Cerebral thrombosis

Objectives: We reviewed our most recent work on the protective effect of adenosine A^sub 2A^ antagonism in cerebral ischemia.

Methods: Focal ischemia was produced in rats by introducing a nylon monofilament pre-coated with silicone through the external carotid artery to occlude the right MCA at its origin.

Results: A^sub 2A^ antagonism was found protective in the model of permanent focal ischemia induced by the monofilament technique. This methodology provides the possibility of evaluating the protection against the outflow of excitatory amino acids and against an acute motor disturbance, i.e. contralateral turning to the ischemic side in the first hours after ischemia in awake rats. Hours later, a definite neurological deficit and necrotic neuronal damage can be evaluated.

Discussion: Our results suggest that A^sub 2A^ antagonism may be protective from the earliest up to several hours after the ischemic event. [Neurol Res 2005; 27: 169-174]

Keywords: Adenosine; A2A receptor; cerebral ischemia; neurological deficit

INTRODUCTION

Stroke is not only the third leading cause of death in major industrialized countries, but also a major cause of long-lasting disabilities. It results from a transient or permanent reduction in cerebral blood flow, which is, in most cases, caused by the occlusion of a cerebral artery, either by an embolus or by local thrombosis. It is defined as 'the rapid development of clinical signs of focal (or global) disturbance of cerebral function, with symptoms lasting 24 hours or longer, or which leads to death' with a mortality rate of around 30%.

Significant efforts have been made by neuroscientists to develop animal models that mimic the neuropathological consequences of stroke and have predictive value for drug neuroprotective effects in humans. Focal occlusion of the middle cerebral artery (MCA) is reported to be the most common cause of stroke in humans. Therefore, animal models of focal ischemia are believed to be the most pertinent in relation to human stroke1,2.

The putative cascade of damaging events in focal ischemia2 consists of very early excitotoxic mechanisms, which quickly cause acute cell death (necrosis), that then trigger or are followed by a number of events: immediately by peri-infarct depolarisations, hours later by inflammatory processes and days later by apoptotic cell death.

ADVANTAGES OF FOCAL ISCHEMIA INDUCED BY OCCLUSION OF THE MCA

In recent years we have developed a method of occlusion of the MCA in the rat originally devised by Zea Longa and co-workers3 and modified by us by coupling it with the microdialysis technique4.

Under deep anesthesia, after cutting the neck skin, the method consists of introducing a nylon monofilament pre-coated with silicone through the external carotid artery to occlude the right MCA at its origin. We thus obtain a focal ischemia. The occlusion of the MCA at its origin also occludes the lenticulostriate end arteries afferent to the striatum. This area therefore is not supplied by collateral flux and is considered the 'ischemic core', while the cortex may be considered a 'penumbral area' because a minimal residual blood flow remains5.

The procedure of the monofilament presents several advantages in comparison to the occlusion technique by electrocoagulation of the MCA. First of all, the rat is not craniectomized and can be kept awake after the MCA occlusion (MCAo). Therefore, in the first hours after ischemia, biochemical parameters such as transmitter outflow can be evaluated in awake animals. In addition, the effects of ischemia on transmitter outflow are not compounded by those of microdialysis probe implantation because the MCAo is performed 24 hours after microdialysis surgery (Table 1).

Soon after ischemia we have found that the outflow of different transmitters is increased. The outflow of excitatory amino acid glutamate and aspartate increase, the inhibitory transmitters GABA, adenosine and even taurine increase. Except for adenosine, which tends to return to basal values 4 hours after ischemia, all other transmitters tend to remain at high level for 4 hours after MCAo4,6.

