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Local oedema and general excitation of cutaneous sensory receptors produced by electrical stimulation of the saphenous nerve in the rat
25 Pain, 2 (1976) 25-34 0 Elsevier/North-Holland,
Amsterdam
- Printed
in The Netherlands
LOCAL OEDEMA AND GENERAL EXCITATION OF CUTANEOUS SENSORY RECEPTORS PRODUCED BY ELECTRICAL STIMULATION OF THE SAPHENOUS NERVE IN THE RAT LORIS A. CHAHL and R.J. LADD Department (Accepted
o~Physio~og3}, U~~Llersityof Queenslal~d, St. Lucia 4067 ~A~stralia~ December
8th, 1975)
SUMMARY
Recordings were made from multifibre strands of the saphenous nerve of rats anaesthetized with urethane and given Evan’s blue intravenously. Stimulation of A plus C fibres of the saphenous nerve, but not A fibres alone, at 10 Hz for 5 min produced dye leakage in the skin of the hind limb. Stimulation of the nerve at A plus C fibre voltages with the stimulating electrodes placed distal to the recording electrodes produced reversible block of nerve impulses at the stimulating electrodes. However, when the stimulating electrodes were placed proximal to the recording electrodes, stimulation of the nerve at A plus C fibre voltages, but not at ,4 fibre voltages, produced an increase in activity in nerve strands. An increase in activity was also observed in experiments where the contralateral saphenous nerve was stimulated. These effects were not abolished by pretreatment of rats with reserpine. The early phase of the local oedema response ‘appeared to be reduced by local pretreatment with compound 48/80 but the excitatory action on nerve terminals was not. The effects of nerve stimulation were not mimicked by intravenous injection of 5HT, ATP or adenosine into the contralateral saphenous vein. It is suggested that several substances might be released on antidromic stimulation of C fibres and that the resulting general excitation of sensory nerve terminals might play a role in modifying the central nervous system response to nociceptive information.
INTRODUCTION
Hinsey and Gasser [9] showed that antidromic vasodilatation in the cat produced by stimulation of the dorsal roots was mediated by C fibres. More recently it has been shown that oedema also occurred on electrical stimulation of the rat saphenous nerve [lo] and that a substance which was released into the venous effluent after stimulation of the rat saphenous nerve pro-
26 duced the vasodilatation and oedema in the skin of the stimulated region [6]. The identity of the substance is not known but Garcia Leme and Hamamura [6] showed that it could be distinguished from the known chemical mediators of inflammation. Garcia Leme and Lice [7] reported that this substance caused depression of cortical potentials evoked by tooth pulp stimulation in the rat. However, Chapman et al. [3] showed that the perfusate collected during antidromic vasodilatation produced pain on intradermal injection. The present study was undertaken to investigate the effects of substance(s) released by electrical stimulation of the saphenous nerve on cutaneous sensory receptors in the rat. METHODS
Electrophysiological experiments Male albino rats, 200-250 g, were anaesthetized with urethane (12501500 mg/kg), the trachea and jugular vein were cannulated and the right hind limb was fixed in position by partially embedding it in gypsum. A paraffin pool was made over the saphenous nerve and extracellular recordings were made from multifibre strands of the nerve using silver wire electrodes. The signals were amplified and simultaneously displayed on an oscilloscope and recorded on magnetic tape. In the first series of 9 experiments electrodes for stimulation of the nerve were placed 1 cm distal to the recording electrodes so that the compound action potential of fine strands could be observed (Fig. la). In the second series of experiments the stimulating electrodes were placed proximal to the recording electrodes. In 3 experiments re-
Fig. 1. Positions of stimulating (S) and recording (R) electrodes. a: in the first series of experiments the stimulating electrodes were placed distal to the recording electrodes. b: in the second series the stimulating electrodes were proximal to the recording electrodes (see text). c: in the third series the stimulating electrodes were placed under the decentralised, contralateral nerve and the recording electrodes under decentralised strands.
27 cordings were made from large nerve strands which were continuous between the stimulating and recording electrodes and in 5 experiments recordings were made from finer strands which were not across the stimulating electrodes (Fig. lb). In the third series of 7 experiments the stimulating electrodes were placed under the decentralised, contralateral saphenous nerve and recordings were made from decentralised strands (Fig. lc). In all 3 series of experiments an injection of 2% Evan’s blue solution (50 mg/kg) was given into the jugular vein after all dissection was complete. The activity in the strands was observed for several minutes before the stimulation period and the response to mechanical stimulation by stroking the skin was observed. In the first series of experiments where the compound action potential could be observed, the nerve was stimulated to produce maximal elevation of the A waves in the compound action potential with no C elevation (0.1 msec duration, l-2 V) at 1 Hz (3 experiments) or 10 Hz (6 experiments), for 5 min. The activity in the strand foIlowing the period of stimulation was observed for lo-15 min and at various intervals the response to mechanical stimulation by stroking the skin was observed. The nerve was then stimulated to excite both A and C fibres for 5 min, and the activity in the strand observed for at ieast 15 min following the period of stimulation. In 3 experiments the stimulus used was 15-20 V, 0.5 msec duration whereas in the other experiments 3-8 V, 2 msec duration was used. Frequencies of 1 Hz or 10 Hz were used. In the second and third series of experiments a similar procedure was followed, except that the compound action potential was not observed, and the stimuli used were those which were found to produce reliably A fibre stimulation (0.1 msec duration, 2 V), or A plus C fibre stimulation (2 msec duration, 20 V), in the first series of experiments. In 2 experiments in each series the frequency was 1 Hz but in all other experiments a frequency of 10 Hz was used. In the third series, 6 experiments were done on rats with the contralateral saphenous vein ligated. Pharmacological experiments Reserpine. Four experiments of the first series design (see above), and one experiment each of the second and third series designs were performed on rats which had been pretreated with reserpine 10 mg/kg, 24 h previously. Compound 48/80. Eight rats were anaesthetized with urethane, the jugular vein cannulated and a cannula placed in the femoral artery distal to the branching of the saphenous artery so that retrograde intra-arterial injections could be given into the skin of the hind limb without occluding its blood supply. Three intraarterial injections of compound 48180, 0.2 ml of 100 pg/ ml solution in Tyrode were given at hourly intervals, and 1 h after the final injection Evan’s blue solution was given into the jugular vein. Stimulation of both saphenous nerves was then carried out at A plus C fibre voltages for 5 min. In two of these experiments recordings were made from strands of one saphenous nerve before and after the injections of compound 48/80 into the contralateral hind limb, and before and after stimulation of the contralateral saphenous nerve at A plus C fibre voltages.
