Tissue microarray, high throughput automated microscopy and high content screening facility


Dr Nasim Mehrabi (pictured) and Dr Malvindar Singh-Bains manage the TMA facility.

The tissue microarray (TMA), high throughput automated microscopy and high content screening (HCS) facility at the CBR was established, and is directed by, Professor Mike Dragunow. Funding for establishing the facility was initially obtained from the National Research Centre for Growth and Development/Gravida (a Centre of Excellence), Freemasons Mount Roskill, as well as the Coker Charitable Trust and most recently Brain Research New Zealand.

The facility focuses on developing and using human brain tissue microarray technology to undertake research into brain disorders.

The TMA/HCS facility is run in conjunction with the Neurological Foundation Human Brain Bank, the Hugh Green Biobank and Neurovalida and provides a high throughput facility for molecular pathological analysis of the human brain. Through Neurovalida TMA and HCS are used to help Pharmaceutical and Biotech companies to validate their candidate drugs targets in human brain and in the disease of interest. This work contributes to the development of medications to treat brain disorders.

Deidre Microtome Cutting of Paraffin blocks (1)

 

What is TMA?

TMA is a technique of arraying cores of tissue (in our case human brain tissue) into blocks that can then be cut and used for histological, immunohistochemical, immunofluorescence and in situ hybridization studies. For example, TMA’s designed to study Alzheimer’s disease (AD) comprise 30 cores of middle temporal gyrus (a region affected severely in AD) from AD donors and 30 cores from neurologically normal aged- and sex-matched controls. Thin sections of these arrays are then cut on a microtome and adhered to standard microscope slides and processed in many ways to compare expression of desired proteins in AD.

Because only small cores are used this TMA method saves on precious donated human brain tissue and also because all cores from all 60 donors are studied in one slide any differences between AD and controls are more likely to be discovered as there is very little experimental error involved.

 

What is automated scanning?

Once the slides are processed using a particular staining method (eg: immunohistochemistry) they are then put into an automated slide scanner. The initial scanner to acquire images from TMA’s was the Discovery-1 high content analysis microscope purchased by a grant to Professor Dragunow from the National Research Centre for Growth and Development in 2003, the first facility of its type in Australasia.

More recently, this was updated to the VSlide scanner, based at the BIRU and purchased with a grant to Professor Dragunow from Gravida. This scanner acquires images at high throughput and in an automated standardised fashion. 

TMA images acquired on the VSlide scanner are then analysed using high content analysis/screening with the Metamorph (or other) high throughput image analysis software (also purchased by a grant from the National Research Centre for Growth and Development to Professor Dragunow).

Discovery-1 also acquired images from microplates for high content screening of cells (rather than tissue on slides) and this was the first facility (established in 2004) of its type in Australasia. More recently, Discovery-1 has been replaced by the next generation of cell scanners, with the purchase of ImageXpress micro by the University of Auckland which is housed in BIRU.

What is high content analysis/screening?

High content analysis/screening uses automated image analysis programs to extract simple or complex features from images at high throughput. For example, it can be used to count nuclei from cells for simple cell counts and complex features of nerve cells such as dendritic arborisation. It can also be used to quantify signal transduction processes in cells such as agonist-induced receptor internalisation, nuclear translocation of proteins (eg: NFkB) and cytoplasmic versus nuclear localisation of proteins involved in brain diseases (eg: cytoplasmic tdp-43 inclusions in Motor Neuron Disease).

Essentially HCA/HCS does what our eyes and brain do but more accurately, much more quickly, and in a standardised, objective manner. It allows researchers to put numbers to (ie: quantify) complex features that were previously only analysed qualitatively.

This facility is being used by staff and students of the CBR, BRNZ and other collaborators to study the causes of human brain disorders, and by Neurovalida scientists to identify and validate drugs targets to promote the development of medications for these brain disorders.

 

References that developed the blue-print for the facility

*Dragunow M. The adult human brain in preclinical drug development.  Nature Reviews Drug Discovery. 7, 659-666, 2008. 

*Dragunow M. High content analysis in neuroscience. Nature Reviews Neuroscience 9(10), 779-88, 2008. 

