641

J. Physiol. (1977), 264, pp. 641-664 With 14 text-figure. Printed in Great Britain

RESPONSES OF INTRADENTAL NERVES TO ELECTRICAL AND THERMAL STIMULATION OF TEETH IN DOGS

BY B. MATTHEWS From the Department of Physiology (Oral Biology), The Medical School, University of Bristol, University Walk, Bristol BS8 1TD

(Received 20 January 1976) SUMMARY

1. Experiments were carried out to investigate the mechanism whereby thermal stimuli excite nerves to produce pain from teeth. 2. Recordings have been made from single fibres dissected from the inferior dental nerve in dogs during thermal stimulation of the lower canine tooth. 3. In preliminary experiments, no units were found with thresholds close to the thresholds for pain in man (45 and 270 0) and subsequently, test stimuli of 55° 0, applied for up to 15 sec, and O-50 C were used. 4. Of 117 fibres tested, forty-three responded to cooling but not to heating and nine responded to heating but not to cooling. 5. By applying thermal stimuli direct to the saphenous nerve in cats, it was shown that these responses might have been due to direct excitation of nerves and not to stimulation of specialized receptors. 6. Some units responded to electrical stimulation of the tooth pulp with a latency which decreased abruptly at a critical intensity as the stimulus was increased above threshold. Evidence was obtained which suggested that this was due to branching of the fibres. INTRODUCTION

Hot and cold stimuli applied to teeth in man produce pain and no other sensation, provided the stimuli do not excite receptors in the gingival or periodontal tissue (Hensel & Mann, 1956). With stimuli applied to the enamel, the thresholds for pain are approximately 45 and 270C. The exact location of the receptors responsible for the pain is not known although much of the evidence indicates that they must be either in the inner dentine or in the surface layers of the pulp (Anderson, Hannam & Matthews, 1970). The experiments to be described were carried out in dogs to determine

642 B. MATTHEWS how individual nerves from the dentine or pulp respond to thermal stimulation of dentine at intensities that would cause pain in man. The experiments proved difficult and the results to be reported have been accumulated over several years. As well as searching for fibres that responded to thermal stimulation of teeth, experiments have also been carried out to investigate the mechanism responsible for the latency of the response to electrical stimulation of some units varying according to the stimulus strength. The possibility that some of the responses to thermal stimulation might have been due to direct excitation of nerve fibres, rather than to stimulation of receptors, has also been investigated. Previous studies (Pfaffmann, 1939; Wagers & Smith, 1960; Funakoshi & Zotterman, 1963; Yamada, Suzuta & Higuchi, 1968) have established that thermal stimulation of teeth in cats and dogs evokes responses in dental nerves but little information is available on the properties of individual units. Brief reports on some of the early experiments in the present series have been given elsewhere (Matthews, 1967, 1968, 1969). METHODS

Response8 to thermal stimulation of dentine. The experiments were carried out on adult dogs which were anaesthetized with pentobarbitone sodium. Core temperature was maintained at 370 C with an electric blanket around the animal. Recordings were made from functional single fibres dissected from the inferior dental nerve while thermal stimuli were applied to the lower canine (Fig. 1). The inferior dental nerve was exposed by removing bone from the lower border of the mandible between the posterior mental foramen and the anterior border of the masseter muscle. The skin flaps were sutured to a ring to form a pool which was filled with warm liquid paraffin to cover the nerve. The branches of the inferior dental nerve were cut centrally and lifted on to a small dissecting platform within the pool. Fine filaments were dissected from the nerve and recordings made from them using fine (0-1 mm diameter) platinum wire electrodes and Tektronix 122 or 2A61 preamplifiers. The filaments were subdivided until a single all-or-none action potential was recorded in response to electrical stimulation of the coronal pulp of the lower canine tooth. Units isolated this way were then tested with thermal stimulation of the tooth. Thermal stimuli were applied by passing isotonic saline at different temperatures through a small water-jacket which enclosed most of the crown of the tooth (Fig. 1). The water-jacket was constructed from a piece of Perspex tube which was adapted to fit the tooth accurately with self-curing acrylic resin. A collar of enamel 2 mm wide was left uncovered around the neck of the tooth. Once the acrylic had set, the water-jacket was removed and short lengths of 1-5 mm diameter stainless steel tubing were fixed into it to attach inlet and outlet pipes and a thermocouple was incorporated to record the temperature of the stimulating solutions. Using a slowrunning bur under a constant stream of isotonic saline, the enamel and outermost layer of dentine were removed from the whole crown except for the tip of the cusp and a 3-4 mm wide collar around the neck of the tooth. The water-jacket was then fixed in place with oxyphosphate cement which was applied to the areas of contact between plastic and enamel at the tip of the cusp and around the cervical collar.

