A Selective Histamine H4 Receptor Antagonist, JNJ7777120, Is Protective in a Rat Model of Transient Cerebral Ischemia.

Cerebral ischemia is a multifactorial pathology characterized by different events evolving in time. The acute injury, characterized by excitoxicity, is followed by a secondary brain injury that develops from hours to days after ischemia. Extracellular levels of histamine increase in the ischemic area after focal cerebral ischemia induced by occlusion of the middle cerebral artery (MCAo). The histamine H4 receptor (H4R) is predominantly expressed in cell types of immune system where is involved in the regulation of immunological and inflammatory responses, and in numerous area of the Central Nervous System (CNS) including cortex and striatum. Our aim was to assess the putative neuroprotective effects of the potent and selective H4R antagonist, JNJ7777120 (JNJ), chronically administered (1 mg/kg, i.p., twice/day for 7 days) on damage parameters in a rat model of focal ischemia induced by transient MCAo (tMCAo). Chronic treatment with the H4R antagonist JNJ, significantly protected from the neurological deficit and from body weight loss after tMCAo. Seven days after the ischemic insult, JNJ reduced the volume of the ischemic cortical and striatal damage, the number of activated microglia and astrocytes in the ischemic cortex and striatum and decreased the plasma levels of IL-1ß and TNF-α, while increased the levels of IL-10. Two days after ischemia, JNJ has reduced granulocyte infiltration in the ischemic area. Results demonstrate that the selective antagonist of H4R, JNJ, systemically and chronically administered after ischemia, reduces the ischemic brain damage, improves the neurological deficit and decreases blood pro-inflammatory cytokines, suggesting that H4R is a valuable pharmacological target after focal brain ischemia.

The neuron-astrocyte-microglia triad in CA3 after chronic cerebral hypoperfusion in the rat: Protective effect of dipyridamole.

We investigated the quantitative and morphofunctional alterations of neuron-astrocyte-microglia triads in CA3 hippocampus, in comparison to CA1, after 2 Vessel Occlusion (2VO) and the protective effect of dipyridamole. We evaluated 3 experimental groups: sham-operated rats (sham, n=15), 2VO-operated rats treated with vehicle (2VO-vehicle, n=15), and 2VO-operated rats treated with dipyridamole from day 0 to day 7 (2VO-dipyridamole, n=15), 90days after 2VO. We analyzed Stratum Pyramidalis (SP), Stratum Lucidum (SL) and Stratum Radiatum (SR) of CA3. 1) ectopic neurons increased in SL and SR of 2VO-vehicle, and 2VO-dipyridamole rats; 2) apoptotic neurons increased in SP of 2VO-vehicle rats and dipyridamole reverted this effect; 3) astrocytes increased in SP, SL and SR of 2VO-vehicle and 2VO-dipyridamole rats; 4) TNF-α expression increased in astrocytes, blocked by dipyridamole, and in dendrites in SR of 2VO-vehicle rats; 5) total microglia increased in SL and SR of 2VO-vehicle and 2VO-dipyridamole rats; 6) triads increased in SR of 2VO-vehicle rats and dipyridamole reverted this effect. Microglia cooperated with astrocytes to phagocytosis of apoptotic neurons and debris, and engulfed ectopic non-fragmented neurons in SL of 2VO-vehicle and 2VO-dipyridamole rats, through a new mechanism called phagoptosis. CA3 showed a better adaptive capacity than CA1 to the ischemic insult, possibly due to the different behaviour of astrocytes and microglial cells. Dipyridamole had neuroprotective effects.

Purinergic signalling in brain ischemia.

Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia a primary damage due to the early massive increase of extracellular glutamate is followed by activation of resident immune cells, i.e microglia, and production or activation of inflammation mediators. Protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. Extracellular concentrations of ATP and adenosine in the brain increase dramatically during ischemia in concentrations able to stimulate their respective specific P2 and P1 receptors. Both ATP P2 and adenosine P1 receptor subtypes exert important roles in ischemia. Although adenosine exerts a clear neuroprotective effect through A1 receptors during ischemia, the use of selective A1 agonists is hampered by undesirable peripheral effects. Evidence up to now in literature indicate that A2A receptor antagonists provide protection centrally by reducing excitotoxicity, while agonists at A2A (and possibly also A2B) and A3 receptors provide protection by controlling massive infiltration and neuroinflammation in the hours and days after brain ischemia. Among P2X receptors most evidence indicate that P2X7 receptor contribute to the damage induced by the ischemic insult due to intracellular Ca(2+) loading in central cells and facilitation of glutamate release. Antagonism of P2X7 receptors might represent a new treatment to attenuate brain damage and to promote proliferation and maturation of brain immature resident cells that can promote tissue repair following cerebral ischemia. Among P2Y receptors, antagonists of P2Y12 receptors are of value because of their antiplatelet activity and possibly because of additional anti-inflammatory effects. Moreover strategies that modify adenosine or ATP concentrations at injury sites might be of value to limit damage after ischemia. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.

Role of adenosine in oligodendrocyte precursor maturation.

Differentiation and maturation of oligodendroglial cells are postnatal processes that involve 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 recognized. 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 the 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.

Time-course of protection by the selective A2A receptor antagonist SCH58261 after transient focal cerebral ischemia.

Evidence indicates that the adenosine A2A receptor subtype is of critical importance in stroke. In previous studies, in the model of permanent middle cerebral artery occlusion (pMCAo), the adenosine A2A receptor antagonist, SCH58261, administered soon after ischemia, proved protective against excessive glutamate outflow in the first 4 h after ischemia and against neurological deficit and tissue damage evaluated 24 h after pMCAo. In the present work, we investigated if neuroprotective effect of SCH58261 was maintained 7 days after transient MCAo (tMCAo). SCH58261 (0.01 mg/kg, i.p.), administered twice/day for 7 days, protected from neurological deficit 1 day after tMCAo, but no more after 5 and 7 days. Two days after tMCAo, SCH58261 did not reduce blood cell infiltration, evaluated as HIS-48 positive cells, into ischemic striatal and cortical tissue. Moreover, 7 days after tMCAo, SCH58261 has not protected ischemic areas from damage and has not ameliorated myelin organization into the ischemic striatum. Protection by the A2A receptor antagonist 24 h after ischemia is attributable to reduced excitotoxicity. Seven days after ischemia the early protective effect of the A2A receptor antagonist likely has been overwhelmed by a secondary damage due to blood cell infiltration and neuroinflammation.

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/mm(2)) 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.

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.

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.

Adenosine receptors in cerebral ischemia.

Ischemic stroke is a complex pathology characterized by a sequence of events that evolve over time and space. It is the second leading cause of death and the main cause of adult long-term disability in developed countries. At the moment, there is no promising pharmacotherapy for acute ischemic stroke. Adenosine receptors (A1, A2A, A2B, A3) are important targets for therapeutic implementation in the treatment of stroke because extracellular adenosine concentrations increase dramatically soon after ischemia. Adenosine receptors located both on central nervous system cells and on immune blood cells exert important roles during ischemia. The neuroprotective role of adenosine through A1 receptor subtype during ischemia is accepted, but the use of selective A1 agonists is hampered by undesirable side effects such as sedation, bradycardia, and hypotension. Recently, the A2A receptor subtype emerged as a potential therapeutic attractive target in ischemia. Evidence suggests that A2A receptor has dual role: in a first phase of ischemia, it potentiates excitotoxicity, while hours and days after ischemia, A2A receptors on immune blood cells potentiate cell adhesion mechanisms and infiltration in the ischemic parenchyma. Consistently, the use of A2A receptor agonists/antagonists (administered at doses that do not modify blood pressure and heart rate) should be carefully evaluated in function of time after ischemia. Although much is still to be known about the role of A2B and A3 receptor subtypes in brain ischemia, most consistent information indicates their role in regulation of immunosuppression and inflammation.

Adenosine A2A receptors modulate acute injury and neuroinflammation in brain ischemia.