Four rats were pretreated with an interdermal injection of compound 48/80 (0.05 ml of 10 pg/ml solution (2 rats); 0.05 ml of 100 p&/ml (2 rats)), into the skin of the medial surface of the hind paw at 24, 48 and 72 h prior to the day of the experiment, and control injections of Tyrode solution were given into the other paw. The rats were then anaesthetized with urethane, the jugular vein cannulated, and Evan’s blue was given into the jugular vein. Both saphenous nerves were then stimulated at A plus C fibre voltages for 5 min. ~-~ydroxy~ryptu~z~~e J5HT), adenosine tr~ph~spha~e (ATP), and adenosine. Recordings were made from the saphenous nerves of 4 rats before and after injections of 5HT (0.2 ml of 2.5 X lo-’ M), ATP (0.2 ml of lo-” M), and adenosine (0.2 ml of lo-” M), into the contralateral saphenous vein. Chetnicals The following chemicals were used: adenosine (Merck), adenosine 5’triphosphate disodium salt (Sigma), compound 48/80 (Wellcome), Evan’s blue (Difco Laboratories), 5-hydroxytryptamine creatinine sulphate complex (Sigma). All dilutions of these chemicals which were injected into rats were made in Tyrode solution. RESULTS
Elec~rop~ys~olog~ca~ experiments In the first series of experiments where the stimulating electrodes were placed distal to the recording electrodes it was found that no dye leakage occurred when only the A fibres of the nerve were stimulated. However, when the C fibres were stimulated as well, marked dye leakage occurred in the skin of the leg. The dye leakage was characteristically spotted in appearance, which might reflect the receptive fields of the C fibres of the saphenous nerve. When the stimulus frequency was 10 Hz the effect became obvious between 1 and 2 min after commencement of stimulation. At 1 Hz stimulus frequency the effect became apparent between 2 and 4 min after commencement of stimulation. In this first series of experiments it was also found that the electrical stimulation of the nerve at A plus C fibre voltages for 5 min produced block of all response to mechanical stimulation of the skin. Recovery commenced 5 min after cessation of stimulation of the nerve and was complete by lo15 min. The time course of the recovery suggested that this effect might be due to block at the stimulating electrodes. This was tested in the second series of experiments in which the stimulating electrodes were placed proximal to the recording electrodes. Recordings from large strands which were continuous between the stimulating and recording electrodes showed that, even after stimulation at C fibre voltages, the response in the strand to mechanical stimulation of the skin was unchanged, This showed that the block observed in the first series of experiments had been at the stimulating eiectrodes. In some of these experiments, recordings were made from finer
29
strands which were not across the stimulating electrodes. These strands had not been stimulated but had their receptive fields in the area of dye leakage. The activity in these strands was observed before and after stimulation of the remainder of the nerve with A, and A plus C fibre voltages. It was found that there was little change after A fibres had been stimulated, but after A plus C fibre stimulation there was a marked increase in the activity in the strands and also an after-discharge following mechanical stimulation of the skin was sometimes apparent (Fig. 2). This increased activity and after-discharge effect decreased progressively until 20 min after cessation of electrical stimulation when it had apparently disappeared. Since these strands had their receptive fields in the stimulated oedematous region it was necessary to know whether the increased activity recorded in the nerve strands was due to a
Fig. 2. Activity in a strand of saphenous nerve before and after stimulation of A and A plus C fibres of the remainder of the nerve (stimulating electrodes proximal to recording electrodes). Bars indicate duration of application of mechanical stimulation to the skin. a: activity in strand before stimulation. b: response to mechanical stimulation of skin before stimulation. c: activity after stimulation of A fibres. d: response to mechanical stimulation after stimulation of A fibres. e: activity after stimulation of A plus C fibres. f: response to mechanical stimulation immediately after stimulation of A plus C fibres (note after-discharge). g: section of f on expanded time scale. h-j: responses to mechanical stimulation at 10 min, 15 min and 20 min after stimulation of A plus C fibres.