Another advantage of the filament procedure is that, besides damaging the cortex, it provokes a definite striatal damage and ensuing sensorimotor deficit. Furthermore, since the rat is awake, acute motility disturbance can be evaluated in the first hours after ischemia (Table 7). In contrast to a global ischemic insult7,8, spontaneous locomotor activity was reported to be a relatively insensitive index of sensorimotor functions after induction of focal ischemia9. This is true for focal ischemia that only involves the cortex, but after focal ischemia that involves also the striatum, starting 1 hour after ischemia and in awake animals, we noted6 a clear turning behavior contralateral to the ischemic side. This is an acute effect during the time period in which a significant outflow of excitatory amino acid outflow occurs. This behavior is no longer evident 24 hours after ischemia. This increased motor behavior may well be ascribed to excitatory amino acid outflow. Glutamatergic agonists NMDA and AMPA injected into the intermediate and caudal parts of the caudate-putamen, induce contralateral rotations10. Contralateral turning behavior may be considered a useful predictor of damage and can be used to screen MCA occluded rats from further study where histological verification of the lesion is not possible. This behavior is significantly correlated with the ensuing neurological deficit and histological damage6. It is worth noting that the increases in locomotion after global ischemia or after focal ischemia that damages the cortex and striatum are quite different. While increased turning behavior after MCAo represents an acute disturbance of motility, increased locomotion after global ischemia persists for days after ischemia and is correlated with the damage of the CA1 area of the hippocampus11. After global ischemia this behavior is probably a deficit in habituation or spatial mapping rather than real motor hyperactivity12.

Although the most common method of assessment of damage in ischemia models is the histological measurement of neurodegeneration, lesion size is not always correlated with neurological deficit in different rodent models13. This is not an option for the clinician who will use measures of functional and neurological outcome. Therefore, measures of functional impairment and improvement are an important corollary for studying the putative neuroprotective effect of drugs. In the monofilament suture model, the rats show a clear neurological deficit 24 hours after ischemia, which according to Garcia and co-workers5 takes into account the spontaneous motor activity, symmetry in the movements of the four limbs, forepaw outstretching, climbing, body proprioception and response to the vibrissae touch. The MCA-occluded rats show a clear neurological deficit with a score which is reduced by 50% in comparison to sham-operated animals4,6.

Histological evaluation of the damage, performed 24 hours after ischemia by the Nissl method, shows a pallid area that corresponds to the number of necrotic neurons14. The damage found in the territory supplied by MCA, calculated as percentage of the ischemic hemisphere volume, is 19% in the striatum and 38% in the cortex. Taking into account the volume of the striatum and cortex, the damage involves the entire striatum and the cortex to a minor extent, including mainly the frontal and parietal cortex (Figure 7).

ADENOSINE OUTFLOW DURING MCAo

Our interest was focused on adenosine in this model. Adenosine is a powerful modulator in the CNS which is released during a variety of insults to the brain15. Under hypoxic/ischemic conditions, it is released directly from cells following degradation of intracellular ATP16. In the absence of oxygen and glucose, the imbalance between ATP degradation and ATP resynthesis favors adenosine production, which is transported out of cells by an equilibrative nucleoside transporter. Under normoxic conditions, outside cells adenosine reaches concentrations of around 200 nM as evaluated in vivo4,6 and in vitro15,17,18. During an ischemia-like stimulus in vitro obtained by depriving hippocampal slices of glucose and oxygen, adenosine release is proportional to the duration of the ischemic stimulus and is time-related to the depression of synaptic activity19. We found by a pharmacodynamic-electrophysiological approach in the hippocampus in vitro that adenosine increases proportionally during ischemia over time, reaching an increase of 140-fold at the receptor level 5 minutes thereafter. The concentration of 30 µM is high enough to stimulate all four of the identified adenosine receptor subtypes: A^sub 1^, A^sub 2A^, A^sub 2b^ and A^sub 3^ receptors. Kitagawa and co-workers20 demonstrated that direct perfusion of adenosine into the striatum during transient (90 minutes) focal ischemia protects against both necrotic damage and neurological deficit. The protective effect of adenosine is largely attributed to stimulation of A^sub 1^ receptors, which strongly regulate tissue excitability by reducing the influx of Ca^sub ++^ into neurons, by hyperpolarizing neurons, by reducing the release of excitatory amino acids, therefore inducing a general decrease of neuronal electrical activity, and thus reduction of cellular metabolism and energy consumption and consequently hypothermia21. However, both central and peripheral effects of adenosine or A^sub 1^ agonists hampers their use as protectants in particular sedation, hypotension and bradycardia22.

