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The nervous control of gingival blood flow in cats
MICROVASCULAR
RESEARCH
39, 94- 104 (I!?%)
The Nervous HIROSHI IZUMI, Departments
Control
SHIZUKO
of Physiology
of Gingival
Blood Flow in Cats
KURIWADA, * KEISHIRO KARITA, DAISUKE SANJO*
and * Oral Diagnosis, Tohoku University Machi 4-1, Sendai 980, Japan Received
August
TAKASHI School
SASANO,*
of Dentistry,
AND Seiryo-
17, 1989
The purpose of the present study was to investigate the nervous control of gingival blood flow in cats. Gingival blood flow was measured by laser Doppler flowmeter in 75 cats during electrical stimulation and cutting or ligation of the inferior alveolar nerve and cervical sympathetic nerve without sympathectomy or pretreatment with adrenoceptor blocking agents. Three different patterns of responses in gingival blood flow were observed following electrical stimulation of the inferior alveolar nerve in cats. In 45 cats there was an increase in blood flow, in 4 cats a decrease in blood flow, and in 7 cats a biphasic change consisting of an initial decrease and a successive increase in blood flow. The vasodilator effect was significantly reduced by pretreatment with (o-Pro’, o-Trp7.9)-substance P, tripelennamine, and methysergide. Pretreatment with cimetidine, atropine, hexamethonium, phentolamine, or propranolol had no effect on vasodilatation. The vasoconstrictor response was completely inhibited by pretreatment with phentolamine; in this case the vasodilator response appeared after stimulation of the inferior alveolar nerve. Ligation or cutting of the inferior alveolar nerve always elicited an increase in gingival blood flow. Cutting the cervical sympathetic nerve had no effect on gingival blood flow in 8 of 10 cats and caused an increase in gingival blood flow in 2 cats: however, electrical stimulation of the cervical sympathetic nerve always caused a decrease in gingival blood flow in the cats investigated. The present results suggest that cat gingival blood flow is controlled by sympathetic (Yadrenergic fibers for vasoconstriction and by sensory fibers and mast cells for vasodilatation. 0 19913 Academic Press. Inc.
INTRODUCTION The inferior alveolar nerve, a branch of the mandibular division of the trigeminal nerve, contains afferent fibers from the ipsilateral lower lip, areas of oral mucous membrane, and mandibular teeth (33). Sympathetic fibers have also been reported to travel within the inferior alveolar nerve (2,29,31,33,37). It is generally considered that blood circulation in the oral tissue of mammals, e.g., dental pulp, periodontal tissue, and gingiva, is influenced by sympathetic vasoconstrictor fibers (6,lO). However, it was shown that electrical stimulation of the distal cut end of the inferior alveolar nerve elicited an increase in pulpal blood flow in dog (15,34,37). These findings suggest that there is a neural vasodilator mechanism in the dental pulp and raise the question of whether such neurons are functionally efferent or afferent. It has previously been reported that antidromic stimulation of sensory fibers induces vasodilatation in the skin of various animals (8,26,38), and that the flare 94 Oil26-2862/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All tights of reproduction in any form reserved Printed in U.S.A.
NERVOUS
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95
of arteriolar vasodilatation following stroking or injection of histamine or substance P is induced by activation of axon reflexes in sensory fibers of human skin (1,14,17,20). So far, studies on antidromic vasodilatation have been carried out only in sympathectomized animals or after exclusion of sympathetic vasoconstrictor effects by pretreatment with an adrenoceptor blocking agent such as phentolamine or guanethidine (8,15,26,27); hence, we still know little about the relationship between the sensory fibers and sympathetic fibers in blood flow regulation. Laser Doppler velocimetry is a unique method for continuously and dynamically following cutaneous blood flow and its changes noninvasively (19,25,30,35). Recently, a laser Doppler flowmeter was used to study blood flow in the gingival tissues (3,9). By using this equipment, we could observe the vasodilator and vasoconstrictor responses simultaneously in gingiva following antidromic stimulation of the inferior alveolar nerve in cats. The purpose of the present study was to investigate the nervous control of gingival blood flow in cats by measuring the blood flow with a laser Doppler flowmeter. METHODS
AND MATERIALS
The experiments were conducted on 75 cats, weighing 1.5-3 kg (l-2 years old), anesthetized with ketamine hydrochloride (30 mg/kg, im) initially and then with an iv injection of Nembutal (sodium pentobarbital) at an initial dose of 30 mg/kg, supplemented when necessary with additional doses of 20-30 mg. The trachea was cannulated. The cervical sympathetic or the inferior alveolar nerve was dissected free for electrical stimulation, cutting, or ligation on the side where gingival blood flow was measured. During experiments, nerves were covered with paraffin oil. Bipolar silver electrodes were applied to the distal part of the nerve. The nerves were stimulated for 3 set with 40-100 V and ~-HZ pulses of 2-msec duration using a stimulator (Nihon Koden Model SEN-71103). Identification of the sympathetic nerve was made functionally by observing the dilatation of the ipsilateral pupil and the contraction of the nictating membrane during stimulation. A femoral artery and vein were exposed and catheterized; the vein was used for infusion of the drugs and the artery for continuous recording of systemic arterial pressure with a Statham pressure transducer and recorder. The transducer was calibrated against a mercury manometer. Gingival blood flow under the lower canine tooth was measured by laser Doppler techniques. Because a laser Doppler flowmeter is sensitive to movements of the probe in the measuring area, the tip of the fiber optic probe was maintained at a distance of 0.5 mm from the gingiva surface by insertion into acrylic dental resin attached to the incisor on the same side where gingival blood flow was being measured and thus held motionless relative to the tissue during continuous measurements (Fig. 1). In the present experiments blood flow value has been expressed in terms of the output signal (mV) of the instrument (Med Pacific 5000). A linear relation between blood flow and output signal has previously been reported by Holloway (19), Stern er af. (35), Le-Cong and Zweifach (25), and Nilsson et al. (30).
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IZUMI ET AL.