INTRADENTAL NERVES

643

A pair of silver wire stimulating electrodes (0.2 mm diameter) were applied to the surface of the pulp in the floor of a cavity which was prepared on the labial surface of the tooth in the space between the water-jacket and the gingival margin (Fig. 1). The wires were held in place by fixing them with wax to the lower rim of the waterjacket. The cavity was deepened until just a thin layer of dentine remained and the red glow of the pulp could be seen over the whole of the pulp's diameter. Using a small chip of razor-blade under a dissecting microscope, two small holes were cut through the remaining dentine to expose the pulp at the mesial and distal ends of the cavity and the electrodes were adapted so that their ends rested gently on to the pulp surface. The cavity was filled with liquid paraffin and checked periodically during the experiment. This arrangement for thermal and electrical stimulation allowed the tooth to be moved in its socket for the identification of periodontal mechanoreceptor units.

Water-jacketThermocouple

W

a

g

s

~~~~~~Dentine

Inferior dental nerve

Fig. 1. Diagram of the preparation. S, silver wire stimulating electrodes; R, recording electrodes. The solution which flowed through the water-jacket was selected with four solenoid-operated valves positioned with their common outlet within 2 cm of the tooth. The valves operated by compressing and occluding tubing so that there was no electrical contact with the solutions inside. The solutions used were: isotonic saline at 370 C (the control), 550 C and 0-50 C, and 6 molal CaCl2 at 370 C. The calcium chloride solution was included since in human experiments it had proved to be a potent pain-producing stimulus which appeared to act by an osmotic effect (Anderson & Matthews, 1967). However, it also had a desensitizing effect on human dentine and therefore its application to dentine in dogs was confined to one experiment in which it was the only solution used and to the latter part of a few of the other experiments. To ensure that the solutions reached the tooth at the correct temperature, they were passed from reservoirs to the corresponding valves through counter-current heat exchangers. Each heat exchanger consisted of three concentric polyvinylchloride tubes (Portex nos. 8, 23 and 40H), 6 ft. in length. The test solution flowed from a head of 4 ft. through the centre tube while water at the appropriate temperature

644

B. MATTHEWS

was pumped towards the tooth in the intermediate tube and back in the outer tube. This arrangement ensured that the solutions were electrically isolated from the pumps and heaters and that the temperature of the solution reaching the tooth was constant even when the flow was intermittent. With other forms of thermal stimulation that were tried, lack of adequate electrical isolation affected the excitability of the nerve terminals in the teeth as well as causing interference in the recording system. In some of the initial experiments, a 0 5 mm diameter hole was drilled through the end of the water-jacket and into the tip of the cusp to expose the cornu of the pulp. A fine thermocouple (diameter 75 sm) was inserted into the pulp to record the temperature changes there during thermal stimulation of the overlying dentine. Latency changes with electrical stimulation. The latency of the response of some units to electrical stimulation changed with stimulus strength. To investigate the mechanism responsible for this, animals were prepared as described above except that the construction of the water-jacket and removal of underlying tooth substance was omitted. In some animals, a second pair of stimulating electrodes were applied to the pulp midway along the root of the tooth. For this, the mucous membrane was reflected from over the root, bone was removed and a cavity like that in the crown was prepared to expose the pulp. Thernmal stimulation of nerves. The response of nerves to direct thermal stimulation was investigated using a saphenous nerve preparation in cats. The cats were anaesthetized with sodium pentobarbitone and the saphenous nerve was exposed from the top of the leg to below the knee. The nerve was cut centrally and peripherally. Recordings were made from filaments dissected from its central end and a pair of stimulating electrodes were placed around the nerve peripherally. Approximately midway between the recording and stimulating electrodes, the nerve with its connective tissue sheath intact was dissected free from the adjacent tissues over a length of 1O mm and enclosed within a silver thermode (Fig. 2). The thermode consisted of a 8 x 8 x 3 mm block of silver with a 1 mm slot in one side just large enough to accommodate the nerve. An inlay fitted into the mouth of the slot to completely enclose the nerve. The thermode had an insulating layer of silicone rubber covering its external surface except for a narrow strip underneath. The thermode was earthed and the uninsulated area underneath provided the only earth reference of the animal. A thermocouple was incorporated in the thermode close to the nerve to monitor its temperature. The upper surface of the silver block was soldered to the ends of four brass tubes (diameter 3 mm) and these were also brazed to each other along their lengths. The tubes were interconnected in pairs at the surface of the thermode to provide two independent circulation channels. Hot water at 550 C was pumped through one channel and 10 % ethylene glycol in water at -5° C through the other to heat or cool the thermode. The pumps incorporated small d.c. motors and were controlled from servo-amplifiers according to the difference between the thermode temperature and the required temperature, as indicated by a reference signal. The system was used to apply stimuli in the range 5-48' C. The thermode was supported with a rod fixed with an insulating spacer to the lower ends of the tubes. In positioning the thermode and the stimulating electrodes, care was taken to ensure that these did not interrupt blood flow through the small vessels in the connective tissue around the nerve. The space around the nerve in the thermode was filled with Ringer solution and the nerve and thermode were covered in warm liquid paraffin. Vaseline was injected around each end of the thermode to minimize convection currents. Preliminary experiments (Matthews, 1969) were carried out using a thermode of slightly different specifications. In all but one of the experiments, the selection of fibres was biased towards those

