Adenosine A2A Receptors Modulate Acute Injury and Neuroinflammation in Brain Ischemia.

The extracellular concentration of adenosine in the brain increases dramatically during ischemia. Adenosine A2A receptor is expressed in neurons and glial cells and in inflammatory cells (lymphocytes and granulocytes). Recently, adenosine A2A receptor emerged as a potential therapeutic attractive target in ischemia. Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia the early massive increase of extracellular glutamate is followed by activation of resident immune cells, that is, microglia, and production or activation of inflammation mediators. Proinflammatory cytokines, which upregulate cell adhesion molecules, exert an important role in promoting recruitment of leukocytes that in turn promote expansion of the inflammatory response in ischemic tissue. Protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. A2A receptors present on central cells and on blood cells account for important effects depending on the time-related evolution of the pathological condition. Evidence suggests that A2A receptor antagonists provide early protection via centrally mediated control of excessive excitotoxicity, while A2A receptor agonists provide protracted protection by controlling massive blood cell infiltration in the hours and days after ischemia. Focus on inflammatory responses provides for adenosine A2A receptor agonists a wide therapeutic time-window of hours and even days after stroke

Role of adenosine in oligodendrocyte precursor maturation

Differentiation and maturation of oligodendroglial cells are postnatal processes involving specific morphological changes correlated with the expression of stage-specific surface antigens and functional voltage-gated ion channels. A small fraction of oligodendrocyte progenitor cells (OPCs) generated during development are maintained in an immature and slowly proliferative or quiescent state in the adult central nervous system (CNS) representing an endogenous reservoir of immature cells. Adenosine receptors are expressed by OPCs and a key role of adenosine in oligodendrocyte maturation has been recently recognised. As evaluated on OPC cultures, adenosine by stimulating A1 receptors, promotes oligodendrocyte maturation and inhibits their proliferation; on the contrary by stimulating A2A receptors, it inhibits oligodendrocyte maturation. A1 and A2A receptor-mediated effects are related to opposite modifications of outward delayed rectifying membrane K+ currents (IK) that are involved in regulation of oligodendrocyte differentiation. Brain A1 and A2A receptors might represent new molecular targets for drugs useful in demyelinating pathologies, such as multiple sclerosis (MS), stroke and brain trauma

Temporal profile of cell proliferation in the SGZ of DG.

<p>Quantification of BrdU⁺ cells in the SGZ of the DG at 3, 6 and 24 hours after the end of OGD. Each column shows the total number of BrdU⁺ cells in the SGZ. Bars represent the mean±SEM. In parentheses is the number of slices investigated. *<i>P</i><0.05 and **<i>P</i><0.01 <i>vs</i> control, One-way ANOVA followed by Newman–Keuls post hoc test.</p

The block of P2Y1R counteracts the synaptic failure induced by severe OGD in the DG.

<p>AD was recorded as a negative d.c. shift in response to 9-min OGD in control (<b>a</b>, n = 26). MRS2179 (10 µM) completely prevented the appearance of AD in 23 out of 28 slices during 9-min OGD (<b>b</b>). MRS2179 (10 µM) had no effect in 5 out of 28 slices after 9-min OGD (<b>c</b>, n = 6), in which AD was recorded as a negative d.c. shift. <b>d</b>. example traces taken at the time-points indicated on the graph, immediately before (1), 10 min after the beginning of MRS2179 application (2), 9 minutes after OGD+MRS2179 (3) or 50 min after washout in oxygenated aCSF (4). Inset: example trace taken from the same hippocampal slice 24 hours from the end of OGD applied in the presence of 10 µM MRS2179. In each graph, traces are averages of 3 consecutive responses. Scale bars: 10 ms, 0.5 mV. <b>f</b>. The graph shows the time-course of the effect caused by 9-min OGD on fEPSP amplitude (mean±SEM) in untreated OGD slices (n = 26) and in OGD slices treated with 10 µM MRS2179, in which AD was absent (n = 23) or present (n = 5). fEPSPs amplitude is expressed as percent of the respective pre-ischemic baseline. Open bar: time of drug application.</p

The Selective Antagonism of P2X₇ and P2Y₁ Receptors Prevents Synaptic Failure and Affects Cell Proliferation Induced by Oxygen and Glucose Deprivation in Rat Dentate Gyrus

