$3.00+ 0.00 ooo3-9%9/91
1991 Archs oral Bid. Vol. 36,No. 8, pp. 575-581, Printedin GreatBritain.All rightsreserved
Copyright 0
1991 Pergamon Press pk
INVOLVEMENT OF AFFERENT NERVES IN PULPAL BLOOD-FLOW REACTIONS IN RESPONSE CLINICAL AND EXPERIMENTAL PROCEDURES IN THE CAT
TO
L. OLGART,‘* L. EDWALL* and B. GAZELIUS’ ‘Department of Pharmacology and ‘Department of Endodontics, Karolinska Institute, Stockholm, Sweden (Accepted 21 February 1991) Summary-A unilateral resection of the mandibular nerve (n = 20) was made 10-14 days before investigation of the contribution of afferent nerves in vasodilator reactions in the dental pulp. Lower canine teeth were subjected to various stimuli and pulp blood-flow responses monitored by laser Doppler flowmetry. An absence of response to bipolar electrical (5 impulses, 50 PA, 5 ms, 2 Hz) stimulation on the tooth surface was used to demonstrate a successful chronic nerve lesion. Local application of capsaicin (10e4 M) in a deep dentinal cavity induced a long-lasting increase in pulpal blood flow in control teeth only. Bradykinin (10e3 M) induced significantly larger responses in control than in denervated teeth (58.3 + 9.8% and 24.5 + 4.9%, respectively, p < 0.005, n = 8); in addition, the onset was slower and the duration of the response significantly (60%) shorter than in control teeth. Intermittent grinding of surface dentine instantly increased Row in control teeth by 53.0 f 12.5% (n = 12) whereas in denervated teeth the response was delayed and significantly (70%) smaller. Deeper preparation produced responses of similar magnitude in control and denervated teeth (69 and 50%, respectively) but the onset was delayed in denervated teeth. Low-intensity ultrasonic stimulation caused vasodilation in intact teeth (38% increase) but had no effect in denervated teeth. This effect was abolished after local anaesthetic (mepivacaine) injection. Sympathectomy (n = 3) did not influence stimulation-induced blood-flow responses in the dental pulp. The results show that afferent nerves make an important contribution to haemodynamic reactions in the pulp in response to experimental and clinical procedures directed at the tooth. Key words: dental pulp, cat, blood flow, denervation, afferent nerves, tooth stimulation.
INTRODUCTION
The pulp of cat and human teeth is richly innervated by both myelinated and unmyelinated afferent nerves, the unmyelinated type (C-fibres) containing neuropeptides such as substance P, neurokinin A and calcitonin-gene related peptide (Olgart et al., 1977a; Gazelius et al., 1987; Akai and Wakisaka, 1990). High-intensity electrical stimulation activates the C-fibres and causes release of substance P and vasodilation in the cat dental pulp (Olgart et al., 1977b; Gazelius and Olga& 1980). Such effects have not been reported after activation of the A-fibre population. It is well known that the intradental A-units respond to various types of stimuli affecting dentine such as drilling, probing and air drying (Narhi, Hirvonen and Hakumliki, 1982a; Nlrhi, Jyviisjlrvi and Huopaniemi, 1984; Hirvonen and Narhi, 1986; Narhi, 1985) while the intradental C-units fail to respond to such stimulation (NHrhi et al., 1982b, 1984; Nlrhi and Haegerstam, 1983; Nlrhi, 1985). However, intradental C-fibre units have been shown to share many properties with polymodal C-nociceptors in the skin (NSirhi, 1989), and it has been shown in dermal tissues that noxious external stimulation induces the well-known flare reaction mediated by the vasoactive neuropeptides released from the afferent C-fibre nerve *To whom all correspondence
should be addressed.
endings (see Lembeck et al., 1980). Thus, it would be of interest to know if clinical dental stimuli could influence blood flow in the pulp and to find out to what extent afferent nerves are involved. Interestingly, preliminary experiments in our laboratory had shown that in cats with unilaterally denervated jaws, deep cavity preparation resulted in hyperaemia in the pulp of innervated teeth but not of the contralateral denervated teeth. We have therefore now investigated the effect of some clinical procedures on pulpal blood flow in denervated and innervated teeth in the cat and compared these results with those obtained with experimental procedures that have well-known, direct and/or indirect, vascular actions. MATERIALS AND METHODS
Operative procedure
Twenty-three young (1-yr-old) male cats weighing 3-4 kg were used. They were anaesthetized with sodium pentobarbital (initial dose, 30 mg/kg intravenously and then as needed). The inferior alveolar nerve was sectioned unilaterally in 20 cats. Access to the nerve was through an incision over the inferior border of one side of the mandible. The periosteum was reflected from the medial aspect of the bone, exposing the nerve as it entered the mandibular canal. 575
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After careful separation from the inferior alveolar artery and vein, 3-4mm of the nerve were resected and the wound sutured. In three separate experiments the sympathetic superior cervical ganglion was resected unilaterally. The ganglion and an adjacent 3-4 mm of the sympathetic nerve were removed and the wound sutured after careful separation from vessels and the vagus nerve. After lo-14 days, which allowed the transected nerves to degenerate, the cats were again anaesthetized. The trachea was intubated and blood pressure in one femoral artery was recorded with a Statham 23 AC transducer. Rectal temperature was kept constant at about 38°C with a thermostat-controlled heating pad. The head and jaws were immobilized by a rigid steel rod attached bilaterally to the molar and premolar teeth by dental acrylic. The experiments were ended by administering a lethal dose of anaesthetic intravenously. Blood-jlow recording
Two laser Doppler flowmeters (Periflux PF2) were used to measure simultaneously pulpal blood flow in the canine teeth of one jaw (Edwall et al., 1987). PF 103 probes were positioned in contact orduring physical stimulation-at a distance of about 0.6 mm from the intact and cleaned tooth surface at the lingual aspect, midway between the gingiva and the tip of the tooth. The flowmeter’s time constant was set at 3 s and band width at 4 kHz. Electrical stimulation and infusion of substance P
To test if degeneration of the pulpal nerves had occurred after sectioning the inferior alveolar nerve, bipolar electrical stimulations (5 imp, 50 PA, 5 ms. 2 Hz) and intravenous infusions of substance P @Ofmol/kg) were made. Electrodes were brought into contact with the intact and dried enamel surface using electrode paste. Substance P was given in the femoral vein as a bolus (0.4ml) injection after the cannula had been flushed with 0.9% NaCl containing 0.2% bovine serum albumin. Lack of increase of pulpal blood flow in response to electrical stimulation was used to indicate that fine-calibre, high-threshold afferent nerves (C-fibres) had degenerated in the pulp. Conversely, an increased blood flow indicated an intact pulpal afferent nerve supply. According to a previous study (Olgart and Gazelius, 1988) vasodilator responses to substance P given intravenously can be expected to be 200-300% larger in inferior alveolar-denervated pulps during the degenerative phase than in innervated pulps. Chemical stimulation
After unilateral denervation, dentinal cavities were prepared bilaterally in the mandibular canine teeth in a group of eight cats. Each pair of cavities was prepared as identically as possible on the buccal aspect of the tooth and located midway between the gingiva and the tip of the crown. A round diamond bur (dia 1.5 mm) was used to penetrate the enamel. Dentine was removed with an end-cutting bur (dia 1.Omm) rotated in a holder held between the fingers. Preparation was made under visual control through a binocular stereo microscope (Zeiss OPM 1) into
the innermost layer of dentine and accompanied by flushing with isotonic saline. At the end of the experiments, adrenaline (5.5 x 10m3M) was applied to each pair of cavities to observe the latency and magnitude of blood-flow reduction. Finally, the floor of each cavity was penetrated with a fine probe in order to estimate the remaining dentine thickness. Capsaicin, bradykinin, aconitine and sodium chloride were applied as local chemical stimuli in the dentinal cavities. Care was taken to apply each substance (2 ~1) as identically as possible. Capsaicin (Fluka Chemie AG, Switzerland) was dissolved in 20% Tween 80 and 10% ethyl alcohol and diluted in isotonic saline to a final concentration of 10m4M. Bradykinin (Sigma, St Louis, MO) was dissolved in isotonic saline and applied in a concentration of 10e3 M. Aconitine (Sigma, St Louis, MO) was dissolved in isotonic saline and applied in a concentration of 10e2 M. Hypertonic sodium chloride was applied as 0.76 and 1.54 M solutions. Physical stimulation
A round diamond instrument (dia 1.5 mm) was operated at slow speed to grind, repetitively, spherical cavities with increasing depth through the enamel and into the dentine in a group of 12 cats. Pressure and speed (6000-8000 rev/min) were similar to those used clinically. The grinding time was, however, considerably shorter-three grindings, each of l-s duration with l-s intervals between-in order to minimize the heat production. The timing of the grinding procedure was signalled by repetitive sounds of 1 s duration. The first grinding-into the most peripheral dentine-was accompanied by flushing with isotonic saline. Grinding was repeated every 5 mitt, or with longer time intervals if either of the teeth showed a blood-flow reaction. As before, care was taken to prepare the teeth identically. In this way, each pair of teeth were stimulated by grinding in a series of three times 1 s procedures allowing for recording of blood flow for at least 5 min between the stimulations. Physical stimulation was also applied by an ultrasonic dental instrument (Piezon, Electromedical Systems AS, Le Sentier, Switzerland). The tip of the instrument was held perpendicularly in contact with the incisal third of the canine tooth for 1 s. A rest of 5 min was allowed before stimulation was repeated. As above, care was taken to apply the vibration (30 kHz) identically on both sides. Two different levels of vibration amplitude were used. On the instrument’s scale of 10 units, a setting of 3-5 was regarded as low intensity and 8 as high. The third type of physical stimulation was percussion, which was applied in a series of three, one every second, with at least a 5min interval for recording in between. A 30 g steel instrument was allowed to fall from a height of 9 cm and hit the tip of the tooth crown, perpendicularly to its long axis. Dental nerve blockade by local injection of mepivacaine chloride 30 mg/ml (Astra, Sweden) was made either submucosally or subperiosteally in the apical region-to obtain periodontal nerve blockade-or, intraosseously-to obtain pulpal nerve blockade. The response to electrical tooth stimulation was checked before each physical stimulus was applied.
