Abstract
ObjectiveComplement activation is instrumental in the pathogenesis of Hypoxic-ischemic encephalopathy (HIE), a significant cause of neonatal mortality and disability worldwide. Therapeutic hypothermia (HT), the only available treatment for HIE, only modestly improves outcomes. Complement modulation as a therapeutic adjunct to HT has been considered, but is challenging due to the wide-ranging role of the complement system in neuroinflammation, homeostasis and neurogenesis in the developing brain. We sought to identify potential therapeutic targets by measuring the impact of treatment with HT on complement effector expression in neurons and glia in neonatal HIE, with particular emphasis on the interactions between microglia and C1q.MethodsThe Vannucci model was used to induce HIE in term-equivalent rat pups. At P10-12, pups were randomly assigned to three different treatment groups: Sham (control), normothermia (NT), and hypothermia (HT) treatment. Local and systemic complement expression and neuronal apoptosis were measured by ELISA, TUNEL and immunofluorescence labeling, and differences compared between groups.ResultsTreatment with HT is associated with decreased systemic and microglial expression of C1q, decreased systemic C5a levels, and decreased microglial and neuronal deposition of C3 and C9. The effect of HT on cytokines was variable with decreased expression of pro and anti-inflammatory effectors. HT treatment was associated with decreased C1q binding on cells undergoing apoptosis.ConclusionOur data demonstrate the extreme complexity of the immune response in neonatal HIE. We propose modulation of downstream effectors C3a and C5a as a therapeutic adjunct to HT to enhance neuroprotection in the developing brain.
Highlights
Neonatal hypoxic ischemic encephalopathy (HIE) is a significant worldwide public health problem affecting 1–2/1000 live-births in the developed world, with as much as an eight-fold higher incidence in low and middle income countries with limited access to healthcare (Kurinczuk et al, 2010)
In the Vannucci rat model, we confirmed that HT treatment after HIE is neuroprotective, as demonstrated by decreased brain infarct size on Tetrazolium Chloride (TTC) staining (Figures 3a,b)
Complement Whole brain C1q levels in NT animals were significantly higher than HT treated animals at 8, 12, and 24 h (P < 0.05) after the initial insult (Figure 4a)
Summary
Neonatal hypoxic ischemic encephalopathy (HIE) is a significant worldwide public health problem affecting 1–2/1000 live-births in the developed world, with as much as an eight-fold higher incidence in low and middle income countries with limited access to healthcare (Kurinczuk et al, 2010). The limited success of HT may be attributable to its variable modulation of inflammatory mediators of reperfusion injury (Rocha-Ferreira et al, 2017). The complement system is the most potent inflammatory cascade in humans and plays a major role in innate immune defense as well as many inflammatory diseases, including ischemia-reperfusion injuries such as HIE (Arumugam et al, 2006; Liu and McCullough, 2013). The classical complement cascade is initiated by C1q, binding to antigen-antibody immune complexes on the cell surface, or binding to specific receptors expressed during apoptosis. Experimental studies have shown that C1q is highly expressed in the brain following ischemia (Schafer et al, 2000), and that classical complement pathway activation via C1 generates pro-inflammatory mediators such as C5a, which are associated with HI brain injury (Rocha-Ferreira and Hristova, 2015).
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