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Simultaneous measurement of parasympathetic reflex vasodilator and arterial blood pressure responses in the cat
Brain Research 952 (2002) 61–70 www.elsevier.com / locate / bres
Research report
Simultaneous measurement of parasympathetic reflex vasodilator and arterial blood pressure responses in the cat Hiroshi Izumi a , *, Kentaro Mizuta a , Satoshi Kuchiiwa b a
Department of Orofacial Functions, Tohoku University Graduate School of Dentistry, Sendai 980 -8575, Japan b Department of Anatomy, Faculty of Medicine, Kagoshima University, Kagoshima 890 -8520, Japan Accepted 22 April 2002
Abstract We measured the changes in lower lip blood flow and systemic arterial blood pressure evoked by lingual nerve or trigeminal spinal nucleus (Vsp) stimulation to gain an insight into the brainstem integration of sympathetic and parasympathetic responses to nociceptive stimulation. We used artificially ventilated, cervically vago-sympathectomized cats deeply anesthetized with a-chloralose and urethane. A lip blood flow increase occurred in an intensity- and frequency-dependent manner following electrical stimulation of Vsp or lingual nerve regardless of whether systemic arterial blood pressure increased or decreased. In contrast, there was no apparent optimal frequency for the changes in systemic arterial blood pressure elicited by electrical stimulation of Vsp or lingual nerve. No relationship was found between the amplitude of the lip blood flow increase and that of the systemic arterial blood pressure change. Microinjection of lidocaine or kainic acid into the Vsp evoked, respectively, reversible and irreversible inhibition of the lip blood flow increase and systemic arterial blood pressure change evoked by lingual nerve stimulation. When microinjected unilaterally directly into the ipsilateral Vsp, the GABA agonist muscimol abolished both lingual nerve-evoked effects (increase in lip blood flow and changes in systemic arterial blood pressure) without changing basal systemic arterial blood pressure, suggesting the presence in the Vsp of GABA receptors serving to modulate both the parasympathetically mediated lip blood flow increase and the sympathetically mediated systemic arterial blood pressure change. Lidocaine microinjection into the salivatory nucleus caused a significant attenuation of the lingual nerve-induced blood flow increase, but had no effect on the lingual nerve-induced systemic arterial blood pressure change. Thus, the neural pathway mediating the lingual nerve-induced lip blood flow increase seems to be simple, requiring a minimum of four neurons: trigeminal afferent–Vsp–parasympathetic preganglionic neurons with cell body located in the inferior salivatory nucleus–otic postganglionic neuron. On the other hand, the pathway underlying the evoked systemic arterial blood pressure changes, presumably mediated via altered sympathetic activity, seems to be more complicated and could be affected by more numerous factors. 2002 Elsevier Science B.V. All rights reserved. Keywords: Autonomic reflex; Nociceptive trigeminal stimulation; Vasodilatation; Arterial blood pressure
1. Introduction The patterns of vasomotor and other cardiovascular responses elicited by noxious stimulation seem to be dependent on the site at which the noxious stimulation is applied. Such noxious stimulation as electrical stimulation of the sciatic nerve, surgical stress, or pinching the body skin evokes a skin blood flow decrease as well as other cardiovascular responses (i.e., increases in systemic arterial *Corresponding author. Tel.: 181-22-717-8321; fax: 181-22-7178322. E-mail address: [email protected] (H. Izumi).
blood pressure and heart rate) in anesthetized animals and humans [24,25,29]. These are thought to be mediated via activation of a sympathetic reflex mechanism serving to maintain hemodynamic and metabolic homeostasis, the so-called somato-sympathetic reflex [5,7,8,34]. However, noxious stimulation of orofacial areas, such as the tongue and nasal mucosa, does not evoke a sympathetically mediated blood flow decrease; instead, there are blood flow increases in the lip, tongue, and salivary gland in anesthetized cats and rats, responses that are mediated via a somato-parasympathetic mechanism [9,11]. In this response, systemic arterial blood pressure is reportedly decreased, probably due to withdrawal of sympathetic
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vasomotor tone to femoral, superior mesenteric, and renal vascular beds [21–23,26]. Although these observations suggest that trigeminally mediated nociceptive stimulation affects both the sympathetic and parasympathetic systems, few actual data are available concerning the way in which the two systems may be affected by this form of stimulation. We examined changes in both lower lip blood flow and systemic arterial blood pressure following nociceptive trigeminal stimulation to gain an insight into the integration of the sympathetic and parasympathetic components of the response in the brainstem of the cat. To this end, we electrically stimulated (using various stimulus conditions) the central cut end of the lingual nerve (‘lingual nerve’) and the trigeminal spinal nucleus (Vsp), which is known to be a brainstem relay for the nociceptive inputs from the orofacial area that travel via the trigeminal nerve [2,27], and we measured the systemic arterial blood pressure changes and lower lip blood flow changes. In our cats, in which the cervical sympathetics and cervical vagi are cut, we took these changes to be indicators of changes in sympathetic nerve activity and parasympathetic nerve activity, respectively, since the systemic arterial blood pressure changes are known to be markedly attenuated by antiadrenergic agents (phentolamine and propranolol) and by an autonomic ganglionic blocking agent (hexamethonium) [4,15,21] and since the lip blood flow changes are abolished by ipsilateral section of the glossopharyngeal nerve root [15]. We also made microinjections within the brain stem to produce nonselective, reversible local anesthesia (lidocaine) or soma-selective, irreversible neurotoxic damage (kainic acid) to examine the contribution to these responses made by the Vsp and the salivatory nucleus, which is known to give rise to preganglionic parasympathetic vasodilator fibers.
was cannulated for the measurement of systemic arterial blood pressure. One cephalic vein was cannulated to allow drug injection. The anesthetized animals were intubated, paralysed by intravenous injection of pancuronium bromide (Mioblock; Organon, Teknika, The Netherlands; 0.4 mg / kg initially, supplemented with 0.2 mg / kg every hour or so after testing the level of anesthesia; see below) and artificially ventilated via the tracheal cannula with a mixture of 50% air–50% O 2 . The ventilator (Model SN480-6; Shinano, Tokyo, Japan) was set to deliver a tidal volume of 10–12 cm 3 / kg at a rate of 20 breaths / min, and the end-tidal concentration of CO 2 was determined by means of an infrared analyzer (Capnomac Ultima; Datex, Helsinki, Finland) as reported previously [10,12,17]. Endtidal CO 2 was kept at 35–40 mmHg. Ringer solution (Otsuka Pharmaceutical, Tokyo, Japan) was continuously infused at a rate of approximately 5 ml / h. Rectal temperature was maintained at 37–38 8C using a heating pad. In all experiments, the cervical vagi and superior cervical sympathetic trunks were cut bilaterally in the neck prior to any stimulation to eliminate reflex effects on the cardiovascular system via the vagus nerve and sympathetic vasoconstrictor effects on the orofacial area, respectively. The criterion for the maintenance of an adequate depth of anesthesia was the absence of any precipitate changes in systemic arterial blood pressure as reflex responses to a fairly minor noxious stimulus (such as pinching the upper lip for approximately 2 s). If the depth of anesthesia was considered inadequate, additional a-chloralose and urethane (i.e., intermittent doses of 5 and 10 mg / kg, i.v., respectively) were administered. Once an adequate depth of anesthesia had been attained, supplementary doses of pancuronium were given approximately every 60 min to maintain immobilisation during periods of stimulation.
2.2. Electrical stimulation of lingual nerve 2. Materials and methods
2.1. Preparation of animals The experimental protocols were reviewed by the Committee on the Ethics of Animal Experiments in Tohoku University School of Medicine, and they were carried out in accordance with both the Guidelines for Animal Experiments issued by the Tohoku University School of Medicine and The Law (No. 105) and Notification (No. 6) issued by the Japanese Government. Twenty-eight cats, unselected as to sex and of 2.0–4.5 kg body wt (approximate age 2–4 years), were initially sedated with ketamine hydrochloride (30 mg / kg, i.m.) and then anesthetized with a mixture of a-chloralose (50 mg / kg, i.v.) and urethane (100 mg / kg, i.v.). These anesthetics were supplemented if and when necessary throughout the experiment (see below). Local anesthesia (2% lidocaine; 1–2 ml) was applied to all skin incisions. A femoral artery
To elicit a parasympathetic reflex vasodilatation in the lower lip, the central cut end of lingual nerve was electrically stimulated (Fig. 1). The routine stimulus parameters were: a 20-s train of 2-ms rectangular pulses at a frequency of 10 Hz and at supramaximal intensity (usually 30 V), as described previously [10,12,17]. A bipolar silver electrode attached to a Nihon Kohden Model SEN-7103 Stimulator (Tokyo, Japan) was used for this stimulation.
