- Email: [email protected]
Vasodilator responses following intracranial stimulation of the trigeminal, facial and glossopharyngeal nerves in the cat gingiva
Brain Research, 560 (1991) 71-75 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939116973M
71
BRES 16973
Vasodilator responses following intracranial stimulation of the trigeminal, facial and glossopharyngeal nerves in the cat gingiva Hiroshi Izumi and Keishiro Karita Department of Physiology, Tohoku University School of Dentistry, Sendai (Japan) (Accepted 30 April 1991)
Key words: Vasodilatation; Trigeminal nerve; Facial nerve; Glossopharyngeal nerve; Parasympathetic nerve; Gingiva; Cat
The effects of electrical stimulation of the trigeminal, facial and glossopharyngeal nerves on gingival blood flow in the cat were studied. The intracranial part of these nerves was stimulated electrically, and gingival blood flow was measured by the laser Doppler technique. Electrical stimulation of the trigeminal, facial and glossopharyngeal nerves caused blood flow to increase in the ipsilateral gingiva both with the cranial nerve intact and after cutting it to the medulla. Stimulation of the distal cut ends of the facial and glossopharyngeal nerves elicited an increase in blood flow but no increase in systemic blood pressure. Pretreatment with hexamethonium reduced the increase in blood flow elicited by electrical stimulation of the facial and glossopharyngeal nerves, but had no effect on that elicited by stimulation of the trigeminal nerve. In contrast, pretreatment with tripelennamine attenuated the trigeminal nerve-stimulated blood flow increase, but not that elicited by stimulation of the facial and glossopharyngeal nerves. Atropine, propranolol and phentolamine had no effect on these responses. These results suggest that the autonomic nervous system, particularly the parasympathetic nervous system, is responsible for the blood flow increase elicited by facial and glossopharyngeal nerve stimulation, and that the trigeminal nerve-stimulated blood flow increase is induced by antidromic vasodilatation of the trigeminal sensory nerve. INTRODUCTION
We have recently reported that electrical stimulation of the distal cut end of the inferior alveolar nerve produces vasodilatation and vasoconstriction in the cat gingiva 12"13. The vasodilator response seems to be mediated partly through trigeminal sensory fibers and mast cells; however, other mechanisms seem to be involved since antagonists for this trigeminal system such as (D-Pro2,D Try7'9)-substance P (substance P-receptor antagonist) and tripelennamine (histamine H i - r e c e p t o r antagonist) elicited no more than 20-30% attenuation of this vasodilatation. O n the other hand, the vasoconstrictor response was completely reduced by phentolamine, an a-adrenoceptor antagonist, indicating that the vasoconstrictor response occurs almost entirely via sympathetic a-adrenergic receptors. If sympathetic innervation of the gingiva does exist, it is not improbable that parasympathetic fibers would reach this region and have a complementary antagonistic function. However, the existence of parasympathetic vasodilator fibers in the cat gingiva remains to be clarified. Gonzalez et al. lo have previously reported the existence of a vasodilator system in the cat facial area; most of this emerges from the brainstem with the facial nerve,
while some run directly with the trigeminal nerve to be distributed it in a divisional pattern. Recent histological and immunohistochemical studies suggested the possible existence of parasympathetic fibers originating from the otic ganglion in cat tooth pulp 21 and rat lip 14. These studies prompted us to investigate whether parasympathetic vasodilator fibers are present in cat gingiva. Therefore, we stimulated the trigeminal, facial, and glossopharyngeal nerves intracranially in order to determine how these cranial nerves control blood flow in the cat gingiva. MATERIALS AND METHODS The experiments were conducted on 30 cats, weighing 1.2-3.5 kg, anesthetized with ketamine hydrochloride (30 mg/kg, i.m.) initially and then with an i.v. injection of Nembutal (sodium pentobarbital) at an initial dose of 30 mg/kg, supplemented, when necessary, with additional doses of 20-30 mg. Polyethylene catheters were inserted into a femoral artery for the purpose of monitoring systemic blood pressure, and into the cephalic vein for the injection of anesthetics and drugs. The animals were intubated, paralyzed with pancuronim bromide, and artificial ventilation was maintained with a Harvard respirator. The skull of the animal was firmly fixed in a stereotaxic frame. The roots of the trigeminal (fifth), facial (seventh) and glossopharyngeal (ninth) cranial nerves were exposed from their origins at the brainstem after an extensive craniectomy and aspiration of cerebellum. Electrical stimulation of each cranial nerve was carded out intracranially. A bipolar electrode was inserted under visual control into the exposed cranial nerves after cutting the two vagal
Correspondence: H. Izumi, Department of Physiology, Tohoku University School of Dentistry, Seiryo-machi 4-1, Aoba, Sendai 980, Japan.
72
A GBF (re ft)
B
C
D i ! (~}
; -.•..
~'.:.:. .
' '.
[
:
,.J-
,
'-:5,
J () GBF
(rig h ~)-
::-'::::~::": :::
t
;:'2'~:~":;";::"'" ; '~';,,;:k"
t
:.:.i:~;,/}:.:...~:~:::..~,~:,::>:.:,:~.:. . . . . t;..-:%, :
t
t
": :: :,::.":.-::~
I 5o (%)
o
Fig. 1. Changes of the ipsilateral (left) and contralateral (right) gingival blood flow (GBF) in response to electrical stimulation of the left trigeminal (A), facial (B) and glossopharyngeal (C) nerves with the cranial nerve intact and of the distal cut end of the left facial nerve after cutting it to the medulla (D) in the cat. Electrical stimulation with 40 Hz, 2 ms, 40 V for 10 s, as indicated by arrows. Abscissa: time (min). Ordinate: GBF expressed as a percentage.
nerves in the neck (cervical). The eighth and tenth nerves may be stimulated during stimulation of the seventh and ninth nerves if the roots of these nerves are not completely isolated. The roots of the cranial nerves were stimulated for 10 s with 20-100 V and 40 Hz pulses of 2 ms duration using a stimulator (Nihon Koden Model SEN-7103). Gingival blood flow under the lower canine tooth was measured by a laser Doppler flowmeter (Canon LC-1, Tokyo, Japan). This equipment gives no absolute value, but permits measurements of relative changes in blood flow. Electrical calibration for zero blood flow was performed, and an appropriate gain was selected. In the figures, the maximal output at a given level was set at 100%. Output from the flowmeter was recorded on a multichannel chart recorder at a speed of 10 mm/min. For analysis, the increase in blood flow following stimulation of the cranial nerves was calculated by measuring the area using a planimeter. Changes in blood flow were expressed as percentages of the control response. Results are expressed as means -+ S.E.M. The significance of changes in blood flow data was tested with the Wilcoxon signedrank test and paired t-test. Differences were considered significant at a level of 0.05. Tripelennamine hydrochloride was generously supplied by CibaGeigy (Basel, Switzerland). DL-Propranolol hydrochloride was purchased from Sigma Chemicals (St. Louis, MO, U.S.A.). Hexamethonium bromide was kindly donated by Squibb and Sons (New Brunswick, NJ, U.S.A.). Phentolamine mesylate (Regitin) was purchased from Japan Ciba-Geigy (Takarazuka, Japan). Atropine sulfate was obtained from Iwaki Pharmaceuticals (Tokyo, Japan). All other chemicals were of reagent grade and were obtained from commercial sources.
