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

Abstract

Background: 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. Methods: The Cell Injury Controller II (CICII), a validated model of cellular neurotrauma, was used to induce a mechanical stretch-injury in primary rat microglia. Statistical analysis utilized student t-test and one and two way-ANOVAs with Tukey’s and Sidak’s multiple comparisons, respectively. Results: 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 production nitric oxide (NO), identified by media assay and immunocytochemistry, at 6, 12, 18, and 48 hours post-injury. Furthermore, gene expression analysis revealed the expression of inflammatory cytokines IL-6 and IL-10 and enzyme arginase-1 was significantly down-regulated at 12 hours 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 hours 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 hours. Conclusions: Taken together, the data demonstrate that stretch-injury produces significant alterations in microglial function, which may have marked impact on their response to injury or their interaction with other cells.