Selected references using this facility

Human brain tissue microarray

Coppieters N, Dieriks BV, Lill C, Faull RLM, Curtis MA, Dragunow M. Global changes in DNA methylation and hydroxymethylation in Alzheimer’s disease human brain. Neurobiology of Aging, 35, 1334-1344, 2014.

Narayan P, Lill C, Faull RLM, Curtis M, Dragunow M. Increased acetyl and total histone levels in post-mortem Alzheimer’s disease brain. Neurobiology of Disease 74, 281-294, 2015.

Narayan P, Kim, S-L, Lill C, Feng S, Faull RLM, Curtis MA, Dragunow M. Assessing fibrinogen extravasation into Alzheimer’s disease brain using high content screening of brain tissue microarrays. Journal of Neuroscience Methods 247, 41-49, 2015.

Yang P, Waldvogel H, Turner C, Faull RLM, Dragunow M, Guan J. Vascular remodelling is impaired in Parkinson’s disease. Journal of Alzheimer's Disease & Parkinsonism 7:313 doi: 10.4172/2161-0460.1000313, 2017.

Yang P, Min, Mohammadi M, Turner C, Faull R, Waldvogel H, Dragunow M and Guan J. Endothelial Degeneration of Parkinson Disease is related to Alpha-Synuclein Aggregation. Alzheimer's Disease & Parkinsonism DOI: 10.4172/2161-0460.1000370, 2017.

Dominy SS, Lynch C, Ermini F, Benedyk M, Marczyk A, konradi A, Nguyen M, Haditsch U, Raha D, Griffin C, Holsinger LJ, Arastu-Kapur S, Kaba S, Lee A, Ryder MI, Potempa B, Mydel P, Hellvard A, Adamowicz K, Hasturk H, Walker GD, Reynolds EC, Faull RLM, Curtis M, Dragunow M, Potempa J. Porphyromonas gingivalis in Alzheimer’s disease brains: evidence for disease causation and treatment with small-molecule inhibitors. Science Advances 23 Jan 2019: Vol. 5, no. 1, eaau3333 DOI: 10.1126/sciadv.aau3333

High content analysis

Dragunow M, Trzoss M, Brimble M, Cameron R, Beuzenberg V, Holland P and Mountford D. Investigations into the cellular actions of the shellfish toxin gymnodimine and analogues. Environmental Toxicology and Pharmacology 20, 305-312, 2005.

Dragunow M, Greenwood J, Cameron R, Narayan P, O’Carroll S, Pearson A, Gibbons H. Valproic acid induces caspase 3-mediated apoptosis in microglial cells. Neuroscience 140 (4) 1149-1156, 2006.

Brimble M, Robinson J, Merten J, Beuzenberg V, Dragunow M, Holland P, and Mountford D. A novel bromine-induced ring expansion of the spiroimine moiety of the shellfish toxin Gymnodimine. SYNLETT 10: 1610-1612 (2006).

Lind CRP, Gray CW, Pearson AG, Cameron RE, O’Carroll S, Narayan P, Lim J, Dragunow M. The MEK 1/2 inhibitor U0126 induces GFAP expression and reduces the proliferation and migration of C6 glioma cells. Neuroscience 141(4): 1925-1933, 2006.

Graham ES, Ball NB, Scotter EL, Narayan P, Dragunow M, Glass M. Induction of krox 24 by endogenous CB1 receptors in Neuro2a cells is mediated by the MEK-ERK MAPK pathway and is suppressed by the PI3K pathway. Journal of Biological Chemistry 281(39):29085-95, 2006.

Curtis MA, Kam M, Nannmark U, Anderson MF, Zetterstrom Axell M, Wikkelso C, Holtås S, van Roon-Mom W, Björk-Eriksson T, Nordborg C, Frisen J, Dragunow M, Faull R, Eriksson P. Human Neuroblasts Migrate to the Olfactory bulb via a Lateral Ventricular Extension. Science 315 (5816) 1243-1249, 2007 (Research Article).

Wilkie RP, Vissers M, Dragunow M, Hampton M. A Functional NADPH Oxidase Prevents Caspase Involvement In The Clearance Of Phagocytic Neutrophils. Infection & Immunity 75 (7) 3256-3263, 2007.

Narayan P, Gibbons H, Mee E, Faull R, Dragunow M. High throughput quantification of cells with complex morphology in mixed cultures. Journal of Neuroscience Methods 164(2) 339-349, 2007.