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645

INTRADENTAL NERVES

which responded to thermal stimulation. The general procedure adopted was that a fine filament was teased from the nerve and recordings made from it during heating and cooling of the nerve for 15 see each. If either stimuli produced a response, the filament was subdivided as necessary to identify the response of a single unit, or in the case of C fibres, a small group of units. The conduction velocities of the fibres at 370 C and the temperature at which conduction block occurred during cooling were also measured.

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-NervE - Silver

11

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Insulation7 0 1 2mm

Fig. 2. Diagram of the thermode. The inlay was removed from the mouth of the slit to allow the thermode to be placed around the nerve. The thermode was supported by a bar (not shown) which was attached with an insulated spacer to the lower ends of the brass tubes. The thermode was brought into position around the nerve with a micromanipulator. This was done with a dissecting microscope with care to avoid tension or compression on the nerve or any interruption in its blood supply. RESULTS

The criteria used to identify an intradental fibre in the inferior dental nerve were: (1) an all-or-none response to bipolar electrical stimulation of the tooth pulp using a stimulus of 1 msec duration and less than 10 V amplitude; and (2) no response to mechanical stimulation of the tooth or adjacent mucous membrane. In fact, none of the units that responded to the electrical stimulus also responded to mechanical stimulation. Furthermore, periodontal mechanoreceptor units were shown not to respond to a 100 V, 1 msec, bipolar, pulp stimulus. These results are consistent with those of previous studies (Greenwood, Horiuchi & Matthews, 1972; Horiuchi & Matthews, 1974). The term 'pulpal fibre' will be used to refer to any fibre which responded to electrical stimulation of the coronal pulp although some of the fibres may have been passing through the pulp to terminate in dentine.

B. MATTHEWS In preliminary experiments on the effects of thermal stimuli, no pulpal nerves could be found which responded to stimulation of either enamel or cavities cut into dentine with saline at temperatures down to 150 C or up to 500 C, although the thresholds for pain in man are within these limits. The technique which appeared to give the best chances of evoking a response, without producing irreversible damage, was to use cold stimuli of 0-5° C and hot stimuli of 550 C, the latter being applied for no longer than 15 sec at a time and to apply the stimuli to as large an area of the crown as possible. Although all the alternatives were not investigated, this technique was found to give some positive results and was therefore used as the standard test procedure. No attempt was made to define the threshold of units by finding the minimum temperature change which evoked a response. When a thermal stimulus of 55 or 00 C was applied, more than 90 % of the final change in water-jacket temperature occurred within the first second of the stimulus. Simultaneous measurements of water-jacket and pulp temperature in some of the early experiments showed that, with a 550 C stimulus, the pulpal temperature was approximately 440 C after 3 sec, 48° C after 5 sec, 500 C after 7 see and 52° C after 12 sec. With a 00 C stimulus, the corresponding values were: 220 C after 3 see, 14° C after 5 see, 10° C after 7 see and 70 C after 12 sec. Of 117 pulpal units tested in seventeen dogs, fifty-two responded to thermal stimulation. These thermo-sensitive units were of two main types, forty-three which responded to cooling and nine which responded to heating. A typical example of a unit that responded to cooling is shown in Fig. 3. The response to a 50 C stimulus of 15 sec duration (Fig. 3A) consisted of a short latency discharge which ceased after less than 4 sec and a single impulse during re-warming. From data obtained in other experiments it would be expected that the pulpal temperature had fallen to approximately 290 C when the first impulse was recorded. These units generally gave poorly reproducible responses; for example, with one unit, repeated applications of a 15 sec cold stimulus at 4 min intervals gave total impulse counts of: 6, 0, 0, 4 and 2. There was a progressive decrease in the mean number of impulses evoked if less than 4 min was allowed between stimuli. To obtain a response to cooling with the tooth initially at 370 C, it was necessary to achieve a rapid temperature change in the waterjacket. These units only rarely discharged impulses during heating above 370 C but in every case when a cold stimulus was applied after a hot stimulus (Fig. 3b) the response to cooling was much greater than that obtained with cooling from 370 C. Even brief heating for 4 sec was sufficient to potentiate the response; for example, with the unit referred to 646

