Pharmacological Characterization of P626, a Novel Dual Adenosine A2A/A2B Receptor Antagonist, on Synaptic Plasticity and during an Ischemic-like Insult in CA1 Rat Hippocampus.

In recent years, the use of multi-target compounds has become an increasingly pursued strategy to treat complex pathologies, including cerebral ischemia. Adenosine and its receptors (A1AR, A2AAR, A2BAR, A3AR) are known to play a crucial role in synaptic transmission either in normoxic or ischemic-like conditions. Previous data demonstrate that the selective antagonism of A2AAR or A2BAR delays anoxic depolarization (AD) appearance, an unequivocal sign of neuronal injury induced by a severe oxygen-glucose deprivation (OGD) insult in the hippocampus. Furthermore, the stimulation of A2AARs or A2BARs by respective selective agonists, CGS21680 and BAY60-6583, increases pre-synaptic neurotransmitter release, as shown by the decrease in paired-pulse facilitation (PPF) at Schaffer collateral-CA1 synapses. In the present research, we investigated the effect/s of the newly synthesized dual A2AAR/A2BAR antagonist, P626, in preventing A2AAR- and/or A2BAR-mediated effects by extracellular recordings of synaptic potentials in the CA1 rat hippocampal slices. We demonstrated that P626 prevented PPF reduction induced by CGS21680 or BAY60-6583 and delayed, in a concentration-dependent manner, AD appearance during a severe OGD. In conclusion, P626 may represent a putative neuroprotective compound for stroke treatment with the possible translational advantage of reducing side effects and bypassing differences in pharmacokinetics due to combined treatment.

Harmonization of sensorimotor deficit assessment in a registered multicentre pre-clinical randomized controlled trial using two models of ischemic stroke.

Multicentre preclinical randomized controlled trials (pRCTs) are a valuable tool to improve experimental stroke research, but are challenging and therefore underused. A common challenge regards the standardization of procedures across centres. We here present the harmonization phase for the quantification of sensorimotor deficits by composite neuroscore, which was the primary outcome of two multicentre pRCTs assessing remote ischemic conditioning in rodent models of ischemic stroke. Ischemic stroke was induced by middle cerebral artery occlusion for 30, 45 or 60 min in mice and 50, 75 or 100 min in rats, allowing sufficient variability. Eleven animals per species were video recorded during neurobehavioural tasks and evaluated with neuroscore by eight independent raters, remotely and blindly. We aimed at reaching an intraclass correlation coefficient (ICC) ≥0.60 as satisfactory interrater agreement. After a first remote training we obtained ICC = 0.50 for mice and ICC = 0.49 for rats. Errors were identified in animal handling and test execution. After a second remote training, we reached the target interrater agreement for mice (ICC = 0.64) and rats (ICC = 0.69). In conclusion, a multi-step, online harmonization phase proved to be feasible, easy to implement and highly effective to align each centre's behavioral evaluations before project's interventional phase.

Therapeutic Potential of Highly Selective A3 Adenosine Receptor Ligands in the Central and Peripheral Nervous System.

Ligands of the Gi protein-coupled adenosine A3 receptor (A3R) are receiving increasing interest as attractive therapeutic tools for the treatment of a number of pathological conditions of the central and peripheral nervous systems (CNS and PNS, respectively). Their safe pharmacological profiles emerging from clinical trials on different pathologies (e.g., rheumatoid arthritis, psoriasis and fatty liver diseases) confer a realistic translational potential to these compounds, thus encouraging the investigation of highly selective agonists and antagonists of A3R. The present review summarizes information on the effect of latest-generation A3R ligands, not yet available in commerce, obtained by using different in vitro and in vivo models of various PNS- or CNS-related disorders. This review places particular focus on brain ischemia insults and colitis, where the prototypical A3R agonist, Cl-IB-MECA, and antagonist, MRS1523, have been used in research studies as reference compounds to explore the effects of latest-generation ligands on this receptor. The advantages and weaknesses of these compounds in terms of therapeutic potential are discussed.

