Neurophysiology, Sahlgren's Hospital, University of Goteborg, Sweden

Transcription

1 J. Physiol. (1987), 382, pp With 11 text figures Printed in Great Britain DIRECT EVIDENCE OF NEURALLY MEDIATED VASODILATATION IN HAIRY SKIN OF THE HUMAN FOOT BY H. BLUMBERG* AND B. GUNNAR WALLINt From the * Department of Clinical Neurology and Neurophysiology, University of Freiburg, Hansastr. 9, 7800 Freiburg, F.R.G. and the t Department of Clinical Neurophysiology, Sahlgren's Hospital, University of Goteborg, Sweden (Received 12 December 1985) SUMMARY 1. Intraneural stimulation (i.n.s.) was made in the superficial peroneal nerve at the ankle in seventeen healthy subjects. The effect on skin blood flow was monitored by laser-doppler flowmeters and photo-electrical pulse plethysmographs inside and outside the innervation zone of the stimulated nerve fascicle. I.n.s. was applied before and after proximal local anaesthesia of the stimulated nerve. 2. Painful i.n.s. (stimulation strength V) induced skin vasodilatation with the following characteristics: (a) it occurred on the dorsal side of both feet, (b) the blood flow increase on the opposite foot was blocked by local anaesthesia of the nerve supplying the skin area under study, (c) the blood flow increase on the stimulated foot was abolished by proximal local anaesthesia of the stimulated nerve. The findings show that the vasodilatation was due to activation of a reflex pathway. 3. The reflex vasodilatation was bigger in the stimulated than in the opposite foot. At the same time there were signs of skin vasoconstriction in the fingers. The reflex vasodilatation in the foot was enhanced by body cooling. It was unaffected by atropine or propranolol. 4. After local anaesthesia of the nerve proximal to the stimulation site, i.n.s. with 2-6 times increased stimulation strength produced skin vasodilatation restricted to the innervation zone of the stimulated nerve fascicle. This response had greater amplitude and longer duration than the reflex vasodilatation. 5. Intravenously administered atropine and propranolol did not affect the local dilatation to i.n.s. but after chronic treatment of the skin with capsaicin (1 % in ethanol), i.n.s. after a proximal nerve block induced skin vasoconstriction. In five of seven experiments subcutaneous injection of terbutaline (0'25 mg) in the innervation zone abolished the local dilatation. 6. It is suggested that (a) the reflex vasodilatation is of sympathetic nature and is induced by stimulation of thin (Ad?) afferent fibres, (b) the local vasodilatation is due to centrifugally conducted impulses in (afferent?) non-myelinated fibres. INTRODUCTION Different neural mechanisms participate in regulation of skin blood flow in man. Cutaneous arterioles and arterio-venous (a.v.) anastomoses are supplied by adrenergic vasoconstrictor neurones and reflex vasoconstriction is induced by body cooling and

2 106 H. BLUMBERG AND B. GUNNAR WALLIN also by arousal and emotional actions (Folkow, 1955; Delius, Hagbarth, Hongell & Wallin, 1972). Body warming reduces vasoconstrictor activity (Bini, Hagbarth, Hynninen & Wallin, 1980) and in addition may activate a neurally mediated non-cholinergic vasodilator mechanism, the exact nature of which is unknown (Rowell, 1981). In addition to these generalized mechanisms there is evidence that local vasodilatation may be induced by an axon reflex on stimulation of cutaneous polymodal nociceptor fibres (Bayliss, 1901; Lewis, 1927; Celander & Folkow, 1953; Kenins, 1982). Recently it was proposed that this dilatation is due to antidromic impulses releasing a peptide (e.g. substance P) from the sensory nerve terminals (Hdkfelt, Johannson, Kellerth, Ljungdahl, Nilsson, Nygards & Pernow, 1977; Foremann & Jordan, 1983; Helme & McKernan, 1983). In a previous study in man we stimulated cutaneous nerves electrically via an intraneural micro-electrode and recorded vasomotor responses within the innervation zone with a photo-electric pulse plethysmograph (Wallin, Blumberg & Hynninen, 1983). In many experiments little or no skin vasoconstriction was detected although recordings of skin electrical resistance showed that the stimulation was effective in activating non-myelinated post-ganglionic sudomotor fibres. A possible explanation for the weak vasoconstrictor responses would be that the stimuli activated both vasoconstrictor and vasodilator mechanisms which opposed each other. To test this hypothesis the present study was undertaken in order to search for evidence that intraneural stimulation (i.n.s.) induces cutaneous vasodilatation. METHODS With the consent of each subject, investigations were made on seventeen healthy adults, ten men and seven women, whose age ranged from nineteen to forty-three years. The investigation was approved by the ethical committee of the Medical Faculty of the University of Freiburg. Nerve electrodes, recording, stimulation and display systems Micro-electrode recordings and intraneural stimulations were made with tungsten microelectrodes in cutaneous fascicles of the superficial peroneal nerve at the ankle (for technical details see Vallbo, Hagbarth, Torebjork & Wallin, 1979). The electrode, insulated by Volta lacquer, had a shaft diameter of 0-2 mm and a tip diameter of a few micrometres. The reference electrode, similar but with a larger uninsulated tip, was inserted subcutaneously 1-2 cm away. For recordings the signal was amplified in two steps with a total gain of 2 or 5x 104. Signal-to-noise ratio was improved by the use of a Hz bandpass filter and an amplitude discriminator. An RC-integrating network with a time constant of 0 1 s was used to obtain a mean voltage display of the multi-unit nerve activity. The mean voltage neurogram was stored together with other parameters (see below) on a seven channel FM tape recorder (Kyberna 601). During experiments neural activity was monitored continuously on a storage oscilloscope and a loudspeaker. For intraneural stimulation the same electrode was connected (via a switch on the pre-amplifier) to the output of a constant-voltage stimulator (T6nnies), delivering square-wave pulses (01-15 V, 0-2ms). With electrode impedances of kq this would correspond to currents of 3-30,uA. Skin blood flow. Skin blood flow was monitored on the dorsal surface of the feet by laser-doppler flowmeters (Periflux, Perimed AB, Stockholm, Sweden). Electrical calibration for zero blood flow was made in all recordings. Several gains for the analogue output were selectable by switches. The maximum output of a given gain level (defined electrically) was taken as 100 %. In some figures the signal has been subject to further amplification to facilitate comparisons between recordings with different gains. The analogue output of this equipment gives no absolute values but relative changes of cutaneous blood flow (for technical details and evaluation of the laser-doppler flowmeter see Holloway & Watkins, 1977; Nilsson, Tenland & Oberg, 1980; Johnson, Taylor, Shepherd &

