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.
Highlights
Mechanical stretch-injury is a prominent force involved in the etiology of traumatic brain injury (TBI)
Following a 20% stretch-injury, media was assayed for lactate dehydrogenase (LDH) at 1, 6, 12, 18, 24, 48, and 72 hours post-injury and showed no significant differences in media LDH content between stretch-injured and control cells at any time point (Fig. 1A)
To further assess microglia viability, an immunocytochemistry evaluation of cleaved caspase-3, a marker of apoptosis, was conducted on fixed cells at the 12 hours post-injury time-point and revealed no significant difference between injured and non-injured cells
Summary
Mechanical stretch-injury is a prominent force involved in the etiology of traumatic brain injury (TBI). The deleterious effects of stretch-injury on microglia, the brain’s primary immunocompetent cell, are currently unknown. The primary injury results from the impact or acceleration events generating mechanical forces that act upon the sensitive brain tissue, resulting in immediate, irreparable damage and dysfunction. A prominent secondary injury process is the inflammatory response, primarily mediated by activated microglia, the brain’s immunocompetent cell [3]. The initial immune response is thought to be a protective and restorative process; there are multiple experimental models [4,5,6,7] and human [8, 9] cases of TBI that implicate prolonged microglia activation in deleterious outcomes.
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