Abstract
Background: Perinatal asphyxia, leading to neonatal encephalopathy, is one of the leading causes for child mortality and long-term morbidities. Neonatal encephalopathy rates are significantly increased in newborns with perinatal infection. Therapeutic hypothermia is only neuroprotective in 50% of cooled asphyxiated newborns. As shown experimentally, cooling has failed to be neuroprotective after inflammation-sensitized hypoxic ischemic (HI) brain injury. Microglia are thought to be key players after inflammation-sensitized HI brain injury. We performed this study investigating early microglia phenotype polarization in our newborn animal model of inflammation-sensitized HI brain injury, better understanding the underlying pathophysiological processes.Methods: Seven days old Wistar rat pups were injected with either vehicle (NaCl 0.9%) or E. coli lipopolysaccharide (LPS), followed by left carotid ligation combined with global hypoxia inducing a mild unilateral hypoxic-ischemic injury. Pups were randomized to (1) Sham group (n = 41), (2) LPS only group (n = 37), (3) Veh/HI group (n = 56), and (4) LPS/HI group (n = 79). On postnatal days 8 and 14 gene-expression analysis or immunohistochemistry was performed describing early microglia polarization in our model.Results: We confirmed that LPS pre-sensitization significantly increases brain area loss and induced microglia activation and neuronal injury after mild hypoxia-ischemia. Additionally, we show that microglia upregulate pro-inflammatory genes involving NLRP-3 inflammasome gene expression 24 h after inflammation-sensitized hypoxic-ischemic brain injury.Conclusion: These results demonstrate that microglia are early key mediators of the inflammatory response following inflammation-sensitized HI brain injury and that they polarize into a predominant pro-inflammatory phenotype 24 h post HI. This may lead to new treatment options altering microglia phenotype polarization early after HI brain injury.
Highlights
Perinatal asphyxia is one of the leading causes of neonatal mortality and long-term mental and motor disabilities, including cerebral palsy (Jacobs et al, 2013)
After we found that pro-inflammatory gene expression was upregulated in total brain lysates of the LPS/HI group, we wanted to analyze the role of CD11b/c microglia, as microglia are supposed to be a major source of pro-inflammatory gene expression (Hellstrom Erkenstam et al, 2016)
As in total brain lysates, we observed a significant increase in M1 associated proinflammatory gene expression for inducible nitric oxide synthase (iNOS) (p < 0.0001) and IL-1beta (p = 0.0029) in microglia of the LPS/HI group compared to the LPS and Sham group (Figure 5). iNOS was significantly upregulated in the Veh/HI group, compared to the sham group (p = 0.0015)
Summary
Perinatal asphyxia is one of the leading causes of neonatal mortality and long-term mental and motor disabilities, including cerebral palsy (Jacobs et al, 2013). We have previously shown that TH is not neuroprotective in our established animal model of inflammation-sensitized hypoxic-ischemic brain injury (Osredkar et al, 2013, 2015). Perinatal asphyxia, leading to neonatal encephalopathy, is one of the leading causes for child mortality and long-term morbidities. Neonatal encephalopathy rates are significantly increased in newborns with perinatal infection. As shown experimentally, cooling has failed to be neuroprotective after inflammation-sensitized hypoxic ischemic (HI) brain injury. We performed this study investigating early microglia phenotype polarization in our newborn animal model of inflammation-sensitized HI brain injury, better understanding the underlying pathophysiological processes
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