Properties of single nerve fibres that evoke blood flow changes in cat dental pulp

Transcription

1 (2002), 542.3, pp DOI: /jphysiol The Physiological Society Properties of single nerve fibres that evoke blood flow changes in cat dental pulp D. Andrew and B. Matthews Department of Physiology, University of Bristol, Bristol BS8 1TD, UK Single nerve fibres innervating tooth pulp were isolated from filaments dissected from the inferior alveolar nerve in 17 anaesthetized cats. The fibres were studied to determine whether electrical stimulation of single units produced detectable changes in pulpal blood flow. Single pulpal nerve fibres were electrically stimulated at just above their thresholds and blood flow was recorded with a laser-doppler flow meter from the pulp of the ipsilateral canine. The thresholds of single fibres in dissected filaments were determined either by recording antidromic action potentials from the tooth or by using a novel technique based on collision. Units that produced blood flow changes were further characterized by recording their response to hot, cold, osmotic and hydrostatic pressure, and mechanical stimulation of exposed dentine and to drying the dentine. Of 93 units isolated, 14 produced changes in pulpal blood flow when stimulated electrically at 1 or 10 Hz. All had conduction velocities ( m s _1 ) in the C-fibre range. Ten produced vasodilatation and the remaining four, vasoconstriction. Five of the fibres that produced vasodilatation also responded to the hot stimulus, suggesting that they may form part of an axon reflex or similar mechanism. The four vasoconstrictor units did not respond to any form of stimulus other than electrical and were presumed to be sympathetic post-ganglionic fibres. (Resubmitted 20 April 2002; accepted 17 May 2002) Corresponding author D. Andrew: Spinal Cord Group, West Medical Building, Institute of Biomedical and Life Sciences, West Medical Building, University of Glasgow, Glasgow G12 8QQ, UK. d.andrew@bio.gla.ac.uk Electrical stimulation of the peripheral end of the cut inferior alveolar nerve (IAN) evokes vasodilatation in the dental pulps of the ipsilateral mandibular teeth in cats (Gazelius & Olgart, 1980; Vongsavan & Matthews, 1992a,b) and dogs (Tønder & Næss, 1978). Recordings of the antidromic compound action potential show that this effect occurs at stimulus intensities that recruit a group of slow Ad fibres (Vongsavan & Matthews, 1992a). We have carried out experiments to determine if detectable changes in pulpal blood flow can be evoked by electrical stimulation of single pulpal nerve fibres. Recordings were also made from these fibres to determine if any responded to pain-producing stimuli. This would support the existence of either axon reflex or axon response type vasodilator mechanisms in pulp (for review see Lisney & Bharali, 1989). Preliminary findings and details of the methods used have been published in abstract form (Andrew & Matthews, 1996; Andrew et al. 1996). METHODS General preparation of animals All experimental procedures were licensed by the UK Home Office Animal Inspectorate. Young adult cats ( kg, n = 17) were anaesthetized with sodium pentobarbitone (Sagatal; Rhóne Mérieux, Harlow, UK; 42 mg kg _1 I.P.) and cannulae inserted into the trachea, a saphenous vein and a femoral artery. Supplemental doses of anaesthetic (5 mg kg _1 ) were given intravenously to maintain areflexia. Systemic blood pressure was recorded with a pressure transducer connected to the arterial cannula. Body temperature was maintained at 38.0 ± 0.5 C with an electric blanket controlled from a rectal thermistor. The animals were placed on their side, and the head supported by a metal rod attached to the skull over the frontal sinus with self-tapping screws and self-curing acrylic resin. The rod was clamped to a heavy metal table to stabilize the preparation. The mandible was immobilized by fixing it to the maxilla with hypodermic tubing inserted through holes drilled in the molar teeth and reinforced with acrylic resin. Following completion of the experiments the animals were killed with an overdose of anaesthetic. Tooth preparation Dentine was exposed by removing 1 mm from the tip of a mandibular canine tooth with a rotating diamond disc under Ringer irrigation. The exposed dentine surface was etched with 46 mmol l _1 orthophosphoric acid for 30 s to ensure that the ends of the dentinal tubules were open. Cap and cervical dentine electrodes (Horiuchi & Matthews, 1974; Cadden et al. 1983) were used to electrically stimulate and record from intradental nerves innervating the tooth pulp. They consisted of a pair of Ag AgCl electrodes, one in an acrylic resin cap which was cemented over the tip of the tooth and a second in a cervical cavity in the tooth, 1 mm above the gingival margin (Fig. 1). The cap was filled with Ringer solution to maintain electrical contact between its electrode and the exposed dentine. Recording pulpal blood flow Pulpal blood flow was recorded non-invasively with a laser- Doppler flowmeter (Moor Instruments Ltd, Axminster, Devon,