2.3. Microinjections of lidocaine, kainic acid, and muscimol To determine whether the vasodilator response elicited by lingual nerve stimulation was mediated via Vsp or the salivatory nucleus, lidocaine (2% / site), kainic acid (10 mM / site), or muscimol (13.2 mM / site) was microinjected into Vsp or salivatory nucleus in a volume of 1.0 ml / site via an injection cannula (0.50 mm o.d.) inserted through an
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evoked vasodilatation or on resting cardiovascular parameters. The magnitude of the lingual nerve-evoked response obtained after microinjection of a given agent was expressed as a percentage of the control response recorded before its administration (mean6S.E.).
2.4. Measurement of lower lip blood flow and of SABP Blood flow changes in the lower lip were monitored (Fig. 1) using a laser-Doppler flowmeter (LDF; model ALF21D; Advance, Tokyo, Japan), as described before [10,12,17]. The probe was placed against the lower lip without exerting any pressure on the tissue. The blood flow changes were assessed by measuring the height of the response on the chart. In the figures, flow levels are expressed in arbitrary units. Systemic arterial blood pressure was recorded from the femoral catheter via a Statham pressure transducer. A tachograph (model AT-610G; Nihon Kohden) triggered by the arterial pulse was used to monitor heart rate.
2.5. Histology
Fig. 1. Schematic representation of the sites used for electrical stimulation, blood flow measurements, and microinjections of lidocaine, kainic acid, or muscimol. Stimulation site: central cut end of the lingual nerve (LN; A). Blood flow measurement site: lower lip (by laser-Doppler flowmeter, LDF; B). Microinjection sites: Vsp (C) and salivatory nucleus (SN, D). The reflex pathways relating to the present study are indicated by solid and broken lines. Abbreviations: OG, otic ganglion; SN, salivatory nucleus; NTS, nucleus tractus solitarius; SSN, V, trigeminal nerve root; Vsp, trigeminal spinal nucleus; Vt, trigeminal spinal tract; VII, facial nerve root; IX, glossopharyngeal nerve root.
implanted guide cannula. Briefly, the animal was mounted in a stereotaxic frame (Narishige, Tokyo, Japan), and after a partial craniotomy, part of the tentorium cerebelli was removed by drilling. As indicated schematically in Fig. 1, a guide cannula (1.00 mm o.d.) was positioned within the Vsp (P, 10.0–11.0; L, 5.0–6.0; H, 5.0–6.0 mm; coordinates of Berman [1]) or the salivatory nucleus (P, 8.0–9.0; L, 2.5–3.5; H, 5.0–6.0 mm) via a small burr-hole in the skull. This was achieved with the aid of a micromanipulator and without removing any part of the brain. The stimulating electrode was interchangeable with the injection cannula. Both were of equal length and each extended 5.0 mm beyond the tip of the guide cannula. Thus, microinjection and electrical stimulation were carried out at the same sites. Saline (1.0 ml) was used for control injections. It never produced any significant effect on the lingual nerve-
Animals were given an overdose of pentobarbital (60 mg / kg) by i.v. infusion and perfused through the ascending aorta with 1.0–2.0 l of saline (0.9%) followed immediately by 2 l of 10% formalin. Then, the brain stem and upper cervical spinal cord were removed and stored for 1–4 days in buffered 30% sucrose. After storage, sections 50 mm thick were cut on a freezing microtome and collected in 0.1 M phosphate buffer (pH 7.4). Sections were mounted on gelatin-coated slides and stained with thionin. Photomicrographs of representative coronal sections showing sites used for electrical stimulation or microinjection of lidocaine, kainic acid, or muscimol into the Vsp or the SN can be seen in Fig. 6. Visualization of the spread of the HRP injected into the salivatory nucleus was achieved using the peroxidase–DAB reaction (Fig. 7).
2.6. Statistical analysis All numerical data are given as mean6S.E. The significance of changes in the test responses was assessed using an analysis of variance (ANOVA) followed by a contrast test. Differences were considered significant at the level P,0.05. Data were analyzed using a Macintosh Computer with StatView 5.0 and Super ANOVA.
3. Results The resting mean arterial blood pressure of the cats used in this study lay within the range 68.367.5 to 121.7610.5 mmHg, the average value for the group being 86.168.1 mmHg.
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3.1. Electrical stimulation of lingual nerve and Vsp on lip blood flow and arterial blood pressure Fig. 2 shows the effects of electrical stimulation of the central cut end of lingual nerve and of Vsp on blood flow in the ipsilateral lower lip when the stimulation was delivered at various intensities (lingual nerve, 1–50 V; Vsp, 2–200 mA; Fig. 2A) and at various frequencies (0.1–100 Hz; Fig. 2B). Electrical stimulation of lingual nerve or Vsp at ,1 V or ,10 mA had no significant effects, whereas increasing the stimulus intensity from 2 up to 10 V or from 10 up to 100 mA produced successively bigger vasodilator responses, the response being saturated at 10 V or 100 mA, respectively. Electrical stimulation of lingual nerve or Vsp
needed to be at .0.2 Hz to have significant effects on lip blood flow, and the response was saturated at 10 Hz in each case. The optimal frequency for vasodilatation in response to either lingual nerve or Vsp stimulation was 10 Hz (Fig. 2B). The effect of electrical stimulation of either lingual nerve (Fig. 3A,B) or Vsp (Fig. 3C,D) on systemic arterial blood pressure varied from animal to animal, both qualitatively and quantitatively. No optimal frequency could be identified when either lingual nerve or Vsp was electrically stimulated at various frequencies from 1 to 100 Hz; in fact, no clear relationships emerged between systemic arterial blood pressure changes and the frequency or intensity of lingual nerve or Vsp stimulation.
3.2. Effects of microinjection of lidocaine, kainic acid, and muscimol
Fig. 2. Stimulus intensity–response (A) and frequency–response (B) relationships for changes in lower lip blood flow evoked by electrical stimulation of the central cut end of the lingual nerve (LN; open circles) and of the trigeminal spinal nucleus (Vsp; filled circles). Stimulation was at various intensities (1–50 V for LN and 2–200 mA for Vsp) and various frequencies (0.1–100 Hz). Intensity–response curves were generated using stimulus trains at 10 Hz. Frequency–response curves were generated using stimulus trains at 30 V for LN and 100 mA for Vsp. Each value is given as mean6S.E. Number of animals used was 8 for LN, 10 for Vsp.
Local anesthesia of the ipsilateral Vsp by microinjection of lidocaine was produced to examine the contribution made by this area to the blood flow increases and systemic arterial blood pressure changes evoked by lingual nerve stimulation (Fig. 4). Fig. 4A shows a typical recording of the time course (before and 10–60 min after microinjection) of the effects of such lidocaine microinjection (mean data are shown in Fig. 5). Control lingual nerve stimulation elicited a blood flow increase in the ipsilateral lower lip and a change (decrease or increase) in systemic arterial blood pressure. Both changes had disappeared when the stimulation was repeated 10 min after microinjection of lidocaine into the Vsp, but the response recovered in a time-dependent manner, indicating that the inhibitory effects of lidocaine were reversible. A typical microinjection site is shown in Fig. 6. Kainic acid (10 mM / site, 1 ml) was microinjected into Vsp at sites at which microinjection of lidocaine had previously evoked a marked reduction in the vasodilator response evoked by stimulation of the ipsilateral lingual nerve (Fig. 4B). This was done to examine the contribution made by cell bodies in this area to the response to lingual nerve stimulation. Kainic acid was given 20–30 min after the lingual nerve-evoked response had completely recovered from the effects of lidocaine. The mean data (Fig. 5) show that both the LBF increase and the systemic arterial blood pressure change were significantly reduced at t510 and t560 min (t50 is the time at which blood flow returned to the basal level following the increase (duration 4.962.3 min; n56 animals) induced by microinjection of kainic acid into Vsp). Thus, these effects of kainic acid were irreversible (at least within the time-frame of the experiment). Fig. 4C shows typical recordings of effects of microinjection of muscimol (13.2 mM / site, 1 ml) into Vsp on the lip blood flow increase and systemic arterial blood pressure change evoked by electrical stimulation of lingual nerve. This was done to examine the involvement of GABA receptors in Vsp on these responses. The mean data (Fig. 5) show that muscimol microinjection elicited time-
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Fig. 3. Stimulus intensity–response (A,C) and frequency–response (B,D) relationships for changes in systemic arterial blood pressure (SABP) evoked by electrical stimulation of the central cut end of the lingual nerve (LN, A,B) and of the trigeminal spinal nucleus (Vsp, C,D). Stimulation was at various intensities (1–50 V for LN and 2–200 mA for Vsp) and various frequencies (0.1–100 Hz). Intensity–response curves were generated using stimulus trains at 10 Hz. Frequency–response curves were generated using stimulus trains at 30 V for LN and 100 mA for Vsp. Each value is given as mean6S.E. Number of animals used was 10 for LN, 11 for Vsp.