THE PROTECTIVE ROLE OF ADENOSINE A^sub 2A^ ANTAGONISTS

According to the definition of Ribeiro et al.23 adenosine plays a 'fine tuning' role in the CNS by stimulation of its receptors.

A second adenosine receptor strongly involved in ischemia is the A^sub 2A^ receptor. Since the first observations of Gao and Phillis7 in 1994 that the A^sub 2A^ antagonist CGS 15943 reduced cerebral ischemic injury in the gerbil, a number of reports have suggested that adenosine A^sub 2A^ antagonist may be protective during ischemia24-26. Jones and coworkers have elegantly underlined that A^sub 2A^ antagonists are protective by centrally reducing excitotoxicity. Furthermore, generation of A^sub 2A^ knockout mice has added the novel information that these mice are protected against cerebral infarction and also the neurological outcome of focal ischemia29.

Most recently, we have demonstrated6 in the MCA suture model that the A^sub 2A^ antagonist SCH 58261 given i.p. 5 minutes after MCAo at the concentration of 0.01 mg/kg significantly decreases, by about 35%, the outflow of glutamate and aspartate in the striatum during the 4 hours after ischemia (Figure 2). An unexpected and original observation was that the drug completely inhibits acute motor disturbance consisting of turning contralateral to the ischemic side (Figure 3). Since increased motor behavior is likely due to excitatory amino acid outflow10, the protective effect of SCH 58261 may well be ascribed to antagonism of the glutamate outflow and therefore of excitotoxic phenomena, which develop in the first hours after ischemia. The A^sub 2A^ antagonist induced reduction of infarct volume in the penumbral area, i.e. the cortex, but it did not improve the neurological outcome 24 hours after MCAo.

In response to a criticism regarding the utility of the neurological scoring system as a corollary of neuroprotection in rat models of focal ischemia, this is obviously highlighted in drug studies where reduced ischemic damage has been associated with improved neurological outcome30. On the other hand, not all ischemia models allow such a strict relationship15 and we have ascribed the failure of the A^sub 2A^ antagonist to improve the neurological deficit 24 hours after ischemia to the lack of significant striatal protection. The test battery used according to the protocol of Garcia and co-workers5 greatly involves regulation by the basal nuclei.

We made a further attempt to verify if the A^sub 2A^ antagonist was able to improve neurological deficit in the model of transient focal ischemia. Considering that in humans neurological symptoms tend to improve during the first week after insult31, transient focal ischemia in rats could be considered a suitable model to investigate a putative protective pharmacological effect from a neurological deficit. In this model, Garcia et al.5 showed that cerebral damage and neurological impairment are lower and that the sensory-motor functions tend to spontaneously recover starting from 3 days after occlusion. In agreement with results reported by Garcia and co-workers5, we observed that the neurological score significantly improves starting on the 4th day after ischemia. However, in the model of 1 hour transient cerebral ischemia, the A^sub 2A^ antagonist SCH 58261, given i.p. 5 minutes after MCAo at the concentration of 0.01 mg/kg, failed to improve the neurological deficit or to anticipate recovery (Table 2).

We next considered that protective effects of the A^sub 2A^ antagonist, besides protection against excitotoxicity, may be attributed also to blockade of adenosine A^sub 2A^ receptors located on microglial cells32 and therefore to possible anti-inflammatory effects. Upon neurotoxic insult, microglial cells start to proliferate and, by production of pro-inflammatory cytokines, may contribute to the inflammatory response that follows ischemic insult, hence aggravating brain damage2. While excitotoxicity is an early event after ischemia, inflammatory processes tend to develop during hours or days after ischemia. Considering that the half-life of the SCH 58261 is reported in the 2-3 hour range33, in the next study, the drug was subchronically administered at the dose of 0.01 mg/kg i.p. 5 minutes, 6 and 15 hours after ischemia. It is worth noting that SCH 58261 does not induce hemodynamic changes in the rat up to the dose of 1 mg/kg i.p. Preliminary data (unpublished data), obtained in the model of permanent focal ischemia, indicate that subchronic administration of the A^sub 2A^ antagonist significantly protects against the neurological deficit evaluated 24 hours after ischemia and from the histologically assessed damage both in the 'penumbral area', i.e. the cortex, and in the 'ischemic core', i.e. the striatum.