L user Doppler flowmeter
rl
Recorder
FIG. 1. Diagram of the experimental by laser Doppler flowmeter.
model illustrating measurement of cat gingival blood flow
Drugs
(u-Pro*, o-Trp7’9)-substance P was obtained from the Peptide Institute, Inc. (Osaka, Japan). m-Propranolol hydrochloride and cimetidine were purchased from Sigma Chemical Co. (St. Louis, MO). Methysergide maleate was kindly donated by Sandoz Pharmaceuticals (E. Hanover, NJ). Tripelennamine hydrochloride and phentolamine hydrochloride were generously supplied by CibaGeigy Corp. (Base& Switzerland). All other chemicals were reagent grade obtained from general commercial sources. Drug Administration
Drugs, prepared daily in Ringer solution, were injected and second stimulation at the described dose.
iv between the first
Statistical Analysis
All numerical data are given as the means t SEM. The significance of the differences was evaluated using paired or unpaired Student’s t test and Welch analysis. RESULTS In preliminary experiments under our experimental conditions, it was found that the increase in blood flow was dependent on the duration (0.2-10 set) and the intensity (lo-100 V) of electrical stimulation (5 Hz, 2 msec) of the inferior alveolar nerve. Stimulation did not cause any change in systemic arterial blood pressure. Gingival blood flow remained fairly constant during the experiments over several hours. When stimulation was repeated at 5- to IO-min intervals under the same conditions, the increase in blood flow was reproducible at least 10 times.
NERVOUS
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As shown in Fig. 2, stimulation of the distal end of cut inferior alveolar nerve elicited an inconsistent effect on gingival blood flow in cats. Three different patterns of response, i.e., the increase in blood flow (45 cats), the biphasic responses (7 cats), and the decrease in blood flow (4 cats) in a total of 56 cats, were observed by stimulation of the inferior alveolar nerve. In the case of blood flow increase (vasodilatation) (Fig. 2A), stimulation of the inferior alveolar nerve (2 msec, 5 Hz, 60 V) for 3 set resulted in a prompt increase in gingival blood flow. Blood flow reached its peak about 14.8 & 1.7 set (means -+ SEM, N = 10) and returned to its base level at 81.3 2 15.2 set (means & SEM, N = 10) after the start of the stimulation. On the other hand, when blood flow was decreased by stimulation (vasoconstriction) (Fig. 2C), the onset, its peak (9.1 31.0 set) (means f SEM, N = 12), and the recovery (34.2 + 3.6 set) (means + SEM, N = 11) of blood flow decrease were more rapid than those of blood flow increase. The nature of the vasodilator or vasoconstrictor mechanism was explored by
A
s1 . 0
B
(80%)
(13%)
+
a
(7%)
c
lOOm V
I min
llcl FIG. 2. Three different blood flow changes in gingiva following electrical stimulation (5 HZ, 2 msec, 30-100 V) of the distal end of the cut inferior alveolar nerve for 3 sec. The inferior alveolar nerve was electrically stimulated as indicated by solid circles. Abscissa: time (min). Ordinate: gingival blood flow expressed as millivolts. The percentage in the parentheses indicates the rate of appearance of the vasoresponse following electrical stimulation of the inferior alveolar nerve in a total of 56 cats.
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pharmacological means. Blocking agents were introduced by the systemic route between the first and second stimulations in one series of experiments. As shown in Figs. 3A-3C, the increase in gingival blood flow elicited by stimulation of the inferior alveolar nerve was significantly reduced by pretreatment with (o-Pro*, o-Trp7~9)-substance P (substance P receptor antagonist), tripelennamine (histamine Hl-receptor antagonist), or methysergide (serotonin receptor antagonist). The blood flow values, expressed as percentage of control after the treatments of (n-Pro*, n-Trp7’9)-substance P at a dose of 0.2 mg/kg, tripelennamine at doses of 1.O or 5.0 mg/kg, and methysergide at doses of 0.5 or I .O mg/kg, were 75.7% (P < 0.05, N = 7), 75.6% (P < 0.01, N = 8), 67.0% (P < 0.001, N = 6), 66.5% (P < 0.01, N = 6), and 46.5% (P < 0.001, N = 7), respectively. Atropine
A
fiLL .
.
.
.
.
E9Lk
c .
FIG. 3. Influence of inferior alveolar nerve stimulation (5 Hz, 2 msec, 30-100 V) for 3 set on gingival blood flow before and after systemic administration of (A) tripelennamine (5.0 mg/kg), (B) methysergide (0.5 mg/kg), (C) (o-Pro*, o-Trp7.9)-substance P (0.2 mg/kg), and (D) phentolamine (1.0 mg/kg). The inferior alveolar nerve was electrically stimulated as indicated by solid circles. Abscissa: time (min). Ordinate: gingival blood flow expressed as millivolts.
NERVOUS
CONTROL
OF GINGIVAL
BLOOD
99
FLOW
(muscarinic receptor antagonist) (3.0 mg/kg), phentolamine (a-adrenoceptor antagonist) (1 .O mg/kg), propranolol (P-adrenoceptor antagonist) (1 .O mg/kg), hexamethonium (autonomic ganglion blocker) (5.0 mg/kg), and cimetidine (histamine H2-receptor antagonist) (5.0 mg/kg) had no effect on this vasodilator action. On the other hand, the decrease in gingival blood flow following stimulation of the inferior alveolar nerve was completely reduced by pretreatment with phentolamine (1 .O mg/kg, iv) (Fig. 3D). When the vasoconstriction was abolished by pretreatment with phentolamine, the pattern of response was changed and vasodilation occurred after stimulation of the inferior alveolar nerve. Figure 4 shows the effects of cutting or ligation of the inferior alveolar nerve on gingival blood flow. Both treatments consistently elicited an increase in gingival blood flow in all nine cats investigated. Cutting or ligation itself caused no change in systemic arterial blood pressure. Figure 5 shows the effects of electrical stimulation of the cervical sympathetic nerve and of cutting of the nerve on gingival blood flow. The cutting of the nerve had an inconsistent effect on gingival blood flow; no effect in 8 out of 10 cats and a sustained increase in gingival blood flow in 2 cats. Electrical stimulation of the nerve always produced a decrease in gingival blood flow in all 10 cats examined. DISCUSSION Much data have recently been accumulated regarding the physiological role of the sensory nerve in peripheral blood flow control (8,15,26,37). In all these experiments, except that of Tflnder and Ness (37), the animals either had been sympathectomized or were pretreated during experiments with an cY-adrenoceptor blocking agent, such as phentolamine, since the vasodilator effect was suppressed by a severe vasoconstriction elicited by electrical stimulation of sympathetic
1OOmV
Cutting of IAN
Ligation of IAN
FIG. 4. Effects of cutting or ligation of the inferior alveolar nerve (IAN) on gingival blood flow. The ligation was performed at the distal end of cut IAN. Abscissa: time (min). Ordinate: gingival blood flow expressed as millivolts.