dependent reductions in both responses, and that there was no recovery within 60 min after the microinjection. Fig. 4D shows typical recordings of the effects of microinjection of lidocaine into the salivatory nucleus on the lip blood flow increase and systemic arterial blood pressure change evoked by electrical stimulation of lingual nerve. A typical microinjection site is shown in Fig. 6B. The mean data (Fig. 5) show that although lidocaine microinjection elicited a statistically significant attenuation of the lingual nerve-induced blood flow increase, it had no effect on the lingual nerve-induced systemic arterial blood pressure change. In the present study, to examine the possibility that the changes in lip blood flow and systemic arterial blood pressure evoked by lingual nerve stimulation might be mediated via the Vsp and / or salivatory nucleus in the medulla oblongata, we inactivated some or all neural function in these regions using unilateral microinjections of lidocaine, kainic acid, or muscimol. A histological marker (HRP; 1 ml, 10%) was injected into the salivatory nucleus to indicate how far the injections might have spread. As shown in Fig. 7, the spread of HRP was approximately 1–1.5 mm in radius (this was also the spread in the rostral direction and in the caudal direction). This spread of HRP suggests that the effects
produced by microinjecting lidocaine, kainic acid, or muscimol into the Vsp or salivatory nucleus were not a consequence of spread of these drugs to the other site (i.e., Vsp injection did not spread to salivatory nucleus) since (i) the salivatory nucleus microinjection site is nearly 1.5 mm rostral and 2.5 mm medial to the Vsp site, (ii) lidocaine microinjection into the salivatory nucleus did not attenuate the lingual nerve-mediated increase in systemic arterial blood pressure, which is mediated via Vsp, and (iii) microinjection of lidocaine into the salivatory nucleus reduced the palate blood flow increase only on the ipsilateral side, not on the contralateral side, when lingual nerve stimulation was used to evoke a bilateral increase in palate blood flow (data not shown).
4. Discussion It is well known that autonomic responses elicited by stimulation of somatic and visceral afferent nerves are mediated by reflex mechanisms. Numerous reports have described somato-sympathetic responses, such as arterial blood pressure increases, and somato-parasympathetic responses, such as salivary secretion [9,11,20,30,31]. How-
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Fig. 4. Typical examples of effects produced by microinjection of (A) lidocaine (2%, 1 ml / site), (B) kainic acid (10 mM, 1 ml / site), and (C) muscimol (13.2 mM, 1 ml / site) into the ipsilateral trigeminal spinal nucleus (Vsp), and of (D) lidocaine (2%, 1 ml / site) into the ipsilateral salivatory nucleus (SN) on the blood flow increase in lower lip (LBF) (in arbitrary units (au)) and changes in systemic arterial blood pressure (SABP, mmHg) elicited by electrical stimulation of the central cut end of the lingual nerve (LN). Electrical stimulation of the LN was at 30 V, 10 Hz, 2-ms pulse duration for 20 s. Kainic acid and lidocaine were microinjected at the same site in Vsp (see Section 3 for further details). Electrical stimulation of LN was carried out before and at 10–60 min after each microinjection (30 V, 10 Hz, 2-ms pulse duration for 20 s). In the kainic acid experiment, electrical stimulation of LN was not resumed until after blood flow had returned to the basal level following the increase induced by microinjection of kainic acid into Vsp (i.e., the times ‘10 min’ to ‘60 min’ refer to the interval after restoration of blood flow to the basal level). In the particular experiment illustrated in (B), the duration of the kainic acid-induced blood flow increase was 7 min.
ever, few previous experimental studies have examined both types of somato-autonomic response simultaneously. The possibility that the increases in lip blood flow described here were the passive result of evoked increases in blood pressure seems remote: since such blood flow increases (1) occur only on the ipsilateral side of the lip, not on the contralateral side [15], and (2) are abolished by
ipsilateral section of the glossopharyngeal nerve root and by the autonomic ganglionic blocker, hexamethonium [15]. Microinjections of lidocaine or kainic acid into the Vsp attenuated (reversibly and irreversibly, respectively) not only the trigeminally (lingual nerve stimulation) elicited lip blood flow increase, but also the accompanying changes in systemic arterial blood pressure (Fig. 4A,B). These results
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Fig. 5. Effects produced by microinjection (1.0 ml / site) of lidocaine (2% / site), kainic acid (10 mM / site), and muscimol (13.2 mM / site) into the trigeminal spinal nucleus (Vsp) on the blood flow increase in lower lip (LBF) and change in systemic arterial blood pressure (SABP, mmHg) elicited by electrical stimulation of the central cut end of the lingual nerve (LN) (ipsilateral to the microinjection site). Electrical stimulation of the LN was at 30 V, 10 Hz, 2-ms pulse duration for 20 s. In the kainic acid experiment, electrical stimulation of LN was not resumed until after blood flow had returned to the basal level following the increase induced by microinjection of kainic acid into Vsp (i.e., the times ‘10 min’ to ‘60 min’ refer to the interval after restoration of blood flow to the basal level). Open and hatched bars indicate, respectively, LBF and SABP responses. The ‘control’ alterations in SABP evoked by stimulation of the lingual nerve or Vsp are expressed as 100% regardless of whether the alteration was a decrease or increase in SABP. The responses illustrated were obtained before, and 10 and 60 min after treatment with lidocaine, kainic acid, or muscimol. Each value is expressed as a percentage of the pretreatment response and is given as mean6S.E. Statistical significance of difference from control was assessed by means of ANOVA followed by a contrast test (*P,0.05, **P,0.01, ***P,0.001). Number of animals used was five.
Fig. 6. Photomicrographs of representative coronal sections through the medulla oblongata of the cat showing sites at which microinjections of lidocaine or kainic acid were delivered to the trigeminal spinal nucleus (Vsp; A) and salivatory nucleus (SN; B). Thionin stain; scale bar represents 1.0 mm. Abbreviations: FN, facial nucleus; IO, inferior olivary nucleus; SN, salivatory nucleus; PH, nucleus praepositus hypoglossi; RB, restiform body; VIN, inferior vestibular nucleus; VLD, lateral vestibular nucleus; Vsp, trigeminal spinal nucleus; Vt, trigeminal spinal tract.