DRUG AND MECHANISM OF ACTION

A^sub 2A^ receptor mRNA is expressed on neurons at high levels in the striatum and at lower levels in the cortex and hippocampus34. In the striatum the bulk of A^sub 2A^ receptors are present on GABA-enkephalin striatopallidal neurons35, where they are localized with dopamine D2 receptors36. To a lesser extent, A^sub 2A^ receptors are located on corticostriatal glutamatergic terminals37. Stimulation by selective A^sub 2A^ agonists of striatal glutamate outflow under normoxic conditions38-40 or from the ischemic cortex41 is attributed to A^sub 2A^ pre-synaptic receptors. In agreement, the decreasing effect on glutamate outflow by A^sub 2A^ antagonists, under both normoxic42 and ischemic conditions6,41, is attributed to antagonism of A^sub 2A^ pre-synaptic receptors. Interestingly, we have recently found that the A^sub 2A^ antagonist SCH 58261 reduces striatal glutamate outflow in a different neurodegenerative disease model, i.e. in transgenic Huntington disease mice43. This observation indicates that in the striatum of transgenic mice the subpopulation of A^sub 2A^ receptors located on pre-synaptic glutamatergic terminals is not reduced while, as expected, A^sub 2A^ post-synaptic receptors located on GABA-enkephalin spiny neurons are reduced44. Since it was postulated that increased glutamatergic excitoxicity could play a role in the etiology of this disease45, antagonism of A^sub 2A^ receptors might be regarded as potentially useful in the treatment of Huntington disease.

Although most evidence indicates that A^sub 2A^ antagonists are protective in ischemia by reducing excitotoxicity, different mechanisms may account for their protective effects. Besides neurons, A^sub 2A^ receptors are located in microglia32. It has been shown that A^sub 2A^ receptor activation stimulates the proliferation of rat microglial cells in culture32 and that the A^sub 2A^ receptor antagonist DMPX protects hippocampal neurons in culture from death induced by microglial secretory products46.

Furthermore, adenosine activating different intracellular signaling pathways, depending on the receptor subtype activated, may stimulate both cell survival 49 and death50. Activation of adenosine A^sub 2A^ receptors increases intracellular cAMP with ensuing protein kinase A activation51, stimulates phosphatidylinositol, with ensuing protein kinase C activation, and increases intracellular Ca^sup ++^51/52. Recently, most studies demonstrate that stimulation of A^sub 2A^ receptors activates mitogen-activated protein kinase (MAPK) ERK1/2 in different cell preparations. Such activation may involve both Gs or different coupling mechanisms53. All these intracellular pathways are involved in cell proliferation, or cell suffering and death. The intracellular pathways that may be involved in neuronal survival, brought about by adenosine A^sub 2A^ antagonism during ischemia, are still to be elucidated.

CONCLUSIONS

Most evidence indicates now that adenosine A^sub 2A^ antagonism is a therapeutic approach that is worth considering after cerebral ischemia. In particular the model of permanent focal ischemia induced by the monofilament technique we describe here, permits the investigation of definite parameters of impairment: clear acute motor impairment, clear neurological deficit, definite necrotic damage in the cortex and striatum. All these parameters may be considered in relation to early modifications of glutamate outflow and therefore of excitotoxicity.

