Sara Cipriani

The selective A2A receptor antagonist SCH 58261 protects from neurological deficit, brain damage and activation of p38 MAPK in rat focal cerebral ischemia

We investigated the protective effect of subchronic treatment of the A2A receptor antagonist, SCH 58261 (0.01 mg/kg, i.p.), administered 5 min, 6 h and 15 h after permanent right middle cerebral artery occlusion (MCAo). Twenty-four hours after ischemia, an extensive pallid area, evaluated by cresyl violet staining, is evident in the vascular territories supplied by the MCA, the striatum and the sensory motor cortex. The pallid area reflects the extent of necrotic neurons. Soon after waking, rats showed a definite contralateral turning behavior which was significantly reduced by SCH 58261 treatment. Twenty-four hours after MCAo, SCH 58261 significantly improved the neurological deficit and reduced ischemic damage in the striatum and cortex. Phospho-p38 mitogen-activated protein kinase (MAPK), evaluated by Western Blot, increased by 500% in the ischemic striatum 24 h after MCAo. SCH 58261 treatment significantly reduced phospho-p38 MAPK by 70%. Microglia was immunostained using the OX-42 antibody. Phospho-p38 MAPK and OX-42-immunoreactive cells are localized in the ventral striatum and frontoparietal cortex. Furthermore, both OX-42 and phospho-p38 MAPK-immunoreactive cells have overlapping morphological features, typical of reactive microglia. SCH 58261 reduced phospho-p38 MAPK immunoreactivity in the striatum and in the cortex without changing the microglial cell morphology. These results indicate that the protective effect of the adenosine antagonist SCH 58261 during ischemia is not due to reduced microglial activation but involves inhibition of phospho-p38 MAPK and suggest that treatment with the A2A antagonist from the first hour to several hours after ischemia may be a useful therapeutic approach in cerebral ischemia.

Selective adenosine A2a receptor antagonism reduces JNK activation in oligodendrocytes after cerebral ischaemia

Adenosine is a potent biological mediator, the concentration of which increases dramatically following brain ischaemia. During ischaemia, adenosine is in a concentration range (muM) that stimulates all four adenosine receptor subtypes (A(1), A(2A), A(2B) and A(3)). In recent years, evidence has indicated that the A(2A) receptor subtype is of critical importance in stroke. We have previously shown that 24 h after medial cerebral artery occlusion (MCAo), A(2A) receptors up-regulate on neurons and microglia of ischaemic striatum and cortex and that subchronically administered adenosine A(2A) receptor antagonists protect against brain damage and neurological deficit and reduce activation of p38 mitogen-activated protein kinase (MAPK) in microglial cells. The mechanisms by which A(2A) receptors are noxious during ischaemia still remain elusive. The objective of the present study was to investigate whether the adenosine A(2A) antagonist SCH58261 affects JNK and MEK1/ERK MAPK activation. A further aim was to investigate cell types expressing activated JNK and MEK1/ERK MAPK after ischaemia. We hereby report that the selective adenosine A(2A) receptor antagonist, SCH58261, administered subchronically (0.01 mg/kg i.p) 5 min, 6 and 20 h after MCAo in male Wistar rats, reduced JNK MAPK activation (immunoblot analysis: phospho-JNK54 isoform by 81% and phospho-JNK46 isoform by 60%) in the ischaemic striatum. Twenty-four hours after MCAo, the Olig2 transcription factor of oligodendroglial progenitor cells and mature oligodendrocytes was highly expressed in cell bodies in the ischaemic striatum. Immunofluorescence staining showed that JNK MAPK is maximally expressed in Olig2-stained oligodendrocytes and in a few NeuN stained neurons. Striatal cell fractioning into nuclear and extra-nuclear fractions demonstrated the presence of Olig2 transcription factor and JNK MAPK in both fractions. The A(2A) antagonist reduced striatal Olig 2 transcription factor (immunoblot analysis: by 55%) and prevented myelin disorganization, assessed by myelin-associated glycoprotein staining. Twenty-four hours after MCAo, ERK1/2 MAPK was highly activated in the ischaemic striatum, mostly in microglia, while it was reduced in the ischaemic cortex. The A(2A) antagonist did not affect activation of the ERK1/2 pathway. The efficacy of A(2A) receptor antagonism in reducing activation of JNK MAPK in oligodendrocytes suggests a mechanism of protection consisting of scarring oligodendrocyte inhibitory molecules that can hinder myelin reconstitution and neuron functionality.