3 HUMAN SKIN VASODILATATION Park, 1984.) On the plantar side of toes and on the palmar side of fingers vasomotor reactions were monitored by photo-electrical pulse plethysmographs (modified van Gough type ILP/7 A). Skin resistance changes. Skin resistance changes (galvanic skin response, g.s.r.) were monitored by a modified van Gough g.s.r. module (type IGSR/7A a.c. coupled) with Ag/AgCl electrodes fixed to the skin on the plantar side of toes or on the palmar side of fingers. Skin temperature. Measurements were made with a multichannel thermometer (Exacon 2000), the thermocouple probes of which were taped to the skin at the tip of the big toes. The electrocardiogram (e.c.g.) was monitored via surface electrodes on the chest. Changes in ambient temperature. Before the experiment started subjects were put into a thermal suit, lined with plastic tubing in which running water of different temperatures was used to cool or warm the subject. Pain rating. In several experiments the pain evoked by intraneural stimulation was monitored using a ten point visual analogue scale. Drugs. In a few experiments atropine (0 04 mg/kg body weight) and propranolol (0-15 mg/kg body weight) were given intravenously to achieve full cholinergic and fl-adrenergic blockade, respectively. The,-2-mimetic substance terbutaline (0-25 mg) was injected subcutaneously in some experiments into the skin area under the laser-doppler flowmeter probe. Local anaesthesia. For local anaesthesia (5 % bupivacain 1-5 ml) the nerve was localized by electrical stimulation delivered through an injection needle, which was Teflon coated except at the tip. The effectiveness of anaesthesia was confirmed by testing (a) that mechanical skin stimuli within the innervation zone of the anaesthetized nerve were not felt by the subject, and (b) that subjects did not feel electrical stimuli which previously had been painful. Capsaicin treatment. In one subject a skin area on the dorsum of the foot innervated by the superficial peroneal nerve was treated (eleven times) for 3 days with capsaicin (1 % in ethanol) before the actual experiment. For the first two applications the skin was pre-treated with dimethyl sulphoxide (DMSO). Experimental procedure Subjects were tested while lying comfortably in the thermal suit. In most experiments water temperature in the suit was adjusted so that the skin temperatures were C on the tip of the big toe. The micro-electrode was inserted manually and its position adjusted until a skin nerve fascicle was impaled, as judged by (1) the appearance of an insertion shower of impulses accompanied by skin paraesthesia and (2) mechanoreceptive afferent impulses arising in response to touch stimuli within the fascicular receptive field. The occurrence and nature of sympathetic activity was determined as described elsewhere (Vallbo et al. 1979). I.n.s. was made irrespective of whether or not sympathetic activity was recorded. Blood flow probes were put on the skin inside the innervation zone and on the corresponding skin area of the contralateral foot. The probes of the pulse plethysmograph were fixed to palmar and plantar sides of the ipsilateral big toe or to an ipsilateral finger. For intraneural stimulation the recording micro-electrode was connected to the stimulator. The magnitude of perceived pain elicited by i.n.s. was judged by the subject using an analogue visual scale. Pain ratings were given by memory 1-2 min after i.n.s. in order to ensure that flow responses were not affected by the rating procedure. During experiments 15 s-2 min periods of intraneural stimulation at 2-10 Hz were applied at different stimulation strengths and under the various conditions described in the Results. 107 RESULTS Most receptive fields of afferent fibres of the impaled nerve fascicles were situated on the dorsum of the foot and only in few cases on the medial side of the foot. Twenty-seven recording sites were obtained in twenty-three experiments. In sixteen sites spontaneous and/or reflexly evoked sympathetic post-ganglionic activity was recorded but in eleven cases no clear sympathetic activity occurred. No differences were found with respect to the blood flow responses whether sympathetic activity was recorded or not.