Pulpal blood flow
In two experiments the inferior alveolar nerve was sectioned unilaterally on the day of the experiment. This procedure did not change the pattern of response to the stimuli as compared with those obtained on the control side. All data were stored on a video tape recorder and on an on-line computer and analysed by commercial software (Perisoft, Perimed KB). The following measures were recorded and expressed as follows. Laser Doppler flowmeter output values from the dental pulps were expressed in perfusion units (PU). The use of this unit (10 mV) makes comparison possible with results obtained using recently available automatic devices. As the flowmeter probes monitored pulpal blood flow through the intact enamel and dentine (Edwall et al., 1987) baseline PU values could differ between teeth (see Fig. l)-or in the same tooth, when the probe was repositioned-due to different laser beam penetration through the mineralized tissues. For this reason and as the flowmeter monitors relative changes in the flux of blood cells, i.e. the number of cells times their velocity, effects obtained were calculated as percentages of baseline values. The duration of a response was expressed in minutes. Mean arterial blood pressure and heart rate were monitored and did not change during the experimental procedures. The flowmetric results were expressed as mean f SEM and evaluated with the paired, and when necessary unpaired, Student’s t-test; p-values less than 0.05 were considered to be significant. RESULTS
In the unilaterally sympathectomized cats the magnitude of pulpal blood-flow changes in response to electrical and ultrasonic stimulation as well as to percussion and cavity preparation was similar on both sides. Therefore, the term “denervated teeth” discussed below refers to chronically inferior alveolar nerve-sectioned cats. Electrical
stimulation
and substance P
In all 20 cats with resected inferior alveolar nerves, electrical stimulation of the lower canine teeth on the control side resulted, within lo-20 s, in a blood-flow increase (111 f 15%) lasting 8-15 min, whereas there were no responses on the denervated side [Fig. l(A)]. In contrast, infusion of substance P (80 fmol/kg) induced blood-flow responses in the denervated pulps (81 k 15%) exceeding those obtained in control pulps (28 + 2%, p < 0.01) as has been reported previously (Olgart and Gazelius, 1988) [Fig. l(A)].
517 20 16 A
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Fig. I. Blood-flow changes (laser Doppler perfusion units) in innervated (c) and denervated (d) lower canine teeth of the cat in response to: (A) bipolar (50PA, 5 ms; 5 imp) electrical stimulation on intact tooth surface, (B) capsaicin (tom4 M) and (C) bradykinin (lo-) M) applied in a deep dentinal cavity. (A) are local stimulations, (A) intravenous bolus injections of substance P (80fmol/kg). Steep spikes [(B) and (C)] are artefacts due to application of stimuli or cavity washing. appeared later and the duration of the response was shorter (4.3 + 1.5 min) than in control teeth (9.6 k 1.2 min, p < 0.05, n = 8) (Fig. 2).
Application of hypertonic sodium chloride 0.76 and 1.54 M gave increased pulpal blood flow but with variable results and without significant differences between sides. Aconitine was without effect on pulpal blood flow. Physical stimulation
Cavity preparation in the outer half of dentine in control teeth always caused an instant increase in flow (53.0 f 12.5%, n = 12), whereas the presence of a response in denervated teeth varied and, when observed, was delayed and significantly smaller (15.6 f 8.9%, p < 0.05, n = 10) with a shorter duration
n=6
16
Chemical stimulation
Capsaicin induced a powerful and long-lasting (> 30 min) vasodilation within IO-30 s in innervated teeth, whereas in the contralateral denervated teeth no such responses were observed apart from small transient reductions in flow values [Fig. l(B)]. Application of bradykinin induced increased flow in both denervated and control teeth within lo-30 s. However, the responses in innervated teeth (58.2 f 9.8%) were significantly larger than those in contralateral denervated teeth (24.5 f 4.8%, p < 0.005, n = 8) (Fig. 2). Typically, as can be seen in Fig. l(C), the maximum of the response in denervated teeth
60 ti B z Y
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Fig. 2. Vasodilator effects in control (0) and denervated teeth (m) of the cat in response to bradykinin application (IO-’ M) in a deep dentinal cavity. Pulpal blood flow was measured with laser Doppler flowmetry (LDF); **I, < 0.01.
L. OLGARTet al.
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Fig. 3. Effects of grinding of dentine on pulpal blood flow measured by laser Doppler flowmetry (LDF) in innervated (0) and denervated (W) lower canine teeth in the cat. (1) Grinding 3 x 1 s in outer half of dentine and (2) in inner half of dentine. Figures within parentheses are number of procedures. *p < 0.05.
Fig. 5. The effect of ultrasonic stimulation (1 s), measured by laser Doppler flowmetry (LDF) with high and low amplitudes on enamel surface of lower canine teeth on innervated (0) and denervated (m) side. The maximal increase (Basal-Peak) and duration are shown. Figures within parentheses are number of procedures. ***p < 0.001.
(8.7 + 2.1 and 3.4 & 2.1 min, respectively). In the first trial, cutting through the enamel, blood-flow responses were obtained only in innervated teeth. In deeper preparations there was only a slight difference in the magnitude and duration of responses between sides (Fig. 3). However, the responses in denervated teeth were much delayed and the maximum effect was reached after several minutes [Fig. 4(A)]. As can also be seen, responses to substance P after such procedures had a similar appearance on both sides but showed a higher percentage increase in denervated teeth. Ultrasonic stimulation at the highest amplitudes caused increased blood flow in control (67.4 f 11.5%) as well as in denervated teeth (45.4 k 10.6%) with similar durations (Fig. 5). As for grinding in deep cavities, the first phase of the response in denervated teeth was delayed as compared with that in controls
[Fig. 4(B)]. However, at lower amplitudes, there was a clear-cut difference in the overall increase between control and denervated sides (38.2 f 5.6%, n = 34 and 1.4 k 0.7%, n = 31, respectively, p < 0.005) (Fig. 5). In fact, in the group of denervated teeth, only one single procedure resulted in a small, short-lasting response. To elucidate whether pulpal and/or periodontal nerves were responsible for the ultrasonically induced vasodilation, local anaesthetics were used to block the effects. First, buccal and lingual infiltrations, unable to block intradental vasodilation induced by electrical stimulation of the tooth crown, were used. Pulpal vasodilation to subsequent low-amplitude ultrasonic stimulation was then almost abolished (1.3 f 0.9% increase) as compared with initial control responses (58.7 + 17.3%, p < 0.01, n = 8). Responses to ultrasonic stimulation with high amplitudes were only slightly changed (Fig. 6). After additional apical injections resulting in abolished responses to electrical tooth stimulation, high-amplitude ultrasonic stimulations produced only small vasodilatory responses in some teeth; 43.0 f 10.1% increase before and 6.0 f 3.1% after injection, p < 0.001, n = 11) (Fig. 6).
