Temporal Characterization of Microglia/Macrophage Phenotypes in a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury

Abstract

Immune cells display a high degree of phenotypic plasticity, which may facilitate their participation in both the progression and resolution of injury-induced inflammation. The purpose of this study was to investigate the temporal expression of genes associated with classical and alternative polarization phenotypes described for macrophages and to identify related cell populations in the brain following neonatal hypoxia-ischemia (HI). HI was induced in 9-day old mice and brain tissue was collected up to 7 days post-insult to investigate expression of genes associated with macrophage activation. Using cell-markers, CD86 (classic activation) and CD206 (alternative activation), we assessed temporal changes of CD11b+ cell populations in the brain and studied the protein expression of the immunomodulatory factor galectin-3 in these cells. HI induced a rapid regulation (6 h) of genes associated with both classical and alternative polarization phenotypes in the injured hemisphere. FACS analysis showed a marked increase in the number of CD11b+CD86+ cells at 24 h after HI (+3667%), which was coupled with a relative suppression of CD11b+CD206+ cells and cells that did not express neither CD86 nor CD206. The CD11b+CD206+ population was mixed with some cells also expressing CD86. Confocal microscopy confirmed that a subset of cells expressed both CD86 and CD206, particularly in injured gray and white matter. Protein concentration of galectin-3 was markedly increased mainly in the cell population lacking CD86 or CD206 in the injured hemisphere. These cells were predominantly resident microglia as very few galectin-3 positive cells co-localized with infiltrating myeloid cells in Lys-EGFP-ki mice after HI. In summary, HI was characterized by an early mixed gene response, but with a large expansion of mainly the CD86 positive population during the first day. However, the injured hemisphere also contained a subset of cells expressing both CD86 and CD206 and a large population that expressed neither activation marker CD86 nor CD206. Interestingly, these cells expressed the highest levels of galectin-3 and were found to be predominantly resident microglia. Galectin-3 is a protein involved in chemotaxis and macrophage polarization suggesting a novel role in cell infiltration and immunomodulation for this cell population after neonatal injury.

Aggravated brain injury after neonatal hypoxic ischemia in microglia-depleted mice

“…In our study, analyses at 72 h after the injury showed no significant differences in the levels of several pro-inflammatory cytokines. This could be attributed to our experimental design of the evaluation of pro-inflammatory cytokines at the end of the sub-acute phase [3,62]. We found differences in the antiinflammatory cytokines between DTA − and DTA + mice.…”

“…Neonatal hypoxic-ischemic encephalopathy (HIE) is a major worldwide cause of neonatal death and long-term disability, including mental retardation, visual motor or visual perceptive dysfunction, hyperactivity, cerebral palsy, and epilepsy. HI insults are known to induce inflammatory reactions within hours in rodent models with microglia playing an important role [1][2][3][4][5].…”

Lipopolysaccharide‐induced alteration of mitochondrial morphology induces a metabolic shift in microglia modulating the inflammatory response in vitro and in vivo

“…Neuroinflammation includes complex changes in microglial phenotypes, mediated by gene expression changes leading to the production of cytokines and chemokines and production of ROS. Altogether this triggers oxidative and nitrosative stress in the brain (Bolouri et al, ; Hellström Erkenstam et al, ). We observed as expected that LPS‐activated microglia produced a plethora of chemokines and cytokines and ROS.…”

Purmorphamine Attenuates Neuro-Inflammation and Synaptic Impairments After Hypoxic-Ischemic Injury in Neonatal Mice via Shh Signaling

“…In the HI brain, there is also a considerable contribution of infiltrating peripheral immune cells to the brain after injury (Hellstrom Erkenstam et al, 2016). Next, we investigated the effect of PUR administration on infiltrating monocytes/neutrophils (CD11b + /CD45 high cells).…”