4 108 H. BLUMBERG AND B. GUNNAR WALLIN In twenty-one of the twenty-three experiments (twenty-four of twenty-seven recording sites) continuous i.n.s. for 30 s-1 min led to a reproducible increase of skin blood flow within the innervation zone of impaled fascicle. The dilatory responses had varying time courses (Fig. 1). In most stimulations the dilatation started after a delay of approximately 15 s and reached its peak within the period 100- AStim. 2 V i Flow BC,', B % A Ail probe A v C 100 %ls 2-5 V 0-75 V 1 min Fig. 1. Cutaneous blood flow increases of different time courses recorded with laser-doppler flowmeter during i.n.s. (horizontal bar) in the superficial peroneal nerve (frequency 2 Hz, pulse duration 0-2 ms). Upward deflexion indicates flow increase in this and all subsequent Figures. Receptive fields of the recorded afferent fibres indicated by shaded areas on the foot and * indicates position of flow probe. A and C obtained in the same experiment from different electrode sites, B from another subject. of stimulation (Fig. 1 A). In a few cases maximum dilatation occurred after the end of stimulation (Fig. 1 B) and in a single case magnitude and duration of the dilatory response was markedly greater than in all other observations (Fig. 1 C). It is worthy of notice that Fig. 1 A and C were obtained in different electrode sites in the same experiment and that stimulation strength was lower in Fig. 1C but perceived pain was nevertheless greater with a diffuse, dull component. In principle the blood flow increase could be caused by a sympathetic reflex, antidromic activation of afferent fibres or direct stimulation of sympathetic fibres. Experiments were carried out to differentiate between these possibilities. Evidence for reflex vasodilatation In twelve experiments bilateral laser-doppler recordings of skin blood flow showed that painful intraneural stimulation on one side induced a short-lasting increase of blood flow not only within the innervation zone of the stimulated nerve but also in the skin on the dorsal side of the opposite foot (Fig. 2 A). In two experiments

5 HUMAN SKIN VASODILATATION 109 bupivacain was injected around the opposite superficial peroneal nerve to anaesthetize the skin under the opposite flow probe. After anaesthesia intraneural stimulation no longer evoked vasodilatation in this area (Fig. 2B) but in a non-anaesthetized area of the same foot the blood flow increase still occurred (Fig. 2C). Local anaesthesia A B 100 Le Stim. l.a. Stim. C l~~~~~~~~a. l~~~~~~~~~~~~~.a. ~a l~a.4~stim. D Xtim. 10J - Ki [- l-i I--4 Fig. 2. Proof of reflex vasodilatation. A, i.n.s. in left superficial peroneal nerve induced cutaneous blood flow increases in both feet. B, reflex nature of vasodilatation in right foot proven by elimination of flow increase after local anaesthesia (l.a.) of right superficial peroneal nerve with 2 ml 5% bupivacain. C, outside anaesthetized area in right foot i.n.s. still induced blood flow increase. D, reflex nature of vasodilatation in left foot proven by elimination of flow increase after injection of 2 ml 5 % bupivacain around the nerve proximal to stimulation site. Stimulation parameters 1.1 V, 5 Hz, 02 ms for 1 min. Symbols as in Fig. 1. of the stimulated superficial peroneal nerve proximal to the stimulation site abolished responses to i.n.s. in both feet (Fig. 2D). The findings, which were similar in both experiments, prove that in both feet the blood flow increases were of a reflex nature. The reflex nature of the dilatory response within the innervation zone of the stimulated nerve was tested in six other experiments. In five of these local anaesthesia of the nerve proximal to the stimulation site abolished the blood flow increase (see below). In the sixth experiment a weak dilatation still remained after the anaesthesia suggesting that a minor part of the response was a local effect induced by direct stimulation of the distal part of the nerve. Variation of stimulation parameters. In six experiments stimulation strength was increased in steps from perception threshold to well above pain threshold. In eight out of nine fascicles no dilatation occurred if stimulation strength was below pain threshold. In one case a weak dilatation was seen at a non-painful stimulation

6 110 H. BLUMBERG AND B. GUNNAR WALLIN 'v,4 I Stim. enstim. B blood flow A C I Is D Pain rating v 1.5 V 1 75 V 1 min Fig. 3. A-C, increase of reflex vasodilatation and of pain rating with increasing strength of i.n.s. D, local anaesthesia (l.a.) of the superficial peroneal nerve proximal to site of stimulation abolished flow increase and perceived pain. Following each stimulation period subjects indicated magnitude of perceived pain by an analogue visual scale (lower tracing). Stimulation parameters 2 Hz, 0-2 ms for 1 min. Symbols as in Fig. 1. A B C C C Blood flow Pleth.hand" 0 Pain rating 0 09V 09V 1O0V 1 min Fig. 4. Effect of ambient temperature upon reflex dilatation. I.n.s. at 2 Hz, 0-9 V, 02 ms, in the superficial peroneal nerve induced clear increase of skin blood flow on the dorsal side of the stimulated foot when subject was cool (A) but not when warm (B) (similar pain rating in A and B). With increase of stimulation strength to 10 V (higher pain rating) vasodilatation re-appeared (C). Note that during stimulation period in C photo-electric pulse plethysmogram from tip of thumb (pleth i.l. hand) showed reduced pulse amplitudes (indicating finger vasoconstriction) coinciding with blood flow increase in foot. Increase of basal foot blood flow and finger pulse amplitudes between A and B related to increase of skin temperature from 19-2 to 33 TC in big toe. I