INTRADENTAL NERVES 647 above, preceding the cold stimulus each time with a 5 sec hot stimulus gave total impulse counts of 31, 11, 9, 11 and 12. The response sometimes began before the water-jacket temperature record had fallen below 370 C, indicating that, with a large temperature gradient across the dentine and A

I sec

B

55. oc

37 5s

]0-5 mV 1 sec

Fig. 3. A, response of a single pulpal fibre to a 15 sec cold stimulus. B, response of the same unit when the cold stimulus was preceded by a 5 sec hot stimulus. Spikes re-touched.

rapid cooling, these units were capable of responding at surface temperatures above 370 C. There was no discharge from any of the units with the tooth equilibrated to 370 C. A typical example of a unit that responded to heating is shown in Fig. 4.

648 B. MA TTHE WS In response to a 55° C stimulus of 12 see duration (Fig. 4A) a high frequency, irregular discharge appeared with a latency of 7 see and continued for 3 see after the water-jacket temperature had returned to 370 C. The latency of the responses varied from 6 to 12 see in different units,

A

1 sec

B

55

-

37

-

°c

1 sec

02 mV

Fig. 4. A, response of a single fibre to a 12 sec hot stimulus. B, response of same fibre when the hot stimulus was preceded by a 12 sec cold stimulus. Spikes re-touched.

corresponding with pulp temperatures of 49-52° C. The responses often consisted of irregular high frequency bursts of impulses which were not synchronized to the heart-beat. In general, the responses tended to be more reproducible than those of the units that responded to cooling although repeated stimulation at intervals of less than 4 min also resulted

INTRADENTAL NERVES 649 in a progressive decrease in the number of impulses evoked. The continuation of the discharge after the stimulus was removed appears to have been due to lag in the temperature change at the site at which the impulses were generated and not, as was thought earlier (Matthews, 1967), to the cooling of the dentine surface. These units never responded to cooling from 37 to 50 C and a 50 C stimulus applied after a hot one reduced the after discharge. Cooling the tooth also reduced the response to subsequent heating (Fig. 4B). Two of these units discharged occasional impulses when the tooth was equilibrated to 370 C. 20

15

10

Conduction velocity (mlsec) of the conduction velocities of 117 pulpal fibres tested 5. Distribution Fig. wihhot and cold stimuli: heat sensitive, stippled columns; cold sensitive, filled columns.

Twelve units in one experiment were tested only with 6 molal CaCl2 for 15 sec and none responded. In other experiments, five units which responded to cooling, one that responded to heating, and one that responded to neither cooling nor heating were tested with 6 molal CaCl2 and none

responded. The estimated conduction velocities of all the fibres tested with hot and cold stimuli are shown in Fig. 5. In those cases in which the latency of the response of a unit to electrical stimulation changed with stimulus strength (see below), the conduction velocity was estimated on the basis of the shortest latency.

B. MATTHEWS

650

A 48 V 50 52 5.4

56 2 mV

58 2 msec

B

1 4 mV

IT

4

msec

Ss

ss

Fig. 6. A, response of a unit to bipolar electrical stimulation of the pulp at the intensities shown. The latency changed from 8-2 to 4-2 msec. The stimulus duration was 50 ,llsec and the threshold of the unit was 4*8 V. B, responses recorded from the same unit when pairs of stimuli were applied at different intervals. In the upper trace, a stimulus (SI) adjusted to evoke a long latency response was applied 9 msec before a stimulus (S.) adjusted to evoke a short latency response. In each of the subsequent traces, S, was delayed by 1 msec, that in the last trace S. preceded S, by 5 msec. By recording the responses with smaller steps, it was shown that the shortest interval between stimuli which produced a long latency response followed by a short latency response was 6 1 msec (between traces 3 and 4). The refractory period of the short latency response was 1P7 msec and the conduction distance was 57 mm. so

INTRADENTAL NERVES

651

Latency changes with electrical stimulation An example of a unit which responded to electrical stimulation with different latencies, depending upon the stimulus strength, is shown in Fig. 6A. With a stimulus duration of 50,usec, the threshold of the unit was 4-8 V. As the stimulus was increased above this level, the latency changed little up to just above 5-2 V, at which point the latency decreased abruptly to a new stable value. It was not possible to obtain responses 100

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Fig. 7. The relationship between the shortest latency (L.) and the change in latency (L,- L.) for forty-nine units in which the latency of the response to electrical stimulation of the tooth decreased as the stimulus was increased above threshold. Both scales are logarithmic. For ten units in which there were three possible latencies: the decrease from the longest to the shortest is shown by a filled square and the decrease from the intermediate to the shortest is shown by a filled circle. For the other units where there was a single change, this is shown by a filled circle.

with intermediate latencies and both responses were never present with a single stimulus. When the stimulus was varied above and below the threshold of the short latency response, the latency changed in a stable,

reproducible manner. In the example in Fig 6A, the change was 4-0 msec, from 8-2 to 4-2 msec. In others, a change of up to 100 msee was recorded. The relationship between the short latency and the change in latency which occurred when the long latency response was replaced by the short latency response 22

PH Y

264