Uncovering the Mechanisms of Adenosine Receptor-Mediated Pain Control: Focus on the A3 Receptor Subtype.

Agonists of the Gi protein-coupled A3 adenosine receptor (A3AR) have shown important pain-relieving properties in preclinical settings of several pain models. Active as a monotherapy against chronic pain, A3AR agonists can also be used in combination with classic opioid analgesics. Their safe pharmacological profile, as shown by clinical trials for other pathologies, i.e., rheumatoid arthritis, psoriasis and fatty liver diseases, confers a realistic translational potential, thus encouraging research studies on the molecular mechanisms underpinning their antinociceptive actions. A number of pathways, involving central and peripheral mechanisms, have been proposed. Recent evidence showed that the prototypical A3AR agonist Cl-IB-MECA and the new, highly selective, A3AR agonist MRS5980 inhibit neuronal (N-type) voltage-dependent Ca2+ currents in dorsal root ganglia, a known pain-related mechanism. Other proposed pathways involve reduced cytokine production, immune cell-mediated responses, as well as reduced microglia and astrocyte activation in the spinal cord. The aim of this review is to summarize up-to-date information on A3AR in the context of pain, including cellular and molecular mechanisms underlying this effect. Based on their safety profile shown in clinical trials for other pathologies, A3AR agonists are proposed as novel, promising non-narcotic agents for pain control.

Role of Carbonic Anhydrase in Cerebral Ischemia and Carbonic Anhydrase Inhibitors as Putative Protective Agents.

Ischemic stroke is a leading cause of death and disability worldwide. The only pharmacological treatment available to date for cerebral ischemia is tissue plasminogen activator (t-PA) and the search for successful therapeutic strategies still remains a major challenge. The loss of cerebral blood flow leads to reduced oxygen and glucose supply and a subsequent switch to the glycolytic pathway, which leads to tissue acidification. Carbonic anhydrase (CA, EC 4.2.1.1) is the enzyme responsible for converting carbon dioxide into a protons and bicarbonate, thus contributing to pH regulation and metabolism, with many CA isoforms present in the brain. Recently, numerous studies have shed light on several classes of carbonic anhydrase inhibitor (CAI) as possible new pharmacological agents for the management of brain ischemia. In the present review we summarized pharmacological, preclinical and clinical findings regarding the role of CAIs in strokes and we discuss their potential protective mechanisms.

A2 B Adenosine Receptors and Sphingosine 1-Phosphate Signaling Cross-Talk in Oligodendrogliogenesis.

Oligodendrocyte-formed myelin sheaths allow fast synaptic transmission in the brain. Impairments in the process of myelination, or demyelinating insults, might cause chronic diseases such as multiple sclerosis (MS). Under physiological conditions, remyelination is an ongoing process throughout adult life consisting in the differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes (OLs). During pathological events, this process fails due to unfavorable environment. Adenosine and sphingosine kinase/sphingosine 1-phosphate signaling axes (SphK/S1P) play important roles in remyelination processes. Remarkably, fingolimod (FTY720), a sphingosine analog recently approved for MS treatment, plays important roles in OPC maturation. We recently demonstrated that the selective stimulation of A2 B adenosine receptors (A2 B Rs) inhibit OPC differentiation in vitro and reduce voltage-dependent outward K+ currents (I K ) necessary to OPC maturation, whereas specific SphK1 or SphK2 inhibition exerts the opposite effect. During OPC differentiation A2 B R expression increases, this effect being prevented by SphK1/2 blockade. Furthermore, selective silencing of A2 B R in OPC cultures prompts maturation and, intriguingly, enhances the expression of S1P lyase, the enzyme responsible for irreversible S1P catabolism. Finally, the existence of an interplay between SphK1/S1P pathway and A2 B Rs in OPCs was confirmed since acute stimulation of A2 B Rs activates SphK1 by increasing its phosphorylation. Here the role of A2 B R and SphK/S1P signaling during oligodendrogenesis is reviewed in detail, with the purpose to shed new light on the interaction between A2 B Rs and S1P signaling, as eventual innovative targets for the treatment of demyelinating disorders.