100
IZUMI
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AL.
(100%) I
cutting
of CSN
1
.
.
min -1$71”
.
.
.
.
.-,
.
(80%) t
Cutting
1 1OOmV
of CSN
FIG. 5. Effects of cutting or stimulation of cervical sympathetic nerve (CSN) flow. Electrical stimulation with 5 Hz, 3 msec, IO-50 V for 10 set, as indicated Abscissa: time (min). Ordinate: gingival blood flow expressed as millivolts. The parentheses indicates the rate of appearance of vasoresponse following electrical CSN in a total of 10 cats.
on gingival blood by solid circles. percentage in the stimulation of cut
fibers in the inferior alveolar nerve, saphenous nerve, or trigeminal nerve. In these experiments, radioisotope- or gas-clearance or blood perfusion has been used for the measurement of blood flow. These measurements for the determination of blood flow, however, have some drawbacks such as reproducibility, sensitivity, difficulties in data analysis, and invasion of the tissues. A new instrument for measurement of tissue blood flow based on the laser Doppler principle makes it possible to continuously and instantaneously follow rapid changes in blood flow under noninvasive conditions (19,25,30,35). Using this instrument, we investigated the influence of stimulation of the inferior alveolar nerve on gingival blood flow without sympathectomy or pretreatment with adrenoceptor a-blocker. As shown in Fig. 2, three different patterns in blood flow changes were observed following the electrical stimulation of the cut inferior alveolar nerve: an increase in blood flow (80%), a decrease in blood flow (7%), and a biphasic response (13%) in a total of 56 cats. As systemic arterial blood pressure was not affected during the electrical stimulation of the inferior alveolar nerve, it is probable that the increase or decrease in blood flow is due to the vasodilatation or vasoconstriction of blood vessels in gingiva. This suggests that the inferior alveolar nerve contains both vasodilator and vasoconstrictor fibers and that blood flow in cat gingiva is influenced by both fibers. However, at present, we are unable to determine which pattern of response would occur after stimulation of the inferior alveolar nerve. This may imply the complex geometry of the gingival microvasculature and the complexity or diversity of the nervous control of gingival blood flow. For these reasons, to understand how gingival blood flow is regulated, further investigation is required.
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As shown in Fig. 3, an increase in gingival blood flow was significantly attenuated by pretreatment with substance P receptor antagonist ((~-Pro*, D-Trp7’9)substance P), histamine Hl-receptor antagonist (tripelennamine), or serotonin receptor antagonist (methysergide). Since cimetidine had no effect on vasodilatation, the action of histamine is probably mediated by the histamine Hlreceptor. Atropine (0.3 mg/kg, iv) and propranolol (0.5 mg/kg, iv) had no effect on this result, suggesting that the muscarinic receptor and adrenoceptor P-receptor are not involved in vasodilatation. The present results suggest that the vasodilator response is induced by antidromic activation of sensory fibers, particularly nociceptive C-fibers, in the inferior alveolar nerve, after which substance P releases histamine or serotonin from mast cells and then causes vasodilatation in gingiva. This is supported by the following: (1) substance P is released from the peripheral end of sensory fibers following electrical stimulation of the sensory nerve (5,7,32); (2) substance P is able to release not only histamine but also serotonin from mast cells (11,13,17,22); and (3) histamine and serotonin possess a powerful vasodilator action (4,16,24). However, the direct vasodilator action of substance P released from sensory terminals could not be excluded at present, since the vasodilatation elicited by the stimulation of inferior alveolar nerve was only partially inhibited by pretreatment with histamine or serotonin receptor antagonist (Fig. 3) and since substance P possesses a powerful vasodilator action (12,18). On the other hand, a decrease in gingival blood flow was completely reduced by pretreatment with adrenoceptor a-antagonist, phentolamine, and then an increase of blood flow appeared by stimulation of the inferior alveolar nerve (Fig. 3). These results indicate that the inferior alveolar nerve contains sympathetic vasoconstrictor fibers and sensory fibers which cause vasodilatation when activated and suggest that cat gingival blood flow might be controlled by sympathetic a-adrenergic fibers for a decrease in blood flow and by sensory fibers and mast cells for an increase in blood flow. At present, we cannot explain why other investigators observed the vasodilator response only when the vasoconstrictor response was suppressed and why they could not obtain highly reproducible responses during repeated stimulation (8,15,26,28,34). These authors reported that a l- to 30-min electrical stimulation was necessary to elicit antidromic vasodilatation. It is possible that the prolonged electrical stimulation produced the severe vasoconstriction, which can mask the vasodilator response. Furthermore, electrical stimulation of sensory fibers is known to increase vascular permeability in skin, eye, and tooth pulp (8,21,23,26,27,36). This effect usually occurs some minutes after the commencement of stimulation. It is therefore likely that prolonged antidromic electrical stimulation of sensory fibers causes not only vasodilatation but also increased vascular permeability. Gazelius and Olgart (15) have previously speculated that an increase in vascular permeability and protein exudation would impair the capillary exchange of solutes from the tissue and thus reduce the rate of disappearance of the tracer from the cavity-tissue depot. Such a phenomenon may explain the markedly less reproducible responses in the antidromic vasodilatation reported by previous investigators (8,15). Our experimental data support the above speculation. As shown in Fig. 2, a few seconds of electrical stimulation of the inferior alveolar nerve is enough to cause both the responses of vasodi-
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latation and vasoconstriction. This duration is remarkably short compared to those of other reports described above, suggesting a small possibility of an increase in vascular permeability under the present experimental conditions. The Laser Doppler flowmeter offers the advantages of continuous measurement of the rate of blood flow and detection of the slightest change in blood flow. It seems likely that methods of blood flow measurement may be of great importance in investigations of the nervous control of blood flow. Ligation or cutting of the inferior alveolar nerve has produced a prompt increase in ipsilateral gingival blood flow in all cats examined (Fig. 4). Tender and Ness (37) and Gazelius and Olgart (15) have previously reported that cutting of the inferior alveolar nerve caused a rise in the ipsilateral pulpal blood flow in dog and cat. However, these authors frequently failed to observe the increase in blood flow produced by nerve section. Although no satisfactory explanation can be offered for this failure at present, it might be due to differences in the sensitivities of the methods of blood flow measurements between the present experiments and those of other authors. It seems likely that the increase in gingival blood flow after ligation or cutting of the inferior alveolar nerve (Fig. 4) is due to nerve excitation elicited by mechanical antidromic stimulation of sensory trigeminal fibers. As shown in Fig. 5, the effects of cutting the cervical sympathetic nerve on gingival blood flow were inconsistent; cutting the nerve had no effect on gingival blood flow in 8 of 10 cats, but caused an increase in gingival blood flow in 2 cats, implying that the vasoconstrictor effect was not elicited by the cutting of the cervical sympathetic nerve in the present experiments. On the other hand, electrical stimulation of the cervical sympathetic nerve always produced a decrease in ipsilateral gingival blood flow in all experiments (Fig. 5). These results indicate that there is no nerve excitation evoked by mechanical stimulation of the vasoconstrictor fibers by cervical sympathetic nerve section, contrary to that of sensory vasodilator fibers. In view of the results, we suggest that sympathetic nervous basal tone in the gingiva is fundamentally low under the present experimental conditions. However, further investigation is necessary to reach a definite conclusion on this point because an increase in gingival blood flow was occasionally observed after the cutting of cervical sympathetic nerves. REFERENCES 1. ANAND, P., BLOOM, S. R., AND MCGREGOR, G. P. (1983). Topical capsaicin pretreatment inhibits axon reflex vasodilatation caused by somatostatin and vasoactive intestinal polypeptide in human skin.
