 9, pages 323–336 (2014)Cite this article

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A Corrigendum to this article was published on 25 September 2014

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Abstract

The cerebral circulation is highly specialized, both structurally and functionally, and it provides a fine-tuned supply of oxygen and nutrients to active regions of the brain. Our understanding of blood flow regulation by cerebral arterioles has evolved rapidly. Recent work has opened new avenues in microvascular research; for example, it has been demonstrated that contractile pericytes found on capillary walls induce capillary diameter changes in response to neurotransmitters, suggesting that pericytes could have a role in neurovascular coupling. This concept is at odds with traditional models of brain blood flow regulation, which assume that only arterioles control cerebral blood flow. The investigation of mechanisms underlying neurovascular coupling at the capillary level requires a range of approaches, which involve unique technical challenges. Here we provide detailed protocols for the successful physiological and immunohistochemical study of pericytes and capillaries in brain slices and isolated retinae, allowing investigators to probe the role of capillaries in neurovascular coupling. This protocol can be completed within 6–8 h; however, immunohistochemical experiments may take 3–6 d.

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Figure 1: Neuronal activity results in the release of neurotransmitters that stimulate astrocytes via receptors on these glial cells.
Figure 2: Vessels in a live, acutely isolated retina preparation from an NG2DsRedBAC mouse, in the absence of any drugs.
Figure 3: Examples of healthy and unhealthy capillaries in ex vivo preparations in the absence of any drugs.
Figure 4: Removal and dissection of the brain for preparation of acute neocortical slices.
Figure 5: Pericytes regulate capillary diameter in response to neurotransmitters.
Figure 6: Immunohistochemical labeling of brain pericytes and capillaries in 200-μm-thick fixed cortical slices from the NG2DsRedBAC mouse.

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Change history

  • 02 May 2014

     In the version of this article initially published, the authors omitted the following from the acknowledgments: "We thank A. Nishiyama and D. Dietrich for providing NG2-DsRed mice". The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

This work was supported by the Wellcome Trust, Fondation Leducq, the European Research Council, the Medical Research Council and Rosetrees Trust. We thank A. Nishiyama and D. Dietrich for providing NG2-DsRed mice. We acknowledge C. Howarth, P. Mobbs and C. Peppiatt-Wildman, whose work paved the way for the development of these protocols.

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Author notes

  1. Anusha Mishra and Fergus M O'Farrell: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neuroscience, Physiology and Pharmacology, University College London, London, UK

    Anusha Mishra, Fergus M O'Farrell, Clare Reynell, Nicola B Hamilton, Catherine N Hall & David Attwell

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  1. Anusha Mishra
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Contributions

A.M., F.M.O., C.R., N.B.H. and C.N.H. prepared the figures and wrote the paper. A.M., C.R., N.B.H. and C.N.H. developed the experimental protocols and performed experiments. C.N.H. and D.A. supervised the project. All authors discussed the results and commented on the manuscript at all stages.

Corresponding authors

Correspondence to Catherine N Hall or David Attwell.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Number of healthy capillaries found per unit area in the surface 50 μm of brain slices from P12 and P30 rats.

P30 slices contain very few capillaries when sliced in standard slicing solution, but significantly more healthy capillaries are found when sliced using NMDG-based slicing solution (two-way ANOVA, with Tukey's post hoc test, mean ± sem). Numbers on bars indicate number of brain slices.

Supplementary Figure 2 Slice incubation chamber constructed of Perspex blocks for use when imaging brain slices.

The slice sits on the glass coverslip, under a harp, and is submerged in perfusate flowing through the chamber from the inlet to the outlet (also see Fig. 4g).

Supplementary information

Supplementary Figure 1

Number of healthy capillaries found per unit area in the surface 50 μm of brain slices from P12 and P30 rats (PDF 89 kb)

Supplementary Figure 2

Slice incubation chamber constructed of Perspex blocks for use when imaging brain slices. (PDF 59 kb)

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Mishra, A., O'Farrell, F., Reynell, C. et al. Imaging pericytes and capillary diameter in brain slices and isolated retinae. Nat Protoc 9, 323–336 (2014). https://doi.org/10.1038/nprot.2014.019

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