Protective effects of carbonic anhydrase inhibition in brain ischaemia in vitro and in vivo models.

Ischaemic stroke is a leading cause of death and disability. One of the major pathogenic mechanisms after ischaemia includes the switch to the glycolytic pathway, leading to tissue acidification. Carbonic anhydrase (CA) contributes to pH regulation. A new generation of CA inhibitors, AN11-740 and AN6-277 and the reference compound acetazolamide (ACTZ) were investigated in two models of brain ischaemia: in rat hippocampal acute slices exposed to severe oxygen, glucose deprivation (OGD) and in an in vivo model of focal cerebral ischaemia induced by permanent occlusion of the middle cerebral artery (pMCAo) in the rat. In vitro, the application of selective CAIs significantly delayed the appearance of anoxic depolarisation induced by OGD. In vivo, sub-chronic systemic treatment with AN11-740 and ACTZ significantly reduced the neurological deficit and decreased the infarct volume after pMCAo. CAIs counteracted neuronal loss, reduced microglia activation and partially counteracted astrocytes degeneration inducing protection from functional and tissue damage.

Acetylcholine modulates K+ and Na+ currents in human basal forebrain cholinergic neuroblasts through an autocrine/paracrine mechanism.

The Nucleus Basalis of Meynert (NBM) is the main source of cholinergic neurons in the basal forebrain to be crucially involved in cognitive functions and whose degeneration correlates with cognitive decline in major degenerative pathologies as Alzheimer's and Parkinson's diseases. However, knowledge concerning NBM neurons derived from human brain is very limited to date. We recently characterized a primary culture of proliferating neuroblasts isolated from the human fetal NBM (hfNBM) as immature cholinergic neurons expressing the machinery to synthetize and release acetylcholine. Here we studied in detail electrophysiological features and cholinergic effects in this cell culture by patch-clamp recordings. Our data demonstrate that atropine-blocked muscarinic receptor activation by acetylcholine or carbachol enhanced IK and reduced INa currents by stimulating Gi -coupled M2 or phospholipase C-coupled M3 receptors, respectively. Inhibition of acetylcholine esterase activity by neostigmine unveiled a spontaneous acetylcholine release from hfNBM neuroblasts that might account for an autocrine/paracrine signaling during human brain development. Present data provide the first description of cholinergic effects in human NBM neurons and point to a role of acetylcholine as an autocrine/paracrine modulator of voltage-dependent channels. Our research could be of relevance in understanding the mechanisms of cholinergic system development and functions in the human brain, either in health or disease.

A2B Adenosine Receptors: When Outsiders May Become an Attractive Target to Treat Brain Ischemia or Demyelination.

Adenosine is a signaling molecule, which, by activating its receptors, acts as an important player after cerebral ischemia. Here, we review data in the literature describing A2BR-mediated effects in models of cerebral ischemia obtained in vivo by the occlusion of the middle cerebral artery (MCAo) or in vitro by oxygen-glucose deprivation (OGD) in hippocampal slices. Adenosine plays an apparently contradictory role in this receptor subtype depending on whether it is activated on neuro-glial cells or peripheral blood vessels and/or inflammatory cells after ischemia. Indeed, A2BRs participate in the early glutamate-mediated excitotoxicity responsible for neuronal and synaptic loss in the CA1 hippocampus. On the contrary, later after ischemia, the same receptors have a protective role in tissue damage and functional impairments, reducing inflammatory cell infiltration and neuroinflammation by central and/or peripheral mechanisms. Of note, demyelination following brain ischemia, or autoimmune neuroinflammatory reactions, are also profoundly affected by A2BRs since they are expressed by oligodendroglia where their activation inhibits cell maturation and expression of myelin-related proteins. In conclusion, data in the literature indicate the A2BRs as putative therapeutic targets for the still unmet treatment of stroke or demyelinating diseases.

Acute visceral pain relief mediated by A3AR agonists in rats: involvement of N-type voltage-gated calcium channels.

