Regulatory mechanism of parasympathetic vasodilatation of masseter muscle in rat

Objectives: We have previously reported that electrical stimulation of the trigeminal nerve such as lingual nerve (LN) induced blood flow increase of the masseter muscle (MBF) via an activation of parasympathetic reflex mechanism in rat. Administration of atropine, a muscarinic receptor antagonist, reduced this response by nearly 40% (P<0.01), suggesting possible presence of noncholinergic vasodilatation mechanism. The purpose of the present study was to examine the involvement of vasoactive intestinal peptide (VIP), a major noncholinergic neurotransmitter of many tissues, on the parasympathetic mediated MBF increase, and to investigate an interaction of ACh and VIP on this response. Methods: Frozen sections of the masseter muscle and otic ganglion were immunohistochemicaly stained by the antibodies of VIP, and/or vesicular acetylcholine transporter (VAChT), a cholinergic marker. Changes in the MBF in the urethane-anesthetized, artificially ventilated rats were monitored on the ipsilateral side using a laser-Doppler flowmeter. Effect of the atropine and/or [D-p-Cl-Phe6, Leu17] VIP, a VIP receptor antagonist, were evaluated. Results: VAChT- and VIP-immunoreactions were detected in the otic ganglion on all and nearly 80% neuronal cell bodies, respectively. VAChT- and VIP-immunoreactions were co-localized on the nerve fibers innervating vessels in the masseter muscle. These results suggest that most of the parasympathetic nerve innervating the vessels of the masseter muscle contained both ACh and VIP. Simultaneous administration of atropine and [D-p-Cl-Phe6, Leu17] VIP almost abolished the MBF increase (80% inhibition, P<0.001), although inhibitory effect of atropine or [D-p-Cl-Phe6, Leu17] VIP alone were 40% or 5% (not significant), respectively. Conclusions: The present study implies that 1) VIP and ACh coexist in the parasympathetic vasodilator fibers in the rat masseter muscle and regulate the MBF, coordinately. 2) Inhibitory effect of [D-p-Cl-Phe6, Leu17] VIP occurred only in the presence of atropine.