The role of ATP and adenosine in the brain under normoxic and ischemic conditions.

By taking advantage of some recently synthesized compounds that are able to block ecto-ATPase activity, we demonstrated that adenosine triphosphate (ATP) in the hippocampus exerts an inhibitory action independent of its degradation to adenosine. In addition, tonic activation of P2 receptors contributes to the normally recorded excitatory neurotransmission. The role of P2 receptors becomes critical during ischemia when extracellular ATP concentrations increase. Under such conditions, P2 antagonism is protective. Although ATP exerts a detrimental role under ischemia, it also exerts a trophic role in terms of cell division and differentiation. We recently reported that ATP is spontaneously released from human mesenchymal stem cells (hMSCs) in culture. Moreover, it decreases hMSC proliferation rate at early stages of culture. Increased hMSC differentiation could account for an ATP-induced decrease in cell proliferation. ATP as a homeostatic regulator might exert a different effect on cell trophism according to the rate of its efflux and receptor expression during the cell life cycle. During ischemia, adenosine formed by intracellular ATP escapes from cells through the equilibrative transporter. The protective role of adenosine A1 receptors during ischemia is well accepted. However, the use of selective A1 agonists is hampered by unwanted peripheral effects, thus attention has been focused on A2A and A3 receptors. The protective effects of A2A antagonists in brain ischemia may be largely due to reduced glutamate outflow from neurones and glial cells. Reduced activation of p38 mitogen-activated protein kinases that are involved in neuronal death through transcriptional mechanisms may also contribute to protection by A2A antagonism. Evidence that A3 receptor antagonism may be protective after ischemia is also reported.

Selective adenosine A2A receptor agonists and antagonists protect against spinal cord injury through peripheral and central effects.

BackgroundPermanent functional deficits following spinal cord injury (SCI) arise both from mechanical injury and from secondary tissue reactions involving inflammation. Enhanced release of adenosine and glutamate soon after SCI represents a component in the sequelae that may be responsible for resulting functional deficits. The role of adenosine A2A receptor in central ischemia/trauma is still to be elucidated. In our previous studies we have demonstrated that the adenosine A2A receptor-selective agonist CGS21680, systemically administered after SCI, protects from tissue damage, locomotor dysfunction and different inflammatory readouts. In this work we studied the effect of the adenosine A2A receptor antagonist SCH58261, systemically administered after SCI, on the same parameters. We investigated the hypothesis that the main action mechanism of agonists and antagonists is at peripheral or central sites.MethodsSpinal trauma was induced by extradural compression of SC exposed via a four-level T5-T8 laminectomy in mouse. Three drug-dosing protocols were utilized: a short-term systemic administration by intraperitoneal injection, a chronic administration via osmotic minipump, and direct injection into the spinal cord.ResultsSCH58261, systemically administered (0.01 mg/kg intraperitoneal. 1, 6 and 10 hours after SCI), reduced demyelination and levels of TNF-α, Fas-L, PAR, Bax expression and activation of JNK mitogen-activated protein kinase (MAPK) 24 hours after SCI. Chronic SCH58261 administration, by mini-osmotic pump delivery for 10 days, improved the neurological deficit up to 10 days after SCI. Adenosine A2A receptors are physiologically expressed in the spinal cord by astrocytes, microglia and oligodendrocytes. Soon after SCI (24 hours), these receptors showed enhanced expression in neurons. Both the A2A agonist and antagonist, administered intraperitoneally, reduced expression of the A2A receptor, ruling out the possibility that the neuroprotective effects of the A2A agonist are due to A2A receptor desensitization. When the A2A antagonist and agonist were centrally injected into injured SC, only SCH58261 appeared neuroprotective, while CGS21680 was ineffective.ConclusionsOur results indicate that the A2A antagonist protects against SCI by acting on centrally located A2A receptors. It is likely that blockade of A2A receptors reduces excitotoxicity. In contrast, neuroprotection afforded by the A2A agonist may be primarily due to peripheral effects.

