This study was conducted by the Department of Dentistry and Oral Biology at Hokkaido University of Health SciencesTakeharu NiiokaTeam published in《Archives of Oral Biology》Journal. The research focuses on the masseter muscle of rats and revolves around three questions:（1）Does the parasympathetic nervous system that controls blood vessels have vasoactive intestinal peptides（VIP）Immunoreactivity; （2）Intravenous injectionVIPWill it induce vasodilation; （3）Selective in the presence or absence of atropineVIPReceptor antagonist[4Cl-D-Phe ⁶,Leu¹⁷]VIPThe impact on parasympathetic vasodilation.

summary

It is known that parasympathetic vasodilator fibers innervate the blood vessels of rat masseter muscle through both cholinergic and non cholinergic mechanisms, but the non cholinergic mechanism is not yet clear. In recent years, vasoactive intestinal peptide（VIP）It has been confirmed to be involved in the vasodilation process of the parasympathetic nervous system in the oral and maxillofacial region (such as the submandibular gland and lower lip), but research on the masseter muscle in rats is extremely limited. This study focuses on the masseter muscle of rats and aims to evaluate:（1）Does the parasympathetic nervous system that controls blood vessels haveVIPImmunoreactivity; （2）Intravenous injectionVIPWill it induce vasodilation; （3）Selective in the presence or absence of atropineVIPReceptor antagonist[4Cl-D-Phe ⁶,Leu¹⁷]VIPThe impact on parasympathetic vasodilation. Research has found that in the parasympathetic ear ganglion（OG）And both the nerve fibers around the blood vessels were detectedVIPImmune reactivity. intravenous injectionVIPCan induce vasodilation, and[4Cl-D-Phe ⁶,Leu¹⁷]VIPCan weakenVIPThe vasodilation effect induced by injection. use alone[4Cl-D-Phe ⁶,Leu¹⁷]VIPUnable to inhibit parasympathetic vasodilation, but when[4Cl-D-Phe ⁶,Leu¹⁷]VIPWhen used in combination with atropine, it can weaken parasympathetic vasodilation. These results indicate that:（1）There are parasympathetic nerves that innervate the masseter muscle blood vessels in the postganglionic regionVIP；（2）Intravenous injectionVIPCan induce vasodilation of masseter muscle blood vessels; （3）When muscarinic cholinergic receptors are blocked by atropine or acetylcholine（ACh）When the release is inhibited and deactivated,VIPMay participate in the parasympathetic vasodilation process of the masseter muscle.

experimentMaterials and Instruments

（1） Experimental materials

1. Experimental animal: Adult maleWistarRats;

2. Tracers and reagents:2%Fluorescent gold, sterile physiological salineVIPSelective selectionVIPReceptor antagonist[4Cl-D-Phe ⁶,Leu¹⁷]VIPAtropine, Uratan, Pankul ammonium bromide;

3. Antibody and staining related reagents:VIPAntibodies, vesicular acetylcholine transporter antibodiesAlexa-488Mark donkey anti rabbitIgGAntibodiesAlexa-568Mark donkey against goatIgGAntibody and fluorescein labeled castor bean agglutininI、1%Non immune donkey serum0.3%TritonX-100、0.3%Bovine serum albumin4%Polyformaldehyde0.1MPhosphate buffer solution30%Sucrose;

（2） Experimental instruments

4. OmegawaveFLO-C1 Laser Doppler flowmeter、fluorescence microscope、Confocal laser scanning microscope、cryostatTheHamiltonmicrosyringe、heating pad、Artificial ventilator、Catheter (used for femoral vein and femoral artery catheterization)

OmegawaveFLO-C1Laser Doppler flowmeter

Experimental Procedure

1. Animal feeding and ethical approval

2. Fluorescence dual labeling experiment

3. Immunohistochemical staining

4. Measurement of masseter muscle blood flowCut off the bilateral cervical vagus nerve and superior cervical sympathetic trunk before the experiment to eliminate the influence of vagus nerve and sympathetic nerve. Inducing reflex vasodilation of the parasympathetic nerve in the masseter muscle by electrically stimulating the left central cutting end of the lingual nerveOmegawaveFLO-C1 Laser Doppler flowmeterMonitor and record masseter muscle blood flow（MBF）Change. Inject different doses intravenously separatelyVIP, observeMBFChange; Injecting100ng/kg VIPfront20Minutes, continuous infusion[4Cl-D-Phe ⁶,Leu¹⁷]VIPObserve its impactVIPinduceMBFThe impact of elevation. In addition, after intravenous injection of atropine and continuous infusion[4Cl-D-Phe ⁶,Leu¹⁷]VIPWhen used in combination, electrically stimulate the lingual nerve and record the resultsMBFChange, expressed as the percentage of control response before medication.

OmegawaveFLO-C1Laser Doppler flowmeter

（A）Lingual nerve（LN）Electrical stimulation (control) and intravenous injection of vasoactive intestinal peptide（VIPThe doses are respectively1、10 and100 ng/kg）Inducing average blood flow（MBF）Typical examples of additions.

（B）Summarized injections 0(physiological saline only)1、10 and100 ng/kg VIPPost inducedMBFIncrease situation（n = 4）. Data inLNThe percentage of control responses induced by electrical stimulation.

（C）（C）In terms of selectivityVIPReceptor antagonist（[4 -chlorine- D -Phenylalanine ⁶,Leucine ¹⁷]Vasoactive intestinal peptide,[4Cl-D-Phe⁶, Leu¹⁷] VIP）Inject when present or absent100 ng/kg VIPinduceMBFTypical examples of additions.

（D）（D）[4Cl-D-Phe⁶, Leu¹⁷] VIPto100 ng/kg VIPInduced by injectionMBFIncreased impact, expressed as a percentage of pre-processing reactions（n = 5）.

5. statistical analysis

empirical conclusion

1. The presence of postganglionic parasympathetic nervous system innervating the masseter muscle blood vessels in ratsVIPImmunoreactivity, andVIPandVAChTCholinergic markers in the ear ganglionFGThere is co localization on labeled neurons and nerve fibers surrounding the masseter muscle blood vessels;

2. intravenous injectionVIPInducing vasodilation of rat masseter muscle in a dose-dependent manner without affecting systemic arterial blood pressure, selectivelyVIPReceptor antagonist[4Cl-D-Phe ⁶,Leu¹⁷]VIPCan be suppressedVIPThe induced vasodilation effect indicates thatVIPBy activating the blood vessel wall of the masseter muscleVIPThe receptor plays a role;

3. use alone[4Cl-D-Phe ⁶,Leu¹⁷]VIPThere is no effect on the parasympathetic vasodilation of the masseter muscle induced by tongue nerve stimulation, and the use of atropine alone can partially weaken this vasodilation effect; And when[4Cl-D-Phe ⁶,Leu¹⁷]VIPWhen used in combination with atropine, it can inhibit the vasodilation effect, indicating that when muscarinic receptors are blocked by atropine or acetylcholine release is inhibited,VIPParticipate in the parasympathetic vasodilation regulation of the masseter muscle.