Caffeine Modulates Spontaneous Adenosine and Oxygen Changes during Ischemia and Reperfusion

Abstract

Adenosine is an endogenous neuroprotectant that modulates vasodilation in the central nervous system. Oxygen changes occur when there is an increase in local cerebral blood flow and thus are a measure of vasodilation. Transient oxygen events following rapid adenosine events have been recently discovered, but the relationship between adenosine and blood flow change during ischemia/reperfusion (I/R) has not been characterized. Caffeine is a nonselective adenosine receptor antagonist that can modulate the effects of adenosine in the brain, but how it affects adenosine and oxygen levels during I/R is also unknown. In this study, extracellular changes in adenosine and oxygen were simultaneously monitored using fast-scan cyclic voltammetry during bilateral common carotid artery occlusion (BCCAO) and the effects of a specific A_2A antagonist, SCH 442416, or general antagonist, caffeine, were studied. Measurements were made in the caudate-putamen for 1 h of normoxia, followed by 30 min of BCCAO and 30 min of reperfusion. The frequency and number of both adenosine and oxygen transient events significantly increased during I/R. The specific A_2A antagonist, SCH 442416 (3 mg/kg, i.p.), eliminated the increase in adenosine and oxygen events caused by I/R. The general adenosine receptor antagonist, caffeine (100 mg/kg, i.p.), decreased the frequency of adenosine and oxygen transient events during I/R. These results demonstrate that, during BCCAO, there are more rapid release events of the neuromodulator adenosine and correlated local oxygen changes, and these rapid, local effects are dampened by caffeine and other A_2A antagonists.

Keywords: A2A receptor; Adenosine; BCCAO; FSCV; blood flow; caffeine; fast-scan cyclic voltammetry; in vivo; ischemia/reperfusion injury; oxygen.

Figure 1.. Spontaneous adenosine and oxygen changes in the caudate-putamen.

(A) CV for adenosine and oxygen events collected in vivo. The primary oxidation of adenosine occurs at 1.27 V on the cathodic scan and the secondary oxidation occurs at 1.26 V on the anodic scan. The reduction of oxygen occurs at −1.3 V on the cathodic scan. (B) Concentration vs time traces of adenosine (bottom) and oxygen (top) events. (C) Color plot of adenosine and oxygen events in vivo. One oxygen and three adenosine events are starred, where one adenosine is correlated with an oxygen event. Adenosine oxidations are the green/purple in the middle of the plot while reduction of oxygen is dark blue at the top. (D) Microelectrode recording locations (red dots) in the caudate putamen. Drawing from the stereotaxic atlas of Paxinos and Watson.

Figure 2.. Cerebral blood flow changes in rat brain.

(A) Blood flow change during normoxia and I/R (n = 3 animals). Blood flow decreased during BCCAO and is restored, but not to baseline, during reperfusion. (B) Blood flow change during I/R with SCH 442416 (3 mg/kg) administration is similar to plot without drug (n = 3 animals). (C) Blood flow change during I/R with caffeine (100 mg/kg) administration is similar to that without caffeine. (D) Caffeine control (n = 3 animals). Blood flow was measured before and after caffeine (100 mg/kg) during normoxia. Blood flow did not change. (E) Average changes in blood flow. Blood flow significantly decreased during 30 min of ischemia (I) (*p = 0.027), SCH+ ischemia (*p = 0.034) and caffeine + ischemia (*p = 0.023) groups compared to 30 min of normoxia (Norm) Caffeine administration itself did not affect blood flow under normoxia (p = 0.94). Statistical analyses are paired t-test, where each treatment is compared with its own control.

Figure 3.. Spontaneous adenosine and oxygen changes in the caudate-putamen during I/R injury.