2 922 D. Andrew and B. Matthews J. Physiol UK; model MBF3D). The probe of the flowmeter was stabilized by inserting it into a short length of stainless steel tubing that was stuck to the tooth, with its centre 2 mm above the gingival margin, using cyanoacrylate adhesive. The sensitivity of the instrument was standardized as described previously (Vongsavan & Matthews, 1993) and recordings made with an upper bandwidth setting of 14.9 khz and a time constant of 0.1 s. Single unit stimulation and recording The ipsilateral IAN was exposed after removal of the masseter muscle and the bone overlying the inferior alveolar canal (Horiuchi & Matthews, 1974). The nerve was carefully separated from the inferior alveolar vessels, cut centrally and placed on a small dissecting platform in a pool of liquid paraffin. To limit the number of functional units supplying other non-pulpal tissues that would contaminate recordings from pulpal fibres, the ipsilateral mental nerves were tied and cut. Also, the periodontal ligament was removed from parts of the buccal and mesial surfaces of the tooth root with a rotating bur under Ringer irrigation. Initially, the whole of the distal end of the nerve was placed on a pair of platinum wire electrodes (0.15 mm diameter) and stimulated with constant voltage electrical pulses of 0.1 ms duration at either 1 or 10 Hz for 30 s. The stimulus intensity was increased to determine the threshold for pulpal vasodilatation. The amplitude of the vasodilatation evoked by supramaximal stimulation of A-fibres (10 V; 0.1 ms) was also determined. Single fibres innervating the pulp of the canine tooth were identified in filaments dissected from the IAN either by recording from the tooth during electrical stimulation of a filament or by recording from a filament during electrical stimulation of the tooth. Either platinum wire (0.15 mm diameter) or paper-in-glass wick electrodes (see below) were used for stimulating and recording from the filaments. Recordings were made differentially (bandpass 10 Hz 1 khz) from pairs of filaments, to obtain the best signal-to-noise ratio for discriminating the activity of single units. Once a single pulpal unit had been isolated, it was stimulated electrically and changes in pulpal blood flow recorded. In those cases in which there was a change in blood flow, recordings were made from the unit during the application to dentine of a range of stimuli that cause pain in man. With some units, a difficulty was encountered in determining their electrical thresholds during stimulation of a filament. This was not a problem with most of the faster conducting pulpal fibres because it was possible to record an antidromic, all-or-none potential from the tooth with the dentine electrodes when the threshold of the unit was exceeded (Horiuchi & Matthews, 1974). With slower conducting Ad and C-fibres, the single unit potentials cannot usually be resolved in the dentine record, even with averaging. In such cases, an alternative strategy for determining a unit s threshold was adopted. Method for determining the threshold of a single pulpal axon in a dissected nerve filament The method is based on the collision technique and depends on being able to record from a filament within half a millisecond or so after applying an electrical stimulus to it. Technically, it is not difficult to switch the electrodes this rapidly but, with wire electrodes, the stimulus tends to leave the electrodes polarized and we have been unable to discharge the resultant voltage difference between the electrodes and avoid overloading the amplifier in time for the small unitary potentials to be recorded. The solution was to use a symmetrical pair of Ag AgCl wick electrodes (nonpolarizing and having a large area of metal electrolyte contact) and place a filament on each. One filament contained the unit under study and the other was selected to match its resistance. Wick electrodes were constructed from dental paper points placed inside glass micropipettes. The tip (length 10 mm) of a dental paper point (Kent Dental, Gillingham, Kent, UK; size 10) was pushed into a glass micropipette to form a tight fit at the shoulder. The pipette tip was then broken back to expose just the end of the paper point and the pipette was filled