RESULTS Fig. 1 s h o w s t h a t e l e c t r i c a l s t i m u l a t i o n o f e i t h e r t h e t r i g e m i n a l (Fig. 1 A ) , facial (Fig. 1B) o r g l o s s o p h a r y n geal n e r v e (Fig. 1C) w i t h t h e c r a n i a l n e r v e i n t a c t elici t e d i n c r e a s e d b l o o d flow in t h e i p s i l a t e r a l c a t gingiva. T h e s e i n c r e a s e s in b l o o d flow w e r e also o b s e r v e d following s t i m u l a t i o n o f t h e p e r i p h e r a l e n d o f e a c h c r a n i a l n e r v e a f t e r c u t t i n g it t o t h e m e d u l l a . T h e r e s u l t o f a typical e x p e r i m e n t o n e l e c t r i c a l s t i m u l a t i o n o f t h e d i s t a l cut e n d o f t h e facial n e r v e is s h o w n in Fig. 1D.
Fig. 2 A s h o w s t h e effects o f facial n e r v e s t i m u l a t i o n at v a r i o u s i n t e n s i t i e s o n i p s i l a t e r a l o r c o n t r a l a t e r a l gingival b l o o d flow a n d s y s t e m i c b l o o d p r e s s u r e . A s s h o w n in t h e figure, e l e c t r i c a l s t i m u l a t i o n o f less t h a n 20 V h a d n o e f f e c t o n g i n g i v a l b l o o d flow o n e i t h e r side o r o n syst e m i c b l o o d p r e s s u r e . A t m o r e t h a n 40 V, a l t h o u g h a n i n t e n s i t y - d e p e n d e n t i n c r e a s e in b l o o d flow was o b s e r v e d f o l l o w i n g s t i m u l a t i o n o f t h e facial n e r v e , t h e e f f e c t s o f
............................ t
r~:
t
~,,
t
% --
I ¸~¸ ic
Fig. 2. Changes of ipsilateral (left) or contralateral (right) gingival blood flow (GBF) and blood pressure in response to electrical stimulation of the facial or glossopharyngeal nerve in the cats. A: effects of the left facial nerve stimulation with the cranial nerve intact at different intensities (indicated below arrows). B: effects of the left facial nerve stimulation both with the cranial nerve intact (left tracing) and after cutting it to the medulla (right tracing)• Electrical stimulation with 40 Hz, 2 ms, 80 V for 10 s, as indicated by arrows. C: effects of the left glossopharyngeal nerve stimulation both with the cranial nerve intact (left tracing) and after cutting it to the medulla (right tracing). Electrical stimulation with 40 Hz, 2 ms, 40 V for 10 s, as indicated by arrows. Abscissa: time (min). Ordinate: GBF and blood pressure were shown as a percentage and systemic arterial blood pressure (mm Hg), respectively.
73 stimulation on systemic blood pressure were diverse; stimulation of the facial nerve at a low intensity (40 V) elicited a decrease in systemic blood pressure, whereas stimulation at a high intensity (100 V) produced an increase in systemic blood pressure (Fig. 2A, right tracing). However, the increase in systemic blood pressure was no longer observed when the peripheral end of the facial and glossopharyngeal nerves was stimulated after cutting it to the medulla (Fig. 2B,C). When blood pressure was increased by stimulation, the onset of the increase after the start of stimulation (6.7 -+ 0.65 s; mean
A 100
._L
II xX
3
~X
o
XX XX
50
xx Kx
D
L9
KX i (12)
(6)
(4)
(4)
(4)
B 100
_L
_J_
~o
X~ XX XX ,KX ~X
¢11t ¢/11
o
¢11t
50
¢11t
o
llll fllJ fllJ II11
(4)
_L
(6)
(5)
Kx (5)
, (4)
- S.E.M., n = 6) was almost the same as the onset of the increase in blood flow (5.74 - 0.63 s; n = 6). In these cases, a slight increase in contralateral gingival blood flow was sometimes observed following stimulation of the facial nerve (Fig. 2A, right tracing). On the other hand, when blood pressure was decreased by stimulation the onset of the increase in blood flow (6.70 -+ 0.98 s; n = 10) preceded the decrease in blood pressure (12.00 ___ 0.77 s; n = 10, P < 0.01). The nature of the vasodilator mechanism activated by electrical stimulation of the cranial nerves was examined by pharmacological means. Blocking agents were introduced by the systemic route between the first and the second stimulation in one series of experiments. As shown in Fig. 3, pretreatment with hexamethonium (C6), an autonomic ganglionic blocker, caused a marked reduction in the gingival blood flow increase following stimulation of the facial and glossopharyngeal nerves, but had no effect on the trigeminal nerve-induced blood flow increase. The result of a typical experiment on the effect of hexamethonium on the increase in blood flow elicited by stimulation of the glossopharyngeal nerve is shown in Fig. 4. Hexamethonium at a dose of 1 mg/kg produced time-dependent inhibition of the increase in blood flow. By 45 min after its application, hexamethonium no longer had an inhibitory effect on this response. Pretreatment with tripelennamine, a histamine Hi-receptor antagonist, caused a significant reduction of the blood flow increase elicited by stimulation of the trigeminal nerve, but had no effect on the increases caused by stimulation of the facial and glossopharyngeal nerves (Fig. 3). Atropine (muscarinic receptor antagonist; 1.0 mg/kg), propranolol (fl-adrenoceptor antagonist; 1.0 mg/ kg) and phentolamine (a-adrenoceptor antagonist; 1.0 mg/kg) had no effect on these responses (Fig. 3).
C v 3=
100
_t
rn
o
~
,
_L.~
-L,
×>
5o
o
c
III
ql (4)
IX~
L~_ (6)
R~
,LI~
(4)
(5)
i
illll
A
B
C
D 100
.'.,,
.~.
