Please use this identifier to cite or link to this item: https://hdl.handle.net/2440/119630
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Type: Journal article
Title: Visualizing neuroinflammation with fluorescence and luminescent lanthanide-based in situ hybridization
Author: Parker, L.M.
Sayyadi, N.
Staikopoulos, V.
Shrestha, A.
Hutchinson, M.R.
Packer, N.H.
Citation: Journal of Neuroinflammation, 2019; 16(1):65-1-65-13
Publisher: Springer Nature
Issue Date: 2019
ISSN: 1742-2094
1742-2094
Statement of
Responsibility: 
Lindsay M. Parker, Nima Sayyadi, Vasiliki Staikopoulos, Ashish Shrestha, Mark R. Hutchinson, and Nicolle H. Packer
Abstract: Background: Neurokine signaling via the release of neurally active cytokines arises from glial reactivity and is mechanistically implicated in central nervous system (CNS) pathologies such as chronic pain, trauma, neurodegenerative diseases, and complex psychiatric illnesses. Despite significant advancements in the methodologies used to conjugate, incorporate, and visualize fluorescent molecules, imaging of rare yet high potency events within the CNS is restricted by the low signal to noise ratio experienced within the CNS. The brain and spinal cord have high cellular autofluorescence, making the imaging of critical neurokine signaling and permissive transcriptional cellular events unreliable and difficult in many cases. Methods: In this manuscript, we developed a method for background-free imaging of the transcriptional events that precede neurokine signaling using targeted mRNA transcripts labeled with luminescent lanthanide chelates and imaged via time-gated microscopy. To provide examples of the usefulness this method can offer to the field, the mRNA expression of toll-like receptor 4 (TLR4) was visualized with traditional fluorescent in situ hybridization (FISH) or luminescent lanthanide chelate-based in situ hybridization (LISH) in mouse BV2 microglia or J774 macrophage phenotype cells following lipopolysaccharide stimulation. TLR4 mRNA staining using LISH- and FISH-based methods was also visualized in fixed spinal cord tissues from BALB/c mice with a chronic constriction model of neuropathic pain or a surgical sham model in order to demonstrate the application of this new methodology in CNS tissue samples. Results: Significant increases in TLR4 mRNA expression and autofluorescence were visualized over time in mouse BV2 microglia or mouse J774 macrophage phenotype cells following lipopolysaccharide (LPS) stimulation. When imaged in a background-free environment with LISH-based detection and time-gated microscopy, increased TLR4 mRNA was observed in BV2 microglia cells 4 h following LPS stimulation, which returned to near baseline levels by 24 h. Background-free imaging of mouse spinal cord tissues with LISH-based detection and time-gated microscopy demonstrated a high degree of regional TLR4 mRNA expression in BALB/c mice with a chronic constriction model of neuropathic pain compared to the surgical sham model. Conclusions: Advantages offered by adopting this novel methodology for visualizing neurokine signaling with time-gated microscopy compared to traditional fluorescent microscopy are provided.
Keywords: Neuroinflammation; time-gating microscopy; neurokine; microglia; macrophages; chronic pain
Rights: © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
DOI: 10.1186/s12974-019-1451-2
Grant ID: http://purl.org/au-research/grants/arc/CE140100003
http://purl.org/au-research/grants/arc/DE180100206
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