Opposite reactivity of meningeal versus cortical microvessels to the nitric oxide donor glyceryl trinitrate evaluated in vivo with two-photon imaging

Abstract

Vascular changes underlying headache in migraine patients induced by Glyceryl trinitrate (GTN) were previously studied with various imaging techniques. Despite the long history of medical and experimental use of GTN, its effects on the brain vasculature are still poorly understood presumably due to low spatial resolution of the imaging modalities used so far. We took advantage of the micrometer-scale vertical resolution of two-photon microscopy to differentiate between the vasodynamic effects of GTN on meningeal versus cortical vessels imaged simultaneously in anesthetized rats through either thinned skull or glass-sealed cranial window. Intermediate and small calibre vessels were visualized in vivo by imaging intravascular fluorescent dextran, and detection of blood flow direction allowed identification of individual arterioles and venules. We found that i.p.-injected GTN induced a transient constriction of meningeal arterioles, while their cortical counterparts were, in contrast, dilated. These opposing effects of GTN were restricted to arterioles, whereas the effects on venules were insignificant. Interestingly, the NO synthase inhibitor L-NAME did not affect the diameter of meningeal vessels but induced a constriction of cortical vessels. The different cellular environment in cortex versus meninges as well as distinct vessel wall anatomical features probably play crucial role in the observed phenomena. These findings highlight differential region- and vessel-type-specific effects of GTN on cranial vessels, and may implicate new vascular mechanisms of NO-mediated primary headaches.

Figure 1. Upper (A) and side (B) views of 3D reconstruction of meningeal and cortical arterioles (red color) and venules (blue color) obtained from a typical z-stack of brain images.

Note the clear gap between meningeal (dural) and cortical vessels in the side view projection shown (B). Scale bar 50 µm.

Figure 2. Effects of GTN on cortical vessels in the open cranial window preparation.

A, examples of cortical vessels before (upper image) and after GTN administration (lower image). B, the normalized diameter of cortical arterioles after injection of the GTN (filled circles) or vehicle (empty circles), real diameter before application 36.1±6 µm for cortical arterioles and 38±8 µm for cortical venules. C, comparison of changes in the area under curve (AUC) in GTN versus vehicle in cortical arterioles (n = 6 and n = 3, respectively). D and E, the same for cortical venules (n = 8 and n = 4, respectively). Note that GTN significantly changed the diameter of arterioles (*  =  P<0.05) but not venules (P = 0.09). Scale bar 25 µm.

Figure 3. The action of of GTN on meningeal vessels in open cranial window preparation.

A, examples of meningeal vessels before (upper image) and after GTN administration (lower image). B, the normalized diameter of meningeal arterioles after injection of the GTN (filled circles) or vehicle (empty circles), real diameter before application 17.4±2 µm for meningeal arterioles and 22.8±4 µm for meningeal venules. C, comparison of changes in the area under curve in GTN vs vehicle in meningeal arterioles (n = 6 and n = 4, respectively, ***  =  P<0.001), D and E, the same for meningeal venules (n = 5 and n = 4, respectively). Note that GTN changed the diameter of arterioles but not venules. Scale bar 40 µm.

Figure 4. The action of L-NAME (filled circles) and vehicle (open circles) on the diameter of cortical and meningeal vessels in open cranial window preparation.

A, the time of changes in cortical arterioles (real diameter 38.1±6 µm), B, cortical venules (real diameter 46.4±13 µm), D, meningeal arterioles (real diameter 20.7±5 µm) and E, meningeal venules (real diameter 24.1±7 µm), respectively. C and F, quantification of results (n = 9, 4, 8, 8 for meningeal arterioles, meningeal venules, cortical arterioles and cortical venules, respectively, *** = P<0.001, ** = P<0.01). Notice constriction of cortical but not of meningeal vessels.

Figure 5. Schematic presentation of potential mechanisms for opposite modulation of dura mater and pial/cortical vessels by GTN.

In dura mater occupied by mast cells and densely innervated by trigeminal and autonomous nerves GTN can induce vasoconstriction of small vessels either directly, or via release of vasoconstrictory agents from mast cells or through the neuronal control. The functional outcome depends on the combination of vasodilatatory versus vasoconstrictory agents and on the receptor profile. The dilatatory effect of GTN in pial/cortical vessels could be due to the direct action of this agent on the vessel wall or mediated via astrocytes releasing NO.