652 B.MATTHEWS in forty-nine units is shown in Fig. 7. Only those units have been included in which the change in latency was at least 1-0 msec and more than fivetimes the shortest stimulus duration that could be used to produce the change. The distribution of the conduction velocities of all 214 units isolated in these experiments and those in which thermal stimuli were applied, including the forty-nine units which showed a change in latency, are shown in Fig. 8. The range was 03-32-1 m/sec. The conduction velocities of the variable latency units have been calculated from their shortest latencies. With ten of these units, three or more stable latencies could be obtained at different stimulus intensities and in each case the latency decreased as the stimulus was increased. 35 30

-

252 20 .0

6 15

z

10

0

4

8

20 24 12 16 Conduction velocity (m/sec)

28

32

Fig. 8. The distribution of the conduction velocities of all the fibres studied in both groups of experiments on dogs. The conduction velocities of the variable latency units (shown by hatched columns) are based upon their shortest latencies. The rejection of units in which there was only a small decrease in latency with increase in stimulus intensity could account for the apparent lack of variable latency units at the upper end of the conduction velocity range.

With several of the units, a change in latency could only be obtained with stimuli 05 msec or longer in duration and, with short duration stimuli, the short latency response only could be produced. The strengthduration curves of the two responses in these nerves intersected, with the

INTRADENTAL NERVES 653 long latency response apparently having the higher threshold at short stimulus durations. Two possible explanations were considered to account for the change in latency: (a) that it was due to stimulus spread to a point proximal to the stimulating electrodes (Fig. 9A); or (b) that the fibres were branched (Fig. 9B). For these to account for the observations, the following assumptions had to be made. In (a), an increase in stimulus intensity did not cause the point of excitation to spread progressively along the nerve but produced a step-like shift in the point of excitation. This could have S

I

II

1

I

Fig. 9. Diagrammatic representation of the hypotheses to account for the variable latency responses: 1, site of origin of long latency response; 8, site of origin of short latency response; S, stimulating electrodes; and R, recording electrodes.

arisen if the nerve looped back on itself so that the points 1 and 8 were close together, or if there was a relatively low resistance path for current spread to the more proximal point. In (b), it would have to be assumed that the long latency pathway involved a branch which was either finer in diameter or longer than the short latency pathway. If it were of finer diameter, the fact that it had the lower threshold would have to be accounted for by it being closer to the stimulating electrodes. Also in (b), it would have to be assumed that an impulse propagated towards the branching point from I caused impulses to be propagated both towards the recording electrodes in the stem of the fibre and also antidromically in the other branch. Under these conditions, in neither (a) nor (b) would a single stimulus be expected to produce both short and long latency responses. In (a), as the stimulus intensity was increased from zero, a long latency response would be produced when the threshold at I was reached and this would be replaced by a short latency response when the stimulus was increased to exceed the threshold at 8. The impulse generated at I with a stimulus suprathreshold for 8 would collide with an antidromic impulse from 8. A similar sequence would occur in (b). To investigate which, if either, of these might be the mechanism responsible for the latency change, the interference between long and short latency responses was investigated by applying a stimulus adjusted to evoke a long latency response at various intervals before or after one adjusted to give a short latency response (Fig. 6B). The principal 22-2

654 B. MATTHEWS difference between the hypotheses which could be tested by this experiment was the minimum interval between stimuli that would produce a long latency (L1) response followed by a short latency (L.) response. In (a), this interval minus the refractory period of the nerve at s (RB) would be equal to the difference between the latencies of the two responses when evoked singly, whereas in (b) the minimum interval minus the refractory period at s would be greater than the difference between the latencies. This was investigated in twelve units and the results are summarized in Fig. 10. 50

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L, -L,(msec) Fig. 10. The relationship between the difference between long and short latencies (L, - L8) and the minimum interval between stimuli that produced a long latency response followed by a short latency response, minimum interval (L,+ L8), minus the refractory period at 8 (R.). The interrupted line is the line of equality. The closest point to the line represents the unit shown in Fig. 6.

In every case, the minimum interval minus the refractory period was greater than the difference between the latencies, indicating that the phenomenon was not due to stimulus spread from the electrodes to a more proximal point on the nerve. For eight of the units, the observations could be explained on the basis of the nerves being branched and the delay in each branch and the stem calculated. However, with two of the units the minimum interval between stimuli that would produce a long latency response followed by a short latency response, minus the refractory period at a, was greater than the sum of the short and long latencies. This would be impossible on the basis of the simple branched nerve hypothesis.

