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Preparation, construction and high-throughput automated analysis of human brain tissue microarrays for neurodegenerative disease drug development

Nature Protocols volume 16, pages 2308–2343 (2021)Cite this article

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Abstract

A major challenge in the treatment of neurodegenerative disorders is the translation of effective therapies from the lab to the clinic. One approach to improve this process is the use of human brain tissue microarray (HBTMA) technology to aid in the discovery and validation of drug targets for brain disorders. In this protocol we describe a platform for the production of high-quality HBTMAs that can be used for drug target discovery and validation. We provide examples of the use of this platform and describe detailed protocols for HBTMA design, construction and use for both protein and mRNA detection. This platform requires less tissue and reagents than single-slide approaches, greatly increasing throughput and capacity, enabling samples to be compared in a more consistent way. It takes 4 d to construct a 60 core HBTMA. Immunohistochemistry and in situ hybridization take a further 2 d. Imaging of each HBTMA slide takes 15 min, with subsequent high-content analysis taking 30 min–2 h.

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Fig. 1: Overview of preparation, construction and high-throughput automated analysis of HBTMAs.
Fig. 2: Process of constructing TMAs.
Fig. 3: Examples of IHC and ISH staining of a TMA.
Fig. 4: Integrated optical density (scaled): measurement of total core area.
Fig. 5: Count nuclei: object number and protein expression (integrated intensity).
Fig. 6: Neurite outgrowth: cellular processes and neurite length/branching.
Fig. 7: Blood vessel quantification: vessel number, area, bifurcation.
Fig. 8: Examples of IHC staining in TMA with several unsuitable cores for analysis.

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Data availability

Datasets generated and described in our supporting primary papers can be made available from the corresponding author upon reasonable request. An example of data generated using this protocol is available in Supplementary Table 1.

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Acknowledgements

We express our appreciation to all donor families in New Zealand, who through their generosity have enabled their invaluable tissue donation to the Neurological Foundation Human Brain Bank for the construction of HBTMAs. We thank M. A. Curtis, Deputy Director of the Neurological Foundation Human Brain Bank. We thank Senior Histologist S. Amirapu for her contribution to tissue processing and paraffin embedding protocols for TMA preparation. We thank C. Turner for his expert neuropathological assessments of the cases utilized for TMA construction. We acknowledge the excellent work and assistance of M. Eszes (Human Brain Bank), K. Hubbard and C. Lill (Research Technicians). We acknowledge the staff at the Biomedical Imaging Research Unit (R. Kurian and P. V. Anekal) for VSlide Scanner support. We acknowledge P. J. Narayan and E. L. Scotter for their contributions to the development of the TMA imaging and analysis protocols (references cited in the manuscript). We acknowledge M. D. R. Austria for his contribution to the presubmission enquiry. This work was supported by a Programme Grant from the Health Research Council of New Zealand, Brain Research New Zealand, Neurological Foundation of New Zealand, the Hugh Green Foundation, The Coker Trust, the Sir Thomas and Lady Duncan Trust and the Freemasons Foundation of New Zealand. MKSB was supported by the Leo Nilon Huntington’s Disease Research Fellowship.

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Author notes

  1. These authors contributed equally: Malvindar K. Singh-Bains, Nasim F. Mehrabi.

Authors and Affiliations

  1. Centre for Brain Research, University of Auckland, Auckland, New Zealand

    Malvindar K. Singh-Bains, Nasim F. Mehrabi, Adelie Y. S. Tan, Richard L. M. Faull & Mike Dragunow

  2. Department of Anatomy and Medical Imaging, University of Auckland, Auckland, New Zealand

    Malvindar K. Singh-Bains, Adelie Y. S. Tan & Richard L. M. Faull

  3. Department of Pharmacology and Clinical Pharmacology, University of Auckland, Auckland, New Zealand

    Nasim F. Mehrabi & Mike Dragunow

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  1. Malvindar K. Singh-Bains
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Contributions

M.K.S.B. and N.F.M. contributed equally to manuscript production, figure production, HBTMA design, construction and analysis protocols. A.Y.S.T. contributed to figure production and HBTMA analysis protocols. R.L.M.F. supervised the ethical collection, donor interaction, clinical assessment and processing of human brain tissue for the HBTMA platform. M.D. conceived, established and directs the HBTMA platform and high-content-analysis platform. M.K.S.B. and N.F.M. wrote the draft and completed the final manuscript with the contributions, edits and approvals of the final manuscript from A.Y.S.T., R.L.M.F. and M.D.

Corresponding author

Correspondence to Mike Dragunow.

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Competing interests

M.D. and R.L.M.F. have developed a platform called Neurovalida for commercial use of human brain tissue microarray.

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Peer review information Nature Protocols thanks Christel Herold-Mende and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Key references using this protocol

Dominy, S. S. et al. Sci. Adv. 5, eaau3333 (2019): https://doi.org/10.1126/sciadv.aau3333

Singh-Bains, M. K. et al. Neurobiol. Dis. 132, 104589 (2019): https://doi.org/10.1016/j.nbd.2019.104589

Narayan, P. J. et al. J. Neurosci. Methods 247, 41–49 (2015): https://doi.org/10.1016/j.jneumeth.2015.03.017

Supplementary information

Supplementary Table 1

Supplementary Table 1 contains example data outputs generated from count nuclei (gray heading), neurite outgrowth (pink heading) and optical density scaled (yellow heading) Metamorph journals

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Singh-Bains, M.K., Mehrabi, N.F., Tan, A.Y.S. et al. Preparation, construction and high-throughput automated analysis of human brain tissue microarrays for neurodegenerative disease drug development. Nat Protoc 16, 2308–2343 (2021). https://doi.org/10.1038/s41596-021-00503-7

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