Abstract
Neuroinflammation has a key role in the pathogenesis of perinatal brain injury. Caffeine, a nonspecific antagonist of adenosine receptors (ARs), is widely used to treat apnea of prematurity and has been linked to a decrease in the incidence of cerebral palsy in premature infants. The mechanisms explaining its neuroprotective effect have not yet been elucidated. The objective of this study was to characterize the expression of adenosine and ARs in two neonatal rat models of neuroinflammation and to determine the effect of A2aR blockade on microglial activation assessed through inflammatory cytokine gene expression. We have used two rat models of microglial activation: the gestational low protein diet (LPD) model, associated with chronic brain injury, and postnatal ibotenate intracerebral injections, responsible for acute excitotoxicity injury. Adenosine blood levels have been measured by Tandem Mass Spectrometry. The expression of ARs in vivo was assessed using qPCR and immunohistochemistry. In vivo models have been replicated in vitro on primary microglial cell cultures exposed to A2aR agonist CGS-21680 or antagonist SCH-58261. The effects of these treatments have been assessed on the M1/M2 cytokine expressions measured by RT-qPCR. LPD during pregnancy was associated with higher adenosine levels in pups at postnatal day 1 and 4. A2aR mRNA expression was significantly increased in both cortex and magnetically sorted microglial cells from LPD animals compared to controls. CD73 expression, responsible for extracellular production of brain adenosine, was significantly increased in LPD cortex and sorted microglia cells. Moreover, CD73 protein level was increased in ibotenate treated animals. In vitro experiments confirmed that LPD or control microglial cells exposed to ibotenate display an increased expression, at both protein and molecular levels, of A2aR and M1 markers (IL-1β, IL-6, iNOS, TNFα). This pro-inflammatory profile was significantly reduced by SCH-58261, which reduces M1 markers in both LPD and ibotenate-exposed cells, with no effect on control cells. In the same experimental conditions, a partial increased of M1 cytokines was observed in response to A2aR agonist CGS-21680. These results support the involvement of adenosine and particularly of its receptor A2aR in the regulation of microglia in two different animal models of neuroinflammation.
Highlights
Brain injury is one of the most important complication related to preterm birth [1]
Prenatal low protein diet (LPD)-induced malnutrition resulted in the absence of postnatal mortality but fetal growth restriction with body weight of the pups significantly lower from P0 to at least P4 compared to the controls (Supplemental Figure 4)
LPD pups at P1 and P4 showed significantly higher adenosine blood levels compared to the control pups (0.822 ± 0.088 μM in Controls vs. 1.850 ± 0.355 μM in LPD at P1, 0.598 ± 0.051 μM in Controls vs. 1.464 ± 0.215 μM LPD at P4, see Figure 1)
Summary
Preclinical and clinical studies show that neuroinflammation plays a central role in the pathogenesis of perinatal brain damage [3,4,5,6,7,8]. One of the first events following neuroinflammation is activation of microglia cells [9], that assume different phenotypes, conventionally classified as M1 (pro-inflammatory) and M2 (anti-inflammatory, reparative) [10, 11]. The most implicated adenosine receptor in neuroinflammation is A2aR [14]. Its expression in microglia is usually low but increases following brain insults. A2aR antagonists suppress microglia activation, as described using in vitro [17, 18] and in vivo [18] studies
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