GBF (%)
I
(4)
Fig. 3. Effects of tripelennamine (hatched column), hexamethonium (filled column), atropine (dotted column), propranolol (crosshatched column) and phentolamine (stripped column) on trigeminal (A), facial (B) or glossopharyngeal (C) nerve-stimulated gingival blood flow (GBF) increase in the cats. Open column represents the increase in blood flow following stimulation of the cranial nerve in the absence of compounds and is considered as 100%. Results are expressed as means _+ S.E.M. (vertical bars). The number of cats used is shown in parentheses. *P < 0.05.
0
/ C6
rain
2 rain
15 rain
4 5 rain
Fig. 4. Effects of hexamethonium (C6) on the glossopharyngeal nerve-stimulated blood flow increase. The glossopharyngeal nerve was electrically stimulated with 40 Hz, 2 ms, 40 V for 10 s as indicated by arrows before (A) and after i.v. administration of hexamethonium at 2 (B), 15 (C) and 45 (D) min. Abscissa: time (rain). Ordinate: GBF expressed as a percentage.
74
DISCUSSION Recent data suggest the presence of at least 4 vasodilator mechanisms in the peripheral vessels of the skin, gingiva, mucosa, tongue and pia of mammals, namely; (1) inhibition of sympathetic vasoconstrictor tone7'8'25; (2) sympathetic vasodilatation3"4"19"2°; (3) parasympathetic vasodilatation ~6 18; and (4) antidromic vasodilatation mediated by sensory nerves 11-~3. Our previous observation showing that electrical stimulation of the cut inferior alveolar nerve elicits vasodilatation and vasoconstriction in cat gingiva indicated innervation of both vasodilator and vasoconstrictor fibers in this region ~3. Pharmacological analysis suggested the involvement of sensory fibers and sympathetic a-adrenergic fibers in these vasoresponses. Although specific sympathetic vasodilator fibers to human foot and forearm skin have recently been reported by many investigators 3'4'19'2°, the existence of sympathetic vasodilator fibers was not found in the present experiment; electrical stimulation of the cervical sympathetic nerves elicited only a vasoconstrictor response in this tissue. We have previously reported that atropine, a parasympathetic muscarinic receptor antagonist, did not produce significant attenuation of inferior alveolar nervestimulated vasodilatation ~2"~3. This suggests that there is no cholinergic innervation of the peripheral blood vessels in cat gingiva. However, this does not imply that there are no parasympathetic fibers, because non-cholinergic parasympathetic fibers have been shown to be distributed in cephalic artery, tongue, nasal mucosa and submandibular gland in cat 2'~'~6-~s. in addition, electrical stimulation of the facial (or chordalingual) and glossopharyngeal nerves elicits vasodilatation in these tis-
suesl,%6,16 18. As the laser Doppler technique measures the velocity of moving objects, in this case basically red cells, changes in systemic blood pressure may modify the value for gingival blood flow. Therefore, it is necessary to minimize changes, particularly increases, in systemic blood pressure when measuring blood flow by laser Doppler flowmeter. When the facial nerve was stimulated at a high intensity (100 V), simultaneous increases in contralateral blood flow and systemic blood pressure occurred following cranial nerve stimulation (Fig. 2A, right tracing). At the present time, it is unclear whether contralateral blood flow increased as a result of an increase in blood pressure or vasodilatation itself. The changes in systemic blood pressure following electrical stimulation of the cranial nerves varied from animal to animal and may reflect different degrees of current spread in the electrical stimulation, which may stimulate the pressure center at the brainstem. For these reasons, we chose a lower intensity
as far as possible when stimulating the cranial nerves in the present experiments. As shown in Fig 1, an ipsilateral increase in blood flow was observed following electrical stimulation of the trigeminal, facial and glossopharyngeal nerves, both with the cranial nerve intact and after cutting it to the medulla. It seems reasonable to suppose that the increase in gingival blood flow and blood pressure are both the result of cranial nerve stimulation but are nevertheless independent. Evidence that this is indeed the case was obtained in our experiments in which stimulating the facial cranial nerve at a relatively low intensity (40 V) was shown to cause increased blood flow in gingiva without a concomitant increase in blood pressure (Fig. 2A, middle tracing). Furthermore, blood flow increased with no concomitant increase in systemic blood pressure when the peripheral end of each cranial nerve was stimulated (Fig. 2B,C, right tracing). These data suggest that blood flow increase in gingiva is the result of a neurogenic response, and that the blood circulation in the cat gingiva is affected by 3 different cranial nerves, namely, the trigeminal, facial and glossopharyngeal. Pretreatment with hexamethonium, an autonomic ganglionic blocking agent, reduced the increase in blood flow elicited by electrical stimulation of the facial and glossopharyngeal nerves, but had no significant effect on the increase in blood flow elicited by stimulation of the trigeminal nerve (Fig. 3). These results suggest that the autonomic nervous system is responsible for the increase in blood flow elicited by facial and glossopharyngeal nerve stimulation. Neither response was affected by pretreatment with atropine. It seems likely that the mechanism of both responses is atropine-resistant and hexamethonium-sensitive. This type of blood flow increase has been suggested to be mediated by vasoactive intestinal peptide (VIP) in various tissues such as salivary gland 16,t7 and tongue 18. Recently, Kaji et al. 14 have reported that VIP-immunoreactive positive fibers are present in the cutaneous blood vessels of rat lower lip and are completely lost on removal of the parasympathetic ganglion. These findings suggest parasympathetic control of blood circulation in the cat gingiva. VIP is a potent vasodilator in many vascular beds 15'2~. If VIP were a true parasympathetic nerve fiber transmitter in cat gingiva and were released from nerve terminals by stimulation of these fibers, it would be possible to speculate that the decrease in blood pressure following stimulation of the facial and glossopharyngeal nerves, as shown in Fig. 2, may be due to an increase of VIP in systemic blood. This is supported by the present observation that the onset of blood flow increase preceded that of blood pressure decrease. However, further investigations are necessary to conclusively establish the neurotransmitter involved in these
75 vasodilatations. In addition, it is still u n k n o w n why dif-
lated blood flow increases, suggesting sensory nerve in-
ferent parasympathetic fibers originating in the facial and
volvement in the blood flow increase caused by trigem-
glossopharyngeal nerves regulate blood circulation in the
inal nerve stimulation. The present results are thus different from those obtained by Gonzalez et al. 10, who
same gingival area of the cat. I n this respect, Segade and Squarez-Quintanilla22 have recently reported that in the maxillary division of guinea pig there is a partial overlapping of the territories innervated by the pterygopala-
have shown a vasodilator system emerging from the brainstem with the facial nerve, which reaches the trigeminal ganglion by way of the greater superficial
tine and otic ganglia. A similar overlapping of nerve fibers of pterygopalatine and ciliary origins has b e e n
petrosal nerve; a moderate n u m b e r of vasodilator fibers
reported in the ophthalmic division of rats 23. O n the other hand, p r e t r e a t m e n t with t r i p e l e n n a m i n e ,
trigeminal nerve. For these reasons, they ruled out the possibility that sensory nerves are responsible for the
a
also appear to leave the brainstem directly with the
the
cutaneous vasodilatation that occurs after stimulation of
trigeminal nerve-stimulated blood flow increase but had
the trigeminal root. A t present no satisfactory explana-
no effect on facial and glossopharyngeal nerve-stimu-
tion can be offered for this discrepancy.