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Fig. 7. Diagram derived from a control section through the salivatory nucleus injection site region, with the injection site (arrow) marked by the peroxidase–DAB reaction. Scale bar represents 1.0 mm. Abbreviations: FN, facial nucleus; FTG, gigantocellar tegmental field; PH, nucleus praepositus hypoglossi; RB, restiform body; RFN, retrofacial nucleus; SA, Stria acustica; SN, salivatory nucleus; V4, fourth ventricle; VIN, inferior vestibular nucleus; VLD, lateral vestibular nucleus; Vsp, trigeminal spinal nucleus; Vt, trigeminal spinal tract.
suggest that both the increase in lip blood flow and the changes in systemic arterial blood pressure evoked by lingual nerve stimulation are mediated by neurons situated within the Vsp. On the other hand, microinjection of either lidocaine (Figs. 4D and 5) or kainic acid (data not shown) into the salivatory nucleus significantly reduced only the lip blood flow increase; it had no effect on the magnitude of the evoked changes in systemic arterial blood pressure regardless of whether systemic arterial blood pressure increased or decreased. These results suggest (i) that the salivatory nucleus represents a critical bulbar relay site for the lingual nerve-induced lip blood flow increase, but not for the lingual nerve-evoked changes in systemic arterial blood pressure, (ii) that neural pathways mediating these two responses diverge after traffic derived from nociceptive trigeminal information leaves the Vsp, and (iii) that the lingual nerve-induced changes in systemic arterial blood pressure are not secondary to changes in blood flow in the orofacial area, even though lingual nerve stimulation evokes blood flow increases in nearly all orofacial regions [11]. It has previously been reported that in the anesthetized rabbit, electrical or chemical stimulation of the trigeminal nerve or the spinal trigeminal nucleus, or pinching of the
lip with forceps caused a profound fall in systemic arterial blood pressure, and Blessing and co-workers suggested that the trigeminal depressor response depends on a net inhibition of spinal sympathetic preganglionic neurons via the brainstem [2–4,36]. Kumada et al. [21–23] showed that the hypotensive effect elicited by electrical stimulation of the brain (the spinal trigeminal complex) or a peripheral nerve (branches of the trigeminal nerve) was optimal at frequencies between 5 and 10 Hz. This band of frequencies is similar to the one we found to be optimal for the parasympathetic vasodilatation elicited by electrical stimulation of both Vsp and lingual nerve (Fig. 2B). However, no optimal frequency could be established for the changes in systemic arterial blood pressure elicited by electrical stimulation of the Vsp or lingual nerve (Fig. 3). Similarly, no intensity dependence of the changes in systemic arterial blood pressure was observed on electrical stimulation of either Vsp or lingual nerve (Fig. 3A,C). In their lack of dependence on stimulus frequency, the systemic arterial blood pressure changes we elicited by electrical stimulation of the lingual nerve and Vsp differ from those reported long ago by Gruber [7] who reported that systemic arterial blood pressure decreased when spinal sensory nerves such as the saphenous, peroneal, ulnar, radial, median or popliteal nerves were electrically stimulated at low frequency (4 Hz), but increased when the same nerves were stimulated at high frequency (20 Hz). The neural pathways mediating the lip blood flow increase induced by lingual nerve stimulation would seem to be quite simple, requiring a minimum of only four neurons: trigeminal afferent–Vsp–parasympathetic preganglionic neuron with cell body located in the inferior salivatory nucleus–otic postganglionic neuron [27]. This notion is supported by anatomical and physiological studies showing that (i) trigeminal afferents running in lingual nerve pass to Vsp [28,32], (ii) the parasympathetic preganglionic neurons in salivatory nucleus receive projections from Vsp (30), (iii) cutting the glossopharyngeal nerve root abolished the nociceptive trigeminal stimulation-induced blood flow increase in the lower lip [33], and (iv) the lip blood flow increase evoked by electrical stimulation of the glossopharyngeal nerve root was abolished by prior treatment with the autonomic ganglionic blocker hexamethonium [13,15]. On the other hand, neural mediation of the lingual nerve-induced systemic arterial blood pressure changes, presumably involving alterations in sympathetic activity, would seem to be more complicated and probably subject to influence by more numerous factors since consistent systemic arterial blood pressure changes were not observed in our cats in response to nociceptive trigeminal stimulation. Blessing and co-workers [3,4,36] have reported that the systemic arterial blood pressure fall evoked by electrical stimulation of Vsp / spinal tract of the trigeminal nerve in the rabbit occurred as a result of withdrawal of sympathetic vasomotor tone to femoral, mesenteric, renal,
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and skeletal muscle resistance vessels. A systemic arterial blood pressure fall has also been reported to occur on electrical stimulation of the lingual nerve, the inferior alveolar nerve, and the supraorbital nerve in rabbit and cat [6,21]. However, under our experimental conditions consistent systemic arterial blood pressure changes were not evoked by electrical stimulation of either lingual nerve or Vsp in the cat despite the same anesthetic (urethane and a-chloralose) being used in each animal. In some preliminary experiments in our laboratory, we found evidence of a species difference in the direction of the systemic arterial blood pressure changes evoked by lingual nerve stimulation: pressure always rose in the rat, but fell in rabbit and guinea-pig, even though the same anesthetics (urethane and a-chloralose) were used (data not shown). The above results suggest that there are numerous factors besides the type of anesthetic agents used that can affect the systemic arterial blood pressure changes evoked by stimulation of somatic afferents under anesthesia, and that the neural pathways in the brainstem responsible for mediating such responses may differ somewhat among species. As a further point, the above results also indicate that the changes in systemic arterial blood pressure evoked by nociceptive trigeminal stimulation reflect not just sympathetic activation, but presumably sympatho-inhibition and parasympathetic activation (at least in the orofacial region). Unilateral microinjection of the GABA agonist, muscimol, directly into the ipsilateral Vsp abolished the lingual nerve-evoked increase in lip blood flow and change in systemic arterial blood pressure without changing basal systemic arterial blood pressure (Figs. 4C and 5). This result suggests that GABA receptors serve to mediate or modulate the passage of traffic in the parasympathetic and sympathetic reflex pathways running via the Vsp, although the precise location of these receptors remains unknown. Further investigations will be needed to establish the origin of the neurons that release GABA within the Vsp. The trigeminal system appears to participate in the control of autonomic functions such as salivation, lacrimation, vasomotor responses, and other cardiovascular responses [4,16,22,35], and this seems not to depend on actual perception of somatosensory information since these responses can be observed in the unconscious individual, for example during surgical anesthesia, or even in a decerebrate animal preparation [4,18,22]. This is supported by our previous observations that the narcotic drug, morphine, had no effect on the lingual nerve stimulationinduced lip blood flow increase in the anesthetized cat [18] We need always to remember that systemic arterial blood pressure is a dependent variable. That is to say, it is determined by a number of factors (peripheral resistance in multiple beds, cardiac activity), each of which is in turn subject to many influences. Further, a quite marked redistribution of regional blood flow may be concealed behind even a moderate change in systemic arterial blood pressure.
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We do not yet know the nature of the adequate stimulus, or indeed the identity of the sensory receptors, for the lingual nerve-induced increase in lip blood flow and change in systemic arterial blood pressure, or the characteristics of the afferent fibers that mediate it. However, C-polymodal nociceptors have been put forward as strong candidates for the primary afferents partaking in these responses since electrical stimulation at higher intensities, topical application of capsaicin, and radiant heat stimulation to the tongue all cause an increase in ipsilateral lip blood flow [14,15,19], and since the lip blood flow increases evoked by all of these stimuli are significantly attenuated by pretreatment with an autonomic ganglionic blocker, hexamethonium, [14,15,19]. Since the above responses can be evoked from all branches of the trigeminal nerve, the afferents are unlikely to arise from a specific cranial organ (e.g., eye, teeth, nasal mucosa, or tongue), and they are not to a specific division of the trigeminal nerve. This suggests that the parasympathetic reflex vasodilatation that occurs in response to trigeminal stimulation does not arise from proprioceptors.
Acknowledgements This study was partly supported by a Grant-in Aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (No. 12671797; H. Izumi).