7 HUMAN SKIN VASODILATATION strength. The response was smaller than those in the same experiment that were elicited by painful stimulation. When strength (seventeen experiments) or frequency (ten experiments) of stimulation was varied there was a good correlation between the subject's pain rating and the degree of blood flow increase. This is illustrated in Fig. 3 which shows that increase of stimulation voltage from 1 V (A) to 1-5 V (B) and 1-75 V (C) elicited successively bigger dilatory responses. To prove that the vasodilation, which in this case was observed inside the innervation zone was of reflex nature the nerve was anaesthetized proximal to the stimulation site. Following anaesthesia the blood flow increase disappeared (D). Increase of frequency (2-5 Hz) at a given voltage also induced higher pain ratings and bigger flow increases. Variation of ambient temperature. In six experiments we studied the effect of cooling or warming upon reflexly evoked skin vasodilatation. In these experiments warming diminished the vasodilatory response whereas cooling enhanced it. Fig. 4A shows that when the subject was cold a typical reflex dilatation was elicited on the ipsilateral foot. When the subject was warmed basal skin blood flow increased both in the foot (laser-doppler flowmeter) and the hand (pulse plethysmogram) but no flow increase was induced by intraneural stimulation although the pain rating was unchanged (Fig. 4B). When stimulation strength was increased, however, a clear vasodilatation occurred, accompanied by a higher pain rating (Fig. 4C). Fig. 4 also shows that when skin vasodilatation occurred in the foot, clear signs of vasoconstriction were seen in the hand, indicated by reduced amplitudes in the plethysmogram. Spatial organization. When blood flow responses to i.n.s. recorded with the laser-doppler technique were compared between corresponding skin areas on the two feet (twelve experiments) responses on the ipsilateral side always had a higher peak amplitude. The difference was the same whether the flow recording on the stimulated side was made inside or outside the innervation zone. A typical example is shown in Fig. 5A. In seven experiments plethysmographic finger pulsations were large enough (i.e. skin temperature high enough) to permit evaluation of vasomotor effects in the finger to intraneural stimulation in the peroneal nerve. In all these experiments the stimulation evoked blood flow increases on the dorsal side of the foot (laserdoppler) and pulse amplitude reductions in the finger indicating that vasodilatation occurred in the foot and vasoconstriction in the hand (Fig. 5B, see also Fig. 4C). Pharmacological effects. To test whether cholinergic or fl-adrenergic mechanisms might be involved in the vasodilatory response to i.n.s. an experiment was carried out in which atropine (004 mg/kg) and propranolol (0 15 mg/kg) were given intravenously to block peripheral cholinergic or fl-adrenergic effects. Before giving any drug, i.n.s. elicited vasodilatation on the opposite foot accompanied by skin resistance changes (Fig. 6A). Resting heart rate oscillated around 60 beats/min. Following i.v. injection of atropine heart rate increased to 120 beats/min and spontaneous and evoked skin resistance changes disappeared. I.n.s. still evoked vasodilatation (Fig. 6B). Injection of propranolol led to a decrease of heart rate to around 100 beats/min but did not change the dilatory response to i.n.s. (Fig. 6C). III

8 112 H. BLUMBERG AND B. GUNNAR WALLIN A B i1% Stim. in -Stim. I ~~~~~~ % Fig. 5. Spatial organization of reflex vasodilatation. A, greater increase of skin blood flow induced by i.n.s. in stimulated than in opposite foot. B, reduction of pulse amplitudes indicating vasoconstriction in finger (lower trace) and increase of skin blood flow in foot (upper trace) in response to i.n.s. of superficial peroneal nerve. Stimulation parameters, 2 Hz, 1-5 V, 0-2 ms for 1 min in both A and B (same subject). Symbols as in Fig Blood flow %, 0 g.s.r. 2 R-R interval 0 S I K A B C Atropine Propranolol I 0-04 mg/kg 0-15 mg/kg 1 min Fig. 6. Reflex dilatation to i.n.s. in the opposite superficial peroneal nerve (A), remaining after i.v. injection of atropine, 0 04 mg/kg (B), and propranolol, 0-15 mg/kg (C). Cholinergic blockade indicated by decreased cardiac interval (increase of heart rate from 120 to 100 beats/min) and elimination of skin resistance variations (g.s.r.) in the sole of the foot. After propranolol cardiac interval increased (heart rate decreased from 120 to 100 beats/min). The skin resistance changes at the end of panel (A) occurred when the subject was talking (when zero calibrations of R-R interval were made). A-C from the same experiment. Stimulation parameters 1-85 V, 5 Hz, 0-2 ins. Localized vasodilatation As demonstrated above, nerve blockade by local anaesthesia proximal to the stimulation site usually abolished skin vasodilatory responses within the innervation zone of the stimulated nerve fascicle. If, however, stimulation strength was raised 2-6-fold after nerve block, vasodilatation occurred again (Fig. 7). The test was made in ten experiments and all results were similar, except in the experiment with a capsaicin pre-treated skin (see below). As seen in Fig. 7 C, the blood flow increase after proximal nerve block was strong and long lasting and similar to that observed in one case without proximal nerve block (Fig. 1 C).

9 HUMAN SKIN VASODILATATION 113 Stim. Stim. 50 ILa. _. Stim. 100] J 1 min 6 V Fig. 7. Comparison of cutaneous blood flow increase to i.n.s. before and after proximal local anaesthesia. Before local anaesthesia (l.a.) i.n.s. at 1 V induced a small and shortlasting flow increase (A). Anaesthesia proximal to the stimulated site abolished the dilatory response to i.n.s. (B). The reason for the post-stimulus dilatation in B, not observed in other cases, is unknown. Increase of stimulation strength (6 V) induced more pronounced long-lasting blood flow increase (C). Time between vertical black bars = 10 min. Note blood flow oscillations after peak of flow (see also Figs. 9 and 10). Stimulation parameters 2 Hz, 0-2 ms. Symbols as in Fig. 1. In contrast to the reflex vasodilatation the blood flow increase elicited after proximal nerve block was localized. It never occurred on the opposite side (nine experiments) and in two experiments when blood flow was monitored at two sites on the dorsum of the stimulated foot, the flow increase occurred only within the innervation zone of the stimulated nerve fascicle. This is illustrated in Fig. 8. The local dilatory responses had a different time course than the reflex dilatation. The delays to onset of dilatation were similar ( s, mean + S.D., twenty-eight observations for the local and s, sixty-seven observations for the reflex response, difference not statistically significant) whereas time of maximum dilatation from start of i.n.s. differed markedly ( s for the local and s for the reflex response, significant difference, P < t test). No systematic analysis was made for the difference in duration and magnitude between local and reflex vasodilatation. In general, the reflex response lasted less than 2-5 min whereas local dilatory responses always lasted more than 10 min and in some cases up to 45 min. As for the reflex vasodilatory response an increased stimulation strength led to an augmented local vasodilatation (Fig. 9). As seen in Fig. 9A stimulation with 1-1 V evoked no dilatory response. Stepwise increases in stimulation strength to 1-5 V (B), 2 V (C) and 4 V (D) led to successively bigger dilatory responses. After the peak response has been reached, skin blood flow often tended to oscillate (C, D, see also