histamine H i - r e c e p t o r
antagonist,
attenuated
REFERENCES 1 Anggard, A., The effects of parasympathetic nerve stimulation on the microcirculation and secretion in the nasal mucosa of the cat, Acta Otolaryng. 78 (1974) 98-105. 2 Baecker, B., Yanaihara, N. and Frossmann, W.G., VIP innervation of the tongue in vertebrates, Anat. Embryol., 167 (1983) 173-189. 3 Bell, C., Janig, W., Kummel, H. and Xu, H., Differentiation of vasodilator and sudomotor responses in the cat paw pad to preganglionic sympathetic stimulation, J. Physiol., 364 (1985) 93-104. 4 Biumberg, H. and Wallin, B.G., Direct evidence of neurally mediated vasodilatation in hairy skin of the human foot, J. Physiol., 382 (1987) 105-121. 5 Chorobski, J. and Penfield, W., Cerebral vasodilator nerves and their pathway from the medulla oblongata: with observations on the pial and intracerebral vascular plexus, Arch. Neurol. Psychiatry, 28 (1932) 1257-1289. 6 Cobb, S. and Finesnger, J.E., Cerebral circulation. XIX. The vagal pathway of the vasodilator impulses, Arch. Neurol. Psychiatry, 28 (1932) 1243-1256. 7 Edwall, L. and Kindlova, M., The effect of sympathetic nerve stimulation on the rate of disappearance of tracers from various tissues, Acta Odontol. Scand., 29 (1971) 387-400. 8 Fox, R.H., Goldsmith, R. and Kidd, D.J., Cutaneous vasomotor control in the human head, neck and upper chest, J. Physiol., 161 (1962) 298-312. 9 Gibbins, I.L., Brayden, J.E. and Bevan, J.A., Perivascular nerves with immunoreactivity to vasoactive intestinal polypeptide in cephalic arteries of the cat, distribution, possible origins and functional implications, Neuroscience, 13 (1984) 1327-1346. 10 Gonzalez, G., Onofrio, B.M. and Kerr, F.W., Vasodilator system for the face, J. Neurosurg., 42 (1975) 696-703. 11 Izumi, H. and Karita, K., Investigation of mechanisms of the flare and wheal reactions in human skin by band method, Brain Research, 449 (1988) 328-331. 12 Izumi, H. and Karita, K., The effects of capsaicin applied topically to inferior alveolar nerve on antidromic vasodilatation in cat gingiva, Neurosci. Lett., 112 (1990) 65-69. 13 Izumi, H., Kuriwada, S., Karita, K., Sasano, T. and Sanjo, D., The nervous control of gingival blood flow, Microvasc. Res., 39 (1990) 94-104. 14 Kaji, A., Shigematsu, H., Fujita, K., Maeda, T. and Watanabe, S., Parasympathetic innervation of cutaneous blood vessels by
15
t6
17
18
19
20 21 22
23
24 25
vasoactive intestinal polypeptide-immunoreactive and acetylcholinesterase-positive nerves: histochemical and experimental study on rat lower lip, Neuroscience, 25 (1988) 353-362. Larsson, L.-I., Edvinsson, L., Fahrenkrug, J., Hakanson, R., Owman, C.H., Schaffalitzky de Muckadell, O. and Sundler, E, Immunohistochemical localization of vasodilatory polypeptide (VIP) in cerebrovaseular nerves, Brain Research, 113 (1976) 400-404. Lundberg, J.M,, Anggard, A. and Fahrenkrug, J., Complementary role of vasoactive intestinal polypeptide (VIP) and acetylcholine for cat submandibular gland blood flow and secretion, I. VIP release, Acta Physiol. Scand., 113 (1981) 317-327. Lundberg, J.M., Anggard, A. and Fahrenkrug, J., Complementary role of vasoactive intestinal polypeptide (VIP) and acetylcholine for cat submandibular blood flow and secretion, II. Effects of cholinergic antagonists and VIP antiserum, Acta Physiol. Scand,, 114 (1982) 329-337. Lundberg, J.A., Anggard, A. and Fahrenkrug, J., VIP as a mediator of hexamethonium-sensitive, atropine-resistant vasodilatation in the cat tongue, Acta Physiol. Scand., 116 (1982) 387-392. Lundberg, J., Norgren, L., Ribbe, E., Rosen, I., Steen, S., Thrne, J. and Wallken, B.G., Direct evidence of active sympathetic vasodilatation in the skin of the human foot, J. Physiol., 417 (1989) 437-446. Nordin, M., Sympathetic discharges in the human supraorbital nerve and their relation to sudo- and vasomotor responses, J. Physiol., 423 (1990) 241-255. Segade, L.A.G. and Squarez-Quintanilla, D., Otic ganglion parasympathetic neurons innervate the pulp of the mandibular incisor of the guinea-pig, Neurosci. Lea., 90 (1988) 33-38. Segadc, K.A.G. and Squarez-Quintanilla, D., Distribution of postganglionic parasympathetic fibers originating in the pterygopalatine ganglion in the maxillary and ophthalmic nerve branches of the trigeminal nerve: HRP and WGA-HRP study in the guinea pig, Brain Research, 522 (1990) 327-332. Tem Tusscher, M.P.M., Klooster, J., Baljet, B., Van der Weft, E and Vrensen, G.EL.M., Pre- and post-ganglionic nerve fibres of the pterygopalatine ganglion and their allocation to the eyeball of rats, Brain Research, 517 (1990) 315-323. Uddman, R., Alumets, J., Edvinsson, L., Hakanson, R. Loren, I. and Sundler, F., VIP nerve fibres around peripheral blood vessels, Acta Physiol. Scand., 112 (1981) 65-70. Willems, J.L. and Bogaert, M., Neurogenic vasodilatation, Gen. Pharmacol., 9 (1978) 223-227.
