Quantifying Spinal Cord Neuroinflammation

To analyse AFSM accumulation and glial activation in the grey matter (GFAP‐positive astrocytes + CD68‐positive microglia), markers of oligodendrocyte precursors (Olig2 and NG2) in the white matter and CGRP‐positive fibres in laminae III‐IV of the dorsal horn of the spinal cord, semiautomated thresholding image analysis was performed using Image‐Pro Premier (Media Cybernetics) [17]. This involved collecting slide‐scanned images at 10× magnification from each animal followed by demarcation of regions of interest. Images were subsequently analysed using Image‐Pro Premier (Media Cybernetics) using an appropriate threshold that selected the foreground immunoreactivity above background. Due to the density of interneurons in laminae I‐III of the dorsal horn [20, 21, 22], image densitometry for the average pixel luminance data was gathered for those antigens that showed a higher density of staining (calbindin, calretinin and parvalbumin) and for other antigens with higher staining densities (CGRP, Substance P). Slide‐scanned images at 10x magnification were collected, followed by collecting the mean luminance data across all pixels from regions of interest delineated using StereoInvestigator (MBF Bioscience).

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Nelvagal H.R., Dearborn J.T., Ostergaard J.R., Sands M.S, & Cooper J.D. (2020). Spinal manifestations of CLN1 disease start during the early postnatal period. Neuropathology and Applied Neurobiology, 47(2), 251-267.

Publication 2020

Image‐Pro Premier StereoInvestigator

Animal Antigens Astrocytes Calbindin Calretinin Densitometry Dorsal horn Fibres Gfap Glial Grey matter Interneurons Laminae iii Laminae iv Microglia Olig2 Oligodendrocyte precursors Parvalbumin Substance p White matter

Corresponding Organization : Washington University in St. Louis

Other organizations : Aarhus University Hospital

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

AFSM accumulation in the grey matter
Glial activation (GFAP-positive astrocytes and CD68-positive microglia) in the grey matter
Markers of oligodendrocyte precursors (Olig2 and NG2) in the white matter
CGRP-positive fibres in laminae III-IV of the dorsal horn of the spinal cord

control variables

None explicitly mentioned

controls

No positive or negative controls were specified by the authors.

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