80
I?(8)
(6)
(11)
I
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Fig. 4. Blood flow changes (laser Doppler perfusion units) in innervated (c) and denervated (d) lower canine teeth of the cat in response to: (A) grinding (3 x 1 s) of dentine (inner half of dentine), (B) high-amplitude ultrasonic (1 s) stimulation on the intact tooth surface. (A) local stimulation, (A) intravenous bolus injection of substance P (80 fmol/kg).
HIGH
b
Fig. 6. Vasodilation in the dental pulp, measured by laser Doppler flowmetry (LDF) in response to high- and lowamplitude ultrasonic stimulation before (0) and after (W) periapical injection with mepivacaine. Section (a) is comparison when electrically induced vasodilation in the pulp was unaffected by mepivacaine and (b) when such stimulusevoked pulpal response was blocked by the local anaesthetic. Figures within parentheses are number of procedures.
Pulpal blood flow 4
3 2
E 2 f
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DURATION
Fig. 7. Percussion-induced increase in pulpal blood flow measured with laser Doppler flowmetry (LDF) in control (0) and denervated (m) lower canine teeth of cats. Maximal increase (Basal-Peak) and duration are shown. **p < 0.01.
Percussion resulted in increased blood flow in control teeth (29.6 + 7.6%) with a duration of 1.7 + 0.4 min. Corresponding values were lower in denervated teeth; 3.9 f 3.0% and 0.3 f 0.2 min, respectively, p < 0.005, n = 15 (Fig. 7). DISCUSSION
We show that a variety of stimuli with different modes of action cause pulpal vasodilation when applied to the intact tooth or into a deep dentinal cavity. Furthermore, we show that this effect to a large extent depends on activation of afferent nerves; there was no evidence that sympathetic nerves participated. The proposed mechanism of action is interesting in view of the rich supply of vasoactive neuropeptides (e.g. calcitonin-gene related peptide, substance P and neurokinin A) stored in afferent fibres in the dental pulp of the cat and other species, including man (Olgart et al., 1977a; Gazelius et al., 1987; Akai and Wakisaka, 1990). It has previously been shown that substance P is released in the cat pulp when the inferior alveolar nerve is electrically stimulated (Olgart et al., 1977b; Brodin et al., 1981). There is also a long-lasting vasodilation in the pulp after such stimulation (Tiinder and Naess, 1978; Gazelius and Olgart, 1980). Both these events occur at a current intensity high enough to activate unmyelinated C-fibres. However, A-fibres are also activated and an interesting observation was recently made by JSinig and Lisney (1989) suggesting that cutaneous vasodilation in rats can be brought about by stimulation of AG-fibres in the saphenous nerve. However, there is to date no evidence that pulpal A-fibres contain vasoactive substances or that they contribute to vascular effects upon stimulation. On the contrary, stimulation of the tooth crown using current intensities which activate only A-fibres, according to Ntirhi et al. (1983), results in digastric reflexes but not in pulpal vasodilation (Olgart, Edwall and Gazelius, 1989). We found further evidence that afferent C-fibres are involved in pulpal vasodilation, as capsaicin caused increased pulpal blood flow mimicking the electrically induced blood-flow response. Capsaicin, the pungent constituent of some red peppers, is regarded as a selective activator of unmyelinated afferent nerves (see Lynn, 1990) and has been shown by local application in the cat tooth to activate pulpal C-fibres but not
579
A-fibres (Narhi, 1985). That A-fibres do not seem to be involved in the vascular effects is also indicated by the absence of a response to application of aconitine, a substance suggested to stimulate A-fibres in the pulp (Haegerstam, 1976). Inferior alveolar denervation with the same technique and timing as ours has previously been shown by immunohistochemical methods to abolish tachykinin-positive nerve endings in the pulp (Olgart et al., 1977a; Akai and Wakisaka, 1990) and by radioimmunoassay to deplete almost totally substance P-like immunorectivity in the pulp (Gazelius et al., 1983). In accordance with these findings, we found that there were no vascular responses to electrical stimulation in denervated teeth, suggesting that an important part of the afferent innervation had degenerated. Whether in these teeth the weak but persisting vascular responses to physical stimulation were partly dependent on remaining nerves, too few, or less prone to be electrically excited, and originating from other sources, cannot be excluded. For example, there is evidence that the lower canine teeth in cats may receive some afferent fibres from the lingual nerve (Robinson, 1980). In denervated teeth the absence of afferent nervemediated vasodilation was also demonstrated by using capsaicin. Instead of a vasodilatory effect, capsaicin induced a slight reduction in blood flow in the denervated teeth. This sort of vasoconstrictor effect has been found after nerve degeneration in different tissues (Duckles, 1986) and was recently suggested to be due to a direct effect of capsaicin on the vasculature (Edvinsson et al., 1990); in our study it shows that cavity depth and diffusion were not critical factors in denervated teeth. Great attention was given to preparing cavities of equal depth on control and denervated sides. The pink colour of the pulp observed through a thin layer of dentine in the microscope has been used as a guide in judging the amount of remaining dentine in normal teeth (Olgart, 1974). However the intensity of the colour is evidently related to hyperaemia in response to preparation and was, as mentioned earlier, not so significant in denervated teeth. This resulted in somewhat deeper preparations in denervated teeth. Thus, topical application of adrenaline in cavities of denervated teeth produced a more rapid vasoconstriction in the pulp, and probing with a sharp instrument at the termination of experiments more easily caused a pulp exposure in these cavities than in control cavities. Bradykinin was recently shown to increase pulpal blood flow in the cat when applied locally in deep dentinal cavities (Okabe, Todoki and Ito, 1988), and its action on the vasculature was suggested to be mediated via prostaglandin formation. According to our results the vasodilator effect of bradykinin is to a considerable part dependent on activation of afferent nerve endings. In denervated teeth the onset was delayed and the magnitude and duration of the response were much reduced. The appearance of the bradykinin response in innervated teeth resembled that obtained by electrical stimulation and capsaicin: an almost instant rise in blood flow with a long duration. Thus, a nerve-mediated effect could well be at hand and is supported by previous findings showing that local application of bradykinin selectively
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OLGART
activates intradental C-fibres in the cat (Narhi et al., 1984; N&hi, 1985) and causes a dull pain when applied to human teeth (Ahlquist et al., 1985). In addition, bradykinin produces vascular effects and miosis in the rabbit eye and vascular effects in the rat paw that can be explained partly by nerve-mediated release of substance P (Bynke et al., 1983; Ueda, Muramatsu and Fujiwara, 1984; Shibata et al., 1986). It has also been shown recently that bradykinin acts via different receptors as regards nerve-mediated and direct vascular effects (Mizumura et al., 1990). Thus, in addition to exerting a direct effect on the pulpal vessels, as shown in the denervated teeth, bradykinin has important vascular effects mediated by afferent C-fibres via release of neuropeptides. In our study this nervemediated effect may have been underestimated in relation to the direct vascular effect, due to the above mentioned difference in cavity depth between the denervated and control sides. Also, preparation of dentine with a slowly rotating diamond bur seemed to have two actions leading to increased pulpal blood flow. An afferent nervemediated effect can probably explain the rapid onset of the response in control teeth, whereas a rise in temperature reaching the pulpal vasculature by drilling in deeper dentine in denervated teeth may explain the gradual increase in blood flow in these teeth. However, when grinding in superficial dentine, nerve-induced vasodilation may dominate, as a response, rapid in onset, was seen only in control teeth. Whether C-fibres in this situation were excited indirectly via the dentine tubules by a hydrodynamic mechanism or by a rise in temperature is not known. A slow heating of dentine of cat teeth has been shown to activate intradental C-fibres (Narhi et al., 1982b; Hirvonen ef al., 1989). As the production and transfer of heat to the pulp should be minimal from the enameldentine region when water cooling was used, an effect of temperature directly on nerves is unlikely. This finding may seem contradictory to results obtained in single-fibre recordings from cat teeth showing that A-fibres but not C-fibres in the pulp are activated by drilling in dentine (NHrhi and Haegerstam, 1983; Narhi et al., 1984). However, recent findings by Wakisaka et al. (1984, 1985, 1987) show that peptide-containing C-fibres are present in the odontoblastic layer of feline teeth and in the predentine zone of rat and human teeth. These findings indicate that not only A-fibres but also C-fibre nerve endings are located in a position to receive information from external stimuli affecting dentine and odontoblasts. In agreement with our results are those of Olgart, Gazelius and Sundstriim (1988) showing that mechanical deformation of dentine results in instant vasodilation in the pulp. Thus, the rich supply and strategic position of C-fibres in the pulp may result in a vascular defence reaction when dentine is reached by an intense mechanical stimulus. Ultrasonic stimulation at low amplitude increased pulpal blood flow by stimulating periodontal fibres. This implies that nerve endings, mechanically excited in the periodontal tissues, can conduct impulses antidromically in branches supplying the pulp. This is consistent with findings in the cat that both pulpal A-fibres (Lisney and Matthews, 1979) and C-fibres (B. Gazelius and L. Olgart, unpublished results) have
ef
d.