with Ringer solution. Electrical contact with the Ringer solution was made with a chlorided silver wire. The shank of the electrode was sealed with wax to prevent drying. Electrical stimuli (50 ms duration) were applied between an indifferent electrode (earth) and both electrodes plus filaments in parallel. Differential recordings were made between the two electrodes (Fig. 2). A pair of optically coupled switches (International Rectifier, El Segundo, CA, USA; type PVA3354) was used to connect the stimulator to the electrodes during a stimulus pulse (Fig. 2). The switches also shorted together the amplifier inputs during the stimulus. Even with this system, the mismatch in electrode polarization could be sufficient to interfere with unitary recordings, particularly with fine, high resistance filaments. In such cases, a potentiometer was used to place a resistance of between 1 and 6 MV in parallel with each filament (Fig. 2) to balance the voltage at the amplifier inputs. The balancing potentiometer was switched into the circuit and adjusted so that the settling time of the amplifier after a stimulus was minimal. The procedure used to measure the threshold of a unit was first to identify the unit in a filament by recording its response during electrical stimulation of the tooth. Then a suprathreshold stimulus of constant intensity was applied to the tooth and the method outlined above used to apply a stimulus of variable intensity to the filament immediately before each stimulus to the tooth. When the stimulus to the filament excited the unit, and the interval between the stimuli was less than the conduction time and refractory period of the unit, the unit s action potential was absent from the response evoked from the tooth (Fig. 3). Figure 1. Diagram of the method used to record from and to stimulate single fibres innervating the pulp of a lower canine tooth in cats A laser-doppler flow meter was used to record blood flow in the pulp of the tooth during electrical stimulation of single fibres in filaments dissected from the inferior alveolar nerve. R/S: recording from, and stimulation of, intradental nerve fibres.

3 J. Physiol Vasomotor fibres in tooth pulp 923 A unit was considered to affect pulpal blood flow if stimulation of the filament at an intensity just above the threshold of the unit (50 ms duration) at 1 or 10 Hz for 30 s evoked reproducible changes in the blood flow signal. To confirm that the effects were due to the unit, the stimuli were also applied just below the unit s threshold. Non-electrical stimulation of the tooth The properties of the sensory receptors associated with an identified vasomotor fibre were determined by recording from the filament while stimulating the exposed dentine with hot (55 C) and cold (5 C) Ringer solution, 4 M dextrose at 30 C, hydrostatic pressures in the range ± 500 mmhg applied through Ringer solution, mechanical stimuli using the fire-polished tip of a glass probe (tip diameter 0.5 mm), and by drying the dentine surface with a stream of air. Recordings of the temperature of the solutions in the cap were obtained with a thermocouple (Fig. 1). The duration of test stimuli was limited to 3 5 s to minimize cumulative damage and/or sensitization of the preparation. Data acquisition Records of neural activity, blood pressure, blood flow and temperature were displayed on an oscilloscope and digitized with a computer interface (Cambridge Electronic Design, Cambridge, UK; model 1401) for storage and analysis. Each channel was sampled at either 5 or 12 khz with 12-bit accuracy. RESULTS The effects of surgery and of electrical stimulation of the whole IAN on pulpal blood flow The average resting blood flow signal recorded from the canine tooth pulp prior to exposure of the ipsilateral IAN was 7.8 perfusion units (P.U.; range , S.D. 2.6 P.U., n = 17). Recordings were interrupted during surgery but 30 min or more after exposure and section of the nerve, the blood flow signal had decreased significantly to an average of 5.4 P.U. (range , S.D. 2.5 P.U., n = 15; P < 0.05, Student s paired t test). In the remaining two preparations, the reduction in blood flow signal was so great (resting levels < 1 P.U.) that the experiment was terminated. In all 15 preparations it was possible to record an increase in pulpal blood flow in the canine tooth during electrical stimulation of the whole IAN. Since this response was not associated with an increase in systemic arterial blood pressure, it was assumed to be due to pulpal vasodilatation. Its threshold was in the range V (mean 2.9, S.D. 0.9 V) with stimuli of 0.1 ms duration. In these preparations, recordings of the compound action potential