10 114 H. BLUMBERG AND B. GUNNAR WALLIN 30 I I~~~~~~ % A Stim, 0 V V _ DO I I 30 % ILa. +: 0 stim.- I75 1 min Fig. 8. Evidence for localized character of the dilatory responses following proximal local anaesthesia. A, before anaesthesia, i.n.s. (079 V) induced cutaneous blood flow increase *~~~~~~~~~~~~~~~ inside and outside the receptive field of the impaled nerve fascicle. B, after local anaesthesia (l.a.) of the nerve proximal to the stimulation electrode, i.n.s. (3 V) induced blood flow increase only within the receptive field (lower trace). Stimulation parameters 2 Hz, 0-2 ms for I min. Figs. 9 and 10). The frequency of this oscillation was unrelated to heart rate or to respiration. Pharmacological effects. In two experiments atropine and propranolol were injected intravenously to achieve complete cholinergic and s-adrenergic receptor blockade. Following injection of atropine, 0s04 mg/kg, i.n.s. still evoked a typical long-lasting vasodilatation and the response remained unchanged also after propranolol 0215 mg/kg. The effectiveness of the receptor blockades was indicated by absence of sweating and the occurrence of tachycardia after atropine and heart rate reduction after propranolol. In seven experiments (six subjects) 0a25 mg of the 2-mimetic substance terbutaline was injected subcutaneously under the flow probe. In five experiments the injection caused some increase of basal blood flow (possibly a directkmimetic effect) but the vasodilatory response to i.n.s. disappeared. In four of these experiments stimulation after terbutaline even resulted in vasoconstriction. The result from one

11 HUMAN SKIN VASODILATATION 115 I.a.- A B C :1l 1-1 V 1 l I % D Of 4l 1 min Fig. 9. Relationship between stimulation strength and degree of blood flow increase within receptive field after local anaesthesia (l.a.) of the nerve proximal to the stimulation site. Stimulation parameters 2 Hz, 0-2 ms. Symbols as in Fig. 1. Id Q /I D100 Stim. Jo 100,, O~~~~~~~ 30 s 30Os 1 min Terbutaline 0-25 mg s.c. Fig. 10. Effect of terbutaline upon the localized vasodilatation. A, i.n.s. following local anaesthesia (l.a.) induced flow increases at two skin areas inside the innervation zone of the stimulated nerve fascicle. B. subcutaneous injection of 0-25 mg terbutaline under the distal flow probe led to an increased basal flow probe in that skin area (lower signal) but i.n.s. no longer evoked a dilatory response. Flow still increased under the proximal flow probe (upper signal). C, 35 min later i.n.s. resulted in a flow reduction in both skin areas. Note that before the stimulation in C basal blood flow had increased also at the proximal probe, probably due to spread of terbutaline from injection site to the skin area of the proximal flow probe. Stimulation parameters 10 V, 10 Hz, 0-2 ms. Symbols as in Fig. 1. such experiment is shown in Fig. 10. Before terbutaline was injected stimulation induced an increase of skin blood flow under both probes with somewhat different time courses (Fig. IOA). 1-2 min after terbutaline has been injected under the distal flow probe i.n.s. no longer evoked a flow increase at this probe, whereas at the proximal probe the blood flow increase remained unchanged (Fig. lob). 35 min later

12 116 H. BLUMBERG AND B. GUNNAR WALLIN renewed i.n.s. resulted in a transient decrease of flow at both probes. Subcutaneous injection of 0 9 % NaCl (two experiments) did not influence the effect of i.n.s. In one experiment the skin on the dorsal side of the foot had been treated repeatedly by capsaicin before i.n.s. was applied. Reflex vasodilatation could still be elicited inside and outside the pre-treated skin area, but after proximal nerve block high voltage i.n.s. caused a blood flow decrease. This is illustrated in Fig stim. L~a. X Stim. A B C Tmin 1mm ' 2 Hz/4 V 2 Hz/4 V 5 Hz/15 V Fig. 11. Effect of i.n.s. upon cutaneous blood flow in a skin area pre-treated with capsaicin (stippled area). A, stimulation at 2 Hz, 4 V, 0-2 ms led to short-lasting increase of skin blood flow which disappeared after proximal local anaesthesia (l.a.) (B) proving its reflex nature. C, stimulation at 5 Hz, 15 V, 0-2 ms, induced decrease of skin blood flow. Symbols as in Fig. 1. DISCUSSION The present results provide direct evidence of two types of neurally mediated vasodilatation in hairy skin of the human foot. Both types were evoked by intraneural stimulation but there is evidence that one was a reflex effect induced by stimulation of afferent nerve fibres and the other a local effect due to centrifugal conduction of impulses to the innervation zone of the impaled nerve fascicle. Reflex dilatation J.n.s. of one superficial peroneal nerve caused short-lasting cutaneous vasodilatation on the dorsal side of both feet, irrespective of whether the electrode site allowed recording of sympathetic impulses or not. For unknown reasons, reflex vasodilatation could only be obtained in twenty-four of twenty-seven electrode sites. Since the dilatation disappeared when the corresponding skin nerve was anaesthetized, a humoral mechanism was excluded. In most cases no reflex dilatation occurred until stimulation strength was high enough to cause sharp and localized pain. This suggests that the afferent limb of the reflex may include Ad fibres (Adriaensen, Gybels, Handwerker & van Hees, 1983). Whether only pain fibre stimulation was important or if cold fibres which also belong to the Ad group contributed to the reflex dilatation is unclear. With the exception of warm receptor stimulation no other