an extensive branching with functional connections between teeth, gingiva and lip. Stimulation at high amplitude activated nerves in the pulp, as revealed by local anaesthesia blocking both ultrasonic and electrically induced pulpal vasodilation. It is surprising that a short (1 s) stimulation induced this effect. Whether it is due to heat or some other noxious action in the pulp is not known. According to Narhi (1990) C-fibres in the pulp are polymodal and are thus also excited by physical stimuli if strong enough. As highintensity ultrasonic stimulation induced some vasodilation in denervated teeth and after local anaesthesia, some other non-neural mechanisms must also have been triggered. Whether percussion caused vasodilation in the pulp by activation of periodontal and/or pulpal fibres was not resolved. We assumed that application of hypertonic NaCl should have different effects in control and denervated sides. Hypertonic sodium solutions are known to excite pulpal A-fibres (Olgart, 1974) in a non-specific manner (Orchardson, 1977) and our test solution would therefore be expected to evoke discharges also in intradental C-fibres. There was, however, no significant difference between denervated and control teeth, and in both cases a weak vasodilation developed slowly as compared with the other chemical stimulations. The inability to induce a clear-cut, nervemediated vasodilation may be due to insufficient nerve excitation and a direct vasodilatory action by the hypertonic solution. The latter suggestion is supported by previous findings showing that locally induced hyperosmolarity inhibits myogenic activity in precapillary vessels resulting in hyperaemia (Mellander et al., 1967). Our findings give a novel aspect on the functional role of afferent nerves in the pulp in connection with clinical stimulation of teeth. Thus, afferent C-fibres can mediate vasodilation that occurs almost instantly when
activated by such stimuli, but there is also a direct effect on the vessels by heat formation and/or noxious actions which, in teeth that have lost the nerve supply, leads to delayed and reduced vasodilation. Acknowledgemenrs-This study was supported by the Swedish Medical Research Council (B83-24X-816-20C) and Hedlunds Foundation
REFERENCES Ahlquist M. L., Franzen 0. G., Edwall L. G., Fors U. G. and Haegetstam G. A. T. (1985) Quality of pain sensations following local application of algogenic agents on the exposed human tooth pulp: a psychophysical and electrophysiological study. In Advances in Pain Research and Therapy (Eds Fields H. L., Dubner R. and Cervero F.), pp. 351-359. Raven Press, New York. Akai M. and Wakisaka S. (1990) Distribution of peptidergic nerves. In Dynamic Aspects of Denral Pulp (Eds Inoki R., Kudo T. and Olgart L.), pp. 337-348. Chapman and Hall, London. Brodin E., Gazelius B., Lundberg J. M. and Olgart L. (1981) Substance P in trigeminal nerve endings. Acta physiol. stand. 111, 501-503.
Bynke G., HHkansson R., Horig J. and Leander S. (1983) Bradykinin contracts the pupillary sphincter and evokes ocular inflammation through release of neuronal substance P. Eur. J. Pharmac. 91, 469-475.
Pulpal blood flow Duckles S. P. (1986) Effects of capsaicin on vascular smooth muscle. Nuunyn-Schmiedebergs Arch. Pharmuc. 333, 59-64. Edvinsson L., Jansen I., Kinaman T. A. and McCulloch J. (1990) Cerebrovascular responses to capsaicin in vitro and in situ. Br. J. Pharmac. 100, 312-318. Edwall B., Gazelius B., Berg J. O., Edwall L., Hellander K. and Olgart L. (1987) Blood flow changes in the dental pulp of the cat and rat measured simultaneously by laser Doppler flowmetry and local lzsI clearance. Acru physiol. scund. 131, 81-91. Gazelius B. and Olgart L. (1980) Vasodilatation in the dental pulp produced by electrical stimulation of the inferior alveolar nerve in the cat. Acta physiol. stand. 108, 181-186. Gazelius B., Brodin E., Kahan T., Panopoulos P. and Olgart L. (1983) Local application of capsaicin to a peripheral nerve: effects on substance P and noradrenaline levels in peripheral nerve endings and vascular responses to nerve stimulation. In Substance P (Eds Skrabanek P. and Powell D.), pp. 241-242. Boole Press, Dublin. Gazelius B., Edwall B., Olgart L., Lundberg J. M., Hokfelt T. and Fisher J. A. (1987) Vasodilatory effects and coexistence of calcitonin gene-related peptide (CGRP) and substance P in sensory neurons of cat dental pulp. Acta physiol. stand. 130, 33340. Haegerstam G. (1976) The effect of veratrine and aconitine on the excitability of sensory units in the tooth of the cat. Acta physiol. stand. 98, 1-7.
Hirvonen T. and Niirhi M. (1986) The effect of dentinal stimulation on pulp nerve function and pulp morphology in the dog. J. dent. Res. 65, 1290-1293. Hirvonen T., N&hi M., Jyvisjirvi E. and Ngassapa D. (1989) Intradental nerve activation and jaw muscle regex responses to stimulation of dentine in the cat. Proc. Finn. de&. Sot. 85 (Suppl), 28. Janie W. and Lisnev S. J. W. (1989) Small diameter mielinated afferents produce vasodilatation but not plasma extravasation in rat skin. .r. Fhysiol. 415, 477-486. Lembeck F., Gamse R., Holzer P. and Molnar A. (1980) Substance P and chemosensitive neurones. In Neuropeprides and Neural Transmission (Eds Marsan A. and Traczyk W. Z.). pp. 51-72. Raven Press, New York. Lisnev S. J. W. and Matthews B. (1979) Branched afferent nerves supplying tooth pulp in the cat. J. Physiol. Land. 279, 509-5 17.
N&hi M. V. O., Hirvonen T. J. and Hakumaki M. 0. K. (1982a) Activation of intradental nerves in the doa to some stimuli applied to the dentine. Archs oral Biol:27, 1053-1058. Narhi M., Jyv%jlrvi E., Hirvonen T. and Huopaniemi T. (1982b) Activation of heat-sensitive nerve fibres in the dental pulp of the cat. Pain 14, 317-326. NLrhi M., Jyvasjarvi E. and Huopaniemi T. H. T. (1984) Functional differences in intradental A- and C-nerve units in the cat. Pain Suppl 2, S242. Narhi M.. Virtanen A.. Hirvonen 1. and Huooaniemi T. (1983) Comparison of electrical thresholds of intradental nerves and jaw-opening reflex in the cat. Acta physiol. stand. 119, 399-403.