from the tooth showed that at the threshold of the vasodilatation a group of nerve fibres with Ad conduction velocities were recruited, as previously reported (Vongsavan & Matthews, 1992a). An example of the effect of stimulus intensity on pulpal blood flow is shown in Fig. 4. Electrical stimulation of the nerve at 1 Hz and at intensities that were supramaximal for the A-fibres, evoked increases in blood flow signal in the range % above resting levels (mean 76.9, S.D %). In each preparation, neither the amplitude nor the duration of the increase in blood flow evoked by stimulation at 10 Hz was significantly different from that evoked by stimulation at 1 Hz (P > 0.05, Wilcoxon matched pairs test). Since it is not possible to resolve a C-fibre component in the compound action potential recorded under these experimental conditions, even with averaging and high intensity stimulation of the inferior alveolar nerve (Cadden et al. 1983), it was not possible to relate the magnitude of the vasodilatation to the recruitment of C-fibres. Figure 2. Block diagram of the method used to stimulate and, near simultaneously, record from single pulpal nerve fibres in filaments dissected from the inferior alveolar nerve A stimulus could also be applied to the tooth at a variable interval after the stimulus to the filament. The method enabled the threshold of the unit to electrical stimulation of the filament to be determined using the collision technique (see text for details). Amp, amplifier; CRO, cathode ray oscilloscope; gnd, ground (earth).

4 924 D. Andrew and B. Matthews J. Physiol Figure 3. Illustration of the method used to determine the electrical threshold of a single pulpal fibre in a filament dissected from the inferior alveolar nerve Top trace, response recorded from the filament when the tooth was stimulated with a single pulse of 1 ma in intensity and 0.1 ms duration. The stimulus was applied at the start of the trace. The conduction distance was 34 mm. The conduction velocity of the unit was 1.2 m s _1. Middle trace,record from the same filament. The same stimulus was applied to the tooth but 20 ms before this, a 1.5 V, 50 ms stimulus was applied to the filament. The stimulus to the filament did not excite the unit as there was no collision and the response from the tooth was recorded as before. The stimulus to the filament was applied at the start of the trace. Bottom trace, same as middle trace but the intensity of the stimulus to the filament was increased to 2 V, 50 ms. The response from the tooth was blocked as a result of collision with an impulse evoked in the same fibre in the filament. The threshold of the unit in the filament was therefore between 1.5 and 2 V. A and B refer to stimulation (Stim) and recording (Rec) points. Properties of single pulpal units Data were obtained from 93 single pulpal nerves (conduction velocity, CV m s _1 ) in eight cats. Twenty were Ab fibres (CV m s _1 ), 52 were Ab or Ad fibres (CV m s _1 ) and 21 were C-fibres (CV m s _1 ). Fibres in the intermediate group were classified as Ab or Ad to allow for slowing of conduction in the nerve terminals in the pulp (see Cadden et al. 1983). On electrical stimulation, ten of the C-fibres (CV m s _1, mean 1.8, S.D. 0.4,m s _1 ) produced an increase in pulpal blood flow and four (CV 0.9, 1.5, 1.5 and 1.7ms _1 ) produced a decrease in blood flow. Stimulation of none of the 72 fibres with conduction velocities in the Ab or Ad ranges produced a significant change in blood flow. The maximum number of C-fibres found in any one experiment was four, and the largest number that produced vasodilatation was two. The thresholds to electrical stimulation of the 10 units that evoked an increase in blood flow were V (mean 3.9, S.D. 2.1 V) at a stimulus duration of 50 ms. There was no consistent relationship between a unit s threshold in a dissected filament and its conduction velocity: C-fibres could have thresholds below those of Ab fibres. This was a cause for some confusion in seven preliminary experiments Figure 4. Graph showing the relationship between stimulus intensity and blood flow recorded from the pulp of a cat canine tooth Trains of electrical stimuli (0.1 ms stimulus duration, 1 per s for 30 s) were applied to the peripheral cut end of the inferior alveolar nerve. The blood flow record obtained at each stimulus intensity was integrated with respect to time for the duration of the response and expressed as a percentage of a corresponding length of record obtained under resting conditions. The fall off in the response at high stimulus intensities can be attributed to the recruitment of postganglionic sympathetic vasoconstrictor fibres.