71
BRES 16973
Vasodilator responses following intracranial stimulation of the trigeminal, facial and glossopharyngeal nerves in the cat gingiva Hiroshi Izumi and Keishiro Karita Department of Physiology, Tohoku University School of Dentistry, Sendai (Japan) (Accepted 30 April 1991)
Key words: Vasodilatation; Trigeminal nerve; Facial nerve; Glossopharyngeal nerve; Parasympathetic nerve; Gingiva; Cat
The effects of electrical stimulation of the trigeminal, facial and glossopharyngeal nerves on gingival blood flow in the cat were studied. The intracranial part of these nerves was stimulated electrically, and gingival blood flow was measured by the laser Doppler technique. Electrical stimulation of the trigeminal, facial and glossopharyngeal nerves caused blood flow to increase in the ipsilateral gingiva both with the cranial nerve intact and after cutting it to the medulla. Stimulation of the distal cut ends of the facial and glossopharyngeal nerves elicited an increase in blood flow but no increase in systemic blood pressure. Pretreatment with hexamethonium reduced the increase in blood flow elicited by electrical stimulation of the facial and glossopharyngeal nerves, but had no effect on that elicited by stimulation of the trigeminal nerve. In contrast, pretreatment with tripelennamine attenuated the trigeminal nerve-stimulated blood flow increase, but not that elicited by stimulation of the facial and glossopharyngeal nerves. Atropine, propranolol and phentolamine had no effect on these responses. These results suggest that the autonomic nervous system, particularly the parasympathetic nervous system, is responsible for the blood flow increase elicited by facial and glossopharyngeal nerve stimulation, and that the trigeminal nerve-stimulated blood flow increase is induced by antidromic vasodilatation of the trigeminal sensory nerve. INTRODUCTION
We have recently reported that electrical stimulation of the distal cut end of the inferior alveolar nerve produces vasodilatation and vasoconstriction in the cat gingiva 12"13. The vasodilator response seems to be mediated partly through trigeminal sensory fibers and mast cells; however, other mechanisms seem to be involved since antagonists for this trigeminal system such as (D-Pro2,D Try7'9)-substance P (substance P-receptor antagonist) and tripelennamine (histamine H i - r e c e p t o r antagonist) elicited no more than 20-30% attenuation of this vasodilatation. O n the other hand, the vasoconstrictor response was completely reduced by phentolamine, an a-adrenoceptor antagonist, indicating that the vasoconstrictor response occurs almost entirely via sympathetic a-adrenergic receptors. If sympathetic innervation of the gingiva does exist, it is not improbable that parasympathetic fibers would reach this region and have a complementary antagonistic function. However, the existence of parasympathetic vasodilator fibers in the cat gingiva remains to be clarified. Gonzalez et al. lo have previously reported the existence of a vasodilator system in the cat facial area; most of this emerges from the brainstem with the facial nerve,
while some run directly with the trigeminal nerve to be distributed it in a divisional pattern. Recent histological and immunohistochemical studies suggested the possible existence of parasympathetic fibers originating from the otic ganglion in cat tooth pulp 21 and rat lip 14. These studies prompted us to investigate whether parasympathetic vasodilator fibers are present in cat gingiva. Therefore, we stimulated the trigeminal, facial, and glossopharyngeal nerves intracranially in order to determine how these cranial nerves control blood flow in the cat gingiva. MATERIALS AND METHODS The experiments were conducted on 30 cats, weighing 1.2-3.5 kg, anesthetized with ketamine hydrochloride (30 mg/kg, i.m.) initially and then with an i.v. injection of Nembutal (sodium pentobarbital) at an initial dose of 30 mg/kg, supplemented, when necessary, with additional doses of 20-30 mg. Polyethylene catheters were inserted into a femoral artery for the purpose of monitoring systemic blood pressure, and into the cephalic vein for the injection of anesthetics and drugs. The animals were intubated, paralyzed with pancuronim bromide, and artificial ventilation was maintained with a Harvard respirator. The skull of the animal was firmly fixed in a stereotaxic frame. The roots of the trigeminal (fifth), facial (seventh) and glossopharyngeal (ninth) cranial nerves were exposed from their origins at the brainstem after an extensive craniectomy and aspiration of cerebellum. Electrical stimulation of each cranial nerve was carded out intracranially. A bipolar electrode was inserted under visual control into the exposed cranial nerves after cutting the two vagal
Correspondence: H. Izumi, Department of Physiology, Tohoku University School of Dentistry, Seiryo-machi 4-1, Aoba, Sendai 980, Japan.
72
A GBF (re ft)
B
C
D i ! (~}
; -.•..
~'.:.:. .
' '.
[
:
,.J-
,
'-:5,
J () GBF
(rig h ~)-
::-'::::~::": :::
t
;:'2'~:~":;";::"'" ; '~';,,;:k"
t
:.:.i:~;,/}:.:...~:~:::..~,~:,::>:.:,:~.:. . . . . t;..-:%, :
t
t
": :: :,::.":.-::~
I 5o (%)
o
Fig. 1. Changes of the ipsilateral (left) and contralateral (right) gingival blood flow (GBF) in response to electrical stimulation of the left trigeminal (A), facial (B) and glossopharyngeal (C) nerves with the cranial nerve intact and of the distal cut end of the left facial nerve after cutting it to the medulla (D) in the cat. Electrical stimulation with 40 Hz, 2 ms, 40 V for 10 s, as indicated by arrows. Abscissa: time (min). Ordinate: GBF expressed as a percentage.
nerves in the neck (cervical). The eighth and tenth nerves may be stimulated during stimulation of the seventh and ninth nerves if the roots of these nerves are not completely isolated. The roots of the cranial nerves were stimulated for 10 s with 20-100 V and 40 Hz pulses of 2 ms duration using a stimulator (Nihon Koden Model SEN-7103). Gingival blood flow under the lower canine tooth was measured by a laser Doppler flowmeter (Canon LC-1, Tokyo, Japan). This equipment gives no absolute value, but permits measurements of relative changes in blood flow. Electrical calibration for zero blood flow was performed, and an appropriate gain was selected. In the figures, the maximal output at a given level was set at 100%. Output from the flowmeter was recorded on a multichannel chart recorder at a speed of 10 mm/min. For analysis, the increase in blood flow following stimulation of the cranial nerves was calculated by measuring the area using a planimeter. Changes in blood flow were expressed as percentages of the control response. Results are expressed as means -+ S.E.M. The significance of changes in blood flow data was tested with the Wilcoxon signedrank test and paired t-test. Differences were considered significant at a level of 0.05. Tripelennamine hydrochloride was generously supplied by CibaGeigy (Basel, Switzerland). DL-Propranolol hydrochloride was purchased from Sigma Chemicals (St. Louis, MO, U.S.A.). Hexamethonium bromide was kindly donated by Squibb and Sons (New Brunswick, NJ, U.S.A.). Phentolamine mesylate (Regitin) was purchased from Japan Ciba-Geigy (Takarazuka, Japan). Atropine sulfate was obtained from Iwaki Pharmaceuticals (Tokyo, Japan). All other chemicals were of reagent grade and were obtained from commercial sources.