Okabe E., Todoki K. and Ito H. (1988) Direct pharmacological action of vasoactive substances on pulpal blood flow: an analysis and critique. J. Ended. Is, 473-477. Olgart L. (1974) Excitation of intradental sensory units bv ~ha~a~olo~~l agents. Aetu physiol. scund. $2, 48-55: Olgart L. and Gazelius B. (1988) Enhanced vasodilation in sensory denervated tooth pulps by substance P (SP). Regulat. Peptides 22, 137.
Olgart L., Hokfelt T., Nilsson G. and Pernow B. (1977a) Localization of substance P-like immunoreactivity in nerves in the tooth pulp. Pain 4, 153-159. Olgart L., Gazelius B., Brodin E. and Nilsson G. (1977b) Release of substance P-like immunoreacti~ty from the dental pulp. Ac(a physiol. stand. 101, 510-512. Olgart L., Gazelius B. and Sundstriim F. (1988) Intradental nerve activity and jaw-opening reflex in response to mechanical deformation of cat teeth. Acra physiol. stand. 133, 399-406.
Olgart L., Edwall B. and Gazelius B. (1989) Neurogenic mediators in control of pulpal blood flow. J. ~ndod. 15, 409-412.
Orchardson R. (1977) Ion sensitivity of intradental nerves in the cat. In Pain in the Trigeminal Region (Eds Anderson D. J. and Matthews B.), pp. 7782. Elsevier/North-Holland Biomedical Press, New York. Robinson P. P. (1980) An electrophysiologicat study of the pathways of pulpal nerves from mandibular teeth in the cat. Archs oral Biof. 25, 825-829. Shibata M., Ohkubo T., Takahashi H. and Inoki R. (1986) Interaction of bradvkinin with substance P on vascular permeability and pain response. Jap. J. Pharmac. 41, 427-429.
Lynn B. (1990) Capsaicin: actions on nociceptive C-fibres and therapeutic potential. Pain 41, 61-69. Mellander S., Johansson B., Gray S., Jonsson D. and Lundvall J. (1967) The effects of hypero~ola~ty on intact and isolated vascular smooth muscle. Possible role in exercise hyperaemia. Angiologica 4, 310-322. Mizumura K., Minagawa M., Tsujii Y. and Kumazawa T. (1990) The effects of bradykinin agonists and antagonists on visceral polymodal receptor activities. Pain 40,221-227. Narhi M. V. 0. (1985) The characteristics of intradental sensory units and their responses to stimulation. J. dent. Res. 44, Xv-57
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Nirhi M. (1989) Interaction between the autonomic and sensory nerves in the dental pulp. Proc. Finn. Dent. Sot. 85, 389-393.
Narhi M. (1990) Intradental sensory units. In Dynamic Aspects of Dental Pulo (Eds Inoki R., Kudo T. and Olaart L.), pp. 137-149. Chapman and Hall, London. Niirhi M. and Haegerstam G. (1983) Intradental nerve activity induced by reduced pressure applied to exposed dentine in the cat. Acfa physiol. stand. 119, 381-386.
Tender K. H. and Naess G. (1978) Control of blood flow in the dental pulp in dogs. Acfa physiol. scund. 104, 13-23. Ueda N., Muramatsu I. and Fujiwara M. (1984) Capsaicin and bradykinin-induced substance P-ergic responses in the iris sphincter muscle of the rabbit. J. Phannac. e.rp. Ther. 23& 469-473.
Wakisaka S., Ichikawa H., Nishimoto T., Matsuo S., Yamamoto K., Nakata T. and Akaki M. (1984) Substance P-like immunoreactivity in the pulp-dentine zone of human molar teeth demonstrated by indirect immunofluore~ence. Archs oral Biol. 29, 73-75.
Wakisaka S., Nishikawa S., Ichikawa H., Matsou S.. Takano Y. and Akai M. (1985) The distribution and origin of substance P-like immunoreactivity in rat molar pulp and periodontal tissues. Archs oral Biol. 30, 813-818.
Wakisaka S., Ichikawa Takano Y. and Akai origin of calcitonin nerve fibres in feline 585-589.
H., Nishikawa S., Matsuo S., M. (1987) The dist~bution and gene-related peptide-containing dental pulp. Histochemisrr~ 86,
1991 Archs oral Bid. Vol. 36,No. 8, pp. 575-581, Printedin GreatBritain.All rightsreserved
Copyright 0
1991 Pergamon Press pk
INVOLVEMENT OF AFFERENT NERVES IN PULPAL BLOOD-FLOW REACTIONS IN RESPONSE CLINICAL AND EXPERIMENTAL PROCEDURES IN THE CAT
TO
L. OLGART,‘* L. EDWALL* and B. GAZELIUS’ ‘Department of Pharmacology and ‘Department of Endodontics, Karolinska Institute, Stockholm, Sweden (Accepted 21 February 1991) Summary-A unilateral resection of the mandibular nerve (n = 20) was made 10-14 days before investigation of the contribution of afferent nerves in vasodilator reactions in the dental pulp. Lower canine teeth were subjected to various stimuli and pulp blood-flow responses monitored by laser Doppler flowmetry. An absence of response to bipolar electrical (5 impulses, 50 PA, 5 ms, 2 Hz) stimulation on the tooth surface was used to demonstrate a successful chronic nerve lesion. Local application of capsaicin (10e4 M) in a deep dentinal cavity induced a long-lasting increase in pulpal blood flow in control teeth only. Bradykinin (10e3 M) induced significantly larger responses in control than in denervated teeth (58.3 + 9.8% and 24.5 + 4.9%, respectively, p < 0.005, n = 8); in addition, the onset was slower and the duration of the response significantly (60%) shorter than in control teeth. Intermittent grinding of surface dentine instantly increased Row in control teeth by 53.0 f 12.5% (n = 12) whereas in denervated teeth the response was delayed and significantly (70%) smaller. Deeper preparation produced responses of similar magnitude in control and denervated teeth (69 and 50%, respectively) but the onset was delayed in denervated teeth. Low-intensity ultrasonic stimulation caused vasodilation in intact teeth (38% increase) but had no effect in denervated teeth. This effect was abolished after local anaesthetic (mepivacaine) injection. Sympathectomy (n = 3) did not influence stimulation-induced blood-flow responses in the dental pulp. The results show that afferent nerves make an important contribution to haemodynamic reactions in the pulp in response to experimental and clinical procedures directed at the tooth. Key words: dental pulp, cat, blood flow, denervation, afferent nerves, tooth stimulation.
INTRODUCTION
The pulp of cat and human teeth is richly innervated by both myelinated and unmyelinated afferent nerves, the unmyelinated type (C-fibres) containing neuropeptides such as substance P, neurokinin A and calcitonin-gene related peptide (Olgart et al., 1977a; Gazelius et al., 1987; Akai and Wakisaka, 1990). High-intensity electrical stimulation activates the C-fibres and causes release of substance P and vasodilation in the cat dental pulp (Olgart et al., 1977b; Gazelius and Olga& 1980). Such effects have not been reported after activation of the A-fibre population. It is well known that the intradental A-units respond to various types of stimuli affecting dentine such as drilling, probing and air drying (Narhi, Hirvonen and Hakumliki, 1982a; Nlrhi, Jyviisjlrvi and Huopaniemi, 1984; Hirvonen and Narhi, 1986; Narhi, 1985) while the intradental C-units fail to respond to such stimulation (NHrhi et al., 1982b, 1984; Nlrhi and Haegerstam, 1983; Nlrhi, 1985). However, intradental C-fibre units have been shown to share many properties with polymodal C-nociceptors in the skin (NSirhi, 1989), and it has been shown in dermal tissues that noxious external stimulation induces the well-known flare reaction mediated by the vasoactive neuropeptides released from the afferent C-fibre nerve *To whom all correspondence
should be addressed.