Brit.
J. Pharmacol.
78, 665-669.
2. ANDERSON,D. J. A., AND LINDEN, R. W. A. (1977). Sympathetic modulation of intraoral mechanoreceptor activity. J. Dent. Res. 56, D125. 3. BAAB, D. A., ~BERG, P. A., AND HOLLOWAY, G. A. (1986). Gingival blood flow measured with a laser doppler flowmeter. J. Periodontal Res. 21, 73-85. 4. BIBER, B., FARA, .I., AND LUNDGREN, 0. (1973). Intestinal vascular responses to S-hydroxytryptamine. Acta Physiol. Stand. 87, 526-534. 5. BILL, A., STJERNSCHANTZ, J., MANDAHL, A., BRODIN, E., AND NILSSON, G. (1979). Substance P: Release on trigeminal nerve stimulation, effect in the eye. Acta Physiol. Stand. 106, 371-373. 6. BISHOP, J. G., AND DORMAN, H. L. (1968). Control of blood circulation in oral tissue. In “Advances in Oral Biology” (P. H. Staple, Ed.), Vol. 3, pp. l-44. Academic Press, New York/London. 7. BRODIN, E., GAZELIUS, B., LUNDBERG, J. M., AND OLGART, L. (1981). Substance P in trigeminal nerve endings: Occurrence and release. Acta Physiol. Scand. 111, 501-503.
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R., AND CUELLO, A. C. (1984). Studies on the trigeminal antidromic vasodilatation and plasma extravasation in the rat. J. PhysioI. 346, 273-285. 9. DE RJJK, W. G., BOWEN, P. D., AND BONNER, R. F. (1980). Preliminary results with laser Doppler velocimetry (LDV) in gingival tissues. J. Dent. Res. 59(Abst. 232), 325. 10. EDWALL, L., AND KINDLOVA, M. (1971). The effect of sympathetic nerve stimulation on the rate of disappearance of tracers from various oral tissues. Actu Odontol. &and. 29, 387-400. 11. ERJAVEC, F., LEMBECK, F., FLORJANC-IRMAN, T., SKOFITSCH, G., DONNERER, J., SARIA, A., AND HOLZER, P. (1981). Release of histamine by substance P. Nuunyn-SchmiedeberR’s Arch. Phar8. COUTURE,
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12. EULER, U. ST., VON, AND GADDUM, J. H. (1931). An unidentified depressor substance in certain tissue extracts. J. Physiol. 72, 74-87. 13. FEWTRELL, C. M. S., FOREMAN, J. C., JORDAN, C. C., OEHME, P., RENNER, H., AND STEWART, J. M. (1982). The effects of substance P on histamine and 5hydroxytryptamine release in the rat. .I. Physiol. 330, 393-411. 14. FOREMAN, J. C., JORDAN, C. C., OEHME, P., AND RENNER, H. (1983). Structure-activity relationships for some substance P-related peptides that cause wheal and flare reactions in human skin. J. Physiol. 335, 449-465. 15. GAZELIUS, B., AND OLGART, L. (1980). Vasodilatation in the dental pulp produced by electrical stimulation of the inferior alveolar nerve in the cats. Acta Physiol. Scund. 108, 181-186. 16. HADDY, F. J., GORWN, P., AND EMANUEL, D. A. (1959). The influence of tone upon responses of small and large vessels to serotonin. Circ. Res. 7, 123-130. 17. H~GERMARK, 8., HBKFELT, T., AND PERNOW, B. (1978). Flare and itch induced by substance P in human skin. J. Invest. Dermatol. 71, 233-235. 18. HALLBERG, D., AND PERNOW, B. (1975). Effect of substance P on various vascular beds in the dog. Acta Physiol. Stand. 93, 277-285. 19. HOLLOWAY, G. A. (1980). Cutaneous blood flow responses to injection trauma measured by laser Doppler velocimetry. J. Invest. Dermatol. 74, l-4. 20. IZUMI, H., AND KARITA, K. (1988). Investigation of mechanisms of the flare and wheal reactions in human skin by band method. Bruin Res. 449, 328-331. 21. JANCS~, N., JANCS~GABOR, A., AND SZOLCSANYI, J. (1967). Direct evidence for neurogenic inflammation and its prevention by denervation and by pretreatment with capsaicin. Brit. J. Phnrmucol. 31, 138-151. 22. JOHNSON, A. R., AND ERD~S, E. (1973). Release of histamine from mast cells by vasoactive peptides. Proc. Sot. Exp. Biol. Med. 142, 1251-1256. 23. KENINS, P. (1981). Identification of the unmyelinated sensory nerves which evoke plasma extravasation in response to antidromic stimulation. Neurosci. Lett. 25, 137-141. 24. LANG, W. J., AND WOODMAN, 0. L. (1982). Comparison of the vasodilator action of dopamine and dopamine agonists in the renal and coronary beds of the dog. Brir. J. Pharmacol. 77, 2328. 25. LE-CONG, 26.
27. 28. 29. 30.
31.