30 secondary effect of the oedema in the skin or due to a direct effect of substance(s) released from the stimulated sensory nerve terminals. In the third series of experiments the stimulating electrodes were placed under the decentralised, contralateral saphenous nerve and recordings were made from decentralised strands. In these experiments an increase in the activity in the strands was found after stimulation of the contralateral saphenous nerve at A plus C fibre voltages (Fig. 3) but not after stimulation at A fibre voltages. The effect was quite persistent in some experiments and in Fig. 3 it can be seen that even after an hour the activity in the strand was still greater than at the commencement of the experiment. It was found that a second period of stimulation was less effective in producing increased activity in the strands. Similar responses were observed in the second and third series of experiments with stimulus frequencies of 1 and 10 Hz and stimulus durations of 0.5 msec and 2 msec. The response did not occur in the 6 rats which had the contralateral saphenous vein ligated. Pharmacological experiments Reserpine. Dye leakage and increased activity in the strands following stimulation of A plus C fibres occurred in rats which had been pretreated with reserpine.
Fig. 3. Activity in a strand of saphenous nerve before and after stimulation of A plus C fibres of the decentralised, contralateral saphenous nerve. Bars indicate duration of application of mechanical stimulation to the skin. a: response to mechanical stimulation of skin before stimulation. b: activity in strand before stimulation. c: activity immediately after stimulation of A plus C fibres. d-h: activity at 5 min, 10 min, 15 min, 20 min and 1 h after stimulation of A plus C fibres.
31 Compound 48/80. Stimulation of A plus C fibres did not produce dye leakage in rats which had been given 3 intraarterial injections of compound 48/80 to deplete mast cell amine stores. The intraarterial injections of compound 48/80 produced systemic effects since stimulation of the other saphenous nerve did not produce dye leakage in 5 out of 6 rats. In rats which had been pretreated with intradermal injections of compound 48/80 there was a zone of reduced dye leakage in the injected area of the treated paw, which was apparent at 2-3 min after commencement of stimulation of A plus C fibres, but only in 2 out of these animals (one from each dose level of compound 48/80) was this still marked by the end of the 5 min stimulation period. It was also noticed in all 4 rats that the dye leakage in the other areas of the treated paw was more marked that in the control paw. In 2 experiments in which the activity in the contralateral saphenous nerve was monitored during injections of compound 48/80, it was found that there was an increase in activity especially following the second and third injections of compound 48/80. This effect was less marked after the first injection of compound 48/80, probably because intense vasoconstriction occurred which might have prevented the escape of substances into the general circulation. Stimulation of the saphenous nerve following the 3 injections of compound 48/80 did not produce any dye leakage in the stimulated hind limb but increase in activity was recorded in the contralateral saphenous nerve, and marked after-discharge was observed in one of the experiments. 5HT, ATP and adenosine. In 4 experiments, each of these chemicals, when injected into the contralateral saphenous vein, produced increase in activity in saphenous nerve strands. They produced transient effects, the responses commencing within 1 min after the injections and disappearing between 3-6 min. After-discharge was not observed following injection of these chemicals. DISCUSSION
The experiments reported here confirm and extend the observations by Jansco et al. [lo] and Garcia Leme and Hamamura [6] that a substance is released on electrical stimulation of the saphenous nerve in rats which causes local oedema. In addition we have shown that the substance is released from C fibres and also that it causes general excitation of afferent units in regions of the body remote from the receptive field of the stimulated nerve. This effect (as observed by increased activity recorded from the other saphenous nerve) was not observed when the saphenous vein draining the stimulated hind limb skin was occluded, thus demonstrating that the substance reached the afferent nerve terminals via the blood stream. These experiments also showed that the substance was released into the blood from the skin and not from the muscles of the stimulated hind limb. Results from our experiments offer one possible explanation for the finding of reduction in cortical evoked potentials produced by antidromic stimulation of the rat saphenous nerve, which was observed by Garcia Leme and Lice [ 71. It is suggested that the substance released from the afferent nerves
32 might alter the evoked response to nociceptive stimulation by causing an increase in the input to the central nervous system from the peripheral receptors throughout the body, thus altering central processing of nociceptive information. The types of afferent units which were stimulated by the substance have not been characterised, but in each multifibre strand a number of units were excited which had the size and shape of A spikes. The units which responded with an after-discharge appeared to be slowly-adapting mechanoreceptors. Measurements of conduction velocities were not made in these experiments because it was necessary to limit electrical stimulation of the nerve. It is not known whether the effects produced by electrical stimulation of C fibres would ever be mimicked by other, more natural forms of C fibre stimulation. Jansco et al. [lo] showed that the counterirritant, capsaicin, produced neurogenic oedema similar to that produced by electrical stimulation of the rat saphenous nerve. It is still not known whether any of the phlogistic effects of the natural mediators of inflammation are of neurogenic origin. It has not been established which C fibres release the ‘sensory nerve substance’ on electrical stimulation and it, can only be assumed at present that release occurs from nociceptive C fibres. However, Celander and Folkow [l] found that vasodilatation in the skin of cats occurred with noxious stimulation but not with non-noxious thermal stimulation. If severe noxious stimulation did result in release of the substance then, from the present findings, it would be reasonable to postulate that the substance, by a purely peripheral action, might play a role in modifying the central nervous system response to nociception. The identity of the substance released on electrical stimulation of C fibres is not yet known. Dale [5] proposed that the agent responsible for antidromic vasodilatation might be the transmitter at the primary afferent nerve terminals in the spinal cord. However, the putative amino acid transmitters GABA, glutamate, aspartate and glycine did not produce marked oedema in rat skin (Chahl, L.A., unpublished observations) or degranulation of rat mast cells [ll]. Although the sympathetic nerves were intact in these experiments the effects observed were not produced by noradrenaline since they were not abolished by pretreatment with reserpine at a dose shown by Haggendal and Dahlstrom [8] to deplete other rat tissues of noradrenaline. However, this might not preclude the release of substances other than noradrenaline from the intact adrenergic nerves. Results from experiments with compound 48/80 were not conclusive. Intra-arterial injections of compound 48/80 appeared to block completely the response to stimulation of A plus C fibres whereas intradermal injections produced definite areas of reduced dye leakage in only 2 out of 4 rats at the end of 5 min stimulation. It is possible that the intradermal injections did not achieve adequate depletion of the amines, or that the intraarterial injections produced non-specific inhibition of the dye leakage effect. Nevertheless it appeared that part of the dye leakage was due to the effect of amines released from mast cells, particularly during the early phase of the response.