5 J. Physiol Vasomotor fibres in tooth pulp 925 in which we did not use high intensity electrical stimulation of the tooth that would excite C-fibres in order to avoid producing prolonged pulpal vasodilatation. In those experiments it sometimes appeared that electrical stimulation of Ab fibres caused vasodilatation. The median latency of the blood flow increase evoked by single unit stimulation at 1 Hz was 6.7 s (range , S.D. 2.7 s). This was not significantly different from the corresponding value with stimulation of the same unit at 10 Hz (median 6.3, S.D. 3.0 s; P > 0.05, Wilcoxon matched pairs test) or with stimulation of the whole IAN at 1 or 10 Hz (1 Hz: median 7.0, S.D. 1.6 s; 10 Hz: median 6.1, S.D. 2.1 s; P > 0.1 for both stimulus frequencies, Wilcoxon matched pairs test). The increases in the blood flow signal that resulted from electrical stimulation of these units at 1 Hz ranged from 7.1 to 70.4 % (median 21.7, S.D %) of the resting levels. The effect of stimulation at 10 Hz was not significantly different from that at 1 Hz (median 26.4, S.D %; P > 0.1, Wilcoxon matched pairs test). These responses to single unit stimulation represented between 12.4 and 63.8 % (median 38.3, S.D %) of the effect produced by supramaximal stimulation of the whole IAN. There was no significant difference in this respect between the effects of stimulation at 1 and 10 Hz (P > 0.08, Wilcoxon matched pairs test). An example of a unit that produced vasodilatation is shown in Fig. 5. Of the ten units that evoked vasodilatation, five responded to the application of the hot stimulus to dentine but not to the other forms of non-electrical stimulation tested. The average latency of the responses to heating was 1.9 s (range , S.D. 0.6 s). The remaining five units did not respond to any form of non-electrical stimulus. The response of the unit shown in Fig. 5 to thermal stimulation of dentine is shown in Fig. 6. Note that the brief hot stimulus did not produce a change in pulpal blood flow. The four C-fibre units that evoked vasoconstriction were presumed to be sympathetic, post-ganglionic fibres. Electrical stimulation of these units at 1 Hz reduced the blood flow signal to 67.5, 71.2, 78.1 and 81.5 % of the corresponding resting values. These changes are significant (P = 0.02, Wilcoxon matched pairs test). Stimulation of the same filaments at 10 Hz produced greater effects, to 48.4, 41.9, 52.8 and 58.5 %, respectively, of resting values. These values are significantly different from the corresponding values with stimulation at 1 Hz (P = 0.009, Mann Whitney U test). An example of the effects of electrical stimulation of one of these presumed sympathetic Figure 5. Record of the effects on pulpal blood flow of electrical stimulation of a single intradental nerve fibre The unit was the same one as shown in Fig. 3. The traces are, from the top downwards, the record from the filament (stimulus artefacts), arterial blood pressure, and pulpal blood flow. During the period indicated by the bar, the filament was stimulated with electrical pulses of 2 V intensity, 50 ms duration at 10 Hz. There was no change in pulpal blood flow when this procedure was repeated but with the stimulus intensity reduced to 1.5 V.

