The Role of Endothelium and Nitric Oxide in Rat Pial Arteriolar Dilatory Responses to CO2 in vivo

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Abstract
Using a closed cranial window system and intravital microscopy/videometry, we studied the rat pial arteriolar (30–60 μm) responses to CO2 before and following a light/dye (L/D) endothelial injury or topical application of the nitric oxide synthase (NOS) inhibitor, nitro-l-arginine (L-NA) or its inactive form, D-NA. L/D treatment consisted of intravenous injection of sodium fluorescein and the illumination (for 90 s) of arteriolar discrete segments on the cortical surface with light from a mercury lamp. Functional changes in pial arteriolar endothelium were characterized by evaluating responses to topical application of acetylcholine (Ach, 5 × 10−4 M) and to intravenous (i.v.) oxotremorine (OXO, a stable blood–brain barrier permeant muscarinic agonist, 1 μg kg−1 min−1). After the L/D injury, dilation to Ach was absent whereas dilations to the NO donor, S-nitrosoacetyl-penicillamine (SNAP, 10−5 M) and to CO2 (5%) were unchanged (Paco2 = 70 mm Hg). Loss of Ach response but intact SNAP response confirmed functional endothelial injury and intact smooth-muscle function. The global endothelium-dependent vasodilation induced by i.v. OXO was markedly attenuated when expanding the L/D injury field from 300 μm to 6 mm in diameter. However, the global vasodilation induced by inhalation of CO2 was still unaffected by this increase in the area of light exposure. This provides evidence that the expanded exposure was capable of impairing global vasodilation resulting from endothelium-dependent stimuli but not from inhalation of CO2. The intact CO2 response despite an endothelial dysfunction suggests that the reported NO dependence of hypercapnia-induced cerebral hyperemia in rats cannot be attributed to an endothelial NO source. Topical suffusion of L-NA (1 m M) for 45–60 min in our preparation blocked the pial arteriolar response to Ach, whereas CO2 and SNAP responses were unaffected. An attenuation (by 50%) of the response to CO2 was achieved if suffusion of L-NA was given for ≥2 h. Suffusion of D-NA, applied in the same manner, did not influence responses to any of the above applications. This demonstrates that there is a NO-dependent component for hypercapnic cerebral vasodilation even at the pial arteriolar level. The strikingly different time-related effect of topical L-NA on the Ach and CO2 responses, together with the lack of effect of endothelial injury on CO2-induced dilation, strongly suggest a nonendothelial source of NO in hypercapnic cerebrovascular dilation.