endings (see Lembeck et al., 1980). Thus, it would be of interest to know if clinical dental stimuli could influence blood flow in the pulp and to find out to what extent afferent nerves are involved. Interestingly, preliminary experiments in our laboratory had shown that in cats with unilaterally denervated jaws, deep cavity preparation resulted in hyperaemia in the pulp of innervated teeth but not of the contralateral denervated teeth. We have therefore now investigated the effect of some clinical procedures on pulpal blood flow in denervated and innervated teeth in the cat and compared these results with those obtained with experimental procedures that have well-known, direct and/or indirect, vascular actions. MATERIALS AND METHODS
Operative procedure
Twenty-three young (1-yr-old) male cats weighing 3-4 kg were used. They were anaesthetized with sodium pentobarbital (initial dose, 30 mg/kg intravenously and then as needed). The inferior alveolar nerve was sectioned unilaterally in 20 cats. Access to the nerve was through an incision over the inferior border of one side of the mandible. The periosteum was reflected from the medial aspect of the bone, exposing the nerve as it entered the mandibular canal. 575
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After careful separation from the inferior alveolar artery and vein, 3-4mm of the nerve were resected and the wound sutured. In three separate experiments the sympathetic superior cervical ganglion was resected unilaterally. The ganglion and an adjacent 3-4 mm of the sympathetic nerve were removed and the wound sutured after careful separation from vessels and the vagus nerve. After lo-14 days, which allowed the transected nerves to degenerate, the cats were again anaesthetized. The trachea was intubated and blood pressure in one femoral artery was recorded with a Statham 23 AC transducer. Rectal temperature was kept constant at about 38°C with a thermostat-controlled heating pad. The head and jaws were immobilized by a rigid steel rod attached bilaterally to the molar and premolar teeth by dental acrylic. The experiments were ended by administering a lethal dose of anaesthetic intravenously. Blood-jlow recording
Two laser Doppler flowmeters (Periflux PF2) were used to measure simultaneously pulpal blood flow in the canine teeth of one jaw (Edwall et al., 1987). PF 103 probes were positioned in contact orduring physical stimulation-at a distance of about 0.6 mm from the intact and cleaned tooth surface at the lingual aspect, midway between the gingiva and the tip of the tooth. The flowmeter’s time constant was set at 3 s and band width at 4 kHz. Electrical stimulation and infusion of substance P
To test if degeneration of the pulpal nerves had occurred after sectioning the inferior alveolar nerve, bipolar electrical stimulations (5 imp, 50 PA, 5 ms. 2 Hz) and intravenous infusions of substance P @Ofmol/kg) were made. Electrodes were brought into contact with the intact and dried enamel surface using electrode paste. Substance P was given in the femoral vein as a bolus (0.4ml) injection after the cannula had been flushed with 0.9% NaCl containing 0.2% bovine serum albumin. Lack of increase of pulpal blood flow in response to electrical stimulation was used to indicate that fine-calibre, high-threshold afferent nerves (C-fibres) had degenerated in the pulp. Conversely, an increased blood flow indicated an intact pulpal afferent nerve supply. According to a previous study (Olgart and Gazelius, 1988) vasodilator responses to substance P given intravenously can be expected to be 200-300% larger in inferior alveolar-denervated pulps during the degenerative phase than in innervated pulps. Chemical stimulation
After unilateral denervation, dentinal cavities were prepared bilaterally in the mandibular canine teeth in a group of eight cats. Each pair of cavities was prepared as identically as possible on the buccal aspect of the tooth and located midway between the gingiva and the tip of the crown. A round diamond bur (dia 1.5 mm) was used to penetrate the enamel. Dentine was removed with an end-cutting bur (dia 1.Omm) rotated in a holder held between the fingers. Preparation was made under visual control through a binocular stereo microscope (Zeiss OPM 1) into
the innermost layer of dentine and accompanied by flushing with isotonic saline. At the end of the experiments, adrenaline (5.5 x 10m3M) was applied to each pair of cavities to observe the latency and magnitude of blood-flow reduction. Finally, the floor of each cavity was penetrated with a fine probe in order to estimate the remaining dentine thickness. Capsaicin, bradykinin, aconitine and sodium chloride were applied as local chemical stimuli in the dentinal cavities. Care was taken to apply each substance (2 ~1) as identically as possible. Capsaicin (Fluka Chemie AG, Switzerland) was dissolved in 20% Tween 80 and 10% ethyl alcohol and diluted in isotonic saline to a final concentration of 10m4M. Bradykinin (Sigma, St Louis, MO) was dissolved in isotonic saline and applied in a concentration of 10e3 M. Aconitine (Sigma, St Louis, MO) was dissolved in isotonic saline and applied in a concentration of 10e2 M. Hypertonic sodium chloride was applied as 0.76 and 1.54 M solutions. Physical stimulation
A round diamond instrument (dia 1.5 mm) was operated at slow speed to grind, repetitively, spherical cavities with increasing depth through the enamel and into the dentine in a group of 12 cats. Pressure and speed (6000-8000 rev/min) were similar to those used clinically. The grinding time was, however, considerably shorter-three grindings, each of l-s duration with l-s intervals between-in order to minimize the heat production. The timing of the grinding procedure was signalled by repetitive sounds of 1 s duration. The first grinding-into the most peripheral dentine-was accompanied by flushing with isotonic saline. Grinding was repeated every 5 mitt, or with longer time intervals if either of the teeth showed a blood-flow reaction. As before, care was taken to prepare the teeth identically. In this way, each pair of teeth were stimulated by grinding in a series of three times 1 s procedures allowing for recording of blood flow for at least 5 min between the stimulations. Physical stimulation was also applied by an ultrasonic dental instrument (Piezon, Electromedical Systems AS, Le Sentier, Switzerland). The tip of the instrument was held perpendicularly in contact with the incisal third of the canine tooth for 1 s. A rest of 5 min was allowed before stimulation was repeated. As above, care was taken to apply the vibration (30 kHz) identically on both sides. Two different levels of vibration amplitude were used. On the instrument’s scale of 10 units, a setting of 3-5 was regarded as low intensity and 8 as high. The third type of physical stimulation was percussion, which was applied in a series of three, one every second, with at least a 5min interval for recording in between. A 30 g steel instrument was allowed to fall from a height of 9 cm and hit the tip of the tooth crown, perpendicularly to its long axis. Dental nerve blockade by local injection of mepivacaine chloride 30 mg/ml (Astra, Sweden) was made either submucosally or subperiosteally in the apical region-to obtain periodontal nerve blockade-or, intraosseously-to obtain pulpal nerve blockade. The response to electrical tooth stimulation was checked before each physical stimulus was applied.
Pulpal blood flow
In two experiments the inferior alveolar nerve was sectioned unilaterally on the day of the experiment. This procedure did not change the pattern of response to the stimuli as compared with those obtained on the control side. All data were stored on a video tape recorder and on an on-line computer and analysed by commercial software (Perisoft, Perimed KB). The following measures were recorded and expressed as follows. Laser Doppler flowmeter output values from the dental pulps were expressed in perfusion units (PU). The use of this unit (10 mV) makes comparison possible with results obtained using recently available automatic devices. As the flowmeter probes monitored pulpal blood flow through the intact enamel and dentine (Edwall et al., 1987) baseline PU values could differ between teeth (see Fig. l)-or in the same tooth, when the probe was repositioned-due to different laser beam penetration through the mineralized tissues. For this reason and as the flowmeter monitors relative changes in the flux of blood cells, i.e. the number of cells times their velocity, effects obtained were calculated as percentages of baseline values. The duration of a response was expressed in minutes. Mean arterial blood pressure and heart rate were monitored and did not change during the experimental procedures. The flowmetric results were expressed as mean f SEM and evaluated with the paired, and when necessary unpaired, Student’s t-test; p-values less than 0.05 were considered to be significant. RESULTS
In the unilaterally sympathectomized cats the magnitude of pulpal blood-flow changes in response to electrical and ultrasonic stimulation as well as to percussion and cavity preparation was similar on both sides. Therefore, the term “denervated teeth” discussed below refers to chronically inferior alveolar nerve-sectioned cats. Electrical
stimulation
and substance P
In all 20 cats with resected inferior alveolar nerves, electrical stimulation of the lower canine teeth on the control side resulted, within lo-20 s, in a blood-flow increase (111 f 15%) lasting 8-15 min, whereas there were no responses on the denervated side [Fig. l(A)]. In contrast, infusion of substance P (80 fmol/kg) induced blood-flow responses in the denervated pulps (81 k 15%) exceeding those obtained in control pulps (28 + 2%, p < 0.01) as has been reported previously (Olgart and Gazelius, 1988) [Fig. l(A)].