INTRADENTAL NERVES

655

A

37| °cC I sec

B

37

-]

10 L

1° 1 sec|

C

37 -l oc

~[I

I I mV

10 _e I sec|

Fig. 11. A, response of a single saphenous nerve fibre to cooling of the nerve to 15° C. B, response of the same fibre to cooling to 10° C. C, the 100 C stimulus was repeated at the same time as electrical stimuli were applied, once a second, peripheral to the thermode. This demonstrated that conduction block occurred at the time the discharge evoked by cooling ceased. Spikes re-touched.

656 B. MATTHEWS Observations were made on forty-six units in five dogs by stimulating the pulp in the crown and in the root. Eighteen of these units gave responses of more than one latency at either or both sites and in four of them a longer latency response could be obtained by stimulating the root than by stimulating the crown. All the possible combinations of collision experiments to investigate the interference between these responses was not undertaken but it was shown that, with stimuli applied simultaneously, a long latency response from the root was always blocked by a shorter latency response from the crown and visa versa. Estimates of the conduction velocities of the fibres indicated that the fibres tended to have significantly slower conduction velocities within the tooth than outside. 20

16

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z 8

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~ 12

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14 16 18 Temperature (0 C)

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Fig. 12. The relationship between the total number of impulses recorded from one unit and the lowest temperatures reached with 15 sec cold stimuli applied to the saphenous nerve. The fall off in the response at the lower end of the range was associated with conduction block during the stimulus.

Thermal stimulation of nerves A total of 194 fibres were isolated from the saphenous nerve in eight cats and tested with thermal stimuli. The characteristics of the responses were similar in many respects to those of pulpal nerves. Seventeen fibres were isolated that were excited by cooling but not by heating. Their thresholds ranged from 13 to 200 a and their conduction velocities, from 0'9 to 12 m/sec. The responses of one unit to cooling of the nerve from

657 INTRADENTAL NERVES 370 C to 15 and 10° C are shown in Fig. l lA and B. By stimulating the nerve electrically peripheral to the thermode at the same time as applying the thermal stimuli, it was shown that conduction through the cooled region had been blocked when the discharge evoked by cooling to 1O° C stopped (Fig. tIC). Unlike the dental units, the response of the units

A -1

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c

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m01mV 4 sec

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0-1 my 0 2 sec

Fig. 13. A, the response of a small group of C-fibres to a 470 C stimulus. There was no response to a 460 C stimulus. B, part of a response to a 470 C stimulus on a faster time base to show the irregularity of the discharge. The record was obtained from the same filament as that in A.

could not be potentiated by pre-heating the nerve and their threshold appeared to be independent of the rate of cooling at rates down to 10 C/sec. The relationship between the total number of impulses evoked by a 15 sec stimulus and the minimum temperature reached in one unit is shown in Fig. 12. Twenty units were isolated that responded to heating but not to cooling.

658 B. MA TTHE WS Their thresholds ranged from 45 to 470 C and their conduction velocities were all below 1-5 m/sec. The response of a small group of C-fibres to a stimulus of 470 C is shown in Fig. 13A. As the mean frequency of the discharge increased, the impulses tended to be grouped together in high frequency bursts (Fig. 13B), as was also seen in the responses of dental nerves. B

A 100

s0 .

20

6 z 40

.20 0

J 10

20

30

40

50 60 0 10 20 30 40 50 60 Conduction velocity (m/sec) Fig. 14. A, the distribution of the conduction velocities of 123 units tested with hot and cold stimuli in one animal. These fibres were selected prior to testing with thermal stimuli: cold sensitive, filled columns; heat sensitive, stippled columns. B, the distribution of all 214 units tested with thermal stimuli, including those in A. Only two of the units shown in the 0-5 mlsec range had conduction velocities greater than 1-5 m/sec. 0

In one animal, 123 units were isolatedwithout biasing the sample to those responding to thermal stimuli. Of this group, two responded to cooling and six to heating. The distribution of the conduction velocities of this group are shown in Fig. 14A, with those which responded to thermal stimulation indicated. This gives some indication of the proportion of the fibre in the nerve which responded to thermal stimulation. The distribution of the conduction velocities of all the 194 fibres tested in the whole series is shown in Fig. 14B, with those which responded to heating or cooling indicated. The mean blocking temperature of thirty-four myelinated fibres during slow cooling (0.10 C/sec) was 12 1' C (S.D. of observations 1.9). There was no significant correlation between the blocking temperature and the conduction velocity of the fibres. Many of the fibres with conduction

659 INTRADENTAL NERVES velocities less than 1-5 m/sec were not blocked at 50 C, the lowest temperature which could be maintained with the thermode. The slow rate of cooling used in determining blocking temperature only rarely evoked a discharge. DISCUSSION