P., AND ZWEIFACH, B. W. (1979). In vivo and in vitro velocity measurements in microvasculature with a laser. Microvasc. Res. 17, 131-141. LEMBECK, F., AND HOLZER, P. (1979). Substance P as neurogenic mediator of antidromic vasodilation and neurogenic plasma extravasation. Nauyn-Schmiedeberg’s Arch. Pharmucol. 310, 175-183. LUNDBERG, J. M., AND SARIA, A. (1982). Capsaicin-sensitive vagal neurons involved in control of vascular permeability in rat trachea. Acta Physiol. Stand. 115, 521-523. LUNDBLAD, L., WNCC~RD, A., AND LUNDBERG, J. M. (1982). Vasodilation in the cat nasal mucosa induced by antidromic trigeminal nerve stimulation. Actu Physiol. &and. 115, 517-519. MATTHEWS, B., AND ROBINSON, P. P. (1980). The course of post-ganglionic sympathetic fibres distributed with the trigeminal nerve in the cat. J. Physiol. 303, 391-401. NILSSON, G. E., TENLAND, T., AND OBERG, P. A. (1980). Evaluation of a laser Doppler flowmeter for measurement of tissue blood flow. IEEE Trans. Bio-Med. Eng. BME-27, 597-604. OGILVIE, R. W. (1969). The vasomotor innervation of the cat’s lower right canine tooth pulp.
Anut. Rec. 163, 237. 32. OLGART, L., GAZELIUS,
B., BRODIN, E., AND NILSSON, G. (1977). Release of substance P-like immunoreactivity from the dental pulp. Acta Physiol. Scund. 101, 510-512. 33. ROBINSON, P. P. (1979). The course, relations and distribution of the inferior alveolar nerve and its branches in the cat. Anut. Rec. 195, 265-272.
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34. ROSELL, S., OLGART, L., GAZELIUS, B., PANOPOULOS, P., FOLKERS, K., AND HGRIG, J. (1981). Inhibition of antidromic and substance P-induced vasodilatation by a substance P antagonist. Acta Physio/. Stand. 111, 381-382. 35. STERN, D. M., LAPPE, D. L., BOWEN, P. D., CHIMOSKY, J. E., HOLLOWAY, G. A., KEISER, H. R., AND BOWMAN, R. L. (1977). Continuous measurement of tissue blood flow by laser-Doppler spectroscopy. Amer. 1. Physiol. 232, H441-H448. 36. STJERNSCHANTZ, J., GEUER, C., AND BILL, A. (1979). Electrical stimulation of the fifth cranial nerve in rabbits: Effects on ocular blood flow, extravascular albumin content and intraocular pressure. Exp. Eye Res. 28, 229-238. 37. TBNDER, K. H., AND NESS, G. (1978). Nervous control of blood flow in the dental pulp in the dogs. Acta Physiol. Stand. 104, 13-23. 38. Uv~jcs, B. (1954). Antidromic vasodilatation in the paw of the cat. Pharmacol. Rev. 6, 99-101.
RESEARCH
39, 94- 104 (I!?%)
The Nervous HIROSHI IZUMI, Departments
Control
SHIZUKO
of Physiology
of Gingival
Blood Flow in Cats
KURIWADA, * KEISHIRO KARITA, DAISUKE SANJO*
and * Oral Diagnosis, Tohoku University Machi 4-1, Sendai 980, Japan Received
August
TAKASHI School
SASANO,*
of Dentistry,
AND Seiryo-
17, 1989
The purpose of the present study was to investigate the nervous control of gingival blood flow in cats. Gingival blood flow was measured by laser Doppler flowmeter in 75 cats during electrical stimulation and cutting or ligation of the inferior alveolar nerve and cervical sympathetic nerve without sympathectomy or pretreatment with adrenoceptor blocking agents. Three different patterns of responses in gingival blood flow were observed following electrical stimulation of the inferior alveolar nerve in cats. In 45 cats there was an increase in blood flow, in 4 cats a decrease in blood flow, and in 7 cats a biphasic change consisting of an initial decrease and a successive increase in blood flow. The vasodilator effect was significantly reduced by pretreatment with (o-Pro’, o-Trp7.9)-substance P, tripelennamine, and methysergide. Pretreatment with cimetidine, atropine, hexamethonium, phentolamine, or propranolol had no effect on vasodilatation. The vasoconstrictor response was completely inhibited by pretreatment with phentolamine; in this case the vasodilator response appeared after stimulation of the inferior alveolar nerve. Ligation or cutting of the inferior alveolar nerve always elicited an increase in gingival blood flow. Cutting the cervical sympathetic nerve had no effect on gingival blood flow in 8 of 10 cats and caused an increase in gingival blood flow in 2 cats: however, electrical stimulation of the cervical sympathetic nerve always caused a decrease in gingival blood flow in the cats investigated. The present results suggest that cat gingival blood flow is controlled by sympathetic (Yadrenergic fibers for vasoconstriction and by sensory fibers and mast cells for vasodilatation. 0 19913 Academic Press. Inc.
INTRODUCTION The inferior alveolar nerve, a branch of the mandibular division of the trigeminal nerve, contains afferent fibers from the ipsilateral lower lip, areas of oral mucous membrane, and mandibular teeth (33). Sympathetic fibers have also been reported to travel within the inferior alveolar nerve (2,29,31,33,37). It is generally considered that blood circulation in the oral tissue of mammals, e.g., dental pulp, periodontal tissue, and gingiva, is influenced by sympathetic vasoconstrictor fibers (6,lO). However, it was shown that electrical stimulation of the distal cut end of the inferior alveolar nerve elicited an increase in pulpal blood flow in dog (15,34,37). These findings suggest that there is a neural vasodilator mechanism in the dental pulp and raise the question of whether such neurons are functionally efferent or afferent. It has previously been reported that antidromic stimulation of sensory fibers induces vasodilatation in the skin of various animals (8,26,38), and that the flare 94 Oil26-2862/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All tights of reproduction in any form reserved Printed in U.S.A.