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[25] T. Mashimo, P. Zhang, T. Kamibayashi, Y. Inagaki, A. Ohara, A. Yamatodani, I. Yoshiya, Laser Doppler skin blood flow and sympathetic nervous responses to surgical incision during halothane and isoflurane anesthesia, Anesth. Analg. 85 (1997) 291–298. [26] R.B. McCall, L.T. Harris, Sympathetic alterations after midline medullary raphe lesions, Am. J. Physiol. 253 (1987) R91–R100. [27] K. Mizuta, S. Kuchiiwa, T. Saito, H. Mayanagi, K. Karita, H. Izumi, Involvement of trigeminal spinal nucleus in parasympathetic reflex vasodilatation in cat lower lip, Am. J. Physiol. (Reg. Integ. Comp. Physiol.) 282 (2002) R492–R500. [28] S. Nomura, Y. Yasui, M. Takada, A. Konishi, N. Mizuni, Trigeminal nerve branches containing primary afferent fibers to the dorsal division of the principal sensory trigeminal nucleus: a transganglionic horseradish peroxidase study in the cat, Neurosci. Lett. 6 (1986) 221–225. [29] O.S. Possas, O.U. Lopes, S.L. Cravo, Glutamatergic and GABAergic inputs to the RVL mediate cardiovascular adjustments to noxious stimulation, Am. J. Physiol. (Reg. Integ. Comp. Physiol.) 280 (2001) R434–R440. [30] A. Sato, Y. Sato, R.F. Schmidt, The impact of somatosensory input on autonomic functions, Rev. Physiol. Biochem. Pharmacol. 130 (1997) 1–328. [31] A. Sato, R.F. Schmidt, Somatosympathetic reflexes: afferent fibers, central pathways, discharge characteristics, Physiol. Rev. 53 (1973) 916–947. [32] Y. Shigenaga, T. Okamoto, T. Nishimori, S. Suemune, I.D. Nasution, I.C. Chen, K. Tsuru, A. Yoshida, K. Tabuchi, M. Hosoi, Oral and facial representation in the trigeminal principal and rostral spinal nuclei of the cat, J. Comp. Neurol. 244 (1986) 1–18. [33] S.E. Spencer, W.B. Sawyer, H. Wada, K.B. Platt, A.D. Loewy, CNS projections to the pterygopalatine parasympathetic preganglionic neurons in the rat: a retrograde transneuronal viral cell body labeling study, Brain Res. 534 (1990) 149–169. [34] C. Weissman, The metabolic response to stress: an overview and update, Anesthesiology 73 (1990) 308–327. [35] T. Yasui, K. Karita, H. Izumi, M. Tamai, Correlation between vasodilatation and secretion in the lacrimal gland elicited by stimulation of the cornea and facial nerve root of the cat, Invest. Ophthalmol. Vis. Sci. 38 (1997) 2476–2482. [36] Y.H. Yu, W.W. Blessing, Constriction of the ear pinna vascular bed accompanies the trigeminal depressor response in rabbits, Neurosci. Lett. 255 (1998) 172–174.
Research report
Simultaneous measurement of parasympathetic reflex vasodilator and arterial blood pressure responses in the cat Hiroshi Izumi a , *, Kentaro Mizuta a , Satoshi Kuchiiwa b a
Department of Orofacial Functions, Tohoku University Graduate School of Dentistry, Sendai 980 -8575, Japan b Department of Anatomy, Faculty of Medicine, Kagoshima University, Kagoshima 890 -8520, Japan Accepted 22 April 2002
Abstract We measured the changes in lower lip blood flow and systemic arterial blood pressure evoked by lingual nerve or trigeminal spinal nucleus (Vsp) stimulation to gain an insight into the brainstem integration of sympathetic and parasympathetic responses to nociceptive stimulation. We used artificially ventilated, cervically vago-sympathectomized cats deeply anesthetized with a-chloralose and urethane. A lip blood flow increase occurred in an intensity- and frequency-dependent manner following electrical stimulation of Vsp or lingual nerve regardless of whether systemic arterial blood pressure increased or decreased. In contrast, there was no apparent optimal frequency for the changes in systemic arterial blood pressure elicited by electrical stimulation of Vsp or lingual nerve. No relationship was found between the amplitude of the lip blood flow increase and that of the systemic arterial blood pressure change. Microinjection of lidocaine or kainic acid into the Vsp evoked, respectively, reversible and irreversible inhibition of the lip blood flow increase and systemic arterial blood pressure change evoked by lingual nerve stimulation. When microinjected unilaterally directly into the ipsilateral Vsp, the GABA agonist muscimol abolished both lingual nerve-evoked effects (increase in lip blood flow and changes in systemic arterial blood pressure) without changing basal systemic arterial blood pressure, suggesting the presence in the Vsp of GABA receptors serving to modulate both the parasympathetically mediated lip blood flow increase and the sympathetically mediated systemic arterial blood pressure change. Lidocaine microinjection into the salivatory nucleus caused a significant attenuation of the lingual nerve-induced blood flow increase, but had no effect on the lingual nerve-induced systemic arterial blood pressure change. Thus, the neural pathway mediating the lingual nerve-induced lip blood flow increase seems to be simple, requiring a minimum of four neurons: trigeminal afferent–Vsp–parasympathetic preganglionic neurons with cell body located in the inferior salivatory nucleus–otic postganglionic neuron. On the other hand, the pathway underlying the evoked systemic arterial blood pressure changes, presumably mediated via altered sympathetic activity, seems to be more complicated and could be affected by more numerous factors. 2002 Elsevier Science B.V. All rights reserved. Keywords: Autonomic reflex; Nociceptive trigeminal stimulation; Vasodilatation; Arterial blood pressure
1. Introduction The patterns of vasomotor and other cardiovascular responses elicited by noxious stimulation seem to be dependent on the site at which the noxious stimulation is applied. Such noxious stimulation as electrical stimulation of the sciatic nerve, surgical stress, or pinching the body skin evokes a skin blood flow decrease as well as other cardiovascular responses (i.e., increases in systemic arterial *Corresponding author. Tel.: 181-22-717-8321; fax: 181-22-7178322. E-mail address: [email protected] (H. Izumi).
blood pressure and heart rate) in anesthetized animals and humans [24,25,29]. These are thought to be mediated via activation of a sympathetic reflex mechanism serving to maintain hemodynamic and metabolic homeostasis, the so-called somato-sympathetic reflex [5,7,8,34]. However, noxious stimulation of orofacial areas, such as the tongue and nasal mucosa, does not evoke a sympathetically mediated blood flow decrease; instead, there are blood flow increases in the lip, tongue, and salivary gland in anesthetized cats and rats, responses that are mediated via a somato-parasympathetic mechanism [9,11]. In this response, systemic arterial blood pressure is reportedly decreased, probably due to withdrawal of sympathetic
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03196-7
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vasomotor tone to femoral, superior mesenteric, and renal vascular beds [21–23,26]. Although these observations suggest that trigeminally mediated nociceptive stimulation affects both the sympathetic and parasympathetic systems, few actual data are available concerning the way in which the two systems may be affected by this form of stimulation. We examined changes in both lower lip blood flow and systemic arterial blood pressure following nociceptive trigeminal stimulation to gain an insight into the integration of the sympathetic and parasympathetic components of the response in the brainstem of the cat. To this end, we electrically stimulated (using various stimulus conditions) the central cut end of the lingual nerve (‘lingual nerve’) and the trigeminal spinal nucleus (Vsp), which is known to be a brainstem relay for the nociceptive inputs from the orofacial area that travel via the trigeminal nerve [2,27], and we measured the systemic arterial blood pressure changes and lower lip blood flow changes. In our cats, in which the cervical sympathetics and cervical vagi are cut, we took these changes to be indicators of changes in sympathetic nerve activity and parasympathetic nerve activity, respectively, since the systemic arterial blood pressure changes are known to be markedly attenuated by antiadrenergic agents (phentolamine and propranolol) and by an autonomic ganglionic blocking agent (hexamethonium) [4,15,21] and since the lip blood flow changes are abolished by ipsilateral section of the glossopharyngeal nerve root [15]. We also made microinjections within the brain stem to produce nonselective, reversible local anesthesia (lidocaine) or soma-selective, irreversible neurotoxic damage (kainic acid) to examine the contribution to these responses made by the Vsp and the salivatory nucleus, which is known to give rise to preganglionic parasympathetic vasodilator fibers.
was cannulated for the measurement of systemic arterial blood pressure. One cephalic vein was cannulated to allow drug injection. The anesthetized animals were intubated, paralysed by intravenous injection of pancuronium bromide (Mioblock; Organon, Teknika, The Netherlands; 0.4 mg / kg initially, supplemented with 0.2 mg / kg every hour or so after testing the level of anesthesia; see below) and artificially ventilated via the tracheal cannula with a mixture of 50% air–50% O 2 . The ventilator (Model SN480-6; Shinano, Tokyo, Japan) was set to deliver a tidal volume of 10–12 cm 3 / kg at a rate of 20 breaths / min, and the end-tidal concentration of CO 2 was determined by means of an infrared analyzer (Capnomac Ultima; Datex, Helsinki, Finland) as reported previously [10,12,17]. Endtidal CO 2 was kept at 35–40 mmHg. Ringer solution (Otsuka Pharmaceutical, Tokyo, Japan) was continuously infused at a rate of approximately 5 ml / h. Rectal temperature was maintained at 37–38 8C using a heating pad. In all experiments, the cervical vagi and superior cervical sympathetic trunks were cut bilaterally in the neck prior to any stimulation to eliminate reflex effects on the cardiovascular system via the vagus nerve and sympathetic vasoconstrictor effects on the orofacial area, respectively. The criterion for the maintenance of an adequate depth of anesthesia was the absence of any precipitate changes in systemic arterial blood pressure as reflex responses to a fairly minor noxious stimulus (such as pinching the upper lip for approximately 2 s). If the depth of anesthesia was considered inadequate, additional a-chloralose and urethane (i.e., intermittent doses of 5 and 10 mg / kg, i.v., respectively) were administered. Once an adequate depth of anesthesia had been attained, supplementary doses of pancuronium were given approximately every 60 min to maintain immobilisation during periods of stimulation.