13 HUMAN SKIN VASODILATATION 117 stimulus is known to elicit reflex skin vasodilatation in healthy humans. In patients suffering from reflex sympathetic dystrophy, however, reflex vasodilatation can be evoked by noxious heat stimulation of the skin (H. Blumberg, F. Hibler & M. Hollerbach, unpublished observation). The only known efferent skin fibres are sympathetic. Thus we conclude that our reflex dilatation is a sympathetic reflex induced by stimulation of afferent Ad fibres. This conclusion is supported by the observation that we were not able to elicit reflex dilatation in a sympathectomized skin area (H. Blumberg & B. G. Wallin, unpublished observation). There are three types of sympathetic fibres that could mediate our reflex dilatation, (a) vasoconstrictor, (b) vasodilatator and (c) sudomotor neurones. (a) To get clear vasodilatory responses to i.n.s. subjects had to be cooled and under cool conditions skin vasoconstrictor activity is high (Bini et al. 1980). Therefore if i.n.s. caused inhibition of vasoconstrictor nerve traffic, vasodilatation would occur. This possibility would agree with findings in animals. In cats, noxious skin stimulation caused reflex inhibition of skin vasoconstrictor activity which was stronger on the stimulated than on the opposite side (Horeyseck & Jinig, 1974; Jinig, 1975; Grosse & JUnig, 1976). In rabbits, electrical stimulation of the central end of cutaneous nerves (n. auricularis, n. dorsalis pedis and n. tibialis) induced dilatation of cutaneous vessels bilateraly which was more pronounced on the stimulated than the opposite side (Loven, 1866). The results of both studies agree with our findings of a more pronounced blood flow increase on the stimulated compared to the opposite foot. On the other hand, we found i.n.s. to cause reflex vasodilatation on the dorsal side of the foot and vasoconstriction in the palm of the hand (Figs. 4 and 5) and in previous studies reflex changes of skin sympathetic activity have always occurred in parallel in all four extremities (Vallbo et al. 1979; Wallin, 1981; Janig, Sundlof & Wallin, 1983; Blumberg & Oberle, 1984). (b) Activation of vasodilator neurones could also be responsible for the observed reflex dilatation. So far, no direct evidence for the existence of skin vasodilator neurones is available in humans (Rowell, 1981). Indirect evidence was presented by Shepherd and by Edholm who found a decrease of skin blood flow after sympathectomy (Shepherd, 1963) or when skin nerves were anaesthetized (Edholm, Fox & MacPherson, 1957) after maximal warming of the subject. From these experiments the authors concluded that vasodilator neurones must have contributed to the vasodilated state before nerve section or block. In cats, stimulation of the lumbar sympathetic trunk induced skin vasodilatation, indicating the existence of sympathetic skin vasodilator neurones in this species (Bell, Janig, Kiummel & Xu, 1985). It is not known, however, if such neurones can be activated by noxious skin stimulation. (c) Sympathetic sudomotor neurones are activated reflexly by noxious stimulation in cats and man but there is no report about spatial organization of such reflexes (Janig & Rath, 1977; Janig et al. 1983). Whether vasodilatation can be induced by sudomotor activation and mediated by the release of substances in the sweat is controversial (for review see Rowell, 1981). Brengelmann, Freund, Rowell, Olerud & Kraning (1981) found that in patients with congenital absence of sweat glands heat stimulation did not induce skin vasodilatation. However, it was not proved that the vasoconstrictor system was normal in these patients.

14 118 H. BLUMBERG AND B. GUNNAR WALLIN From our data we cannot exclude that vasodilator or sudomotor neurones contribute to the observed reflex dilatation. If so, the preserved blood flow increase after atropine and propranolol suggests a non-cholinergic, non-,f-adrenergic mechanism. This is consistent with the observation of Bell et al. (1985) who found that atropine blocked sweating but not skin vasodilatation in response to stimulation of the lumbar sympathetic trunk. Our data give no evidence concerning the physiological role of reflex vasodilatation. Baroreceptor-induced inhibition of vasoconstrictor activity (in response to a blood pressure increase induced by i.n.s.) cannot be excluded but seems unlikely since arterial baroreceptor influence on skin vasoconstrictor activity is weak or absent (Bini, Hagbarth & Wallin, 1981; Fagius, Wallin, Sundldf, Nerhed & Engelsson, 1985). Furthermore, such a mechanism would not be expected to have different effects on different extremities. An arousal reaction is also unlikely since previously, only vasoconstriction has been described in response to arousal stimuli (Jinig et al. 1983). Local dilatation Vasodilatation restricted to the receptive field of the impaled nerve fascicle was obtained with i.n.s. following a proximal nerve block. The stimulation strength, necessary to induce this type of vasodilatation, had to be considerably higher (2-6-fold) than the stimulation strength which induced reflex dilatation. We have no direct evidence about the type of fibres mediating the local response. However, since Ad fibres had to be stimulated for reflex dilatation to occur, it is likely that the increased stimulation strength activated also C fibres. This is supported by two kinds of evidence. First, from previous experiments using an identical technique we know that non-myelinated sudomotor neurones will be stimulated by i.n.s. (Wallin et al. 1983). Secondly, vasoconstrictor neurones are probably also excited since i.n.s. induced local vasoconstriction after subcutaneous injection of terbutaline (Fig. 10) and after capsaicin pre-treatment (Fig. 11). C fibres include post-ganglionic sympathetic vasoconstrictor neurones, sudomotor and vasodilator fibres as well as nonmyelinated afferent fibres. Again, as mentioned in the discussion of the reflex dilatation, if the vasodilatation was induced by sympathetic neurones it must have been a non-cholinergic, non-,l-adrenergic mechanism, since the response was unchanged after atropine and propranolol. By definition, stimulation of vasoconstrictor neurones does not cause vasodilatation, but we cannot exclude that sudomotor or vasodilator neurones contribute. Recently Bell et al. (1985) found evidence of skin sympathetic vasodilator neurones in cats but in man it is not known if they exist. Recently we found that i.n.s. induced long-lasting vasodilatation in a sympathectomized hand (H. Blumberg & B. G. Wallin, unpublished observation) indicating that local vasodilatation can be induced in the absence of sympathetic neurones. Therefore we suggest that the local dilatation is induced by antidromic stimulation of afferent C fibres. The disappearance of the vasodilatation after capsaicin pre-treatment also agrees with this interpretation (see below). Bell et al. (1985) found in cats that antidromic stimulation of the peripheral end of cut dorsal roots resulted in a localized skin vasodilatation which was not blocked by atropine or hexamethonium. Couture & Cuello (1984) showed in rats that