NERVOUS
CONTROL
OF GINGIVAL
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95
of arteriolar vasodilatation following stroking or injection of histamine or substance P is induced by activation of axon reflexes in sensory fibers of human skin (1,14,17,20). So far, studies on antidromic vasodilatation have been carried out only in sympathectomized animals or after exclusion of sympathetic vasoconstrictor effects by pretreatment with an adrenoceptor blocking agent such as phentolamine or guanethidine (8,15,26,27); hence, we still know little about the relationship between the sensory fibers and sympathetic fibers in blood flow regulation. Laser Doppler velocimetry is a unique method for continuously and dynamically following cutaneous blood flow and its changes noninvasively (19,25,30,35). Recently, a laser Doppler flowmeter was used to study blood flow in the gingival tissues (3,9). By using this equipment, we could observe the vasodilator and vasoconstrictor responses simultaneously in gingiva following antidromic stimulation of the inferior alveolar nerve in cats. The purpose of the present study was to investigate the nervous control of gingival blood flow in cats by measuring the blood flow with a laser Doppler flowmeter. METHODS
AND MATERIALS
The experiments were conducted on 75 cats, weighing 1.5-3 kg (l-2 years old), anesthetized with ketamine hydrochloride (30 mg/kg, im) initially and then with an iv injection of Nembutal (sodium pentobarbital) at an initial dose of 30 mg/kg, supplemented when necessary with additional doses of 20-30 mg. The trachea was cannulated. The cervical sympathetic or the inferior alveolar nerve was dissected free for electrical stimulation, cutting, or ligation on the side where gingival blood flow was measured. During experiments, nerves were covered with paraffin oil. Bipolar silver electrodes were applied to the distal part of the nerve. The nerves were stimulated for 3 set with 40-100 V and ~-HZ pulses of 2-msec duration using a stimulator (Nihon Koden Model SEN-71103). Identification of the sympathetic nerve was made functionally by observing the dilatation of the ipsilateral pupil and the contraction of the nictating membrane during stimulation. A femoral artery and vein were exposed and catheterized; the vein was used for infusion of the drugs and the artery for continuous recording of systemic arterial pressure with a Statham pressure transducer and recorder. The transducer was calibrated against a mercury manometer. Gingival blood flow under the lower canine tooth was measured by laser Doppler techniques. Because a laser Doppler flowmeter is sensitive to movements of the probe in the measuring area, the tip of the fiber optic probe was maintained at a distance of 0.5 mm from the gingiva surface by insertion into acrylic dental resin attached to the incisor on the same side where gingival blood flow was being measured and thus held motionless relative to the tissue during continuous measurements (Fig. 1). In the present experiments blood flow value has been expressed in terms of the output signal (mV) of the instrument (Med Pacific 5000). A linear relation between blood flow and output signal has previously been reported by Holloway (19), Stern er af. (35), Le-Cong and Zweifach (25), and Nilsson et al. (30).
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L user Doppler flowmeter
rl
Recorder
FIG. 1. Diagram of the experimental by laser Doppler flowmeter.
model illustrating measurement of cat gingival blood flow
Drugs
(u-Pro*, o-Trp7’9)-substance P was obtained from the Peptide Institute, Inc. (Osaka, Japan). m-Propranolol hydrochloride and cimetidine were purchased from Sigma Chemical Co. (St. Louis, MO). Methysergide maleate was kindly donated by Sandoz Pharmaceuticals (E. Hanover, NJ). Tripelennamine hydrochloride and phentolamine hydrochloride were generously supplied by CibaGeigy Corp. (Base& Switzerland). All other chemicals were reagent grade obtained from general commercial sources. Drug Administration
Drugs, prepared daily in Ringer solution, were injected and second stimulation at the described dose.
iv between the first
Statistical Analysis
All numerical data are given as the means t SEM. The significance of the differences was evaluated using paired or unpaired Student’s t test and Welch analysis. RESULTS In preliminary experiments under our experimental conditions, it was found that the increase in blood flow was dependent on the duration (0.2-10 set) and the intensity (lo-100 V) of electrical stimulation (5 Hz, 2 msec) of the inferior alveolar nerve. Stimulation did not cause any change in systemic arterial blood pressure. Gingival blood flow remained fairly constant during the experiments over several hours. When stimulation was repeated at 5- to IO-min intervals under the same conditions, the increase in blood flow was reproducible at least 10 times.
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As shown in Fig. 2, stimulation of the distal end of cut inferior alveolar nerve elicited an inconsistent effect on gingival blood flow in cats. Three different patterns of response, i.e., the increase in blood flow (45 cats), the biphasic responses (7 cats), and the decrease in blood flow (4 cats) in a total of 56 cats, were observed by stimulation of the inferior alveolar nerve. In the case of blood flow increase (vasodilatation) (Fig. 2A), stimulation of the inferior alveolar nerve (2 msec, 5 Hz, 60 V) for 3 set resulted in a prompt increase in gingival blood flow. Blood flow reached its peak about 14.8 & 1.7 set (means -+ SEM, N = 10) and returned to its base level at 81.3 2 15.2 set (means & SEM, N = 10) after the start of the stimulation. On the other hand, when blood flow was decreased by stimulation (vasoconstriction) (Fig. 2C), the onset, its peak (9.1 31.0 set) (means f SEM, N = 12), and the recovery (34.2 + 3.6 set) (means + SEM, N = 11) of blood flow decrease were more rapid than those of blood flow increase. The nature of the vasodilator or vasoconstrictor mechanism was explored by
A
s1 . 0
B
(80%)
(13%)
+
a
(7%)
c
lOOm V
I min
llcl FIG. 2. Three different blood flow changes in gingiva following electrical stimulation (5 HZ, 2 msec, 30-100 V) of the distal end of the cut inferior alveolar nerve for 3 sec. The inferior alveolar nerve was electrically stimulated as indicated by solid circles. Abscissa: time (min). Ordinate: gingival blood flow expressed as millivolts. The percentage in the parentheses indicates the rate of appearance of the vasoresponse following electrical stimulation of the inferior alveolar nerve in a total of 56 cats.
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pharmacological means. Blocking agents were introduced by the systemic route between the first and second stimulations in one series of experiments. As shown in Figs. 3A-3C, the increase in gingival blood flow elicited by stimulation of the inferior alveolar nerve was significantly reduced by pretreatment with (o-Pro*, o-Trp7~9)-substance P (substance P receptor antagonist), tripelennamine (histamine Hl-receptor antagonist), or methysergide (serotonin receptor antagonist). The blood flow values, expressed as percentage of control after the treatments of (n-Pro*, n-Trp7’9)-substance P at a dose of 0.2 mg/kg, tripelennamine at doses of 1.O or 5.0 mg/kg, and methysergide at doses of 0.5 or I .O mg/kg, were 75.7% (P < 0.05, N = 7), 75.6% (P < 0.01, N = 8), 67.0% (P < 0.001, N = 6), 66.5% (P < 0.01, N = 6), and 46.5% (P < 0.001, N = 7), respectively. Atropine
A
fiLL .
.
.
.
