| Objective: The adenosine plays an important role in the induction of brain ischemic tolerance (BIT). It has been illustrated that cerebral ischemic preconditioning (CIP) leads to increases in synthesis and release of adenosine. The released adenosine binds to its specific receptors to play protective effects on neurons. However, the enhanced level of adenosine induced by CIP might not last to the occurrence of the subsequent severe ischemic insult, because there are efficient inactivating mechanisms for adenosine. So, compared with adenosine, changes in abundance and function of adenosine receptors (AR) may play more important role during the induction of BIT. Indeed, our previous study using radioligand binding method has showed increases in amount and affinity of the adenosine receptor after CIP. These increases could last 1-7 d after ischemic insult, which coincided with the window of induction and maintenance of BIT. Furthermore, it was indicated in our followed study using immunohistochemistry that the expression of adenosine receptor A1 subtype was upregulated on the pyramidal neurons of the hippocampus after CIP. Thererfore, in order to further confirm the role of up-regulation of adenosine receptors A1 subtype in the induction of BIT, the present study was designed to observe the effect of A1 adenosine receptor antisense oligodexynucleatide (ARA1 As-ODN), which can inhibit upregulation of the adenosine receptor A1 subtype, on the induction of BIT by CIP.Methods: The experiment was performed using rat four-vessel occlusion global cerebral ischemic model. Forty-two Wistar rats with permanently occlusion of the bilateral vertebral arteries were divided into 8 groups:1 Sham group: exposing bilateral carotid arteries but without blocking blood flow;2 CIP group: the bilateral carotid arteries were clamped for 3 min;3 Ischemic insult group: the bilateral carotid arteries were clamped for 8 min;4 CIP+ Ischemic insult group: the bilateral carotid arteries were clamped for 3 min as CIP, and then a clamping of the arteries in a relatively long period 8 min was given 48 h after the CIP;5 Distilled water + CIP + Ischemic insult group: distilled water was injected into the right lateral cerebral ventricle 12 h before CIP, and 12 h, 36 h after CIP(5μl in each time). Other procedures were the same as those in CIP+ Ischemic insult group;6 ARA1 As-ODN group: ARA1 As-ODN was injected into the right lateral cerebral ventricle 12 h before and 12 h, 36 h after exposing bilateral carotid arteries (20 nmol each time). Other procedures were the same as those in Sham group;7 ARA1 As-ODN + CIP group: ARA1 As-ODN was injected into the right lateral cerebral ventricle 12 h before and 12 h, 36 h after the CIP (20 nmol each time). Other procedures were the same as those in CIP group;8 ARA1 As-ODN + CIP + Ischemic insult group: ARA1 As-ODN was injected into the right lateral cerebral ventricle 12 h before and 12 h, 36 h after CIP. Two doses of ARA1 As-ODN in 10 nmol/5μl and 20 nmol/5μl were used. Other procedures were the same as those in CIP + Ischemic insult group.Histological changes of the CA1 region of the hippocampus were examined 7 d after the sham operation or the last time of ischemia under thionin staining. Histological changes were divided into the following 4 grades (histological grade, HG) under light microscope: grade 0, no neuron death; grade 1, scattered single neuron death; grade 2, death of many neurons; grade 3, death of almost complete neurons. The average HG of the bilateral hippocampus was counted as statistical data. The neuronal density (ND) of the hippocampal CA1 subfield was determined by counting the number of surviving pyramidal neurons with intact cell membrane, full nucleus and clear nucleolus within 1 mm linear length of the CA1. The average of the number of pyramidal neurons in 4 areas of the hippocampal CA1 subfield was calculated as value of ND. Results: It was found that the rats in the Sham and CIP groups showed no significant neuronal damage in the CA1 subfield of the hippocampus (grade 0～1), the ND of CA1 was 185.8±16.7 and 178.3±12.4 respectively. Ischemic insult caused obvious destruction of CA1 subfield (grade 2～3), the ND was 23.6±23.5, which was much low as compared with that of the Sham and CIP groups (p<0.05). CIP + Ischemic insult group exhibited no obvious neuronal damage in CA1 subfield, the histological grade (grade 0~1) was significantly low, and the ND (174.2±8.8) was much higher compared with those of ischemic insult group (p<0.05). This result indicated that the CIP played neuroprotective effect against cerebral injury normally induced by ischemic insult. Histological change in distilled water + CIP + Ischemic insult group, ARA1 As-ODN group and ARA1 As-ODN + CIP group was similar to that in CIP + Ischemic insult group, Sham-operated group and CIP + Ischemic insult group, respectively. These results indicated the injection of distilled water or ARA1 As-ODN itself had no harmful effect on the neuronal survival in rats subjected to the sham operation or CIP. In ARA1 As-ODN + CIP + Ischemic insult group, 10 nmol/5μl subgroup showed no significant neuronal damage in the CA1 subfield of the hippocampus, in which the histological grade was 0～1 and the ND was 169.7±12.7, which were similar to those in CIP + Ischemic insult group. While the injection of ARA1 As-ODN in a dose of 20 nmol/5μl in ARA1 As-ODN + CIP + Ischemic insult group caused obvious destruction of CA1 subfield. The histological grade was 2～3, and the ND was 49.7±29.8, which were significantly different from those in the CIP + Ischemic insult group (p<0.05). The results indicated that the neuroprotective effect of CIP against cerebral injury normally induced by ischemic insult was blocked by the administration of ARA1 As-ODN.Conclusion: The ARA1 As-ODN itself injected into the lateral cerebral ventricle had no harmful effect on the neuronal survival in rats subjected to the sham operation or CIP. But the injection of the ARA1 As-ODN significantly inhibited the neuroprotective effect of CIP against cerebral injury normally induced by ischemic insult. The above results further demonstrated the association of up-regulation of A1 adenosine receptors with the induction of CIP-mediated BIT.
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