The extracellular concentration of adenosine in the brain increases dramatically during ischemia. Adenosine A(2A) receptor is expressed in neurons and glial cells and in inflammatory cells (lymphocytes and granulocytes). Recently, adenosine A(2A) 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. A(2A) 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 A(2A) receptor antagonists provide early protection via centrally mediated control of excessive excitotoxicity, while A(2A) 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 A(2A) receptor agonists a wide therapeutic time-window of hours and even days after stroke.

Low doses of the selective adenosine A2A receptor agonist CGS21680 are protective in a rat model of transient cerebral ischemia.

Evidence indicate that adenosine A2A receptor subtype is of critical importance in stroke. An overexpression of A2A adenosine receptors occurs at central level on neurons and microglia of ischemic striatum and cortex after focal ischemia. Adenosine A2A receptor subtype is localized not only at central level but also peripherally on blood cells, where it is known to exert antiinflammatory effect. Purpose of the present work was to investigate the putative neuroprotective effect of the adenosine A2A receptor agonist CGS21680 in a rat model of transient medial cerebral artery occlusion (MCAo). Transient cerebral ischemia was induced by 1h occlusion of MCA. CGS21680 (0.01 and 0.1mg/kg, i.p.) was administered starting 4h after ischemia according to a chronic protocol (twice/day for 7 days). CGS21680, at the dose of 0.1mg/kg transiently increased heart frequency but did not modify blood pressure. At the dose of 0.01mg/kg the drug did not modify either heart frequency or blood pressure. Following transient MCAo, CGS21680 at both doses protected from neurological deficit from the first day up to 7 days thereafter. At this time, it has reduced microgliosis, astrogliosis and improved myelin organization in the striatum and cytoarchitecture of the ischemic cortex and striatum. Two days after transient MCAo, CGS21680 has reduced the number of infiltrated granulocytes into the ischemic tissue. Data indicate that CGS21680 systemically administered is protective by immunosuppressive effects.

Adenosine is present in rat brain synaptic vesicles.

Evidences in the central nervous system are in favor that adenosine under basal conditions is released by a direct excitation-secretion modality. However, till now, there is no direct evidence that adenosine is contained in synaptic vesicles. Eight synaptic vesicle fractions were recovered on a discontinuous sucrose gradient after ultracentrifugation of the crude synaptosomal fraction (pellet P2) of rat brain. The adenosine content in each fraction was measured by high-performance liquid chromatography coupled to a fluorescence detector (minimum sensitivity 10 femtomoles). The immunoblot analysis, to detect synaptophysin, a molecular marker for the vesicle membrane, showed that fractions from 3 to 8 were rich in synaptophysin. The sum of adenosine found in fractions 3-8 was (mean ± SEM, n = 4) 3325.6 ± 94.6 pmol/mg of tissue protein. We proved that adenosine measured in synaptic vesicle fractions was not contaminated by cytosolic adenosine, as adenosine exogenously added to the P2 preferentially distributed in fractions 1 and 2 that are synaptophysin-free and did not contaminate the vesicle pellet P3. Data provide direct demonstration that adenosine is present in rat brain synaptic vesicle fractions. This information is consistent with the notion that adenosine is stored in synaptic vesicles and is released under normoxic physiological conditions by an excitation-secretion mechanism typical of neuronal cells.

Ecto-ATPase inhibition: ATP and adenosine release under physiological and ischemic in vivo conditions in the rat striatum.

In the central nervous system (CNS) ATP and adenosine act as transmitters and neuromodulators on their own receptors but it is still unknown which part of extracellular adenosine derives per se from cells and which part is formed from the hydrolysis of released ATP. In this study extracellular concentrations of adenosine and ATP from the rat striatum were estimated by the microdialysis technique under in vivo physiological conditions and after focal ischemia induced by medial cerebral artery occlusion. Under physiological conditions, adenosine and ATP concentrations were in the range of 130 nmol/L and 40 nmol/L, respectively. In the presence of the novel ecto-ATPase inhibitor, PV4 (100 nmol/L), the extracellular concentration of ATP increased 12-fold to ~360 nmol/L but the adenosine concentration was not altered. This demonstrates that, under physiological conditions, adenosine is not a product of extracellular ATP. In the first 4h after ischemia, adenosine increased to ~690 nmol/L and ATP to ~50 nmol/L. In the presence of PV4 the extracellular concentration of ATP was in the range of 450 nmol/L and a significant decrease in extracellular adenosine (to ~270 nmol/L) was measured. The contribution of extracellular ATP to extracellular adenosine was maximal in the first 20 min after ischemia onset. Furthermore we demonstrated, by immunoelectron microscopy, the presence of the concentrative nucleoside transporter CNT2 on plasma and vesicle membranes isolated from the rat striatum. These results are in favor that adenosine is transported in vesicles and is released in an excitation-secretion manner under in vivo physiological conditions. Early after ischemia, extracellular ATP is hydrolyzed by ecto-nucleotidases which significantly contribute to the increase in extracellular adenosine. To establish the contribution of extracellular ATP to adenosine might constitute the basis for devising a correct putative purinergic strategy aimed at protection from ischemic damage.