2.2. Electrical stimulation of lingual nerve 2. Materials and methods
2.1. Preparation of animals The experimental protocols were reviewed by the Committee on the Ethics of Animal Experiments in Tohoku University School of Medicine, and they were carried out in accordance with both the Guidelines for Animal Experiments issued by the Tohoku University School of Medicine and The Law (No. 105) and Notification (No. 6) issued by the Japanese Government. Twenty-eight cats, unselected as to sex and of 2.0–4.5 kg body wt (approximate age 2–4 years), were initially sedated with ketamine hydrochloride (30 mg / kg, i.m.) and then anesthetized with a mixture of a-chloralose (50 mg / kg, i.v.) and urethane (100 mg / kg, i.v.). These anesthetics were supplemented if and when necessary throughout the experiment (see below). Local anesthesia (2% lidocaine; 1–2 ml) was applied to all skin incisions. A femoral artery
To elicit a parasympathetic reflex vasodilatation in the lower lip, the central cut end of lingual nerve was electrically stimulated (Fig. 1). The routine stimulus parameters were: a 20-s train of 2-ms rectangular pulses at a frequency of 10 Hz and at supramaximal intensity (usually 30 V), as described previously [10,12,17]. A bipolar silver electrode attached to a Nihon Kohden Model SEN-7103 Stimulator (Tokyo, Japan) was used for this stimulation.
2.3. Microinjections of lidocaine, kainic acid, and muscimol To determine whether the vasodilator response elicited by lingual nerve stimulation was mediated via Vsp or the salivatory nucleus, lidocaine (2% / site), kainic acid (10 mM / site), or muscimol (13.2 mM / site) was microinjected into Vsp or salivatory nucleus in a volume of 1.0 ml / site via an injection cannula (0.50 mm o.d.) inserted through an
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evoked vasodilatation or on resting cardiovascular parameters. The magnitude of the lingual nerve-evoked response obtained after microinjection of a given agent was expressed as a percentage of the control response recorded before its administration (mean6S.E.).
2.4. Measurement of lower lip blood flow and of SABP Blood flow changes in the lower lip were monitored (Fig. 1) using a laser-Doppler flowmeter (LDF; model ALF21D; Advance, Tokyo, Japan), as described before [10,12,17]. The probe was placed against the lower lip without exerting any pressure on the tissue. The blood flow changes were assessed by measuring the height of the response on the chart. In the figures, flow levels are expressed in arbitrary units. Systemic arterial blood pressure was recorded from the femoral catheter via a Statham pressure transducer. A tachograph (model AT-610G; Nihon Kohden) triggered by the arterial pulse was used to monitor heart rate.
2.5. Histology
Fig. 1. Schematic representation of the sites used for electrical stimulation, blood flow measurements, and microinjections of lidocaine, kainic acid, or muscimol. Stimulation site: central cut end of the lingual nerve (LN; A). Blood flow measurement site: lower lip (by laser-Doppler flowmeter, LDF; B). Microinjection sites: Vsp (C) and salivatory nucleus (SN, D). The reflex pathways relating to the present study are indicated by solid and broken lines. Abbreviations: OG, otic ganglion; SN, salivatory nucleus; NTS, nucleus tractus solitarius; SSN, V, trigeminal nerve root; Vsp, trigeminal spinal nucleus; Vt, trigeminal spinal tract; VII, facial nerve root; IX, glossopharyngeal nerve root.
implanted guide cannula. Briefly, the animal was mounted in a stereotaxic frame (Narishige, Tokyo, Japan), and after a partial craniotomy, part of the tentorium cerebelli was removed by drilling. As indicated schematically in Fig. 1, a guide cannula (1.00 mm o.d.) was positioned within the Vsp (P, 10.0–11.0; L, 5.0–6.0; H, 5.0–6.0 mm; coordinates of Berman [1]) or the salivatory nucleus (P, 8.0–9.0; L, 2.5–3.5; H, 5.0–6.0 mm) via a small burr-hole in the skull. This was achieved with the aid of a micromanipulator and without removing any part of the brain. The stimulating electrode was interchangeable with the injection cannula. Both were of equal length and each extended 5.0 mm beyond the tip of the guide cannula. Thus, microinjection and electrical stimulation were carried out at the same sites. Saline (1.0 ml) was used for control injections. It never produced any significant effect on the lingual nerve-
Animals were given an overdose of pentobarbital (60 mg / kg) by i.v. infusion and perfused through the ascending aorta with 1.0–2.0 l of saline (0.9%) followed immediately by 2 l of 10% formalin. Then, the brain stem and upper cervical spinal cord were removed and stored for 1–4 days in buffered 30% sucrose. After storage, sections 50 mm thick were cut on a freezing microtome and collected in 0.1 M phosphate buffer (pH 7.4). Sections were mounted on gelatin-coated slides and stained with thionin. Photomicrographs of representative coronal sections showing sites used for electrical stimulation or microinjection of lidocaine, kainic acid, or muscimol into the Vsp or the SN can be seen in Fig. 6. Visualization of the spread of the HRP injected into the salivatory nucleus was achieved using the peroxidase–DAB reaction (Fig. 7).
2.6. Statistical analysis All numerical data are given as mean6S.E. The significance of changes in the test responses was assessed using an analysis of variance (ANOVA) followed by a contrast test. Differences were considered significant at the level P,0.05. Data were analyzed using a Macintosh Computer with StatView 5.0 and Super ANOVA.
3. Results The resting mean arterial blood pressure of the cats used in this study lay within the range 68.367.5 to 121.7610.5 mmHg, the average value for the group being 86.168.1 mmHg.
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3.1. Electrical stimulation of lingual nerve and Vsp on lip blood flow and arterial blood pressure Fig. 2 shows the effects of electrical stimulation of the central cut end of lingual nerve and of Vsp on blood flow in the ipsilateral lower lip when the stimulation was delivered at various intensities (lingual nerve, 1–50 V; Vsp, 2–200 mA; Fig. 2A) and at various frequencies (0.1–100 Hz; Fig. 2B). Electrical stimulation of lingual nerve or Vsp at ,1 V or ,10 mA had no significant effects, whereas increasing the stimulus intensity from 2 up to 10 V or from 10 up to 100 mA produced successively bigger vasodilator responses, the response being saturated at 10 V or 100 mA, respectively. Electrical stimulation of lingual nerve or Vsp
needed to be at .0.2 Hz to have significant effects on lip blood flow, and the response was saturated at 10 Hz in each case. The optimal frequency for vasodilatation in response to either lingual nerve or Vsp stimulation was 10 Hz (Fig. 2B). The effect of electrical stimulation of either lingual nerve (Fig. 3A,B) or Vsp (Fig. 3C,D) on systemic arterial blood pressure varied from animal to animal, both qualitatively and quantitatively. No optimal frequency could be identified when either lingual nerve or Vsp was electrically stimulated at various frequencies from 1 to 100 Hz; in fact, no clear relationships emerged between systemic arterial blood pressure changes and the frequency or intensity of lingual nerve or Vsp stimulation.
3.2. Effects of microinjection of lidocaine, kainic acid, and muscimol
Fig. 2. Stimulus intensity–response (A) and frequency–response (B) relationships for changes in lower lip blood flow evoked by electrical stimulation of the central cut end of the lingual nerve (LN; open circles) and of the trigeminal spinal nucleus (Vsp; filled circles). Stimulation was at various intensities (1–50 V for LN and 2–200 mA for Vsp) and various frequencies (0.1–100 Hz). Intensity–response curves were generated using stimulus trains at 10 Hz. Frequency–response curves were generated using stimulus trains at 30 V for LN and 100 mA for Vsp. Each value is given as mean6S.E. Number of animals used was 8 for LN, 10 for Vsp.
