Goat cerebrovascular reactivity to ADP after ischemia–reperfusion. Role of nitric oxide, prostanoids and reactive oxygen species

Abstract

To analyze the cerebrovascular effects of ischemia–reperfusion, cerebrovascular reactivity to ADP was studied after inducing 60-min occlusion followed by 60-min reperfusion of the left middle cerebral artery (MCA) in anesthetized goats. In 12 goats, at the end of reperfusion, left MCA resistance was decreased by 19%, and reactive hyperemia to 5- and 10-s occlusions as well as the cerebral vasodilatation to ADP (0.03–0.3 μg) but not to sodium nitroprusside (0.3–3 μg) was decreased. In 28 animals, killed at the end of reperfusion, segments 3-mm long were obtained from the left (ischemic) and right (control) MCA, prepared for isometric tension recording, and precontracted with the thromboxane A₂ analogue U46619. The relaxation to ADP (10^− 8 to 10^− 5 M) but not to sodium nitroprusside (10^− 8 to 10^− 4 M) was lower in ischemic arteries. l-NAME (inhibitor of nitric oxide synthesis, 10^− 4 M), charybdotoxin (10^− 7 M) + apamin (10^− 6 M) (blockers of K_Ca), or catalase (1000 U/ml) reduced the relaxation to ADP only in control arteries. Charybdotoxin + apamin further augmented the l-NAME-induced reduction in the relaxation to ADP in control arteries. The inhibitor of cyclooxygenase meclofenamate (10^− 5 M) increased the relaxation to ADP only in ischemic arteries. The superoxide dismutase mimetic tiron (10^− 2 M) increased the ADP-induced relaxation only in ischemic arteries. Therefore, it is suggested that ischemia–reperfusion produces cerebrovascular endothelial dysfunction, which may be associated with decreased nitric oxide bioavailability, decreased release of an EDHF, and increased production of vasoconstrictor prostanoids. All these alterations may be related in part with an increased production of superoxide anion.

Introduction

Brain ischemia–reperfusion can produce damage and dysfunction of cerebral vessels, in addition to that of nervous tissue. The function of cerebral vessels is critical for maintenance of cerebral blood supply and minimizes damage to ischemic brain regions during reperfusion, and the vascular endothelium plays a main role in the regulation of cerebral blood flow by releasing vasodilator substances (nitric oxide, prostacyclin, endothelium-derived hyperpolarizing factor).

The studies performed to examine the cerebrovascular effects of ischemia–reperfusion are scarce, and mechanisms involved in these effects remain uncertain. There are studies showing that in cerebral vessels ischemia alone (Rosenblum, 1997) or ischemia followed by reperfusion (Mayhan et al., 1988, Nelson et al., 1992) reduces endothelium-dependent vasodilatation and that after 10 min of reperfusion the impairment of the response to acetylcholine remains whereas the response to another endothelium-dependent vasodilator, bradykinin, recovered (Rosenblum and Wormley, 1995). Furthermore, ischemia–reperfusion may potentiate the EDHF-mediated cerebral vasodilatation in response to UTP (Marrelli et al., 2003) by augmenting endothelial calcium responses (Marrelli, 2002). On the other hand, complete or partial cerebral ischemia followed by reperfusion results in increased production of reactive oxygen species which is accompanied by vasodilatation and decreased endothelium-dependent responses (Kontos, 2001). Pretreatment with scavengers of oxygen radicals as superoxide dismutase and catalase inhibits the cerebral vasodilatation and improves the abnormal endothelium-dependent responses after ischemia–reperfusion indicating the importance of reactive oxygen species in these abnormalities (Nelson et al., 1992). Therefore, it could be of interest to explore further the role of reactive oxygen species in the effects of ischemia–reperfusion on cerebrovascular reactivity.

The present study was performed to study the cerebrovascular effects of ischemia–reperfusion by examining the in vivo and in vitro cerebrovascular reactivity to ADP and analyzing the role of nitric oxide, prostanoids and reactive oxygen species in this reactivity. ADP has been considered as a regulator of cerebral blood flow, and large amounts of this nucleotide could be released from damaged brain under ischemia (Bryan, 2002), and ADP can produce endothelium-dependent cerebral vasodilatation (Faraci, 1992) which may be mediated in part by nitric oxide and an EDHF (Mayhan, 1992, You et al., 1997). Ischemia–reperfusion was induced in anesthetized goats in which the left MCA was subjected to 60-min occlusion followed by 60 min reperfusion. In vivo experiments were performed in anesthetized goats where left MCA flow was electromagnetically measured, and vasodilator responses to brief arterial occlusions (reactive hyperemia) and local injections of ADP and sodium nitroprusside were tested before (control) and after ischemia–reperfusion. In vitro experiments were performed by testing the responses to ADP and sodium nitroprusside of isolated segments from pial branches of left MCA (previously exposed to ischemia—reperfusion) and right MCA (control arteries), analyzing the role of nitric oxide, prostanoids and reactive oxygen species in these responses.