Modulation of Microglial Activation by Adenosine A2a Receptor in Animal Models of Perinatal Brain Injury

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.

Defining a Time Window for Neuroprotection and Glia Modulation by Caffeine After Neonatal Hypoxia-Ischaemia

“…Even though this reduced inflammation could be the result of a contained lesion due to the caffeine treatment, a direct caffeine-induced modulation in microglia has been suggested. Recent findings have demonstrated that adenosine agonists in microglia culture lead to an increase of M1 marker expression [34], and moreover, activation of the A2aR in microglia was associated with cytokine release and process retraction in vivo suggestive of an activation state [35,36]. In contrast, caffeine suppresses the generation of proinflammatory mediators [37], and the blockade of A2aR was indeed reported to reduce microglial activation both in vivo and in vitro [38], thus confirming the present findings.…”

Antioxidant and Neuroprotective Effects of Caffeine against Alzheimer’s and Parkinson’s Disease: Insight into the Role of Nrf-2 and A2AR Signaling

“…Antioxidants 2020, 9, x FOR PEER REVIEW 6 of 21 inflammatory cytokines [105], and changes the microglia into amoeboid shapes [106]. Conversely, A2AR antagonists suppress microglia activation, as highlighted previously [104]. Figure 2 depicts the role of A2A in AD.…”

“…Nrf2 induces the expression of antioxidants and cytoprotective genes, which provoke an anti-inflammatory response that is critical for the healing process [103]. Apart from the different sorts of kinases (like MAP kinases), there are some receptors such as adenosine receptors that play a role in neuroinflammation, such as adenosine A2A receptors [104]. At the microglial level, the expression of A2AR is usually low; it activates with brain insults and facilitates the release of the inflammatory cytokines [105], and changes the microglia into amoeboid shapes [106].…”

Regulation of Microglial Functions by Purinergic Mechanisms in the Healthy and Diseased CNS

“…The enhancement of neuroinflammatory reactions after perinatal brain injury was observed in rats due to A2AR activation and the consequent upregulation of M1 microglial markers (IL-β, IL-6); an antagonist of A2ARs was beneficial under these conditions [156]. Developmental risk factors, such as the exposure of high levels of glucocorticoids, may contribute to the pathogenesis of anxiety disorders [157].…”