RESULTS Fig. 1 s h o w s t h a t e l e c t r i c a l s t i m u l a t i o n o f e i t h e r t h e t r i g e m i n a l (Fig. 1 A ) , facial (Fig. 1B) o r g l o s s o p h a r y n geal n e r v e (Fig. 1C) w i t h t h e c r a n i a l n e r v e i n t a c t elici t e d i n c r e a s e d b l o o d flow in t h e i p s i l a t e r a l c a t gingiva. T h e s e i n c r e a s e s in b l o o d flow w e r e also o b s e r v e d following s t i m u l a t i o n o f t h e p e r i p h e r a l e n d o f e a c h c r a n i a l n e r v e a f t e r c u t t i n g it t o t h e m e d u l l a . T h e r e s u l t o f a typical e x p e r i m e n t o n e l e c t r i c a l s t i m u l a t i o n o f t h e d i s t a l cut e n d o f t h e facial n e r v e is s h o w n in Fig. 1D.
Fig. 2 A s h o w s t h e effects o f facial n e r v e s t i m u l a t i o n at v a r i o u s i n t e n s i t i e s o n i p s i l a t e r a l o r c o n t r a l a t e r a l gingival b l o o d flow a n d s y s t e m i c b l o o d p r e s s u r e . A s s h o w n in t h e figure, e l e c t r i c a l s t i m u l a t i o n o f less t h a n 20 V h a d n o e f f e c t o n g i n g i v a l b l o o d flow o n e i t h e r side o r o n syst e m i c b l o o d p r e s s u r e . A t m o r e t h a n 40 V, a l t h o u g h a n i n t e n s i t y - d e p e n d e n t i n c r e a s e in b l o o d flow was o b s e r v e d f o l l o w i n g s t i m u l a t i o n o f t h e facial n e r v e , t h e e f f e c t s o f
............................ t
r~:
t
~,,
t
% --
I ¸~¸ ic
Fig. 2. Changes of ipsilateral (left) or contralateral (right) gingival blood flow (GBF) and blood pressure in response to electrical stimulation of the facial or glossopharyngeal nerve in the cats. A: effects of the left facial nerve stimulation with the cranial nerve intact at different intensities (indicated below arrows). B: effects of the left facial nerve stimulation both with the cranial nerve intact (left tracing) and after cutting it to the medulla (right tracing)• Electrical stimulation with 40 Hz, 2 ms, 80 V for 10 s, as indicated by arrows. C: effects of the left glossopharyngeal nerve stimulation both with the cranial nerve intact (left tracing) and after cutting it to the medulla (right tracing). Electrical stimulation with 40 Hz, 2 ms, 40 V for 10 s, as indicated by arrows. Abscissa: time (min). Ordinate: GBF and blood pressure were shown as a percentage and systemic arterial blood pressure (mm Hg), respectively.
73 stimulation on systemic blood pressure were diverse; stimulation of the facial nerve at a low intensity (40 V) elicited a decrease in systemic blood pressure, whereas stimulation at a high intensity (100 V) produced an increase in systemic blood pressure (Fig. 2A, right tracing). However, the increase in systemic blood pressure was no longer observed when the peripheral end of the facial and glossopharyngeal nerves was stimulated after cutting it to the medulla (Fig. 2B,C). When blood pressure was increased by stimulation, the onset of the increase after the start of stimulation (6.7 -+ 0.65 s; mean
A 100
._L
II xX
3
~X
o
XX XX
50
xx Kx
D
L9
KX i (12)
(6)
(4)
(4)
(4)
B 100
_L
_J_
~o
X~ XX XX ,KX ~X
¢11t ¢/11
o
¢11t
50
¢11t
o
llll fllJ fllJ II11
(4)
_L
(6)
(5)
Kx (5)
, (4)
- S.E.M., n = 6) was almost the same as the onset of the increase in blood flow (5.74 - 0.63 s; n = 6). In these cases, a slight increase in contralateral gingival blood flow was sometimes observed following stimulation of the facial nerve (Fig. 2A, right tracing). On the other hand, when blood pressure was decreased by stimulation the onset of the increase in blood flow (6.70 -+ 0.98 s; n = 10) preceded the decrease in blood pressure (12.00 ___ 0.77 s; n = 10, P < 0.01). The nature of the vasodilator mechanism activated by electrical stimulation of the cranial nerves was examined by pharmacological means. Blocking agents were introduced by the systemic route between the first and the second stimulation in one series of experiments. As shown in Fig. 3, pretreatment with hexamethonium (C6), an autonomic ganglionic blocker, caused a marked reduction in the gingival blood flow increase following stimulation of the facial and glossopharyngeal nerves, but had no effect on the trigeminal nerve-induced blood flow increase. The result of a typical experiment on the effect of hexamethonium on the increase in blood flow elicited by stimulation of the glossopharyngeal nerve is shown in Fig. 4. Hexamethonium at a dose of 1 mg/kg produced time-dependent inhibition of the increase in blood flow. By 45 min after its application, hexamethonium no longer had an inhibitory effect on this response. Pretreatment with tripelennamine, a histamine Hi-receptor antagonist, caused a significant reduction of the blood flow increase elicited by stimulation of the trigeminal nerve, but had no effect on the increases caused by stimulation of the facial and glossopharyngeal nerves (Fig. 3). Atropine (muscarinic receptor antagonist; 1.0 mg/kg), propranolol (fl-adrenoceptor antagonist; 1.0 mg/ kg) and phentolamine (a-adrenoceptor antagonist; 1.0 mg/kg) had no effect on these responses (Fig. 3).
C v 3=
100
_t
rn
o
~
,
_L.~
-L,
×>
5o
o
c
III
ql (4)
IX~
L~_ (6)
R~
,LI~
(4)
(5)
i
illll
A
B
C
D 100
.'.,,
.~.