It has been shown that there are two groups of intradental nerves which respond to thermal stimulation of dentine in dogs; one which is excited by cooling and another which is excited by heating. No response was obtained with either hot or cold stimuli from 56 % of the fibres tested. In similar experiments, Wagers & Smith (1960) recorded responses to hot and cold stimuli in a small number of pulpal nerves and isolated two single fibre preparations that responded to hot stimuli but not to cold and one that responded only to cold stimuli. They applied hot stimuli of up to 81° C and cold stimuli below 50 C to intact enamel. Funakoshi & Zotterman (1963) isolated sixteen fibres, mostly from deciduous teeth in dogs, which responded to hot stimuli. The thresholds of the fibres, expressed in terms of pulpal temperature, were from 45 to 650 C. One of these fibres only appears to have been tested with a cold stimulus and it did not respond. One multifibre preparation responded to cooling but not to heating. Scott and his colleagues (e.g. Scott, 1966) recorded from dentine in cats during thermal stimulation of the tooth and obtained responses which they attributed to intradental nerves. The temperature of the pulp in some of these experiments must have exceeded 58° C, since this temperature was reached on the opposite side of the tooth from the stimulus. In the present experiments the maximum temperature reached at the dentine surface was 550 C and the duration of the stimuli was limited to a maximum of 15 sec to minimize the risk of producing irreversible damage. When pulpal temperature was monitored, it reached 520 C at the end of such a stimulus. While this would be expected to cause some denaturation of protein, it was necessary to use a stimulus of this intensity to produce a response. Repeated stimulation produced no significant decrease or increase in the sensitivity of a unit as long as at least 4 min were allowed for recovery between stimuli. With some of the units there was a progressive decrease in the response with intervals less than 4 min. The observations on the thermosensitivity of saphenous nerve fibres correspond with the observations of von Euler (1947), Granit & Lundberg (1947) and Dodt (1953), except that no units were found that responded to cooling when the final temperature of the nerve was above 200 C (cf. Dodt, 1953). The mechanism responsible for the responses and the reason for the differences in properties between fibres of different diameter have not been established. In the present experiments, no correlation was

660 B. MATTHE WS possible between the thermosensitivity of a fibre and the properties of its receptors as the nerve trunk was cut peripherally. This was done to be sure that the responses recorded were not due to inadvertent stimulation of thermoreceptors. The responses of the saphenous nerve fibres to thermal stimulation were very similar to the responses recorded from dental nerves. Some responded to cooling and others to heating. The thresholds of the fibres which responded to heating corresponded approximately to the pulpal temperatures estimated to have been present at the time the first impulses were recorded from pulpal fibres during heating of the tooth form 370 C. On the other hand, the thresholds of the saphenous nerve fibres that responded to cooling were lower than the pulpal temperatures estimated to have been present when pulpal fibres responded to cooling the tooth from 370 C. The temperature at the pulp dentine junction would have been closer to the threshold of the saphenous nerve fibres. The responses recorded from pulpal nerves can therefore largely be explained on the basis of direct excitation of nerve fibres or endings in the pulp or close to the pulp-dentine junction, without involving any specialized receptor mechanisms. The principal difference between the saphenous and pulpal fibres was that the response of the dental nerves to cooling was potentiated by pre-heating the tooth, whereas this could not be demonstrated for the saphenous nerve fibres. It appeared that the rate of change of temperature or the temperature gradient across the dentine contributed to the response of the dental nerves to cooling. In some respects, these nerves had properties similar to the mechanosensitive fibres studied by Dodt (1953) which responded optimally to cooling to final temperatures close to 370 C. The correlation between the properties of the dental units studied in the present experiments and the pain sensation produced by thermal stimuli in man is not such as to permit the conclusion that fibres of the type found in the dog are responsible for pain in man. Hensel & Mann (1956) concluded that thermal stimuli produced pain in man by exciting structures in the dentine. They showed that stimuli of approximately 45 or 270 C applied to enamel produced pain after a long latency. With more intense stimuli, the latency of the pain was shorter and they predicted that under these conditions the temperature of the outer dentine at the onset of pain was always about 48 or 280 C. Hensel & Mann also produced evidence that, at threshold, the temperature of the pulp had changed by less than 1° . A stimulus of 400 C could be applied to the tooth surface for 2 min without causing pain and this produced a rise in pulpal temperature of 200. Naylor (1963) also showed that, with cold stimuli, the threshold for pain was reached before the pulp temperature had changed by 10 0. Attempts to find units with corresponding properties in the initial experiments of the

