A(2A) adenosine receptor deficiency attenuates brain injury induced by transient focal ischemia in mice

Abstract

Extracellular adenosine critically modulates ischemic brain injury, at least in part through activation of the A(1) adenosine receptor. However, the role played by the A(2A) receptor has been obscured by intrinsic limitations of A(2A) adenosinergic agents. To overcome these pharmacological limitations, we explored the consequences of deleting the A(2A) adenosine receptor on brain damage after transient focal ischemia. Cerebral morphology, as well as vascular and physiological measures (before, during, and after ischemia) did not differ between A(2A) receptor knock-out and wild-type littermates. The volume of cerebral infarction, as well as the associated neurological deficit induced by transient filament occlusion of the middle cerebral artery, were significantly attenuated in A(2A) receptor knock-out mice. This neuroprotective phenotype of A(2A) receptor-deficient mice was observed in different genetic backgrounds, confirming A(2A) receptor disruption as its cause. Together with complimentary pharmacological studies, these data suggest that A(2A) receptors play a prominent role in the development of ischemic injury within brain and demonstrate the potential for anatomical and functional neuroprotection against stroke by A(2A) receptor antagonists.

Fig. 1.

Generation of A_2A KO mice with target inactivation of the A_2A receptor.A, Schematic diagram of the A_2A receptor targeting vector; a standard replacement-type vector was constructed with 5 and 4.5 kb A_2A receptor genomic fragments split by a positive selection marker (Neo cassette), which replaced the 3′ end of exon 2 (E2) and the adjacent 5′ splice junction and intron sequences. Digestion of wild-type and mutant A_2A receptor genes with BamHI (at sites labeled B) generates 7.5 and 5.0 kb fragments, respectively, that can be distinguished using a nonoverlapping 3′ probe (as in B). B, Genomic Southern analysis of WT (+/+), heterozygous (+/−), and homozygous (−/−) mice with respect to the A_2A receptor gene was performed as described in Materials and Methods, using the 3′ nonoverlapping probe illustrated in A. WT mice displayed a single 7.5 kb band, whereas homozygous A_2A KO mice showed a single 5.0 kb band corresponding to the restriction fragments for WT and mutant alleles, respectively. Heterozygous mice showed both 7.5 and 5.0 kb bands.C, Homozygous A_2A receptor KO mice are defi-cient in A_2A receptors detected by receptor autoradiography; A_2A receptor binding was determined using³H-CGS 21680 as a ligand. A representative coronal brain section from a WT mouse shows specific labeling of A_2Areceptors in striatum (caudate putamen, CP; nucleus accumbens, NA) and olfactory tubercle (OT), whereas that from a homozygous mouse shows no ³H-CGS 21680 binding. D, Behavioral responses to the A_2A agonist CGS 21680 in WT and A_2A KO mice; ambulation was measured in WT and A_2A KO mice (n = 14–16) before and after challenge with CGS 21680 (0.2 mg/kg, i.p.) by recording contiguous photobeam interruptions (ambulation) for 60 min. Error bars represent the mean ± SEM. *p < 0.05 (Student's t test) when compared with ambulation in the WT mice before treatment.

Fig. 2.

Neurochemical markers of striatal and cortical development in the absence of A_2A receptor. Representative coronal sections through corresponding levels of cortex and striatum are shown for A_2A WT (+/+, top) and KO (−/−, bottom) adult mice. Immunohistochemistry for enkephalin (Enk-IR) or dynorphin (Dyn-IR) and receptor autoradiography for NMDA receptor (³H-MK-801) in striatum and cortex were performed on brain sections as described in Materials and Methods. The characteristic striosomal pattern of dynorphin in striatum was indistinguishable between A_2A WT and KO mice (arrows).

Fig. 3.

Inactivation of A_2A receptors attenuated MCA occlusion-induced infarction. A, Twenty-two hours after reperfusion, infarct volumes were determined using hematoxylin and eosin staining as described in Materials and Methods for A_2A KO and WT mice with hybrid C57BL/6 × 129-Steel genetic background (n = 6) as well as with pure 129-Steel genetic background (n = 11–12). B, Regional infarct volume was analyzed with respect to cerebral cortex and striatum of pure 129-Steel substrain mice (n = 11–12). *p < 0.05 when comparing infarct volumes of A_2A KO mice with those of WT littermates (Student's t test).

Fig. 4.

Inactivation of A_2A receptors enhances neurological function after MCA occlusion. Neurological deficit behavioral scores were assessed by a trained observer in a blinded manner as described in Materials and Methods. Neurological deficits were determined for A_2A KO and WT mice with the hybrid C57BL/6 × 129-Steel strain (n = 6) and with the pure 129-Steel strain (n = 11–12). *p < 0.05 when comparing neurological deficit scores of A_2A KO mice with those of their WT littermates (Mann–Whitney U test).