GBF (%)
I
(4)
Fig. 3. Effects of tripelennamine (hatched column), hexamethonium (filled column), atropine (dotted column), propranolol (crosshatched column) and phentolamine (stripped column) on trigeminal (A), facial (B) or glossopharyngeal (C) nerve-stimulated gingival blood flow (GBF) increase in the cats. Open column represents the increase in blood flow following stimulation of the cranial nerve in the absence of compounds and is considered as 100%. Results are expressed as means _+ S.E.M. (vertical bars). The number of cats used is shown in parentheses. *P < 0.05.
0
/ C6
rain
2 rain
15 rain
4 5 rain
Fig. 4. Effects of hexamethonium (C6) on the glossopharyngeal nerve-stimulated blood flow increase. The glossopharyngeal nerve was electrically stimulated with 40 Hz, 2 ms, 40 V for 10 s as indicated by arrows before (A) and after i.v. administration of hexamethonium at 2 (B), 15 (C) and 45 (D) min. Abscissa: time (rain). Ordinate: GBF expressed as a percentage.
74
DISCUSSION Recent data suggest the presence of at least 4 vasodilator mechanisms in the peripheral vessels of the skin, gingiva, mucosa, tongue and pia of mammals, namely; (1) inhibition of sympathetic vasoconstrictor tone7'8'25; (2) sympathetic vasodilatation3"4"19"2°; (3) parasympathetic vasodilatation ~6 18; and (4) antidromic vasodilatation mediated by sensory nerves 11-~3. Our previous observation showing that electrical stimulation of the cut inferior alveolar nerve elicits vasodilatation and vasoconstriction in cat gingiva indicated innervation of both vasodilator and vasoconstrictor fibers in this region ~3. Pharmacological analysis suggested the involvement of sensory fibers and sympathetic a-adrenergic fibers in these vasoresponses. Although specific sympathetic vasodilator fibers to human foot and forearm skin have recently been reported by many investigators 3'4'19'2°, the existence of sympathetic vasodilator fibers was not found in the present experiment; electrical stimulation of the cervical sympathetic nerves elicited only a vasoconstrictor response in this tissue. We have previously reported that atropine, a parasympathetic muscarinic receptor antagonist, did not produce significant attenuation of inferior alveolar nervestimulated vasodilatation ~2"~3. This suggests that there is no cholinergic innervation of the peripheral blood vessels in cat gingiva. However, this does not imply that there are no parasympathetic fibers, because non-cholinergic parasympathetic fibers have been shown to be distributed in cephalic artery, tongue, nasal mucosa and submandibular gland in cat 2'~'~6-~s. in addition, electrical stimulation of the facial (or chordalingual) and glossopharyngeal nerves elicits vasodilatation in these tis-
suesl,%6,16 18. As the laser Doppler technique measures the velocity of moving objects, in this case basically red cells, changes in systemic blood pressure may modify the value for gingival blood flow. Therefore, it is necessary to minimize changes, particularly increases, in systemic blood pressure when measuring blood flow by laser Doppler flowmeter. When the facial nerve was stimulated at a high intensity (100 V), simultaneous increases in contralateral blood flow and systemic blood pressure occurred following cranial nerve stimulation (Fig. 2A, right tracing). At the present time, it is unclear whether contralateral blood flow increased as a result of an increase in blood pressure or vasodilatation itself. The changes in systemic blood pressure following electrical stimulation of the cranial nerves varied from animal to animal and may reflect different degrees of current spread in the electrical stimulation, which may stimulate the pressure center at the brainstem. For these reasons, we chose a lower intensity
as far as possible when stimulating the cranial nerves in the present experiments. As shown in Fig 1, an ipsilateral increase in blood flow was observed following electrical stimulation of the trigeminal, facial and glossopharyngeal nerves, both with the cranial nerve intact and after cutting it to the medulla. It seems reasonable to suppose that the increase in gingival blood flow and blood pressure are both the result of cranial nerve stimulation but are nevertheless independent. Evidence that this is indeed the case was obtained in our experiments in which stimulating the facial cranial nerve at a relatively low intensity (40 V) was shown to cause increased blood flow in gingiva without a concomitant increase in blood pressure (Fig. 2A, middle tracing). Furthermore, blood flow increased with no concomitant increase in systemic blood pressure when the peripheral end of each cranial nerve was stimulated (Fig. 2B,C, right tracing). These data suggest that blood flow increase in gingiva is the result of a neurogenic response, and that the blood circulation in the cat gingiva is affected by 3 different cranial nerves, namely, the trigeminal, facial and glossopharyngeal. Pretreatment with hexamethonium, an autonomic ganglionic blocking agent, reduced the increase in blood flow elicited by electrical stimulation of the facial and glossopharyngeal nerves, but had no significant effect on the increase in blood flow elicited by stimulation of the trigeminal nerve (Fig. 3). These results suggest that the autonomic nervous system is responsible for the increase in blood flow elicited by facial and glossopharyngeal nerve stimulation. Neither response was affected by pretreatment with atropine. It seems likely that the mechanism of both responses is atropine-resistant and hexamethonium-sensitive. This type of blood flow increase has been suggested to be mediated by vasoactive intestinal peptide (VIP) in various tissues such as salivary gland 16,t7 and tongue 18. Recently, Kaji et al. 14 have reported that VIP-immunoreactive positive fibers are present in the cutaneous blood vessels of rat lower lip and are completely lost on removal of the parasympathetic ganglion. These findings suggest parasympathetic control of blood circulation in the cat gingiva. VIP is a potent vasodilator in many vascular beds 15'2~. If VIP were a true parasympathetic nerve fiber transmitter in cat gingiva and were released from nerve terminals by stimulation of these fibers, it would be possible to speculate that the decrease in blood pressure following stimulation of the facial and glossopharyngeal nerves, as shown in Fig. 2, may be due to an increase of VIP in systemic blood. This is supported by the present observation that the onset of blood flow increase preceded that of blood pressure decrease. However, further investigations are necessary to conclusively establish the neurotransmitter involved in these
75 vasodilatations. In addition, it is still u n k n o w n why dif-
lated blood flow increases, suggesting sensory nerve in-
ferent parasympathetic fibers originating in the facial and
volvement in the blood flow increase caused by trigem-
glossopharyngeal nerves regulate blood circulation in the
inal nerve stimulation. The present results are thus different from those obtained by Gonzalez et al. 10, who
same gingival area of the cat. I n this respect, Segade and Squarez-Quintanilla22 have recently reported that in the maxillary division of guinea pig there is a partial overlapping of the territories innervated by the pterygopala-
have shown a vasodilator system emerging from the brainstem with the facial nerve, which reaches the trigeminal ganglion by way of the greater superficial
tine and otic ganglia. A similar overlapping of nerve fibers of pterygopalatine and ciliary origins has b e e n
petrosal nerve; a moderate n u m b e r of vasodilator fibers
reported in the ophthalmic division of rats 23. O n the other hand, p r e t r e a t m e n t with t r i p e l e n n a m i n e ,
trigeminal nerve. For these reasons, they ruled out the possibility that sensory nerves are responsible for the
a
also appear to leave the brainstem directly with the
the
cutaneous vasodilatation that occurs after stimulation of
trigeminal nerve-stimulated blood flow increase but had
the trigeminal root. A t present no satisfactory explana-
no effect on facial and glossopharyngeal nerve-stimu-
tion can be offered for this discrepancy.