15 HUMAN SKIN VASODILATATION antidromic stimulation ofsensory trigeminal branches led to cutaneous vasodilatation which was not altered by decentralization or sympathectomy. They suggested that cholinergic, histaminergic and serotoninergic mechanisms as well as substance P may contribute to the neurogenic vasodilatory response. Antidromic vasodilatation has also been induced in man by electrical stimulation of single dorsal roots (Foerster, 1933). Since the studies of Lewis (1927) it is known that afferent C fibres may induce skin vasodilatation in man by release of a vasodilatory substance via an axon reflex. So far, the most likely candidate to induce antidromic skin vasodilatation seems to be substance P (Lembeck & Gamse, 1982; Foremann & Jordan, 1983). This would agree with our finding that local dilatation did not occur after chronic treatment of the skin with capsaicin, a substance known to deplete non-myelinated nociceptor afferent fibres of substance P (Bernstein, Swift, Soltani & Lorincz, 1981; Helme & McKernan, 1983; Szolesanyi, 1983). Substance P may induce vasodilatation by acting directly on the vessel, by exciting non-myelinated nerve endings and by releasing histamine, via mast-cell activation (Erjavec, Lembeck, Florjan-Irman, Skofitsch, Donnerer, Saria & Holzer, 1981; Foremann & Jordan, 1983). The f,- 2-mimetic terbutaline is known to prevent histamine release from mast cells (Kennes, De Maubeuge & Delespesse, 1977; Gronneberg, Strandberg & Hagermark, 1980; Ting, Zweimann & Lavker, 1983) and this may explain why in some of our experiments i.n.s. no longer evoked local vasodilatation after injection of terbutaline. The transformation of the dilatory response into vasoconstriction (Fig. 10) suggests that before terbutaline, i.n.s., in addition to causing local vasodilatation, also activated a vasoconstrictor mechanism (probably direct stimulation of vasoconstrictor fibres) which was unmasked by the effect of the drug. We thank Professor R. Westermann, Monash University, Australia, for helpful suggestions and being subject in the capsaicin experiment. The skilful technical assistance of A. Boger-Koch and J. Oberle, is gratefully acknowledged. Supported by Swedish Medical Research Council, Grant No. B85-04X A and Swedish Workers Health Fund, Grant No REFERENCES ADRIAENSEN, H., GYBELS, J., HANDWERKER, H. 0. & VAN HEES, J. (1983). Response properties of thin myelinated (A-delta) fibres in human skin nerves. Journal of Neurophysiology 49, BAYLISS, W. M. (1901). On the origin from the spinal cord of the vasodilator fibres of the hind limb, and on the nature of these fibres. Journal of Phy8iology 26, BELL, C., JANIG, W., KfUMMEL, H. & Xu, H. (1985). Differentiation of vasodilatator and sudomotor responses in the cat paw pad to preganglionic sympathetic stimulation. Journal of Physiology 365, BERNSTEIN, J. E., SWIFT, R. M., SOLTANI, K. & LORINCZ, A. L. (1981). Inhibition of axon reflex vasodilatation by topically applied capsaicin. Journal of Investigative Dermatology 76, BINI, G., HAGBARTH, K. E., HYNNINEN, P. & WALLIN, B. G. (1980). Thermoregulatory and rhythm-generating mechanisms governing the sudomotor and vasoconstrictor outflow in human cutaneous nerves. Journal of Physiology 306, BINI, G., HAGBARTH, K. E. & WALLIN, B. B. (1981). Cardiac rhythmicity of skin sympathetic activity recorded from peripheral nerves in man. Journal of the Autonomic Nervous System 4, BLUMBERG, H. & OBERLE, J. (1984). Changes of activity in sympathetic postganglionic neurones supplying skin induced by stimulation of cutaneous nociceptors in humans. Pfluigers Archiv 400, suppl., R 52.