33 Kiernan [13] found that compound 48/80 blocked axon reflex vasodilatation and has suggested that ATP is released from sensory nerve endings and produces mast cell degranulation [12]. In contrast to these results Garcia Leme and Hamamura [6] and Jansco et al. [lo] found that anti-histamine and anti-5HT drugs did not block the increase in vascular permeability produced by saphenous nerve stimulation. This might be explained by the long period of stimulation of the nerve used by these workers (20 min) allowing enough time for the activation of mediators of later phases in the inflammatory response which would not be antagonized by anti-histamines or anti5HT drugs. Garcia Leme and Hamamura [6] did find kinins in perfusates collected after high intensity stimulation. Further studies are required on the role of chemical mediators in antidromic oedema. The question arises as to whether the released amines or the ‘sensory nerve substance’ itself produced excitation of afferent units. Results from our experiments, where depletion of mast cells by compound 48/80 was carried out prior to stimulation of the contralateral saphenous nerve, indicated that the released amines were not responsible for the excitation of afferent units since the effect was not abolished. This conclusion was supported by the finding that injections of 5HT, ATP and adenosine into the contralateral saphenous vein produced only transient increases in activity in the saphenous nerve, whereas the response to contralateral nerve stimulation was usually of longer duration. It could be argued firstly, that a more prolonged effect from these chemicals might occur if they were slowly leaked into the circulation rather than reaching it as a bolus, or secondly, that when they are released in high concentration in close proximity to nerve terminals, as would occur when the nerve is stimulated, they might produce a positive feedback effect by acting back on the nerve terminals to produce further release, or activation of other systems, e.g. the kinin system. It is not known whether prostaglandins are released from sensory nerve terminals, but PGEl has been shown to release amines from mast cells in rat skin [4] and produce prolonged excitation of some afferent units [2] and could therefore be a candidate for the production of both oedema and excitation of afferent nerve terminals. Garcia Leme and Hamamura [6] found that pretreatment of rats with indomethacin, which blocks prostaglandin synthesis [ 141, did not prevent the increased vascular permeability produced by saphenous nerve stimulation and they therefore concluded that prostaglandins were not responsible for this effect. In contrast, Kiernan 1131 found that aspirin blocked the degranulation of rat dermal mast cells following injury. In conclusion, it seems from our experiments, that the early phase of the local oedema resulting from electrical stimulation of C fibres may be produced by amines released from mast cells, but the excitation of afferent nerve terminals at remote sites is produced by some other substance. The possibility must be considered that several agents might be released either simultaneously or sequentially from afferent nerve terminals and mast cells which produce their effects by acting in concert.
34 ACKNOWLEDGEMENTS
We are indebted Technical assistance
to Professor A. Iggo for helpful discussion of this work. by Miss Anne Schafferius is gratefully acknowledged.