histamine H i - r e c e p t o r
antagonist,
attenuated
REFERENCES 1 Anggard, A., The effects of parasympathetic nerve stimulation on the microcirculation and secretion in the nasal mucosa of the cat, Acta Otolaryng. 78 (1974) 98-105. 2 Baecker, B., Yanaihara, N. and Frossmann, W.G., VIP innervation of the tongue in vertebrates, Anat. Embryol., 167 (1983) 173-189. 3 Bell, C., Janig, W., Kummel, H. and Xu, H., Differentiation of vasodilator and sudomotor responses in the cat paw pad to preganglionic sympathetic stimulation, J. Physiol., 364 (1985) 93-104. 4 Biumberg, H. and Wallin, B.G., Direct evidence of neurally mediated vasodilatation in hairy skin of the human foot, J. Physiol., 382 (1987) 105-121. 5 Chorobski, J. and Penfield, W., Cerebral vasodilator nerves and their pathway from the medulla oblongata: with observations on the pial and intracerebral vascular plexus, Arch. Neurol. Psychiatry, 28 (1932) 1257-1289. 6 Cobb, S. and Finesnger, J.E., Cerebral circulation. XIX. The vagal pathway of the vasodilator impulses, Arch. Neurol. Psychiatry, 28 (1932) 1243-1256. 7 Edwall, L. and Kindlova, M., The effect of sympathetic nerve stimulation on the rate of disappearance of tracers from various tissues, Acta Odontol. Scand., 29 (1971) 387-400. 8 Fox, R.H., Goldsmith, R. and Kidd, D.J., Cutaneous vasomotor control in the human head, neck and upper chest, J. Physiol., 161 (1962) 298-312. 9 Gibbins, I.L., Brayden, J.E. and Bevan, J.A., Perivascular nerves with immunoreactivity to vasoactive intestinal polypeptide in cephalic arteries of the cat, distribution, possible origins and functional implications, Neuroscience, 13 (1984) 1327-1346. 10 Gonzalez, G., Onofrio, B.M. and Kerr, F.W., Vasodilator system for the face, J. Neurosurg., 42 (1975) 696-703. 11 Izumi, H. and Karita, K., Investigation of mechanisms of the flare and wheal reactions in human skin by band method, Brain Research, 449 (1988) 328-331. 12 Izumi, H. and Karita, K., The effects of capsaicin applied topically to inferior alveolar nerve on antidromic vasodilatation in cat gingiva, Neurosci. Lett., 112 (1990) 65-69. 13 Izumi, H., Kuriwada, S., Karita, K., Sasano, T. and Sanjo, D., The nervous control of gingival blood flow, Microvasc. Res., 39 (1990) 94-104. 14 Kaji, A., Shigematsu, H., Fujita, K., Maeda, T. and Watanabe, S., Parasympathetic innervation of cutaneous blood vessels by
15
t6
17
18
19
20 21 22
23
24 25
vasoactive intestinal polypeptide-immunoreactive and acetylcholinesterase-positive nerves: histochemical and experimental study on rat lower lip, Neuroscience, 25 (1988) 353-362. Larsson, L.-I., Edvinsson, L., Fahrenkrug, J., Hakanson, R., Owman, C.H., Schaffalitzky de Muckadell, O. and Sundler, E, Immunohistochemical localization of vasodilatory polypeptide (VIP) in cerebrovaseular nerves, Brain Research, 113 (1976) 400-404. Lundberg, J.M,, Anggard, A. and Fahrenkrug, J., Complementary role of vasoactive intestinal polypeptide (VIP) and acetylcholine for cat submandibular gland blood flow and secretion, I. VIP release, Acta Physiol. Scand., 113 (1981) 317-327. Lundberg, J.M., Anggard, A. and Fahrenkrug, J., Complementary role of vasoactive intestinal polypeptide (VIP) and acetylcholine for cat submandibular blood flow and secretion, II. Effects of cholinergic antagonists and VIP antiserum, Acta Physiol. Scand,, 114 (1982) 329-337. Lundberg, J.A., Anggard, A. and Fahrenkrug, J., VIP as a mediator of hexamethonium-sensitive, atropine-resistant vasodilatation in the cat tongue, Acta Physiol. Scand., 116 (1982) 387-392. Lundberg, J., Norgren, L., Ribbe, E., Rosen, I., Steen, S., Thrne, J. and Wallken, B.G., Direct evidence of active sympathetic vasodilatation in the skin of the human foot, J. Physiol., 417 (1989) 437-446. Nordin, M., Sympathetic discharges in the human supraorbital nerve and their relation to sudo- and vasomotor responses, J. Physiol., 423 (1990) 241-255. Segade, L.A.G. and Squarez-Quintanilla, D., Otic ganglion parasympathetic neurons innervate the pulp of the mandibular incisor of the guinea-pig, Neurosci. Lea., 90 (1988) 33-38. Segadc, K.A.G. and Squarez-Quintanilla, D., Distribution of postganglionic parasympathetic fibers originating in the pterygopalatine ganglion in the maxillary and ophthalmic nerve branches of the trigeminal nerve: HRP and WGA-HRP study in the guinea pig, Brain Research, 522 (1990) 327-332. Tem Tusscher, M.P.M., Klooster, J., Baljet, B., Van der Weft, E and Vrensen, G.EL.M., Pre- and post-ganglionic nerve fibres of the pterygopalatine ganglion and their allocation to the eyeball of rats, Brain Research, 517 (1990) 315-323. Uddman, R., Alumets, J., Edvinsson, L., Hakanson, R. Loren, I. and Sundler, F., VIP nerve fibres around peripheral blood vessels, Acta Physiol. Scand., 112 (1981) 65-70. Willems, J.L. and Bogaert, M., Neurogenic vasodilatation, Gen. Pharmacol., 9 (1978) 223-227.