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Assessment of the Effects of Stretch-Injury on Primary Rat Microglia

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Abstract

Mechanical stretch-injury is a prominent force involved in the etiology of traumatic brain injury (TBI). It is known to directly cause damage and dysfunction in neurons, astrocytes, and endothelial cells. However, the deleterious effects of stretch-injury on microglia, the brain’s primary immunocompetent cell, are currently unknown. The Cell Injury Controller II (CICII), a validated cellular neurotrauma model, was used to induce a mechanical stretch-injury in primary rat microglia. Statistical analysis utilized Student’s t test and one- and two-way ANOVAs with Tukey’s and Sidak’s multiple comparisons, respectively. Cells exposed to stretch-injury showed no signs of membrane permeability, necrosis, or apoptosis, as measured by media-derived lactate dehydrogenase (LDH) and cleaved-caspase 3 immunocytochemistry, respectively. Interestingly, injured cells displayed a functional deficit in nitric oxide production (NO), identified by media assay and immunocytochemistry, at 6, 12, 18, and 48 h post-injury. Furthermore, gene expression analysis revealed the expression of inflammatory cytokines IL-6 and IL-10, and enzyme arginase-1 was significantly downregulated at 12 h post-injury. Time course evaluation of migration, using a cell exclusion zone assay, showed stretch-injured cells display decreased migration into the exclusion zone at 48- and 72-h post-stretch. Lastly, coinciding with the functional immune deficits was a significant change in morphology, with process length decreasing and cell diameter increasing following an injury at 12 h. Taken together, the data demonstrate that stretch-injury produces significant alterations in microglial function, which may have a marked impact on their response to injury or their interaction with other cells.

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Data Availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Funding

This work was supported by the Defense Health Program, Congressionally Directed Medical Research Programs through the Spinal Cord Injury Research Program under Award No. SC170244. Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Defense Health Program, Congressionally Directed Medical Research Programs, the Department of Defense, or the Uniformed Services University. Additional funding was provided by the Center for the Study of Traumatic Stress (CSTS) at USUHS.

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  1. Neuroscience Program, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD, 20814, USA

    Mike Shaughness & Kimberly Byrnes

  2. Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, MD, USA

    Kimberly Byrnes

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MC designed and performed experiments, analyzed data, and drafted the manuscript. KB co-conceived the study, participated in its design and coordination, and helped draft the manuscript. All authors have read and approved the final version of the manuscript.

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Shaughness, M., Byrnes, K. Assessment of the Effects of Stretch-Injury on Primary Rat Microglia. Mol Neurobiol 58, 3545–3560 (2021). https://doi.org/10.1007/s12035-021-02362-5

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