Regulation of A(2A) adenosine receptor expression and functioning following permanent focal ischemia in rat brain.

Ischemia, through modulation of adenosine receptors (ARs), may influence adenosine‐mediated‐cellular responses. In the present study, we investigated the modulation of rat A2A receptor expression and functioning, in rat cerebral cortex and striatum, following in vivo focal ischemia (24 h). In cortex, middle cerebral artery occlusion did not induce any alterations in A2A receptor binding and functioning. On the contrary, in striatum, a significant decrease in A2A ligand affinity, associated with an increase in receptor density, were detected. In striatum, ischemia also induced a significant reduction both in G protein pool and in A2A receptor‐G protein coupling. On the contrary, A2A receptor functional responsiveness, measured as stimulation of adenylyl cyclise, was not affected by ischemia, suggesting receptor up‐regulation may represent a compensatory mechanism to maintain receptor functioning during cerebral damage. Immunohistochemical study showed that following 24 h middle cerebral artery occlusion, A2A ARs were definitely expressed both on neurons and activated microglia in ischemic striatum and cortex, but were not detected on astrocytes. In the non‐ischemic hemisphere and in sham‐operated rats A2A ARs were barely detected. Modifications of ARs may play a significant role in determining adenosine effects during ischemia and therefore should be taken into account when evaluating time‐dependent protective effects of specific A2A active compounds.

The adenosine A2A receptor antagonist ZM241385 enhances neuronal survival after oxygen-glucose deprivation in rat CA1 hippocampal slices

Background and purpose:  Activation of adenosine A2A receptors in the CA1 region of rat hippocampal slices during oxygen‐glucose deprivation (OGD), a model of cerebral ischaemia, was investigated.

Adenosine A2A receptor antagonism increases nNOS-immunoreactive neurons in the striatum of Huntington transgenic mice.

Medium spiny GABAergic projection neurons are progressively lost in Huntingtons disease (HD), whereas there is preferential sparing of the few interneurons co-expressing NPY, somatostatin and neuronal nitric oxide synthase. We investigated the effect of the selective adenosine A(2A) receptor antagonist SCH58261 (0.01 mg/kg, acutely and chronically administered i.p.) on nNOS striatal expression and motor impairment in R6/2 transgenic mice in clearly symptomatic phase (10-11-week old). SCH58261 chronically administered increased the number of nNOS-immunoreactive neurons (nNOS-IR) in the striatum of R6/2 mice. No glial activation was detected in the striatum or cortex. SCH58261 also improved walking in the inclined plane test but not motor capability evaluated by the rotarod test. These findings demonstrate for the first time a role of adenosine A(2A) receptors in regulating nNOS expression in the striatum. We suggest that the protective effect of A(2A) antagonism in HD is related to the increase in striatal nNOS-IR neurons.

P2 receptor antagonists prevent synaptic failure and extracellular signal-regulated kinase 1/2 activation induced by oxygen and glucose deprivation in rat CA1 hippocampus in vitro.