REFERENCES
1 Celander, 0. and Folkow, 2 3 4 5 6
7
8
9 10
11 12 13
14
B., The nature and the distribution of afferent fibres provided with the axon reflex arrangement, Acta physiol. stand., 29 (1953) 359-370. Chahl, L.A. and Iggo, A., The effect of prostaglandin Et on cutaneous afferent nerve fibres of the rat, Proc. Aust. Physiol. Pharmac. Sot., 5 (1974) 180-181. Chapman, L.F., Ramos, A.O., Goodell, H. and Wolff, H.G., Neurohumoral features of afferent fibres in man, Arch. Neurol. (Chic.), 4 (1961) 617-650. Crunkhorn, P. and Willis, A.L., Cutaneous reactions to intradermal prostaglandins, Brit. J. Pharmacol., 41 (1971) 49-56. Dale, H.H., Pharmacology and nerve endings, Proc. roy. Sot. Med., 28 (1935) 319~332. Garcia Leme, J. and Hamamura, L., Formation of a factor increasing vascular permeability during electrical stimulation of the saphenous nerve in rats, Brit. J. Pharmacol., 51 (1974) 383-389. Garcia Leme, J. and Lice, M., Cortical potentials evoked by nociceptive stimuli: their depression by a factor released during saphenous nerve stimulation in the rat, Brit. J. Pharmacol., 51 (1974) 491-496. HPggendal, J. and Dahlstriim, A., The recovery of noradrenaline in adrenergic nerve terminals of the rat after reserpine treatment, J. Pharm. Pharmacol., 23 (1971) 8189. Hinsey, J.C. and Gasser, H.S., The component of the dorsal root mediating vasodilatation and the Sherrington contracture, Amer. J. Physiol., 92 (1930) 679-689. inJansco, N., Jansco-Ggbor, A. and Szolcsanyi, J., Direct evidence for neurogenic flammation and its prevention by denervation and by pretreatment with capsaicin, Brit. J. Pharmacol., 31 (1967) 138-151. Kiernan, J.A., Effects of known and suspected neurotransmitter substances and of some nucleotides on isolated mast cells, Experientia (Basel), 28 (1972) 653-655. Kiernan, J.A., The involvement of mast cells in vasodilatation due to axon reflexes in injured skin, Quart. J. exp. Physiol., 57 (1972) 311-317. Kiernan, J.A., A pharmacological and histological investigation of the involvement of mast cells in cutaneous axon reflex vasodilatation, Quart J. exp. Physiol., 60 (1975) 123-130. Vane, J.R., Inhibition of prostaglandin synthesis as a mechanism of action for aspirinlike drugs, Nature New Biol., 231 (1971) 232-235.
Amsterdam
- Printed
in The Netherlands
LOCAL OEDEMA AND GENERAL EXCITATION OF CUTANEOUS SENSORY RECEPTORS PRODUCED BY ELECTRICAL STIMULATION OF THE SAPHENOUS NERVE IN THE RAT LORIS A. CHAHL and R.J. LADD Department (Accepted
o~Physio~og3}, U~~Llersityof Queenslal~d, St. Lucia 4067 ~A~stralia~ December
8th, 1975)
SUMMARY
Recordings were made from multifibre strands of the saphenous nerve of rats anaesthetized with urethane and given Evan’s blue intravenously. Stimulation of A plus C fibres of the saphenous nerve, but not A fibres alone, at 10 Hz for 5 min produced dye leakage in the skin of the hind limb. Stimulation of the nerve at A plus C fibre voltages with the stimulating electrodes placed distal to the recording electrodes produced reversible block of nerve impulses at the stimulating electrodes. However, when the stimulating electrodes were placed proximal to the recording electrodes, stimulation of the nerve at A plus C fibre voltages, but not at ,4 fibre voltages, produced an increase in activity in nerve strands. An increase in activity was also observed in experiments where the contralateral saphenous nerve was stimulated. These effects were not abolished by pretreatment of rats with reserpine. The early phase of the local oedema response ‘appeared to be reduced by local pretreatment with compound 48/80 but the excitatory action on nerve terminals was not. The effects of nerve stimulation were not mimicked by intravenous injection of 5HT, ATP or adenosine into the contralateral saphenous vein. It is suggested that several substances might be released on antidromic stimulation of C fibres and that the resulting general excitation of sensory nerve terminals might play a role in modifying the central nervous system response to nociceptive information.
INTRODUCTION
Hinsey and Gasser [9] showed that antidromic vasodilatation in the cat produced by stimulation of the dorsal roots was mediated by C fibres. More recently it has been shown that oedema also occurred on electrical stimulation of the rat saphenous nerve [lo] and that a substance which was released into the venous effluent after stimulation of the rat saphenous nerve pro-
26 duced the vasodilatation and oedema in the skin of the stimulated region [6]. The identity of the substance is not known but Garcia Leme and Hamamura [6] showed that it could be distinguished from the known chemical mediators of inflammation. Garcia Leme and Lice [7] reported that this substance caused depression of cortical potentials evoked by tooth pulp stimulation in the rat. However, Chapman et al. [3] showed that the perfusate collected during antidromic vasodilatation produced pain on intradermal injection. The present study was undertaken to investigate the effects of substance(s) released by electrical stimulation of the saphenous nerve on cutaneous sensory receptors in the rat. METHODS
Electrophysiological experiments Male albino rats, 200-250 g, were anaesthetized with urethane (12501500 mg/kg), the trachea and jugular vein were cannulated and the right hind limb was fixed in position by partially embedding it in gypsum. A paraffin pool was made over the saphenous nerve and extracellular recordings were made from multifibre strands of the nerve using silver wire electrodes. The signals were amplified and simultaneously displayed on an oscilloscope and recorded on magnetic tape. In the first series of 9 experiments electrodes for stimulation of the nerve were placed 1 cm distal to the recording electrodes so that the compound action potential of fine strands could be observed (Fig. la). In the second series of experiments the stimulating electrodes were placed proximal to the recording electrodes. In 3 experiments re-
Fig. 1. Positions of stimulating (S) and recording (R) electrodes. a: in the first series of experiments the stimulating electrodes were placed distal to the recording electrodes. b: in the second series the stimulating electrodes were proximal to the recording electrodes (see text). c: in the third series the stimulating electrodes were placed under the decentralised, contralateral nerve and the recording electrodes under decentralised strands.
