This study is conducted byToshiya SatoTeam development,andPublished in《American Journal of Physiology - Regulatory, Integrative and Comparative Physiology》In the journal.

研究以Uratan anesthesiaThe rat（That is, ethyl carbamate）As experimental subjects, foritsLingual nerve（LN）Perform electrical stimulation on the central cut-off end and simultaneously measure the submandibular gland blood flow（SMGBF）Sublingual gland blood flow（SLGBF）The aim is to explore the differences in parasympathetic vasodilation control between submandibular gland and sublingual gland in rats.

summary

This study used a laser speckle imaging hemocytometer to measure blood flowUratan anesthesiaIn rats, the submandibular gland blood flow was detected by electrical stimulation of the central cutting end of the lingual nerve（SMGBF）Sublingual gland blood flow（SLGBF）The change. The results showed that tongue nerve stimulation can cause an intensity dependent and frequency dependent increase in submandibular gland blood flow and sublingual gland blood flow, with the increase in submandibular gland blood flow being greater than that in sublingual gland blood flow. Intravenous injection of autonomic cholinergic ganglion blocker hexamethonium（hexamethonium）It can significantly inhibit the increase in blood flow to both types of glands. Atropine, an anti muscarinic drug（atropine）It can significantly inhibit the increase of submandibular gland blood flow, but only partially inhibit the increase of sublingual gland blood flow. When using atropine, vasoactive intestinal peptide（VIP）Receptor antagonists can significantly inhibit the increase in sublingual gland blood flow that atropine cannot block, while the use of vasoactive intestinal peptide receptor antagonists alone has no such effect. After tongue nerve stimulation, the time required for glandular blood flow to recover to baseline levels is shorter than after injection of vasoactive intestinal peptide. However, after injection of atropine, the recovery time of glandular blood flow caused by tongue nerve stimulation is significantly prolonged in a dose-dependent manner, ultimately reaching the same level as the recovery time after injection of vasoactive intestinal peptide.

The results of this study indicate that:1）Tongue nerve stimulation can cause an increase in submandibular gland blood flow mediated by the parasympathetic nervous system (mainly caused by cholinergic fibers) and an increase in sublingual gland blood flow mediated by the parasympathetic nervous system (caused by both cholinergic and non cholinergic fibers);2）The vasoactive intestinal peptide mechanism is involved in the increase of sublingual gland blood flow mediated by non cholinergic fibers and is activated when the muscarinic mechanism is inactivated.

experimentMaterials and Instruments

（1） Experimental materials

laboratory animal：11-16Age and weight325-490gThe maleWistarRats.

(II)experimentinstrument

1. Instruments related to vascular catheterizationInstruments used for femoral vein and femoral artery catheterization.

2. Artificial ventilation equipment: Ventilator, tracheal intubation, infrared analyzer.

3. Blood flow monitoring instrument：Omegawave OZ-1Laser speckle flow imaging instrument（Used to monitor submandibular gland and sublingual gland blood flow and obtain high-resolution two-dimensional blood flow images.

4. Neural stimulation instrumentBipolar silver electrode, electrical stimulator (to electrically stimulate the central cutting end of the tongue nerve), binocular microscope (to separate the tongue nerve).

5. Physiological parameter monitoring instrument: Used for monitoring systemic arterial blood pressure（SABP）And heart rate（HR）The equipment.

Experimental Procedure

（1） Animal preparation

（2） Laser speckle flow imaging

Place the anesthetized rat in a horizontal position and place the submandibular gland and sublingual gland on a black polyurethane pad,useOmegawave OZ-1Laser speckle flow imaging instrumentof780nmSemiconductor lasers provide diffuse illumination for it. After filtering, the scattered light is transmitted through a charge coupled device located above the neck（CCD）Camera detection. Record the raw speckle images related to the number and speed of moving red blood cells, and transmit them to the computer for analysis.

Omegawave OZ-1Laser speckle flow imaging instrument

Electrical stimulation of left lingual nerve（LN）Central cutting end to bilateral submandibular glands（SMG）And sublingual gland（SLG）The impact of blood flow

APhoto shows bilateral submandibular glands（SMG）And sublingual gland（SLG）Placed on a black Uratan pad, as well as before stimulation (resting state), on the lingual nerve（LN）After the stimulation begins12 Seconds and20 Typical speckle image time series in seconds.

BBilateral submandibular glands（SMG）And sublingual gland（SLG）The blood flow（au, any unit), vascular conductivity（VC）And systemic arterial blood pressure（SABP）A typical example of change.

CBilateral sublingual glands（SLGSolid column and submandibular gland（SMGHollow cylinder vascular conductivity（VC）The average value of change (±)SE, standard error)（n=8The sample size is8）. Perform analysis of varianceTukeyTest and evaluate vascular conductivity（VC）Statistical significance of differences between changes.*P<0.01(The difference has extremely significant statistical significance).