Lim J, Gibbons HM, O’Carroll S, Narayan P, Faull RLM, and Dragunow M. Extracellular signal-regulated kinase involvement in human astrocyte migration. Brain Research 1164: 1-13, 2007.

Gibbons H, Hughes S, Van Roon-Mom W, Greenwood J, Narayan P, Teoh H,  Bergin P, Wood P, Mee E, Faull R, Dragunow, M. Cellular composition of human glial cultures from adult biopsy brain tissue. Journal of Neuroscience Methods 166, 89-98, 2007.

Dragunow M, Cameron R, Narayan P, and O’Carroll S. Image-based high-through-put quantification of cellular fat accumulation. Journal of Biomolecular Screening 12(7) 999-1005, 2007.

Scotter E, Narayan P, Glass M, Dragunow M. High Throughput Quantification of Mutant Huntington Aggregates. Journal of Neuroscience Methods 171, 174-179, 2008.

Grimsey N, Narayan P, Dragunow M, Glass M. Novel high-throughput assay for the quantitative assessment of receptor trafficking. Clinical and Experimental Pharmacology and Physiology 35(11), 1377-1382, 2008.

Byrne U, Bond J, Faull RLM, Dragunow M. High-throughput quantification of Alzheimer’s disease pathological markers in the post-mortem human brain. Journal of Neuroscience Methods, 176, 298-309, 2009.

Shinjyo N, Stahlberg A, Dragunow M, Pekny M, Pekna M. Complement-derived anaphylatoxin C3a regulates in vitro differentiation and migration of neural progenitor cells. Stem Cells 27, 2824-2832, 2009.

Scotter E, Goodfellow C, Graham E, Dragunow M, Glass M. Neuroprotective Potential of CB1 Receptor Agonists in an in vitro model of Huntington’s Disease. British Journal of Pharmacology 160 (3), 747-761, 2010.

Smith A, Gibbons H, Dragunow M. Valproic acid enhances microglial phagocytosis of amyloid 1-42. Neuroscience 169(1), 505-515, 2010.

Grimsey N, Graham S, Dragunow M, Glass M. Cannabinoid receptor trafficking and the role of the intracellular pool: implications for therapeutics. Biochemical Pharmacology 80(7):1050-1062, 2010.

Narayan P Dragunow M. High content analysis of histone acetylation in human cells and tissues. Journal of Neuroscience Methods 193, 54-61, 2010.

Gibbons H, Smith A, Teoh H, Bergin P, Mee E, Faull RLM, Dragunow M. Valproic acid induces microglial dysfunction, not apoptosis, in human glial cultures. Neurobiology of Disease 41, 96-103, 2011.

Grimsey N, Goodfellow C, Dragunow M, Glass M. Cannabinoid Receptor 2 undergoes Rab5-mediated internalization and recycles via a Rab11-dependent pathway. BBA Molecular Cell Research 1813: 1554-1560, 2011.

Monzo H, Park T, Montgomery J, Faull RLM, Dragunow M, Curtis M. A method for generating high-yield enriched neuronal cultures from P19 embryonal carcinoma cells. Journal of Neuroscience Methods 204(1):87-103, 2011.

Jowers C, Kim J, Taberner A, Dragunow M, Anderson I. The Cell Injury Device: A High-Throughput Platform for Traumatic Brain Injury Research. Journal of Neurotrauma 28: A17-A18, June 2011.

Park T, Monzo H, Mee EW, Bergin PS, Teoh HH, Montgomery JM, Faull RLM, Curtis MA, Dragunow M. Adult Human Brain Neural Progenitor Cells (NPCs) and Fibroblast-Like Cells Have Similar Properties In Vitro but Only NPCs Differentiate Into Neurons. PLoS One, 92(4):841-9, 2012.

Parker H, Dragunow M, Hampton M, Kettle A, Winterbourn C. Requirements for NADPH Oxidase and Myeloperoxidase in Neutrophil Extracellular Trap Formation Differ Depending on Stimulus. Journal of Leukocyte Biology 92, 841-849, 2012.