27 cordings were made from large nerve strands which were continuous between the stimulating and recording electrodes and in 5 experiments recordings were made from finer strands which were not across the stimulating electrodes (Fig. lb). In the third series of 7 experiments the stimulating electrodes were placed under the decentralised, contralateral saphenous nerve and recordings were made from decentralised strands (Fig. lc). In all 3 series of experiments an injection of 2% Evan’s blue solution (50 mg/kg) was given into the jugular vein after all dissection was complete. The activity in the strands was observed for several minutes before the stimulation period and the response to mechanical stimulation by stroking the skin was observed. In the first series of experiments where the compound action potential could be observed, the nerve was stimulated to produce maximal elevation of the A waves in the compound action potential with no C elevation (0.1 msec duration, l-2 V) at 1 Hz (3 experiments) or 10 Hz (6 experiments), for 5 min. The activity in the strand foIlowing the period of stimulation was observed for lo-15 min and at various intervals the response to mechanical stimulation by stroking the skin was observed. The nerve was then stimulated to excite both A and C fibres for 5 min, and the activity in the strand observed for at ieast 15 min following the period of stimulation. In 3 experiments the stimulus used was 15-20 V, 0.5 msec duration whereas in the other experiments 3-8 V, 2 msec duration was used. Frequencies of 1 Hz or 10 Hz were used. In the second and third series of experiments a similar procedure was followed, except that the compound action potential was not observed, and the stimuli used were those which were found to produce reliably A fibre stimulation (0.1 msec duration, 2 V), or A plus C fibre stimulation (2 msec duration, 20 V), in the first series of experiments. In 2 experiments in each series the frequency was 1 Hz but in all other experiments a frequency of 10 Hz was used. In the third series, 6 experiments were done on rats with the contralateral saphenous vein ligated. Pharmacological experiments Reserpine. Four experiments of the first series design (see above), and one experiment each of the second and third series designs were performed on rats which had been pretreated with reserpine 10 mg/kg, 24 h previously. Compound 48/80. Eight rats were anaesthetized with urethane, the jugular vein cannulated and a cannula placed in the femoral artery distal to the branching of the saphenous artery so that retrograde intra-arterial injections could be given into the skin of the hind limb without occluding its blood supply. Three intraarterial injections of compound 48180, 0.2 ml of 100 pg/ ml solution in Tyrode were given at hourly intervals, and 1 h after the final injection Evan’s blue solution was given into the jugular vein. Stimulation of both saphenous nerves was then carried out at A plus C fibre voltages for 5 min. In two of these experiments recordings were made from strands of one saphenous nerve before and after the injections of compound 48/80 into the contralateral hind limb, and before and after stimulation of the contralateral saphenous nerve at A plus C fibre voltages.
Four rats were pretreated with an interdermal injection of compound 48/80 (0.05 ml of 10 pg/ml solution (2 rats); 0.05 ml of 100 p&/ml (2 rats)), into the skin of the medial surface of the hind paw at 24, 48 and 72 h prior to the day of the experiment, and control injections of Tyrode solution were given into the other paw. The rats were then anaesthetized with urethane, the jugular vein cannulated, and Evan’s blue was given into the jugular vein. Both saphenous nerves were then stimulated at A plus C fibre voltages for 5 min. ~-~ydroxy~ryptu~z~~e J5HT), adenosine tr~ph~spha~e (ATP), and adenosine. Recordings were made from the saphenous nerves of 4 rats before and after injections of 5HT (0.2 ml of 2.5 X lo-’ M), ATP (0.2 ml of lo-” M), and adenosine (0.2 ml of lo-” M), into the contralateral saphenous vein. Chetnicals The following chemicals were used: adenosine (Merck), adenosine 5’triphosphate disodium salt (Sigma), compound 48/80 (Wellcome), Evan’s blue (Difco Laboratories), 5-hydroxytryptamine creatinine sulphate complex (Sigma). All dilutions of these chemicals which were injected into rats were made in Tyrode solution. RESULTS
Elec~rop~ys~olog~ca~ experiments In the first series of experiments where the stimulating electrodes were placed distal to the recording electrodes it was found that no dye leakage occurred when only the A fibres of the nerve were stimulated. However, when the C fibres were stimulated as well, marked dye leakage occurred in the skin of the leg. The dye leakage was characteristically spotted in appearance, which might reflect the receptive fields of the C fibres of the saphenous nerve. When the stimulus frequency was 10 Hz the effect became obvious between 1 and 2 min after commencement of stimulation. At 1 Hz stimulus frequency the effect became apparent between 2 and 4 min after commencement of stimulation. In this first series of experiments it was also found that the electrical stimulation of the nerve at A plus C fibre voltages for 5 min produced block of all response to mechanical stimulation of the skin. Recovery commenced 5 min after cessation of stimulation of the nerve and was complete by lo15 min. The time course of the recovery suggested that this effect might be due to block at the stimulating electrodes. This was tested in the second series of experiments in which the stimulating electrodes were placed proximal to the recording electrodes. Recordings from large strands which were continuous between the stimulating and recording electrodes showed that, even after stimulation at C fibre voltages, the response in the strand to mechanical stimulation of the skin was unchanged, This showed that the block observed in the first series of experiments had been at the stimulating eiectrodes. In some of these experiments, recordings were made from finer
29
strands which were not across the stimulating electrodes. These strands had not been stimulated but had their receptive fields in the area of dye leakage. The activity in these strands was observed before and after stimulation of the remainder of the nerve with A, and A plus C fibre voltages. It was found that there was little change after A fibres had been stimulated, but after A plus C fibre stimulation there was a marked increase in the activity in the strands and also an after-discharge following mechanical stimulation of the skin was sometimes apparent (Fig. 2). This increased activity and after-discharge effect decreased progressively until 20 min after cessation of electrical stimulation when it had apparently disappeared. Since these strands had their receptive fields in the stimulated oedematous region it was necessary to know whether the increased activity recorded in the nerve strands was due to a
Fig. 2. Activity in a strand of saphenous nerve before and after stimulation of A and A plus C fibres of the remainder of the nerve (stimulating electrodes proximal to recording electrodes). Bars indicate duration of application of mechanical stimulation to the skin. a: activity in strand before stimulation. b: response to mechanical stimulation of skin before stimulation. c: activity after stimulation of A fibres. d: response to mechanical stimulation after stimulation of A fibres. e: activity after stimulation of A plus C fibres. f: response to mechanical stimulation immediately after stimulation of A plus C fibres (note after-discharge). g: section of f on expanded time scale. h-j: responses to mechanical stimulation at 10 min, 15 min and 20 min after stimulation of A plus C fibres.