517 20 16 A
12 -J---C
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Fig. I. Blood-flow changes (laser Doppler perfusion units) in innervated (c) and denervated (d) lower canine teeth of the cat in response to: (A) bipolar (50PA, 5 ms; 5 imp) electrical stimulation on intact tooth surface, (B) capsaicin (tom4 M) and (C) bradykinin (lo-) M) applied in a deep dentinal cavity. (A) are local stimulations, (A) intravenous bolus injections of substance P (80fmol/kg). Steep spikes [(B) and (C)] are artefacts due to application of stimuli or cavity washing. appeared later and the duration of the response was shorter (4.3 + 1.5 min) than in control teeth (9.6 k 1.2 min, p < 0.05, n = 8) (Fig. 2).
Application of hypertonic sodium chloride 0.76 and 1.54 M gave increased pulpal blood flow but with variable results and without significant differences between sides. Aconitine was without effect on pulpal blood flow. Physical stimulation
Cavity preparation in the outer half of dentine in control teeth always caused an instant increase in flow (53.0 f 12.5%, n = 12), whereas the presence of a response in denervated teeth varied and, when observed, was delayed and significantly smaller (15.6 f 8.9%, p < 0.05, n = 10) with a shorter duration
n=6
16
Chemical stimulation
Capsaicin induced a powerful and long-lasting (> 30 min) vasodilation within IO-30 s in innervated teeth, whereas in the contralateral denervated teeth no such responses were observed apart from small transient reductions in flow values [Fig. l(B)]. Application of bradykinin induced increased flow in both denervated and control teeth within lo-30 s. However, the responses in innervated teeth (58.2 f 9.8%) were significantly larger than those in contralateral denervated teeth (24.5 f 4.8%, p < 0.005, n = 8) (Fig. 2). Typically, as can be seen in Fig. l(C), the maximum of the response in denervated teeth
60 ti B z Y
40
20
4 0
BASAL-PEAK
DURATION
Fig. 2. Vasodilator effects in control (0) and denervated teeth (m) of the cat in response to bradykinin application (IO-’ M) in a deep dentinal cavity. Pulpal blood flow was measured with laser Doppler flowmetry (LDF); **I, < 0.01.
L. OLGARTet al.
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Fig. 3. Effects of grinding of dentine on pulpal blood flow measured by laser Doppler flowmetry (LDF) in innervated (0) and denervated (W) lower canine teeth in the cat. (1) Grinding 3 x 1 s in outer half of dentine and (2) in inner half of dentine. Figures within parentheses are number of procedures. *p < 0.05.
Fig. 5. The effect of ultrasonic stimulation (1 s), measured by laser Doppler flowmetry (LDF) with high and low amplitudes on enamel surface of lower canine teeth on innervated (0) and denervated (m) side. The maximal increase (Basal-Peak) and duration are shown. Figures within parentheses are number of procedures. ***p < 0.001.
(8.7 + 2.1 and 3.4 & 2.1 min, respectively). In the first trial, cutting through the enamel, blood-flow responses were obtained only in innervated teeth. In deeper preparations there was only a slight difference in the magnitude and duration of responses between sides (Fig. 3). However, the responses in denervated teeth were much delayed and the maximum effect was reached after several minutes [Fig. 4(A)]. As can also be seen, responses to substance P after such procedures had a similar appearance on both sides but showed a higher percentage increase in denervated teeth. Ultrasonic stimulation at the highest amplitudes caused increased blood flow in control (67.4 f 11.5%) as well as in denervated teeth (45.4 k 10.6%) with similar durations (Fig. 5). As for grinding in deep cavities, the first phase of the response in denervated teeth was delayed as compared with that in controls
[Fig. 4(B)]. However, at lower amplitudes, there was a clear-cut difference in the overall increase between control and denervated sides (38.2 f 5.6%, n = 34 and 1.4 k 0.7%, n = 31, respectively, p < 0.005) (Fig. 5). In fact, in the group of denervated teeth, only one single procedure resulted in a small, short-lasting response. To elucidate whether pulpal and/or periodontal nerves were responsible for the ultrasonically induced vasodilation, local anaesthetics were used to block the effects. First, buccal and lingual infiltrations, unable to block intradental vasodilation induced by electrical stimulation of the tooth crown, were used. Pulpal vasodilation to subsequent low-amplitude ultrasonic stimulation was then almost abolished (1.3 f 0.9% increase) as compared with initial control responses (58.7 + 17.3%, p < 0.01, n = 8). Responses to ultrasonic stimulation with high amplitudes were only slightly changed (Fig. 6). After additional apical injections resulting in abolished responses to electrical tooth stimulation, high-amplitude ultrasonic stimulations produced only small vasodilatory responses in some teeth; 43.0 f 10.1% increase before and 6.0 f 3.1% after injection, p < 0.001, n = 11) (Fig. 6).
80
I?(8)
(6)
(11)
I
l
l
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LOW
a
min
Fig. 4. Blood flow changes (laser Doppler perfusion units) in innervated (c) and denervated (d) lower canine teeth of the cat in response to: (A) grinding (3 x 1 s) of dentine (inner half of dentine), (B) high-amplitude ultrasonic (1 s) stimulation on the intact tooth surface. (A) local stimulation, (A) intravenous bolus injection of substance P (80 fmol/kg).
HIGH
b
Fig. 6. Vasodilation in the dental pulp, measured by laser Doppler flowmetry (LDF) in response to high- and lowamplitude ultrasonic stimulation before (0) and after (W) periapical injection with mepivacaine. Section (a) is comparison when electrically induced vasodilation in the pulp was unaffected by mepivacaine and (b) when such stimulusevoked pulpal response was blocked by the local anaesthetic. Figures within parentheses are number of procedures.
Pulpal blood flow 4
3 2
E 2 f
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DURATION
Fig. 7. Percussion-induced increase in pulpal blood flow measured with laser Doppler flowmetry (LDF) in control (0) and denervated (m) lower canine teeth of cats. Maximal increase (Basal-Peak) and duration are shown. **p < 0.01.