Smith A, Gibbons H, Oldfield RL, Bergin PM, Mee EW, Faull RLM, Dragunow M. The transcription factor PU.1 is critical for viability and function of human brain microglia. Glia 61(6):929-42, 2013.

Smith AM, Graham ES, Dragunow M. Differential regulation of HLA and IP-10 in adult human glia. 11th International Congress of Neuroimmunology (ISNI), Boston, MA, 04 Nov 2012 - 08 Nov 2012. Journal of Neuroimmunology 253: 115-115. 15 Dec 2012 (Conference Abstract).

Jowers C, Taberner A, Dragunow M, Anderson I. The Cell Injury Device: A High Throughput Platform for Traumatic Brain Injury Research. Journal of Neuroscience Methods 218, 1-8, 2013.

Vance C, Scotter E, Nishimura A, Troakes C, Mitchell J, Kathe C, Urwin H, Manser C, Miller C, Hortobagyi T, Dragunow M, Rogelj B, Shaw CE. ALS mutant FUS disrupts nuclear localisation and sequesters wild-type FUS within cytoplasmic stress granules. Human Molecular Genetics 22(13): 2676-88, 2013

Jarlestedt K, Rousset C, Stahlberg A, Sourkova H, Atkins A, Thomton C, Barnum S, Wetsel R, Dragunow M, Pekny M, Mallard C, Hagberg H, Pekna M. Receptor for complement peptide C3a: a therapeutic target for neonatal hypoxic-ischemic brain injury. FASEB J 27(9) 3797-3804, 2013.

Heapy A, Dragunow M, Brimble M. Synthesis of the cysteine protease inhibitors CPI-2081a and CPI-2081b using a controlled SPPS by-product forming reaction. SYNLETT: LETTER 24, 1818-1824, 2013.

Smith A, Gibbons H, Oldfield R, Bergin P, Mee E, Curtis M, Faull RLM, Dragunow M. M-CSF increases proliferation and phagocytosis while modulating receptor and transcription factor expression in adult human microglia. Journal of Neuroinflammation 10: 85. doi: 10.1186/1742-2094-10-85, 2013

Park T, Waldvogel H, Montgomery J, Mee E, Bergin P, Faull RLM, Dragunow M, Curtis M. Chapter 27. Identifying neural progenitor cells in the adult human brain. In Neural Progenitor Cells: Methods and Protocols B Reynolds and L. Deleyrolle (Eds.). Springer Science+Business Media New York, 1059:195-225, 2013.

Smith A, Gibbons H, Lill C, Faull RLM, Dragunow M. Isolation and culture of adult human microglia within mixed glial cultures for functional experimentation and high content analysis. In Microglia: Methods in Molecular Biology Edited by Bertrand Joseph and José Luis Venero, Humana Press, NY, Volume 1041, 2013, pp 41-51.

Graham S, Woo K, Aalderink M, Fry S, Greenwood J, Glass M, Dragunow M. M1 muscarinic receptor activation mediates cell death in M1-HEK293 Cells. PLoS One 2013 Sep 2; 8(9):e72011.

Smith A, Graham S, Feng S, Oldfield R, Bergin P, Mee E, Faull RLM, Curtis M, Dragunow M. Adult human glia, pericytes and meningeal fibroblasts respond similarly to IFNg but not to TGFβ1 or M-CSF. PLoS One 2013 Dec 5;8(12):e80463

Jansson D, Rustenhoven J, Feng S, Hurley D, Oldfield RL, Bergin PS, Mee EW, Faull RLM, Dragunow M. A role for human brain pericytes in neuroinflammation. Journal of Neuroinflammation 11:104 DOI: 10.1186/1742-2094-11-104, 2014.

Wassink G, Barrett RD, Davidson JO, Bennet L, Galinsky R, Dragunow M, Gunn A. Greater hypothermic neuroprotection is associated with better recovery of spectral edge frequency after asphyxia in preterm fetal sheep. Stroke 46, 585-587, 2015.

Rustenhoven J, Scotter E, Jansson D, Kho D, Oldfield R, Bergin PS, Mee E, Faull RLM, Curtis M, Graham S, Park T, Dragunow M. An anti-inflammatory role for CEBPδ in human brain pericytes. Scientific Reports 5:12132. doi: 10.1038/srep12132, 2015.