ABSTRACT: Pharmacological tools for chronic visceral pain management are still limited and inadequate. A3 adenosine receptor (A3AR) agonists are effective in different models of persistent pain. Recently, their activity has been related to the block of N-type voltage-gated Ca2+ channels (Cav2.2) in dorsal root ganglia (DRG) neurons. The present work aimed to evaluate the efficacy of A3AR agonists in reducing postinflammatory visceral hypersensitivity in both male and female rats. Colitis was induced by the intracolonic instillation of 2,4-dinitrobenzenesulfonic acid (DNBS; 30 mg in 0.25 mL 50% EtOH). Visceral hypersensitivity was assessed by measuring the visceromotor response and the abdominal withdrawal reflex to colorectal distension. The effects of A3AR agonists (MRS5980 and Cl-IB-MECA) were evaluated over time after DNBS injection and compared to that of the selective Cav2.2 blocker PD173212, and the clinically used drug linaclotide. A3AR agonists significantly reduced DNBS-evoked visceral pain both in the postinflammatory (14 and 21 days after DNBS injection) and persistence (28 and 35 days after DNBS) phases. Efficacy was comparable to effects induced by linaclotide. PD173212 fully reduced abdominal hypersensitivity to control values, highlighting the role of Cav2.2. The effects of MRS5980 and Cl-IB-MECA were completely abolished by the selective A3AR antagonist MRS1523. Furthermore, patch-clamp recordings showed that A3AR agonists inhibited Cav2.2 in dorsal root ganglia neurons isolated from either control or DNBS-treated rats. The effect on Ca2+ current was PD173212-sensitive and prevented by MRS1523. A3AR agonists are effective in relieving visceral hypersensitivity induced by DNBS, suggesting a potential therapeutic role against abdominal pain.

Adenosine A2B receptors inhibit K+ currents and cell differentiation in cultured oligodendrocyte precursor cells and modulate sphingosine-1-phosphate signaling pathway.

Oligodendrocytes are the only myelinating cells in the brain and differentiate from their progenitors (OPCs) throughout adult life. However, this process fails in demyelinating pathologies. Adenosine is emerging as an important player in OPC differentiation and we recently demonstrated that adenosine A2A receptors inhibit cell maturation by reducing voltage-dependent K+ currents. No data are available to date about the A2B receptor (A2BR) subtype. The bioactive lipid mediator sphingosine-1-phosphate (S1P) and its receptors (S1P1-5) are also crucial modulators of OPC development. An interaction between this pathway and the A2BR is reported in peripheral cells. We studied the role of A2BRs in modulating K+ currents and cell differentiation in OPC cultures and we investigated a possible interplay with S1P signaling. Our data indicate that the A2BR agonist BAY60-6583 and its new analogue P453 inhibit K+ currents in cultured OPC and the effect was prevented by the A2BR antagonist MRS1706, by K+ channel blockers and was differently modulated by the S1P analogue FTY720-P. An acute (10 min) exposure of OPCs to BAY60-6583 also increased the phosphorylated form of sphingosine kinase 1 (SphK1). A chronic (7 days) treatment with the same agonist decreased OPC differentiation whereas SphK1/2 inhibition exerted the opposite effect. Furthermore, A2BR was overexpressed during OPC differentiation, an effect prevented by the pan SphK1/2 inhibitor VPC69047. Finally, A2BR silenced cells showed increased cell maturation, decreased SphK1 expression and enhanced S1P lyase levels. We conclude that A2BRs inhibit K+ currents and cell differentiation and positively modulate S1P synthesis in cultured OPCs.

Protective Effect of Adenosine A2B Receptor Agonist, BAY60-6583, Against Transient Focal Brain Ischemia in Rat.