Data recently obtained in our laboratory indicate that A^sub 2A^ antagonism may be a useful therapeutic approach, starting from the first hour up to several hours after ischemia, suggesting that A^sub 2A^ antagonists may be drugs suitable for clinical experimentation. In clinical experimentation drugs can be administered several hours or days after the ischemic event. A^sub 2A^ antagonism improves functional and neurological outcome after ischemia. This is an option appropriate to the clinician and is an important corollary for the studying of the putative neuroprotective effect of drugs.

ACKNOWLEDGEMENTS

Investigations described in this paper were supported by grants from Italian MIUR (ex 40%) and from "Ente Cassa di Risparmio" of Florence, Italy.

REFERENCES

1 Hunter AJ, Green AR, Cross AJ. Animal models of acute ischaemic stroke: can they predict clinically successful neuroprotective drugs? Trends Pharmacol Sci 1995; 16: 123-128

2 Dirnagl U, ladecola C, Moskowitz MA. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 1999; 22: 391-397

3 Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 1989; 20: 84-91

4 Melani A, Pantoni L, Corsi C, et al. Striatal outflow of adenosine, excitatory amino acids, gamma-aminobutyric acid, and taurine in awake freely moving rats after middle cerebral artery occlusion: correlations with neurological deficit and histopathological damage. Stroke 1999; 30: 2448-2454

5 Garcia JH, Wagner S, Liu KF, et al. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats. Statistical validation. Stroke 1995; 26: 627-634

6 Melani A, Pantoni L, Bordoni F, et al. The selective A^sub 2A^ receptor antagonist SCH 58261 reduces Striatal transmitter outflow, turning behavior and ischemic brain damage induced by permanent focal ischemia in the rat. Brain Res 2003; 959: 243-250

7 Gao Y, Phillis JW. CGS 15943, an adenosine A^sub 2^ receptor antagonist, reduces cerebral ischemic injury in the Mongolian gerbil. Life Sci 1994; 55: L61-L65

8 Phillis JW. The effects of selective A^sub 1^ and A^sub 2A^ adenosine receptor antagonists on cerebral ischemic injury in the gerbil. Brain Res 1995; 705: 79-84

9 Hunter AJ, Mackay KB, Rogers DC. To what extent have functional studies of ischaemia in animals been useful in the assessment of potential neuroprotective agents? Trends Pharmacol Sd 1998; 19: 59-66

10 Ossowska K. Interaction between Striatal excitatory amino acid and gamma- aminobutyric acid (GABA) receptors in the turning behaviour of rats. Neurosci Lett 1995; 202: 57-60

11 Mileson BE, Schwartz RD. The use of locomotor activity as a behavioral screen for neuronal damage following transient forebrain ischemia in gerbils. Neurosci Lett 1991; 128: 71-76

12 Babcock AM, Baker DA, Lovec R. Locomotor activity in the ischemic gerbil. Brain Res 1993; 625: 351-354

13 Rogers DC, Campbell CA, Stretton JL, et al. Correlation between motor impairment and infarct volume after permanent and transient middle cerebral artery occlusion in the rat. Stroke 1997; 28: 2060-2065

14 Garcia JH, Liu KF, Ho KL. Neuronal necrosis after middle cerebral artery occlusion in Wistar rats progresses at different time intervals in the caudoputamen and the cortex. Stroke 1995; 26: 636-642

15 Latini S, Pedata F. Adenosine in the central nervous system: release mechanisms and extracellular concentrations. J Neurochem 2001; 79: 463-484

16 Latini S, Corsi C, Pedata F, et al. The source of brain adenosine outflow during ischemia and electrical stimulation. Neurochem Int 1995; 27: 239-244

17 Dunwiddie TV, Diao L. Extracellular adenosine concentrations in hippocampal brain slices and the tonic inhibitory modulation of evoked excitatory responses. J Pharmacol Exp Ther 1994; 268: 537-545

18 Latini S, Bordoni F, Pedata F, et al. Extracellular adenosine concentrations during in vitro ischaemia in rat hippocampal slices. Br J Pharmacol 1999; 127: 729-739