（3） Tongue nerve electrical stimulation

Separate the left lingual nerve under binocular microscope（LN）After cutting off, use bipolar silver electrodes connected to the electrical stimulator to electrically stimulate the central cutting end. In each rat, at different voltages and frequencies2msPulse duration for unilateral stimulation of the lingual nerve, with each stimulation lasting20 Seconds, stimulus interval15minute.

（4） Drug action testing

1. All drugs were dissolved in sterile physiological saline and administered via the femoral vein.

2. To detect submandibular gland blood flow induced by electrical stimulation of the lingual nerve（SMGBF）Sublingual gland blood flow（SLGBF）Whether the increase is mediated by the autonomic nervous system, muscarinic receptors, and vasoactive intestinal peptide receptors, perform the following operations:

◦intravenous injection10mg/kgHexamethylammonium bromide, when administered at least5 Minutes (waiting)SMGBFTheSLGBFandSABPAfter reaching a stable state, the blood flow changes caused by tongue nerve stimulation were detected, and the same volume of physiological saline was used as a control.

◦Use an injection pump to2ml/hThe flow rate of intravenous infusion0.1mg/mlAtropine sulfate and0.2mg/mlVasoactive intestinal peptide receptor antagonist, starting infusion at least once10 After minutes, detect the blood flow changes caused by tongue nerve stimulation.

（5） Time course analysis of increased sublingual gland blood flow

Cut off the left lingual nerve center using20VThe20HzThe2msPulse continuous electrical stimulation20 Seconds, or intravenous injection100ng/kgAcetylcholine bromide（ACh）The10ng/kgVasoactive intestinal peptide（VIP）Triggering sublingual gland blood flow（SLGBF）Increase. Intravenous injection of different doses（1-100μg/kg）Atropine sulfate (volume)0.1ml）Using the same volume of physiological saline as a control.

empirical conclusion

1. Lingual nerve（LN）Stimulation can cause submandibular gland blood flow（SMGBF）Sublingual gland blood flow（SLGBF）Increased in intensity and frequency dependence, with submandibular gland blood flow increasing more than sublingual gland blood flow, and systemic arterial blood pressure（SABP）Significantly elevated, heart rate（HR）There was no significant change, and the increase in blood flow of the ipsilateral gland was significantly greater than that of the contralateral gland, indicating that this increase in blood flow is not passively caused by changes in blood pressure, but rather“vasodilation”The result of the action.

2. Intravenous injection of the autonomic cholinergic ganglion blocker hexamethonium can significantly inhibit the increase in blood flow to the submandibular and sublingual glands, indicating that the increase in blood flow to the two glands caused by tongue nerve stimulation is mainly mediated by the parasympathetic nervous system.

3. Atropine, an anti muscarinic drug, can significantly inhibit the increase of submandibular gland blood flow and only partially inhibit the increase of sublingual gland blood flow. Moreover, when used alone, vasoactive intestinal peptide can also be used（VIP）Receptor antagonists have no effect on the increase of blood flow in both types of glands, but in the case of atropine use, vasoactive intestinal peptide receptor antagonists can significantly inhibit the increase in sublingual gland blood flow that atropine cannot block, indicating that the increase in submandibular gland blood flow is mainly caused by cholinergic fibers, and the increase in sublingual gland blood flow is jointly caused by cholinergic fibers and non cholinergic fibers, and the vasoactive intestinal peptide mechanism is involved in the increase of sublingual gland blood flow mediated by non cholinergic fibers.

4. The time for glandular blood flow to recover to baseline levels after tongue nerve stimulation is shorter than after injection of vasoactive intestinal peptide. However, after injection of atropine, the recovery time of glandular blood flow caused by tongue nerve stimulation is significantly prolonged in a dose-dependent manner, ultimately consistent with the recovery time after injection of vasoactive intestinal peptide. This indicates that when the muscarinic mechanism is inactivated, the vasoactive intestinal peptide mechanism is activated, and the mechanism of increased sublingual glandular blood flow will shift from cholinergic to vasoactive intestinal peptide, which depends on the activation state of muscarinic receptors.

5. The results of this study are consistent with previous reports on the vasodilation of the parasympathetic nervous system in the submandibular gland of rats (the increase in blood flow mediated by the parasympathetic nervous system in the submandibular gland is mainly caused by cholinergic fibers). At the same time, it was found that there is an increase in blood flow mediated by both cholinergic and non cholinergic fibers in the sublingual gland. It is speculated that there may be muscarinic self receptors in the parasympathetic nervous system of the salivary gland in rats, which can negatively regulate the release of vasoactive intestinal peptides in the sublingual gland, but not in the submandibular gland. This may have important significance in the hemodynamic regulation of the oral and facial regions (when acetylcholine release is inhibited).