Cerebral ischemia is a multifactorial pathology characterized first by an acute injury, due to excitotoxicity, followed by a secondary brain injury that develops hours to days after ischemia. During ischemia, adenosine acts as an endogenous neuroprotectant. Few studies have investigated the role of A2B receptor in brain ischemia because of the low potency of adenosine for it and the few selective ligands developed so far. A2B receptors are scarcely but widely distributed in the brain on neurons, glial and endothelial cells and on hematopoietic cells, lymphocytes and neutrophils, where they exert mainly anti-inflammatory effects, inhibiting vascular adhesion and inflammatory cells migration. Aim of this work was to verify whether chronic administration of the A2B agonist, BAY60-6583 (0.1 mg/kg i.p., twice/day), starting 4 h after focal ischemia induced by transient (1 h) Middle Cerebral Artery occlusion (tMCAo) in the rat, was protective after the ischemic insult. BAY60-6583 improved the neurological deficit up to 7 days after tMCAo. Seven days after ischemia BAY60-6583 reduced significantly the ischemic brain damage in cortex and striatum, counteracted ischemia-induced neuronal death, reduced microglia activation and astrocytes alteration. Moreover, it decreased the expression of TNF-α and increased that of IL-10 in peripheral plasma. Two days after ischemia BAY60-6583 reduced blood cell infiltration in the ischemic cortex. The present study indicates that A2B receptors stimulation can attenuate the neuroinflammation that develops after ischemia, suggesting that A2B receptors may represent a new interesting pharmacological target to protect from degeneration after brain ischemia.

Multicentre translational Trial of Remote Ischaemic Conditioning in Acute Ischaemic Stroke (TRICS): protocol of multicentre, parallel group, randomised, preclinical trial in female and male rat and mouse from the Italian Stroke Organization (ISO) Basic Science network.

INTRODUCTION: Multicentre preclinical randomised controlled trials (pRCT) are emerging as a necessary step to confirm efficacy and improve translation into the clinic. The aim of this project is to perform two multicentre pRCTs (one in rats and one in mice) to investigate the efficacy of remote ischaemic conditioning (RIC) in an experimental model of severe ischaemic stroke. METHODS AND ANALYSIS: Seven research laboratories within the Italian Stroke Organization (ISO) Basic Science network will participate in the study. Transient endovascular occlusion of the proximal right middle cerebral artery will be performed in two species (rats and mice) and in both sexes. Animals will be randomised to receive RIC by transient surgical occlusion of the right femoral artery, or sham surgery, after reperfusion. Blinded outcome assessment will be performed for dichotomised functional neuroscore (primary endpoint) and infarct volume (secondary endpoint) at 48 hours. A sample size of 80 animals per species will yield 82% power to detect a significant difference of 30% in the primary outcome in both pRCTs. Analyses will be performed in a blind status and according to an intention-to-treat paradigm. The results of this study will provide robust, translationally oriented, high-quality evidence on the efficacy of RIC in multiple species of rodents with large ischaemic stroke. ETHICS AND DISSEMINATION: This is approved by the Animal Welfare Regulatory Body of the University of Milano Bicocca, under project license from the Italian Ministry of Health. Trial results will be subject to publication according to the definition of the outcome presented in this protocol. TRIAL REGISTRATION NUMBER: PCTE0000177.

Functional characterization of a novel adenosine A2B receptor agonist on short-term plasticity and synaptic inhibition during oxygen and glucose deprivation in the rat CA1 hippocampus.

Adenosine is an endogenous neuromodulator exerting its biological functions via four receptor subtypes, A1, A2A, A2B, and A3. A2B receptors (A2BRs) are expressed at hippocampal level where they are known to inhibit paired pulse facilitation (PPF), whose reduction reflects an increase in presynaptic glutamate release. The effect of A2BRs on PPF is known to be sensitive not only to A2BR blockade but also to the A1R antagonist DPCPX, indicating that it involves A1R activation. In this study we provide the first functional characterization of the newly synthesized non-nucleoside like A2BR agonist P453, belonging to the amino-3,5-dicyanopyridine series. By extracellular electrophysiological recordings, we demonstrated that P453 mimicked the effect of the prototypical A2BR agonist BAY60-6583 in decreasing PPF at Schaffer collateral-CA1 synapses in rat acute hippocampal slices. This effect was prevented by two different A2BR antagonists, PSB603 and MRS1754, and by the A1R antagonist DPCPX. We also investigated the functional role of A2BR during a 2 min of oxygen and glucose deprivation (OGD) insult, known to produce a reversible fEPSP inhibition due to adenosine A1R activation. We found that P453 and BAY60-6583 significantly delayed the onset of fEPSP reduction induced by OGD and the effect was blocked by PSB603. We conclude that P453 is a functional A2BR agonist whose activation decreases PPF by increasing glutamate release at presynaptic terminals and delays A1R-mediated fEPSP inhibition during a 2-minute OGD insult.