6 926 D. Andrew and B. Matthews J. Physiol Figure 6. Response of a single vasodilator pulpal nerve fibre to thermal stimulation of dentine Response of the same unit as in Figs 3 and 5 to thermal stimulation of dentine. The traces are, from the top downwards, multi-fibre activity recorded from the tooth, single unit activity recorded from a filament dissected from the inferior alveolar nerve, the temperature of the Ringer solution in the tooth cap, and pulpal blood flow. The unit responded to the hot but not the cold stimulus. Figure 7. Example of a pulpal vasoconstrictor nerve fibre Record of the effects on pulpal blood flow of electrical stimulation of a filament containing a single pulpal C-fibre (CV 0.9 m s _1 ; threshold 8.0 V, 50 ms). The traces are, from the top downwards, the record from the filament (stimulus artefacts), arterial blood pressure, and pulpal blood flow. During the period indicated by the bars, the filament was stimulated with electrical pulses of 8 V intensity, 50 ms duration at 1 and 10 Hz. There was no change in pulpal blood flow when the stimulus intensity was reduced below the threshold of the unit.

7 J. Physiol Vasomotor fibres in tooth pulp 927 vasoconstrictor fibres is shown in Fig. 7. None of these units responded to any form of non-electrical stimulation of dentine. DISCUSSION These experiments demonstrate that it is possible to record either an increase or a decrease in pulpal blood flow by electrical stimulation of single pulpal nerve fibres in the cat. Since there was no associated change in arterial blood pressure, it can be assumed that these effects were due to pulpal vasodilatation or vasoconstriction. The relatively large proportion of the vasodilatation evoked by stimulation of the whole IAN that could be evoked by single fibre stimulation was surprising. This could be explained if, due to the low compliance of the pulpal micro-circulation, there was a maximum vasodilatation that could be produced by the release of neuropeptides following antidromic stimulation and that this maximum effect could be achieved by only a small amount of neuropeptide, not much greater than the quantity released from the terminals of some single units. The volume of the pulp of a lower canine tooth in a young adult cat is approximately 4.2 ml. An alternative explanation is that only a small number of fibres in any one tooth are capable of producing significant vasodilatation. But in that case, we would have to have been very lucky to have found them in the small sample that we isolated. The number of true C-fibres (i.e. those with non-myelinated axons throughout their length, see Cadden et al. 1983) is not known. From the sample we obtained, it would appear that a high proportion of pulpal C-fibres are capable of affecting blood flow in dental pulp. The other possible explanation, that whole nerve stimulation activated both vasodilator and vasoconstrictor fibres and thus produced less vasodilatation than would have occurred with vasodilator stimulation alone, is unlikely since Olgart et al. (1991) found that the vasodilatation produced by electrical stimulation of tooth pulp was not increased following chronic sympathectomy. This is also consistent with the effects we observed with graded stimulation of the inferior alveolar nerve (see Fig. 4). The observation that some of the units that produced vasodilatation responded to the application of painproducing stimuli to dentine suggests that they formed part of an axon reflex or axon response mechanism. Consistent with this, Olgart et al. (1991) showed that topical applications of capsaicin or bradykinin to pulp, which are known to excite pulpal C-fibres (Närhi, 1985), produced pulpal vasodilatation in normal, but not in chronically denervated teeth. Heat stimulation did not cause vasodilatation in the present experiments but this can be accounted for by the very short duration of these stimuli. In other experiments (V. Sirimaharaj & B. Matthews, unpublished observations) 30 s heat stimuli have been shown to increase pulpal blood flow. Antidromic stimulation of heat-sensitive pulpal afferents for 30 s in the present experiments also produced vasodilatation. All of the single fibres that produced a detectable vasodilatation had conduction velocities in the C-fibre range. It was surprising that none of the Ad fibres caused vasodilatation since, during electrical stimulation of the whole IAN, the threshold for vasodilatation coincided with that of fibres in the Ad range, as reported previously (Vongsavan & Matthews, 1992a). Also Matthews & Vongsavan (1994) have shown that mechanical stimulation of exposed dentine, which selectively activates A-fibres (Närhi, 1985), evokes pulpal vasodilatation in cats, and many of the nerve terminals in dentinal tubules that are assumed to be sensitive to mechanical stimuli, contain calcitonin gene-related peptide, CGRP (Heyeraas et al. 1993). Olgart et al. (1991) also showed that drilling outer dentine produced vasodilatation in control but not in chronically denervated teeth. Maybe, like the C-fibres, there is only a small number of Ad fibres that evoke vasodilatation in any one tooth and these were not included in the sample isolated in the current experiments. Alternatively, there may have been overlap in the thresholds of Ad and C-fibres when the whole IAN was stimulated, so that some C-fibres were recruited at the threshold of vasodilatation. The response of a small number of C-fibres would not be detectable in the compound action potential recorded from a tooth. Jacobsen & Heyeraas (1997) demonstrated a resting vasodilator tone in ferret dental pulp that was abolished by sectioning the inferior alveolar nerve but not by cervical sympathectomy, indicating that it was of sensory origin. Since pulpal afferents do not discharge impulses spontaneously, and since pulpal blood flow does not change following acute denervation once the acute effects subside (Vongsavan & Matthews, 1992a), it appears that the resting vasodilator tone observed by Jacobsen & Heyeraas may be due to chemical mediators released spontaneously from peripheral sensory nerve terminals. Lisney and colleagues have made similar observations on vasodilatation produced by Ad and C-fibre stimulation in rat skin: Jänig & Lisney (1989) showed that electrical stimulation of the peripheral end of the cut saphenous nerve, at intensities that evoked an Ad wave but not a C-wave in the compound action potential, produced a small transient increase in blood flow. Kolston & Lisney (1993) extended this study by recording blood flow while stimulating filaments that contained single Ad fibres. They studied 39 fibres and none produced a detectable increase in skin blood flow. To account for these results, they suggested that the amount of neurotransmitter released by a single Ad fibre might be too small to produce a significant relaxation of local arteriolar smooth muscle, and that simultaneous activation of several such units was necessary

8 928 D. Andrew and B. Matthews J. Physiol to produce a detectable increase in blood flow. Such an explanation could also account for the results of the present experiments. Lynn et al. (1996) and Gee et al. (1997), who used the method developed for the current experiments to determine the electrical thresholds of single units in nerve filaments, demonstrated that stimulation of single C-fibres could produce detectable vasodilatation in skin. Lynn et al. (1996) showed that, in the pig, antidromic stimulation of single, heat-specific nociceptors produced vasodilatation in the area of the receptive field of the fibre and that stimulation of other classes of C-fibre had no such effect. However, Gee et al. (1997) showed that vasodilatation was also produced by polymodal nociceptors in rats and rabbits. While half the C-fibres that produced vasodilatation in the present experiments were heat sensitive, such fibres have also been shown to respond to chemical stimuli (Närhi et al. 1992). Of the four vasoconstrictor units identified, none responded to non-electrical stimulation of dentine. This is consistent with them being sympathetic fibres without sensory receptors. The observation that pulpal blood flow had decreased when recording resumed after exposure and section of the IAN, and in some cases had ceased altogether, indicates that, despite great care to avoid damage to the inferior alveolar artery, the surgery interfered with the arterial supply to the tooth. This observation indicates that blood flow in tissues supplied by the inferior alveolar artery should be recorded routinely before and after exposure of the IAN. REFERENCES ANDREW, D. & MATTHEWS, B. (1996). Properties of single nerve fibres which evoke antidromic vasodilatation in cat dental pulp. Journal of Physiology 491.P, 27P. ANDREW, D., MATTHEWS, B. & COATES, T. W. (1996). A method for determining the electrical thresholds of single fibres in dissected nerve filaments. 493.P, 6 7P. CADDEN, S. 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Antidromic vasodilatation in the dental pulp and lip of cats. Journal of Dental Research 71, 534. VONGSAVAN, N. & MATTHEWS, B. (1993). Experiments on extracted teeth into the validity of using laser Doppler techniques for recording pulpal blood flow. Archives of Oral Biology 38, Acknowledgements This work was supported by the Wellcome Trust.