(A) Example adenosine and oxygen events during normoxia. Three adenosine and one oxygen events (starred) were observed. (B) Example adenosine and oxygen changes during ischemia, where five adenosine and two oxygen events were observed. (C) Number of adenosine events, in 6 min bins, during normoxia and I/R injury. Inset: Average number of adenosine events increases during I/R (paired t-test, n = 7 animals, **p = 0.0066 <0.01). (D) Number of oxygen events in 6 min bins. Inset: Oxygen events increased during I/R injury (paired t-test, n = 7 animals, *p = 0.022 < 0.01). (E) Inter-event time histogram for adenosine. Underlying distributions were significantly different between normoxia and I/R injury (KS test, n = 7 animals, ****p < 0.0001). (F) Oxygen inter-event time distributions were significantly different for I/R injury (KS test, n = 7 animals, ****p < 0.0001). (G) Mean event concentration of adenosine events was not different during I/R injury for all adenosine events or adenosine events with oxygen event (ADO w/O₂)(paired t-test, n = 7 animals, p = 0.62 and p = 0.42, respectively). (H) The mean concentration of each oxygen event was not significantly different during I/R injury (paired t-test, n = 7 animals, p = 0.92).

Figure 4.. Percent of transient adenosine events that correlate with an oxygen event.

Each animal was used as its own control to compare percentage of correlated adenosine and oxygen events during normoxia and I/R with/without drug treatment. The percent of transient adenosine events that correlates with an oxygen event did not significantly change under I/R injury (Paired t-test, n = 7 animals, p = 0.34) and I/R injury with SCH 442416 administration (Paired t-test, n = 6 animals, p = 0.059). However, the percent of adenosine events that correlates with an oxygen event significantly decreased under I/R injury with caffeine administration (Paired t-test, n = 6 animals, *p = 0.043).

Figure 5.. Effect of the A_2A antagonist, SCH 442416 (3 mg/kg, i.p.), on adenosine and oxygen changes during I/R injury.

(A) Number of adenosine event release traces during every 6 min during normoxia and I/R injury. Inset: No change in number of adenosine events between normoxia and I/R injury (paired t-test, n = 6 animals, p = 0.64). (B) Number of oxygen events in 6 min. bins. Inset: Number of oxygen events did not change during I/R (paired t-test, n = 6 animals, p = 0.24). (C) For adenosine, there are no differences in inter-event times between normoxia and I/R injury (KS test, n = 6 animals, p = 0.63). (D) For oxygen, there were no changes in inter-event times during I/R (KS test, n = 6 animals, p = 0.46). (E) Mean event concentration of all adenosine (ADO) and ADO w/O₂ were not significantly different during I/R injury (paired t-test, n = 6 animals, p = 0.55 and p = 0.63, respectively). (F) The mean event concentration of oxygen was not significantly different during I/R injury (paired t-test, n = 6 animals, p = 0.96).

Figure 6.. Effect of caffeine (100 mg/kg, i.p.) on adenosine and oxygen changes during I/R injury.

(A) Example adenosine and oxygen changes during normoxia, where four adenosine and two oxygen events (starred) were observed. (B) Example adenosine and oxygen changes during I/R injury with caffeine treatment, where two adenosine and one oxygen events were observed. (C) Number of adenosine event release traces during every 6 min during normoxia and I/R injury. Inset: Average number of adenosine release events decreased during caffeine+I/R (paired t-test, n = 6 animals, *p = 0.022). (D) Number of oxygen events in 6 min bins. Inset: Average number of oxygen events decreased during caffeine+ I/R injury (paired t-test, n = 6 animals, *p = <0.01). (E) For adenosine, underlying distributions of inter-event times were significantly different between normoxia and caffeine + I/R injury (KS test, n = 6 animals, ****p < 0.0001). (F) For oxygen, the distributions of inter-event times were significantly different for caffeine+I/R (KS test, n = 6 animals, ****p < 0.0001). (G) Average event concentration of adenosine events was not significantly different (paired t-test, n = 6 animals, all ADO: p = 0.75 and ADO w/O₂: p =0.56). (H) Average oxygen events concentration was not significantly (paired t-test, n = 6 animals, p = 0.96).