Local anesthesia of the ipsilateral Vsp by microinjection of lidocaine was produced to examine the contribution made by this area to the blood flow increases and systemic arterial blood pressure changes evoked by lingual nerve stimulation (Fig. 4). Fig. 4A shows a typical recording of the time course (before and 10–60 min after microinjection) of the effects of such lidocaine microinjection (mean data are shown in Fig. 5). Control lingual nerve stimulation elicited a blood flow increase in the ipsilateral lower lip and a change (decrease or increase) in systemic arterial blood pressure. Both changes had disappeared when the stimulation was repeated 10 min after microinjection of lidocaine into the Vsp, but the response recovered in a time-dependent manner, indicating that the inhibitory effects of lidocaine were reversible. A typical microinjection site is shown in Fig. 6. Kainic acid (10 mM / site, 1 ml) was microinjected into Vsp at sites at which microinjection of lidocaine had previously evoked a marked reduction in the vasodilator response evoked by stimulation of the ipsilateral lingual nerve (Fig. 4B). This was done to examine the contribution made by cell bodies in this area to the response to lingual nerve stimulation. Kainic acid was given 20–30 min after the lingual nerve-evoked response had completely recovered from the effects of lidocaine. The mean data (Fig. 5) show that both the LBF increase and the systemic arterial blood pressure change were significantly reduced at t510 and t560 min (t50 is the time at which blood flow returned to the basal level following the increase (duration 4.962.3 min; n56 animals) induced by microinjection of kainic acid into Vsp). Thus, these effects of kainic acid were irreversible (at least within the time-frame of the experiment). Fig. 4C shows typical recordings of effects of microinjection of muscimol (13.2 mM / site, 1 ml) into Vsp on the lip blood flow increase and systemic arterial blood pressure change evoked by electrical stimulation of lingual nerve. This was done to examine the involvement of GABA receptors in Vsp on these responses. The mean data (Fig. 5) show that muscimol microinjection elicited time-
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Fig. 3. Stimulus intensity–response (A,C) and frequency–response (B,D) relationships for changes in systemic arterial blood pressure (SABP) evoked by electrical stimulation of the central cut end of the lingual nerve (LN, A,B) and of the trigeminal spinal nucleus (Vsp, C,D). Stimulation was at various intensities (1–50 V for LN and 2–200 mA for Vsp) and various frequencies (0.1–100 Hz). Intensity–response curves were generated using stimulus trains at 10 Hz. Frequency–response curves were generated using stimulus trains at 30 V for LN and 100 mA for Vsp. Each value is given as mean6S.E. Number of animals used was 10 for LN, 11 for Vsp.
dependent reductions in both responses, and that there was no recovery within 60 min after the microinjection. Fig. 4D shows typical recordings of the effects of microinjection of lidocaine into the salivatory nucleus on the lip blood flow increase and systemic arterial blood pressure change evoked by electrical stimulation of lingual nerve. A typical microinjection site is shown in Fig. 6B. The mean data (Fig. 5) show that although lidocaine microinjection elicited a statistically significant attenuation of the lingual nerve-induced blood flow increase, it had no effect on the lingual nerve-induced systemic arterial blood pressure change. In the present study, to examine the possibility that the changes in lip blood flow and systemic arterial blood pressure evoked by lingual nerve stimulation might be mediated via the Vsp and / or salivatory nucleus in the medulla oblongata, we inactivated some or all neural function in these regions using unilateral microinjections of lidocaine, kainic acid, or muscimol. A histological marker (HRP; 1 ml, 10%) was injected into the salivatory nucleus to indicate how far the injections might have spread. As shown in Fig. 7, the spread of HRP was approximately 1–1.5 mm in radius (this was also the spread in the rostral direction and in the caudal direction). This spread of HRP suggests that the effects
produced by microinjecting lidocaine, kainic acid, or muscimol into the Vsp or salivatory nucleus were not a consequence of spread of these drugs to the other site (i.e., Vsp injection did not spread to salivatory nucleus) since (i) the salivatory nucleus microinjection site is nearly 1.5 mm rostral and 2.5 mm medial to the Vsp site, (ii) lidocaine microinjection into the salivatory nucleus did not attenuate the lingual nerve-mediated increase in systemic arterial blood pressure, which is mediated via Vsp, and (iii) microinjection of lidocaine into the salivatory nucleus reduced the palate blood flow increase only on the ipsilateral side, not on the contralateral side, when lingual nerve stimulation was used to evoke a bilateral increase in palate blood flow (data not shown).
4. Discussion It is well known that autonomic responses elicited by stimulation of somatic and visceral afferent nerves are mediated by reflex mechanisms. Numerous reports have described somato-sympathetic responses, such as arterial blood pressure increases, and somato-parasympathetic responses, such as salivary secretion [9,11,20,30,31]. How-
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Fig. 4. Typical examples of effects produced by microinjection of (A) lidocaine (2%, 1 ml / site), (B) kainic acid (10 mM, 1 ml / site), and (C) muscimol (13.2 mM, 1 ml / site) into the ipsilateral trigeminal spinal nucleus (Vsp), and of (D) lidocaine (2%, 1 ml / site) into the ipsilateral salivatory nucleus (SN) on the blood flow increase in lower lip (LBF) (in arbitrary units (au)) and changes in systemic arterial blood pressure (SABP, mmHg) elicited by electrical stimulation of the central cut end of the lingual nerve (LN). Electrical stimulation of the LN was at 30 V, 10 Hz, 2-ms pulse duration for 20 s. Kainic acid and lidocaine were microinjected at the same site in Vsp (see Section 3 for further details). Electrical stimulation of LN was carried out before and at 10–60 min after each microinjection (30 V, 10 Hz, 2-ms pulse duration for 20 s). In the kainic acid experiment, electrical stimulation of LN was not resumed until after blood flow had returned to the basal level following the increase induced by microinjection of kainic acid into Vsp (i.e., the times ‘10 min’ to ‘60 min’ refer to the interval after restoration of blood flow to the basal level). In the particular experiment illustrated in (B), the duration of the kainic acid-induced blood flow increase was 7 min.
ever, few previous experimental studies have examined both types of somato-autonomic response simultaneously. The possibility that the increases in lip blood flow described here were the passive result of evoked increases in blood pressure seems remote: since such blood flow increases (1) occur only on the ipsilateral side of the lip, not on the contralateral side [15], and (2) are abolished by
ipsilateral section of the glossopharyngeal nerve root and by the autonomic ganglionic blocker, hexamethonium [15]. Microinjections of lidocaine or kainic acid into the Vsp attenuated (reversibly and irreversibly, respectively) not only the trigeminally (lingual nerve stimulation) elicited lip blood flow increase, but also the accompanying changes in systemic arterial blood pressure (Fig. 4A,B). These results
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Fig. 5. Effects produced by microinjection (1.0 ml / site) of lidocaine (2% / site), kainic acid (10 mM / site), and muscimol (13.2 mM / site) into the trigeminal spinal nucleus (Vsp) on the blood flow increase in lower lip (LBF) and change in systemic arterial blood pressure (SABP, mmHg) elicited by electrical stimulation of the central cut end of the lingual nerve (LN) (ipsilateral to the microinjection site). Electrical stimulation of the LN was at 30 V, 10 Hz, 2-ms pulse duration for 20 s. In the kainic acid experiment, electrical stimulation of LN was not resumed until after blood flow had returned to the basal level following the increase induced by microinjection of kainic acid into Vsp (i.e., the times ‘10 min’ to ‘60 min’ refer to the interval after restoration of blood flow to the basal level). Open and hatched bars indicate, respectively, LBF and SABP responses. The ‘control’ alterations in SABP evoked by stimulation of the lingual nerve or Vsp are expressed as 100% regardless of whether the alteration was a decrease or increase in SABP. The responses illustrated were obtained before, and 10 and 60 min after treatment with lidocaine, kainic acid, or muscimol. Each value is expressed as a percentage of the pretreatment response and is given as mean6S.E. Statistical significance of difference from control was assessed by means of ANOVA followed by a contrast test (*P,0.05, **P,0.01, ***P,0.001). Number of animals used was five.
Fig. 6. Photomicrographs of representative coronal sections through the medulla oblongata of the cat showing sites at which microinjections of lidocaine or kainic acid were delivered to the trigeminal spinal nucleus (Vsp; A) and salivatory nucleus (SN; B). Thionin stain; scale bar represents 1.0 mm. Abbreviations: FN, facial nucleus; IO, inferior olivary nucleus; SN, salivatory nucleus; PH, nucleus praepositus hypoglossi; RB, restiform body; VIN, inferior vestibular nucleus; VLD, lateral vestibular nucleus; Vsp, trigeminal spinal nucleus; Vt, trigeminal spinal tract.
