Abstract
BackgroundAfter central nervous system injury, inflammatory macrophages (M1) predominate over anti-inflammatory macrophages (M2). The temporal profile of M1/M2 phenotypes in macrophages and microglia after traumatic brain injury (TBI) in rats is unknown. We subjected female rats to severe controlled cortical impact (CCI) and examined the postinjury M1/M2 time course in their brains.MethodsThe motor cortex (2.5 mm left laterally and 1.0 mm anteriorly from the bregma) of anesthetized female Wistar rats (ages 8 to 10 weeks; N = 72) underwent histologically moderate to severe CCI with a 5-mm impactor tip. Separate cohorts of rats had their brains dissociated into cells for flow cytometry, perfusion-fixed for immunohistochemistry (IHC) and ex vivo magnetic resonance imaging or flash-frozen for RNA and protein analysis. For each analytical method used, separate postinjury times were included for 24 hours; 3 or 5 days; or 1, 2, 4 or 8 weeks.ResultsBy IHC, we found that the macrophagic and microglial responses peaked at 5 to 7 days post-TBI with characteristics of mixed populations of M1 and M2 phenotypes. Upon flow cytometry examination of immunological cells isolated from brain tissue, we observed that peak M2-associated staining occurred at 5 days post-TBI. Chemokine analysis by multiplex assay showed statistically significant increases in macrophage inflammatory protein 1α and keratinocyte chemoattractant/growth-related oncogene on the ipsilateral side within the first 24 hours after injury relative to controls and to the contralateral side. Quantitative RT-PCR analysis demonstrated expression of both M1- and M2-associated markers, which peaked at 5 days post-TBI.ConclusionsThe responses of macrophagic and microglial cells to histologically severe CCI in the female rat are maximal between days 3 and 7 postinjury. The response to injury is a mixture of M1 and M2 phenotypes.
Highlights
After central nervous system injury, inflammatory macrophages (M1) predominate over anti-inflammatory macrophages (M2)
The rat brains were harvested for ex vivo magnetic resonance imaging (MRI), and tissues were processed for flow cytometry, IHC and RNA and protein analysis
Expression of the M2 marker CD163 was significantly different relative to baseline at 3 and 5 days postinjury (DPI) (CD163 expression as percentage of cells counted: control = 0.9 ± 0.2%, 3 DPI = 12 ± 3%, 5 DPI = 20 ± 2%; F = 55.70, P ≤ 0.0001 by one-way analysis of variance (ANOVA))
Summary
After central nervous system injury, inflammatory macrophages (M1) predominate over anti-inflammatory macrophages (M2). The temporal profile of M1/M2 phenotypes in macrophages and microglia after traumatic brain injury (TBI) in rats is unknown. The initial trauma to the brain triggers an inflammatory response, Similar to the phenotypes of T cells in autoimmune and inflammatory diseases, different subsets of peripheral macrophages (inflammatory macrophages (M1) and antiinflammatory macrophages (M2a and M2c)) have been identified in gliomas [5] and in breast and lung cancer [6,7], as well as after injury caused by acute peripheral nerve transection [8] and spinal cord injury [9]. Macrophages and microglia do not exist as immutable subsets; rather, they are sensitive to their host tissue microenvironments, with their phenotypes determined in part by their surroundings and the length of time after injury [9,12,13,14,15]. The M2 subset results from activation of macrophages with interleukin 4 (IL-4) or IL-13 and can support reparative and regenerative processes [10,11,17,18]
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