.
E9Lk
c .
FIG. 3. Influence of inferior alveolar nerve stimulation (5 Hz, 2 msec, 30-100 V) for 3 set on gingival blood flow before and after systemic administration of (A) tripelennamine (5.0 mg/kg), (B) methysergide (0.5 mg/kg), (C) (o-Pro*, o-Trp7.9)-substance P (0.2 mg/kg), and (D) phentolamine (1.0 mg/kg). The inferior alveolar nerve was electrically stimulated as indicated by solid circles. Abscissa: time (min). Ordinate: gingival blood flow expressed as millivolts.
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FLOW
(muscarinic receptor antagonist) (3.0 mg/kg), phentolamine (a-adrenoceptor antagonist) (1 .O mg/kg), propranolol (P-adrenoceptor antagonist) (1 .O mg/kg), hexamethonium (autonomic ganglion blocker) (5.0 mg/kg), and cimetidine (histamine H2-receptor antagonist) (5.0 mg/kg) had no effect on this vasodilator action. On the other hand, the decrease in gingival blood flow following stimulation of the inferior alveolar nerve was completely reduced by pretreatment with phentolamine (1 .O mg/kg, iv) (Fig. 3D). When the vasoconstriction was abolished by pretreatment with phentolamine, the pattern of response was changed and vasodilation occurred after stimulation of the inferior alveolar nerve. Figure 4 shows the effects of cutting or ligation of the inferior alveolar nerve on gingival blood flow. Both treatments consistently elicited an increase in gingival blood flow in all nine cats investigated. Cutting or ligation itself caused no change in systemic arterial blood pressure. Figure 5 shows the effects of electrical stimulation of the cervical sympathetic nerve and of cutting of the nerve on gingival blood flow. The cutting of the nerve had an inconsistent effect on gingival blood flow; no effect in 8 out of 10 cats and a sustained increase in gingival blood flow in 2 cats. Electrical stimulation of the nerve always produced a decrease in gingival blood flow in all 10 cats examined. DISCUSSION Much data have recently been accumulated regarding the physiological role of the sensory nerve in peripheral blood flow control (8,15,26,37). In all these experiments, except that of Tflnder and Ness (37), the animals either had been sympathectomized or were pretreated during experiments with an cY-adrenoceptor blocking agent, such as phentolamine, since the vasodilator effect was suppressed by a severe vasoconstriction elicited by electrical stimulation of sympathetic
1OOmV
Cutting of IAN
Ligation of IAN
FIG. 4. Effects of cutting or ligation of the inferior alveolar nerve (IAN) on gingival blood flow. The ligation was performed at the distal end of cut IAN. Abscissa: time (min). Ordinate: gingival blood flow expressed as millivolts.
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(100%) I
cutting
of CSN
1
.
.
min -1$71”
.
.
.
.
.-,
.
(80%) t
Cutting
1 1OOmV
of CSN
FIG. 5. Effects of cutting or stimulation of cervical sympathetic nerve (CSN) flow. Electrical stimulation with 5 Hz, 3 msec, IO-50 V for 10 set, as indicated Abscissa: time (min). Ordinate: gingival blood flow expressed as millivolts. The parentheses indicates the rate of appearance of vasoresponse following electrical CSN in a total of 10 cats.
on gingival blood by solid circles. percentage in the stimulation of cut
fibers in the inferior alveolar nerve, saphenous nerve, or trigeminal nerve. In these experiments, radioisotope- or gas-clearance or blood perfusion has been used for the measurement of blood flow. These measurements for the determination of blood flow, however, have some drawbacks such as reproducibility, sensitivity, difficulties in data analysis, and invasion of the tissues. A new instrument for measurement of tissue blood flow based on the laser Doppler principle makes it possible to continuously and instantaneously follow rapid changes in blood flow under noninvasive conditions (19,25,30,35). Using this instrument, we investigated the influence of stimulation of the inferior alveolar nerve on gingival blood flow without sympathectomy or pretreatment with adrenoceptor a-blocker. As shown in Fig. 2, three different patterns in blood flow changes were observed following the electrical stimulation of the cut inferior alveolar nerve: an increase in blood flow (80%), a decrease in blood flow (7%), and a biphasic response (13%) in a total of 56 cats. As systemic arterial blood pressure was not affected during the electrical stimulation of the inferior alveolar nerve, it is probable that the increase or decrease in blood flow is due to the vasodilatation or vasoconstriction of blood vessels in gingiva. This suggests that the inferior alveolar nerve contains both vasodilator and vasoconstrictor fibers and that blood flow in cat gingiva is influenced by both fibers. However, at present, we are unable to determine which pattern of response would occur after stimulation of the inferior alveolar nerve. This may imply the complex geometry of the gingival microvasculature and the complexity or diversity of the nervous control of gingival blood flow. For these reasons, to understand how gingival blood flow is regulated, further investigation is required.