Adenosine A3 receptor activation inhibits pronociceptive N-type Ca2+ currents and cell excitability in dorsal root ganglion neurons.

Recently, studies have focused on the antihyperalgesic activity of the A3 adenosine receptor (A3AR) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Here, we investigated the expression and functional effects of A3AR on the excitability of small- to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats. Real-time quantitative polymerase chain reaction experiments and immunofluorescence analysis revealed A3AR expression in DRG neurons. Patch-clamp experiments demonstrated that 2 distinct A3AR agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca-activated K (KCa) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca influx via N-type voltage-dependent Ca channels, as Cl-IB-MECA-induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca currents, and its effect was inhibited by 56% in the presence of A3AR antagonist MRS1523, demonstrating that the majority of adenosine's effect is mediated by this receptor subtype. Current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A3AR activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca measurements confirmed the inhibitory role of A3AR on DRG neuronal firing. We conclude that pain-relieving effects observed on A3AR activation could be mediated through N-type Ca channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A3AR-based therapy as a viable approach to alleviate pain in different pathologies.

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 Selective Antagonism of Adenosine A2B Receptors Reduces the Synaptic Failure and Neuronal Death Induced by Oxygen and Glucose Deprivation in Rat CA1 Hippocampus in Vitro.

Ischemia is a multifactorial pathology characterized by different events evolving in time. Immediately after the ischemic insult, primary brain damage is due to the massive increase of extracellular glutamate. Adenosine in the brain increases dramatically during ischemia in concentrations able to stimulate all its receptors, A1, A2A, A2B, and A3. Although adenosine exerts clear neuroprotective effects through A1 receptors during ischemia, the use of selective A1 receptor agonists is hampered by their undesirable peripheral side effects. So far, no evidence is available on the involvement of adenosine A2B receptors in cerebral ischemia. This study explored the role of adenosine A2B receptors on synaptic and cellular responses during oxygen and glucose deprivation (OGD) in the CA1 region of rat hippocampus in vitro. We conducted extracellular recordings of CA1 field excitatory post-synaptic potentials (fEPSPs); the extent of damage on neurons and glia was assessed by immunohistochemistry. Seven min OGD induced anoxic depolarization (AD) in all hippocampal slices tested and completely abolished fEPSPs that did not recover after return to normoxic condition. Seven minutes OGD was applied in the presence of the selective adenosine A2B receptor antagonists MRS1754 (500 nM) or PSB603 (50 nM), separately administered 15 min before, during and 5 min after OGD. Both antagonists were able to prevent or delay the appearance of AD and to modify synaptic responses after OGD, allowing significant recovery of neurotransmission. Adenosine A2B receptor antagonism also counteracted the reduction of neuronal density in CA1 stratum pyramidale, decreased apoptosis at least up to 3 h after the end of OGD, and maintained activated mTOR levels similar to those of controls, thus sparing neurons from the degenerative effects caused by the simil-ischemic conditions. Astrocytes significantly proliferated in CA1 stratum radiatum already 3 h after the end of OGD, possibly due to increased glutamate release. A2Breceptor antagonism significantly prevented astrocyte modifications. Both A2B receptor antagonists did not protect CA1 neurons from the neurodegeneration induced by glutamate application, indicating that the antagonistic effect is upstream of glutamate release. The selective antagonists of the adenosine A2B receptor subtype may thus represent a new class of neuroprotective drugs in 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.