<div><p>Purinergic P2X and P2Y receptors are broadly expressed on both neurons and glial cells in the central nervous system (CNS), including dentate gyrus (DG). The aim of this research was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of P2X₇ and P2Y₁ receptor antagonism to recovery of synaptic activity after OGD. Extracellular field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG were recorded from rat hippocampal slices. Nine-min OGD elicited an irreversible loss of fEPSP and was invariably followed by the appearance of anoxic depolarization (AD). Application of MRS2179 (selective antagonist of P2Y₁ receptor) and BBG (selective antagonist of P2X₇ receptor), before and during OGD, prevented AD appearance and allowed a significant recovery of neurotransmission after 9-min OGD. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone (SGZ) of slices prepared from rats treated with 5-Bromo-2′-deoxyuridine (BrdU) were investigated. Slices were further incubated with an immature neuron marker, doublecortin (DCX). The number of BrdU⁺ cells in the SGZ was significantly decreased 6 hours after OGD. This effect was antagonized by BBG, but not by MRS2179. Twenty-four hours after 9-min OGD, the number of BrdU⁺ cells returned to control values and a significant increase of DCX immunofluorescence was observed. This phenomenon was still evident when BBG, but not MRS2179, was applied during OGD. Furthermore, the P2Y₁ antagonist reduced the number of BrdU⁺ cells at this time. The data demonstrate that P2X₇ and P2Y₁ activation contributes to early damage induced by OGD in the DG. At later stages after the insult, P2Y₁ receptors might play an additional and different role in promoting cell proliferation and maturation in the DG.</p></div

The P2X7R antagonist, BBG, prevents the synaptic failure induced by 9-min OGD in the DG.

<p>AD was recorded as a negative d.c. shift in response to 9-min OGD in control conditions (<b>a</b>, n = 21). BBG (10 µM) completely prevented the appearance of AD in 22 out of 28 slices during 9-min OGD (<b>b</b>). BBG (10 µM) had no effect in 6 out of 28 slices after 9-min OGD (<b>c</b>, n = 6), in which AD was recorded as a negative d.c. shift. <b>d</b>. example traces taken at the time-points indicated on the graph, immediately before (1), 10 min after the beginning of BBG application (2), 9 minutes after OGD+BBG (3) or 50 min after washout in oxygenated aCSF (4). Inset: example trace taken from the same hippocampal slice 24 hours from the end of OGD applied in the presence of 10 µM BBG. In each graph, traces are averages of 3 consecutive responses. Scale bars: 10 ms, 0.5 mV. <b>f</b>. The graph shows the time-course of the effect caused by 9-min OGD on fEPSP amplitude (mean ± SEM) in untreated OGD slices (n = 21) and in OGD slices treated with 10 µM BBG, in which AD was absent (n = 22) or present (n = 6). fEPSPs amplitude is expressed as percent of the respective pre-ischemic baseline. Open bar: time of drug application.</p

AD development and synaptic failure in the DG of rat hippocampal slices after 9-min OGD.

<p><b>a</b>. Upper panel: fEPSP taken from a typical experiment at the time-points indicated on the graph in a control hippocampal slice (Ctr) continuously superfused with oxygenated aCSF. Lower panel: example traces taken at the time-points indicated on the graph recorded from a hippocampal slice before (4), at the end of 9-min OGD (5) or 50 min after washout in oxygenated aCSF (6). <b>b</b>. The graph shows the time-course of the fEPSP amplitude, expressed as percent of pre-ischemic baseline in the DG (mean±SEM) in a group of control slices (Ctr, n = 7) or in a group of slices subjected to 9-min OGD (OGD, n = 34). Note that, while a stable fEPSP was recorded in control slices, the ischemic insult elicited a gradual reduction of fEPSPs amplitude, which completely disappears and does not recover even after prolonged washing in oxygenated aCSF. Inset: example traces taken from the same respective hippocampal slices shown in panel <b>a</b>, in control (Ctr, recorded 24 hours from slice preparation) or 24 hours from the end of OGD. In each graph, traces are averages of 3 consecutive responses. Scale bars: 10 ms, 0.5 mV. <b>c</b>. Anoxic depolarization (AD) was recorded as a negative d.c. shift in response to 9-min OGD. The d.c. shift was always recorded (n = 34) during 9-min OGD. <b>d</b>. Each column represents the mean±SEM of AD latency or AD amplitude recorded in the DG during 9-min OGD. AD latency was measured from the beginning of OGD insult.</p