Hypothermia modulates myeloid cell polarization in neonatal hypoxic–ischemic brain injury

Abstract

Background Neonatal encephalopathy due to hypoxia–ischemia (HI) is a leading cause of death and disability in term newborns. Therapeutic hypothermia (HT) is the only recommended therapy. However, 30% still suffer from neurological deficits. Inflammation is a major hallmark of HI pathophysiology with myeloid cells being key players, participating either in progression or in resolution of injury-induced inflammation. In the present study, we investigated the impact of HT on the temporal and spatial dynamics of microglia/macrophage polarization after neonatal HI in newborn mice. Methods Nine-day-old C57BL/6 mice were exposed to HI through occlusion of the right common carotid artery followed by 1 h hypoxia. Immediately after HI, animals were cooled for 4 h or kept at physiological body core temperature. Analyses were performed at 1, 3 and 7 days post HI. Brain injury, neuronal cell loss, apoptosis and microglia activation were assessed by immunohistochemistry. A broad set of typical genes associated with classical (M1) and alternative (M2) myeloid cell activation was analyzed by real time PCR in ex vivo isolated CD11b+ microglia/macrophages. Purity and composition of isolated cells was determined by flow cytometry. Results Immediate HT significantly reduced HI-induced brain injury and neuronal loss 7 days post HI, whereas only mild non-significant protection from HI-induced apoptosis and neuronal loss were observed 1 and 3 days after HI. Microglia activation, i.e., Iba-1 immunoreactivity peaked 3 days after HI and was not modulated by HT. However, ex vivo isolated CD11b+ cells revealed a strong upregulation of the majority of M1 but also M2 marker genes at day 1, which was significantly reduced by HT and rapidly declined at day 3. HI induced a significant increase in the frequency of peripheral macrophages in sorted CD11b+ cells at day 1, which deteriorated until day 7 and was significantly decreased by HT. Conclusion Our data demonstrate that HT-induced neuroprotection is preceded by acute suppression of HI-induced upregulation of inflammatory genes in myeloid cells and decreased infiltration of peripheral macrophages, both representing potential important effector mechanisms of HT.

Persistent cortical and white matter inflammation after therapeutic hypothermia for ischemia in near-term fetal sheep

“…We speculate that maximal hypothermia-mediated shift to M2 polarization in white matter may only be achieved with earlier initiation of hypothermia. This hypothesis is consistent with evidence in P9 mice that after common carotid ligation and hypoxia, hypothermia started immediately after the insult was associated with downregulated CD86 expression at day 1 [ 17 ].…”

“…The net effect of microglial induction seems to be beneficial as shown by exacerbation of brain injury when microglia were depleted in postnatal day 10 (P10) mice after common carotid artery ligation plus hypoxia [ 15 ]. Nevertheless, in studies in P9 mice, HI was associated with significant upregulation of both pro and anti-inflammatory genes from 24 h [ 16 , 17 ]. Supporting the hypothesis that activation of these pro-inflammatory genes is deleterious, in P10 mice, administration of the anti-inflammatory factor colony-stimulating factor 1 after HI was neuroprotective [ 18 ].…”

“…Supporting the hypothesis that activation of these pro-inflammatory genes is deleterious, in P10 mice, administration of the anti-inflammatory factor colony-stimulating factor 1 after HI was neuroprotective [ 18 ]. Potentially, these changes may be a target for therapeutic hypothermia; in P9 mice HI was associated with upregulation of the “pro-inflammatory” marker CD86 and the “anti-inflammatory” marker CD206 at day 1, while immediate hypothermia for just 4 h after HI was associated with suppression of the pro-inflammatory response with reduced infiltration of peripheral macrophages [ 17 ].…”

Systemic Maternal Human sFLT1 Overexpression Leads to an Impaired Foetal Brain Development of Growth-Restricted Foetuses upon Experimental Preeclampsia

“…Microglia are highly dynamic cells, and their activation can be functionally differentiated to a proinflammatory M1-phenotype or an alternative anti-inflammatory M2-phenotype. M2-microglia typically secrete anti-inflammatory cytokines and growth factors which rather promote repair and regeneration of damaged tissue, whereas M1-microglia specifically release proinflammatory cytokines and chemokines and contribute to neurodegeneration [ 61 ]. To check which phenotype is predominant in offspring of our PE model, cellular distribution and morphology of microglia as well as the expression of pro- and anti-inflammatory cytokine chemokines and growth factors need to be characterized in future studies.…”

Targeting Persistent Neuroinflammation after Hypoxic-Ischemic Encephalopathy—Is Exendin-4 the Answer?

“…There is recent evidence to suggest that hypothermia may also play a role in modulating the polarization of microglia. Hypothermia started immediately after common carotid artery ligation and hypoxia was associated with suppressing the expression of “pro-inflammatory” CD 86 and increased expression of “anti-inflammatory” CD206 in P9 rats [ 62 ]. Recently, it has been reported that hypothermia partially reduced CD86 expression in the cortex, but not in the white matter in near-term fetal sheep at 7 days after global cerebral ischemia [ 11 ].…”