Age-Dependent Microglial Activation in Immature Brains After Hypoxia- Ischemia

Abstract

In the present study, we tested whether the ongoing differentiation of microglia in the immature brain results in more robust microglial activation and pro-inflammatory responses than juvenile brains following hypoxia-ischemia (HI). Under normoxic conditions, microglial activation profiles were assessed in postnatal day 9 and postnatal day 30 mice (P9 and P30) by analyzing relative expression levels of CD45 in CD11b+/CD45+ microglia/macrophages. Flow cytometry analysis revealed that the hippocampi of P9 and P30 brains exhibited higher levels of CD45 expression in CD11b+/CD45+ cells than in the cortex and striatum. In response to HI, there was an early increase in number of CD11b+/CD45+ microglia/macrophages in the ipsilateral hippocampus of P9 mice. These cells transformed from a “ramified” to an “amoeboid” morphology in the CA1 region, which was accompanied by a loss of microtubule-associated protein 2 immunostaining in this brain region. The peak response of microglial activation in the ipsilateral hippocampus of P9 mice occurred on day 2 post-HI, which was in contrast to a delayed and persistent microglial activation in the cortex and striatum (peak on day 9 post-HI). P9 brains demonstrated a 2–3 fold greater increase in microglia counts than P30 brains in each region (hippocampus, cortex, and striatum) during day 1–17 post-HI. P9 brains also showed more robust expression of pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1β) than P30 brains. Taken together, compared to P30 mice, P9 mice demonstrated differences in microglial activation and pro-inflammatory responses after HI, which may be important in brain damage and tissue repair.

Suppression of microglia activation after hypoxia–ischemia results in age-dependent improvements in neurologic injury

“…Ipsilateral microglia counts were normalized to the counts in the corresponding contralateral hemisphere (IL/CL), ( Figure 1 ). At 2 days post-HI, HI induced a robust increase in microglia counts in the ipsilateral hippocampus of non-treated P9 mice (~ 9.5 fold), and less microglia proliferation in P30 hippocampus (~5.7 fold) ( Figure 1 A ), similar to what we reported previously(Ferrazzano, Chanana, 2013a). By comparison, in cortex and striatum the increase in ipsilateral microglial population in P9 and P30 at day 2 post-HI was comparatively modest (1.7 - 3.5 times) ( Figure 1 B, C ).…”

“…As previously described, CD45 expression can be used to characterize the activation state of microglia (Campanella et al, 2002, Denker et al, 2007, Ferrazzano, Chanana, 2013a, Stevens et al, 2002), where low signal is associated with quiescent microglia, medium intensity with activated microglia and high signal labeling blood-born monocytes. In the current study, less than 1% of the CD11b + /CD45 + population demonstrated CD45 high expression, similar to our prior studies(Ferrazzano, Chanana, 2013a). Figure 2 A-C, shows the percentage of CD45 medium activated microglia in P9 and P30 brains at day 2 and day 9 post-HI in hippocampus, cortex and striatum.…”

“…We previously demonstrated that microglia in the P9 brain respond to HI with greater proliferation and pro-inflammatory cytokine release compared to P30 brains (Ferrazzano, Chanana, 2013a). In the current study, we similarly found that P9 mice demonstrated a greater increase in microglia counts compared to P30 mice in the hippocampus early after injury.…”

“…Using the Vannucci model of neonatal hypoxia-ischemia (Gurd et al, 1999), HI was induced in P9 and P30 mice as described previously (Ferrazzano et al, 2013b). C57BL/6J mice (P9 and P30) were anesthetized with isoflurane (3% for induction, 1.5% for maintenance), in 30% O 2 and 70% N 2 O.…”

“…We previously described that microglia in the neonatal brain respond to hypoxia-ischemia (HI) with increased activation, proliferation, and release of pro-inflammatory mediators compared to juveniles (Ferrazzano et al, 2013a). How the microglial response aggravates or ameliorates injury after hypoxia-ischemia in the developing brain has remained poorly understood.…”