30 secondary effect of the oedema in the skin or due to a direct effect of substance(s) released from the stimulated sensory nerve terminals. In the third series of experiments the stimulating electrodes were placed under the decentralised, contralateral saphenous nerve and recordings were made from decentralised strands. In these experiments an increase in the activity in the strands was found after stimulation of the contralateral saphenous nerve at A plus C fibre voltages (Fig. 3) but not after stimulation at A fibre voltages. The effect was quite persistent in some experiments and in Fig. 3 it can be seen that even after an hour the activity in the strand was still greater than at the commencement of the experiment. It was found that a second period of stimulation was less effective in producing increased activity in the strands. Similar responses were observed in the second and third series of experiments with stimulus frequencies of 1 and 10 Hz and stimulus durations of 0.5 msec and 2 msec. The response did not occur in the 6 rats which had the contralateral saphenous vein ligated. Pharmacological experiments Reserpine. Dye leakage and increased activity in the strands following stimulation of A plus C fibres occurred in rats which had been pretreated with reserpine.
Fig. 3. Activity in a strand of saphenous nerve before and after stimulation of A plus C fibres of the decentralised, contralateral saphenous nerve. Bars indicate duration of application of mechanical stimulation to the skin. a: response to mechanical stimulation of skin before stimulation. b: activity in strand before stimulation. c: activity immediately after stimulation of A plus C fibres. d-h: activity at 5 min, 10 min, 15 min, 20 min and 1 h after stimulation of A plus C fibres.
31 Compound 48/80. Stimulation of A plus C fibres did not produce dye leakage in rats which had been given 3 intraarterial injections of compound 48/80 to deplete mast cell amine stores. The intraarterial injections of compound 48/80 produced systemic effects since stimulation of the other saphenous nerve did not produce dye leakage in 5 out of 6 rats. In rats which had been pretreated with intradermal injections of compound 48/80 there was a zone of reduced dye leakage in the injected area of the treated paw, which was apparent at 2-3 min after commencement of stimulation of A plus C fibres, but only in 2 out of these animals (one from each dose level of compound 48/80) was this still marked by the end of the 5 min stimulation period. It was also noticed in all 4 rats that the dye leakage in the other areas of the treated paw was more marked that in the control paw. In 2 experiments in which the activity in the contralateral saphenous nerve was monitored during injections of compound 48/80, it was found that there was an increase in activity especially following the second and third injections of compound 48/80. This effect was less marked after the first injection of compound 48/80, probably because intense vasoconstriction occurred which might have prevented the escape of substances into the general circulation. Stimulation of the saphenous nerve following the 3 injections of compound 48/80 did not produce any dye leakage in the stimulated hind limb but increase in activity was recorded in the contralateral saphenous nerve, and marked after-discharge was observed in one of the experiments. 5HT, ATP and adenosine. In 4 experiments, each of these chemicals, when injected into the contralateral saphenous vein, produced increase in activity in saphenous nerve strands. They produced transient effects, the responses commencing within 1 min after the injections and disappearing between 3-6 min. After-discharge was not observed following injection of these chemicals. DISCUSSION
The experiments reported here confirm and extend the observations by Jansco et al. [lo] and Garcia Leme and Hamamura [6] that a substance is released on electrical stimulation of the saphenous nerve in rats which causes local oedema. In addition we have shown that the substance is released from C fibres and also that it causes general excitation of afferent units in regions of the body remote from the receptive field of the stimulated nerve. This effect (as observed by increased activity recorded from the other saphenous nerve) was not observed when the saphenous vein draining the stimulated hind limb skin was occluded, thus demonstrating that the substance reached the afferent nerve terminals via the blood stream. These experiments also showed that the substance was released into the blood from the skin and not from the muscles of the stimulated hind limb. Results from our experiments offer one possible explanation for the finding of reduction in cortical evoked potentials produced by antidromic stimulation of the rat saphenous nerve, which was observed by Garcia Leme and Lice [ 71. It is suggested that the substance released from the afferent nerves
32 might alter the evoked response to nociceptive stimulation by causing an increase in the input to the central nervous system from the peripheral receptors throughout the body, thus altering central processing of nociceptive information. The types of afferent units which were stimulated by the substance have not been characterised, but in each multifibre strand a number of units were excited which had the size and shape of A spikes. The units which responded with an after-discharge appeared to be slowly-adapting mechanoreceptors. Measurements of conduction velocities were not made in these experiments because it was necessary to limit electrical stimulation of the nerve. It is not known whether the effects produced by electrical stimulation of C fibres would ever be mimicked by other, more natural forms of C fibre stimulation. Jansco et al. [lo] showed that the counterirritant, capsaicin, produced neurogenic