CGS 21680, an agonist of the adenosine (A2A) receptor, reduces progression of murine type II collagen-induced arthritis.

OBJECTIVE: The aim of our study was to investigate the effect of an adenosine A2A receptor agonist, 2-[p-(2-carboxyethyl)phenylethylamino]-50 ethylcarboxamidoadenosine (CGS 21680), on modulation of the inflammatory response in mice subjected to collagen-induced arthritis (CIA). METHODS: CIA was induced by intradermal injection of 100 µl of emulsion containing 100 µg of bovine type II collagen (CII) and complete Freund's adjuvant (CFA) at the base of the tail. On Day 21, a second injection of CII in CFA was administered. Immunized mice developed erosive hind paw arthritis. Macroscopic clinical evidence of CIA first appeared as periarticular erythema and edema in the hind paws. The incidence of CIA was 100% by Day 27 in the CII challenged mice and the severity of CIA progressed over a 35-day period, with radiographic evaluation revealing focal resorption of bone. The histopathology of CIA included erosion of cartilage at the joint margins. RESULTS: Treatment of mice with CGS 21680 starting at the onset of arthritis (Day 25) ameliorated the clinical signs at Days 26-35 and improved histological status in the joint and paw. The degree of oxidative and nitrosative damage was significantly reduced in CGS 21680-treated mice as indicated by elevated levels of malondialdehyde, formation of nitrotyrosine, and activation of poly(ADP-ribose) polymerase. Plasma levels of proinflammatory cytokines such as tumor necrosis factor, interleukin 1ß (IL-1ß) and IL-6 were also reduced by CGS 21680. Treatment with CGS 21680 also decreased the expression of inducible nitric oxide synthase and cyclooxygenase-2. CONCLUSION: We demonstrate that CGS 21680 exerts an antiinflammatory effect during chronic inflammation and ameliorates the tissue damage associated with CIA.

CGS 21680, an agonist of the adenosine (A2A) receptor, decreases acute lung inflammation.

Adenosine A(2A) receptor agonists may be important regulators of inflammation. The aim of this study was to investigate the effects of CGS 21680 (0.1mg/kgi.p.), an agonist of the adenosine (A(2A)) receptor, in a mouse model of carrageenan-induced pleurisy. Injection of carrageenan into the pleural cavity of mice elicited an acute inflammatory response characterised by: infiltration of neutrophils in lung tissues and subsequent lipid peroxidation, increased production of nitric oxide (NO), cytokines such as tumour necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) and increased expression of intercellular adhesion molecule (ICAM-1) and platelet-adhesion molecule (P-selectin). Furthermore, carrageenan induced the expression of nuclear factor-κB (NF-κB), inducible nitric oxide synthase (iNOS), nitrotyrosine, the activation of poly-ADP-ribosyl polymerase (PARP), as well as induced apoptosis (FAS-ligand expression, Bax and Bcl-2 expression) in the lung tissues. Administration of CGS 21680, 30 min prior to challenge with carrageenan, caused a significant reduction of all the parameters of inflammation measured. In addition, to confirm the anti-inflammatory effect of CGS 21680, we have also evaluated the effects of CGS 21680 post-treatment (30 min after the challenge with carrageenan) and we have demonstrated that also it caused a reduction of neutrophil infiltration and the degree of lung injury. Thus, based on these findings we propose that adenosine A(2A) receptor agonists such as CGS 21680 may be useful in the treatment of various inflammatory diseases.