Percussion resulted in increased blood flow in control teeth (29.6 + 7.6%) with a duration of 1.7 + 0.4 min. Corresponding values were lower in denervated teeth; 3.9 f 3.0% and 0.3 f 0.2 min, respectively, p < 0.005, n = 15 (Fig. 7). DISCUSSION
We show that a variety of stimuli with different modes of action cause pulpal vasodilation when applied to the intact tooth or into a deep dentinal cavity. Furthermore, we show that this effect to a large extent depends on activation of afferent nerves; there was no evidence that sympathetic nerves participated. The proposed mechanism of action is interesting in view of the rich supply of vasoactive neuropeptides (e.g. calcitonin-gene related peptide, substance P and neurokinin A) stored in afferent fibres in the dental pulp of the cat and other species, including man (Olgart et al., 1977a; Gazelius et al., 1987; Akai and Wakisaka, 1990). It has previously been shown that substance P is released in the cat pulp when the inferior alveolar nerve is electrically stimulated (Olgart et al., 1977b; Brodin et al., 1981). There is also a long-lasting vasodilation in the pulp after such stimulation (Tiinder and Naess, 1978; Gazelius and Olgart, 1980). Both these events occur at a current intensity high enough to activate unmyelinated C-fibres. However, A-fibres are also activated and an interesting observation was recently made by JSinig and Lisney (1989) suggesting that cutaneous vasodilation in rats can be brought about by stimulation of AG-fibres in the saphenous nerve. However, there is to date no evidence that pulpal A-fibres contain vasoactive substances or that they contribute to vascular effects upon stimulation. On the contrary, stimulation of the tooth crown using current intensities which activate only A-fibres, according to Ntirhi et al. (1983), results in digastric reflexes but not in pulpal vasodilation (Olgart, Edwall and Gazelius, 1989). We found further evidence that afferent C-fibres are involved in pulpal vasodilation, as capsaicin caused increased pulpal blood flow mimicking the electrically induced blood-flow response. Capsaicin, the pungent constituent of some red peppers, is regarded as a selective activator of unmyelinated afferent nerves (see Lynn, 1990) and has been shown by local application in the cat tooth to activate pulpal C-fibres but not
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A-fibres (Narhi, 1985). That A-fibres do not seem to be involved in the vascular effects is also indicated by the absence of a response to application of aconitine, a substance suggested to stimulate A-fibres in the pulp (Haegerstam, 1976). Inferior alveolar denervation with the same technique and timing as ours has previously been shown by immunohistochemical methods to abolish tachykinin-positive nerve endings in the pulp (Olgart et al., 1977a; Akai and Wakisaka, 1990) and by radioimmunoassay to deplete almost totally substance P-like immunorectivity in the pulp (Gazelius et al., 1983). In accordance with these findings, we found that there were no vascular responses to electrical stimulation in denervated teeth, suggesting that an important part of the afferent innervation had degenerated. Whether in these teeth the weak but persisting vascular responses to physical stimulation were partly dependent on remaining nerves, too few, or less prone to be electrically excited, and originating from other sources, cannot be excluded. For example, there is evidence that the lower canine teeth in cats may receive some afferent fibres from the lingual nerve (Robinson, 1980). In denervated teeth the absence of afferent nervemediated vasodilation was also demonstrated by using capsaicin. Instead of a vasodilatory effect, capsaicin induced a slight reduction in blood flow in the denervated teeth. This sort of vasoconstrictor effect has been found after nerve degeneration in different tissues (Duckles, 1986) and was recently suggested to be due to a direct effect of capsaicin on the vasculature (Edvinsson et al., 1990); in our study it shows that cavity depth and diffusion were not critical factors in denervated teeth. Great attention was given to preparing cavities of equal depth on control and denervated sides. The pink colour of the pulp observed through a thin layer of dentine in the microscope has been used as a guide in judging the amount of remaining dentine in normal teeth (Olgart, 1974). However the intensity of the colour is evidently related to hyperaemia in response to preparation and was, as mentioned earlier, not so significant in denervated teeth. This resulted in somewhat deeper preparations in denervated teeth. Thus, topical application of adrenaline in cavities of denervated teeth produced a more rapid vasoconstriction in the pulp, and probing with a sharp instrument at the termination of experiments more easily caused a pulp exposure in these cavities than in control cavities. Bradykinin was recently shown to increase pulpal blood flow in the cat when applied locally in deep dentinal cavities (Okabe, Todoki and Ito, 1988), and its action on the vasculature was suggested to be mediated via prostaglandin formation. According to our results the vasodilator effect of bradykinin is to a considerable part dependent on activation of afferent nerve endings. In denervated teeth the onset was delayed and the magnitude and duration of the response were much reduced. The appearance of the bradykinin response in innervated teeth resembled that obtained by electrical stimulation and capsaicin: an almost instant rise in blood flow with a long duration. Thus, a nerve-mediated effect could well be at hand and is supported by previous findings showing that local application of bradykinin selectively
580
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OLGART
activates intradental C-fibres in the cat (Narhi et al., 1984; N&hi, 1985) and causes a dull pain when applied to human teeth (Ahlquist et al., 1985). In addition, bradykinin produces vascular effects and miosis in the rabbit eye and vascular effects in the rat paw that can be explained partly by nerve-mediated release of substance P (Bynke et al., 1983; Ueda, Muramatsu and Fujiwara, 1984; Shibata et al., 1986). It has also been shown recently that bradykinin acts via different receptors as regards nerve-mediated and direct vascular effects (Mizumura et al., 1990). Thus, in addition to exerting a direct effect on the pulpal vessels, as shown in the denervated teeth, bradykinin has important vascular effects mediated by afferent C-fibres via release of neuropeptides. In our study this nervemediated effect may have been underestimated in relation to the direct vascular effect, due to the above mentioned difference in cavity depth between the denervated and control sides. Also, preparation of dentine with a slowly rotating diamond bur seemed to have two actions leading to increased pulpal blood flow. An afferent nervemediated effect can probably explain the rapid onset of the response in control teeth, whereas a rise in temperature reaching the pulpal vasculature by drilling in deeper dentine in denervated teeth may explain the gradual increase in blood flow in these teeth. However, when grinding in superficial dentine, nerve-induced vasodilation may dominate, as a response, rapid in onset, was seen only in control teeth. Whether C-fibres in this situation were excited indirectly via the dentine tubules by a hydrodynamic mechanism or by a rise in temperature is not known. A slow heating of dentine of cat teeth has been shown to activate intradental C-fibres (Narhi et al., 1982b; Hirvonen ef al., 1989). As the production and transfer of heat to the pulp should be minimal from the enameldentine region when water cooling was used, an effect of temperature directly on nerves is unlikely. This finding may seem contradictory to results obtained in single-fibre recordings from cat teeth showing that A-fibres but not C-fibres in the pulp are activated by drilling in dentine (NHrhi and Haegerstam, 1983; Narhi et al., 1984). However, recent findings by Wakisaka et al. (1984, 1985, 1987) show that peptide-containing C-fibres are present in the odontoblastic layer of feline teeth and in the predentine zone of rat and human teeth. These findings indicate that not only A-fibres but also C-fibre nerve endings are located in a position to receive information from external stimuli affecting dentine and odontoblasts. In agreement with our results are those of Olgart, Gazelius and Sundstriim (1988) showing that mechanical deformation of dentine results in instant vasodilation in the pulp. Thus, the rich supply and strategic position of C-fibres in the pulp may result in a vascular defence reaction when dentine is reached by an intense mechanical stimulus. Ultrasonic stimulation at low amplitude increased pulpal blood flow by stimulating periodontal fibres. This implies that nerve endings, mechanically excited in the periodontal tissues, can conduct impulses antidromically in branches supplying the pulp. This is consistent with findings in the cat that both pulpal A-fibres (Lisney and Matthews, 1979) and C-fibres (B. Gazelius and L. Olgart, unpublished results) have
ef
d.
an extensive branching with functional connections between teeth, gingiva and lip. Stimulation at high amplitude activated nerves in the pulp, as revealed by local anaesthesia blocking both ultrasonic and electrically induced pulpal vasodilation. It is surprising that a short (1 s) stimulation induced this effect. Whether it is due to heat or some other noxious action in the pulp is not known. According to Narhi (1990) C-fibres in the pulp are polymodal and are thus also excited by physical stimuli if strong enough. As highintensity ultrasonic stimulation induced some vasodilation in denervated teeth and after local anaesthesia, some other non-neural mechanisms must also have been triggered. Whether percussion caused vasodilation in the pulp by activation of periodontal and/or pulpal fibres was not resolved. We assumed that application of hypertonic NaCl should have different effects in control and denervated sides. Hypertonic sodium solutions are known to excite pulpal A-fibres (Olgart, 1974) in a non-specific manner (Orchardson, 1977) and our test solution would therefore be expected to evoke discharges also in intradental C-fibres. There was, however, no significant difference between denervated and control teeth, and in both cases a weak vasodilation developed slowly as compared with the other chemical stimulations. The inability to induce a clear-cut, nervemediated vasodilation may be due to insufficient nerve excitation and a direct vasodilatory action by the hypertonic solution. The latter suggestion is supported by previous findings showing that locally induced hyperosmolarity inhibits myogenic activity in precapillary vessels resulting in hyperaemia (Mellander et al., 1967). Our findings give a novel aspect on the functional role of afferent nerves in the pulp in connection with clinical stimulation of teeth. Thus, afferent C-fibres can mediate vasodilation that occurs almost instantly when
activated by such stimuli, but there is also a direct effect on the vessels by heat formation and/or noxious actions which, in teeth that have lost the nerve supply, leads to delayed and reduced vasodilation. Acknowledgemenrs-This study was supported by the Swedish Medical Research Council (B83-24X-816-20C) and Hedlunds Foundation
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