19 Latini S, Bordoni F, Corradetti R, et al. Temporal correlation between adenosine outflow and synaptic potential inhibition in rat hippocampal slices during ischemia-like conditions. Brain Res 1998; 794: 325-328

20 Kitagawa H, Mori A, Shimada J, et al. Intracerebral adenosine infusion improves neurological outcome after transient focal ischemia in rats. Neurol Res 2002; 24: 317-323

21 von Lubitz DK. Adenosine and cerebral ischemia: therapeutic future or death of a brave concept? Eur J Pharmacol 1999; 365: 925

22 Olsson RA, PearsonJD. Cardiovascular purinoceptors. Physiol Rev 1990; 70: 761-845

23 Ribeiro JA, Sebastiao AM, cle Mendonca A. Participation of adenosine receptors in neuroprotection. Drug News Perspect 2003; 16: 80-86

24 von Lubitz DK, Lin RC, lacobson KA. Cerebral ischemia in gerbils: effects of acute and chronic treatment with adenosine A^sub 2A^ receptor agonist and antagonist. Eur J Pharmacol 1995; 287: 295-302

25 Bona E, Aden U, Gill and E, et al. Neonatal cerebral hypoxiaischemia: the effect of adenosine receptor antagonists. Neuropharmacology 1997; 36: 1327-1338

26 Monopoli A, Lozza G, Forlani A, et al. Blockade of adenosine A^sub 2A^ receptors by SCH 58261 results in neuroprotective effects in cerebral ischaemia in rats. Neuroreport 1998; 9: 3955-3959

27 Jones PA, Smith RA, Stone TW. Protection against kainate-induced excitotoxicity by adenosine A^sub 2A^ receptor agonists and antagonists. Neuroscience 1998; 85: 229-237

28 Jones PA, Smith RA, Stone TW. Protection against hippocampal kainate excitotoxicity by intracerebral administration of an adenosine A^sub 2A^ receptor antagonist. Brain Res 1998; 800: 328-335

29 Chen JF, Huang Z, Ma J, et al. A^sub 2A^ adenosine receptor deficiency attenuates brain injury induced by transient focal ischemia in mice. J Neurosci 1999; 19: 9192-9200

30 Belayev L, Alonso OF, Busto R, ct al. Middle cerebral artery occlusion in the rat by intraluminal suture. Neurological and pathological evaluation of an improved model. Stroke 1996; 27: 1616-1622

31 Loddick SA, Rothwell NJ. Neuroprotective effects of human recombinant interleukin-1 receptor antagonist in focal cerebral ischaemia in the rat. J Cereb Blood Flow Metab 1996; 16: 932-940

32 Fiebich BL, Biber K, Lieb K, et al. Cyclooxygenase-2 expression in rat microglia is induced by adenosine A^sub 2A^-receptors. Glia 1996; 18: 152-160

33 Ongini E. SCH 58261: a selective A^sub 2A^ adenosine receptor antagonist. Drug Develop Res 1997; 42: 53-70

34 Dixon AK, Gubitz AK, Sirinathsinghji DJ, et al. Tissue distribution of adenosine receptor mRNAs in the rat. Br J Pharmacol 1996; 118: 1461-1468

35 Schiffmann SN, Jacobs O, Vanderhaeghen JJ. Striatal restricted adenosine A^sub 2^ receptor (RDC8) is expressed by enkephalin but not by substance P neurons: an in situ hybridization histochemistry study. J Neurochem 1991; 57: 1062-1067

36 Fink JS, Weaver DR, Rivkees SA, et al. Molecular cloning of the rat A^sub 2^ adenosine receptor: selective co-expression with D2 dopamine receptors in rat striatum. Brain Res Mol Brain Res 1992; 14: 186195

37 Hettinger BD, Lee A, Linden J, et al. Ultrastructural localization of adenosine A^sub 2A^ receptors suggests multiple cellular sites for modulation of GABAergic neurons in rat striatum. J Comp Neurol 2001; 431: 331-346