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

BACKGROUND: Permanent 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. METHODS: Spinal 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. RESULTS: SCH58261, 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. CONCLUSIONS: Our 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.

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 dipyridamole's anti-inflammatory properties.

Adenosine A2A receptor-selective stimulation reduces signaling pathways involved in the development of intestine ischemia and reperfusion injury.

In the present study, we tested the efficacy of treatment with the selective adenosine A2A receptor agonist 2-[p-(2-carboxyethyl)phenylethylamino]-50-ethylcarboxamidoadenosine (CGS 21680) on ischemia and reperfusion injury of the multivisceral organs. Ischemia and reperfusion injury was induced in mice by clamping both the superior mesenteric artery and the celiac artery for 30 min, followed thereafter by reperfusion. Sixty minutes after reperfusion, animals were killed for histological examination and biochemical studies. Injured vehicle-treated mice developed a significant increase of ileum TNF-alpha levels, myeloperoxidase activity, and marked histological injury and apoptosis. Ischemia and reperfusion injury of the multivisceral organs was also associated with significant mortality. Reperfused ileum sections from injured vehicle-treated mice showed positive staining for P-selectin and intercellular adhesion molecule 1. The intensity and degree of P-selectin and intercellular adhesion molecule 1 were markedly reduced in tissue sections from injured CGS 21680-treated mice. Ischemia and reperfusion-injured mice that have been treated with CGS 21680 showed also a significant reduction of neutrophil infiltration into the intestine, a reduction of apoptosis, and improved histological status of the intestine and survival. Taken together, our results clearly demonstrate that selective activation of adenosine A2A receptors plays an important role in the regulation of ischemia and reperfusion injury and results put forward the hypothesis that selective activation of adenosine A2A receptors may represent a novel and possible strategy.

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 selective adenosine A2A receptor agonist CGS 21680 reduces JNK MAPK activation in oligodendrocytes in injured spinal cord.

Permanent functional deficit after spinal cord injury (SCI) arises from both mechanical injury and from secondary tissue reactions involving inflammation. Adenosine is an important regulator of inflammatory mechanisms. Although functional studies indicate a protective effect of adenosine A2A receptor agonists in SCI, the basic molecular mechanisms accounting for the their protective effects from SCI have to be fully elucidated. In this study, we investigated if the selective A2A receptor agonist 2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680) administered after SCI has protective effects against tissue damage, motor deficit, and different inflammatory readouts. Spinal cord injury was induced in mice by extradural compression of a section of the SC exposed via a four-level T5-T8 laminectomy. CGS 21680, administered by subcutaneously implanted osmotic minipumps after SCI, clearly reduced motor deficit for up to 19 days after operation. The drug repeatedly administered intraperitoneally after SCI reduced tissue damage, influx of myeloperoxidase-positive leukocytes, nuclear factor-kappaB activation and iNOS expression in injured spinal cord tissue 24 h after SCI. Enhanced immunoreactivity of microglia, astrocytes, and oligodendrocytes (stained by anti-CD11/B, anti-glial fibrillary acidic protein, and anti-Olig2 antibodies, respectively) was also observed 24 h after SCI. Neurons lose immunoreactivity in the nucleus. c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase, quantified by Western blot, was definitely activated in injured tissue. CGS 21680 treatment significantly reduced JNK phosporylation. Phospho-JNK mitogen-activated protein kinase was de novo expressed selectively in oligodendrocytes. CGS 21680 reduced phospho-JNK immunostaining in oligodendrocytes. Data indicate that protection by the A2A agonist is secondary to reduced leukocyte recruitment in the damaged area. A reducing effect of JNK activation in oligodendrocytes might account for protective effect of the A2A agonist against SCI-induced demyelination.

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 A(2A) 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 A(2A) receptor binding and functioning. On the contrary, in striatum, a significant decrease in A(2A) 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 A(2A) receptor-G protein coupling. On the contrary, A(2A) 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, A(2A) 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 A(2A) 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 A(2A) active compounds.