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Fig. 7. Diagram derived from a control section through the salivatory nucleus injection site region, with the injection site (arrow) marked by the peroxidase–DAB reaction. Scale bar represents 1.0 mm. Abbreviations: FN, facial nucleus; FTG, gigantocellar tegmental field; PH, nucleus praepositus hypoglossi; RB, restiform body; RFN, retrofacial nucleus; SA, Stria acustica; SN, salivatory nucleus; V4, fourth ventricle; VIN, inferior vestibular nucleus; VLD, lateral vestibular nucleus; Vsp, trigeminal spinal nucleus; Vt, trigeminal spinal tract.
suggest that both the increase in lip blood flow and the changes in systemic arterial blood pressure evoked by lingual nerve stimulation are mediated by neurons situated within the Vsp. On the other hand, microinjection of either lidocaine (Figs. 4D and 5) or kainic acid (data not shown) into the salivatory nucleus significantly reduced only the lip blood flow increase; it had no effect on the magnitude of the evoked changes in systemic arterial blood pressure regardless of whether systemic arterial blood pressure increased or decreased. These results suggest (i) that the salivatory nucleus represents a critical bulbar relay site for the lingual nerve-induced lip blood flow increase, but not for the lingual nerve-evoked changes in systemic arterial blood pressure, (ii) that neural pathways mediating these two responses diverge after traffic derived from nociceptive trigeminal information leaves the Vsp, and (iii) that the lingual nerve-induced changes in systemic arterial blood pressure are not secondary to changes in blood flow in the orofacial area, even though lingual nerve stimulation evokes blood flow increases in nearly all orofacial regions [11]. It has previously been reported that in the anesthetized rabbit, electrical or chemical stimulation of the trigeminal nerve or the spinal trigeminal nucleus, or pinching of the
lip with forceps caused a profound fall in systemic arterial blood pressure, and Blessing and co-workers suggested that the trigeminal depressor response depends on a net inhibition of spinal sympathetic preganglionic neurons via the brainstem [2–4,36]. Kumada et al. [21–23] showed that the hypotensive effect elicited by electrical stimulation of the brain (the spinal trigeminal complex) or a peripheral nerve (branches of the trigeminal nerve) was optimal at frequencies between 5 and 10 Hz. This band of frequencies is similar to the one we found to be optimal for the parasympathetic vasodilatation elicited by electrical stimulation of both Vsp and lingual nerve (Fig. 2B). However, no optimal frequency could be established for the changes in systemic arterial blood pressure elicited by electrical stimulation of the Vsp or lingual nerve (Fig. 3). Similarly, no intensity dependence of the changes in systemic arterial blood pressure was observed on electrical stimulation of either Vsp or lingual nerve (Fig. 3A,C). In their lack of dependence on stimulus frequency, the systemic arterial blood pressure changes we elicited by electrical stimulation of the lingual nerve and Vsp differ from those reported long ago by Gruber [7] who reported that systemic arterial blood pressure decreased when spinal sensory nerves such as the saphenous, peroneal, ulnar, radial, median or popliteal nerves were electrically stimulated at low frequency (4 Hz), but increased when the same nerves were stimulated at high frequency (20 Hz). The neural pathways mediating the lip blood flow increase induced by lingual nerve stimulation would seem to be quite simple, requiring a minimum of only four neurons: trigeminal afferent–Vsp–parasympathetic preganglionic neuron with cell body located in the inferior salivatory nucleus–otic postganglionic neuron [27]. This notion is supported by anatomical and physiological studies showing that (i) trigeminal afferents running in lingual nerve pass to Vsp [28,32], (ii) the parasympathetic preganglionic neurons in salivatory nucleus receive projections from Vsp (30), (iii) cutting the glossopharyngeal nerve root abolished the nociceptive trigeminal stimulation-induced blood flow increase in the lower lip [33], and (iv) the lip blood flow increase evoked by electrical stimulation of the glossopharyngeal nerve root was abolished by prior treatment with the autonomic ganglionic blocker hexamethonium [13,15]. On the other hand, neural mediation of the lingual nerve-induced systemic arterial blood pressure changes, presumably involving alterations in sympathetic activity, would seem to be more complicated and probably subject to influence by more numerous factors since consistent systemic arterial blood pressure changes were not observed in our cats in response to nociceptive trigeminal stimulation. Blessing and co-workers [3,4,36] have reported that the systemic arterial blood pressure fall evoked by electrical stimulation of Vsp / spinal tract of the trigeminal nerve in the rabbit occurred as a result of withdrawal of sympathetic vasomotor tone to femoral, mesenteric, renal,
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and skeletal muscle resistance vessels. A systemic arterial blood pressure fall has also been reported to occur on electrical stimulation of the lingual nerve, the inferior alveolar nerve, and the supraorbital nerve in rabbit and cat [6,21]. However, under our experimental conditions consistent systemic arterial blood pressure changes were not evoked by electrical stimulation of either lingual nerve or Vsp in the cat despite the same anesthetic (urethane and a-chloralose) being used in each animal. In some preliminary experiments in our laboratory, we found evidence of a species difference in the direction of the systemic arterial blood pressure changes evoked by lingual nerve stimulation: pressure always rose in the rat, but fell in rabbit and guinea-pig, even though the same anesthetics (urethane and a-chloralose) were used (data not shown). The above results suggest that there are numerous factors besides the type of anesthetic agents used that can affect the systemic arterial blood pressure changes evoked by stimulation of somatic afferents under anesthesia, and that the neural pathways in the brainstem responsible for mediating such responses may differ somewhat among species. As a further point, the above results also indicate that the changes in systemic arterial blood pressure evoked by nociceptive trigeminal stimulation reflect not just sympathetic activation, but presumably sympatho-inhibition and parasympathetic activation (at least in the orofacial region). Unilateral microinjection of the GABA agonist, muscimol, directly into the ipsilateral Vsp abolished the lingual nerve-evoked increase in lip blood flow and change in systemic arterial blood pressure without changing basal systemic arterial blood pressure (Figs. 4C and 5). This result suggests that GABA receptors serve to mediate or modulate the passage of traffic in the parasympathetic and sympathetic reflex pathways running via the Vsp, although the precise location of these receptors remains unknown. Further investigations will be needed to establish the origin of the neurons that release GABA within the Vsp. The trigeminal system appears to participate in the control of autonomic functions such as salivation, lacrimation, vasomotor responses, and other cardiovascular responses [4,16,22,35], and this seems not to depend on actual perception of somatosensory information since these responses can be observed in the unconscious individual, for example during surgical anesthesia, or even in a decerebrate animal preparation [4,18,22]. This is supported by our previous observations that the narcotic drug, morphine, had no effect on the lingual nerve stimulationinduced lip blood flow increase in the anesthetized cat [18] We need always to remember that systemic arterial blood pressure is a dependent variable. That is to say, it is determined by a number of factors (peripheral resistance in multiple beds, cardiac activity), each of which is in turn subject to many influences. Further, a quite marked redistribution of regional blood flow may be concealed behind even a moderate change in systemic arterial blood pressure.
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We do not yet know the nature of the adequate stimulus, or indeed the identity of the sensory receptors, for the lingual nerve-induced increase in lip blood flow and change in systemic arterial blood pressure, or the characteristics of the afferent fibers that mediate it. However, C-polymodal nociceptors have been put forward as strong candidates for the primary afferents partaking in these responses since electrical stimulation at higher intensities, topical application of capsaicin, and radiant heat stimulation to the tongue all cause an increase in ipsilateral lip blood flow [14,15,19], and since the lip blood flow increases evoked by all of these stimuli are significantly attenuated by pretreatment with an autonomic ganglionic blocker, hexamethonium, [14,15,19]. Since the above responses can be evoked from all branches of the trigeminal nerve, the afferents are unlikely to arise from a specific cranial organ (e.g., eye, teeth, nasal mucosa, or tongue), and they are not to a specific division of the trigeminal nerve. This suggests that the parasympathetic reflex vasodilatation that occurs in response to trigeminal stimulation does not arise from proprioceptors.
Acknowledgements This study was partly supported by a Grant-in Aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (No. 12671797; H. Izumi).
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