To investigate the role of purinergic P2 receptors under ischemia, we studied the effect of P2 receptor antagonists on synaptic transmission and mitogen‐activated protein kinase (MAPK) activation under oxygen and glucose deprivation (OGD) in rat hippocampal slices. The effect of the P2 antagonists pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonate (PPADS, unselective, 30 μm), N 6‐methyl‐2′‐deoxyadenosine‐3′,5′‐bisphosphate (MRS2179, selective for P2Y1 receptor, 10 μm), Brilliant Blue G (BBG, selective for P2X7 receptor, 1 μm), and 5‐[[[(3‐phenoxyphenyl)methyl][(1S)‐1,2,3,4‐tetrahydro‐1‐naphthalenyl]amino]carbonyl]‐1,2,4‐benzenetricarboxylic acid (A‐317491, selective for P2X3 receptor, 10 μm), and of the newly synthesized P2X3 receptor antagonists 2‐amino‐9‐(5‐iodo‐2‐isopropyl‐4‐methoxybenzyl)adenine (PX21, 1 μm) and 2‐amino‐9‐(5‐iodo‐2‐isopropyl‐4‐methoxybenzyl)‐N 6‐methyladenine (PX24, 1 μm), on the depression of field excitatory postsynaptic potentials (fEPSPs) and anoxic depolarization (AD) elicited by 7 min of OGD were evaluated. All antagonists significantly prevented these effects. The extent of CA1 cell injury was assessed 3 h after the end of 7 min of OGD by propidium iodide staining. Substantial CA1 pyramidal neuronal damage, detected in untreated slices exposed to OGD injury, was significantly prevented by PPADS (30 μm), MRS2179 (10 μm), and BBG (1 μm). Western blot analysis showed that, 10 min after the end of the 7 min of OGD, extracellular signal‐regulated kinase (ERK)1/2 MAPK activation was significantly increased. MRS2179, BBG, PPADS and A‐317491 significantly counteracted ERK1/2 activation. Hippocampal slices incubated with the ERK1/2 inhibitors 1,4‐diamino‐2,3‐dicyano‐1,4‐bis(2‐aminophenylthio)butadiene (U0126, 10 μm) and α‐[amino[(4‐aminophenyl)thio]methylene]‐2‐(trifluoromethyl) benzeneacetonitrile (SL327, 10 μm) showed significant fEPSP recovery after OGD and delayed AD, supporting the involvement of ERK1/2 in neuronal damage induced by OGD. These results indicate that subtypes of hippocampal P2 purinergic receptors have a harmful effect on neurotransmission in the CA1 hippocampus by participating in AD appearance and activation of ERK1/2.

The neuron-astrocyte-microglia triad in a rat model of chronic cerebral hypoperfusion: protective effect of dipyridamole.

Chronic cerebral hypoperfusion during aging may cause progressive neurodegeneration as ischemic conditions persist. Proper functioning of the interplay between neurons and glia is fundamental for the functional organization of the brain. The aim of our research was to study the pathophysiological mechanisms, and particularly the derangement of the interplay between neurons and astrocytes-microglia with the formation of “triads,” in a model of chronic cerebral hypoperfusion induced by the two-vessel occlusion (2VO) in adult Wistar rats (n = 15). The protective effect of dipyridamole given during the early phases after 2VO (4 mg/kg/day i.v., the first 7 days after 2VO) was verified (n = 15). Sham-operated rats (n = 15) were used as controls. Immunofluorescent triple staining of neurons (NeuN), astrocytes (GFAP), and microglia (IBA1) was performed 90 days after 2VO. We found significantly higher amount of “ectopic” neurons, neuronal debris and apoptotic neurons in CA1 Str. Radiatum and Str. Pyramidale of 2VO rats. In CA1 Str. Radiatum of 2VO rats the amount of astrocytes (cells/mm2) did not increase. In some instances several astrocytes surrounded ectopic neurons and formed a “micro scar” around them. Astrocyte branches could infiltrate the cell body of ectopic neurons, and, together with activated microglia cells formed the “triads.” In the triad, significantly more numerous in CA1 Str. Radiatum of 2VO than in sham rats, astrocytes and microglia cooperated in the phagocytosis of ectopic neurons. These events might be common mechanisms underlying many neurodegenerative processes. The frequency to which they appear might depend upon, or might be the cause of, the burden and severity of neurodegeneration. Dypiridamole significantly reverted all the above described events. The protective effect of chronic administration of dipyridamole might be a consequence of its vasodilatory, antioxidant and anti-inflammatory role during the early phases after 2VO.

Effect of intravenous administration of dipyridamole in a rat model of chronic cerebral ischemia

Pharmacological therapy able to improve the cognitive performances of patients with chronic vascular pathologies currently remains unavailable. Many studies of chronic cerebral hypotension in rodents have revealed alterations in reference memory and learning. Dipyridamole was introduced into clinical medicine in the early 1960s as a coronary vasodilator. It is a potent inhibitor of platelet activation and reduces formation of thrombi in vivo. In addition, it is an antithrombotic agent used for secondary stroke prevention in combination with aspirin. Recent evidence indicates that dipyridamole has anti‐inflammatory properties. Bilateral common carotid artery occlusion (2VO) in the rat is recognized as a valid model of chronic cerebral hypotension, also defined as the “vascular cognitive impairment rat model.” Here, we report that dipyridamole reverses the impairment of spatial working memory 90 days after 2VO. This protective effect might be in relation to dipyridamoles anti‐inflammatory properties.