16 120 H. BLUMBERG AND B. GUNNAR WALLIN BRENGELMANN, G. L., FREUND, P. R., ROWELL, L. B., OLERUD, J. E. & KRANING, K. K. (1981). Absence of active cutaneous vasodilatation associated with congenital absence of sweat glands in man. American Journal of Physiology 240, H CELANDER, 0. & FOLKOW, B. (1953). The nature and the distribution of afferent fibres provided with the axon reflex arrangement. Acta physiologica scandinavica 29, COUTURE, R. & CUELLO, A. C. (1984). Studies on the trigeminal antidromic vasodilatation and plasma extravasation in the rat. Journal of Physiology 346, DELIUS, W., HAGBARTH, K.-E., HONGELL, A. & WALLIN, B. G. (1972). Manoeuvres affecting sympathetic outflow in human skin nerves. Acta physiologica scandinavica 84, EDHOLM, 0. G., Fox, R. H. & MACPHERSON, R. K. (1957). Vasomotor control of the cutaneous blood vessels in the human forearm. Journal of Physiology 139, ERJAVEC, F., LEMBECK, F., FLORJAN-IRMAN, T., SKoFITsCH, G., DONNERER, J., SARIA, A. & HOLZER, P. (1981). Release of histamine by substance P. Naunyn Schmiedebergs Archives of Pharmacology 317, FAGIUS, J., WALLIN, B. G., SUNDL6F, G., NERHED, C. & ENGELSSON, S. (1985) Sympathetic outflow in man after anaesthesia of the glossopharyngeal and vagus nerves. Brain 108, FOERSTER, 0. (1933). The dermatomes in man. Brain 56, FOLKOW, B. (1955). Nervous control of blood vessels. Physiological Reviews 35, FOREMANN, J. & JORDAN, C. (1983). Histamine release and vascular changes induced by neuropeptides. Agents and Actions 13, GRONNEBERG, R., STRANDBERG, K. & HAGERMARK, 0. (1980). Cutaneous response to allergen after local pretreatment with beta adrenoceptor stimulating and blocking agents. Allergy 35, GROSSE, M. & JANIG, W. (1976). Vasoconstrictor and pilomotor fibres in skin nerves to the cat's tail. Pfluigers Archiv 361, HELME, R. D. & McKERNAN, S. (1983). Flare responses in man following topical application of capsaicin. In Antidromic Vasodilatation and Neurogenic Inflammation, ed. CHAHL, L. A., SzoLCsANYI, J. & LEMBECK, F., pp Budapest: Akademiae Kiado. HOKFELT, T., JOHANNSON, O., KELLERTH, J. O., LJUNGDAHL, A., NILSSON, G., NYGARDS, A. & PERNOW, B. (1977). Immunohistochemical distribution of substance P. In Substance P, ed. VON EULER, U. S. & PERNOW, B. pp New York: Raven Press. HOLLOWAY, G. A. & WATKINS, D. W. (1977). Laser-doppler measurement of cutaneous blood flow. Journal of Investigative Dermatology 69, HOREYSECK, G. & JANIG, W. (1974). Reflexes in postganglionic fibres within skin and muscle nerves after noxious stimulation of skin. Experimental Brain Research 20, JiNIG, W. (1975). Central organization of somatosympathetic reflexes in vasoconstrictor neurones. Brain Research 85, JXNIG, W. & RXTH, B. (1977). Electrodermal reflexes in the cats paws elicited by natural stimulation of skin. Pfluigers Archiv 369, JXNIG, W., SUNDL6F, G. & WALLIN, B. G. (1983). Discharge patterns of sympathetic neurones supplying skeletal muscle and skin in man and cat. Journal of the Autonomic Nervous System 7, JOHNSON, J. M., TAYLOR, W. F., SHEPHERD, P. & PARK, M. K. (1984). Laser-doppler measurements of skin blood flow: comparison with plethysmography. Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology 56, KENINS, P. (1982). Response of single nerve fibres to capsaicin applied to the skin. Neuroscience Letters 29, KENNES, B., DE MAUBEUGE, J. & DELESPESSE, G. (1977) Treatment of chronic urticaria with a beta-2-adrenergic stimulant. Clinical Allergy 7, LEMBECK, F. & GAMSE, R. (1982). Substance P in peripheral sensory processes. In Substance P in the Nervous System, Ciba Symposium 91, pp London: Pitman. LEWIS, T. (1927). The Blood Vessels of the Human Skin and their Responses. London: Shaw and Sons. LOVEN, C. (1866).Uber die Erweiterung von Arterien in Folge einer Nervenerregung. Berichte fiber die Verhandlungen der koniglich sachsischen Gesellschaft der Wissenschaften zu Leipzig. Mathematisch-physische Classe 18, NILSSON, G. E., TENLAND, T. & OBERG, P. A. (1980). Evaluation of a laser doppler flowmeter for measurement of tissue blood flow. The Institute of Electrical and Electronics Engineers, New York, Transactions on Biomedical Engineering 27,

17 HUMAN SKIN VASODILATATION 121 ROWELL, L. B. (1981). Active neurogenic vasodilatation in man. In Vasodilatation ed. VANHOUTTE, P. M. & LEUSEN, I., pp New York: Raven Press. SHEPHERD, J. T. (1963). Physiology of the Circulation in Humans Limbs in Health and Disease. Philadelphia: Saunders. SZOLCSANYI, J. (1983). Capsaicin-sensitive chemoreceptive neural system with dual sensory-efferent function. In Antidromic Vasodilatation and Neurogenic Inflammation, ed. CHAL, L. A., SZOLCSANYI, J. & LEMBECK, F., pp Budapest: Akademia Kiado. TING, S., ZWEIMANN, B. & LAVKER, R. (1983). Terbutaline modulation of human allergic skin reactions. The Journal of Allergy and Clinical Immunology 71, VALLBO, A. B., HAGBARTH, K.-E., TOREBJORK, H. E. & WALLIN, B. G. (1979). Somatosensory proprioceptive and sympathetic activity in human peripheral nerves. Physiological Reviews 59, WALLIN, B. G. (1981). New aspects of sympathetic function in man. In Butterworth International Medical Reviews, Neurology 1, Clinical Neurophysiology, pp London: Butterworth. WALLIN, B. G., BLUMBERG, H. & HYNNINEN, P. (1983). Intraneural stimulation as a method to study sympathetic function in the human skin. Neuroscience Letters 36,