oedema similar to that produced by electrical stimulation of the rat saphenous nerve. It is still not known whether any of the phlogistic effects of the natural mediators of inflammation are of neurogenic origin. It has not been established which C fibres release the ‘sensory nerve substance’ on electrical stimulation and it, can only be assumed at present that release occurs from nociceptive C fibres. However, Celander and Folkow [l] found that vasodilatation in the skin of cats occurred with noxious stimulation but not with non-noxious thermal stimulation. If severe noxious stimulation did result in release of the substance then, from the present findings, it would be reasonable to postulate that the substance, by a purely peripheral action, might play a role in modifying the central nervous system response to nociception. The identity of the substance released on electrical stimulation of C fibres is not yet known. Dale [5] proposed that the agent responsible for antidromic vasodilatation might be the transmitter at the primary afferent nerve terminals in the spinal cord. However, the putative amino acid transmitters GABA, glutamate, aspartate and glycine did not produce marked oedema in rat skin (Chahl, L.A., unpublished observations) or degranulation of rat mast cells [ll]. Although the sympathetic nerves were intact in these experiments the effects observed were not produced by noradrenaline since they were not abolished by pretreatment with reserpine at a dose shown by Haggendal and Dahlstrom [8] to deplete other rat tissues of noradrenaline. However, this might not preclude the release of substances other than noradrenaline from the intact adrenergic nerves. Results from experiments with compound 48/80 were not conclusive. Intra-arterial injections of compound 48/80 appeared to block completely the response to stimulation of A plus C fibres whereas intradermal injections produced definite areas of reduced dye leakage in only 2 out of 4 rats at the end of 5 min stimulation. It is possible that the intradermal injections did not achieve adequate depletion of the amines, or that the intraarterial injections produced non-specific inhibition of the dye leakage effect. Nevertheless it appeared that part of the dye leakage was due to the effect of amines released from mast cells, particularly during the early phase of the response.
33 Kiernan [13] found that compound 48/80 blocked axon reflex vasodilatation and has suggested that ATP is released from sensory nerve endings and produces mast cell degranulation [12]. In contrast to these results Garcia Leme and Hamamura [6] and Jansco et al. [lo] found that anti-histamine and anti-5HT drugs did not block the increase in vascular permeability produced by saphenous nerve stimulation. This might be explained by the long period of stimulation of the nerve used by these workers (20 min) allowing enough time for the activation of mediators of later phases in the inflammatory response which would not be antagonized by anti-histamines or anti5HT drugs. Garcia Leme and Hamamura [6] did find kinins in perfusates collected after high intensity stimulation. Further studies are required on the role of chemical mediators in antidromic oedema. The question arises as to whether the released amines or the ‘sensory nerve substance’ itself produced excitation of afferent units. Results from our experiments, where depletion of mast cells by compound 48/80 was carried out prior to stimulation of the contralateral saphenous nerve, indicated that the released amines were not responsible for the excitation of afferent units since the effect was not abolished. This conclusion was supported by the finding that injections of 5HT, ATP and adenosine into the contralateral saphenous vein produced only transient increases in activity in the saphenous nerve, whereas the response to contralateral nerve stimulation was usually of longer duration. It could be argued firstly, that a more prolonged effect from these chemicals might occur if they were slowly leaked into the circulation rather than reaching it as a bolus, or secondly, that when they are released in high concentration in close proximity to nerve terminals, as would occur when the nerve is stimulated, they might produce a positive feedback effect by acting back on the nerve terminals to produce further release, or activation of other systems, e.g. the kinin system. It is not known whether prostaglandins are released from sensory nerve terminals, but PGEl has been shown to release amines from mast cells in rat skin [4] and produce prolonged excitation of some afferent units [2] and could therefore be a candidate for the production of both oedema and excitation of afferent nerve terminals. Garcia Leme and Hamamura [6] found that pretreatment of rats with indomethacin, which blocks prostaglandin synthesis [ 141, did not prevent the increased vascular permeability produced by saphenous nerve stimulation and they therefore concluded that prostaglandins were not responsible for this effect. In contrast, Kiernan 1131 found that aspirin blocked the degranulation of rat dermal mast cells following injury. In conclusion, it seems from our experiments, that the early phase of the local oedema resulting from electrical stimulation of C fibres may be produced by amines released from mast cells, but the excitation of afferent nerve terminals at remote sites is produced by some other substance. The possibility must be considered that several agents might be released either simultaneously or sequentially from afferent nerve terminals and mast cells which produce their effects by acting in concert.
34 ACKNOWLEDGEMENTS
We are indebted Technical assistance
to Professor A. Iggo for helpful discussion of this work. by Miss Anne Schafferius is gratefully acknowledged.
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