INTRADENTAL NER VES 661 present series were unsuccessful and it was subsequently shown that when heating or cooling a tooth from 370 C, pulpal nerves responded when the temperature of the pulp reached approximately 49 or 290 C. Pulpal temperature was not recorded in all the experiments because the inevitable trauma of inserting the thermocouple into the pulp. Histological evidence indicates that the innervation of dentine is sparse in all species (see Anderson et al. 1970; Matthews, 1976), and in view of this it has been suggested (Anderson et al. 1970) that movement of tubule contents and consequent excitation of nerve endings near the surface of the pulp might account for the short latency of pain with thermal stimuli in man as well as lack of correlation between the reaction time to a cold stimulus and dentine thickness (data from Naylor, 1963). However, the failure of 6 molal CaCI2 to excite nerves which responded to cooling in the present experiments suggests that the fibres were not excited by an outward movement of fluid through the dentinal tubules. Both stimuli have been shown to cause an outward movement of tubule contents in vitro (Horiuchi & Matthews, 1973). The method used to investigate the mechanism responsible for the change in latency of some pulpal fibres was similar to that used by Horrobin (1966) to investigate cells in the lateral cervical nucleus with branched axons in the medial lemniscus. Of the two alternative mechanisms considered in the present experiments, the branched fibre hypothesis fits the data best, although some features of the response suggest that this may not be the complete explanation. For example, some of the long latency responses indicate conduction velocities down to 0*2 m/sec which is much slower than has been found in any other mammalian nerves. The amplitude of the action potentials recorded from some of these fibres was that normally associated with small myelinated fibres rather than C-fibres. This may have been because the nerves became myelinated close to the recording electrodes, although the thresholds of the fibres were often those expected of larger fibres also. A further discrepancy was the fact that, in two cases, the apparent time taken for an impulse to travel between the points of origin of the long and short latency responses was impossibly long for a simple branched fibre. These points indicate that some other mechanism may have been involved. Other experiments (Matthews & Holland, 1975; Matthews, 1975) suggest that there may be electrotonic coupling between the terminals of certain pulpal nerves and some of the present observation could also be explained on this basis. The short latency response might have been due to stimulation of the fibre from which recordings were made and the long latency response to stimulation of another fibre, with a lower threshold, to which the fibre on the recording electrodes was coupled.

662 B. MA TTHE WS Histological studies have shown that dental pulp in man and several animals contains both myelinated and non-myelinated fibres which branch near the pulp surface (Fearnhead, 1967). The diameters of the myelinated fibres have a unimodal distribution with a peak at about 2 ,m (Graf & Bj6rlin, 1951; Brookhart, Livingston & Haugen, 1953; Uchizono & Homma, 1959; Bueltmann, Karlsson & Edie, 1972; Johnsen & Karlsson, 1974). The largest fibres are usually between 6 and 7,um, although a few with diameters up to 10 and 13 ,m have been found (Windle, 1927; Brashear, 1936; Graf & Bj6rlin, 1951; Graf & Helmquist, 1955). The conduction velocities determined from single fibre recording in this and other studies (Wagers & Smith, 1960; Funakoshi & Zotterman, 1963; Greenwood et al. 1972; Horiuchi & Matthews, 1974) and from compound action potential recordings (Brookhart et al. 1953; Bessou, Gauthier & Pages, 1970; Anderson & Pearl, 1975) are consistent with the diameters measured histologically. Thus, contrary to what has sometimes been assumed, all pulpal fibres do not have conduction velocities within the range of the C and A6 groups of cutaneous fibres, as defined by Erlanger & Gasser (1937). There is however no evidence that the fibres with conduction velocities corresponding with the Aa and intermediate groups of cutaneous nerves (Whitehorn, Howe, Lessler & Burgess, 1974) form a functionally distinct group; they probably represent the tapering tail of the myelinated fibre distribution which is mainly within the limits of the A& group. As with the pulpal nerves in the present experiments, some of the response which has been recorded from nerves in other tissues in response to large temperature changes may also have been due to direct excitation of nerves rather than receptors; for example, the discharge evoked by stimulating specific cold fibres at temperatures above 450 C (Dodt & Zotterman, 1952) and some of the response of cutaneous nocireceptors to intense thermal stimuli (Bessou & Perl, 1969). The finding that there was no correlation between the blocking temperature of individual myelinated fibres and their conduction velocities agrees with the findings of Franz & Iggo (1968). However, the mean blocking temperature of 12.10 C is significantly higher than that obtained by Franz & Iggo (7. 2 C) and also higher than that obtained for normal fibres with conduction velocities between 11 and 21 m/sec (10.00 C) by Paintal (1965). This suggests that the fibres in the present study may have suffered damage as a result of the 15 sec periods of heating to 48° C which were applied at intervals during an experiment. Particular care was taken to avoid applying compression or tension to the nerve, or interrupting its blood supply. The experiments were supported by a grant from the Medical Research Council.

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Responses of intradental nerves to electrical and thermal stimulation of teeth in dogs.

641 J. Physiol. (1977), 264, pp. 641-664 With 14 text-figure. Printed in Great Britain RESPONSES OF INTRADENTAL NERVES TO ELECTRICAL AND THERMAL STI...
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