Dragunow M, Feng S, Rustenhoven J, Curtis M, Faull RLM. Studying human brain inflammation in leptomeningeal and choroid plexus explant cultures. Neurochemical Research 41(3) 579-588 2016.

Bansal A, Bloomfield F, Connor K, Dragunow M, Thortensen E, Oliver M, Sloboda D, Harding J, Alsweiler J. Glucocorticoid-induced preterm birth and neonatal hyperglycemia alter ovine beta cell development. Endocrinology 156(10) 3763-3776, 2015.

Yang P, Pavlovic D, Waldvogel H, Dragunow M, Synek B, Turner C, Faull RLM, Guan J. String vessel formation is increased in the brain of Parkinson disease. Journal of Parkinsons Disease 5, 821-836, 2015.

Rustenhoven J, Park T, Schweder P, Scotter J, Correia J, Smith A, Gibbons H, Oldfield R, Bergin P, Mee E, Faull RLM, Curtis M, Graham ES, Dragunow M. Isolation of highly enriched primary human microglia for functional studies.  Scientific Reports, 26: 19371. doi: 10.1038/srep19371 2016.

Rustenhoven J, Aalderink M, Scotter E, Oldfield R, Bergin P, Mee E, Graham ES, Faull RLM, Curtis M, Park T, Dragunow M.  TGFβ1 regulates human brain pericyte inflammatory processes involved in neurovascular function. Journal of Neuroinflammation, 6: 26587. doi: 10.1038/srep26587, 2016.

Park T, Feisst V, Brooks A, Rustenhoven J, Monzo H, Feng S, Mee E, Bergin P, Oldfield R, Graham ES, Curtis M, Faull RLM, Dunbar R, Dragunow M. Cultured pericytes from human brain show phenotypic and functional differences with differential CD90 expression. Scientific Reports 6:26587. doi: 10.1038/srep26587, 2016.

Jansson D, Scotter E, Rustenhoven J, Coppieters N, Smyth L, Oldfield RL, Bergin PS, Mee EW, Faull RLM, Dragunow M. Interferon-γ blocks signalling through PDGFRβ in human brain pericytes. Journal of Neuroinflammation. 13(1):249, 2016.

Stokowska A, Atkins AL, Suárez JM, Pekny T, Bulmer L, Pascoe MC, Barnum SR, Wetsel RA, Nilsson JA, Dragunow M, Pekna M.  Complement peptide C3a stimulates neural plasticity after experimental brain ischemia. Brain 140(Pt 2):353-369. doi: 10.1093/brain/aww314, 2017.

Justin Rustenhoven; Leon Smyth; Deidre Jansson; Patrick Schweder; Miranda Aalderink; Emma Scotter; Edward Mee; Richard Faull; Thomas Park; Michael Dragunow. Modelling physiological and pathological conditions to study pericyte biology in brain function and dysfunction. BMC Neuroscience Feb 22; 19(1):6. doi: 10.1186/s12868-018-0405-4, 2018.

Smyth L, Rustenhoven J , Park T, Schweder P, Jansson D, Heppner PA, O?Carroll SJ, Mee EW, Faull RLM, Curtis M, Dragunow M. Unique and shared inflammatory profiles of human brain endothelia and pericytes. Journal of Neuroinflammation, 2018 May 11; 15(1):138. doi: 10.1186/s12974-018-1167-8.

Smyth L, Rustenhoven J, Scotter EL, Schweder P, Faull RLM, Park TIH, Dragunow M. Markers for human brain pericytes and smooth muscle cells, Journal of Chemical Neuroanatomy (in press, 7/6/18, 2018 Jun 7. pii: S0891-0618(18)30007-3. doi: 10.1016/j.jchemneu.2018.06.001).

Justin Rustenhoven; Amy Smith; Leon Smyth; Deidre Jansson; Emma Scotter; Molly Swanson; Miranda Aalderink; Natacha Coppieters; Pritika Narayan; Renee Handley; Chris Overall; Thomas Park; Patrick Schweder; Peter Heppner; Maurice Curtis; Richard Faull; Mike Dragunow. PU.1 regulates Alzheimer's disease-associated genes in primary human microglia. Molecular Neurodegeneration (In press, August 2018, doi: 10.1186/s13024-018-0277-1).