38 Popoli P, Betto P, Reggio R, et al. Adenosine A^sub 2A^ receptor stimulation enhances striatal extracellular glutamate levels in rats. Eur J Pharmacol 1995; 287: 215-217

39 Golembiowska K, Zylewska A. Adenosine receptors-the role in modulation of dopamine and glutamate release in the rat striatum. Pol J Pharmacol 1997; 49: 31 7-322

40 Corsi C, Melani A, Bianchi L, et al. Effect of adenosine A^sub 2A^ receptor stimulation on CABA release from the striatum of young and aged rats in vivo. Neuroreport 1999; 10: 3933-3937

41 Simpson RE, O'Regan MH, Perkins LM, et al. Excitatory transmitter amino acid release from the ischemic rat cerebral cortex: effects of adenosine receptor agonists and antagonists. J Neurochem 1992; 58: 1683-1690

42 Corsi C, Melani A, Bianchi L, et al. Striatal A^sub 2A^ adenosine receptor antagonism differentially modifies striatal glutamate outflow in vivo in young and aged rats. Neuroreport 2000; 11: 2591-2595

43 Gianfriddo M, Melani A, Turchi D, et al. Adenosine and glutamate extracellular concentrations and mitogen-activated protein kinases in the striatum of Huntington transgenic mice. Selective antagonism of adenosine A^sub 2A^ receptors reduces transmitter outflow. Neurobiol Dis 2004; 17: 77-88

44 Cha JH, Frey AS, Alsdorf SA, et al. Altered neurotransmitter receptor expression in transgenic mouse models of Huntington's disease. Phil Trans Roy Soc Lond B Biol Sd 1999; 354: 981-989

45 Zeron MM, Hansson O, Chen N, et al. Increased sensitivity to Nmethyl-D-aspartate receptor-mediated excitotoxicity in a mouse model of Huntington's disease. Neuron 2002; 33: 849-860

46 Flavin MP, Ho LT. Propentofylline protects neurons in culture from death triggered by macrophage or microglial secretory products. J Neurosci Res 1999; 56: 54-59

47 Neary JT, Zhu Q, Kang Y, et al. Extracellular ATP induces formation of AP-1 complexes in astrocytes via P2 purinoceptors. Neuroreport 1996; 7: 2893-2896

48 Vitolo OV, Ciotti MT, Calli C, et al. Adenosine and ADP prevent apoptosis in cultured rat cerebellar granule cells. Brain Res 1998; 809: 297-301

49 Fredholm BB. Adenosine and neuroprotection, Int Rev Neurobiol 1997; 40: 259-280

50 Jacobson KA, Hoffmann C, Cattabeni F, et al. Adenosine-induced cell death: evidence for receptor-mediated signalling. Apoptosis 1999; 4: 197-211

51 Gubitz AK, Widdowson L, Kurokawa M, et al. Dual signalling by the adenosine A^sub 2A^ receptor involves activation of both N- and Ptype calcium channels by different G proteins and protein kinases in the same striatal nerve terminals. J Neurochem 1996; 67: 374381

52 Goncalves ML, Cunha RA, Ribeiro JA. Adenosine A^sub 2A^ receptors facilitate 45Ca^sup 2+^ uptake through class A calcium channels in rat hippocampal CA3 but not CAl synaptosomes. Neurosci Lett 1997; 238: 73-77

53 Schulte G, Fredholm BB. The G(s)-coupled adenosine A^sub 2B^ receptor recruits divergent pathways to regulate ERK1/2 and p38. Exp Cell Res 2003; 290: 1 68-176

F. Pedata, M. Gianfriddo, D. Turchi and A. Melani

Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy

Correspondence and reprint requests to: Professor F. Pedata, Department of Preclinical and Clinical Pharmacology, University of Florence, V.le Pieraccini 6, 50139 Florence, Italy. [felicita.pedata@ unifi.it] Accepted for publication December 2004.

Copyright Maney Publishing Mar 2005
Provided by ProQuest Information and Learning Company. All rights Reserved