M. C. Scott1, O. LeBlanc1, C. Haase1, J. H. Day1, S. D. Olson1, C. S. Cox1 1University Of Texas Health Science Center At Houston, Department Of Pediatric Surgery, Houston, TX, USA
Introduction: Traumatic brain injury is a leading cause of death and morbidity in the trauma population. TBI patients are at risk for complications like sepsis. Microglia are the tissue resident macrophages of the central nervous system; they mount the primary neuroinflammatory response in the secondary phase of TBI. We sought to determine if the inflammatory response to neurologic injury by microglia was exacerbated by a second stimulus.
Methods: Sprague-Dawley rats (age 2-3 weeks) were divided into injured and non-injured groups. Injured rats underwent a controlled cortical impact injury; non-injured rats remained naïve to any injury. At 24 hours, rats were sacrificed. Primary rat microglia were isolated from cerebral tissue and applied to in vitro cultures. Microglia from the ipsilateral and contralateral hemispheres of injured rats were separately isolated. After incubation for 24 hours, pre-specified rat microglia were stimulated with lipopolysaccharide (100ng/mL) or norepinephrine (10μM). Twenty-four hours after stimulation, cell culture supernatant was collected. TNF-α and Il-6 production were measured by standard ELISAs. Statistical analysis was performed using GraphPad Prism.
Results: LPS induced a significant increase in TNF-α production from microglia from non-injured rats (non-activated vs LPS-stimulation mean difference = 411.6 ± 155.1 pg/mL, p = 0.0298) and from microglia isolated from the ipsilateral (1364 ± 155.1, p<0.0001) and contralateral hemispheres (852.3 ± 155.1, p<0.0001) of injured rats. LPS induced a significantly greater production of TNF-α in microglia isolated from the injured ipsilateral (versus non-injured = 938.8 ± 155.1, p<0.0001) and injured contralateral hemispheres (versus non-injured = 426.6 ± 155.1, p<0.0001). LPS increased IL-6 production in post-injury microglia obtained from the injured ipsilateral (mean difference non-activated vs LPS-stimulation = 12317 ± 3016 pg/mL, p = 0.0014) and contralateral hemisphere (17441 ± 3016, p<0.0001). When compared to microglia from non-injured cerebral tissue, IL-6 production was significantly greater after LPS stimulation in the injured ipsilateral hemisphere (mean difference vs non-injured = 9540 ± 3016, p = 0.0101) and the contralateral hemisphere (16700 ± 3016, p<0.0001). Norepinephrine did not have a significant effect on IL-6 or TNF-α production.
Conclusion: Microglia drive neuroinflammation after TBI. LPS-stimulation may amplify the release of pro-inflammatory cytokines from post-injury microglia. This data suggests that post-TBI complications, like sepsis, may propagate neuroinflammation by augmenting the pro-inflammatory response of microglia.