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As shown in Fig. 3, an increase in gingival blood flow was significantly attenuated by pretreatment with substance P receptor antagonist ((~-Pro*, D-Trp7’9)substance P), histamine Hl-receptor antagonist (tripelennamine), or serotonin receptor antagonist (methysergide). Since cimetidine had no effect on vasodilatation, the action of histamine is probably mediated by the histamine Hlreceptor. Atropine (0.3 mg/kg, iv) and propranolol (0.5 mg/kg, iv) had no effect on this result, suggesting that the muscarinic receptor and adrenoceptor P-receptor are not involved in vasodilatation. The present results suggest that the vasodilator response is induced by antidromic activation of sensory fibers, particularly nociceptive C-fibers, in the inferior alveolar nerve, after which substance P releases histamine or serotonin from mast cells and then causes vasodilatation in gingiva. This is supported by the following: (1) substance P is released from the peripheral end of sensory fibers following electrical stimulation of the sensory nerve (5,7,32); (2) substance P is able to release not only histamine but also serotonin from mast cells (11,13,17,22); and (3) histamine and serotonin possess a powerful vasodilator action (4,16,24). However, the direct vasodilator action of substance P released from sensory terminals could not be excluded at present, since the vasodilatation elicited by the stimulation of inferior alveolar nerve was only partially inhibited by pretreatment with histamine or serotonin receptor antagonist (Fig. 3) and since substance P possesses a powerful vasodilator action (12,18). On the other hand, a decrease in gingival blood flow was completely reduced by pretreatment with adrenoceptor a-antagonist, phentolamine, and then an increase of blood flow appeared by stimulation of the inferior alveolar nerve (Fig. 3). These results indicate that the inferior alveolar nerve contains sympathetic vasoconstrictor fibers and sensory fibers which cause vasodilatation when activated and suggest that cat gingival blood flow might be controlled by sympathetic a-adrenergic fibers for a decrease in blood flow and by sensory fibers and mast cells for an increase in blood flow. At present, we cannot explain why other investigators observed the vasodilator response only when the vasoconstrictor response was suppressed and why they could not obtain highly reproducible responses during repeated stimulation (8,15,26,28,34). These authors reported that a l- to 30-min electrical stimulation was necessary to elicit antidromic vasodilatation. It is possible that the prolonged electrical stimulation produced the severe vasoconstriction, which can mask the vasodilator response. Furthermore, electrical stimulation of sensory fibers is known to increase vascular permeability in skin, eye, and tooth pulp (8,21,23,26,27,36). This effect usually occurs some minutes after the commencement of stimulation. It is therefore likely that prolonged antidromic electrical stimulation of sensory fibers causes not only vasodilatation but also increased vascular permeability. Gazelius and Olgart (15) have previously speculated that an increase in vascular permeability and protein exudation would impair the capillary exchange of solutes from the tissue and thus reduce the rate of disappearance of the tracer from the cavity-tissue depot. Such a phenomenon may explain the markedly less reproducible responses in the antidromic vasodilatation reported by previous investigators (8,15). Our experimental data support the above speculation. As shown in Fig. 2, a few seconds of electrical stimulation of the inferior alveolar nerve is enough to cause both the responses of vasodi-
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latation and vasoconstriction. This duration is remarkably short compared to those of other reports described above, suggesting a small possibility of an increase in vascular permeability under the present experimental conditions. The Laser Doppler flowmeter offers the advantages of continuous measurement of the rate of blood flow and detection of the slightest change in blood flow. It seems likely that methods of blood flow measurement may be of great importance in investigations of the nervous control of blood flow. Ligation or cutting of the inferior alveolar nerve has produced a prompt increase in ipsilateral gingival blood flow in all cats examined (Fig. 4). Tender and Ness (37) and Gazelius and Olgart (15) have previously reported that cutting of the inferior alveolar nerve caused a rise in the ipsilateral pulpal blood flow in dog and cat. However, these authors frequently failed to observe the increase in blood flow produced by nerve section. Although no satisfactory explanation can be offered for this failure at present, it might be due to differences in the sensitivities of the methods of blood flow measurements between the present experiments and those of other authors. It seems likely that the increase in gingival blood flow after ligation or cutting of the inferior alveolar nerve (Fig. 4) is due to nerve excitation elicited by mechanical antidromic stimulation of sensory trigeminal fibers. As shown in Fig. 5, the effects of cutting the cervical sympathetic nerve on gingival blood flow were inconsistent; cutting the nerve had no effect on gingival blood flow in 8 of 10 cats, but caused an increase in gingival blood flow in 2 cats, implying that the vasoconstrictor effect was not elicited by the cutting of the cervical sympathetic nerve in the present experiments. On the other hand, electrical stimulation of the cervical sympathetic nerve always produced a decrease in ipsilateral gingival blood flow in all experiments (Fig. 5). These results indicate that there is no nerve excitation evoked by mechanical stimulation of the vasoconstrictor fibers by cervical sympathetic nerve section, contrary to that of sensory vasodilator fibers. In view of the results, we suggest that sympathetic nervous basal tone in the gingiva is fundamentally low under the present experimental conditions. However, further investigation is necessary to reach a definite conclusion on this point because an increase in gingival blood flow was occasionally observed after the cutting of cervical sympathetic nerves. REFERENCES 1. ANAND, P., BLOOM, S. R., AND MCGREGOR, G. P. (1983). Topical capsaicin pretreatment inhibits axon reflex vasodilatation caused by somatostatin and vasoactive intestinal polypeptide in human skin.
Brit.
J. Pharmacol.
78, 665-669.
2. ANDERSON,D. J. A., AND LINDEN, R. W. A. (1977). Sympathetic modulation of intraoral mechanoreceptor activity. J. Dent. Res. 56, D125. 3. BAAB, D. A., ~BERG, P. A., AND HOLLOWAY, G. A. (1986). Gingival blood flow measured with a laser doppler flowmeter. J. Periodontal Res. 21, 73-85. 4. BIBER, B., FARA, .I., AND LUNDGREN, 0. (1973). Intestinal vascular responses to S-hydroxytryptamine. Acta Physiol. Stand. 87, 526-534. 5. BILL, A., STJERNSCHANTZ, J., MANDAHL, A., BRODIN, E., AND NILSSON, G. (1979). Substance P: Release on trigeminal nerve stimulation, effect in the eye. Acta Physiol. Stand. 106, 371-373. 6. BISHOP, J. G., AND DORMAN, H. L. (1968). Control of blood circulation in oral tissue. In “Advances in Oral Biology” (P. H. Staple, Ed.), Vol. 3, pp. l-44. Academic Press, New York/London. 7. BRODIN, E., GAZELIUS, B., LUNDBERG, J. M., AND OLGART, L. (1981). Substance P in trigeminal nerve endings: Occurrence and release. Acta Physiol. Scand. 111, 501-503.
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34. ROSELL, S., OLGART, L., GAZELIUS, B., PANOPOULOS, P., FOLKERS, K., AND HGRIG, J. (1981). Inhibition of antidromic and substance P-induced vasodilatation by a substance P antagonist. Acta Physio/. Stand. 111, 381-382. 35. STERN, D. M., LAPPE, D. L., BOWEN, P. D., CHIMOSKY, J. E., HOLLOWAY, G. A., KEISER, H. R., AND BOWMAN, R. L. (1977). Continuous measurement of tissue blood flow by laser-Doppler spectroscopy. Amer. 1. Physiol. 232, H441-H448. 36. STJERNSCHANTZ, J., GEUER, C., AND BILL, A. (1979). Electrical stimulation of the fifth cranial nerve in rabbits: Effects on ocular blood flow, extravascular albumin content and intraocular pressure. Exp. Eye Res. 28, 229-238. 37. TBNDER, K. H., AND NESS, G. (1978). Nervous control of blood flow in the dental pulp in the dogs. Acta Physiol. Stand. 104, 13-23. 38. Uv~jcs, B. (1954). Antidromic vasodilatation in the paw of the cat. Pharmacol. Rev. 6, 99-101.